# Patent

## Title
Compositions and methods for inhibiting expression of the ALAS1 gene

## Abstract

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the ALAS1 gene, and methods of using such dsRNA compositions to alter (e.g., inhibit) expression of ALAS1.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 61/622,288, filed Apr. 10, 2012, the entire content of which is hereby incorporated in its entirety.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 19, 2013, is named A2038-719610_SL.txt and is 596,719 bytes in size.
FIELD OF THE INVENTION
The invention relates to the specific inhibition of the expression of the ALAS1 gene.
BACKGROUND OF THE INVENTION
The inherited porphyrias are a family of disorders resulting from the deficient activity of specific enzymes in the heme biosynthetic pathway, also referred to herein as the porphyrin pathway. Deficiency in the enzymes of the porphyrin pathway leads to insufficient heme production and to an accumulation of porphyrins, which are toxic to tissue in high concentrations.
Of the inherited porphyrias, acute intermittent porphyria (AIP, e.g., autosomal dominant AIP), variegate porphyria (VP, e.g., autosomal dominant VP), hereditary coproporphyria (copropophyria or HCP, e.g., autosomal dominant HCP), and 5′ aminolevulinic acid (also known as δ-aminolevulinic acid or ALA) dehydratase deficiency porphyria (ADP, e.g., autosomal recessive ADP) are classified as acute hepatic porphyrias and are manifested by acute neurological attacks that can be life threatening. The acute attacks are characterized by autonomic, peripheral, and central nervous symptoms, including severe abdominal pain, hypertension, tachycardias, constipation, motor weakness, paralysis, and seizures. If not treated properly, quadriplegia, respiratory impairment, and death may ensue. Various factors, including cytrochrome P450-inducing drugs, dieting, and hormonoal changes can precipitate acute attacks by increasing the activity of hepatic 5′-aminolevulinic acid synthase 1 (ALAS1), the first and rate-limiting enzyme of the heme biosynthetic pathway. In the acute porphyrias, e.g., AIP, VP, HCP and ADP, the respective enzyme deficiencies result in hepatic production and accumulation of one or more substances (e.g., porphyrins and/or porphyrin precursors, e.g., ALA and/or PBG) that can be neurotoxic and can result in the occurrence of acute attacks. See, e.g., Balwani, M. and Desnick, R. J., Blood, 120:4496-4504, 2012.
The current therapy for the actute neuroloigcal attacks in the intravenous administration of hemin (Panhematin®, Lundbeck or Normosang®, Orphan Europe), which provides exogenous heme for the negative feedback inhibition of ALAS1, and thereby, decreases production of ALA and PBG. Hemin is used for the treatment during an acute attack and for prevention of attacks, particularly in women with the actue porphyrias who experience frequent attacks with the hormonal changes during their menstrual cycles. While patients generally respond well, its effect is slow, typically taking two to four days or longer to normalize urinary ALA and PBG concentrations towards normal levels. As the intravenous hemin is rapidly metabolized, three to four infusions are usually necessary to effectively treat or prevent an acute attack. In addition, repeated infusions may cause iron overload and phlebitis, which may compromise peripheral venous access. Although orthotrophic liver transplantation is curative, this procedure has significant morbidity and mortality and the availability of liver donors is limited. Therefore, an alternative therapeutic approach that is more effective, fast-acting, and safe is needed. It would be particularly advantageous if such treatment could be delivered by subcutaneous administration, as this would preclude the need for infusions and prolonged hospitalization.
AIP, also referred to as porphobilinogen deaminase deficiency (PBGD), or hydroxymethylbilane synthase (HMBS) deficiency, is the most common of the acute hepatic prophyrias. It is an autosomal dominant disorder caused by mutations in the HMB-synthase (HMBS) gene that result in reduced, e.g., half-normal activity of the enzyme. Previously, a mouse model of AIP that has  ̃30% of wildtype HMBS activity was generated by homologous recombination Like human patients, these mice increase hepatic ALAS1 activity and accumulate large quantities of plasma and urinary ALA and PBG when administered porphyrinogenic drugs, such as phenobarbital. Thus, they serve as an excellent model to evaluate the efficacy of novel therapeutics for the acute hepatic porphyrias.
SUMMARY OF THE INVENTION
The present invention describes methods and iRNA compositions for modulating the expression of an ALAS1 gene. In certain embodiments, expression of an ALAS1 gene is reduced or inhibited using an ALAS1-specific iRNA. Such inhibition can be useful in treating disorders related to ALAS1 expression, such as porphyrias.
Accordingly, described herein are compositions and methods that effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the ALAS1 gene, such as in a cell or in a subject (e.g., in a mammal, such as a human subject). Also described are compositions and methods for treating a disorder related to expression of an ALAS1 gene, such as a porphyria, e.g., X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (Doss porphyria or ADP), acute intermittent porphyria (AIP), congenital erythropoietic porphyria (CEP), prophyria cutanea tarda (PCT), hereditary coproporphyria (coproporphyria, or HCP), variegate porphyria (VP), erythropoietic protoporphyria (EPP), or transient erythroporphyria of infancy. In some embodiments, the disorder is an acute hepatic porphyria, e.g., ALA deyhdratase deficiency porphyria (ADP), AIP, HCP, or VP. In certain embodiments, the disorder is ALA deyhdratase deficiency porphyria (ADP) or AIP.
In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.
As used herein, the term “iRNA,” “RNAi”, “iRNA agent,” or “RNAi agent” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript, e.g., via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of ALAS1 expression in a cell or mammal.
The iRNAs included in the compositions featured herein encompass a dsRNA having an RNA strand (the antisense strand) having a region, e.g., a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of an ALAS1 gene (e.g., a mouse or human ALAS1 gene) (also referred to herein as an “ALAS1-specific iRNA”). Alternatively, or in combination, iRNAs encompass a dsRNA having an RNA strand (the antisense strand) having a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of an ALAS1 gene (e.g., a human variant 1 or 2 of an ALAS1 gene) (also referred to herein as a “ALAS1-specific iRNA”).
In embodiments, the iRNA (e.g, dsRNA) described herein comprises an antisense strand having a region that is substantially complementary to a region of a human ALAS1. In embodiments, the human ALAS1 has the sequence of NM—000688.4 (SEQ ID NO:1) or NM—000688.5 (SEQ ID NO:382).
In other embodiments, an iRNA encompasses a dsRNA having an RNA strand (the antisense strand) having a region that is substantially complementary to a portion of an ALAS1 mRNA according to any one of Tables 2, 3, 6, 7, 8, 9, 14, or 15. In one embodiment, the iRNA encompasses a dsRNA having an RNA strand (the antisense strand) having a region that is substantially complementary to a portion of an ALAS1 mRNA, e.g., a human ALAS1 mRNA (e.g., a human ALAS1 mRNA as provided in SEQ ID NO:1 or SEQ ID NO:382).
In one embodiment, an iRNA for inhibiting expression of an ALAS1 gene includes at least two sequences that are complementary to each other. The iRNA includes a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand includes a nucleotide sequence that is substantially complementary to at least part of an mRNA encoding an ALAS1 transcript, and the region of complementarity is 30 nucleotides or less, and at least 15 nucleotides in length. Generally, the iRNA is 19 to 24 nucleotides in length.
In some embodiments, the iRNA is 19-21 nucleotides in length. In some embodiments, the iRNA is 19-21 nucleotides in length and is in a lipid formulation, e.g. a lipid nanoparticle (LNP) formulation (e.g., an LNP11 formulation).
In some embodiments, the iRNA is 21-23 nucleotides in length. In some embodiments, the iRNA is 21-23 nucleotides in length and is in the form of a conjugate, e.g., conjugated to one or more GalNAc derivatives as described herein.
In some embodiments the iRNA is from about 15 to about 25 nucleotides in length, and in other embodiments the iRNA is from about 25 to about 30 nucleotides in length. An iRNA targeting ALAS1, upon contact with a cell expressing ALAS1, inhibits the expression of an ALAS1 gene by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more, such as when assayed by a method as described herein. In one embodiment, the iRNA targeting ALAS1 is formulated in a stable nucleic acid lipid particle (SNALP).
In one embodiment, an iRNA (e.g., a dsRNA) featured herein includes a first sequence of a dsRNA that is selected from the group consisting of the sense sequences of Tables 2, 3, 6, 7, 8, 9, 14, and 15 and a second sequence that is selected from the group consisting of the corresponding antisense sequences of Tables 2, 3, 6, 7, 8, 9, 14 and 15.
In one embodiment, an iRNA (e.g., a dsRNA) featured herein has sense and/or antisense sequences selected from those of AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, AD-59129, AD-59124, AD-58874, AD-59125, AD-59105, AD-59120, AD-59122, AD-59106, AD-59126, and AD-59107 as disclosed herein in the Examples. In embodiments, the iRNA (e.g., dsRNA) has sense and/or antisense sequences selected from those of AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, and AD-59129.
The iRNA molecules featured herein can include naturally occurring nucleotides or can include at least one modified nucleotide, including, but not limited to a 2′-O-methyl modified nucleotide, a nucleotide having a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Such a modified sequence can be based, e.g., on a first sequence of said iRNA selected from the group consisting of the sense sequences of Table 2, and a second sequence selected from the group consisting of the corresponding antisense sequences of Table 2.
In one embodiment, an iRNA (e.g., a dsRNA) featured herein comprises a sense strand comprising a sequence selected from the group consisting of SEQ ID NO:330, SEQ ID NO:334, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:362, SEQ ID NO:366, SEQ ID NO:376, and SEQ ID NO:380.
In one embodiment, an iRNA (e.g., a dsRNA) featured herein comprises an antisense strand comprising a sequence selected from the group consisting of SEQ ID NO:331, SEQ ID NO:335, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:363, SEQ ID NO:367, SEQ ID NO:377, and SEQ ID NO:381.
In one embodiment, an iRNA (e.g., a dsRNA) featured herein comprises a sense strand comprising a sequence selected from the group consisting of SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:172, SEQ ID NO:176, SEQ ID NO:186, and SEQ ID NO:190. In one embodiment, an iRNA (e.g., a dsRNA) featured herein comprises an antisense strand comprising a sequence selected from the group consisting of SEQ ID NO:141, SEQ ID NO:145, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:173, SEQ ID NO:177, SEQ ID NO:187, and SEQ ID NO:191.
In one embodiment, an iRNA as described herein targets a wildtype ALAS1 RNA transcript variant, and in another embodiment, the iRNA targets a mutant transcript (e.g., an ALAS1 RNA carrying an allelic variant). For example, an iRNA featured in the invention can target a polymorphic variant, such as a single nucleotide polymorphism (SNP), of ALAS1. In another embodiment, the iRNA targets both a wildtype and a mutant ALAS1 transcript. In yet another embodiment, the iRNA targets a particular transcript variant of ALAS1 (e.g., human ALAS1 variant 1). In yet another embodiment, the iRNA agent targets multiple transcript variants (e.g., both variant 1 and variant 2 of human ALAS1).
In one embodiment, an iRNA featured in the invention targets a non-coding region of an ALAS1 RNA transcript, such as the 5′ or 3′ untranslated region of a transcript.
In some embodiments, an iRNA as described herein is in the form of a conjugate, e.g., a carbohydrate conjugate, which may serve as a targeting moiety and/or ligand, as described herein. In one embodiment, the conjugate is attached to the 3′ end of the sense strand of the dsRNA. In some embodiments, the conjugate is attached via a linker, e.g., via a bivalent or trivalent branched linker.
In some embodiments, the conjugate comprises one or more N-acetylgalactosamine (GalNAc) derivatives. Such a conjugate is also referred to herein as a GalNAc conjugate. In some embodiments, the conjugate targets the RNAi agent to a particular cell, e.g., a liver cell, e.g., a hepatocyte. The GalNAc derivatives can be attached via a linker, e.g., a bivalent or trivalent branched linker. In particular embodiments, the conjugate is




In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker, e.g., a linker as shown in the following schematic, wherein X is O or S




In some embodiments, X is O. In some embodiments, X is S.
In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below




In an aspect provided herein is a pharmaceutical composition for inhibiting the expression of an ALAS1 gene in an organism, generally a human subject. The composition typically includes one or more of the iRNAs described herein and a pharmaceutically acceptable carrier or delivery vehicle. In one embodiment, the composition is used for treating a porphyria, e.g., AIP.
In one aspect, an iRNA provided herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length and the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1 or 382.
In a further aspect, an iRNA provided herein is a double stranded RNAi (dsRNA) comprising a sense strand complementary to an antisense strand, wherein said antisense strand comprises a region of complementarity to an ALAS1 RNA transcript, wherein each strand has about 14 to about 30 nucleotides, wherein said double stranded RNAi agent is represented by formula (III):







sense:


5′ np-Na-(X X X)i-Nb-Y Y Y-Nb-(Z Z Z)j-Na-nq 3′


 


antisense:







(III)







3′ np′-Na′-(X′X′X′)k-Nb′-Y′Y′Y′-Nb′-(Z′Z′Z′)l-Na′-


nq′ 5′






    
    
        wherein:
        i, j, k, and 1 are each independently 0 or 1;
        p, p′, q, and q′ are each independently 0-6;
        each Na and Na′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides;
        each Nb and Nb′ independently represents an oligonucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof;
        each np, np′, nq, and nq′ independently represents an overhang nucleotide;
        XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides;
        modifications on Nb differ from the modification on Y and modifications on Nb′ differ from the modification on Y′.
    
    


In embodiments, the sense strand is conjugated to at least one ligand.
In embodiments, i is 1; j is 1; or both i and j are 1.
In embodiments, k is 1; l is 1; or both k and l are 1.
In embodiments, XXX is complementary to X′X′X′, YYY is complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.
In embodiments, the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end.
In embodiments, the Y′ is 2′-O-methyl.
In embodiments, the duplex region is 15-30 nucleotide pairs in length.
In embodiments, the duplex region is 17-23 nucleotide pairs in length.
In embodiments, the duplex region is 19-21 nucleotide pairs in length.
In embodiments, the duplex region is 21-23 nucleotide pairs in length.
In embodiments, the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof.
In embodiments, the modifications on the nucleotides are 2′-O-methyl, 2′-fluoro or both.
In embodiments, the ligand comprises a carbohydrate.
In embodiments, the ligand is attached via a linker.
In embodiments, the linker is a bivalent or trivalent branched linker.
In embodiments, the ligand is




In embodiments, the ligand and linker are as shown in Formula XXIV:




In embodiments, the ligand is attached to the 3′ end of the sense strand.
In embodiments, the dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 2 and 3. In embodiments, the dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 2, 3, 6, 7, 8 and 9. In embodiments, the dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15. In embodiments, the dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 14 and 15.
In embodiments, dsRNA has a nucleotide sequence selected from the group of sequences provided in Tables 3 and 8.
In a further aspect, an iRNA provided herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity to an ALAS1 RNA transcript, which antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from one of the antisense sequences listed in any one of Tables 2, 3, 6, 7, 8, 9, 14, or 15. In some such embodiments, the sense and antisense sequences are selected from those of the duplexes AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, AD-59129, AD-59124, AD-58874, AD-59125, AD-59105, AD-59120, AD-59122, AD-59106, AD-59126, and AD-59107 as disclosed herein in the Examples. In embodiments, the sense and antisense sequences are selected from those of the duplexes AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, and AD-59129.
In some embodiments, the dsRNA comprises at least one modified nucleotide.
In some embodiments, at least one of the modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
In some embodiments, the modified nucleotide is chosen from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
In some embodiments, the region of complementarity is at least 17 nucleotides in length.
In some embodiments, the region of complementarity is between 19 and 21 nucleotides in length.
In some embodiments, the region of complementarity is 19 nucleotides in length.
In some embodiments, each strand is no more than 30 nucleotides in length.
In some embodiments, at least one strand comprises a 3′ overhang of at least 1 nucleotide.
In some embodiments, at least one strand comprises a 3′ overhang of at least 2 nucleotides.
In some embodiments, a dsRNA described herein further comprises a ligand.
In some embodiments, the ligand is a GalNAc ligand.
In some embodiments, the ligand targets the dsRNA to hepatocytes.
In some embodiments, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
In some embodiments, the region of complementarity consists of an antisense sequence selected from Table 2 or Table 3. In embodiments, the region of complementarity consists of an antisense sequence selected from Tables 2, 3, 6, 7, 8, 9, 14, or 15. In some embodiments, he region of complementarity consists of an antisense sequence selected from that of AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, AD-59129, AD-59124, AD-58874, AD-59125, AD-59105, AD-59120, AD-59122, AD-59106, AD-59126, or AD-59107 as disclosed herein in the Examples.
In some embodiments, the dsRNA comprises a sense strand consisting of a sense strand sequence selected from Table 2 or Table 3, and an antisense strand consisting of an antisense sequence selected from Table 2 or Table 3.
In some embodiments, the dsRNA comprises a sense strand consisting of a sense strand sequence selected from Tables 2, 3, 6, 7, 8, 9, 14, or 15, and an antisense strand consisting of an antisense sequence selected from Tables 2, 3, 6, 7, 8, 9, 14, or 15. In embodiments, the dsRNA comprises a pair of corresponding sense and antisense sequences selected from those of the duplexes disclosed in Tables 2, 3, 6, 7, 8, 9, 14, and 15.
In one aspect, the invention provides a cell containing at least one of the iRNAs (e.g., dsRNAs) featured herein. The cell is generally a mammalian cell, such as a human cell. In some embodiments, the cell is an erythroid cell. In other embodiments, the cell is a liver cell (e.g., a hepatocyte).
In an aspect provided herein is a pharmaceutical composition for inhibiting expression of an ALAS1 gene, the composition comprising an iRNA (e.g., a dsRNA) described herein.
In embodiments of the pharmaceutical compositions described herein, the iRNA (e.g., dsRNA) is administered in an unbuffered solution. In embodiments, the unbuffered solution is saline or water.
In embodiments of the pharmaceutical compositions described herein, the iRNA (e.g., dsRNA is administered with a buffer solution. In embodiments, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In embodiments, the buffer solution is phosphate buffered saline (PBS).
In embodiments of the pharmaceutical compositions described herein, the iRNA (e.g., dsRNA) is targeted to hepatocytes.
In embodiments of the pharmaceutical compositions described herein, the composition is administered intravenously.
In embodiments of the pharmaceutical compositions described herein, the composition is administered subcutaneously.
In embodiments, a pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that comprises a ligand (e.g., a GalNAc ligand) that targets the iRNA (e.g., dsRNA) to hepatocytes.
In embodiments, a pharmaceutical composition comprises an iRNA (e.g., a dsRNA) described herein that comprises a ligand (e.g., a GalNAc ligand), and the pharmaceutical composition is administered subcutaneously. In embodiments, the ligand targets the iRNA (e.g., dsRNA) to hepatocytes.
In certain embodiments, a pharmaceutical composition, e.g., a composition described herein, includes a lipid formulation. In some embodiments, the RNAi agent is in a LNP formulation, e.g., a MC3 formulation. In some embodiments, the LNP formulation targets the RNAi agent to a particular cell, e.g., a liver cell, e.g., a hepatocyte. In embodiments, the lipid formulation is a LNP11 formulation. In embodiments, the composition is administered intravenously.
In another embodiment, the pharmaceutical composition is formulated for administration according to a dosage regimen described herein, e.g., not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administration of the pharmaceutical composition can be maintained for a month or longer, e.g., one, two, three, or six months, or one year or longer.
In another embodiment, a composition containing an iRNA featured in the invention, e.g., a dsRNA targeting ALAS1, is administered with a non-iRNA therapeutic agent, such as an agent known to treat a porphyria (e.g., AIP), or a symptom of a porphyria (e.g., pain). In another embodiment, a composition containing an iRNA featured in the invention, e.g., a dsRNA targeting AIP, is administered along with a non-iRNA therapeutic regimen, such as hemin or glucose (e.g., glucose infusion (e.g., IV glucose)). For example, an iRNA featured in the invention can be administered before, after, or concurrent with glucose, dextrose, or a similar treatment that serves to restore energy balance (e.g., total parenteral nutrition). An iRNA featured in the invention can also be administered before, after, or concurrent with the administration of a heme product (e.g., hemin, heme arginate, or heme albumin), and optionally also in combination with a glucose (e.g. IV glucose) or the like.
Typically, glucose administered for the treatment of a porphyria is administered intravenously (IV). Administration of glucose intravenously is referred to herein as “IV glucose.” However, alternative embodiments in which glucose is administered by other means are also encompassed.
In one embodiment, an ALAS1 iRNA is administered to a patient, and then the non-iRNA agent or therapeutic regimen (e.g., glucose and/or a heme product) is administered to the patient (or vice versa). In another embodiment, an ALAS1 iRNA and the non-iRNA therapeutic agent or therapeutic regimen are administered at the same time.
In an aspect provided herein is a method of inhibiting ALAS1 expression in a cell, the method comprising: (a) introducing into the cell an iRNA (e.g. a dsRNA) described herein and (b) maintaining the cell of step (a) for a time sufficient to obtain degradation of the mRNA transcript of an ALAS1 gene, thereby inhibiting expression of the ALAS1 gene in the cell.
In an aspect provided herein is a method for reducing or inhibiting the expression of an ALAS1 gene in a cell (e.g., an erythroid cell or a liver cell, such as, e.g., a hepatocyte). The method includes:

    
    
        (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA includes at least two sequences that are complementary to each other. The dsRNA has a sense strand having a first sequence and an antisense strand having a second sequence; the antisense strand has a region of complementarity that is substantially complementary to at least a part of an mRNA encoding ALAS1, and where the region of complementarity is 30 nucleotides or less, i.e., 15-30 nucleotides in length, and generally 19-24 nucleotides in length, and where the dsRNA upon contact with a cell expressing ALAS1, inhibits expression of an ALAS1 gene by at least 10%, e.g., at least 20%, at least 30%, at least 40% or more; and
        (b) maintaining the cell of step (a) for a time sufficient to obtain degradation of the mRNA transcript of the ALAS1 gene, thereby reducing or inhibiting expression of an ALAS1 gene in the cell.
    
    


In embodiments of the foregoing methods of inhibiting ALAS1 expression in a cell, the cell is treated ex vivo, in vitro, or in vivo. In embodiments, the cell is a hepatocyte.
In embodiments, the cell is present in a subject in need of treatment, prevention and/or management of a disorder related to ALAS1 expression.
In embodiments, the disorder is a porphyria. In embodiments, the porphyria is acute intermittent porphyria or ALA-dehydratase deficiency porphyria.
In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.
In embodiments, the expression of ALAS1 is inhibited by at least 30%.
In embodiments, the iRNA (e.g., dsRNA) has an IC50 in the range of 0.01-1 nM.
In certain embodiments, the cell (e.g., the hepatocyte) is a mammalian cell (e.g., a human, non-human primate, or rodent cell).
In one embodiment, the cell is treated ex vivo, in vitro, or in vivo (e.g., the cell is present in a subject (e.g., a patient in need of treatment, prevention and/or management of a disorder related to ALAS1 expression).
In one embodiment, the subject is a mammal (e.g., a human) at risk, or diagnosed with a porphyria, e.g., X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (ADP or Doss porphyria), acute intermittent porphyria (AIP), congenital erythropoietic porphyria (CEP), prophyria cutanea tarda (PCT), hereditary coproporphyria (coproporphyria, or HCP), variegate porphyria (VP), erythropoietic protoporphyria (EPP), or transient erythroporphyria of infancy. In some embodiments, the disorder is an acute hepatic porphyria, e.g., ALA deyhdratase deficiency porphyria (ADP), AIP, HCP, or VP. In specific embodiments, the disorder is ALA deyhdratase deficiency porphyria (ADP) or AIP.
In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.
In one embodiment, the dsRNA introduced reduces or inhibits expression of an ALAS1 gene in the cell.
In one embodiment, the dsRNA introduced reduces or inhibits expression of an ALAS1 gene, or the level of one or more porphyrins or porphyrin precursors (e.g., δ-aminolevulinic acid (ALA), porphopilinogen (PBG), hydroxymethylbilane (HMB), uroporphyrinogen I or III, coproporphyrinogen I or III, protoporphrinogen IX, and protoporphyrin IX) or porphyrin products or metabolites, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more compared to a reference, (e.g., an untreated cell or a cell treated with a non-targeting control dsRNA). Without being bound by theory, ALAS1 is the first enzyme of the porphyrin pathway. Thus, reducing expression of the ALAS1 gene is likely to reduce the level of one or more porphyrin precursors, porphyrins or porphyrin products or metabolites.
In other aspects, the invention provides methods for treating, preventing or managing pathological processes related to ALAS1 expression (e.g., pathological processes involving porphyrins, porphyrin precuorsors, or defects in the porphyrin pathway, such as, for example, porphyrias). In one embodiment, the method includes administering to a subject, e.g., a patient in need of such treatment, prevention or management, an effective (e.g., a therapeutically or prophylactically effective) amount of one or more of the iRNAs featured herein.
In an aspect provided herein is a method of treating and/or preventing a disorder related to ALAS1 expression comprising administering to a subject in need of such treatment a therapeutically effective amount of an iRNA (e.g., a dsRNA) described herein, or a composition comprising an iRNA (e.g., a dsRNA) described herein.
In an aspect provided herein is a method of treating and/or preventing a porphyria comprising administering to a subject in need of such treatment a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length and the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:382.
In one embodiment, subject (e.g., the patient) has a porphyria. In another embodiment, the subject (e.g., patient) is at risk for developing a porphyria. In someembodiments, administration of the iRNA targeting ALAS1 alleviates or relieves the severity of at least one symptom of a disorder related to ALAS1 in the patient.
In one embodiment, the subject is a mammal (e.g., a human) at risk, or that has been diagnosed with, a disorder related to ALAS1 expression, e.g., a porphyria, e.g., X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (Doss porphyria), acute intermittent porphyria (AIP), congenital erythropoietic porphyria (CEP), prophyria cutanea tarda (PCT), hereditary coproporphyria (coproporphyria, or HCP), variegate porphyria (VP), erythropoietic protoporphyria (EPP), or transient erythroporphyria of infancy. In a further embodiment, the porphyria is an acute hepatic porphyria, e.g., ALA deyhdratase deficiency porphyria (ADP), AIP, HCP, or VP. In some such embodiments, the disorder is ALA deyhdratase deficiency porphyria (ADP) or AIP.
In embodiments the subject has, or is at risk for developing, a porphyria. In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.
In embodiments, a porphyria, a symptom of porphyria, a prodrome, or an attack of porphyria is induced by exposure to a precipitating factor, as described herein. In some embodiments, the precipitating factor is a chemical exposure. In some embodiments, the precipitating factor is a drug, e.g., a prescription drug or an over the counter drug. In some embodiments, the precipitating factor is the menstrual cycle, e.g., a particular phase of the menstrual cycle, e.g., the luteal phase.
In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered after an acute attack of porphyria.
In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered during an acute attack of porphyria.
In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered prophylactically to prevent an acute attack of porphyria.
In embodiments, the iRNA (e.g., dsRNA) is formulated as an LNP formulation.
In embodiments, the iRNA (e.g., dsRNA) is in the form of a GalNAc conjugate.
In embodiments, iRNA (e.g., dsRNA) is administered at a dose of 0.05-50 mg/kg.
In embodiments, the iRNA (e.g., dsRNA) is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
In embodiments, the iRNA (e.g., dsRNA) is formulated as an LNP formulation and is administered at a dose of 0.05-5 mg/kg.
In embodiments, the iRNA (e.g., dsRNA) is in the form of a GalNAc conjugate and is administered at a dose of 0.5-50 mg/kg.
In embodiments, the method decreases a level of a porphyrin or a porphyrin precursor in the subject.
In embodiments, the level is decreased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In an embodiment, the level is decreased by at least 30%.
In embodiments, the porphyrin precursor is δ-aminolevulinic acid (ALA) or porphopilinogen (PBG).
In embodiments, the iRNA (e.g., dsRNA) has an IC50 in the range of 0.01-1 nM.
In embodiments, a method described herein

    
    
        (i) ameliorates a symptom associated with an ALAS1 related disorder (e.g., a porphyria)
        (ii) inhibits ALAS1 expression in the subject,
        (iii) decreases a level of a porphyrin precursor (e.g., ALA or PBG) or a porphyrin in the subject,
        (iv) decreases frequency of acute attacks of symptoms associated with a porphyria in the subject, or
        (v) decreases incidence of acute attacks of symptoms associated with a porphyria in the subject when the subject is exposed to a precipitating factor (e.g., the premenstrual phase or the luteal phase).
    
    


In embodiments, the method ameliorates pain and/or progressive neuropathy.
In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered according to a dosing regimen.
In some embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered before or during an acute attack of porphyria. In some embodiments, the iRNA is administered before an acute attack of porphyria.
In some embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered during a prodrome. In embodiments, the prodrome is characterized by abdominal pain, nausea, psychological symptoms (e.g., anxiety), restlessness and/or insomnia.
In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered during a particular phase of the menstrual cycle, e.g., during the luteal phase.
In embodiments, the method ameliorates or prevents cyclical attacks of porphyria, e.g., by reducing the severity, duration, or frequency of attacks. In embodiments, the cyclical attacks are associated with a precipitating factor. In embodiments, the precipitating factor is the menstrual cycle, e.g., a particular phase of the menstrual cycle, e.g., the luteal phase.
In embodiments, the subject has an elevated level of ALA and/or PBG. In embodiments, the subject has or is at risk for developing a porphyria, e.g., a hepatic porphyria. In embodiments, the subject is asymptomatic. In embodiments, the subject carries a genetic alteration (e.g., a gene mutation) associated with a porphyria, as described herein.
In embodiments, the subject has or is at risk for developing a porphyria and suffers from pain (e.g., chronic pain, e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the subject does not suffer from acute attacks but suffers from pain (e.g., chronic pain, e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the pain is abdominal pain.
In embodiments, the subject (a) has an elevated level of ALA and/or PBG and (b) suffers from pain (e.g., chronic pain, e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the pain is abdominal pain.
In embodiments, the subject has a plasma level and/or a urine level of ALA and/or PBG that is elevated. In embodiments, the elevated level of ALA and/or PBG is accompanied by other symptoms, e.g., pain (e.g., chronic pain, e.g., chronic neuropathic pain) or neuropathy (e.g., progressive neuropathy). In embodiments, the pain is abdominal pain. In embodiments, the subject is asymptomatic. In embodiments, the subject has a genetic mutation associated with a porphyria, e.g., a mutation as described herein.
In embodiments, the subject has a level (e.g., a plasma level or a urine level) of a porphyrin precursor, e.g., ALA and/or PBG, that is elevated, e.g., the level is greater than, or greater than or equal to, a reference value. In embodiments, the level is greater than the reference value. In embodiments, the reference value is two standard deviations above the mean level in a sample of healthy individuals. In embodiments, the reference value is an upper reference limit.
In embodiments, the subject has a plasma level and/or a urine level of ALA and/or PBG that is greater than, or greater than or or equal to, 2 times, 3 times, 4 times, or 5 times that of an upper reference limit. As used herein, an “upper reference limit” refers to a level that is the upper limit of the 95% confidence interval for a reference sample, e.g., a sample of normal (e.g., wild type) or healthy individuals, e.g., individuals who do not carry a genetic mutation associated with a porphyria and/or individuals who do not suffer from a porphyria. In embodiments, the subject has a urine level of ALA and/or PBG that is greater than 2 to 4 times that of an upper reference limit. In embodiments, the subject has a urine level of ALA and/or PBG that is greater than 4 times that of an upper reference limit.
In embodiments, the reference value for plasma PBG is 0.12 μmol/L. In embodiments, the subject is a human and has a plasma PBG level that is greater than, or greater than or equal to, 0.12 μmol/L, 0.24 μmol/L, 0.36 μmol/L, 0.48 μmol/L, or 0.60 μmol/L. In embodiments, the subject is a human and has a plasma level of PBG that is greater than, or greater than or equal to, 0.48 μmol/L.
In embodiments, the reference value for urine PBG is 1.2 mmol/mol creatinine. In embodiments, the subject is a human and has a urine PBG level that is greater than, or greater than or equal to, 1.2 mmol/mol creatinine, 2.4 mmol/mol creatinine, 3.6 mmol/mol creatinine, 4.8 mmol/mol creatinine, or 6.0 mmol/mol creatinine. In embodiments, the subject is a human and has a urine level of PBG that is greater than, or greater than or equal to, 4.8 mmol/mol creatinine.
In embodiments, the reference value for plasma ALA is 0.12 mmol/L. In embodiments, the subject is a human and has a plasma ALA level that is greater than, or greater than or equal to, 0.12 μmol/L, 0.24 μmol/L, 0.36 μmol/L, 0.48 μmol/L, or 0.60 μmol/L. In embodiments, the subject is a human and has a plasma ALA level that is greater than, or greater than or equal to 0.48 μmol/L.
In embodiments, the reference value for urine ALA is 3.1 mmol/mol creatinine. In embodiments, the subject is a human and has a urine ALA level that is greater than, or greater than or equal to, 3.1 mmol/mol creatinine, 6.2 mmol/mol creatinine, 9.3 mmol/mol creatinine, 12.4 mmol/mol creatinine, or 15.5 mmol/mol creatinine.
In embodiments, the method decreases an elevated level of ALA and/or PBG. In embodiments, the method decreases pain (e.g., chronic pain, e.g. chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the pain is abdominal pain. In embodiments, the pain is neuropathic pain (e.g., pain associated with the progressive neuropathy of acute porphyrias). The decrease in pain can include, e.g., prevention of pain, delay in the onset of pain, reduction in the frequency of pain, and/or reduction in severity of pain. In embodiments, the method ameliorates or prevents acute attacks of porphyria, e.g., by reducing the severity, duration, or frequency of attacks.
In embodiments, the method decreases or prevents nerve damage.
In embodiments, the method prevents deterioration (e.g., prevents development of abnormalities) of or results in an improvement of clinical measures, e.g., clinical measures of muscle and/or nerve function, e.g., EMG and/or nerve conduction velocities.
In embodiments, the method is effective to reduce a level of ALA and/or PBG (e.g., a plasma or urine level of ALA and/or PBG). In embodiments, the method is effective to produce a predetermined reduction in the elevated level of ALA and/or PBG.
In embodiments, the predetermined reduction is a reduction to a value that is less than or equal to a reference value. In some embodiments, the reference value is an upper reference limit. In some embodiments, the reference value is the value that is two standard deviations above the mean level in a reference sample.
In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered repeatedly, e.g., according to a dosing regimen.
In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered prophylactically to a subject who is at risk for developing a porphyria. In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered prophylactically beginning at puberty. In embodiments, the subject carries a genetic mutation associated with a porphyria and/or has an elevated level of ALA and/or PBG (e.g., an elevated plasma or urine level of ALA and/or PBG). In embodiments, the mutation makes an individual susceptible to an acute attack (e.g., upon exposure to a precipitating factor, e.g., a drug, dieting or other precipitating factor, e.g., a precipitating factor as disclosed herein). In embodiments, the mutation is associated with elevated levels of a porphyrin or a porphyrin precursor (e.g., ALA and/or PBG). In embodiments, the mutation is associated with chronic pain (e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy).
In embodiments, the mutation is a mutation in the ALAS1 gene. In embodiments, the mutation is a mutation in the ALAS1 gene promoter, or in regions upstream or downstream from the ALAS1 gene. In embodiments, the mutation is a mutation in transcription factors or other genes that interact with ALAS1. In embodiments, the mutation is a mutation in a gene that encodes an enzyme in the heme biosynthetic pathway.
In embodiments, the iRNA (e.g., dsRNA) or composition comprising the iRNA is administered subcutaneously. In embodiments, the iRNA is in the form of a GalNAc conjugate. In embodiments, the iRNA (e.g., the dsRNA) is administered at a dose of 0.5-50 mg/kg.
In one aspect provided herein is a method of treating a subject with an elevated level of ALA and/or PBG, the method comprising administering to the subject a double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises a sense strand and an antisense strand 15-30 base pairs in length and the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:382.
In one aspect provided herein is a method of treating a subject with an elevated level of ALA and/or PBG, the method comprising administering to the subject a therapeutically effective amount of an dsRNA or a composition comprising a dsRNA, as described herein.
In some embodiments, the methods described herein are effective to decrease the level of ALA and/or PBG. In some embodiments, the level of ALA and/or PBG is decreased such that it is less than, or less than or equal to, a reference value, e.g., an upper reference limit. In another aspect, the invention provides methods for decreasing a level of a porphyrin or a porphyrin precursor in a cell (e.g., an erythroid cell or a liver cell, such as, e.g., a hepatocyte). In one embodiment, the cell is treated ex vivo, in vitro, or in vivo (e.g., the cell is present in a subject (e.g., a patient in need of treatment, prevention and/or management of a disorder related to ALAS1 expression). The method includes contacting the cell with an effective amount of one or more of the iRNAs targeting ALAS1, e.g., one or more of the iRNAs disclosed herein, thereby decreasing the level of a porphyrin or a porphyrin precursor in the cell; or decreasing the level of a porphyrin or a porphyrin precursor in other cells, tissues, or fluids within a subject in which the cell is located; relative to the level prior to contacting. Such methods can be used to treat (e.g., ameliorate the severity) of disorders related to ALAS1 expression, such as porphyrias, e.g., AIP or ALA dehydratase deficiency porphyria.
In one embodiment, the contacting step is effected ex vivo, in vitro, or in vivo. For example, the cell can be present in a subject, e.g., a mammal (e.g., a human) at risk, or that has been diagnosed with, a porphyria. In an embodiment, the porphyria is an acute hepatic porphyria. In embodiments, the porphyria is a hepatic porphyria, e.g., a porphyria selected from acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria. In embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments, the porphyria is a dual porphyria.
In an aspect provided herein is a method for decreasing a level of a porphyrin or a porphyrin precursor (e.g., ALA or PBG) in a cell, comprising contacting the cell with an iRNA (e.g. a dsRNA), as described herein, in an amount effective to decrease the level of the porphyrin or the porphyrin precursor in the cell. In embodiments, the cell is a hepatocyte. In embodiments, the porphyrin or porphyrin precursor is δ-aminolevulinic acid (ALA), porphopilinogen (PBG), hydroxymethylbilane (HMB), uroporphyrinogen I or III, coproporphyrinogen I or III, protoporphrinogen IX, or protoporphyrin IX. In embodiments, the porphyrin precursor is ALA or PBG.
In one embodiment, the cell is an erythroid cell. In a further embodiment, the cell is a liver cell (e.g., a hepatocyte).
In an aspect provided herein is a vector encoding at least one strand of an iRNA (e.g., a dsRNA) as described herein.
In an aspect provided herein is a vector encoding at least one strand of a dsRNA, wherein said dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ALAS1, wherein said dsRNA is 30 base pairs or less in length, and wherein said dsRNA targets said mRNA for cleavage.
In embodiments, the region of complementarity is at least 15 nucleotides in length. In embodiments, the region of complementarity is 19 to 21 nucleotides in length. In one aspect, the invention provides a vector for inhibiting the expression of an ALAS1 gene in a cell. In one embodiment, the vector comprises an iRNA as described herein. In one embodiment, the vector includes at least one regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of an iRNA as described herein. In one embodiment the vector comprises at least one strand of an ALAS1 iRNA.
In an aspect provided herein is a cell comprising a vector as described herein. In an aspect provided herein is a cell containing a vector for inhibiting the expression of an ALAS1 gene in a cell. The vector includes a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the iRNAs as described herein. In one embodiment, the cell is a liver cell (e.g., a hepatocyte). In another embodiment, the cell is an erythroid cell.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.



DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the heme biosynthetic pathway.
FIG. 2A is a table that summarizes certain porphyrias associated with genetic errors in heme metabolism.
FIG. 2B is a continuation of the table in FIG. 2A.
FIG. 3A depicts nucleotides 1-2280 of the sequence of a human ALAS1 mRNA sequence transcript variant 1 (Ref. Seq. NM—000688.4 (GI:40316942, record dated Nov. 19, 2011), SEQ ID NO: 1).
FIG. 3B depicts nucleotides 2281-2407 of the sequence of a human ALAS1 mRNA sequence transcript variant 1 (Ref. Seq. NM—000688.4 (GI:40316942, record dated Nov. 19, 2011), SEQ ID NO: 1).
FIG. 4A depicts nucleotides 1-2280 of the sequence of a human ALAS1 mRNA sequence transcript variant 2 (Ref. Seq. NM—000688.5 (GI: 362999011, record dated Apr. 1, 2012), SEQ ID NO: 382).
FIG. 4B depicts nucleotides 2281-2458 of the sequence of a human ALAS1 mRNA sequence transcript variant 2 (Ref. Seq. NM—000688.5 (GI: 362999011, record dated Apr. 1, 2012), SEQ ID NO: 382).
FIG. 5 shows the dose-response of the siRNA AD-53558 in suppressing mouse ALAS1 (mALAS1) mRNA relative to a PBS control. Results for a luciferase (LUC) AD-1955 control are also shown.
FIG. 6 shows the dose-response of the siRNA AD-53558 in suppressing ALAS1 mRNA in rats relative to a PBS control. Results for a luciferase (LUC) AD-1955 control are also shown.
FIG. 7 shows the durability of suppression of mouse ALAS1 (mALAS1) mRNA by the siRNA AD-53558 relative to a PBS control.
FIG. 8 shows means±standard deviations of plasma ALA levels (in μM) at baseline, and after phenobarbitol treatment in the experimental (ALAS1 siRNA) and control (LUC siRNA) groups.
FIG. 9 shows the plasma ALA levels (in μM) of individual animals at baseline, and after phenobarbitol treatment in animals that received ALAS1 siRNA and control (LUC siRNA) treatment.
FIG. 10 shows means±standard deviations of plasma PBG levels (in μM) at baseline, and after phenobarbitol treatment in animals that received ALAS1 siRNA and control (LUC siRNA) treatment.
FIG. 11 shows the plasma PBG levels (in μM) of individual animals at baseline, and after phenobarbitol treatment in animals that received ALAS1 siRNA and control (LUC siRNA) treatment.
FIG. 12 shows the relative mALAS1 mRNA level in liver at baseline, and after phenobarbitol treatment in select representative experimental (ALAS1 siRNA) and control (PBS) animals.
FIG. 13 shows the effects of three GalNAc conjugated mALAS1 siRNAs on mALAS1 expression (relative to a PBS control) in mouse liver tissue.
FIG. 14 shows plasma ALA and PBG levels over time after phenobarbitol administration and treatment with ALAS1 siRNA or control LUC siRNA.



DETAILED DESCRIPTION OF THE INVENTION
iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Described herein are iRNAs and methods of using them for inhibiting the expression of an ALAS1 gene in a cell or a mammal where the iRNA targets an ALAS1 gene. Also provided are compositions and methods for disorders related to ALAS1 expression, such as porphyrias (e.g., ALA deyhdratase deficiency porphyria (ADP or Doss porphyria), acute intermittent porphyria, congenital erythropoietic porphyria, prophyria cutanea tarda, hereditary coproporphyria (coproporphyria), variegate porphyria, erythropoietic protoporphyria (EPP), X-linked sideroblastic anemia (XLSA), and transient erythroporphyria of infancy).
Porphyrias are inherited or acquired disorders that can be caused by decreased or enhanced activity of specific enzymes in the heme biosynthetic pathway, also referred to herein as the porphyrin pathway (See FIG. 1). Porphyrins are the main precursors of heme. Porphyrins and porphyrin precursors include δ-aminolevulinic acid (ALA), porphopilinogen (PBG), hydroxymethylbilane (HMB), uroporphyrinogen I or III, coproporphyrinogen I or III, protoporphrinogen IX, and protoporphyrin IX. Heme is an essential part of hemoglobin, myoglobin, catalases, peroxidases, and cytochromes, the latter including the respiratory and P450 liver cytochromes. Heme is synthesized in most or all human cells. About 85% of heme is made in erythroid cells, primarily for hemoglobin. Most of the remaining heme is made in the liver, 80% of which is used for the synthesis of cytochromes. Deficiency of specific enzymes in the porphyrin pathway leads to insufficient heme production and also to an accumulation of porphyrin precursors and/or porphyrins, which can be toxic to cell or organ function in high concentrations.
Porphyrias may manifest with neurological complications (“acute”), skin problems (“cutaneous”) or both. Porphyrias may be classified by the primary site of the overproduction and accumulation of porphyrins or their precursors. In hepatic porphyrias, porphyrins and porphyrin precursors are overproduced predominantly in the liver, whereas in erythropoietic porphyrias, porphyrins are overproduced in the erythroid cells in the bone. The acute or hepatic porphyrias lead to dysfunction of the nervous system and neurologic manifestations that can affect both the central and peripheral nervous system, resulting in symptoms such as, for example, pain (e.g., abdominal pain and/or chronic neuropathic pain), vomiting, neuropathy (e.g., acute neuropathy progressive neuropathy), muscle weakness, seizures, mental disturbances (e.g., hallucinations, depression anxiety, paranoia), cardiac arrhythmias, tachycardia, constipation, and diarrhea. The cutaneous or erythropoietic porphyrias primarily affect the skin, causing symptoms such as photosensitivitythat can be painful, blisters, necrosis, itching, swelling, and increased hair growth on areas such as the forehead. Subsequent infection of skin lesions can lead to bone and tissue loss, as well as scarring, disfigurement, and loss of digits (e.g., fingers, toes). Most porphyrias are caused by mutations that encode enzymes in the heme biosynthetic pathway. A summary of porphyrias associated with genetic errors in heme metabolism is provided in FIG. 2.
Not all porphyrias are genetic. For example, patients with liver disease may develop porphyria as a result of liver dysfunction, and a transient form of erythroporphria (transient erythroporphyria of infancy) has been described in infancy (see Crawford, R. I. et al, J Am Acad Dermatol. 1995 August; 33(2 Pt 2):333-6.) Patients with PCT can acquire the deficient activity of uroporphyrinogen decarboxylase (URO-D), due to the formation of a ORO-D enzyme with lower than normal enzymatic activity (see Phillips et al. Blood, 98:3179-3185, 2001.)
Acute intermittent porphyria (AIP) (also be referred to as porphobilinogen (PBG) deaminase deficiency, or hydroxymethylbilane synthase (HMBS) deficiency), is the most common type of acute hepatic porphyria. Other types of acute hepatic porphyrias include hereditary coproporphyria (HCP), variegate porphyria (VP), and ALA deyhdratase deficiency porphyria (ADP). Acute hepatic porphyrias are described, e.g., in Balwani, M. and Desnick, R. J., Blood, 120:4496-4504, 2012.
AIP is typically an autosomal dominant disease that is characterized by a deficiency of the enzyme porphobilinogen deaminase (PBG deaminase); this enzyme is also known as hydroxymethylbilane synthase (HMB synthase or HMBS). PBG deaminase is the third enzyme of the heme biosynthetic pathway (see FIG. 1) and catalyzes the head to tail condensation of four porphobilinogen molecules into the linear tetrapyrrole, hydroxymethylbilane (HMB). Alternatively spliced transcript variants encoding different isoforms of PBG deaminase have been described. Mutations in the PBG deaminase gene are associated with AIP. Such mutations may lead to decreased amounts of PBG deaminase and/or decreased activity of PBG deaminase (affected individuals typically have a  ̃50% reduction in PBG deaminase activity).
There are at least two different models of the pathophysiology of AIP and other acute hepatic porphyrias (see, e.g., Lin C S-Y et al., Clinical Neurophysiology, 2011; 122:2336-44). According to one model, the decreased heme production resulting from PBG deaminase deficiency causes energy failure and axonal degeneration. According to the other, currently more favored model, the buildup of porphyrin precursors (e.g., ALA and PBG) results in neurotoxicity.
AIP has been found to have a prevalence as high as 1 in 10,000 in certain populations (e.g., in Northern Sweden; see Floderus Y, et al. Clin Genet. 2002; 62:288-97). The prevalence in the general population in United States and Europe, excluding the U.K., is estimated to be about 1 in 10,000 to 1 in 20,000. Clinical disease manifests itself in only approximately 10-15% of individuals who carry mutations that are known to be associated with AIP. However, the penetrance is as high as 40% in individuals with certain mutations (e.g., the W198X mutation). AIP is typically latent prior to puberty. Symptoms are more common in females than in males. The prevalence of the disease is probably underestimated due to its incomplete penetrance and long periods of latency. In the United States, it is estimated that there are about 2000 patients who have suffered at least one attack. It is estimated that there are about 150 active recurrent cases in France, Sweden, the U.K., and Poland; these patients are predominantly young women, with a median age of 30. See, e.g., Elder et al, J Inherit Metab Dis., published online Nov. 1, 2012.
AIP affects, for example, the visceral, peripheral, autonomic, and central nervous systems. Symptoms of AIP are variable and include gastrointestinal symptoms (e.g., severe and poorly localized abdominal pain, nausea/vomiting, constipation, diarrhea, ileus), urinary symptoms (dysuria, urinary retention/incontinence, or dark urine), neurologic symptoms (e.g., sensory neuropathy, motor neuropathy (e.g., affecting the cranial nerves and/or leading to weakness in the arms or legs), seizures, neuropathic pain (e.g., pain associated with progressive neuropathy, e.g., chronic neuropathic pain), neuropsychiatric symptoms (e.g., mental confusion, anxiety, agitation, hallucination, hysteria, delirium, apathy, depression, phobias, psychosis, insomnia, somnolence, coma), autonomic nervous system involvement (resulting e.g., in cardiovascular sysmptoms such as tachycardia, hypertension, and/or arrhythmias, as well as other symptoms, such as, e.g., increased circulating catecholamine levels, sweating, restlessness, and/or tremor), dehydration, and electrolyte abnormalities. The most common symptoms are abdominal pain and tachycardia. In addition, patients frequently have chronic neuropathic pain and develop a progressive neuropathy. Patients with recurring attacks often have a prodrome. Permanent paralysis may occur after a severe attack. Recovery from severe attacks that are not promptly treated may take weeks or months. An acute attack may be fatal, for example, due to paralysis of respiratory muscles or cardiovascular failure from electrolyte imbalance. (See, e.g., Thunell S. Hydroxymethylbilane Synthase Deficiency. 2005 Sep. 27 [Updated 2011 Sep. 1]. In: Pagon R A, Bird T D, Dolan C R, et al., editors. GeneReviewsTM [Internet]. Seattle (WA): University of Washington, Seattle; 1993-(hereinafter Thunell (1993)), which is hereby incorporated by reference in its entirety.) Prior to the availability of Hemin treatments, up to 20% of patients with AIP died from the disease.
In individuals who carry genes for AIP, the risk of hepatocellular cancer is increased. In those with recurrent attacks, the risk of hepatocellular cancer is particularly grave: after the age of 50, the risk is nearly 100-fold greater than in the general population.
Attacks of acute porphyria may be precipitated by endogenous or exogenous factors. The mechanisms by which such factors induce attacks may include, for example, increased demand for hepatic P450 enzymes and/or induction of ALAS1 activity in the liver. Increased demand for hepatic P450 enzymes results in decreased hepatic free heme, thereby inducing the synthesis of hepatic ALAS1.
Precipitating factors include fasting (or other forms of reduced or inadequate caloric intake, due to crash diets, long-distance athletics, etc.), metabolic stresses (e.g., infections, surgery, international air travel, and psychological stress), endogenous hormones (e.g., progesterone), cigarette smoking, lipid-soluble foreign chemicals (including, e.g., chemicals present in tobacco smoke, certain prescription drugs, organic solvents, biocides, components in alcoholic beverages), endocrine factors (e.g., reproductive hormones (women may experience exacerbations during the premenstrual period), synthetic estrogens, progesterones, ovulation stimulants, and hormone replacement therapy). See, for example, Thunell (1993).
Over 1000 drugs are contraindicated in the acute hepatic porphyrias (e.g., AIP, HCP, ADP, and VP) including, for example, alcohol, barbiturates, Carbamazepine, Carisoprodol, Clonazepam (high doses), Danazol, Diclofenac and possibly other NSAIDS, Ergots, estrogens, Ethyclorvynol, Glutethimide, Griseofulvin, Mephenyloin, Meprobamate (also mebutamate and tybutamate), Methyprylon, Metodopramide, Phenyloin, Primidone, progesterone and synthetic progestins, Pyrazinamide, Pyrazolones (aminopyrine and antipyrine), Rifampin, Succinimides (ethosuximide and methsuximide), sulfonamide antibiotics, and Valproic acid.
Objective signs of AIP include discoloration of the urine during an acute attack (the urine may appear red or red-brown), and increased concentrations of PBG and ALA in urine during an acute attack. Molecular genetic testing identifies mutations in the PBG deaminase (also known as HMBS) gene in more than 98% of affected individuals. Thunell (1993).
The differential diagnosis of porphyrias may involve determining the type of porphyria by measuring individual levels of porphyrins or porphyrin precursors (e.g., ALA, PBG) in the urine, feces, and/or plasma (e.g., by chromatography and fluorometry) during an attack. The diagnosis of AIP can be confirmed by establishing that erythrocyte PBG deaminase activity is at 50% or less of the normal level. DNA testing for mutations may be carried out in patients and at-risk family members. The diagnosis of AIP is typically confirmed by DNA testing to identify a specific caustative gene mutation (e.g., an HMBS mutation).
Treatment of acute attacks typically requires hospitalization to control and treat acute sysmptoms, including, e.g., abdominal pain, seizures, dehydration/hyponatremia, nausea/vomiting, tachycardia/hypertension, urinary retention/ileus. For example, abdominal pain may be treated, e.g., with narcotic analgesics, seizures may be treated with seizure precautions and possibly medications (although many anti-seizure medications are contraindicated), nausea/vomiting may be treated, e.g., with phenothiazines, and tachycardia/hypertension may be treated, e.g., with beta blockers. Treatment may include withdrawal of unsafe medications, monitoring of respiratory function, as well as muscle strength and neurological status. Mild attacks (e.g., those with no paresis or hyponatremia) may be treated with at least 300 g intravenous 10% glucose per day, although increasingly hemin is provided immediately. Severe attacks should be treated as soon as possible with intravenous hemin (3-4 mg/kg daily for 4-14 days) and with IV glucose while waiting for the IV hemin to take effect. Typically, attacks are treated with IV hemin for 4 days and with IV glucose while waiting for administration of the IV hemin.
Hemin (Panhematin® or hemin for injection, previously known as hematin) is the only heme product approved for use in the United States and was the first drug approved under the Orphan Drug Act. Panhematin® is hemin derived from processed red blood cells (PRBCs), and is Protoporphyrin IX containing a ferric iron ion (Heme B) with a chloride ligand. Heme acts to limit the hepatic and/or marrow synthesis of porphyrin. The exact mechanism by which hemin produces symptomatic improvement in patients with acute episodes of the hepatic porphyrias has not been elucidated; however, its action is likely due to the (feedback) inhibition of δ-aminolevulinic acid (ALA) synthase, the enzyme which limits the rate of the porphyrin/heme biosynthetic pathway. See Panhematin® product label, Lundbeck, Inc., October 2010. Inhibition of ALA synthase should result in reduced production of ALA and PBG as well as porphyrins and porphyrin intermediates.
Drawbacks of hemin include its delayed impact on clinical symptoms and its failure to prevent the recurrence of attacks. Adverse reactions associated with hemin administration may include thrombophlebitis, anticoagulation, thrombocytopenia, renal shut down, or iron overload, which is particularly likely in patients requiring multiple courses of hemin treatment for recurrent attacks. To prevent phlebitis, an indwelling venous catheter is needed for access in patients with recurrent attacks. Uncommonly reported side effects include fever, aching, malaise, hemolysis, anaphalaxis, and circulatory collapse. See Anderson, K. E., Approaches to Treatment and Prevention of Human Porphyrias, in The Porphyrin Handbook: Medical Aspects of Porphyrins, Edited by Karl M. Kadish, Kevin M. Smith, Roger Guilard (2003) (hereinafter Anderson).
Heme is difficult to prepare in a stable form for intravenous administration. It is insoluble at neutral pH but can be prepared as heme hydroxide at pH 8 or higher. Anderson. Panhematin is a lyophilized hemin preparation. When lyophilized hemin is solubilized for intravenous administration, degradation products form rapidly; these degradation products are responsible for a transient anticoagulant effect and for phlebitis at the site of infusion. Anderson. Heme albumin and heme arginate (Normosang, the European version of hemin) are more stable and may potentially cause less thrombophlebitis. However, heme arginate is not approved for use in the United States. Panhemin may be stabilized by solubilizing it for infusion in 30% human albumin rather than in sterile water; however, albumin adds intravascular volume-expanding effects and increases the cost of treatment as well as risk of pathogens since it is isolated from human blood. See, e.g., Anderson.
The successful treatment of an acute attack does not prevent or delay recurrence. There is a question of whether hemin itself can trigger recurring attacks due to induction of heme oxygenase. Nonetheless, in some areas (especially France), young women with multiply recurrent attacks are being treated with weekly hemin with the goal of achieving prophylaxis.
Limited experience with liver transplantation suggests that if successful, it is an effective treatment for AIP. There have been approximately 12 transplants in Europe in human patients, with curative or varying effects. Liver transplantation can restore normal excretion of ALA and PBG and prevent acute attacks. See, e.g., Dar, F. S. et al. Hepatobiliary Pancreat. Dis. Int., 9(1):93-96 (2010). Furthermore, if the liver of a patient with AIP is transplanted into another patient (“domino transplant”), the patient receiving the transplant may develop AIP.
Among the long-term clinical effects of acute porphyrias is chronic neuropathic pain that may result from a progressive neuropathy due to neurotoxic effects, e.g., of elevated porphyrin precursors (e.g., ALA and/or PBG). Patients may suffer from neuropathic pain prior to or during an acute attack. Older patients may experience increased neuropathic pain with age for which various narcotic drugs are typically prescribed. Electromyogram abnormalities and decreased conduction times have been documented in patients with acute hepatic porphyrias. Of note, untreated, uninduced mice with AIP (PBG deaminase deficiency) develop a progressive motor neuropathy that has been shown to cause progressive quadriceps nerve axon degeneration and loss presumably due to constitutively elevated porphyrin precursor (ALA & PBG) levels, porphyrins and/or heme deficiency (Lindberg et al., J. Clin. Invest., 103(8): 1127-1134, 1999). In patients with acute porphyria (e.g., ADP, AIP, HCP, or VP), levels of porphyrin precursors (ALA & PBG) are often elevated in asymptomatic patients and in symptomatic patients between attacks. Thus, reduction of the porphyrin precursors and resumption of normal heme biosynthesis by reducing the level of ALAS1 expression and/or activity is expected to prevent and/or minimize development of chronic and progressive neuropathy. Treatment, e.g., chronic treatment (e.g., periodic treatment with iRNA as described herein, e.g., treatment according to a dosing regimen as described herein, e.g., weekly or biweekly treatment) can continuously reduce the ALAS1 expression in acute porphyria patients who have elevated levels of porphyrin precursors, porphyrins, porphyrin products or their metabolites. Such treatment may be provided as needed to prevent or reduce the frequency or severity of an individual patient's symptoms (e.g., pain and/or neuropathy) and/or to reduce a level of a porphyrin precursor, porphyrin, porphyrin product or metabolite.
The need exists for identifying novel therapeutics that can be used for the treatment of porphyrias. As discussed above, existing treatments such as hemin have numerous drawbacks. For example, the impact of hemin on clinical symptoms is delayed, it is expensive, and it may have side effects (e.g., thrombophlebitis, anticoagulation, thrombocytopenia, iron overload, renal shutdown). Novel therapeutics such as those described herein can address these drawbacks and the unmet needs of patients by, for example, acting faster, not inducing phlebitis, providing the convenience of subcutaneous administration, successfully preventing recurrent attacks, preventing or ameliorating pain (e.g., chronic neuropathic pain) and/or progressive neuropathy, and/or not causing certain adverse effects associated with hemin (e.g., iron overload, increased risk of hepatocellular cancer).
The present disclosure provides methods and iRNA compositions for modulating the expression of an ALAS1 gene. In certain embodiments, expression of ALAS1 is reduced or inhibited using an ALAS1-specific iRNA, thereby leading to a decreased expression of an ALAS1 gene. Reduced expression of an ALAS1 gene may reduce the level of one or more porphyrin precursors, porphyrins, or porphyrin products or metabolites. Decreased expression of an ALAS1 gene, as well as related decreases in the level of one or more porphyrin precursors and/or porphyrins, can be useful in treating disorders related to ALAS1 expression, e.g., porphyrias.
The iRNAs of the compositions featured herein include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an ALAS1 gene (also referred to herein as an “ALAS1-specific iRNA”). The use of such an iRNA enables the targeted degradation of mRNAs of genes that are implicated in pathologies associated with ALAS1 expression in mammals, e.g., porphyrias such as ALA dehydratase deficiency porphyria (Doss porphyria) or acute intermittent porphyria. Very low dosages of ALAS1-specific iRNAs can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of an ALAS1 gene. iRNAs targeting ALAS1 can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of an ALAS1 gene, e.g., in cell based assays. Thus, methods and compositions including these iRNAs are useful for treating pathological processes related to ALAS1 expression, such as porphyrias (e.g., X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (Doss porphyria), acute intermittent porphyria (AIP), congenital erythropoietic porphyria, prophyria cutanea tarda, hereditary coproporphyria (coproporphyria), variegate porphyria, erythropoietic protoporphyria (EPP), and transient erythroporphyria of infancy).
The following description discloses how to make and use compositions containing iRNAs to inhibit the expression of an ALAS1 gene, as well as compositions and methods for treating diseases and disorders caused by or modulated by the expression of this gene. Embodiments of the pharmaceutical compositions featured in the invention include an iRNA having an antisense strand comprising a region which is 30 nucleotides or less in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part of an RNA transcript of an ALAS1 gene, together with a pharmaceutically acceptable carrier. Embodiments of compositions featured in the invention also include an iRNA having an antisense strand having a region of complementarity which is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of an ALAS1 gene.
Accordingly, in some aspects, pharmaceutical compositions containing an ALAS1 iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of an ALAS1 gene, and methods of using the pharmaceutical compositions to treat disorders related to ALAS1 expression are featured in the invention.
I. DEFINITIONS
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.
As used herein, “ALAS1” (also known as ALAS-1; δ-aminolevulinate synthase 1; δ-ALA synthase 1; 5′-aminolevulinic acid synthase 1; ALAS-H; ALASH; ALAS-N; ALAS3; EC2.3.1.37; 5-aminolevulinate synthase, nonspecific, mitochondrial; ALAS; MIG4; OTTHUMP00000212619; OTTHUMP00000212620; OTTHUMP00000212621; OTTHUMP00000212622; migration-inducing protein 4; EC 2.3.1) refers to a nuclear-encoded mitochondrial enzyme that is the first and typically rate-limiting enzyme in the mammalian heme biosynthetic pathway. ALAS1 catalyzes the condensation of glycine with succinyl-CoA to form δ-aminolevulinic acid (ALA). The human ALAS1 gene is expressed ubiquitously, is found on chromosome 3p21.1 and typically encodes a sequence of 640 amino acids. In contrast, the ALAS-2 gene, which encodes an isozyme, is expressed only in erythrocytes, is found on chromoxome Xp11.21, and typicallyencodes a sequence of 550 amino acids. As used herein an “ALAS1 protein” means any protein variant of ALAS1 from any species (e.g., human, mouse, non-human primate), as well as any mutants and fragments thereof that retain an ALAS1 activity. Similarly, an “ALAS1 transcript” refers to any transcript variant of ALAS1, from any species (e.g., human, mouse, non-human primate). A sequence of a human ALAS1 variant 1 mRNA transcript can be found at NM—000688.4 (FIG. 3; SEQ ID NO:1). Another version, a human ALAS1 variant 2 mRNA transcript, can be found at NM—000688.5 (FIG. 4; SEQ ID NO:382). The level of the mature encoded ALAS1 protein is regulated by heme: high levels of heme down-regulate the mature enzyme in mitochondria while low heme levels up-regulate. Multiple alternatively spliced variants, encoding the same protein, have been identified.
As used herein, the term “iRNA,” “RNAi”, “iRNA agent,” or “RNAi agent” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript, e.g., via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of ALAS1 expression. Inhibition of ALAS1 expression may be assessed based on a reduction in the level of ALAS1 mRNA or a reduction in the level of the ALAS1 protein. As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ALAS1 gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.
The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding an ALAS1 protein). For example, a polynucleotide is complementary to at least a part of an ALAS1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ALAS1. As another example, a polynucleotide is complementary to at least a part of an ALAS1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ALAS1.
The term “double-stranded RNA” or “dsRNA,” as used herein, refers to an iRNA that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA, e.g., through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker.” The term “siRNA” is also used herein to refer to a dsRNA as described above.
In another embodiment, the iRNA agent may be a “single-stranded siRNA” that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein (e.g., sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15) may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.
In another aspect, the RNA agent is a “single-stranded antisense RNA molecule”. An single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. Single-stranded antisense RNA molecules can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Alternatively, the single-stranded antisense molecules inhibit a target mRNA by hydridizing to the target and cleaving the target through an RNaseH cleavage event. The single-stranded antisense RNA molecule may be about 10 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense nucleotide sequences described herein, e.g., sequences provided in any one of Tables 2, 3, 6, 7, 8, 9, 14, and 15.
The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleostide, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA, e.g., via a RISC pathway.
In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. However, it is self evident that under no circumstances is a double stranded DNA molecule encompassed by the term “iRNA.”
In one aspect, an RNA interference agent includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene.
As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA.
In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.
The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817. These applications are incorporated herein by reference in their entirety.
“Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a β-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art.
As used herein, the term “modulate the expression of,” refers to at an least partial “inhibition” or partial “activation” of an ALAS1 gene expression in a cell treated with an iRNA composition as described herein compared to the expression of ALAS1 in a control cell. A control cell includes an untreated cell, or a cell treated with a non-targeting control iRNA.
The terms “activate,” “enhance,” “up-regulate the expression of,” “increase the expression of,” and the like, in so far as they refer to an ALAS1 gene, herein refer to the at least partial activation of the expression of an ALAS1 gene, as manifested by an increase in the amount of ALAS1 mRNA, which may be isolated from or detected in a first cell or group of cells in which an ALAS1 gene is transcribed and which has or have been treated such that the expression of an ALAS1 gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).
In one embodiment, expression of an ALAS1 gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein. In some embodiments, an ALAS1 gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the invention. In some embodiments, expression of an ALAS1 gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein. In some embodiments, the ALAS1 gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and in US20070111963 and US2005226848, each of which is incorporated herein by reference.
The terms “silence,” “inhibit expression of,” “down-regulate expression of,” “suppress expression of,” and the like, in so far as they refer to an ALAS1 gene, herein refer to the at least partial suppression of the expression of an ALAS1 gene, as assessed, e.g., based on on ALAS1 mRNA expression, ALAS1 protein expression, or another parameter functionally linked to ALAS1 gene expression (e.g., ALA or PBG concentrations in plasma or urine). For example, inhibition of ALAS1 expression may be manifested by a reduction of the amount of ALAS1 mRNA which may be isolated from or detected in a first cell or group of cells in which an ALAS1 gene is transcribed and which has or have been treated such that the expression of an ALAS1 gene is inhibited, as compared to a control. The control may be a second cell or group of cells substantially identical to the first cell or group of cells, except that the second cell or group of cells have not been so treated (control cells). The degree of inhibition is usually expressed as a percentage of a control level, e.g.,




  
    
      
        
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Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to ALAS1 gene expression, e.g., the amount of protein encoded by an ALAS1 gene, or the level of one or more porphyrins. The reduction of a parameter functionally linked to ALAS1 gene expression may similarly be expressed as a percentage of a control level. In principle, ALAS1 gene silencing may be determined in any cell expressing ALAS1, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given iRNA inhibits the expression of the ALAS1 gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of an ALAS1 gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA featured in the invention. In some embodiments, an ALAS1 gene is suppressed by at least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA featured in the invention. In some embodiments, an ALAS1 gene is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of an iRNA as described herein.
As used herein in the context of ALAS1 expression, the terms “treat,” “treating,” “treatment,” and the like, refer to relief from or alleviation of pathological processes related to ALAS1 expression (e.g., pathological processes involving porphyrins or defects in the porphyrin pathway, such as, for example, porphyrias). In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes related to ALAS1 expression), the terms “treat,” “treatment,” and the like mean to prevent, relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition. For example, the methods featured herein, when employed to treat porphyria, may serve to reduce or prevent one or more symptoms associated with porphyria (e.g., pain), to reduce the severity or frequency of attacks associated with porphyria, to reduce the likelihood that an attack of one or more symptoms associated with porphyria will occur upon exposure to a precipitating condition, to shorten an attack associated with porphyria, and/or to reduce the risk of developing conditions associated with porphyria (e.g., hepatocellular cancer or neuropathy (e.g., progressive neuropathy),). Thus, unless the context clearly indicates otherwise, the terms “treat,” “treatment,” and the like are intended to encompass prophylaxis, e.g., prevention of disorders and/or symptoms of disorders related to ALAS1 expression.
By “lower” in the context of a disease marker or symptom is meant a statistically or clinically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is typically down to a level accepted as within the range of normal for an individual without such disorder.
As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes related to ALAS1 expression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological process, the patient's history and age, the stage of pathological process, and the administration of other agents.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an iRNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, in a method of treating a disorder related to ALAS1 expression (e.g., in a method of treating a porphyria), an effective amount includes an amount effective to reduce one or more symptoms associated with a porphyria, an amount effective to reduce the frequency of attacks, an amount effective to reduce the likelihood that an attack of one or more symptoms associated with porphyria will occur upon exposure to a precipitating factor, or an amount effective to reduce the risk of developing conditions associated with porphyria (e.g., neuropathy (e.g., progressive neuropathy), hepatocellular cancer). For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter. For example, a therapeutically effective amount of an iRNA targeting ALAS1 can reduce ALAS1 protein levels by any measurable amount, e.g., by at least 10%, 20%, 30%, 40% or 50%.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.
The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary from, for example, between 1% and 15% of the stated number or numerical range.
II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)
Described herein are iRNA agents that inhibit the expression of an ALAS1 gene. In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an ALAS1 gene in a cell or in a subject (e.g., in a mammal, e.g., in a human having a porphyria), where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an ALAS1 gene, and where the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing the ALAS1 gene, inhibits the expression of the ALAS1 gene by at least 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. In one embodiment, the iRNA agent activates the expression of an ALAS1 gene in a cell or mammal Expression of an ALAS1 gene in cell culture, such as in COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured cells or in a biological sample from a subject can be assayed by measuring ALAS1 mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by immunofluorescence analysis, using, for example, Western Blotting or flow cytometric techniques.
A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of an ALAS1 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, e.g., 15-30 nucleotides in length.
One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of 9 to 36, e.g., 15-30 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex of e.g., 15-30 base pairs that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, then, an miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target ALAS1 expression is not generated in the target cell by cleavage of a larger dsRNA.
A dsRNA as described herein may further include one or more single-stranded nucleotide overhangs. The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In one embodiment, an ALAS1 gene is a human ALAS1 gene. In another embodiment the ALAS1 gene is a mouse or a rat ALAS1 gene. In specific embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence from Table 2 or Table 3, and the second sequence is an antisense strand of a dsRNA that includes an antisense sequence from Table 2 or Table 3. In embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence from Table 2, 3, 6, 7, 8, 9, 14, or 15, and the second sequence is an antisense strand of a dsRNA that includes an antisense sequence from Table 2, 3, 6, 7, 8, 9, 14, or 15. Alternative dsRNA agents that target sequences other than those of the dsRNAs of Table 2 or Table 3 can readily be determined using the target sequence and the flanking ALAS1 sequence.
In one aspect, a dsRNA will include at least sense and antisense nucleotide sequences, whereby the sense strand is selected from the groups of sequences provided in Tables 2 and 3, and the corresponding antisense strand of the sense strand is selected from Tables 2 and 3. In a further aspect, a dsRNA will include at least sense and antisense nucleotide sequences, whereby the sense strand is selected from the groups of sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15, and the corresponding antisense strand of the sense strand is selected from Tables 2, 3, 6, 7, 8, 9, 14, and 15. In these aspects, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated by the expression of an ALAS1 gene. As such, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Table 2, 3, 6, 7, 8, 9, 14, or 15, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand from 2, 3, 6, 7, 8, 9, 14, or 15. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.
The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Table 2, 3, 6, 7, 8, 9, 14, or 15 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Table 2, 3, 6, 7, 8, 9, 14, or 15, and differing in their ability to inhibit the expression of an ALAS1 gene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.
In addition, the RNAs provided in Tables 2 and 3, as well as the RNAs provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15, identify a site in an ALAS1 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of such sequences. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least 15 contiguous nucleotides from one of the sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an ALAS1 gene.
While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 2, 3, 6, 7, 8, 9, 14, and 15, represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
Further, it is contemplated that for any sequence identified, e.g., in Tables 2, 3, 6, 7, 8, 9, 14, and 15, further optimization can be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of an ALAS1 gene, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an ALAS1 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an ALAS1 gene is important, especially if the particular region of complementarity in an ALAS1 gene is known to have polymorphic sequence variation within the population.
In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.
Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No. RE39464, each of which is herein incorporated by reference.
Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O-—CH2—N(CH2)2, also described in examples herein below.
Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F) Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.
An iRNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.
Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3′′-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.
iRNA Motifs
In one embodiment, the sense strand sequence may be represented by formula (I):








5′ np-Na-(X X X)i-Nb-Y Y Y-Nb-(Z Z Z)j-Na-nq 3′
(I)






wherein:
i and j are each independently 0 or 1;
p and q are each independently 0-6;
each Na independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
each Nb independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;
each np and nq independently represent an overhang nucleotide;
wherein Nb and Y do not have the same modification; and
XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.
In one embodiment, the Na and/or Nb comprise modifications of alternating pattern.
In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12 or 11, 12, 13) of—the sense strand, the count starting from the 1st nucleotide, from the 5′-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end.
In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:










5′ np-Na-YYY-Nb-ZZZ-Na-nq 3′;
(Ib)



 



5′ np-Na-XXX-Nb-YYY-Na-nq 3′;
(Ic)



or



 



5′ np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq 3′.
(Id)






When the sense strand is represented by formula (Ib), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the sense strand is represented as formula (Ic), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the sense strand is represented as formula (Id), each Nb independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
Each of X, Y and Z may be the same or different from each other.
In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:










5′ np-Na-YYY-Na-nq 3′.
(Ia)






When the sense strand is represented by formula (Ia), each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):







(II)







5′ nq′-Na′-(Z′Z′Z′)k-Nb′-Y′Y′Y′-Nb′-(X′X′X′)l-N′a-


np′ 3′






wherein:
k and l are each independently 0 or 1;
p′ and q′ are each independently 0-6;
each Na′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
each Nb′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;
each np′ and nq′ independently represent an overhang nucleotide;
wherein Nb′ and Y′ do not have the same modification; and
X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.
In one embodiment, the Na′ and/or Nb′ comprise modifications of alternating pattern.
The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotidein length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1st nucleotide, from the 5′-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.
In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.
In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.
The antisense strand can therefore be represented by the following formulas:







(IIb)









5′ nq′-Na′-Z′Z′Z′-Nb′-Y′Y′Y′-Na′-np′ 3′;



 







(IIc)









5′ nq′-Na′-Y′Y′Y′-Nb′-X′X′X′-np′ 3′;



or



 







(IId)









5′ nq′-Na′-Z′Z′Z′-Nb′-Y′Y′Y′-Nb′-X′X′X′-Na′-



np′ 3′.






When the antisense strand is represented by formula (IIb), NE; represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the antisense strand is represented as formula (IIc), Nb′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the antisense strand is represented as formula (IId), each Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6.
In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula:










5′ np′-Na′Y′Y′Y′-Na′-nq′ 3′.
(Ia)






When the antisense strand is represented as formula (IIa), each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
Each of X′, Y′ and Z′ may be the same or different from each other.
Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.
In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1st nucleotide from the 5′-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.
In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 15t nucleotide from the 5′-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.
The sense strand represented by any one of the above formulas (Ia), (IIb), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.
Accordingly, the RNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):







sense:


5′ np-Na-(X X X)i-Nb-Y Y Y-Nb-(Z Z Z)j-Na-nq 3′


 


antisense:







(III)







3′ np′-Na′-(X′X′X′)k-Nb′-Y′Y′Y′-Nb′-(Z′Z′Z′)l-Na′-


nq′ 5′






wherein:
i, j, k, and 1 are each independently 0 or 1;
p, p′, q, and q′ are each independently 0-6;
each Na and Na′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
each Nb and Nb′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;
wherein
each np′, np, nq′, and nq, each of which may or may not be present, independently represents an overhang nucleotide; and
XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.
In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.
Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:







(IIIa)







5′ np-Na-Y Y Y-Na-nq 3′


3′ np′-Na′-Y′Y′Y′-Na′nq′ 5′


 







(IIIb)







5′ np-Na-Y Y Y-Nb-Z Z Z-Na-nq 3′


3′ np′-Na′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′nq′ 5′


 







(IIIc)







5′ np-Na-X X X-Nb-Y Y Y-Na-nq 3′


3′ np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Na′-nq′ 5′


 







(IIId)







5′ np-Na-X X X-Nb-Y Y Y-Nb-Z Z Z-Na-nq 3′


3′ np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Nb′-Z′Z′Z′-Na-nq′ 5′






When the RNAi agent is represented by formula (IIIa), each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the RNAi agent is represented as formula (IIIc), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or Omodified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the RNAi agent is represented as formula (IIId), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na, Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na′, Nb and Nb′ independently comprises modifications of alternating pattern.
Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.
When the RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.
When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.
When the RNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.
In one embodiment, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.
In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications and np′>0 and at least one np′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker. In another embodiment, when the RNAi agent is represented by formula (Ind), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.
In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.
In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.
iRNA Conjugates
The iRNA agents disclosed herein can be in the form of conjugates. The conjugate may be attached at any suitable location in the iRNA molecule, e.g., at the 3′ end or the 5′ end of the sense or the antisense strand. The conjugates are optionally attached via a linker.
In some embodiments, an iRNA agent described herein is chemically linked to one or more ligands, moieties or conjugates, which may confer functionality, e.g., by affecting (e.g., enhancing) the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Typical ligands will not take part in duplex pairing in a duplexed nucleic acid.
Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an α helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.
In some embodiments, the ligand is a GalNAc ligand that comprises one or more N-acetylgalactosamine (GalNAc) derivatives. Additional description of GalNAc ligands is provided in the section titled Carbohydrate Conjugates.
Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.
The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.
Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.
The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
Lipid Conjugates
In one embodiment, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can typically bind a serum protein, such as human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to modulate, e.g., control (e.g., inhibit) the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
In one embodiment, the lipid based ligand binds HSA. For example, the ligand can bind HSA with a sufficient affinity such that distribution of the conjugate to a non-kidney tissue is enhanced. However, the affinity is typically not so strong that the HSA-ligand binding cannot be reversed.
In another embodiment, the lipid based ligand binds HSA weakly or not at all, such that distribution of the conjugate to the kidney is enhanced. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).
Cell Permeation Agents
In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In one embodiment, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is typically an α-helical agent, and can have a lipophilic and a lipophobic phase.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:3367). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:3368)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:3369)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 3370)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.
An RGD peptide moiety can be used to target a particular cell type, e.g., a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing αvβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).
A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).
Carbohydrate Conjugates
In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).
In one embodiment, a carbohydrate conjugate comprises a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates are described, for example, in U.S. Pat. No. 8,106,022, the entire content of which is hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets the iRNA to particular cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).
In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein.
In some embodiments, the GalNAc conjugate is




In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S




In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below




In some embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:
















Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,




when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.
In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,













when one of X or Y is an oligonucleotide, the other is a hydrogen.

Linkers
In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.
The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.
In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):




wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;

Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R′)═C(R′′), CC or C(O);

R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C are each independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), —C(O)—CH(Ra)—NH—, CO, CH═N—O,





or heterocyclyl;

L2A, L2B, L3A, L3B, LL4, L4B, L5A, L5B and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):




    
    
        wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.
    
    


Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.
A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.
Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
Redox Cleavable Linking Groups
In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
Phosphate-Based Cleavable Linking Groups
In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.
Acid Cleavable Linking Groups
In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.
Ester-Based Cleavable Linking Groups
In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.
Peptide-Based Cleavable Linking Groups
In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)— (SEQ ID NO: 13), where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which is herein incorporated by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.
“Chimeric” iRNA compounds, or “chimeras,” in the context of the present invention, are iRNA compounds, e.g., dsRNAs, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm, 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.
Delivery of iRNA
The delivery of an iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.
Direct Delivery
In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo.
Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to other groups, e.g., a lipid or carbohydrate group as described herein. Such conjugates can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes. For example, GalNAc conjugates or lipid (e.g., LNP) formulations can be used to target iRNA to particular cells, e.g., liver cells, e.g., hepatocytes.
Lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol. 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.
Vector Encoded iRNAs
In another aspect, iRNA targeting the ALAS1 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
An iRNA expression vector is typically a DNA plasmid or viral vector. An expression vector compatible with eukaryotic cells, e.g., with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors contain convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
An iRNA expression plasmid can be transfected into a target cell as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKOTM). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.
Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.
Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.
In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.
Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
Another typical viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
III. PHARMACEUTICAL COMPOSITIONS CONTAINING iRNA
In one embodiment, the invention provides pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the iRNA is useful for treating a disease or disorder related to the expression or activity of an ALAS1 gene (e.g., a disorder involving the porphyrin pathway). Such pharmaceutical compositions are formulated based on the mode of delivery. For example, compositions can be formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. In some embodiments, a composition provided herein (e.g., an LNP formulation) is formulated for intravenous delivery. In some embodiments, a composition provided herein (e.g., a composition comprising a GalNAc conjugate) is formulated for subcutaneous delivery.
The pharmaceutical compositions featured herein are administered in a dosage sufficient to inhibit expression of an ALAS1 gene. In general, a suitable dose of iRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as can be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The effect of a single dose on ALAS1 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes related to ALAS1 expression (e.g., pathological processes involving porphyrins or defects in the porphyrin pathway, such as, for example, porphyrias). Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose and/or an effective dosing regimen.
A suitable mouse model is, for example, a mouse containing a transgene expressing human ALAS1. Mice that have knock-in mutations (e.g., mutations that are associated with acute hepatic porphyrias in humans) can be used to determine the therapeutically effective dosage and/or duration of administration of ALAS1 siRNA. The present invention also includes pharmaceutical compositions and formulations that include the iRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.
The iRNA can be delivered in a manner to target a particular tissue, such as a tissue that produces erythrocytes. For example, the iRNA can be delivered to bone marrow, liver (e.g., hepatocyes of liver), lymph glands, spleen, lungs (e.g., pleura of lungs) or spine. In one embodiment, the iRNA is delivered to bone marrow.
Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
Liposomal Formulations
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245) Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Several studies have assessed the topical delivery of liposomal drug formulations to the skin Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising NovasomeTM I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NovasomeTM II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S. T. P. Pharma. Sci., 1994, 4, 6, 466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Nucleic Acid Lipid Particles
In one embodiment, an ALAS1 dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.
The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
In some embodiments, the iRNA is formulated in a lipid nanoparticle (LNP).
LNP01
In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (e.g., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.




LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
Additional exemplary lipid-dsRNA formulations are provided in the following table.







TABLE 10







Examplary lipid formulations











cationic lipid/non-cationic




lipid/cholesterol/PEG-lipid conjugate



Cationic Lipid
Lipid:siRNA ratio





SNALP
1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-



dimethylaminopropane (DLinDMA)
cDMA




(57.1/7.1/34.4/1.4)




lipid:siRNA~7:1


S-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DPPC/Cholesterol/PEG-cDMA



[1,3]-dioxolane (XTC)
57.1/7.1/34.4/1.4




lipid:siRNA~7:1


LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5




lipid:siRNA~6:1


LNP06
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5




lipid:siRNA~11:1


LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
60/7.5/31/1.5,




lipid:siRNA~6:1


LNP08
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
60/7.5/31/1.5,




lipid:siRNA~11:1


LNP09
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG



[1,3]-dioxolane (XTC)
50/10/38.5/1.5




Lipid:siRNA 10:1


LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-
ALN100/DSPC/Cholesterol/PEG-DMG



di((9Z,12Z)-octadeca-9,12-
50/10/38.5/1.5



dienyl)tetrahydro-3aH-
Lipid:siRNA 10:1



cyclopenta[d][1,3]dioxol-5-amine




(ALN100)



LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-
MC-3/DSPC/Cholesterol/PEG-DMG



6,9,28,31-tetraen-19-yl 4-
50/10/38.5/1.5



(dimethylamino)butanoate (MC3)
Lipid:siRNA 10:1


LNP12
1,1′-(2-(4-(2-((2-(bis(2-
C12-200/DSPC/Cholesterol/PEG-DMG



hydroxydodecyl)amino)ethyl)(2-
50/10/38.5/1.5



hydroxydodecyl)amino)ethyl)piperazin-
Lipid:siRNA 10:1



1-yl)ethylazanediyl)didodecan-2-ol




(C12-200)



LNP13
XTC
XTC/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 33:1


LNP14
MC3
MC3/DSPC/Chol/PEG-DMG




40/15/40/5




Lipid:siRNA: 11:1


LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-




PEG-DSG




50/10/35/4.5/0.5




Lipid:siRNA: 11:1


LNP16
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP17
MC3
MC3/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP18
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 12:1


LNP19
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/35/5




Lipid:siRNA: 8:1


LNP20
MC3
MC3/DSPC/Chol/PEG-DPG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP22
XTC
XTC/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1





DSPC: distearoylphosphatidylcholine


DPPC: dipalmitoylphosphatidylcholine


PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)


PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)


PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)


SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.


XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.


MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference.


ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.


C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.






Synthesis of Cationic Lipids
Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles featured in the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.
“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRx(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRx(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.
“Halogen” means fluoro, chloro, bromo and iodo.
In some embodiments, the methods featured in the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.
Synthesis of Formula A
In one embodiments, nucleic acid-lipid particles featured in the invention are formulated using a cationic lipid of formula A:




where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.





Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.




Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
Synthesis of MC3
Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).
Synthesis of ALNY-100
Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:




Synthesis of 515:
To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
Synthesis of 516:
To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] −232.3 (96.94%).
Synthesis of 517A and 517B:
The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction ( ̃3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude
517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]-266.3, [M+NH4+]−4283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.
Synthesis of 518:
Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.
General Procedure for the Synthesis of Compound 519:
A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.
Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
The pharmaceutical formulations featured in the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions featured in the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Additional Formulations
Emulsions
The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.
Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants:
In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Fatty Acids:
Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
Bile Salts:
The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating Agents:
Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of β-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
Non-Chelating Non-Surfactants:
As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of iRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example LipofectamineTM (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000TM (Invitrogen; Carlsbad, Calif.), 293FectinTM (Invitrogen; Carlsbad, Calif.), CellfectinTM (Invitrogen; Carlsbad, Calif.), DMRIE-CTM (Invitrogen; Carlsbad, Calif.), FreeStyleTM MAX (Invitrogen; Carlsbad, Calif.), LipofectamineTM 2000 CD (Invitrogen; Carlsbad, Calif.), LipofectamineTM (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), OligofectamineTM (Invitrogen; Carlsbad, Calif.), OptifectTM (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFastTM Transfection Reagent (Promega; Madison, Wis.), TfxTM-20 Reagent (Promega; Madison, Wis.), TfxTM-50 Reagent (Promega; Madison, Wis.), DreamFectTM (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVecTM/LipoGenTM (Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTERTTM transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFectTM (B-Bridge International, Mountain View, Calif., USA), among others.
Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism. Examples of such biologic agents include agents that interfere with an interaction of ALAS1 and at least one ALAS1 binding partner.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are typical.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of diseases or disorders related to ALAS1 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Methods for Treating Diseases Related to Expression of an ALAS1 Gene
The invention relates in particular to the use of an iRNA targeting ALAS1 to inhibit ALAS1 expression and/or to treat a disease, disorder, or pathological process that is related to ALAS1 expression.
As used herein, “a disorder related to ALAS1 expression,” a “disease related to ALAS1 expression, a “pathological process related to ALAS1 expression,” or the like includes any condition, disorder, or disease in which ALAS1 expression is altered (e.g., elevated), the level of one or more porphyrins is altered (e.g., elevated), the level or activity of one or more enzymes in the heme biosynthetic pathway (porphyrin pathway) is altered, or other mechisms that lead to pathological changes in the heme biosynthetic pathway. For example, an iRNA targeting an ALAS1 gene, or a combination thereof, may be used for treatment of conditions in which levels of a porphyrin or a porphyrin precursor (e.g., ALA or PBG) are elevated (e.g., certain porphyrias), or conditions in which there are defects in the enzymes of the heme biosynthetic pathway (e.g., certain porphyrias). Disorders related to ALAS1 expression include, for example, X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria (Doss porphyria), acute intermittent porphyria (AIP), congenital erythropoietic porphyria, prophyria cutanea tarda, hereditary coproporphyria (coproporphyria), variegate porphyria, erythropoietic protoporphyria (EPP), and transient erythroporphyria of infancy.
As used herein, a “subject” to be treated according to the methods described herein, includes a human or non-human animal, e.g., a mammal. The mammal may be, for example, a rodent (e.g., a rat or mouse) or a primate (e.g., a monkey). In some embodiments, the subject is a human.
In some embodiments, the subject is suffering from a disorder related to ALAS1 expression (e.g., has been diagnosed with a porphyria or has suffered from one or more symptoms of porphyria and is a carrier of a mutation associated with porphyria) or is at risk of developing a disorder related to ALAS1 expression (e.g., a subject with a family history of porphyria, or a subject who is a carrier of a genetic mutation associated with porphyria).
Classifications of porphyrias, including acute hepatic porphyrias, are described, e.g., in Balwani, M. & Desnick, R. J., Blood, 120(23), published online as Blood First Edition paper, July 12, 102; DOI 10.1182/blood-2012-05-423186. As described in Balwain & Desnick, acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP) are autosomal dominant porphyrias and ALA deyhdratase deficiency porphyria (ADP) is autosomal recessive. In rare cases, AIP, HCP, and VP occur as homozygous dominant forms. In addition, there is a rare homozygous recessive form of porphyria cutanea tarda (PCT), which is the single hepatic cutaneous porphyria, and is also known as hepatoerythropoietic porphyria. The clinical and laboratory features of these porphyrias are described in Table 11 below.







TABLE 11







Human hepatic porphyrias: clinical and laboratory features














Enzyme






Principal
activity,












Deficient
symptoms,
% of
Increased porphyrin precursors and/or porphyrins*














Porphyria
enzyme
Inheritance
NV or CP
normal
Erythrocytes
Urine
Stool










Acute hepatic porphyrias














ADP
ALA-
AR
NV
~5
Zn-protoporphyrin
ALA,
—



dehydratase




coproporphyrin








III


AIP
HMB-
AD
NV
~50
—
ALA, PBG,
—



synthase




uroporphyrin


HCP
COPRO-
AD
NV and CP
~50
—
ALA, PBG,
copropor



oxidase




coproporphyrin
phyrin III








III


VP
PROTO-
AD
NV and CP
~50
—
ALA, PBG
copropor



oxidase




coproporphyrin
phyrin III,








III
protopor









phyrin







Hepatic cutaneous porphyrias














PCT
URO-
Sporadic or
CP
<20
—
uroporphyrin,
uroporphy-



decarboxylase
AD



7-carboxylate
rin, 7-








porphyrin
carboxylate









porphyrin





AR indicates autosomal recessive; AD, autosomal dominant; NV, neurovisceral; CP, cutaneous photosensitivity; and —, not applicable.


*Increases that may be important for diagnosis.






In some embodiments, the subject has or is at risk for developing a porphyria, e.g., a hepatic porphyria, e.g., AIP, HCP, VP, ADP, or hepatoerythropoietic porphyria.
In some embodiments, the porphyria is an acute hepatic porphyria, e.g., an acute hepatic porphyria iselected from acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), variegate porphyria (VP), and ALA deyhdratase deficiency porphyria (ADP).
In some embodiments, the porphyria is a dual porphyria, e.g., at least two porphyrias. In some embodiments, the dual porphyria comprises two or more porphyrias selected from acute intermittent porphyria (AIP) hereditary coproporphyria (HCP), variegate porphyria (VP), and ALA deyhdratase deficiency porphyria (ADP).
In some embodiments, the porphyria is a homozygous dominant hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or hepatoerythropoietic porphyria, In some embodiments, the porphyria is AIP, HCP, VP, or hepatoerythropoietic porphyria, or a combination thereof (e.g., a dual porphyria). In embodiments, the AIP, HCP, or VP is either heterozygous dominant or homozygous dominant.
In embodiments, the subject has or is at risk for developing a porphyria, e.g., ADP, and shows an elevated level (e.g., an elevated urine level) of ALA and/or coproporphyrin III. In embodiments, the subject has or is at risk for developing a porphyria, e.g., ADP, and shows an elevated level of erythrocyte Zn-protoporphyrin.
In embodiments, the subject has or is at risk for developing a porphyria, e.g., AIP, and shows an elevated level (e.g., an elevated urine level) of ALA, PBG, and/or uroporphyrin.
In embodiments, the subject has or is at risk for developing a porphyria, e.g., HCP, and shows an elevated level (e.g., an elevated urine level) of ALA, PBG, and/or coproporphyrin III. In embodiments, the subject has or is at risk for developing a porphyria, e.g., HCP, and shows an elevated level (e.g., an elevated stool level) of coproporphyrin III.
In embodiments, the subject has or is at risk for developing a porphyria, e.g., VP, and shows an elevated level (e.g., an elevated urine level) of ALA, PBG, and/or coproporphyrin III.
In embodiments, the subject has or is at risk for developing a porphyria, e.g., HCP, and shows an elevated level (e.g., an elevated stool level) of coproporphyrin III and/or protoporphyrin.
In embodiments, the subject has or is at risk for developing a porphyria, e.g., PCT, (e.g., hepatoerythropoietic porphyria) and shows an elevated level (e.g., an elevated urine level) of uroporphyrin and/or 7-carboxylate porphyrin. In embodiments, the subject has or is at risk for developing a porphyria, e.g., PCT, (e.g., hepatoerythropoietic porphyria) and shows an elevated level (e.g., an elevated stool level) of uroporphyrin and/or 7-carboxylate porphyrin.
A mutation associated with porphyria includes any mutation in a gene encoding an enzyme in the heme biosynthetic pathway (porphyrin pathway) or a gene which alters the expression of a gene in the heme biosynthetic pathway. In many embodiments, the subject carries one or more mutations in an enzyme of the porphyrin pathway (e.g., a mutation in ALA deydratase or PBG deaminase). In some embodiments, the subject is suffereing from an acute porphyria (e.g., AIP, ALA deydratase deficiency porphyria).
In some cases, patients with an acute hepatic porphyria (e.g., AIP), or patients who carry mutations associated with an acute hepatic porphyria (e.g., AIP) but who are asymptomatic, have elevated ALA and/or PBG levels compared with healthy individuals. See, e.g., Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707, 2006; Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007. In such cases, the level of ALA and/or PBG can be elevated even when the patient is not having, or has never had, an attack. In some such cases, the patient is otherwise completely asymptomatic. In some such cases, the patient suffers from pain, e.g., neuropathic pain, which can be chronic pain (e.g., chronic neuropathic pain). In some cases, the patient has a neuropathy. In some cases, the patient has a progressive neuropathy.
In some embodiments, the subject to be treated according to the methods described herein has an elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. Levels of a porphyrin or a porphyrin precursor can be assessed using methods known in the art or methods described herein. For example, methods of assessing uring and plasma ALA and PBG levels, as well as urine and plasma porphyrin levels, are disclosed in Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707, 2006; and Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007, the entire contents of which are hereby incorporated in their entirety.
In some embodiments, the subject is an animal model of a porphyria, e.g., a mouse model of a porphyria (e.g., a mutant mouse as described in Lindberg et al. Nature Genetics, 12: 195-199, 1996). In some embodiments, the subject is a human, e.g., a human who has or is at risk for developing a porphyria, as described herein. In some embodiments, the subject is not having an acute attack of porphyria. In some embodiments, the subject has never had an attack. In some embodiments, the patient suffers from chronic pain. In some embodiments, the patient has nerve damage. In embodiments, the subject has EMG changes and/or changes in nerve conduction velocity. In some embodiments, the subject is asymptomatic. In some embodiments, the subject is at risk for developing a porphyria (e.g., carries a gene mutation associated with a porphyria) and is asymptomatic. In some embodiments, the subject has previously had an acute attack but is asymptomatic at the time of treatment.
In some embodiments, the subject is at risk for developing a porphyria and is treated prophylactically to prevent the development of a porphyria. In some embodiments the subject has an elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. In some embodiments, the prophylactic treatment begins at puberty. In some embodiments the treatment lowers the level (e.g., the plasma level or the urine level) of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. In some embodiments, the treatment prevents the development of an elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. In some embodiments, the treatment prevents the development of, or decreases the frequency or severity of, a symptom associated with a porphyria, e.g., pain or nerve damage.
In some embodiments, the level of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, is elevated, e.g., in a sample of plasma or urine from the subject. In some embodiments, the level of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, in the subject is assessed based on the absolute level of the porphyrin or the porphyrin precursor, e.g., ALA or PBG in a sample from the subject. In some embodiments, the level of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, in the subject is assessed based on the relative level of the porphyrin or porphyrin precursor, e.g., ALA or PBG, in a sample from the subject. In some embodiments, the relative level is relative to the level of another protein or compound, e.g., the level of creatinine, in a sample from the subject. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is a stool sample.
An elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG, can be established, e.g., by showing that the subject has a level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG (e.g., a plasma or urine level of ALA and/or PBG) that is greater than, or greater than or equal to, a reference value. A physician with expertise in the treatment of porphyrias would be able to determine whether the level of a porphyrin or a porphyrin precursor, (e.g., ALA and/or PBG) is elevated, e.g., for the purpose of diagnosing a porphyria or for determining whether a subject is at risk for developing a porphyria, e.g., a subject may be predisposed to an acute attack or to pathology associated with a porphyria, such as, e.g., chronic pain (e.g., neuropathic pain) and neuropathy (e.g., progressive neuropathy).
As used herein, a “reference value” refers to a value from the subject when the subject is not in a disease state, or a value from a normal or healthy subject, or a value from a reference sample or population, e.g., a group of normal or healthy subjects (e.g., a group of subjects that does not carry a mutation associated with a porphyria and/or a group of subjects that does not suffer from symptoms associated with a porphyria).
In some embodiments, the reference value is a pre-disease level in the same individual. In some embodiments, the reference value is a level in a reference sample or population. In some embodiments, the reference value is the mean or median value in a reference sample or population. In some embodiments, the reference value the value that is two standard deviations above the mean in a reference sample or population. In some embodiments, the reference value is the value that is 2.5, 3, 3.5, 4, 4.5, or 5 standard deviations above the mean in a reference sample or population.
In some embodiments, wherein the subject has an elevated level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG, the subject has a level of ALA and/or PBG that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than a reference value. In some embodiments, the subject has a level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG, that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold higher than a reference value.
In some embodiments, the reference value is an upper reference limit. As used herein, an “upper reference limit” refers to a level that is the upper limit of the 95% confidence interval for a reference sample or population, e.g., a group of normal (e.g., wild type) or healthy individuals, e.g., individuals who do not carry a genetic mutation associated with a porphyria and/or individuals who do not suffer from a porphyria. Accordingly, a lower reference limit refers to a level that is the lower limit of the same 95% confidence interval.
In some embodiments wherein the subject has an elevated level, e.g., a plasma level or a urine level, of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, the level is greater than or equal to 2 times, 3 times, 4 times, or 5 times that of a reference value, e.g., an upper reference limit. In some embodiments, the subject has a urine level of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, that is greater than 4 times that of an upper reference limit.
In some embodiments, the reference value is a value provided in Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707, 2006 or Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007. In some embodiments, the reference value is a value provided in Table 1 of Sardh et al.
In some embodiments, the subject is a human and has a urine level of PBG that is greater than or equal to 4.8 mmol/mol creatinine. In certain embodiments, the subject is a human and has a urine level of PBG that is greater than, or greater than or equal to, about 3, 4, 5, 6, 7, or 8 mmol/mol creatinine.
In embodiments, the reference value for plasma PBG is 0.12 μmol/L. In embodiments, the subject is a human and has a plasma PBG level that is greater than, or greater than or equal to, 0.10 μmol/L, 0.12 μmol/L, 0.24 μmol/L, 0.36 μmol/L, 0.48 μmol/L, or 0.60 μmol/L. In embodiments, the subject is a human and has a plasma level of PBG that is greater than, or greater than or equal to, 0.48 μmol/L.
In embodiments, the reference value for urine PBG is 1.2 mmol/mol creatinine. In embodiments, the subject is a human and has a urine PBG level that is greater than, or greater than or equal to, 1.0 mmol/mol creatinine, 1.2 mmol/mol creatinine, 2.4 mmol/mol creatinine, 3.6 mmol/mol creatinine, 4.8 mmol/mol creatinine, or 6.0 mmol/mol creatinine. In embodiments, the subject is a human and has a urine level of PBG that is greater than, or greater than or equal to, 4.8 mmol/mol creatinine.
In embodiments, the reference value for plasma ALA is 0.12 mmol/L. In embodiments, the subject is a human and has a plasma ALA level that is greater than, or greater than or equal to, 0.10 μmol/L, 0.12 μmol/L, 0.24 μmol/L, 0.36 μmol/L, 0.48 μmol/L, or 0.60 μmol/L. In embodiments, the subject is a human and has a plasma ALA level that is greater than, or greater than or equal to 0.48 μmol/L.
In embodiments, the reference value for urine ALA is 3.1 mmol/mol creatinine. In embodiments, the subject is a human and has a urine ALA level that is greater than, or greater than or equal to, 2.5 mmol/mol creatinine, 3.1 mmol/mol creatinine, 6.2 mmol/mol creatinine, 9.3 mmol/mol creatinine, 12.4 mmol/mol creatinine, or 15.5 mmol/mol creatinine.
In embodiments, the reference value for plasma porphyrin is 10 nmol/L. In embodiments, the subject is a human and has a plasma porphyrin level that is greater than, or greater than or equal to, 10 nmol/L. In embodiments, the subject is a human and has a plasma porphyrin level that is greater than, or greater than or equal to, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nmol/L. the subject is a human and has a plasma porphyrin level that is greater than, or greater than or equal to 40 nmol/L. In embodiments, the reference value for urine porphyrin is 25 μmol/mol creatinine. In embodiments, the subject is a human and has a urine porphyrin level that is greater than, or greater than or equal to, 25 μmol/mol creatinine. In embodiments, the subject is a human and has a urine porphyrin level that is greater than, or equal to, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 μmol/mol creatinine.
In some embodiments, the subject has a level, e.g., a plasma level or a urine level, of a porphyrin or a porphyrin precursor, e.g., ALA or PBG, that is greater than that of 99% of individuals in a sample of healthy individuals.
In some embodiments, the subject has a level, e.g., a plasma level or a urine level, of ALA or PBG that is greater than two standard deviations above the mean level in a sample of healthy individuals.
In some embodiments, the subject has a urine level of ALA that is 1.6 or more times that of the mean level in a normal subject (e.g., a subject that does not carry a mutation associated with a porphyria). In some embodiments, the subject has a plasma level of ALA that is 2 or 3 times that of the mean level in a normal subject. In some embodiments, the subject has a urine level of PBG that is four or more times that of the mean level in a normal subject. In some embodiments, the subject has a plasma level of PBG that is four or more times that of the mean level in a normal subject.
In some embodiments, the method is effective to decrease the level of a porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. In embodiments, the method is effective to produce a predetermined reduction in the elevated level of the porphyrin or porphyrin precursor, e.g., ALA or PBG. In some embodiments, the predetermined reduction is a decrease of at least 10%, 20%, 30%, 40%, or 50%. In some embodiments, the predetermined reduction is a reduction that is effective to prevent or ameliorate symptoms, e.g., pain or recurring attacks.
In some embodiments, the predetermined reduction is a reduction that is at least 1, 2, 3, or more standard deviations, wherein the standard deviation is determined based on the values from a reference sample, e.g., a reference sample as described herein.
In some embodiments, the predetermined reduction is a reduction that brings the level of the porphyrin or porphyrin precursor to a level that is less than, or to a level that is less than or equal to, a reference value (e.g., a reference value as described herein).
In some embodiments, the subject to be treated according to the methods described suffers from pain, e.g., chronic pain. In some embodiments, the subject has or is at risk for developing a porphyria, e.g. an acute hepatic porphyria, e.g., AIP. In embodiments, the method is effective to treat the pain, e.g., by reducing the severity of the pain or curing the pain. In embodiments, the method is effective to decrease or prevent nerve damage.
In some embodiments, the subject to be treated according to the methods described herein (a) has an elevated level of ALA and/or PBG and (b) suffers from pain, e.g., chronic pain. In embodiments, the method is effective to decrease an elevated level of ALA and/or PBG and/or to treat the pain, e.g., by reducing the severity of the pain or curing the pain.
In some embodiments, the subject is an animal that serves as a model for a disorder related to ALAS1 expression.
In some embodiments the subject is an animal that serves as a model for porphyria (e.g., a genetically modified animal with one or more mutations. In some embodiments, the porphyria is AIP and the subject is an animal model of AIP. In one such embodiment, the subject is a genetically modified mouse that is deficient in porphobilinogen deaminase, such as, for example, the mouse described in Lindberg et al., Nature Genetics, 12:195-199, 1996, or the homozygous R167Q mouse described in Yasuda, M., Yu, C. Zhang, J., Clayero, S., Edelmann, W., Gan, L., Phillips, J. D., & Desnick, R. J. Acute intermittent porphyria: A severely affected knock-in mouse that mimics the human homozygous dominant phenotype. (Abstract of Presentation on Oct. 14, 2011 at the American Society of Human Genetics; Program No. 1308F; accessed online on Apr. 4, 2012 at ichg2011.org/cgi-bin/showdetail.pl?absno=21167); both of these references are hereby incorporated herein in their entirety. Several knock-in models for mutations causing homozygous dominant AIP in humans have been generated. The mutations employed include, e.g., R167Q, R173Q, and R173W in PBG deaminase. Viable homozygotes included the R167Q/R176Q and R167Q/R173Q, both of which exhibit constitutively elevated ALA and PBG levels analogous to the phenotype in human homozygous dominant AIP; in some embodiments, such a viable homozygous AIP mouse model is the subject.
In one embodiment, a subject to be treated according to the methods described herein, (e.g., a human subject or patient), is at risk of developing, or has been diagnosed, with a disorder related to ALAS1 expression, e.g. a porphyria. In some embodiments, the subject is a subject who has suffered one or more acute attacks of one or more porphyric symptoms. In other embodiments, the subject is a subject who has suffered chronically from one or more symptoms of porphyria (e.g., pain, e.g., neuropathic pain and or neuropathy, e.g., progressive neuropathy). In some embodiments, the subject carries a genetic alteration (e.g., a mutation) as described herein but is otherwise asymptomatic. In some embodiments, the subject has previously been treated with a heme product (e.g., hemin, heme arginate, or heme albumin), as described herein.
In some embodiments, a subject (e.g., a subject with a porphyria, such as, e.g., AIP) to be treated according to the methods described herein has recently experienced or is currently experiencing a prodrome. In some such embodiments, the subject is administered a combination treatment, e.g., an iRNA as described herein, and one or more additional treatments known to be effective against porphyria (e.g., glucose and/or a heme product such as hemin, as described herein) or its associated symptoms.
In one embodiment, an iRNA as described herein is administered in combination with glucose or dextrose. For example, 10-20% dextrose in normal saline may be provided intravenously. Typically, when glucose is administered, at least 300 g of 10% glucose is administered intravenously daily. The iRNA (e.g., an iRNA in an LNP formulation) may also be administered intravenously, as part of the same infusion that is used to administer the glucose or dextrose, or as a separate infusion that is administered before, concurrently, or after the administration of the glucose or dextrose. In some embodiments, the iRNA is administered via a different route of administration (e.g., subcutaneously). In yet another embodiment, the iRNA is administered in combination with total parenteral nutrition. The iRNA may be administered before, concurrent with, or after the administration of total parenteral nutrition.
In one embodiment, the iRNA is administered in combination with a heme product (e.g., hemin, heme arginate, or heme albumin). In a further embodiment, the iRNA is administered in combination with a heme product and glucose, a heme product and dextrose, or a heme product and total parenteral nutrition.
A “prodrome,” as used herein, includes any symptom that the individual subject has previously experienced immediately prior to developing an acute attack. Typical symptoms of a prodrome include, e.g., abdominal pain, nausea, headaches, psychological symptoms (e.g., anxiety), restlessness and/or insomnia. In some embodiments, the subject experiences pain (e.g., abdominal pain and/or a headache) during the prodrome. In some embodiments, the subject experiences nausea during the prodrome. In some embodiments, the subject experiences psychological symptoms (e.g., anxiety) during the prodrome. In some embodiments, the subject becomes restless and/or suffers from insomnia during the prodrome.
An acute “attack” of porphyria involves the onset of one or more symptoms of porphyria, typically in a patient who carries a mutation associated with porphyria (e.g., a mutation in a gene that encodes an enzyme in the porphyrin pathway).
In certain embodiments, administration of an ALAS1 iRNA results in a decrease in the level of one or more porphyrins or porphyrin precursors, as described herein (e.g., ALA and/or PBG). The decrease may be measured relative to any appropriate control or reference value. For example, the decrease in the level of one or more porphyrins or porphyrin precursors may be established in an individual subject, e.g., as a decrease of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more compared with the level prior to treatment (e.g., immediately prior to treatment). A decrease in the level of a porphyrin precursor, a porphyrin, or or a porphyrin metabolite may be measured using any method known in the art. For example, the level of PBG and/or ALA in urine or plasma may be assessed, using the Watson-Schwartz test, ion exchange chromatography, or high-performance liquid chromatography—mass spectrometry. See, e.g., Thunell (1993).
In some embodiments, administration of an ALAS1 siRNA is effective to reduce the level of ALA and/or PBG in the subject. The level of ALA or PBG in the subject can be assessed, e.g., based on the absolute level of ALA or PBG, or based on the relative level of ALA or PBG (e.g., relative to the level of another protein or compound, e.g., the level of creatinine) in a sample from the subject. In some embodiments, the sample is a urine sample. In some embodiments, the sample is a plasma sample.
In certain embodiments, an iRNA that targets ALAS1 is administered in combination one or more additional treatments, e.g., another treatment known to be effective in treating porphyria or symptoms of porphyria. For example, the other treatment may be glucose (e.g., IV glucose) or a heme product (e.g., hemin, heme arginate, or heme albumin). The additional treatment(s) may be administered before, after, or concurrent with the administration of iRNA.
The iRNA and an additional therapeutic agent can be administered in combination in the same composition, e.g., intravenously, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.
In some embodiments, administration of iRNA, or administration of iRNA in combination one or more additional treatments (e.g., glucose, dextrose or the like), decreases the frequency of acute attacks (e.g., by preventing acute attacks so that they no longer occur, or by reducing the number of attacks that occur in a certain time period, e.g., fewer attacks occur per year). In some such embodiments, the iRNA is administered according to a regular dosing regimen, e.g., daily, weekly, biweekly, or monthly.
In some embodiments, the iRNA is administered after an acute attack of porphyria. In some such embodiments, the iRNA is in a composition, e.g. a composition comprising a lipid formulation, e.g. an LNP formulation.
In some embodiments, the iRNA is administered during an acute attack of porphyria. In some such embodiments, the iRNA is in a composition, e.g. a composition comprising a lipid formulation (e.g., an LNP formulation) or a composition comprising a GalNAc conjugate.
In some embodiments, administration of an ALAS1 siRNA is effective to lessen the severity of the attack (e.g., by ameliorating one or more signs or symptoms associated with the attack). In some embodiments, administration of an ALAS1 siRNA is effective to shorten the duration of an attack. In some embodiments, administration of an ALAS1 siRNA is effective to stop an attack. In some embodiments, the iRNA is administered prophylactically to prevent an acute attack of porphyria. In some such embodiments, the iRNA is in the form of a GalNAc conjugate, e.g., in a composition comprising a GalNAc conjugate. In some embodiments, the prophylactic administration is before, during, or after exposure to or occurrence of a precipitating factor. In some embodiments, the subject is at risk of developing porphyria.
In some embodiments, the siRNA is administered during a prodrome. In some embodiments, the prodrome is characterized by pain (e.g., headache and/or abdominal pain), nausea, psychological symptoms (e.g., anxiety), restlessness and/or insomnia.
In some embodiments, the siRNA is administered during a particular phase of the menstrual cycle, e.g., during the luteal phase.
In some embodiments, administration of an ALAS1 siRNA is effective to prevent attacks (e.g., recurrent attacks that are associated with a prodrome and/or with a precipitating factor, e.g., with a particular phase of the menstrual cycle, e.g., the luteal phase). In some embodiments, administration of an ALAS1 siRNA is effective to reduce the frequency of attacks. In embodiments, administration of an ALAS1 siRNA is effective to lessen the severity of the attack (e.g., by ameliorating one or more signs or symptoms associated with the attack). In some embodiments, administration of an ALAS1 siRNA is effective to shorten the duration of an attack. In some embodiments, administration of an ALAS1 siRNA is effective to stop an attack.
In some embodiments administration of an ALAS1 siRNA is effective to prevent or decrease the frequency or severity of pain, e.g., neuropathic pain.
In some embodiments administration of an ALAS1 siRNA is effective to prevent or decrease the frequency or severity of neuropathy
Effects of administration of an ALAS1 siRNA can be established, for example, by comparison with an appropriate control. For example, a decrease in the frequency of acute attacks, as well as a decrease in the level of one or more porphyrins or porphyrin precursors, may be established, for example, in a group of patients with AIP, as a decreased frequency compared with an appropriate control group. A control group (e.g., a group of similar individuals or the same group of individuals in a crossover design) may include, for example, an untreated population, a population that has been treated with a conventional treatment for porphyria (e.g., a conventional treatment for AIP may include glucose, hemin, or both); a population that has been treated with placebo, or a non-targeting iRNA, optionally in combination with one or more conventional treatments for porphyria (e.g., glucose, e.g., IV glucose), and the like.
A subject “at risk” of developing porphyria, as used herein, includes a subject with a family history of porphyria and/or a history of one or more recurring or chronic porphyric symptoms, and/or a subject who carries a genetic alteration (e.g., a mutation) in a gene encoding an enzyme of the heme biosynthetic pathway, and a subject who carries a genetic alteration, e.g., a mutation. known to be associated with porphyria.
In embodiments, the alteration, e.g., the mutation, makes an individual susceptible to an acute attack (e.g., upon exposure to a precipitating factor, e.g., a drug, dieting or other precipitating factor, e.g., a precipitating factor as disclosed herein). In embodiments, the alteration, e.g., the mutation, is associated with elevated levels of a porphyrin or a porphyrin precursor (e.g., ALA and/or PBG). In embodiments, the alteration, e.g., the mutation, is associated with chronic pain (e.g., chronic neuropathic pain) and/or neuropathy (e.g., progressive neuropathy). In embodiments, the, the alteration, e.g., the mutation, is associated with changes in EMG and/or nerve conduction velocities.
In embodiments, the alteration is a mutation in the ALAS1 gene. In embodiments, the alteration is a mutation in the ALAS1 gene promoter, or in regions upstream or downstream from the ALAS1 gene. In embodiments, the alteration is a mutation in transcription factors or other genes that interact with ALAS1. In embodiments, the alteration is an alteration, e.g., a mutation, in a gene that encodes an enzyme in the heme biosynthetic pathway.
In some embodiments, the subject has an genetic alteration as described herein (e.g., a genetic mutation known to be associated with a porphyria). In some such embodiments, the subject has an elevated level (e.g., urine or plasma level) of ALA and/or PBG. In some such embodiments, the subject does not have an elevated level of ALA and/or PBG. In embodiments, the subject has a genetic alteration as described herein and has other symptoms, e.g., chronic pain, EMG changes, changes in nerve conduction velocity, and/or other symptoms associated with a porphyria. In embodiments, the subject has a genetic alteration but does not suffer from acute attacks.
In embodiments, the subject has a mutation associated with AIP, HCP, VP, or ADP.
In some embodiments, the porphyria is AIP. In some such embodiments, the subject has an alteration, e.g., at least one mutation, in the PBG deaminase gene. Many PBG deaminase mutations are known in the art, for example, as reported in Hrdinka, M. et al. Physiological Research, 55 (Suppl 2):S119-136 (2006). In some embodiments, the subject is heterozygous for a PBG deaminase mutation. In other embodiments, the subject is homozygous for a PBG deaminase mutation. A homozygous subject may carry two identical mutations or two different mutations in the PBG deaminase gene.
In some embodiments, the porphyria is HCP. In some such embodiments, the subject has an alteration, e.g., at least one mutation, in the gene that encodes the enzyme coproporphyrinogen III oxidase.
In some embodiments, the porphyria is VP. In some such embodiments, the subject has an alteration, e.g., at least one mutation, in the gene that encodes protoporphrinogen oxidase.
In embodiments, the porphyria is ADP, e.g., autosomal recessive ADP. In some such embodiments, the subject has an alteration, e.g., at least one mutation, in the gene that encodes ALA deydratase.
Methods of treatment provided herein may serve to ameliorate one or more symptoms associated with porphyria, to reduce the frequency of attacks associated with porphyria, to reduce the likelihood that an attack of one or more symptoms associated with porphyria will occur upon exposure to a precipitating factor, or to reduce the risk of developing conditions associated with porphyria (e.g., neuropathy (e.g., progressive neuropathy), hepatocellular cancer). Additionally, the methods provided herein may serve to decrease the level of one or more porphyrin precursors, porphyrins and/or related porphyrin products or metabolites. The level of a porphyrin precursor or a porhyrin may be measured in any biological sample, such as, e.g., urine, blood, feces, cerebrospinal fluid, or a tissue sample. The sample may be present within a subject or may be obtained or extracted from the subject. In some embodiments, the porphyria is AIP, and the level of PBG and/or ALA is decreased. In some embodiments, the porphyrin product or metabolite is porphobilin, porphobilinogen, or uroporphyrin. A decrease in the level of a porphyrin product or metabolite may be measured using any method known in the art. For example, the level of PBG and/or ALA in urine or plasma may be assessed, using the Watson-Schwartz test, ion exchange chromatography, or high-performance liquid chromatography—mass spectrometry. See, e.g., Thunell (1993).
Methods described herein may also serve to reduce chronically elevated levels of porphyrin precursors (e.g., ALA and/or PBG) in subjects suffering from a porphyria (e.g., an acute hepatic porphyria, e.g., AIP) or at risk for developing a porphyria. Methods for assessing plasma and urine levels (e.g., chronically elevated levels) of porphyrin precursors include, e.g., HPLC-mass spectrometry and ion-exchange chromatography. The levels of porphyrin precursors may be expressed as the level relative to another protein or compound, e.g., creatinine. See, e.g., Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707, 2006; Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007
A “precipitating factor” as used herein, refers to an endogenous or exogenous factor that may induce an acute attack of one or more symptoms associated with porphyria. Precipitating factors include fasting (or other forms of reduced or inadequate caloric intake, due to crash diets, long-distance athletics, etc.), metabolic stresses (e.g., infections, surgery, international air travel, and psychological stress), endogenous hormones (e.g., progesterone), cigarette smoking, lipid-soluble foreign chemicals (including, e.g., chemicals present in tobacco smoke, certain prescription drugs, organic solvents, biocides, components in alcoholic beverages), endocrine factors (e.g., reproductive hormones (women may experience exacerbations during the premenstrual period), synthetic estrogens, progesterones, ovulation stimulants, and hormone replacement therapy). See, for example, Thunell (1993). Common precipitating factors include cytochrome P450 inducing drugs and phenobarbitol.
Symptoms associated with porphyria may include abdominal pain or cramping, headaches, effects caused by nervous system abnormalities, and light sensitivity, causing rashes, blistering, and scarring of the skin (photodermatitis). In certain embodiments, the porphyria is AIP. Symptoms of AIP include gastrointestinal symptoms (e.g., severe and poorly localized abdominal pain, nausea/vomiting, constipation, diarrhea, ileus), urinary symptoms (dysuria, urinary retention/incontinence, or dark urine), neurologic symptoms (e.g., sensory neuropathy, motor neuropathy (e.g., affecting the cranial nerves and/or leading to weakness in the arms or legs), seizures, neuropathic pain, progressive neuropathy, headaches, neuropsychiatric symptoms (e.g., mental confusion, anxiety, agitation, hallucination, hysteria, delirium, apathy, depression, phobias, psychosis, insomnia, somnolence, coma), autonomic nervous system involvement (resulting e.g., in cardiovascular sysmptoms such as tachycardia, hypertension, and/or arrhythmias, as well as other symptoms, such as, e.g., increased circulating catecholamine levels, sweating, restlessness, and/or tremor), dehydration, and electrolyte abnormalities.
In some embodiments, an iRNA targeting ALAS1 is administered together with (e.g., before, after, or concurrent with) another treatment that may serve to alleviate one or more of the above symptoms. For example, abdominal pain may be treated, e.g., with narcotic analgesics, seizures may be treated, e.g., with anti-seizure medications, nausea/vomiting may be treated, e.g., with phenothiazines, and tachycardia/hypertension may be treated, e.g., with beta blockers.
The term “decrease” (or “increase”) is intended to refer to a measurable change, e.g., a statistically significant change. The change may be, for example, at least 5%, 10%, 20%, 30%, 40%, 50% or more change (e.g., decrease (or increase) relative to a reference value, e.g., a reference where no iRNA is provided).
The invention further relates to the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a disorder related to ALAS1 expression, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating the disorder. In one embodiment, the iRNA or pharmaceutical composition thereof can be administered in conjunction with a heme product (e.g., hemin, heme arginate, or heme albumin, as described herein) and/or in conjunction with intravenous glucose infusions. In some embodiments, the iRNA or pharmaceutical composition thereof is used prophylactically, e.g., to prevent or ameliorate symptoms of an anticipated attack of acute porphyria. The prophylactic use may be timed according to the exposure or anticipated exposure of the subject to a precipitating factor. As described herein, a precipitating factor may be any endogenous or exogenous factor known to precipitate an acute attack. For example, the premenstrual phase is an endogenous precipitating factor, and a cytochrome P450 inducing drug is an exogenous precipitating factor.
The effective amount for the treatment of a disorder related to ALAS1 expression (e.g., a porphyria such as AIP) depends on the type of disorder to be treated, the severity of the symptoms, the subject being treated, the sex, age and general condition of the subject, the mode of administration and so forth. For any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using routine experimentation. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting ALAS1 or pharmaceutical composition thereof, “effective against” a disorder related to ALAS1 expression indicates that administration in a clinically appropriate manner results in a beneficial effect, e.g., for an individual patient or for at least a fraction of patients, e.g., a statistically significant fraction of patients. Beneficial effects include, e.g., prevention of or reduction of symptoms or other effects. For example, beneficial effects include, e.g., an improvement (e.g., decrease in the severity or frequency) of symptoms, a reduction in the severity or frequency of attacks, a reduced risk of developing associated disease (e.g., neuropathy (e.g., progressive neuropathy), hepatocellular cancer), an improved ability to tolerate a precipitating factor, an improvement in quality of life, a reduction in the expression of ALAS1, a reduction in a level (e.g., a plasma or urine level) of a porphyrin or a porphyrin precursor (e.g., ALA and/or PBG) or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disorder.
A treatment or preventive effect is evident when there is an improvement, e.g., a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, e.g., at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker (e.g., plasma or urinary ALA or PBG) or symptom is observed.
Patients can be administered a therapeutic amount of iRNA. The therapeutic amount can be, e.g., 0.05-50 mg/kg. For example, the therapeutic amount can be 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, or 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/kg dsRNA.
In some embodiments, the iRNA is formulated as a lipid formulation, e.g., an LNP formulation as described herein. In some such embodiments, the therapeutic amount is 0.05-5 mg/kg, e.g., 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg/kg dsRNA. In some embodiments, the lipid formulation, e.g., LNP formulation, is administered intravenously.
In some embodiments, the iRNA is administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
In some embodiments, the iRNA is in the form of a GalNAc conjugate as described herein. In some such embodiments, the therapeutic amount is 0.5-50 mg, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/kg dsRNA. In some embodiments, the GalNAc conjugate is administered subcutaneously.
In some embodiments, the administration is repeated, for example, on a regular basis, such as, daily, biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer.
In some embodiments, the iRNA agent is administered in two or more doses. In some embodiments, the number or amount of subsequent doses is dependent on the achievement of a desired effect, e.g., suppression of a ALAS gene, reduction of a level of a porphyrin or porphyrin precursor (e.g., ALA and/or PBG), or the achievement of a therapeutic or prophylactic effect, e.g., reduction or prevention of one or more symptoms associated with porphyria (e.g., pain, e.g., neuropathic pain), and/or prevention of attacks or reduction in the frequency and/or severity of attacks associated with porphyria.
In some embodiments, the iRNA agent is administered according to a schedule. For example, the iRNA agent may be administered once per week, twice per week, three times per week, four times per week, or five times per week. In some embodiments, the schedule involves regularly spaced administrations, e.g., hourly, every four hours, every six hours, every eight hours, every twelve hours, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, biweekly, or monthly. In embodiments, the iRNA agent is administered weekly or biweekly to achieve a desired effect, e.g., to decrease the level of ALA and/or PBG, to decrease pain, and/or to prevent acute attacks.
In embodiments, the schedule involves closely spaced administrations followed by a longer period of time during which the agent is not administered. For example, the schedule may involve an initial set of doses that are administered in a relatively short period of time (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours) followed by a longer time period (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) during which the iRNA agent is not administered. In one embodiment, the iRNA agent is initially administered hourly and is later administered at a longer interval (e.g., daily, weekly, biweekly, or monthly). In another embodiment, the iRNA agent is initially administered daily and is later administered at a longer interval (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer interval increases over time or is determined based on the achievement of a desired effect. In a specific embodiment, the iRNA agent is administered once daily during an acute attack, followed by weekly dosing starting on the eighth day of administration. In another specific embodiment, the iRNA agent is administered every other day during a first week followed by weekly dosing starting on the eighth day of administration.
In one embodiment, the iRNA agent is administered to prevent or reduce the severity or frequency of recurring attacks, e.g., cyclical attacks associated with a precipitating factor. In some embodiments, the precipitating factor is the menstrual cycle. In some embodiments, the iRNA is administered repeatedly, e.g., at regular intervals to prevent or reduce the severity or frequency of recurring attacks, e.g., cyclical attacks associated with a precipitating factor, e.g., the menstrual cycle, e.g., a particular phase of the menstrual cycle, e.g., the luteal phase. In some embodiments, the iRNA is administered during a particular phase of the menstrual cycle or based on hormone levels of the patient being treated (e.g., based on hormone levels that are associated with a particular phase of the menstrual cycle). In some embodiments, the iRNA is administered on one or more particular days of the menstrual cycle, e.g., on day 1, 2, 3, 4, 5, 6, 7, 8. 9. 10. 11. 12. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or on day 28 (or later day for subjects who have a longer menstrual cycle). In some embodiments, the iRNA is administered during the luteal phase, e.g., on one or more days between days 14-28 of the menstrual cycle (or later, in subjects who have a menstrual cycle longer than 28 days). In some embodiments, ovulation of the subject is assessed (e.g., using a blood or urine test that detects a hormone associated with ovulation, e.g., LH) and the iRNA is administered at a predetermined interval after ovulation. In some embodiments, the iRNA is administered immediately after ovulation. In some embodiments, the iRNA is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days after ovulation. Any of these schedules may optionally be repeated for one or more iterations. The number of iterations may depend on the achievement of a desired effect, e.g., the suppression of a ALAS1 gene and/or the achievement of a therapeutic or prophylactic effect, e.g., reduce or prevent one or more symptoms associated with porphyria, to reduce the frequency of attacks associated with porphyria.
In some embodiments, an initial dose of the iRNA agent is administered and the level of ALA or PBG is tested, e.g., 1-48 hours, e.g., 2, 4, 8, 12, or 24 hours following administration of the initial dose. In some embodiments, if the level of ALA and/or PBG has decreased (e.g., to achieve a predetermined reduction, e.g., a normalization), and/or if the symptoms associated with porphyria (e.g., pain) have improved (e.g., such that the patient is asymptomatic), no further dose is administered, whereas if the level of ALA and/or PBG has not decreased (e.g., has not achieved a predetermined reduction, e.g., has not normalized), a further dose of ALA or PBG is administered. In some embodiments, the further dose is administered 12, 24, 36, 48, 60, or 72 hours after the initial dose. In some embodiments, if the initial dose is not effective to decrease the level of ALA and/or PBG, the further dose is modified, e.g., increased to achieve a desired decrease (e.g., a predetermined reduction, e.g., a normalization) in ALA or PBG levels.
In some embodiments, the predetermined reduction is a decrease of at least 10%, 20%, 30%, 40%, or 50%. In some embodiments, the predetermined reduction is a reduction that is effective to prevent or ameliorate symptoms, e.g., pain, prodromal symptoms, or recurring attacks.
In some embodiments, the predetermined reduction is a reduction of at least 1, 2, 3, or more standard deviations, wherein the standard deviation is determined based on the values from a reference sample, e.g., a reference sample as described herein.
In some embodiments, the predetermined reduction is a reduction that brings the level of the porphyrin or porphyrin precursor to a level that is less than, or to a level that is less than or equal to, a reference value (e.g., a reference value as described herein).
As used herein, a “normalization” in ALA or PBG levels (or a “normal” or “normalized” level) refers to a level (e.g., a urine and/or plasma level) of either ALA, or PBG, or both, that is within the expected range for a healthy individual, an individual who is asymptomatic (e.g., an individual who does not experience pain and/or suffer from neuropathy), or an individual who does not have a mutation associated with a porphyria. For example, in some embodiments, a normalized level is within two standard deviations of the normal mean. In some embodiments, a normalized level is within normal reference limits, e.g., within the 95% confidence interval for an appropriate control sample, e.g., a sample of healthy individuals or individuals who do not carry a gene mutation associated with a porphyria. In some embodiments, the ALA and/or PBG level of the subject (e.g., the urine and/or plasma ALA and/or PBG level) is monitored at intervals, a further dose of the iRNA agent is administered when the level increases above the reference value
Administration of the iRNA may reduce ALAS1 mRNA or protein levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more. Administration of the iRNA may reduce levels of products associated with ALAS1 gene expression, e.g., levels of one or more porphyrins or porphyrin precursors (e.g., the level of ALA and/or PBG). Administration of the iRNA agent may also inhibit or prevent the upregulation of ALAS1 mRNA or protein levels during an acute attack of AIP.
Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion dose, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted effects.
Methods for Modulating Expression of an ALAS1 Gene
In yet another aspect, the invention provides a method for modulating (e.g., inhibiting or activating) the expression of an ALAS1 gene, e.g., in a cell or in a subject. In some embodiments, the cell is ex vivo, in vitro, or in vivo. In some embodiments, the cell is an erythroid cell or a hepatocyte. In some embodiments, the cell is in a subject (e.g., a mammal, such as, for example, a human). In some embodiments, the subject (e.g., the human) is at risk, or is diagnosed with a disease related to ALAS1 expression, as described above.
In one embodiment, the method includes contacting the cell with an iRNA as described herein, in an amount effective to decrease the expression of an ALAS1 gene in the cell. “Contacting,” as used herein, includes directly contacting a cell, as well as indirectly contacting a cell. For example, a cell within a subject (e.g., an erythroid cell or a liver cell, such as a hepatocyte) may be contacted when a composition comprising an iRNA is administered (e.g., intravenously or subcutaneously) to the subject.
The expression of an ALAS1 gene may be assessed based on the level of expression of an ALAS1 mRNA, an ALAS1 protein, or the level of a parameter functionally linked to the level of expression of an ALAS1 gene (e.g., the level of a porphyrin or the incidence or severity of a symptom related to a porphyria). In some embodiments, the expression of ALAS1 is inhibited by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the iRNA has an IC50 in the range of 0.001-0.01 nM, 0.001-0.10 nM, 0.001-1.0 nM, 0.001-10 nM, 0.01-0.05 nM, 0.01-0.50 nM, 0.02-0.60 nM, 0.01-1.0 nM, 0.01-1.5 nM, 0.01-10 nM. The IC50 value may be normalized relative to an appropriate control value, e.g., the IC50 of a non-targeting iRNA.
In some embodiments, the method includes introducing into the cell an iRNA as described herein and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an ALAS1 gene, thereby inhibiting the expression of the ALAS1 gene in the cell.
In one embodiment, the method includes administering a composition described herein, e.g., a composition comprising an iRNA that targets ALAS1, to the mammal such that expression of the target ALAS1 gene is decreased, such as for an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer. In some embodiments, the decrease in expression of ALAS1 is detectable within 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, or 24 hours of the first administration.
In another embodiment, the method includes administering a composition as described herein to a mammal such that expression of the target ALAS1 gene is increased by e.g., at least 10% compared to an untreated animal. In some embodiments, the activation of ALAS1 occurs over an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, four weeks, or more. Without wishing to be bound by theory, an iRNA can activate ALAS1 expression by stabilizing the ALAS1 mRNA transcript, interacting with a promoter in the genome, and/or inhibiting an inhibitor of ALAS1 expression.
The iRNAs useful for the methods and compositions featured in the invention specifically target RNAs (primary or processed) of an ALAS1 gene. Compositions and methods for inhibiting the expression of an ALAS1 gene using iRNAs can be prepared and performed as described elsewhere herein.
In one embodiment, the method includes administering a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the ALAS1 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration.
In certain embodiments, the compositions are administered by intravenous infusion or injection. In some such embodiments, the compositions comprise a lipid formulated siRNA (e.g., an LNP formulation, such as an LNP11 formulation) for intravenous infusion. In particular embodiments, such compositions may be used to treat acute attacks of porphyria and/or for prophylaxis (e.g., to decrease the severity or frequency of attacks).
In other embodiments, the compositions are administered subcutaneously. In some such embodiments, the compositions comprise an iRNA conjugated to a GalNAc ligand. In particular embodiments, such compositions may be used to treat acute attacks of porphyria or for prophylaxis (e.g., to decrease the severity or frequency of attacks).
Methods for Decreasing a Level of a Porphyrin or Porphyrin Precursor
In another aspect, the invention provides a method for decreasing a level of a porphyrin or a porphyrin precursor, e.g., in a cell or in a subject.
In some embodiments, the cell is ex vivo, in vitro, or in vivo. In some embodiments, the cell is an erythroid cell or a hepatocyte. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is in a subject (e.g., a mammal, such as, for example, a human).
In some embodiments, the subject (e.g., the human) is at risk, or is diagnosed with a porphyria, as described herein. In some embodiments, the method is effective to treat a porphyria as described herein (e.g., by ameliorating one or more symptoms associated with a porphyria, reducing the frequency of attacks associated with a porphyria, reducing the likelihood that an attack of one or more symptoms associated with porphyria will occur upon exposure to a precipitating factor, or reducing the risk of developing conditions associated with a porphyria (e.g., neuropathy (e.g., progressive neuropathy), hepatocellular cancer). In one embodiment, the method includes contacting the cell with an RNAi, as described herein, in an amount sufficient to decrease the level of the porphyrin or porphyrin precursor (e.g., ALA or PBG) in the cell, or in another related cell or group of cells, or in the subject. “Contacting,” as used herein, includes directly contacting a cell, as well as indirectly contacting a cell. For example, a cell within a subject (e.g., an erythroid cell or a liver cell, such as a hepatocyte) may be contacted when a composition comprising an RNAi is administered (e.g., intravenously or subcutaneously) to the subject. “Another related cell or group of cells,” as used herein, includes any cell or group of cells in which the level of the porphyrin or porphyrin precursor decreases as a result of the contacting. For example, the cell may be part of a tissue present within a subject (e.g., a liver cell present within a subject), and contacting the cell within the subject (e.g., contacting one or more liver cells present within a subject) with the RNAi may result in a decrease in the level of the porphyrin or porphyrin precursor in another related cell or group of cells (e.g., nerve cells of the subject), or in a tissue or fluid of the subject (e.g., in the urine, blood, plasma, or cerebrospinal fluid of the subject).
In some embodiments, the porphyrin or porphyrin precursor is selected from the group consisting of δ-aminolevulinic acid (ALA), porphopilinogen (PBG), hydroxymethylbilane (HMB), uroporphyrinogen III, coproporphyrinogen III, protoporphrinogen IX, and protoporphyrin IX In some embodiments the porphyrin precursor is ALA. In some embodiments, the porphyrin precursor is PBG. In some embodiments, the method decreases the level of ALA and PBG. The level of a porphyrin or a porphyrin precursor may be measured as described herein and as known in the art.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
siRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
Oligonucleotide Synthesis.
All oligonucleotides are synthesized on an AKTAoligopilot synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH3CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.
3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.
Deprotection I (Nucleobase Deprotection)
After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture is then reduced to  ̃30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.
Deprotection II (Removal of 2′-TBDMS Group)
The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer until purification.
Analysis
The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.
HPLC Purification
The ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH3CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotidess are diluted in water to 150 μL and then pipetted into special vials for CGE and LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.
siRNA Preparation
For the general preparation of siRNA, equimolar amounts of sense and antisense strand are heated in 1×PBS at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1.







TABLE 1







Abbreviations of nucleotide monomers used in nucleic acid sequence representation.


It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 


5′-3′- phosphodiester bonds.








Abbreviation
Nucleotides(s)





A
Adenosine-3′-phosphate


Ab
beta-L-adenosine-3′-phosphate


Abs
beta-L-adenosine-3′-phosphorothioate


Af
2′-fluoroadenosine-3′-phosphate


Afs
2′-fluoroadenosine-3′-phosphorothioate


As
adenosine-3′-phosphorothioate


C
cytidine-3′-phosphate


Cb
beta-L-cytidine-3′-phosphate


Cbs
beta-L-cytidine-3′-phosphorothioate


Cf
2′-fluorocytidine-3′-phosphate


Cfs
2′-fluorocytidine-3′-phosphorothioate


(Chd)
2′-hexadecyl-cytidine-3′-phosphate


(Chds)
2′-O-hexadecyl-cytidine-3′-phosphorothioate


Cs
cytidine-3′-phosphorothioate


G
guanosine-3′-phosphate


Gb
beta-L-guanosine-3′-phosphate


Gbs
beta-L-guanosine-3′-phosphorothioate


Gf
2′-fluoroguanosine-3′-phosphate


Gfs
2′-fluoroguanosine-3′-phosphorothioate


Gs
guanosine-3′-phosphorothioate


T
5′-methyluridine-3′-phosphate


Tb
beta-L-thymidine-3′-phosphate


Tbs
beta-L-thymidine-3′-phosphorothioate


Tf
2′-fluoro-5-methyluridine-3′-phosphate


Tfs
2′-fluoro-5-methyluridine-3′-phosphorothioate


Ts
5-methyluridine-3′-phosphorothioate


U
Uridine-3′-phosphate


Ub
beta-L-uridine-3′-phosphate


Ubs
beta-L-uridine-3′-phosphorothioate


Uf
2′-fluorouridine-3′-phosphate


Ufs
2′-fluorouridine-3′-phosphorothioate


(Uhd)
2′-O-hexadecyl-uridine-3′-phosphate


(Uhds)
2′-O-hexadecyl-uridine-3′-phosphorothioate


Us
uridine-3′-phosphorothioate


N
any nucleotide (G, A, C, T or U)


a
2′-O-methyladenosine-3′-phosphate


as
2′-O-methyladenosine-3′-phosphorothioate


c
2′-O-methylcytidine-3′-phosphate


cs
2′-O-methylcytidine-3′-phosphorothioate


g
2′-O-methylguanosine-3′-phosphate


gs
2′-O-methylguanosine-3′-phosphorothioate


t
2′-O-methyl-5-methyluridine-3′-phosphate


ts
2′-O-methyl-5-methyluridine-3′-phosphorothioate


u
2′-O-methyluridine-3′-phosphate


us
2′-O-methyluridine-3′-phosphorothioate


dA
2′-deoxyadenosine-3′-phosphate


dAs
2′-deoxyadenosine-3′-phosphorothioate


dC
2′-deoxycytidine-3′-phosphate


dCs
2′-deoxycytidine-3′-phosphorothioate


dG
2′-deoxyguanosine-3′-phosphate


dGs
2′-deoxyguanosine-3′-phosphorothioate


dT
2′-deoxythymidine


dTs
2′-deoxythymidine-3′-phosphorothioate


dU
2′-deoxyuridine


s
phosphorothioate linkage


L961
N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol Hyp-(GalNAc-alkyl)3


(Aeo)
2′-O-methoxyethyladenosine-3′-phosphate


(Aeos)
2′-O-methoxyethyladenosine-3′-phosphorothioate


(Geo)
2′-O-methoxyethylguanosine-3′-phosphate


(Geos)
2′-O-methoxyethylguanosine-3′-phosphorothioate


(Teo)
2′-O-methoxyethyl-5-methyluridine-3′-phosphate


(Teos)
2′-O-methoxyethyl-5-methyluridine-3′-phosphorothioate


(m5Ceo)
2′-O-methoxyethyl-5-methylcytidine-3′-phosphate


(m5Ceos)
2′-O-methoxyethyl-5-methylcytidine-3′-phosphorothioate





1The chemical structure of L96 is as follows:












Example 2
ALAS1 siRNA Design and Synthesis
Experimental Methods
Bioinformatics
Transcripts
siRNA design was carried out to identify siRNAs targeting human, rhesus (Macaca mulatta), mouse, and rat ALAS1 transcripts annotated in the NCBI Gene database (http://www.ncbi.nlm nih.gov/gene/). Design used the following transcripts from the NCBI RefSeq collection: Human—NM—000688.4 (see FIG. 3), NM—199166.1; Rhesus—XM—001090440.2, XM—001090675.2; Mouse—NM—020559.2; Rat—NM—024484.2. Due to high primate/rodent sequence divergence, siRNA duplexes were designed in several separate batches, including but not limited to batches containing duplexes matching human and rhesus transcripts only; human, rhesus, mouse, and rat transcripts only; and mouse and rat transcripts only. Most siRNA duplexes were designed that shared 100% identity the listed human transcript and other species transcripts considered in each design batch (above). In some instances, (see Table 8) mismatches between duplex and mRNA target were allowed at the first antisense (last sense) position when the antisense strand:target mRNA complementary basepair was a GC or CG pair. In these cases, duplexes were designed with UA or AU pairs at the first antisense:last sense pair. Thus the duplexes maintained complementarity but were mismatched with respect to target (U:C, U:G, A:C, or A:G). Eighteen of these “UA-swap” duplexes were designed as part of the human/rhesus/mouse/rat set (see duplexes in Table 8 with “C19U”, “G19U”, “C19A”, or “G19A” labels in the Position column)
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were then selected that lacked repeats longer than 7 nucleotides. These 1510 candidate human/rhesus, 114 human/rhesus/mouse/rat, and 717 mouse/rat siRNAs were used in comprehensive searches against the appropriate transcriptomes (defined as the set of NM_and XM_records within the human, rhesus, dog, mouse, or rat NCBI Refseq sets) using an exhaustive “brute-force” algorithm implemented in the python script ‘BruteForce.py’. The script next parsed the transcript-oligo alignments to generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. Each oligo-transcript pair from the brute-force search was given a mismatch score by summing the individual mismatch scores; mismatches in the position 2-9 were counted as 2.8, mismatches in the cleavage site positions 10-11 were counted as 1.2, and mismatches in region 12-19 counted as 1.0. An additional off-target prediction was carried out by comparing the frequency of heptamers and octomers derived from 3 distinct, seed-derived hexamers of each oligo. The hexamers from positions 2-7 relative to the 5′ start is used to create 2 heptamers and one octomer. We create ‘heptamer1’ by adding a 3′ A to the hexamer; we create heptamer2 by adding a 5′ A to the hexamer; we create the octomer by adding an A to both 5′ and 3′ ends of the hexamer. The frequency of octomers and heptamers in the human, rhesus, mouse, or rat 3′UTRome (defined as the subsequence of the transcriptome from NCBI's Refseq database where the end of the coding region, the ‘CDS’, is clearly defined) was pre-calculated. The octomer frequency was normalized to the heptamer frequency using the median value from the range of octomer frequencies. A ‘mirSeedScore’ was then calculated by calculating the sum of ((3×normalized octomer count)+(2×heptamer2 count)+(1×heptamer1 count)).
Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. We sorted by the specificity of the antisense strand. We then selected duplexes whose antisense oligos lacked GC at the first position, lacked G at both positions 13 and 14, and had 3 or more Us or As in the seed region (characteristics of duplexes with high predicted efficacy)
Candidate GalNac-conjugated duplexes, 21 and 23 nucleotides long on the sense and antisense strands respectively, were designed by extending antisense 19mers 4 additional nucleotides in the 3′ direction (preserving perfect complementarity with the target transcript). The sense strand was specified as the reverse complement of the first 21 nucleotides of the antisense 23mer. Duplexes were selected that maintained perfect matches to all selected species transcripts across all 23 nucleotides.
siRNA Sequence Selection
A total of 90 sense and 90 antisense derived human/rhesus, 40 sense and 40 antisense derived human/rhesus/mouse/mouse/rat, and 40 sense and 40 antisense derived mouse/rat siRNA 19mer oligos were synthesized and formed into duplexes. A total of 45 sense and 45 antisense derived human/rhesus 21/23mer oligos were synthesized to yield 45 GalNac-conjugated duplexes.
The sequences of the sense and antisense strands of the modified duplexes are shown in Table 2, and the sequences of the sense and antisense strands of the unmodified duplexes are shown in Table 3.
Synthesis of ALAS1 Sequences
ALAS1 sequences were synthesized on MerMade 192 synthesizer at either 1 or 0.2 umol scale. Single strands were made with 2′O-methyl modifications for in vitro screening using transfection reagents. 3′ GalNAc conjugates were made with sequences containing 2′F and 2′-O-methyl modifications on the sense strand in the 21-23 mer designs for free uptake in cells. For all the 21mer sequences in the list, ‘endolight’ chemistry was applied as detailed below.

    
    
        All pyrimidines (cytosine and uridine) in the sense strand contained 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)
        In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside were replaced with their corresponding 2-O-Methyl nucleosides
        A two base dTsdT extension at 3′ end of both sense and anti sense sequences was introduced
        The sequence file was converted to a text file to make it compatible for loading in the MerMade 192 synthesis software
    
    


For GalNAc conjugated sense strands and complementary antisense sequences, 2′F and other modified nucleosides were introduced in combination with ribo with 2′O-Methyl nucleosides. The synthesis was performed on a GalNAc modified CPG support for the sense strand and CPG modified with universal support on the antisense sequence.
Synthesis, Cleavage and Deprotection:
The synthesis of ALAS1 sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. For 21 mer endolight sequences, a deoxy thymidine CPG was used as the solid support while for the GalNAc conjugates, GalNAc solid support for sense strand and an universal CPG for the antisesense strand were used.
The synthesis of the above sequences was performed at either 1 or 0.2 um scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.
The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. For GalNAc and 2′F nucleoside containing sequences, deprotection conditions were modified. Sequences after cleavage and deprotection were precipitated using acetone:ethanol (80:20) mix and the pellet were re-suspended in 0.2M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.
Purification and Desalting:
ALAS1 sequences were precipitated and purified on AKTA Purifier system using Sephadex column. The ALAS1ess was run at ambient temperature. Sample injection and collection was performed in 96 well (1.8 mL-deep well) plates. A single peak corresponding to the full length sequence was collected in the eluent. The desalted ALAS1 sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The complementary single strands were then combined in a 1:1 stoichiometric ratio to form siRNA duplexes.







TABLE 2







Human ALAS1 Modified Single Strands and Duplex Sequences













SEQ ID






SEQ ID
NO:
Position on


NO:
(anti-
transcript


(sense)
sense)
NM_000688.4
Duplex Name
Sense Sequence (5′-3′)
Antisense Sequence (5′-3′)















2
3
522-540
AD-55078.2
cuccGGccAGuGAGAAAGAdTsdT
UCUUUCUcACUGGCCGGAGdTsdT


 


4
5
669-687
AD-55084.2
uGGcAGcAcAGAuGAAucAdTsdT
UGAUUcAUCUGUGCUGCcAdTsdT


 


6
7
790-808
AD-55090.2
cAGuGuGGuuAGuGuGAAAdTsdT
UUUcAcACuAACcAcACUGdTsdT


 


8
9
853-871
AD-55096.2
cAucAuGcAAAAGcAAAGAdTsdT
UCUUUGCUUUUGcAUGAUGdTsdT


 


10
11
876-894
AD-55102.2
AAAGAGuGucucAucuucudTsdT
AGAAGAUGAGAcACUCUUUdTsdT


 


12
13
877-895
AD-55106.2
AAGAGuGucucAucuucuudTsdT
AAGAAGAUGAGAcACUCUUdTsdT


 


14
15
914-932
AD-55111.2
ucuGuuuccAcuuuucAGudTsdT
ACUGAAAAGUGGAAAcAGAdTsdT


 


16
17
923-941
AD-55073.2
AcuuuucAGuAuGAucGuudTsdT
AACGAUcAuACUGAAAAGUdTsdT


 


18
19
926-944
AD-55079.2
uuucAGuAuGAucGuuucudTsdT
AGAAACGAUcAuACUGAAAdTsdT


 


20
21
927-945
AD-55085.2
uucAGuAuGAucGuuucuudTsdT
AAGAAACGAUcAuACUGAAdTsdT


 


22
23
928-946
AD-55091.2
ucAGuAuGAucGuuucuuudTsdT
AAAGAAACGAUcAuACUGAdTsdT


 


24
25
932-950
AD-55097.2
uAuGAucGuuucuuuGAGAdTsdT
UCUcAAAGAAACGAUcAuAdTsdT


 


26
27
973-991
AD-55103.2
uGAccAcAccuAucGAGuudTsdT
AACUCGAuAGGUGUGGUcAdTsdT


 


28
29
975-993
AD-55107.2
AccAcAccuAucGAGuuuudTsdT
AAAACUCGAuAGGUGUGGUdTsdT


 


30
31
1029-1047
AD-55112.2
uGGcAGAuGAcuAuucAGAdTsdT
UCUGAAuAGUcAUCUGCcAdTsdT


 


32
33
1077-1095
AD-55074.2
ucuGGuGcAGuAAuGAcuAdTsdT
uAGUcAUuACUGcACcAGAdTsdT


 


34
35
1124-1142
AD-55080.2
uGuGGGGcAGuuAuGGAcAdTsdT
UGUCcAuAACUGCCCcAcAdTsdT


 


36
37
1137-1155
AD-55086.2
uGGAcAcuuuGAAAcAAcAdTsdT
UGUUGUUUcAAAGUGUCcAdTsdT


 


38
39
1182-1200
AD-55098.2
AuAuuucuGGAAcuAGuAAdTsdT
UuACuAGUUCcAGAAAuAUdTsdT


 


40
41
1184-1202
AD-55104.2
AuuucuGGAAcuAGuAAAudTsdT
AUUuACuAGUUCcAGAAAUdTsdT


 


42
43
1185-1203
AD-55108.2
uuucuGGAAcuAGuAAAuudTsdT
AAUUuACuAGUUCcAGAAAdTsdT


 


44
45
1188-1206
AD-55113.2
cuGGAAcuAGuAAAuuccAdTsdT
UGGAAUUuACuAGUUCcAGdTsdT


 


46
47
1325-1343
AD-55075.2
uGuGAGAuuuAcucuGAuudTsdT
AAUcAGAGuAAAUCUcAcAdTsdT


 


48
49
1364-1382
AD-55081.2
AuccAAGGGAuucGAAAcAdTsdT
UGUUUCGAAUCCCUUGGAUdTsdT


 


50
51
1382-1400
AD-55087.2
AGccGAGuGccAAAGuAcAdTsdT
UGuACUUUGGcACUCGGCUdTsdT


 


52
53
1478-1496
AD-55093.2
uuuGAAAcuGuccAuucAAdTsdT
UUGAAUGGAcAGUUUcAAAdTsdT


 


54
55
1531-1549
AD-55099.2
uGAuGuGGcccAuGAGuuudTsdT
AAACUcAUGGGCcAcAUcAdTsdT


 


56
57
1631-1649
AD-53573.3
GucAuGccAAAAAuGGAcAdTsdT
UGUCcAUUUUUGGcAUGACdTsdT


 


58
59
1637-1655
AD-55109.2
ccAAAAAuGGAcAucAuuudTsdT
AAAUGAUGUCcAUUUUUGGdTsdT


 


60
61
1706-1724
AD-55114.2
AcGAGuucucuGAuuGAcAdTsdT
UGUcAAUcAGAGAACUCGUdTsdT


 


62
63
1962-1980
AD-55076.2
AAGucuGuGAuGAAcuAAudTsdT
AUuAGUUcAUcAcAGACUUdTsdT


 


64
65
1967-1985
AD-55082.2
uGuGAuGAAcuAAuGAGcAdTsdT
UGCUcAUuAGUUcAUcAcAdTsdT


 


66
67
1977-1995
AD-55088.2
uAAuGAGcAGAcAuAAcAudTsdT
AUGUuAUGUCUGCUcAUuAdTsdT


 


68
69
2189-2207
AD-55094.2
uuuGAAGuGAuGAGuGAAAdTsdT
UUUcACUcAUcACUUcAAAdTsdT


 


70
71
2227-2245
AD-55100.2
AGGcuuGAGcAAGuuGGuAdTsdT
uACcAACUUGCUcAAGCCUdTsdT


 


72
73
2313-2331
AD-55105.2
ucuucAGAGuuGucuuuAudTsdT
AuAAAGAcAACUCUGAAGAdTsdT


 


74
75
2317-2335
AD-55110.2
cAGAGuuGucuuuAuAuGudTsdT
AcAuAuAAAGAcAACUCUGdTsdT


 


76
77
2319-2337
AD-55115.2
GAGuuGucuuuAuAuGuGAdTsdT
UcAcAuAuAAAGAcAACUCdTsdT


 


78
79
2320-2338
AD-55077.2
AGuuGucuuuAuAuGuGAAdTsdT
UUcAcAuAuAAAGAcAACUdTsdT


 


80
81
2344-2362
AD-55083.2
uuAuAuuAAAuuuuAAucudTsdT
AGAUuAAAAUUuAAuAuAAdTsdT


 


82
83
2352-2370
AD-55089.2
AAuuuuAAucuAuAGuAAAdTsdT
UUuACuAuAGAUuAAAAUUdTsdT


 


84
85
2353-2371
AD-55095.2
AuuuuAAucuAuAGuAAAAdTsdT
UUUuACuAuAGAUuAAAAUdTsdT


 


86
87
2376-2394
AD-55101.2
AGuccuGGAAAuAAAuucudTsdT
AGAAUUuAUUUCcAGGACUdTsdT


 


88
89
358-376
AD-53511.1
cuGcccAuucuuAucccGAdTsdT
UCGGGAuAAGAAUGGGcAGdTsdT


 


90
91
789-807
AD-53512.1
ccAGuGuGGuuAGuGuGAAdTsdT
UUcAcACuAACcAcACUGGdTsdT


 


92
93
1076-1094
AD-53513.1
GucuGGuGcAGuAAuGAcudTsdT
AGUcAUuACUGcACcAGACdTsdT


 


94
95
1253-1271
AD-53514.1
GcAcucuuGuuuuccucGudTsdT
ACGAGGAAAAcAAGAGUGCdTsdT


 


96
97
1544-1562
AD-53515.1
GAGuuuGGAGcAAucAccudTsdT
AGGUGAUUGCUCcAAACUCdTsdT


 


98
99
2228-2246
AD-53516.1
GGcuuGAGcAAGuuGGuAudTsdT
AuACcAACUUGCUcAAGCCdTsdT


 


100
101
404-422
AD-53517.1
GGcAAAucucuGuuGuucudTsdT
AGAAcAAcAGAGAUUUGCCdTsdT


 


102
103
404-422
AD-53517.1
GGcAAAucucuGuuGuucudTsdT
AGAAcAAcAGAGAUUUGCCdTsdT


 


104
105
866-884
AD-53518.1
cAAAGAccAGAAAGAGuGudTsdT
AcACUCUUUCUGGUCUUUGdTsdT


 


106
107
1080-1098
AD-53519.1
GGuGcAGuAAuGAcuAccudTsdT
AGGuAGUcAUuACUGcACCdTsdT


 


108
109
1258-1276
AD-53520.1
cuuGuuuuccucGuGcuuudTsdT
AAAGcACGAGGAAAAcAAGdTsdT


 


110
111
1616-1634
AD-53521.1
GGGGAucGGGAuGGAGucAdTsdT
UGACUCcAUCCCGAUCCCCdTsdT


 


112
113
2230-2248
AD-53522.1
cuuGAGcAAGuuGGuAucudTsdT
AGAuACcAACUUGCUcAAGdTsdT


 


114
115
436-454
AD-53523.1
ccccAAGAuGAuGGAAGuudTsdT
AACUUCcAUcAUCUUGGGGdTsdT


 


116
117
436-454
AD-53523.1
ccccAAGAuGAuGGAAGuudTsdT
AACUUCcAUcAUCUUGGGGdTsdT


 


118
119
885-903
AD-53524.1
cucAucuucuucAAGAuAAdTsdT
UuAUCUUGAAGAAGAUGAGdTsdT


 


120
121
1127-1145
AD-53525.1
GGGGcAGuuAuGGAcAcuudTsdT
AAGUGUCcAuAACUGCCCCdTsdT


 


122
123
1315-1333
AD-53526.1
GAuGccAGGcuGuGAGAuudTsdT
AAUCUcAcAGCCUGGcAUCdTsdT


 


124
125
1870-1888
AD-53527.1
GAGAcAGAuGcuAAuGGAudTsdT
AUCcAUuAGcAUCUGUCUCdTsdT


 


126
127
2286-2304
AD-53528.1
ccccAGGccAuuAucAuAudTsdT
AuAUGAuAAUGGCCUGGGGdTsdT


 


128
129
489-507
AD-53529.1
cAGcAGuAcAcuAccAAcAdTsdT
UGUUGGuAGUGuACUGCUGdTsdT


 


130
131
489-507
AD-53529.1
cAGcAGuAcAcuAccAAcAdTsdT
UGUUGGuAGUGuACUGCUGdTsdT


 


132
133
915-933
AD-53530.1
cuGuuuccAcuuuucAGuAdTsdT
uACUGAAAAGUGGAAAcAGdTsdT


 


134
135
1138-1156
AD-53531.1
GGAcAcuuuGAAAcAAcAudTsdT
AUGUUGUUUcAAAGUGUCCdTsdT


 


136
137
1324-1342
AD-53532.1
cuGuGAGAuuuAcucuGAudTsdT
AUcAGAGuAAAUCUcAcAGdTsdT


 


138
139
1927-1945
AD-53533.1
cccuGuGcGGGuuGcAGAudTsdT
AUCUGcAACCCGcAcAGGGdTsdT


 


140
141
2312-2330
AD-53534.1
GucuucAGAGuuGucuuuAdTsdT
uAAAGAcAACUCUGAAGACdTsdT


 


142
143
646-664
AD-53535.1
cAcuGcAAGcAAAuGcccudTsdT
AGGGcAUUUGCUUGcAGUGdTsdT


 


144
145
922-940
AD-53536.1
cAcuuuucAGuAuGAucGudTsdT
ACGAUcAuACUGAAAAGUGdTsdT


 


146
147
1163-1181
AD-53537.1
GGGGcAGGuGGuAcuAGAAdTsdT
UUCuAGuACcACCUGCCCCdTsdT


 


148
149
1347-1365
AD-53538.1
GGAAccAuGccuccAuGAudTsdT
AUcAUGGAGGcAUGGUUCCdTsdT


 


150
151
1964-1982
AD-53539.1
GucuGuGAuGAAcuAAuGAdTsdT
UcAUuAGUUcAUcAcAGACdTsdT


 


152
153
2321-2339
AD-53540.1
GuuGucuuuAuAuGuGAAudTsdT
AUUcAcAuAuAAAGAcAACdTsdT


 


154
155
671-689
AD-53541.1
GcAGcAcAGAuGAAucAGAdTsdT
UCUGAUUcAUCUGUGCUGCdTsdT


 


156
157
924-942
AD-53542.1
cuuuucAGuAuGAucGuuudTsdT
AAACGAUcAuACUGAAAAGdTsdT


 


158
159
1164-1182
AD-53543.1
GGGcAGGuGGuAcuAGAAAdTsdT
UUUCuAGuACcACCUGCCCdTsdT


 


160
161
1460-1478
AD-53544.1
GuccccAAGAuuGuGGcAudTsdT
AUGCcAcAAUCUUGGGGACdTsdT


 


162
163
1976-1994
AD-53545.1
cuAAuGAGcAGAcAuAAcAdTsdT
UGUuAUGUCUGCUcAUuAGdTsdT


 


164
165
786-804
AD-53546.1
GccccAGuGuGGuuAGuGudTsdT
AcACuAACcAcACUGGGGCdTsdT


 


166
167
935-953
AD-53547.1
GAucGuuucuuuGAGAAAAdTsdT
UUUUCUcAAAGAAACGAUCdTsdT


 


168
169
1165-1183
AD-53548.1
GGcAGGuGGuAcuAGAAAudTsdT
AUUUCuAGuACcACCUGCCdTsdT


 


170
171
1530-1548
AD-53549.1
GuGAuGuGGcccAuGAGuudTsdT
AACUcAUGGGCcAcAUcACdTsdT


 


172
173
2003-2021
AD-53550.1
cAAGcAAucAAuuAcccuAdTsdT
uAGGGuAAUUGAUUGCUUGdTsdT


 


174
175
788-806
AD-53551.1
cccAGuGuGGuuAGuGuGAdTsdT
UcAcACuAACcAcACUGGGdTsdT


 


176
177
974-992
AD-53552.1
GAccAcAccuAucGAGuuudTsdT
AAACUCGAuAGGUGUGGUCdTsdT


 


178
179
1191-1209
AD-53553.1
GAAcuAGuAAAuuccAuGudTsdT
AcAUGGAAUUuACuAGUUCdTsdT


 


180
181
1541-1559
AD-53554.1
cAuGAGuuuGGAGcAAucAdTsdT
UGAUUGCUCcAAACUcAUGdTsdT


 


182
183
2075-2093
AD-53555.1
ccccAGAuGAuGAAcuAcudTsdT
AGuAGUUcAUcAUCUGGGGdTsdT


 


184
185
360-378
AD-53561.1
GcccAuucuuAucccGAGudTsdT
ACUCGGGAuAAGAAUGGGCdTsdT


 


186
187
1356-1374
AD-53567.1
ccuccAuGAuccAAGGGAudTsdT
AUCCCUUGGAUcAUGGAGGdTsdT


 


188
189
1631-1649
AD-53573.1
GucAuGccAAAAAuGGAcAdTsdT
UGUCcAUUUUUGGcAUGACdTsdT


 


190
191
1634-1652
AD-53579.1
AuGccAAAAAuGGAcAucAdTsdT
UGAUGUCcAUUUUUGGcAUdTsdT
















TABLE 3







Human ALAS1 Unmodified Single Strands and Duplex Sequences













SEQ ID NO:
Position on





SEQ ID NO:
(anti-
transcript


(sense)
sense)
NM_000688.4
Duplex Name
Sense Sequence (5′-3′)
Antisense Sequence (5′-3′)















192
193
522-540
AD-55078.2
CUCCGGCCAGUGAGAAAGA
UCUUUCUCACUGGCCGGAG


 


194
195
669-687
AD-55084.2
UGGCAGCACAGAUGAAUCA
UGAUUCAUCUGUGCUGCCA


 


196
197
790-808
AD-55090.2
CAGUGUGGUUAGUGUGAAA
UUUCACACUAACCACACUG


 


198
199
853-871
AD-55096.2
CAUCAUGCAAAAGCAAAGA
UCUUUGCUUUUGCAUGAUG


 


200
201
876-894
AD-55102.2
AAAGAGUGUCUCAUCUUCU
AGAAGAUGAGACACUCUUU


 


202
203
877-895
AD-55106.2
AAGAGUGUCUCAUCUUCUU
AAGAAGAUGAGACACUCUU


 


204
205
914-932
AD-55111.2
UCUGUUUCCACUUUUCAGU
ACUGAAAAGUGGAAACAGA


 


206
207
923-941
AD-55073.2
ACUUUUCAGUAUGAUCGUU
AACGAUCAUACUGAAAAGU


 


208
209
926-944
AD-55079.2
UUUCAGUAUGAUCGUUUCU
AGAAACGAUCAUACUGAAA


 


210
211
927-945
AD-55085.2
UUCAGUAUGAUCGUUUCUU
AAGAAACGAUCAUACUGAA


 


212
213
928-946
AD-55091.2
UCAGUAUGAUCGUUUCUUU
AAAGAAACGAUCAUACUGA


 


214
215
932-950
AD-55097.2
UAUGAUCGUUUCUUUGAGA
UCUCAAAGAAACGAUCAUA


 


216
217
973-991
AD-55103.2
UGACCACACCUAUCGAGUU
AACUCGAUAGGUGUGGUCA


 


218
219
975-993
AD-55107.2
ACCACACCUAUCGAGUUUU
AAAACUCGAUAGGUGUGGU


 


220
221
1029-1047
AD-55112.2
UGGCAGAUGACUAUUCAGA
UCUGAAUAGUCAUCUGCCA


 


222
223
1077-1095
AD-55074.2
UCUGGUGCAGUAAUGACUA
UAGUCAUUACUGCACCAGA


 


224
225
1124-1142
AD-55080.2
UGUGGGGCAGUUAUGGACA
UGUCCAUAACUGCCCCACA


 


226
227
1137-1155
AD-55086.2
UGGACACUUUGAAACAACA
UGUUGUUUCAAAGUGUCCA


 


228
229
1182-1200
AD-55098.2
AUAUUUCUGGAACUAGUAA
UUACUAGUUCCAGAAAUAU


 


230
231
1184-1202
AD-55104.2
AUUUCUGGAACUAGUAAAU
AUUUACUAGUUCCAGAAAU


 


232
233
1185-1203
AD-55108.2
UUUCUGGAACUAGUAAAUU
AAUUUACUAGUUCCAGAAA


 


234
235
1188-1206
AD-55113.2
CUGGAACUAGUAAAUUCCA
UGGAAUUUACUAGUUCCAG


 


236
237
1325-1343
AD-55075.2
UGUGAGAUUUACUCUGAUU
AAUCAGAGUAAAUCUCACA


 


238
239
1364-1382
AD-55081.2
AUCCAAGGGAUUCGAAACA
UGUUUCGAAUCCCUUGGAU


 


240
241
1382-1400
AD-55087.2
AGCCGAGUGCCAAAGUACA
UGUACUUUGGCACUCGGCU


 


242
243
1478-1496
AD-55093.2
UUUGAAACUGUCCAUUCAA
UUGAAUGGACAGUUUCAAA


 


244
245
1531-1549
AD-55099.2
UGAUGUGGCCCAUGAGUUU
AAACUCAUGGGCCACAUCA


 


246
247
1631-1649
AD-53573.3
GUCAUGCCAAAAAUGGACA
UGUCCAUUUUUGGCAUGAC


 


248
249
1637-1655
AD-55109.2
CCAAAAAUGGACAUCAUUU
AAAUGAUGUCCAUUUUUGG


 


250
251
1706-1724
AD-55114.2
ACGAGUUCUCUGAUUGACA
UGUCAAUCAGAGAACUCGU


 


252
253
1962-1980
AD-55076.2
AAGUCUGUGAUGAACUAAU
AUUAGUUCAUCACAGACUU


 


254
255
1967-1985
AD-55082.2
UGUGAUGAACUAAUGAGCA
UGCUCAUUAGUUCAUCACA


 


256
257
1977-1995
AD-55088.2
UAAUGAGCAGACAUAACAU
AUGUUAUGUCUGCUCAUUA


 


258
259
2189-2207
AD-55094.2
UUUGAAGUGAUGAGUGAAA
UUUCACUCAUCACUUCAAA


 


260
261
2227-2245
AD-55100.2
AGGCUUGAGCAAGUUGGUA
UACCAACUUGCUCAAGCCU


 


262
263
2313-2331
AD-55105.2
UCUUCAGAGUUGUCUUUAU
AUAAAGACAACUCUGAAGA


 


264
265
2317-2335
AD-55110.2
CAGAGUUGUCUUUAUAUGU
ACAUAUAAAGACAACUCUG


 


266
267
2319-2337
AD-55115.2
GAGUUGUCUUUAUAUGUGA
UCACAUAUAAAGACAACUC


 


268
269
2320-2338
AD-55077.2
AGUUGUCUUUAUAUGUGAA
UUCACAUAUAAAGACAACU


 


270
271
2344-2362
AD-55083.2
UUAUAUUAAAUUUUAAUCU
AGAUUAAAAUUUAAUAUAA


 


272
273
2352-2370
AD-55089.2
AAUUUUAAUCUAUAGUAAA
UUUACUAUAGAUUAAAAUU


 


274
275
2353-2371
AD-55095.2
AUUUUAAUCUAUAGUAAAA
UUUUACUAUAGAUUAAAAU


 


276
277
2376-2394
AD-55101.2
AGUCCUGGAAAUAAAUUCU
AGAAUUUAUUUCCAGGACU


 


278
279
358-376
AD-53511.1
CUGCCCAUUCUUAUCCCGA
UCGGGAUAAGAAUGGGCAG


 


280
281
789-807
AD-53512.1
CCAGUGUGGUUAGUGUGAA
UUCACACUAACCACACUGG


 


282
283
1076-1094
AD-53513.1
GUCUGGUGCAGUAAUGACU
AGUCAUUACUGCACCAGAC


 


284
285
1253-1271
AD-53514.1
GCACUCUUGUUUUCCUCGU
ACGAGGAAAACAAGAGUGC


 


286
287
1544-1562
AD-53515.1
GAGUUUGGAGCAAUCACCU
AGGUGAUUGCUCCAAACUC


 


288
289
2228-2246
AD-53516.1
GGCUUGAGCAAGUUGGUAU
AUACCAACUUGCUCAAGCC


 


290
291
404-422
AD-53517.1
GGCAAAUCUCUGUUGUUCU
AGAACAACAGAGAUUUGCC


 


292
293
404-422
AD-53517.1
GGCAAAUCUCUGUUGUUCU
AGAACAACAGAGAUUUGCC


 


294
295
866-884
AD-53518.1
CAAAGACCAGAAAGAGUGU
ACACUCUUUCUGGUCUUUG


 


296
297
1080-1098
AD-53519.1
GGUGCAGUAAUGACUACCU
AGGUAGUCAUUACUGCACC


 


298
299
1258-1276
AD-53520.1
CUUGUUUUCCUCGUGCUUU
AAAGCACGAGGAAAACAAG


 


300
301
1616-1634
AD-53521.1
GGGGAUCGGGAUGGAGUCA
UGACUCCAUCCCGAUCCCC


 


302
303
2230-2248
AD-53522.1
CUUGAGCAAGUUGGUAUCU
AGAUACCAACUUGCUCAAG


 


304
305
436-454
AD-53523.1
CCCCAAGAUGAUGGAAGUU
AACUUCCAUCAUCUUGGGG


 


306
307
436-454
AD-53523.1
CCCCAAGAUGAUGGAAGUU
AACUUCCAUCAUCUUGGGG


 


308
309
885-903
AD-53524.1
CUCAUCUUCUUCAAGAUAA
UUAUCUUGAAGAAGAUGAG


 


310
311
1127-1145
AD-53525.1
GGGGCAGUUAUGGACACUU
AAGUGUCCAUAACUGCCCC


 


312
313
1315-1333
AD-53526.1
GAUGCCAGGCUGUGAGAUU
AAUCUCACAGCCUGGCAUC


 


314
315
1870-1888
AD-53527.1
GAGACAGAUGCUAAUGGAU
AUCCAUUAGCAUCUGUCUC


 


316
317
2286-2304
AD-53528.1
CCCCAGGCCAUUAUCAUAU
AUAUGAUAAUGGCCUGGGG


 


318
319
489-507
AD-53529.1
CAGCAGUACACUACCAACA
UGUUGGUAGUGUACUGCUG


 


320
321
489-507
AD-53529.1
CAGCAGUACACUACCAACA
UGUUGGUAGUGUACUGCUG


 


322
323
915-933
AD-53530.1
CUGUUUCCACUUUUCAGUA
UACUGAAAAGUGGAAACAG


 


324
325
1138-1156
AD-53531.1
GGACACUUUGAAACAACAU
AUGUUGUUUCAAAGUGUCC


 


326
327
1324-1342
AD-53532.1
CUGUGAGAUUUACUCUGAU
AUCAGAGUAAAUCUCACAG


 


328
329
1927-1945
AD-53533.1
CCCUGUGCGGGUUGCAGAU
AUCUGCAACCCGCACAGGG


 


330
331
2312-2330
AD-53534.1
GUCUUCAGAGUUGUCUUUA
UAAAGACAACUCUGAAGAC


 


332
333
646-664
AD-53535.1
CACUGCAAGCAAAUGCCCU
AGGGCAUUUGCUUGCAGUG


 


334
335
922-940
AD-53536.1
CACUUUUCAGUAUGAUCGU
ACGAUCAUACUGAAAAGUG


 


336
337
1163-1181
AD-53537.1
GGGGCAGGUGGUACUAGAA
UUCUAGUACCACCUGCCCC


 


338
339
1347-1365
AD-53538.1
GGAACCAUGCCUCCAUGAU
AUCAUGGAGGCAUGGUUCC


 


340
341
1964-1982
AD-53539.1
GUCUGUGAUGAACUAAUGA
UCAUUAGUUCAUCACAGAC


 


342
343
2321-2339
AD-53540.1
GUUGUCUUUAUAUGUGAAU
AUUCACAUAUAAAGACAAC


 


344
345
671-689
AD-53541.1
GCAGCACAGAUGAAUCAGA
UCUGAUUCAUCUGUGCUGC


 


346
347
924-942
AD-53542.1
CUUUUCAGUAUGAUCGUUU
AAACGAUCAUACUGAAAAG


 


348
349
1164-1182
AD-53543.1
GGGCAGGUGGUACUAGAAA
UUUCUAGUACCACCUGCCC


 


350
351
1460-1478
AD-53544.1
GUCCCCAAGAUUGUGGCAU
AUGCCACAAUCUUGGGGAC


 


352
353
1976-1994
AD-53545.1
CUAAUGAGCAGACAUAACA
UGUUAUGUCUGCUCAUUAG


 


354
355
786-804
AD-53546.1
GCCCCAGUGUGGUUAGUGU
ACACUAACCACACUGGGGC


 


356
357
935-953
AD-53547.1
GAUCGUUUCUUUGAGAAAA
UUUUCUCAAAGAAACGAUC


 


358
359
1165-1183
AD-53548.1
GGCAGGUGGUACUAGAAAU
AUUUCUAGUACCACCUGCC


 


360
361
1530-1548
AD-53549.1
GUGAUGUGGCCCAUGAGUU
AACUCAUGGGCCACAUCAC


 


362
363
2003-2021
AD-53550.1
CAAGCAAUCAAUUACCCUA
UAGGGUAAUUGAUUGCUUG


 


364
365
788-806
AD-53551.1
CCCAGUGUGGUUAGUGUGA
UCACACUAACCACACUGGG


 


366
367
974-992
AD-53552.1
GACCACACCUAUCGAGUUU
AAACUCGAUAGGUGUGGUC


 


368
369
1191-1209
AD-53553.1
GAACUAGUAAAUUCCAUGU
ACAUGGAAUUUACUAGUUC


 


370
371
1541-1559
AD-53554.1
CAUGAGUUUGGAGCAAUCA
UGAUUGCUCCAAACUCAUG


 


372
373
2075-2093
AD-53555.1
CCCCAGAUGAUGAACUACU
AGUAGUUCAUCAUCUGGGG


 


374
375
360-378
AD-53561.1
GCCCAUUCUUAUCCCGAGU
ACUCGGGAUAAGAAUGGGC


 


376
377
1356-1374
AD-53567.1
CCUCCAUGAUCCAAGGGAU
AUCCCUUGGAUCAUGGAGG


 


378
379
1631-1649
AD-53573.1
GUCAUGCCAAAAAUGGACA
UGUCCAUUUUUGGCAUGAC


 


380
381
1634-1652
AD-53579.1
AUGCCAAAAAUGGACAUCA
UGAUGUCCAUUUUUGGCAU









Example 3
In Vitro Screening of ALAS1 siRNA Duplexes for ALAS1 Knockdown Activity
ALAS1 siRNA duplexes were screened for the ability to knockdown ALAS1 expression in vitro.
In Vitro Screening
Cell Culture and Transfections
Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in MEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media containing  ̃2×104 Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done at 10, 1.67, 0.27, 0.046, 0.0077, 0.0013, 0.00021, 0.00004 nM final duplex concentration.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were captured again and supernatant removed. Beads were then washed with 15 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)
A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR
2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl ALAS1 TaqMan probe (Applied Biosystems cat #Hs00167441_ml) and 50 Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a Roche LC480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections with two biological replicates each, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.
To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose.
In Vitro Knockdown of Endogenous ALAS1 Expression by ALAS1 siRNA Duplexes
Table 4 illustrates the knockdown of ALAS1 in Hep3B cells by ALAS1 modified siRNA duplexes (See Table 2). Silencing is expressed as the fraction RNA message remaining relative to the negative (luciferase) control siRNA AD-1955. Data were generated as described above following transfection of 10 nM or 0.1 nM of each siRNA. qPCR was run using the ALAS1 TaqMan probe Hs00167441_ml.







TABLE 4







ALAS1 expression in Hep3B cells following 


transfection with ALAS1 siRNA











Duplex ID
10 nM Avg
0.1 nM Avg
10 nM STDEV
0.1 nM STDEV














AD-55078.2
0.7
0.87
0.001
0.089


AD-55084.2
0.08
0.3
0
0.04


AD-55090.2
0.06
0.08
0.002
0.003


AD-55096.2
0.61
0.92
0.171
0.34


AD-55102.2
0.63
0.62
0.005
0.069


AD-55106.2
0.07
0.08
0.004
0.027


AD-55111.2
0.06
0.23
0.013
0.062


AD-55073.2
0.21
0.4
0.018
0.061


AD-55079.2
0.17
0.43
0.033
0.089


AD-55085.2
0.13
0.21
0.011
0.019


AD-55091.2
0.27
0.55
0.033
0.009


AD-55097.2
0.31
0.38
0.051
0.059


AD-55103.2
0.05
0.11
0.017
0.006


AD-55107.2
0.12
0.24
0.007
0.008


AD-55112.2
0.15
0.2
0.036
0.025


AD-55074.2
0.16
0.45
0.008
0.002


AD-55080.2
0.79
0.99
0.095
0.304


AD-55086.2
0.09
0.22
0.005
0.035


AD-55098.2
0.25
0.51
0.03
0.07


AD-55104.2
0.06
0.1
0.017
0.001


AD-55108.2
0.47
0.65
0.03
0.015


AD-55113.2
0.38
0.62
0.068
0.039


AD-55075.2
0.12
0.28
0.007
0.051


AD-55081.2
0.21
0.51
0.036
0.066


AD-55087.2
0.1
0.19
0.017
0.02


AD-55093.2
0.24
0.56
0.029
0.053


AD-55099.2
0.05
0.18
0.001
0.038


AD-53573.3
0.67
1.07
0.16
0.153


AD-55109.2
0.07
0.23
0.006
0.052


AD-55114.2
0.08
0.16
0.004
0.017


AD-55076.2
0.05
0.14
0.007
0.035


AD-55082.2
0.08
0.3
0.019
0.016


AD-55088.2
0.06
0.12
0.008
0.02


AD-55094.2
0.06
0.18
0.005
0.023


AD-55100.2
0.45
0.83
0.02
0.05


AD-55105.2
0.02
0.05
0.005
0.004


AD-55110.2
0.15
0.19
0.031
0.016


AD-55115.2
0.35
0.58
0.045
0.052


AD-55077.2
0.14
0.14
0.006
0.019


AD-55083.2
0.56
0.98
0.24
0.188


AD-55089.2
0.62
0.79
0.036
0.094


AD-55095.2
0.59
0.92
0.12
0.079


AD-55101.2
0.71
0.97
0.074
0.097


AD-1955
1.00
1.01
0.03
0.04


AD-53511.1
0.84
1.08
0.028
0.0515


AD-53512.1
0.15
0.65
0.062
0.023


AD-53513.1
0.34
0.86
0.055
0.011


AD-53514.1
0.12
0.61
0.003
0.008


AD-53515.1
0.25
0.66
0.005
0.004


AD-53516.1
1.05
1.02
0.032
0.011


AD-53517.1
0.145
0.725
0.025
0.0155


AD-53518.1
0.72
0.85
0.045
0.028


AD-53519.1
0.18
0.66
0.061
0.004


AD-53520.1
0.18
0.9
0.041
0.001


AD-53521.1
0.97
1.07
0.01
0.003


AD-53522.1
0.87
1.1
0.065
0.112


AD-53523.1
0.48
0.96
0.0305
0.0255


AD-53524.1
0.11
0.66
0.02
0.006


AD-53525.1
0.71
1.03
0.016
0.01


AD-53526.1
0.23
0.85
0.075
0.01


AD-53527.1
0.25
0.83
0.015
0.017


AD-53528.1
0.44
0.93
0.037
0.006


AD-53529.1
0.185
0.73
0.015
0.014


AD-53530.1
0.1
0.62
0.02
0.003


AD-53531.1
0.48
0.93
0.019
0.045


AD-53532.1
0.06
0.17
0
0.003


AD-53533.1
0.36
0.93
0.025
0.034


AD-53534.1
0.1
0.36
0.014
0.012


AD-53535.1
0.58
1.05
0.036
0.071


AD-53536.1
0.12
0.45
0.009
0.026


AD-53537.1
0.73
0.96
0.101
0.015


AD-53538.1
0.74
1.07
0
0.046


AD-53539.1
0.52
0.97
0.057
0.032


AD-53540.1
0.1
0.47
0.017
0.012


AD-53541.1
0.11
0.29
0.026
0.015


AD-53542.1
0.08
0.23
0.008
0.006


AD-53543.1
0.62
1.01
0.027
0.014


AD-53544.1
0.8
1.04
0.002
0.001


AD-53545.1
0.17
0.73
0.007
0.007


AD-53546.1
0.27
0.93
0.058
0.019


AD-53547.1
0.12
0.28
0.008
0.01


AD-53548.1
0.1
0.34
0.022
0.002


AD-53549.1
0.8
1.04
0.011
0.026


AD-53550.1
0.05
0.54
0.02
0.003


AD-53551.1
0.96
1.16
0.029
0.044


AD-53552.1
0.13
0.5
0.002
0.009


AD-53553.1
0.92
1.1
0.027
0.02


AD-53554.1
0.76
0.67
0.005
0.004


AD-53555.1
0.11
0.53
0.009
0.007


AD-53561.1
0.72
0.94
0.014
0.001


AD-53567.1
0.16
0.66
0.019
0.003


AD-53573.1
1.06
1.10
0.019
0.037


AD-53579.1
0.19
0.76
0.036
0.019









IC50s of Select ALAS1 siRNA Duplexes in In Vitro Screen

Table 5 illustrates the IC50s of select ALAS1 siRNA duplexes determined from the knockdown of endogenously expressed ALAS1 in the Hep3B cell line, by ALAS1 modified siRNA duplexes (see Table 2). Data were generated as described above, at 24 or 120 hours following transfection of each siRNA duplex. Silencing of ALAS1 is expressed as the fraction mRNA message remaining relative to the siRNA AD-1955, a non-targeting siRNA that was used as a negative control. Data from replicate transfection experiments were used to fit a single line to determine the IC50. Several of the duplexes (e.g., AD-53541.1, AD-53542.1, and AD-53547.1) had an IC50 as low as about 0.03 nM at 24 hours. Numerous duplexes had an IC50 of less than 0.1 nM (e.g., AD-53534.1, AD-53536.1, AD-53540.1, AD-53541.1, AD-53542.1, AD-53547.1, AD-53548.1, AD-53550.1, AD-53552.1) at 24 hours, and some of these also had an IC50 of less than 0.1 nM (e.g., AD-53534.1, AD-53540.1, AD-53541.1, AD-53542.1, AD-53547.1, AD-53552.1) at 120 hours.







TABLE 5







IC50s of select ALAS1 siRNA 


duplexes normalized to AD-1955











IC50 (nM)











DUPLEX ID
24 hrs
120 hrs















AD-53534.1
0.045
0.076



AD-53536.1
0.049
0.105



AD-53540.1
0.054
0.077



AD-53541.1
0.032
0.062



AD-53542.1
0.028
0.093



AD-53547.1
0.03
0.062



AD-53548.1
0.044
0.101



AD-53550.1
0.085
0.152



AD-53552.1
0.077
0.063



AD-53567.1
0.219
0.357



AD-53579.1
0.217
0.566










Example 4
In Vivo Silencing Using a Mouse/Rat ALAS1 siRNA Formulated as a LNP
The sequences of the modified duplex AD-53558 are shown in Table 6 below.







TABLE 6







Sequences of ALAS1 siRNA Duplex AD-53558.4













SEQ







ID


SEQ ID
NO:
Start Position on


NO:
(anti-
transcript of


(sense)
sense)
NM_020559.2
Duplex Name
Sense Sequence (5′-3′)
Antisense Sequence (5′-3′)





383
384
1184
AD-53558
cuGuGAAAuuuAcucuGAudTsdT
AUcAGAGuAAAUUUcAcAGdTsdT









This duplex was formulated as a LNP11 formulation (see Table 10 above). The LNP-formulated AD-53558 siRNA was tested in in vivo in mice (N=25 animals; 5 animals per group) and rats (N=20 animals; 4 animals per group) and was confirmed to silence ALAS1 mRNA in vivo. The results are shown in FIG. 5 and FIG. 6.
FIG. 5 shows that the siRNA demonstrated a dose-response effect in mice. The expression of mouse ALAS1 (mALAS1) mRNA was reduced by about 78% when the siRNA was administered at 1 mg/kg; mouse ALAS1 mRNA was reduced by about 60% when the siRNA was administered at 0.3 mg/kg; and mouse ALAS1 mRNA was reduced by about 49% when the siRNA was administered at 0.1 mg/kg. These reductions are expressed relative to a PBS control. An AD-1955 LUC control was also employed, as shown in FIG. 5.
Similarly, FIG. 6 shows that the siRNA demonstrated a dose-response effect in rats. The expression of ALAS1 RNA was reduced by about 70% when the when the siRNA was administered at 1 mg/kg; ALAS1 mRNA was reduced by about 62% when the siRNA was administered at 0.3 mg/kg; and ALAS1 mRNA was reduced by about 34% when the siRNA was administered at 0.1 mg/kg.
The durability of silencing was also tested in mice (N=15; 3 animals per timepoint. The results are shown in FIG. 7, which shows that AD-53558 suppressed mALAS1 mRNA by about 80% for at least 9 days. Suppression of at least about 50% persisted for at least 14 days.
Example 5
Efficacy of ALAS1 siRNA in an Animal Model of AIP
The effects of the AD-53558 LNP11 formulation (a mouse/rat ALAS1 siRNA described in the previous example) were investigated in a mouse model of AIP. The PBGD knockout is not viable (−/−, 0% activity). Heterozygous PBGD knockout mice (+/−,  ̃50% activity) are available but do not have the full biochemical phenotype and thus do not recapitulate the human disease phenotype. Thus, a mouse model of AIP has been developed that is a compound heterozygote with T1/T2 alleles, including T1 (+/−) promoter disruption and T2 (−/−) splice-site alteration. These mice have been shown to have hepatic residual PBGD activity that is about  ̃30% of the wild-type level and normal or slightly elevated baseline plasma ALA and PBG levels. The mice have been found to appear normal early in life and to become slightly slower and ataxic with age. By six months of age, the mice have been documented to develop impaired motor coordination and muscular performance and axonal degeneration on pathological examination. Investigation of the pathology of the mouse model has shown axonal degeneration, impaired motor coordination and muscular performance in older mice. Urinary and plasma ALA and PBG have been found to markedly increase with serial i.p. administration of phenobarbital (see Lindberg et al., (1996), Nature Genetics, 12:195-219 and Lindberg et al., (1999), Journal of Clinical Investigation, 103:1127-34). The mice were rescued by AAV-mediated expression of PBGD in the liver (Yasuda et al. (2010), Molecular Medicine, 1:17-22 and Unzu et al. (2011), Molecular Medicine, 2:243-50).
On day 1, the mice were administered 1 mg/kg ALAS1 siRNA (n=5) or LUC AD-1955 control (n=3) by i.v. injection. Three phenobarbitol injections were given (1 injection per day on days 2, 3, and 4) to induce hepatic ALAS1 ande the porphyrin precursors, ALA and PBG. Plasma and overnight urine specimens were collected on day 5 and metabolite levels were measured by LC-MS. Metabolite levels were measured in plasma by LC-MS and were also measured in urine. Baseline levels of metabolites were measured prior to the first treatment on day 1. The results are shown in FIGS. 8-12 and in Tables 12 and 13.
FIG. 8 and FIG. 9 show the plasma ALA levels in μM. Baseline ALA levels were low, (n=4), and phenobarbitol treatment induced significant increases in plasma ALA levels in the control LUC siRNA treated animals (n=3). Treatment with ALAS1 siRNA inhibited the induction of plasma ALA (n=5), as shown in FIG. 8. The ALAS1 siRNA was consistently effective in blocking the induction of plasma ALA in each of the individual animals studied (see FIG. 9). These results indicate that ALAS1 siRNA treatment was effective in preventing the increases in plasma ALA associated with the phenobarbital-induced acute attacks in this AIP animal model.
FIG. 10 and FIG. 11 show the plasma PBG levels in μM. Baseline PBG levels were low (n=4), and phenobarbitol treatment induced significant increases in plasma PBG levels in the control LUC siRNA treated animals (n=3) Treatment with ALAS1 siRNA inhibited the induction of plasma PBG (n=5), as shown in FIG. 10. The ALAS1 siRNA was consistently effective in blocking the induction of plasma PBG in each of the individual animals studied (see FIG. 11). These results indicate that ALAS1 siRNA treatment was effective in preventing the increases in plasma PBG associated with the phenobarbital-induced acute attacks in this AIP animal model.
Tables 12 and 13 shows urine ALA and PBG levels at baseline and after phenobarbitol treatment in LUC siRNA (n=2) control (CTR, which refers to a PBS buffer treated animal, n=1) and ALAS1 siRNA (n=5) treated animals.







TABLE 12







Urine data from individual animals showing prevention of induced acute attack















ALA
PBG

ALA
PBG





(micro
(micro
Creatinine
(microM/mg
(microM/mg


Mouse ID
M/l)
M/L)
(mg/dl)
creatinine)
creatinine)
siRNA
PB

















Ha-17-4-6



29.7
7.9
Baseline
−


Ha-19-5-4/2



15.7
5.1
Baseline
−


Ha-20-39-



28.6
6.7
Baseline
−


4/3


Ha-20-38-4



21.4
4.7
Baseline
−


Ha-21-33-4
934.92
483.71
0.4205
222.33
115.03
Luc
+


Ha-21-36-9
944.08
563.53
0.5055
186.76
111.48
Luc
+


Ha-21-18-8
32.88
8.69
0.133
24.72
6.53
ALAS1;
+








1 mg/kg


Ha-21-33-7
83.07
23.28
0.426
19.50
5.46
ALAS1;
+








1 mg/kg


Ha-21-34-5
59.15
18.41
0.263
22.49
7.00
ALAS1;
+








1 mg/kg





PB stands for phenobarbitol. A “+” indicates that phenobarbitol was administered.













TABLE 13







Average Urine Data








Mean ALA
Mean PBG


(microM/mg creatinine)
(microM/mg creatinine)











23.8
6.1 AIP Baseline


204.55
113.26 Luc-siRNA


22.24
6.33 ALAS1-siRNA









Phenobarbitol treatment induced strong increases ( ̃25-30 fold increases) in urine ALA ( ̃9-fold over baseline levels) and PBG ( ̃19-fold over baseline levels) in the LUC siRNA treated mice, control, whereas such increases were not observed in the ALAS1 siRNA treated animals. Thus, ALAS1 siRNA blocked phenobarbitol-induced increases in urinary ALA and PBG. These results are consistent with the plasma measurements and show that ALAS1 siRNA treatment was effective in preventing increases in urinary metabolites (ALA and PBG) associated with the phenobarbital-induced acute attacks in this AIP animal model.
In further experiments (FIG. 12), it was found that phenobarbitol treatment induced large increases ( ̃25 fold) in ALAS1 mRNA expression in the liver of the mouse model. Administration of ALAS1 siRNA completely blocked this ALAS1 mRNA induction. These results provide further evidence that ALAS1 siRNA is effective in an animal model of AIP.
Collectively, the results provided in this Example show that ALAS1 siRNA was effective in treating acute attacks in an animal model of the acute hepatic porphyria AIP. Multiple outcome measures support this conclusion, including plasma ALA levels, plasma PBG levels, urine ALA levels, urine PBG levels, and liver ALAS1 mRNA expression levels.
Example 6
In Vivo Silencing Using GalNAc-Conjugated Mouse ALAS1 siRNA
The experiments described in this example investigated the in vivo efficacy of three GalNAc-conjugated siRNAs (see Table 7). These siRNAs were designed and produced with methods such as those described in Example 2.







TABLE 7







Sequences AD-57929



















Position








of



SEQ
Position



antisense


SEQ
ID
of sense



seq.


ID
NO:
seq. on



on


NO:
(anti-
transcript
Duplex


transcript


(sense)
sense)
NM_020559.2
Name
Sense Sequence (5′-3′)
Antisense Sequence (5′-3′)
NM_020559.2





385
386
775-795
AD-
AfaGfuCfuGfuUfUfCfcAfcUfuUfuC
uUfgAfaAfaGfuGfgaaAfcAfgAfcUf
773-795





56211
faAfL96
usUfsg


 


387
388
2168-2188
AD-
AfcAfuAfgUfaGfCfCfaGfaAfuUfgU
aGfaCfaAfuUfcUfggcUfaCfuAfuGf
2166-2188





56173
fcUfL96
usGfsg


 


389
390
775-795
AD-
AfsasGfuCfuGfuUfUfCfcAfcUfuU
usUfsgAfaAfaGfuGfgaaAfcAfgAfc
773-795





57929
fuCfaAfL96
Ufususg









The mice (n=40; n=4 per experimental condition) were divided into groups that received PBS or doses of 3 mg/kg, 10 mg/kg, or 30 mg/kg of siRNA administered subcutaneously. The level of mALAS1/mGAPDH mRNA, relative to the PBS control, was determined in liver cells at 72 hours post-administration. The results are shown in FIG. 13. There was not a clear dose-response effect for the siRNAs AD-56211 and AD-56173. In contrast, the ALAS1 siRNA AD-57929 showed a dose-response effect in inhibiting mALAS1 expression. These results demonstrate that an ALAS1 GalNAc conjugate was effective in inhibiting expression of ALAS1 mRNA in vivo and showed a dose-response effect.
Example 7
Human siRNAs
Additional human siRNAs were designed and produced as described in Example 2. The top 45 siRNAs were selected based on their predicted efficacy. The sequences of these 45 siRNAs are provided in Table 8.







TABLE 8







Human ALAS1 siRNA Sense and Antisense Sequences












SEQ ID






NO:
Position on


SEQ ID NO:
(anti-
transcript


(sense)
sense)
NM_000688.4
Sense Sequence (5′-3′)
Antisense Sequence (5′-3′)





391
392
1635-1657
CAUGCCAAAAAUGGACAUCAU
AUGAUGUCCAUUUUUGGCAUGAC


 


393
394
2352-2374
UAAAUUUUAAUCUAUAGUAAA
UUUACUAUAGAUUAAAAUUUAAU


 


395
396
1324-1346
GGCUGUGAGAUUUACUCUGAU
AUCAGAGUAAAUCUCACAGCCUG


 


397
398
1637-1659
UGCCAAAAAUGGACAUCAUUU
AAAUGAUGUCCAUUUUUGGCAUG


 


399
400
1363-1385
AUGAUCCAAGGGAUUCGAAAC
GUUUCGAAUCCCUUGGAUCAUGG


 


401
402
925-947
ACUUUUCAGUAUGAUCGUUUC
GAAACGAUCAUACUGAAAAGUGG


 


403
404
790-812
CCCAGUGUGGUUAGUGUGAAA
UUUCACACUAACCACACUGGGGC


 


405
406
1531-1553
UGUGAUGUGGCCCAUGAGUUU
AAACUCAUGGGCCACAUCACACA


 


407
408
2189-2211
AUUUUGAAGUGAUGAGUGAAA
UUUCACUCAUCACUUCAAAAUGC


 


409
410
929-951
UUCAGUAUGAUCGUUUCUUUG
CAAAGAAACGAUCAUACUGAAAA


 


411
412
872-894
GACCAGAAAGAGUGUCUCAUC
GAUGAGACACUCUUUCUGGUCUU


 


413
414
706-728
UUCUGCAAAGCCAGUCUUGAG
CUCAAGACUGGCUUUGCAGAAGA


 


415
416
1362-1384
CAUGAUCCAAGGGAUUCGAAA
UUUCGAAUCCCUUGGAUCAUGGA


 


417
418
1634-1656
UCAUGCCAAAAAUGGACAUCA
UGAUGUCCAUUUUUGGCAUGACU


 


419
420
1325-1347
GCUGUGAGAUUUACUCUGAUU
AAUCAGAGUAAAUCUCACAGCCU


 


421
422
2208-2230
AAGAGAGAAGUCCUAUUUCUC
GAGAAAUAGGACUUCUCUCUUUC


 


423
424
2344-2366
AGUUAUAUUAAAUUUUAAUCU
AGAUUAAAAUUUAAUAUAACUUA


 


425
426
924-946
CACUUUUCAGUAUGAUCGUUU
AAACGAUCAUACUGAAAAGUGGA


 


427
428
873-895
ACCAGAAAGAGUGUCUCAUCU
AGAUGAGACACUCUUUCUGGUCU


 


429
430
759-781
GAGGAAAGAGGUUGCUGAAAC
GUUUCAGCAACCUCUUUCCUCAC


 


431
432
871-893
AGACCAGAAAGAGUGUCUCAU
AUGAGACACUCUUUCUGGUCUUU


 


433
434
1183-1205
AAUAUUUCUGGAACUAGUAAA
UUUACUAGUUCCAGAAAUAUUUC


 


435
436
2229-2251
AGGCUUGAGCAAGUUGGUAUC
GAUACCAACUUGCUCAAGCCUGA


 


437
438
671-693
UGGCAGCACAGAUGAAUCAGA
UCUGAUUCAUCUGUGCUGCCAGG


 


439
440
2187-2209
GCAUUUUGAAGUGAUGAGUGA
UCACUCAUCACUUCAAAAUGCAG


 


441
442
913-935
AAAUCUGUUUCCACUUUUCAG
CUGAAAAGUGGAAACAGAUUUUG


 


443
444
1977-1999
ACUAAUGAGCAGACAUAACAU
AUGUUAUGUCUGCUCAUUAGUUC


 


445
446
1174-1196
GGUACUAGAAAUAUUUCUGGA
UCCAGAAAUAUUUCUAGUACCAC


 


447
448
1810-1832
AUCCUGAAGAGCGCUGAGGGA
UCCCUCAGCGCUCUUCAGGAUCC


 


449
450
892-914
CUUCUUCAAGAUAACUUGCCA
UGGCAAGUUAUCUUGAAGAAGAU


 


451
452
877-899
GAAAGAGUGUCUCAUCUUCUU
AAGAAGAUGAGACACUCUUUCUG


 


453
454
935-957
AUGAUCGUUUCUUUGAGAAAA
UUUUCUCAAAGAAACGAUCAUAC


 


455
456
1975-1997
GAACUAAUGAGCAGACAUAAC
GUUAUGUCUGCUCAUUAGUUCAU


 


457
458
1478-1500
CAUUUGAAACUGUCCAUUCAA
UUGAAUGGACAGUUUCAAAUGCC


 


459
460
2366-2388
UAGUAAAAACAUAGUCCUGGA
UCCAGGACUAUGUUUUUACUAUA


 


461
462
853-875
GACAUCAUGCAAAAGCAAAGA
UCUUUGCUUUUGCAUGAUGUCCU


 


463
464
1966-1988
GUCUGUGAUGAACUAAUGAGC
GCUCAUUAGUUCAUCACAGACUU


 


465
466
928-950
UUUCAGUAUGAUCGUUUCUUU
AAAGAAACGAUCAUACUGAAAAG


 


467
468
1186-1208
AUUUCUGGAACUAGUAAAUUC
GAAUUUACUAGUUCCAGAAAUAU


 


469
470
1189-1211
UCUGGAACUAGUAAAUUCCAU
AUGGAAUUUACUAGUUCCAGAAA


 


471
472
973-995
AAUGACCACACCUAUCGAGUU
AACUCGAUAGGUGUGGUCAUUCU


 


473
474
 983-1005
CCUAUCGAGUUUUUAAAACUG
CAGUUUUAAAAACUCGAUAGGUG


 


475
476
1185-1207
UAUUUCUGGAACUAGUAAAUU
AAUUUACUAGUUCCAGAAAUAUU


 


477
478
2353-2375
AAAUUUUAAUCUAUAGUAAAA
UUUUACUAUAGAUUAAAAUUUAA


 


479
480
875-897
CAGAAAGAGUGUCUCAUCUUC
GAAGAUGAGACACUCUUUCUGGU


 


481
482
360-378
GCCCAUUCUUAUCCCGAGU
ACUCGGGAUAAGAAUGGGC


 


483
484
428-446
CAAAACUGCCCCAAGAUGA
UCAUCUUGGGGCAGUUUUG


 


485
486
873-891
CAGAAAGAGUGUCUCAUCU
AGAUGAGACACUCUUUCUG


 


487
488
874-892
AGAAAGAGUGUCUCAUCUU
AAGAUGAGACACUCUUUCU


 


489
490
877-895
AAGAGUGUCUCAUCUUCUU
AAGAAGAUGAGACACUCUU


 


491
492
1295-1313
CUCUUCACCCUGGCUAAGA
UCUUAGCCAGGGUGAAGAG


 


493
494
1296-1314
UCUUCACCCUGGCUAAGAU
AUCUUAGCCAGGGUGAAGA


 


495
496
1299-1317
UCACCCUGGCUAAGAUGAU
AUCAUCUUAGCCAGGGUGA


 


497
498
1347-1365
GGAACCAUGCCUCCAUGAU
AUCAUGGAGGCAUGGUUCC


 


499
500
1355-1373
GCCUCCAUGAUCCAAGGGA
UCCCUUGGAUCAUGGAGGC


 


501
502
1356-1374
CCUCCAUGAUCCAAGGGAU
AUCCCUUGGAUCAUGGAGG


 


503
504
1357-1375
CUCCAUGAUCCAAGGGAUU
AAUCCCUUGGAUCAUGGAG


 


505
506
1631-1649
GUCAUGCCAAAAAUGGACA
UGUCCAUUUUUGGCAUGAC


 


507
508
1634-1652
AUGCCAAAAAUGGACAUCA
UGAUGUCCAUUUUUGGCAU


 


509
510
1635-1653
UGCCAAAAAUGGACAUCAU
AUGAUGUCCAUUUUUGGCA


 


511
512
1791-1809
CCCUGGAGUCUGUGCGGAU
AUCCGCACAGACUCCAGGG


 


513
514
1794-1812
UGGAGUCUGUGCGGAUCCU
AGGAUCCGCACAGACUCCA


 


515
516
1921-1939
CAUCAUCCCUGUGCGGGUU
AACCCGCACAGGGAUGAUG


 


517
518
359-377
UGCCCAUUCUUAUCCCGAA
UUCGGGAUAAGAAUGGGCA


 


519
520
362-380
CCAUUCUUAUCCCGAGUCA
UGACUCGGGAUAAGAAUGG


 


521
522
363-381
CAUUCUUAUCCCGAGUCCA
UGGACUCGGGAUAAGAAUG


 


523
524
434-452
UGCCCCAAGAUGAUGGAAU
AUUCCAUCAUCUUGGGGCA


 


525
526
872-890
CCAGAAAGAGUGUCUCAUA
UAUGAGACACUCUUUCUGG


 


527
528
875-893
GAAAGAGUGUCUCAUCUUA
UAAGAUGAGACACUCUUUC


 


529
530
1112-1130
CACCCACGGGUGUGUGGGA
UCCCACACACCCGUGGGUG


 


531
532
1113-1131
ACCCACGGGUGUGUGGGGA
UCCCCACACACCCGUGGGU


 


533
534
1297-1315
CUUCACCCUGGCUAAGAUA
UAUCUUAGCCAGGGUGAAG


 


535
536
1300-1318
CACCCUGGCUAAGAUGAUA
UAUCAUCUUAGCCAGGGUG


 


537
538
1301-1319
ACCCUGGCUAAGAUGAUGA
UCAUCAUCUUAGCCAGGGU


 


539
540
1348-1366
GAACCAUGCCUCCAUGAUA
UAUCAUGGAGGCAUGGUUC


 


541
542
1481-1499
GAAACUGUCCAUUCAAUGA
UCAUUGAAUGGACAGUUUC


 


543
544
1786-1804
UGGAGCCCUGGAGUCUGUA
UACAGACUCCAGGGCUCCA


 


545
546
1795-1813
GGAGUCUGUGCGGAUCCUA
UAGGAUCCGCACAGACUCC


 


547
548
1919-1937
CACAUCAUCCCUGUGCGGA
UCCGCACAGGGAUGAUGUG


 


549
550
1922-1940
AUCAUCCCUGUGCGGGUUA
UAACCCGCACAGGGAUGAU


 


551
552
1923-1941
UCAUCCCUGUGCGGGUUGA
UCAACCCGCACAGGGAUGA









Example 8
Human siRNAs
Additional 19mer human siRNAs were generated. The sequences of these siRNAs are provided in Table 9. These siRNAs can be tested for efficacy using methods described herein and/or methods known in the art.







TABLE 9







Human ALAS1 siRNA Sense and Antisense Sequences












SEQ





SEQ
ID


ID
NO:
Position on


NO:
(anti-
transcript


(sense)
sense)
NM_000688.4
Sense Sequence (5′-3′)
Antisense Sequence (5′-3′)














553
554
 4-22
UAUAUUAAGGCGCCGGCGA
UCGCCGGCGCCUUAAUAUA


 


555
556
 5-23
AUAUUAAGGCGCCGGCGAU
AUCGCCGGCGCCUUAAUAU


 


557
558
 6-24
UAUUAAGGCGCCGGCGAUC
GAUCGCCGGCGCCUUAAUA


 


559
560
 7-25
AUUAAGGCGCCGGCGAUCG
CGAUCGCCGGCGCCUUAAU


 


561
562
 8-26
UUAAGGCGCCGGCGAUCGC
GCGAUCGCCGGCGCCUUAA


 


563
564
 9-27
UAAGGCGCCGGCGAUCGCG
CGCGAUCGCCGGCGCCUUA


 


565
566
10-28
AAGGCGCCGGCGAUCGCGG
CCGCGAUCGCCGGCGCCUU


 


567
568
11-29
AGGCGCCGGCGAUCGCGGC
GCCGCGAUCGCCGGCGCCU


 


569
570
12-30
GGCGCCGGCGAUCGCGGCC
GGCCGCGAUCGCCGGCGCC


 


571
572
13-31
GCGCCGGCGAUCGCGGCCU
AGGCCGCGAUCGCCGGCGC


 


573
574
14-32
CGCCGGCGAUCGCGGCCUG
CAGGCCGCGAUCGCCGGCG


 


575
576
81-99
CUUGAGUGCCCGCCUCCUU
AAGGAGGCGGGCACUCAAG


 


577
578
 82-100
UUGAGUGCCCGCCUCCUUC
GAAGGAGGCGGGCACUCAA


 


579
580
 83-101
UGAGUGCCCGCCUCCUUCG
CGAAGGAGGCGGGCACUCA


 


581
582
 84-102
GAGUGCCCGCCUCCUUCGC
GCGAAGGAGGCGGGCACUC


 


583
584
 85-103
AGUGCCCGCCUCCUUCGCC
GGCGAAGGAGGCGGGCACU


 


585
586
 86-104
GUGCCCGCCUCCUUCGCCG
CGGCGAAGGAGGCGGGCAC


 


587
588
 87-105
UGCCCGCCUCCUUCGCCGC
GCGGCGAAGGAGGCGGGCA


 


589
590
 88-106
GCCCGCCUCCUUCGCCGCC
GGCGGCGAAGGAGGCGGGC


 


591
592
 89-107
CCCGCCUCCUUCGCCGCCG
CGGCGGCGAAGGAGGCGGG


 


593
594
 90-108
CCGCCUCCUUCGCCGCCGC
GCGGCGGCGAAGGAGGCGG


 


595
596
 91-109
CGCCUCCUUCGCCGCCGCC
GGCGGCGGCGAAGGAGGCG


 


597
598
 92-110
GCCUCCUUCGCCGCCGCCU
AGGCGGCGGCGAAGGAGGC


 


599
600
 93-111
CCUCCUUCGCCGCCGCCUC
GAGGCGGCGGCGAAGGAGG


 


601
602
356-374
CGCUGCCCAUUCUUAUCCC
GGGAUAAGAAUGGGCAGCG


 


603
604
357-375
GCUGCCCAUUCUUAUCCCG
CGGGAUAAGAAUGGGCAGC


 


605
606
359-377
UGCCCAUUCUUAUCCCGAG
CUCGGGAUAAGAAUGGGCA


 


607
608
361-379
CCCAUUCUUAUCCCGAGUC
GACUCGGGAUAAGAAUGGG


 


609
610
362-380
CCAUUCUUAUCCCGAGUCC
GGACUCGGGAUAAGAAUGG


 


611
612
363-381
CAUUCUUAUCCCGAGUCCC
GGGACUCGGGAUAAGAAUG


 


613
614
364-382
AUUCUUAUCCCGAGUCCCC
GGGGACUCGGGAUAAGAAU


 


615
616
365-383
UUCUUAUCCCGAGUCCCCC
GGGGGACUCGGGAUAAGAA


 


617
618
366-384
UCUUAUCCCGAGUCCCCCA
UGGGGGACUCGGGAUAAGA


 


619
620
367-385
CUUAUCCCGAGUCCCCCAG
CUGGGGGACUCGGGAUAAG


 


621
622
368-386
UUAUCCCGAGUCCCCCAGG
CCUGGGGGACUCGGGAUAA


 


623
624
369-387
UAUCCCGAGUCCCCCAGGC
GCCUGGGGGACUCGGGAUA


 


625
626
370-388
AUCCCGAGUCCCCCAGGCC
GGCCUGGGGGACUCGGGAU


 


627
628
371-389
UCCCGAGUCCCCCAGGCCU
AGGCCUGGGGGACUCGGGA


 


629
630
372-390
CCCGAGUCCCCCAGGCCUU
AAGGCCUGGGGGACUCGGG


 


631
632
373-391
CCGAGUCCCCCAGGCCUUU
AAAGGCCUGGGGGACUCGG


 


633
634
374-392
CGAGUCCCCCAGGCCUUUC
GAAAGGCCUGGGGGACUCG


 


635
636
375-393
GAGUCCCCCAGGCCUUUCU
AGAAAGGCCUGGGGGACUC


 


637
638
376-394
AGUCCCCCAGGCCUUUCUG
CAGAAAGGCCUGGGGGACU


 


639
640
377-395
GUCCCCCAGGCCUUUCUGC
GCAGAAAGGCCUGGGGGAC


 


641
642
378-396
UCCCCCAGGCCUUUCUGCA
UGCAGAAAGGCCUGGGGGA


 


643
644
379-397
CCCCCAGGCCUUUCUGCAG
CUGCAGAAAGGCCUGGGGG


 


645
646
380-398
CCCCAGGCCUUUCUGCAGA
UCUGCAGAAAGGCCUGGGG


 


647
648
381-399
CCCAGGCCUUUCUGCAGAA
UUCUGCAGAAAGGCCUGGG


 


649
650
382-400
CCAGGCCUUUCUGCAGAAA
UUUCUGCAGAAAGGCCUGG


 


651
652
383-401
CAGGCCUUUCUGCAGAAAG
CUUUCUGCAGAAAGGCCUG


 


653
654
384-402
AGGCCUUUCUGCAGAAAGC
GCUUUCUGCAGAAAGGCCU


 


655
656
385-403
GGCCUUUCUGCAGAAAGCA
UGCUUUCUGCAGAAAGGCC


 


657
658
386-404
GCCUUUCUGCAGAAAGCAG
CUGCUUUCUGCAGAAAGGC


 


659
660
387-405
CCUUUCUGCAGAAAGCAGG
CCUGCUUUCUGCAGAAAGG


 


661
662
388-406
CUUUCUGCAGAAAGCAGGC
GCCUGCUUUCUGCAGAAAG


 


663
664
389-407
UUUCUGCAGAAAGCAGGCA
UGCCUGCUUUCUGCAGAAA


 


665
666
390-408
UUCUGCAGAAAGCAGGCAA
UUGCCUGCUUUCUGCAGAA


 


667
668
391-409
UCUGCAGAAAGCAGGCAAA
UUUGCCUGCUUUCUGCAGA


 


669
670
392-410
CUGCAGAAAGCAGGCAAAU
AUUUGCCUGCUUUCUGCAG


 


671
672
393-411
UGCAGAAAGCAGGCAAAUC
GAUUUGCCUGCUUUCUGCA


 


673
674
394-412
GCAGAAAGCAGGCAAAUCU
AGAUUUGCCUGCUUUCUGC


 


675
676
395-413
CAGAAAGCAGGCAAAUCUC
GAGAUUUGCCUGCUUUCUG


 


677
678
396-414
AGAAAGCAGGCAAAUCUCU
AGAGAUUUGCCUGCUUUCU


 


679
680
397-415
GAAAGCAGGCAAAUCUCUG
CAGAGAUUUGCCUGCUUUC


 


681
682
398-416
AAAGCAGGCAAAUCUCUGU
ACAGAGAUUUGCCUGCUUU


 


683
684
399-417
AAGCAGGCAAAUCUCUGUU
AACAGAGAUUUGCCUGCUU


 


685
686
400-418
AGCAGGCAAAUCUCUGUUG
CAACAGAGAUUUGCCUGCU


 


687
688
401-419
GCAGGCAAAUCUCUGUUGU
ACAACAGAGAUUUGCCUGC


 


689
690
402-420
CAGGCAAAUCUCUGUUGUU
AACAACAGAGAUUUGCCUG


 


691
692
403-421
AGGCAAAUCUCUGUUGUUC
GAACAACAGAGAUUUGCCU


 


693
694
405-423
GCAAAUCUCUGUUGUUCUA
UAGAACAACAGAGAUUUGC


 


695
696
406-424
CAAAUCUCUGUUGUUCUAU
AUAGAACAACAGAGAUUUG


 


697
698
407-425
AAAUCUCUGUUGUUCUAUG
CAUAGAACAACAGAGAUUU


 


699
700
408-426
AAUCUCUGUUGUUCUAUGC
GCAUAGAACAACAGAGAUU


 


701
702
409-427
AUCUCUGUUGUUCUAUGCC
GGCAUAGAACAACAGAGAU


 


703
704
410-428
UCUCUGUUGUUCUAUGCCC
GGGCAUAGAACAACAGAGA


 


705
706
411-429
CUCUGUUGUUCUAUGCCCA
UGGGCAUAGAACAACAGAG


 


707
708
412-430
UCUGUUGUUCUAUGCCCAA
UUGGGCAUAGAACAACAGA


 


709
710
413-431
CUGUUGUUCUAUGCCCAAA
UUUGGGCAUAGAACAACAG


 


711
712
414-432
UGUUGUUCUAUGCCCAAAA
UUUUGGGCAUAGAACAACA


 


713
714
415-433
GUUGUUCUAUGCCCAAAAC
GUUUUGGGCAUAGAACAAC


 


715
716
416-434
UUGUUCUAUGCCCAAAACU
AGUUUUGGGCAUAGAACAA


 


717
718
417-435
UGUUCUAUGCCCAAAACUG
CAGUUUUGGGCAUAGAACA


 


719
720
418-436
GUUCUAUGCCCAAAACUGC
GCAGUUUUGGGCAUAGAAC


 


721
722
419-437
UUCUAUGCCCAAAACUGCC
GGCAGUUUUGGGCAUAGAA


 


723
724
420-438
UCUAUGCCCAAAACUGCCC
GGGCAGUUUUGGGCAUAGA


 


725
726
421-439
CUAUGCCCAAAACUGCCCC
GGGGCAGUUUUGGGCAUAG


 


727
728
422-440
UAUGCCCAAAACUGCCCCA
UGGGGCAGUUUUGGGCAUA


 


729
730
423-441
AUGCCCAAAACUGCCCCAA
UUGGGGCAGUUUUGGGCAU


 


731
732
424-442
UGCCCAAAACUGCCCCAAG
CUUGGGGCAGUUUUGGGCA


 


733
734
425-443
GCCCAAAACUGCCCCAAGA
UCUUGGGGCAGUUUUGGGC


 


735
736
426-444
CCCAAAACUGCCCCAAGAU
AUCUUGGGGCAGUUUUGGG


 


737
738
427-445
CCAAAACUGCCCCAAGAUG
CAUCUUGGGGCAGUUUUGG


 


739
740
429-447
AAAACUGCCCCAAGAUGAU
AUCAUCUUGGGGCAGUUUU


 


741
742
430-448
AAACUGCCCCAAGAUGAUG
CAUCAUCUUGGGGCAGUUU


 


743
744
431-449
AACUGCCCCAAGAUGAUGG
CCAUCAUCUUGGGGCAGUU


 


745
746
432-450
ACUGCCCCAAGAUGAUGGA
UCCAUCAUCUUGGGGCAGU


 


747
748
433-451
CUGCCCCAAGAUGAUGGAA
UUCCAUCAUCUUGGGGCAG


 


749
750
434-452
UGCCCCAAGAUGAUGGAAG
CUUCCAUCAUCUUGGGGCA


 


751
752
435-453
GCCCCAAGAUGAUGGAAGU
ACUUCCAUCAUCUUGGGGC


 


753
754
437-455
CCCAAGAUGAUGGAAGUUG
CAACUUCCAUCAUCUUGGG


 


755
756
438-456
CCAAGAUGAUGGAAGUUGG
CCAACUUCCAUCAUCUUGG


 


757
758
439-457
CAAGAUGAUGGAAGUUGGG
CCCAACUUCCAUCAUCUUG


 


759
760
440-458
AAGAUGAUGGAAGUUGGGG
CCCCAACUUCCAUCAUCUU


 


761
762
441-459
AGAUGAUGGAAGUUGGGGC
GCCCCAACUUCCAUCAUCU


 


763
764
442-460
GAUGAUGGAAGUUGGGGCC
GGCCCCAACUUCCAUCAUC


 


765
766
443-461
AUGAUGGAAGUUGGGGCCA
UGGCCCCAACUUCCAUCAU


 


767
768
444-462
UGAUGGAAGUUGGGGCCAA
UUGGCCCCAACUUCCAUCA


 


769
770
445-463
GAUGGAAGUUGGGGCCAAG
CUUGGCCCCAACUUCCAUC


 


771
772
446-464
AUGGAAGUUGGGGCCAAGC
GCUUGGCCCCAACUUCCAU


 


773
774
447-465
UGGAAGUUGGGGCCAAGCC
GGCUUGGCCCCAACUUCCA


 


775
776
448-466
GGAAGUUGGGGCCAAGCCA
UGGCUUGGCCCCAACUUCC


 


777
778
449-467
GAAGUUGGGGCCAAGCCAG
CUGGCUUGGCCCCAACUUC


 


779
780
450-468
AAGUUGGGGCCAAGCCAGC
GCUGGCUUGGCCCCAACUU


 


781
782
451-469
AGUUGGGGCCAAGCCAGCC
GGCUGGCUUGGCCCCAACU


 


783
784
452-470
GUUGGGGCCAAGCCAGCCC
GGGCUGGCUUGGCCCCAAC


 


785
786
453-471
UUGGGGCCAAGCCAGCCCC
GGGGCUGGCUUGGCCCCAA


 


787
788
454-472
UGGGGCCAAGCCAGCCCCU
AGGGGCUGGCUUGGCCCCA


 


789
790
455-473
GGGGCCAAGCCAGCCCCUC
GAGGGGCUGGCUUGGCCCC


 


791
792
456-474
GGGCCAAGCCAGCCCCUCG
CGAGGGGCUGGCUUGGCCC


 


793
794
457-475
GGCCAAGCCAGCCCCUCGG
CCGAGGGGCUGGCUUGGCC


 


795
796
458-476
GCCAAGCCAGCCCCUCGGG
CCCGAGGGGCUGGCUUGGC


 


797
798
459-477
CCAAGCCAGCCCCUCGGGC
GCCCGAGGGGCUGGCUUGG


 


799
800
460-478
CAAGCCAGCCCCUCGGGCA
UGCCCGAGGGGCUGGCUUG


 


801
802
461-479
AAGCCAGCCCCUCGGGCAU
AUGCCCGAGGGGCUGGCUU


 


803
804
462-480
AGCCAGCCCCUCGGGCAUU
AAUGCCCGAGGGGCUGGCU


 


805
806
463-481
GCCAGCCCCUCGGGCAUUG
CAAUGCCCGAGGGGCUGGC


 


807
808
464-482
CCAGCCCCUCGGGCAUUGU
ACAAUGCCCGAGGGGCUGG


 


809
810
465-483
CAGCCCCUCGGGCAUUGUC
GACAAUGCCCGAGGGGCUG


 


811
812
466-484
AGCCCCUCGGGCAUUGUCC
GGACAAUGCCCGAGGGGCU


 


813
814
467-485
GCCCCUCGGGCAUUGUCCA
UGGACAAUGCCCGAGGGGC


 


815
816
468-486
CCCCUCGGGCAUUGUCCAC
GUGGACAAUGCCCGAGGGG


 


817
818
469-487
CCCUCGGGCAUUGUCCACU
AGUGGACAAUGCCCGAGGG


 


819
820
470-488
CCUCGGGCAUUGUCCACUG
CAGUGGACAAUGCCCGAGG


 


821
822
471-489
CUCGGGCAUUGUCCACUGC
GCAGUGGACAAUGCCCGAG


 


823
824
472-490
UCGGGCAUUGUCCACUGCA
UGCAGUGGACAAUGCCCGA


 


825
826
473-491
CGGGCAUUGUCCACUGCAG
CUGCAGUGGACAAUGCCCG


 


827
828
474-492
GGGCAUUGUCCACUGCAGC
GCUGCAGUGGACAAUGCCC


 


829
830
475-493
GGCAUUGUCCACUGCAGCA
UGCUGCAGUGGACAAUGCC


 


831
832
476-494
GCAUUGUCCACUGCAGCAG
CUGCUGCAGUGGACAAUGC


 


833
834
477-495
CAUUGUCCACUGCAGCAGU
ACUGCUGCAGUGGACAAUG


 


835
836
478-496
AUUGUCCACUGCAGCAGUA
UACUGCUGCAGUGGACAAU


 


837
838
479-497
UUGUCCACUGCAGCAGUAC
GUACUGCUGCAGUGGACAA


 


839
840
480-498
UGUCCACUGCAGCAGUACA
UGUACUGCUGCAGUGGACA


 


841
842
481-499
GUCCACUGCAGCAGUACAC
GUGUACUGCUGCAGUGGAC


 


843
844
482-500
UCCACUGCAGCAGUACACU
AGUGUACUGCUGCAGUGGA


 


845
846
483-501
CCACUGCAGCAGUACACUA
UAGUGUACUGCUGCAGUGG


 


847
848
484-502
CACUGCAGCAGUACACUAC
GUAGUGUACUGCUGCAGUG


 


849
850
485-503
ACUGCAGCAGUACACUACC
GGUAGUGUACUGCUGCAGU


 


851
852
486-504
CUGCAGCAGUACACUACCA
UGGUAGUGUACUGCUGCAG


 


853
854
487-505
UGCAGCAGUACACUACCAA
UUGGUAGUGUACUGCUGCA


 


855
856
488-506
GCAGCAGUACACUACCAAC
GUUGGUAGUGUACUGCUGC


 


857
858
490-508
AGCAGUACACUACCAACAG
CUGUUGGUAGUGUACUGCU


 


859
860
491-509
GCAGUACACUACCAACAGA
UCUGUUGGUAGUGUACUGC


 


861
862
492-510
CAGUACACUACCAACAGAU
AUCUGUUGGUAGUGUACUG


 


863
864
493-511
AGUACACUACCAACAGAUC
GAUCUGUUGGUAGUGUACU


 


865
866
494-512
GUACACUACCAACAGAUCA
UGAUCUGUUGGUAGUGUAC


 


867
868
495-513
UACACUACCAACAGAUCAA
UUGAUCUGUUGGUAGUGUA


 


869
870
496-514
ACACUACCAACAGAUCAAA
UUUGAUCUGUUGGUAGUGU


 


871
872
497-515
CACUACCAACAGAUCAAAG
CUUUGAUCUGUUGGUAGUG


 


873
874
498-516
ACUACCAACAGAUCAAAGA
UCUUUGAUCUGUUGGUAGU


 


875
876
499-517
CUACCAACAGAUCAAAGAA
UUCUUUGAUCUGUUGGUAG


 


877
878
500-518
UACCAACAGAUCAAAGAAA
UUUCUUUGAUCUGUUGGUA


 


879
880
501-519
ACCAACAGAUCAAAGAAAC
GUUUCUUUGAUCUGUUGGU


 


881
882
502-520
CCAACAGAUCAAAGAAACC
GGUUUCUUUGAUCUGUUGG


 


883
884
523-541
UCCGGCCAGUGAGAAAGAC
GUCUUUCUCACUGGCCGGA


 


885
886
524-542
CCGGCCAGUGAGAAAGACA
UGUCUUUCUCACUGGCCGG


 


887
888
525-543
CGGCCAGUGAGAAAGACAA
UUGUCUUUCUCACUGGCCG


 


889
890
526-544
GGCCAGUGAGAAAGACAAA
UUUGUCUUUCUCACUGGCC


 


891
892
527-545
GCCAGUGAGAAAGACAAAA
UUUUGUCUUUCUCACUGGC


 


893
894
528-546
CCAGUGAGAAAGACAAAAC
GUUUUGUCUUUCUCACUGG


 


895
896
529-547
CAGUGAGAAAGACAAAACU
AGUUUUGUCUUUCUCACUG


 


897
898
530-548
AGUGAGAAAGACAAAACUG
CAGUUUUGUCUUUCUCACU


 


899
900
531-549
GUGAGAAAGACAAAACUGC
GCAGUUUUGUCUUUCUCAC


 


901
902
570-588
CUCCUGAUGGAUCCCAGCA
UGCUGGGAUCCAUCAGGAG


 


903
904
571-589
UCCUGAUGGAUCCCAGCAG
CUGCUGGGAUCCAUCAGGA


 


905
906
572-590
CCUGAUGGAUCCCAGCAGA
UCUGCUGGGAUCCAUCAGG


 


907
908
573-591
CUGAUGGAUCCCAGCAGAG
CUCUGCUGGGAUCCAUCAG


 


909
910
574-592
UGAUGGAUCCCAGCAGAGU
ACUCUGCUGGGAUCCAUCA


 


911
912
575-593
GAUGGAUCCCAGCAGAGUC
GACUCUGCUGGGAUCCAUC


 


913
914
576-594
AUGGAUCCCAGCAGAGUCC
GGACUCUGCUGGGAUCCAU


 


915
916
577-595
UGGAUCCCAGCAGAGUCCA
UGGACUCUGCUGGGAUCCA


 


917
918
578-596
GGAUCCCAGCAGAGUCCAG
CUGGACUCUGCUGGGAUCC


 


919
920
579-597
GAUCCCAGCAGAGUCCAGA
UCUGGACUCUGCUGGGAUC


 


921
922
580-598
AUCCCAGCAGAGUCCAGAU
AUCUGGACUCUGCUGGGAU


 


923
924
581-599
UCCCAGCAGAGUCCAGAUG
CAUCUGGACUCUGCUGGGA


 


925
926
582-600
CCCAGCAGAGUCCAGAUGG
CCAUCUGGACUCUGCUGGG


 


927
928
583-601
CCAGCAGAGUCCAGAUGGC
GCCAUCUGGACUCUGCUGG


 


929
930
584-602
CAGCAGAGUCCAGAUGGCA
UGCCAUCUGGACUCUGCUG


 


931
932
585-603
AGCAGAGUCCAGAUGGCAC
GUGCCAUCUGGACUCUGCU


 


933
934
586-604
GCAGAGUCCAGAUGGCACA
UGUGCCAUCUGGACUCUGC


 


935
936
587-605
CAGAGUCCAGAUGGCACAC
GUGUGCCAUCUGGACUCUG


 


937
938
588-606
AGAGUCCAGAUGGCACACA
UGUGUGCCAUCUGGACUCU


 


939
940
589-607
GAGUCCAGAUGGCACACAG
CUGUGUGCCAUCUGGACUC


 


941
942
590-608
AGUCCAGAUGGCACACAGC
GCUGUGUGCCAUCUGGACU


 


943
944
591-609
GUCCAGAUGGCACACAGCU
AGCUGUGUGCCAUCUGGAC


 


945
946
592-610
UCCAGAUGGCACACAGCUU
AAGCUGUGUGCCAUCUGGA


 


947
948
593-611
CCAGAUGGCACACAGCUUC
GAAGCUGUGUGCCAUCUGG


 


949
950
594-612
CAGAUGGCACACAGCUUCC
GGAAGCUGUGUGCCAUCUG


 


951
952
595-613
AGAUGGCACACAGCUUCCG
CGGAAGCUGUGUGCCAUCU


 


953
954
596-614
GAUGGCACACAGCUUCCGU
ACGGAAGCUGUGUGCCAUC


 


955
956
597-615
AUGGCACACAGCUUCCGUC
GACGGAAGCUGUGUGCCAU


 


957
958
598-616
UGGCACACAGCUUCCGUCU
AGACGGAAGCUGUGUGCCA


 


959
960
599-617
GGCACACAGCUUCCGUCUG
CAGACGGAAGCUGUGUGCC


 


961
962
600-618
GCACACAGCUUCCGUCUGG
CCAGACGGAAGCUGUGUGC


 


963
964
601-619
CACACAGCUUCCGUCUGGA
UCCAGACGGAAGCUGUGUG


 


965
966
602-620
ACACAGCUUCCGUCUGGAC
GUCCAGACGGAAGCUGUGU


 


967
968
603-621
CACAGCUUCCGUCUGGACA
UGUCCAGACGGAAGCUGUG


 


969
970
604-622
ACAGCUUCCGUCUGGACAC
GUGUCCAGACGGAAGCUGU


 


971
972
605-623
CAGCUUCCGUCUGGACACC
GGUGUCCAGACGGAAGCUG


 


973
974
606-624
AGCUUCCGUCUGGACACCC
GGGUGUCCAGACGGAAGCU


 


975
976
607-625
GCUUCCGUCUGGACACCCC
GGGGUGUCCAGACGGAAGC


 


977
978
608-626
CUUCCGUCUGGACACCCCU
AGGGGUGUCCAGACGGAAG


 


979
980
609-627
UUCCGUCUGGACACCCCUU
AAGGGGUGUCCAGACGGAA


 


981
982
610-628
UCCGUCUGGACACCCCUUG
CAAGGGGUGUCCAGACGGA


 


983
984
611-629
CCGUCUGGACACCCCUUGC
GCAAGGGGUGUCCAGACGG


 


985
986
612-630
CGUCUGGACACCCCUUGCC
GGCAAGGGGUGUCCAGACG


 


987
988
613-631
GUCUGGACACCCCUUGCCU
AGGCAAGGGGUGUCCAGAC


 


989
990
614-632
UCUGGACACCCCUUGCCUG
CAGGCAAGGGGUGUCCAGA


 


991
992
615-633
CUGGACACCCCUUGCCUGC
GCAGGCAAGGGGUGUCCAG


 


993
994
616-634
UGGACACCCCUUGCCUGCC
GGCAGGCAAGGGGUGUCCA


 


995
996
617-635
GGACACCCCUUGCCUGCCA
UGGCAGGCAAGGGGUGUCC


 


997
998
618-636
GACACCCCUUGCCUGCCAC
GUGGCAGGCAAGGGGUGUC


 


999
1000
619-637
ACACCCCUUGCCUGCCACA
UGUGGCAGGCAAGGGGUGU


 


1001
1002
620-638
CACCCCUUGCCUGCCACAA
UUGUGGCAGGCAAGGGGUG


 


1003
1004
621-639
ACCCCUUGCCUGCCACAAG
CUUGUGGCAGGCAAGGGGU


 


1005
1006
622-640
CCCCUUGCCUGCCACAAGC
GCUUGUGGCAGGCAAGGGG


 


1007
1008
623-641
CCCUUGCCUGCCACAAGCC
GGCUUGUGGCAGGCAAGGG


 


1009
1010
624-642
CCUUGCCUGCCACAAGCCA
UGGCUUGUGGCAGGCAAGG


 


1011
1012
625-643
CUUGCCUGCCACAAGCCAG
CUGGCUUGUGGCAGGCAAG


 


1013
1014
626-644
UUGCCUGCCACAAGCCAGG
CCUGGCUUGUGGCAGGCAA


 


1015
1016
627-645
UGCCUGCCACAAGCCAGGG
CCCUGGCUUGUGGCAGGCA


 


1017
1018
628-646
GCCUGCCACAAGCCAGGGC
GCCCUGGCUUGUGGCAGGC


 


1019
1020
629-647
CCUGCCACAAGCCAGGGCA
UGCCCUGGCUUGUGGCAGG


 


1021
1022
630-648
CUGCCACAAGCCAGGGCAC
GUGCCCUGGCUUGUGGCAG


 


1023
1024
631-649
UGCCACAAGCCAGGGCACU
AGUGCCCUGGCUUGUGGCA


 


1025
1026
632-650
GCCACAAGCCAGGGCACUG
CAGUGCCCUGGCUUGUGGC


 


1027
1028
633-651
CCACAAGCCAGGGCACUGC
GCAGUGCCCUGGCUUGUGG


 


1029
1030
634-652
CACAAGCCAGGGCACUGCA
UGCAGUGCCCUGGCUUGUG


 


1031
1032
635-653
ACAAGCCAGGGCACUGCAA
UUGCAGUGCCCUGGCUUGU


 


1033
1034
636-654
CAAGCCAGGGCACUGCAAG
CUUGCAGUGCCCUGGCUUG


 


1035
1036
637-655
AAGCCAGGGCACUGCAAGC
GCUUGCAGUGCCCUGGCUU


 


1037
1038
638-656
AGCCAGGGCACUGCAAGCA
UGCUUGCAGUGCCCUGGCU


 


1039
1040
639-657
GCCAGGGCACUGCAAGCAA
UUGCUUGCAGUGCCCUGGC


 


1041
1042
640-658
CCAGGGCACUGCAAGCAAA
UUUGCUUGCAGUGCCCUGG


 


1043
1044
641-659
CAGGGCACUGCAAGCAAAU
AUUUGCUUGCAGUGCCCUG


 


1045
1046
642-660
AGGGCACUGCAAGCAAAUG
CAUUUGCUUGCAGUGCCCU


 


1047
1048
643-661
GGGCACUGCAAGCAAAUGC
GCAUUUGCUUGCAGUGCCC


 


1049
1050
644-662
GGCACUGCAAGCAAAUGCC
GGCAUUUGCUUGCAGUGCC


 


1051
1052
645-663
GCACUGCAAGCAAAUGCCC
GGGCAUUUGCUUGCAGUGC


 


1053
1054
647-665
ACUGCAAGCAAAUGCCCUU
AAGGGCAUUUGCUUGCAGU


 


1055
1056
648-666
CUGCAAGCAAAUGCCCUUU
AAAGGGCAUUUGCUUGCAG


 


1057
1058
649-667
UGCAAGCAAAUGCCCUUUC
GAAAGGGCAUUUGCUUGCA


 


1059
1060
650-668
GCAAGCAAAUGCCCUUUCC
GGAAAGGGCAUUUGCUUGC


 


1061
1062
651-669
CAAGCAAAUGCCCUUUCCU
AGGAAAGGGCAUUUGCUUG


 


1063
1064
652-670
AAGCAAAUGCCCUUUCCUG
CAGGAAAGGGCAUUUGCUU


 


1065
1066
653-671
AGCAAAUGCCCUUUCCUGG
CCAGGAAAGGGCAUUUGCU


 


1067
1068
654-672
GCAAAUGCCCUUUCCUGGC
GCCAGGAAAGGGCAUUUGC


 


1069
1070
655-673
CAAAUGCCCUUUCCUGGCA
UGCCAGGAAAGGGCAUUUG


 


1071
1072
656-674
AAAUGCCCUUUCCUGGCAG
CUGCCAGGAAAGGGCAUUU


 


1073
1074
657-675
AAUGCCCUUUCCUGGCAGC
GCUGCCAGGAAAGGGCAUU


 


1075
1076
658-676
AUGCCCUUUCCUGGCAGCA
UGCUGCCAGGAAAGGGCAU


 


1077
1078
659-677
UGCCCUUUCCUGGCAGCAC
GUGCUGCCAGGAAAGGGCA


 


1079
1080
660-678
GCCCUUUCCUGGCAGCACA
UGUGCUGCCAGGAAAGGGC


 


1081
1082
661-679
CCCUUUCCUGGCAGCACAG
CUGUGCUGCCAGGAAAGGG


 


1083
1084
662-680
CCUUUCCUGGCAGCACAGA
UCUGUGCUGCCAGGAAAGG


 


1085
1086
663-681
CUUUCCUGGCAGCACAGAU
AUCUGUGCUGCCAGGAAAG


 


1087
1088
664-682
UUUCCUGGCAGCACAGAUG
CAUCUGUGCUGCCAGGAAA


 


1089
1090
665-683
UUCCUGGCAGCACAGAUGA
UCAUCUGUGCUGCCAGGAA


 


1091
1092
666-684
UCCUGGCAGCACAGAUGAA
UUCAUCUGUGCUGCCAGGA


 


1093
1094
667-685
CCUGGCAGCACAGAUGAAU
AUUCAUCUGUGCUGCCAGG


 


1095
1096
668-686
CUGGCAGCACAGAUGAAUC
GAUUCAUCUGUGCUGCCAG


 


1097
1098
670-688
GGCAGCACAGAUGAAUCAG
CUGAUUCAUCUGUGCUGCC


 


1099
1100
672-690
CAGCACAGAUGAAUCAGAG
CUCUGAUUCAUCUGUGCUG


 


1101
1102
692-710
GGCAGCAGUGUCUUCUGCA
UGCAGAAGACACUGCUGCC


 


1103
1104
693-711
GCAGCAGUGUCUUCUGCAA
UUGCAGAAGACACUGCUGC


 


1105
1106
694-712
CAGCAGUGUCUUCUGCAAA
UUUGCAGAAGACACUGCUG


 


1107
1108
695-713
AGCAGUGUCUUCUGCAAAG
CUUUGCAGAAGACACUGCU


 


1109
1110
696-714
GCAGUGUCUUCUGCAAAGC
GCUUUGCAGAAGACACUGC


 


1111
1112
697-715
CAGUGUCUUCUGCAAAGCC
GGCUUUGCAGAAGACACUG


 


1113
1114
698-716
AGUGUCUUCUGCAAAGCCA
UGGCUUUGCAGAAGACACU


 


1115
1116
699-717
GUGUCUUCUGCAAAGCCAG
CUGGCUUUGCAGAAGACAC


 


1117
1118
700-718
UGUCUUCUGCAAAGCCAGU
ACUGGCUUUGCAGAAGACA


 


1119
1120
701-719
GUCUUCUGCAAAGCCAGUC
GACUGGCUUUGCAGAAGAC


 


1121
1122
702-720
UCUUCUGCAAAGCCAGUCU
AGACUGGCUUUGCAGAAGA


 


1123
1124
703-721
CUUCUGCAAAGCCAGUCUU
AAGACUGGCUUUGCAGAAG


 


1125
1126
704-722
UUCUGCAAAGCCAGUCUUG
CAAGACUGGCUUUGCAGAA


 


1127
1128
705-723
UCUGCAAAGCCAGUCUUGA
UCAAGACUGGCUUUGCAGA


 


1129
1130
706-724
CUGCAAAGCCAGUCUUGAG
CUCAAGACUGGCUUUGCAG


 


1131
1132
707-725
UGCAAAGCCAGUCUUGAGC
GCUCAAGACUGGCUUUGCA


 


1133
1134
708-726
GCAAAGCCAGUCUUGAGCU
AGCUCAAGACUGGCUUUGC


 


1135
1136
709-727
CAAAGCCAGUCUUGAGCUU
AAGCUCAAGACUGGCUUUG


 


1137
1138
710-728
AAAGCCAGUCUUGAGCUUC
GAAGCUCAAGACUGGCUUU


 


1139
1140
711-729
AAGCCAGUCUUGAGCUUCA
UGAAGCUCAAGACUGGCUU


 


1141
1142
712-730
AGCCAGUCUUGAGCUUCAG
CUGAAGCUCAAGACUGGCU


 


1143
1144
713-731
GCCAGUCUUGAGCUUCAGG
CCUGAAGCUCAAGACUGGC


 


1145
1146
714-732
CCAGUCUUGAGCUUCAGGA
UCCUGAAGCUCAAGACUGG


 


1147
1148
715-733
CAGUCUUGAGCUUCAGGAG
CUCCUGAAGCUCAAGACUG


 


1149
1150
716-734
AGUCUUGAGCUUCAGGAGG
CCUCCUGAAGCUCAAGACU


 


1151
1152
717-735
GUCUUGAGCUUCAGGAGGA
UCCUCCUGAAGCUCAAGAC


 


1153
1154
718-736
UCUUGAGCUUCAGGAGGAU
AUCCUCCUGAAGCUCAAGA


 


1155
1156
719-737
CUUGAGCUUCAGGAGGAUG
CAUCCUCCUGAAGCUCAAG


 


1157
1158
720-738
UUGAGCUUCAGGAGGAUGU
ACAUCCUCCUGAAGCUCAA


 


1159
1160
721-739
UGAGCUUCAGGAGGAUGUG
CACAUCCUCCUGAAGCUCA


 


1161
1162
722-740
GAGCUUCAGGAGGAUGUGC
GCACAUCCUCCUGAAGCUC


 


1163
1164
723-741
AGCUUCAGGAGGAUGUGCA
UGCACAUCCUCCUGAAGCU


 


1165
1166
724-742
GCUUCAGGAGGAUGUGCAG
CUGCACAUCCUCCUGAAGC


 


1167
1168
725-743
CUUCAGGAGGAUGUGCAGG
CCUGCACAUCCUCCUGAAG


 


1169
1170
726-744
UUCAGGAGGAUGUGCAGGA
UCCUGCACAUCCUCCUGAA


 


1171
1172
727-745
UCAGGAGGAUGUGCAGGAA
UUCCUGCACAUCCUCCUGA


 


1173
1174
728-746
CAGGAGGAUGUGCAGGAAA
UUUCCUGCACAUCCUCCUG


 


1175
1176
729-747
AGGAGGAUGUGCAGGAAAU
AUUUCCUGCACAUCCUCCU


 


1177
1178
730-748
GGAGGAUGUGCAGGAAAUG
CAUUUCCUGCACAUCCUCC


 


1179
1180
731-749
GAGGAUGUGCAGGAAAUGA
UCAUUUCCUGCACAUCCUC


 


1181
1182
732-750
AGGAUGUGCAGGAAAUGAA
UUCAUUUCCUGCACAUCCU


 


1183
1184
733-751
GGAUGUGCAGGAAAUGAAU
AUUCAUUUCCUGCACAUCC


 


1185
1186
734-752
GAUGUGCAGGAAAUGAAUG
CAUUCAUUUCCUGCACAUC


 


1187
1188
735-753
AUGUGCAGGAAAUGAAUGC
GCAUUCAUUUCCUGCACAU


 


1189
1190
755-773
GUGAGGAAAGAGGUUGCUG
CAGCAACCUCUUUCCUCAC


 


1191
1192
756-774
UGAGGAAAGAGGUUGCUGA
UCAGCAACCUCUUUCCUCA


 


1193
1194
757-775
GAGGAAAGAGGUUGCUGAA
UUCAGCAACCUCUUUCCUC


 


1195
1196
758-776
AGGAAAGAGGUUGCUGAAA
UUUCAGCAACCUCUUUCCU


 


1197
1198
759-777
GGAAAGAGGUUGCUGAAAC
GUUUCAGCAACCUCUUUCC


 


1199
1200
760-778
GAAAGAGGUUGCUGAAACC
GGUUUCAGCAACCUCUUUC


 


1201
1202
761-779
AAAGAGGUUGCUGAAACCU
AGGUUUCAGCAACCUCUUU


 


1203
1204
762-780
AAGAGGUUGCUGAAACCUC
GAGGUUUCAGCAACCUCUU


 


1205
1206
763-781
AGAGGUUGCUGAAACCUCA
UGAGGUUUCAGCAACCUCU


 


1207
1208
764-782
GAGGUUGCUGAAACCUCAG
CUGAGGUUUCAGCAACCUC


 


1209
1210
765-783
AGGUUGCUGAAACCUCAGC
GCUGAGGUUUCAGCAACCU


 


1211
1212
766-784
GGUUGCUGAAACCUCAGCA
UGCUGAGGUUUCAGCAACC


 


1213
1214
787-805
CCCCAGUGUGGUUAGUGUG
CACACUAACCACACUGGGG


 


1215
1216
791-809
AGUGUGGUUAGUGUGAAAA
UUUUCACACUAACCACACU


 


1217
1218
792-810
GUGUGGUUAGUGUGAAAAC
GUUUUCACACUAACCACAC


 


1219
1220
812-830
GAUGGAGGGGAUCCCAGUG
CACUGGGAUCCCCUCCAUC


 


1221
1222
813-831
AUGGAGGGGAUCCCAGUGG
CCACUGGGAUCCCCUCCAU


 


1223
1224
833-851
CUGCUGAAGAACUUCCAGG
CCUGGAAGUUCUUCAGCAG


 


1225
1226
834-852
UGCUGAAGAACUUCCAGGA
UCCUGGAAGUUCUUCAGCA


 


1227
1228
835-853
GCUGAAGAACUUCCAGGAC
GUCCUGGAAGUUCUUCAGC


 


1229
1230
836-854
CUGAAGAACUUCCAGGACA
UGUCCUGGAAGUUCUUCAG


 


1231
1232
837-855
UGAAGAACUUCCAGGACAU
AUGUCCUGGAAGUUCUUCA


 


1233
1234
838-856
GAAGAACUUCCAGGACAUC
GAUGUCCUGGAAGUUCUUC


 


1235
1236
839-857
AAGAACUUCCAGGACAUCA
UGAUGUCCUGGAAGUUCUU


 


1237
1238
840-858
AGAACUUCCAGGACAUCAU
AUGAUGUCCUGGAAGUUCU


 


1239
1240
841-859
GAACUUCCAGGACAUCAUG
CAUGAUGUCCUGGAAGUUC


 


1241
1242
842-860
AACUUCCAGGACAUCAUGC
GCAUGAUGUCCUGGAAGUU


 


1243
1244
843-861
ACUUCCAGGACAUCAUGCA
UGCAUGAUGUCCUGGAAGU


 


1245
1246
844-862
CUUCCAGGACAUCAUGCAA
UUGCAUGAUGUCCUGGAAG


 


1247
1248
845-863
UUCCAGGACAUCAUGCAAA
UUUGCAUGAUGUCCUGGAA


 


1249
1250
846-864
UCCAGGACAUCAUGCAAAA
UUUUGCAUGAUGUCCUGGA


 


1251
1252
847-865
CCAGGACAUCAUGCAAAAG
CUUUUGCAUGAUGUCCUGG


 


1253
1254
848-866
CAGGACAUCAUGCAAAAGC
GCUUUUGCAUGAUGUCCUG


 


1255
1256
849-867
AGGACAUCAUGCAAAAGCA
UGCUUUUGCAUGAUGUCCU


 


1257
1258
850-868
GGACAUCAUGCAAAAGCAA
UUGCUUUUGCAUGAUGUCC


 


1259
1260
851-869
GACAUCAUGCAAAAGCAAA
UUUGCUUUUGCAUGAUGUC


 


1261
1262
852-870
ACAUCAUGCAAAAGCAAAG
CUUUGCUUUUGCAUGAUGU


 


1263
1264
854-872
AUCAUGCAAAAGCAAAGAC
GUCUUUGCUUUUGCAUGAU


 


1265
1266
855-873
UCAUGCAAAAGCAAAGACC
GGUCUUUGCUUUUGCAUGA


 


1267
1268
856-874
CAUGCAAAAGCAAAGACCA
UGGUCUUUGCUUUUGCAUG


 


1269
1270
857-875
AUGCAAAAGCAAAGACCAG
CUGGUCUUUGCUUUUGCAU


 


1271
1272
858-876
UGCAAAAGCAAAGACCAGA
UCUGGUCUUUGCUUUUGCA


 


1273
1274
859-877
GCAAAAGCAAAGACCAGAA
UUCUGGUCUUUGCUUUUGC


 


1275
1276
860-878
CAAAAGCAAAGACCAGAAA
UUUCUGGUCUUUGCUUUUG


 


1277
1278
861-879
AAAAGCAAAGACCAGAAAG
CUUUCUGGUCUUUGCUUUU


 


1279
1280
862-880
AAAGCAAAGACCAGAAAGA
UCUUUCUGGUCUUUGCUUU


 


1281
1282
863-881
AAGCAAAGACCAGAAAGAG
CUCUUUCUGGUCUUUGCUU


 


1283
1284
864-882
AGCAAAGACCAGAAAGAGU
ACUCUUUCUGGUCUUUGCU


 


1285
1286
865-883
GCAAAGACCAGAAAGAGUG
CACUCUUUCUGGUCUUUGC


 


1287
1288
867-885
AAAGACCAGAAAGAGUGUC
GACACUCUUUCUGGUCUUU


 


1289
1290
868-886
AAGACCAGAAAGAGUGUCU
AGACACUCUUUCUGGUCUU


 


1291
1292
869-887
AGACCAGAAAGAGUGUCUC
GAGACACUCUUUCUGGUCU


 


1293
1294
870-888
GACCAGAAAGAGUGUCUCA
UGAGACACUCUUUCUGGUC


 


1295
1296
871-889
ACCAGAAAGAGUGUCUCAU
AUGAGACACUCUUUCUGGU


 


1297
1298
872-890
CCAGAAAGAGUGUCUCAUC
GAUGAGACACUCUUUCUGG


 


1299
1300
875-893
GAAAGAGUGUCUCAUCUUC
GAAGAUGAGACACUCUUUC


 


1301
1302
878-896
AGAGUGUCUCAUCUUCUUC
GAAGAAGAUGAGACACUCU


 


1303
1304
879-897
GAGUGUCUCAUCUUCUUCA
UGAAGAAGAUGAGACACUC


 


1305
1306
880-898
AGUGUCUCAUCUUCUUCAA
UUGAAGAAGAUGAGACACU


 


1307
1308
881-899
GUGUCUCAUCUUCUUCAAG
CUUGAAGAAGAUGAGACAC


 


1309
1310
882-900
UGUCUCAUCUUCUUCAAGA
UCUUGAAGAAGAUGAGACA


 


1311
1312
883-901
GUCUCAUCUUCUUCAAGAU
AUCUUGAAGAAGAUGAGAC


 


1313
1314
884-902
UCUCAUCUUCUUCAAGAUA
UAUCUUGAAGAAGAUGAGA


 


1315
1316
886-904
UCAUCUUCUUCAAGAUAAC
GUUAUCUUGAAGAAGAUGA


 


1317
1318
887-905
CAUCUUCUUCAAGAUAACU
AGUUAUCUUGAAGAAGAUG


 


1319
1320
888-906
AUCUUCUUCAAGAUAACUU
AAGUUAUCUUGAAGAAGAU


 


1321
1322
889-907
UCUUCUUCAAGAUAACUUG
CAAGUUAUCUUGAAGAAGA


 


1323
1324
890-908
CUUCUUCAAGAUAACUUGC
GCAAGUUAUCUUGAAGAAG


 


1325
1326
891-909
UUCUUCAAGAUAACUUGCC
GGCAAGUUAUCUUGAAGAA


 


1327
1328
892-910
UCUUCAAGAUAACUUGCCA
UGGCAAGUUAUCUUGAAGA


 


1329
1330
893-911
CUUCAAGAUAACUUGCCAA
UUGGCAAGUUAUCUUGAAG


 


1331
1332
894-912
UUCAAGAUAACUUGCCAAA
UUUGGCAAGUUAUCUUGAA


 


1333
1334
895-913
UCAAGAUAACUUGCCAAAA
UUUUGGCAAGUUAUCUUGA


 


1335
1336
896-914
CAAGAUAACUUGCCAAAAU
AUUUUGGCAAGUUAUCUUG


 


1337
1338
897-915
AAGAUAACUUGCCAAAAUC
GAUUUUGGCAAGUUAUCUU


 


1339
1340
898-916
AGAUAACUUGCCAAAAUCU
AGAUUUUGGCAAGUUAUCU


 


1341
1342
899-917
GAUAACUUGCCAAAAUCUG
CAGAUUUUGGCAAGUUAUC


 


1343
1344
900-918
AUAACUUGCCAAAAUCUGU
ACAGAUUUUGGCAAGUUAU


 


1345
1346
901-919
UAACUUGCCAAAAUCUGUU
AACAGAUUUUGGCAAGUUA


 


1347
1348
902-920
AACUUGCCAAAAUCUGUUU
AAACAGAUUUUGGCAAGUU


 


1349
1350
903-921
ACUUGCCAAAAUCUGUUUC
GAAACAGAUUUUGGCAAGU


 


1351
1352
904-922
CUUGCCAAAAUCUGUUUCC
GGAAACAGAUUUUGGCAAG


 


1353
1354
905-923
UUGCCAAAAUCUGUUUCCA
UGGAAACAGAUUUUGGCAA


 


1355
1356
906-924
UGCCAAAAUCUGUUUCCAC
GUGGAAACAGAUUUUGGCA


 


1357
1358
907-925
GCCAAAAUCUGUUUCCACU
AGUGGAAACAGAUUUUGGC


 


1359
1360
908-926
CCAAAAUCUGUUUCCACUU
AAGUGGAAACAGAUUUUGG


 


1361
1362
909-927
CAAAAUCUGUUUCCACUUU
AAAGUGGAAACAGAUUUUG


 


1363
1364
910-928
AAAAUCUGUUUCCACUUUU
AAAAGUGGAAACAGAUUUU


 


1365
1366
911-929
AAAUCUGUUUCCACUUUUC
GAAAAGUGGAAACAGAUUU


 


1367
1368
912-930
AAUCUGUUUCCACUUUUCA
UGAAAAGUGGAAACAGAUU


 


1369
1370
913-931
AUCUGUUUCCACUUUUCAG
CUGAAAAGUGGAAACAGAU


 


1371
1372
916-934
UGUUUCCACUUUUCAGUAU
AUACUGAAAAGUGGAAACA


 


1373
1374
917-935
GUUUCCACUUUUCAGUAUG
CAUACUGAAAAGUGGAAAC


 


1375
1376
918-936
UUUCCACUUUUCAGUAUGA
UCAUACUGAAAAGUGGAAA


 


1377
1378
919-937
UUCCACUUUUCAGUAUGAU
AUCAUACUGAAAAGUGGAA


 


1379
1380
920-938
UCCACUUUUCAGUAUGAUC
GAUCAUACUGAAAAGUGGA


 


1381
1382
921-939
CCACUUUUCAGUAUGAUCG
CGAUCAUACUGAAAAGUGG


 


1383
1384
925-943
UUUUCAGUAUGAUCGUUUC
GAAACGAUCAUACUGAAAA


 


1385
1386
929-947
CAGUAUGAUCGUUUCUUUG
CAAAGAAACGAUCAUACUG


 


1387
1388
930-948
AGUAUGAUCGUUUCUUUGA
UCAAAGAAACGAUCAUACU


 


1389
1390
931-949
GUAUGAUCGUUUCUUUGAG
CUCAAAGAAACGAUCAUAC


 


1391
1392
933-951
AUGAUCGUUUCUUUGAGAA
UUCUCAAAGAAACGAUCAU


 


1393
1394
934-952
UGAUCGUUUCUUUGAGAAA
UUUCUCAAAGAAACGAUCA


 


1395
1396
936-954
AUCGUUUCUUUGAGAAAAA
UUUUUCUCAAAGAAACGAU


 


1397
1398
937-955
UCGUUUCUUUGAGAAAAAA
UUUUUUCUCAAAGAAACGA


 


1399
1400
938-956
CGUUUCUUUGAGAAAAAAA
UUUUUUUCUCAAAGAAACG


 


1401
1402
939-957
GUUUCUUUGAGAAAAAAAU
AUUUUUUUCUCAAAGAAAC


 


1403
1404
940-958
UUUCUUUGAGAAAAAAAUU
AAUUUUUUUCUCAAAGAAA


 


1405
1406
941-959
UUCUUUGAGAAAAAAAUUG
CAAUUUUUUUCUCAAAGAA


 


1407
1408
942-960
UCUUUGAGAAAAAAAUUGA
UCAAUUUUUUUCUCAAAGA


 


1409
1410
943-961
CUUUGAGAAAAAAAUUGAU
AUCAAUUUUUUUCUCAAAG


 


1411
1412
944-962
UUUGAGAAAAAAAUUGAUG
CAUCAAUUUUUUUCUCAAA


 


1413
1414
945-963
UUGAGAAAAAAAUUGAUGA
UCAUCAAUUUUUUUCUCAA


 


1415
1416
946-964
UGAGAAAAAAAUUGAUGAG
CUCAUCAAUUUUUUUCUCA


 


1417
1418
947-965
GAGAAAAAAAUUGAUGAGA
UCUCAUCAAUUUUUUUCUC


 


1419
1420
948-966
AGAAAAAAAUUGAUGAGAA
UUCUCAUCAAUUUUUUUCU


 


1421
1422
949-967
GAAAAAAAUUGAUGAGAAA
UUUCUCAUCAAUUUUUUUC


 


1423
1424
950-968
AAAAAAAUUGAUGAGAAAA
UUUUCUCAUCAAUUUUUUU


 


1425
1426
951-969
AAAAAAUUGAUGAGAAAAA
UUUUUCUCAUCAAUUUUUU


 


1427
1428
952-970
AAAAAUUGAUGAGAAAAAG
CUUUUUCUCAUCAAUUUUU


 


1429
1430
953-971
AAAAUUGAUGAGAAAAAGA
UCUUUUUCUCAUCAAUUUU


 


1431
1432
954-972
AAAUUGAUGAGAAAAAGAA
UUCUUUUUCUCAUCAAUUU


 


1433
1434
955-973
AAUUGAUGAGAAAAAGAAU
AUUCUUUUUCUCAUCAAUU


 


1435
1436
956-974
AUUGAUGAGAAAAAGAAUG
CAUUCUUUUUCUCAUCAAU


 


1437
1438
957-975
UUGAUGAGAAAAAGAAUGA
UCAUUCUUUUUCUCAUCAA


 


1439
1440
958-976
UGAUGAGAAAAAGAAUGAC
GUCAUUCUUUUUCUCAUCA


 


1441
1442
959-977
GAUGAGAAAAAGAAUGACC
GGUCAUUCUUUUUCUCAUC


 


1443
1444
960-978
AUGAGAAAAAGAAUGACCA
UGGUCAUUCUUUUUCUCAU


 


1445
1446
961-979
UGAGAAAAAGAAUGACCAC
GUGGUCAUUCUUUUUCUCA


 


1447
1448
962-980
GAGAAAAAGAAUGACCACA
UGUGGUCAUUCUUUUUCUC


 


1449
1450
963-981
AGAAAAAGAAUGACCACAC
GUGUGGUCAUUCUUUUUCU


 


1451
1452
964-982
GAAAAAGAAUGACCACACC
GGUGUGGUCAUUCUUUUUC


 


1453
1454
965-983
AAAAAGAAUGACCACACCU
AGGUGUGGUCAUUCUUUUU


 


1455
1456
966-984
AAAAGAAUGACCACACCUA
UAGGUGUGGUCAUUCUUUU


 


1457
1458
967-985
AAAGAAUGACCACACCUAU
AUAGGUGUGGUCAUUCUUU


 


1459
1460
968-986
AAGAAUGACCACACCUAUC
GAUAGGUGUGGUCAUUCUU


 


1461
1462
969-987
AGAAUGACCACACCUAUCG
CGAUAGGUGUGGUCAUUCU


 


1463
1464
970-988
GAAUGACCACACCUAUCGA
UCGAUAGGUGUGGUCAUUC


 


1465
1466
971-989
AAUGACCACACCUAUCGAG
CUCGAUAGGUGUGGUCAUU


 


1467
1468
972-990
AUGACCACACCUAUCGAGU
ACUCGAUAGGUGUGGUCAU


 


1469
1470
976-994
CCACACCUAUCGAGUUUUU
AAAAACUCGAUAGGUGUGG


 


1471
1472
977-995
CACACCUAUCGAGUUUUUA
UAAAAACUCGAUAGGUGUG


 


1473
1474
978-996
ACACCUAUCGAGUUUUUAA
UUAAAAACUCGAUAGGUGU


 


1475
1476
979-997
CACCUAUCGAGUUUUUAAA
UUUAAAAACUCGAUAGGUG


 


1477
1478
980-998
ACCUAUCGAGUUUUUAAAA
UUUUAAAAACUCGAUAGGU


 


1479
1480
981-999
CCUAUCGAGUUUUUAAAAC
GUUUUAAAAACUCGAUAGG


 


1481
1482
 982-1000
CUAUCGAGUUUUUAAAACU
AGUUUUAAAAACUCGAUAG


 


1483
1484
 983-1001
UAUCGAGUUUUUAAAACUG
CAGUUUUAAAAACUCGAUA


 


1485
1486
 984-1002
AUCGAGUUUUUAAAACUGU
ACAGUUUUAAAAACUCGAU


 


1487
1488
 985-1003
UCGAGUUUUUAAAACUGUG
CACAGUUUUAAAAACUCGA


 


1489
1490
 986-1004
CGAGUUUUUAAAACUGUGA
UCACAGUUUUAAAAACUCG


 


1491
1492
 987-1005
GAGUUUUUAAAACUGUGAA
UUCACAGUUUUAAAAACUC


 


1493
1494
 988-1006
AGUUUUUAAAACUGUGAAC
GUUCACAGUUUUAAAAACU


 


1495
1496
 989-1007
GUUUUUAAAACUGUGAACC
GGUUCACAGUUUUAAAAAC


 


1497
1498
 990-1008
UUUUUAAAACUGUGAACCG
CGGUUCACAGUUUUAAAAA


 


1499
1500
 991-1009
UUUUAAAACUGUGAACCGG
CCGGUUCACAGUUUUAAAA


 


1501
1502
 992-1010
UUUAAAACUGUGAACCGGC
GCCGGUUCACAGUUUUAAA


 


1503
1504
 993-1011
UUAAAACUGUGAACCGGCG
CGCCGGUUCACAGUUUUAA


 


1505
1506
 994-1012
UAAAACUGUGAACCGGCGA
UCGCCGGUUCACAGUUUUA


 


1507
1508
 995-1013
AAAACUGUGAACCGGCGAG
CUCGCCGGUUCACAGUUUU


 


1509
1510
 996-1014
AAACUGUGAACCGGCGAGC
GCUCGCCGGUUCACAGUUU


 


1511
1512
 997-1015
AACUGUGAACCGGCGAGCA
UGCUCGCCGGUUCACAGUU


 


1513
1514
 998-1016
ACUGUGAACCGGCGAGCAC
GUGCUCGCCGGUUCACAGU


 


1515
1516
 999-1017
CUGUGAACCGGCGAGCACA
UGUGCUCGCCGGUUCACAG


 


1517
1518
1000-1018
UGUGAACCGGCGAGCACAC
GUGUGCUCGCCGGUUCACA


 


1519
1520
1001-1019
GUGAACCGGCGAGCACACA
UGUGUGCUCGCCGGUUCAC


 


1521
1522
1002-1020
UGAACCGGCGAGCACACAU
AUGUGUGCUCGCCGGUUCA


 


1523
1524
1003-1021
GAACCGGCGAGCACACAUC
GAUGUGUGCUCGCCGGUUC


 


1525
1526
1004-1022
AACCGGCGAGCACACAUCU
AGAUGUGUGCUCGCCGGUU


 


1527
1528
1005-1023
ACCGGCGAGCACACAUCUU
AAGAUGUGUGCUCGCCGGU


 


1529
1530
1006-1024
CCGGCGAGCACACAUCUUC
GAAGAUGUGUGCUCGCCGG


 


1531
1532
1007-1025
CGGCGAGCACACAUCUUCC
GGAAGAUGUGUGCUCGCCG


 


1533
1534
1008-1026
GGCGAGCACACAUCUUCCC
GGGAAGAUGUGUGCUCGCC


 


1535
1536
1028-1046
AUGGCAGAUGACUAUUCAG
CUGAAUAGUCAUCUGCCAU


 


1537
1538
1030-1048
GGCAGAUGACUAUUCAGAC
GUCUGAAUAGUCAUCUGCC


 


1539
1540
1031-1049
GCAGAUGACUAUUCAGACU
AGUCUGAAUAGUCAUCUGC


 


1541
1542
1032-1050
CAGAUGACUAUUCAGACUC
GAGUCUGAAUAGUCAUCUG


 


1543
1544
1033-1051
AGAUGACUAUUCAGACUCC
GGAGUCUGAAUAGUCAUCU


 


1545
1546
1034-1052
GAUGACUAUUCAGACUCCC
GGGAGUCUGAAUAGUCAUC


 


1547
1548
1035-1053
AUGACUAUUCAGACUCCCU
AGGGAGUCUGAAUAGUCAU


 


1549
1550
1036-1054
UGACUAUUCAGACUCCCUC
GAGGGAGUCUGAAUAGUCA


 


1551
1552
1037-1055
GACUAUUCAGACUCCCUCA
UGAGGGAGUCUGAAUAGUC


 


1553
1554
1038-1056
ACUAUUCAGACUCCCUCAU
AUGAGGGAGUCUGAAUAGU


 


1555
1556
1039-1057
CUAUUCAGACUCCCUCAUC
GAUGAGGGAGUCUGAAUAG


 


1557
1558
1040-1058
UAUUCAGACUCCCUCAUCA
UGAUGAGGGAGUCUGAAUA


 


1559
1560
1041-1059
AUUCAGACUCCCUCAUCAC
GUGAUGAGGGAGUCUGAAU


 


1561
1562
1042-1060
UUCAGACUCCCUCAUCACC
GGUGAUGAGGGAGUCUGAA


 


1563
1564
1043-1061
UCAGACUCCCUCAUCACCA
UGGUGAUGAGGGAGUCUGA


 


1565
1566
1044-1062
CAGACUCCCUCAUCACCAA
UUGGUGAUGAGGGAGUCUG


 


1567
1568
1045-1063
AGACUCCCUCAUCACCAAA
UUUGGUGAUGAGGGAGUCU


 


1569
1570
1046-1064
GACUCCCUCAUCACCAAAA
UUUUGGUGAUGAGGGAGUC


 


1571
1572
1047-1065
ACUCCCUCAUCACCAAAAA
UUUUUGGUGAUGAGGGAGU


 


1573
1574
1048-1066
CUCCCUCAUCACCAAAAAG
CUUUUUGGUGAUGAGGGAG


 


1575
1576
1049-1067
UCCCUCAUCACCAAAAAGC
GCUUUUUGGUGAUGAGGGA


 


1577
1578
1050-1068
CCCUCAUCACCAAAAAGCA
UGCUUUUUGGUGAUGAGGG


 


1579
1580
1070-1088
GUGUCAGUCUGGUGCAGUA
UACUGCACCAGACUGACAC


 


1581
1582
1071-1089
UGUCAGUCUGGUGCAGUAA
UUACUGCACCAGACUGACA


 


1583
1584
1072-1090
GUCAGUCUGGUGCAGUAAU
AUUACUGCACCAGACUGAC


 


1585
1586
1073-1091
UCAGUCUGGUGCAGUAAUG
CAUUACUGCACCAGACUGA


 


1587
1588
1074-1092
CAGUCUGGUGCAGUAAUGA
UCAUUACUGCACCAGACUG


 


1589
1590
1075-1093
AGUCUGGUGCAGUAAUGAC
GUCAUUACUGCACCAGACU


 


1591
1592
1078-1096
CUGGUGCAGUAAUGACUAC
GUAGUCAUUACUGCACCAG


 


1593
1594
1079-1097
UGGUGCAGUAAUGACUACC
GGUAGUCAUUACUGCACCA


 


1595
1596
1081-1099
GUGCAGUAAUGACUACCUA
UAGGUAGUCAUUACUGCAC


 


1597
1598
1082-1100
UGCAGUAAUGACUACCUAG
CUAGGUAGUCAUUACUGCA


 


1599
1600
1083-1101
GCAGUAAUGACUACCUAGG
CCUAGGUAGUCAUUACUGC


 


1601
1602
1084-1102
CAGUAAUGACUACCUAGGA
UCCUAGGUAGUCAUUACUG


 


1603
1604
1085-1103
AGUAAUGACUACCUAGGAA
UUCCUAGGUAGUCAUUACU


 


1605
1606
1086-1104
GUAAUGACUACCUAGGAAU
AUUCCUAGGUAGUCAUUAC


 


1607
1608
1087-1105
UAAUGACUACCUAGGAAUG
CAUUCCUAGGUAGUCAUUA


 


1609
1610
1088-1106
AAUGACUACCUAGGAAUGA
UCAUUCCUAGGUAGUCAUU


 


1611
1612
1089-1107
AUGACUACCUAGGAAUGAG
CUCAUUCCUAGGUAGUCAU


 


1613
1614
1090-1108
UGACUACCUAGGAAUGAGU
ACUCAUUCCUAGGUAGUCA


 


1615
1616
1091-1109
GACUACCUAGGAAUGAGUC
GACUCAUUCCUAGGUAGUC


 


1617
1618
1092-1110
ACUACCUAGGAAUGAGUCG
CGACUCAUUCCUAGGUAGU


 


1619
1620
1093-1111
CUACCUAGGAAUGAGUCGC
GCGACUCAUUCCUAGGUAG


 


1621
1622
1094-1112
UACCUAGGAAUGAGUCGCC
GGCGACUCAUUCCUAGGUA


 


1623
1624
1095-1113
ACCUAGGAAUGAGUCGCCA
UGGCGACUCAUUCCUAGGU


 


1625
1626
1096-1114
CCUAGGAAUGAGUCGCCAC
GUGGCGACUCAUUCCUAGG


 


1627
1628
1097-1115
CUAGGAAUGAGUCGCCACC
GGUGGCGACUCAUUCCUAG


 


1629
1630
1098-1116
UAGGAAUGAGUCGCCACCC
GGGUGGCGACUCAUUCCUA


 


1631
1632
1099-1117
AGGAAUGAGUCGCCACCCA
UGGGUGGCGACUCAUUCCU


 


1633
1634
1100-1118
GGAAUGAGUCGCCACCCAC
GUGGGUGGCGACUCAUUCC


 


1635
1636
1101-1119
GAAUGAGUCGCCACCCACG
CGUGGGUGGCGACUCAUUC


 


1637
1638
1102-1120
AAUGAGUCGCCACCCACGG
CCGUGGGUGGCGACUCAUU


 


1639
1640
1103-1121
AUGAGUCGCCACCCACGGG
CCCGUGGGUGGCGACUCAU


 


1641
1642
1104-1122
UGAGUCGCCACCCACGGGU
ACCCGUGGGUGGCGACUCA


 


1643
1644
1105-1123
GAGUCGCCACCCACGGGUG
CACCCGUGGGUGGCGACUC


 


1645
1646
1106-1124
AGUCGCCACCCACGGGUGU
ACACCCGUGGGUGGCGACU


 


1647
1648
1107-1125
GUCGCCACCCACGGGUGUG
CACACCCGUGGGUGGCGAC


 


1649
1650
1108-1126
UCGCCACCCACGGGUGUGU
ACACACCCGUGGGUGGCGA


 


1651
1652
1109-1127
CGCCACCCACGGGUGUGUG
CACACACCCGUGGGUGGCG


 


1653
1654
1110-1128
GCCACCCACGGGUGUGUGG
CCACACACCCGUGGGUGGC


 


1655
1656
1111-1129
CCACCCACGGGUGUGUGGG
CCCACACACCCGUGGGUGG


 


1657
1658
1112-1130
CACCCACGGGUGUGUGGGG
CCCCACACACCCGUGGGUG


 


1659
1660
1113-1131
ACCCACGGGUGUGUGGGGC
GCCCCACACACCCGUGGGU


 


1661
1662
1114-1132
CCCACGGGUGUGUGGGGCA
UGCCCCACACACCCGUGGG


 


1663
1664
1115-1133
CCACGGGUGUGUGGGGCAG
CUGCCCCACACACCCGUGG


 


1665
1666
1116-1134
CACGGGUGUGUGGGGCAGU
ACUGCCCCACACACCCGUG


 


1667
1668
1117-1135
ACGGGUGUGUGGGGCAGUU
AACUGCCCCACACACCCGU


 


1669
1670
1118-1136
CGGGUGUGUGGGGCAGUUA
UAACUGCCCCACACACCCG


 


1671
1672
1119-1137
GGGUGUGUGGGGCAGUUAU
AUAACUGCCCCACACACCC


 


1673
1674
1120-1138
GGUGUGUGGGGCAGUUAUG
CAUAACUGCCCCACACACC


 


1675
1676
1121-1139
GUGUGUGGGGCAGUUAUGG
CCAUAACUGCCCCACACAC


 


1677
1678
1122-1140
UGUGUGGGGCAGUUAUGGA
UCCAUAACUGCCCCACACA


 


1679
1680
1123-1141
GUGUGGGGCAGUUAUGGAC
GUCCAUAACUGCCCCACAC


 


1681
1682
1125-1143
GUGGGGCAGUUAUGGACAC
GUGUCCAUAACUGCCCCAC


 


1683
1684
1126-1144
UGGGGCAGUUAUGGACACU
AGUGUCCAUAACUGCCCCA


 


1685
1686
1128-1146
GGGCAGUUAUGGACACUUU
AAAGUGUCCAUAACUGCCC


 


1687
1688
1129-1147
GGCAGUUAUGGACACUUUG
CAAAGUGUCCAUAACUGCC


 


1689
1690
1130-1148
GCAGUUAUGGACACUUUGA
UCAAAGUGUCCAUAACUGC


 


1691
1692
1131-1149
CAGUUAUGGACACUUUGAA
UUCAAAGUGUCCAUAACUG


 


1693
1694
1132-1150
AGUUAUGGACACUUUGAAA
UUUCAAAGUGUCCAUAACU


 


1695
1696
1133-1151
GUUAUGGACACUUUGAAAC
GUUUCAAAGUGUCCAUAAC


 


1697
1698
1134-1152
UUAUGGACACUUUGAAACA
UGUUUCAAAGUGUCCAUAA


 


1699
1700
1135-1153
UAUGGACACUUUGAAACAA
UUGUUUCAAAGUGUCCAUA


 


1701
1702
1136-1154
AUGGACACUUUGAAACAAC
GUUGUUUCAAAGUGUCCAU


 


1703
1704
1139-1157
GACACUUUGAAACAACAUG
CAUGUUGUUUCAAAGUGUC


 


1705
1706
1140-1158
ACACUUUGAAACAACAUGG
CCAUGUUGUUUCAAAGUGU


 


1707
1708
1141-1159
CACUUUGAAACAACAUGGU
ACCAUGUUGUUUCAAAGUG


 


1709
1710
1142-1160
ACUUUGAAACAACAUGGUG
CACCAUGUUGUUUCAAAGU


 


1711
1712
1143-1161
CUUUGAAACAACAUGGUGC
GCACCAUGUUGUUUCAAAG


 


1713
1714
1144-1162
UUUGAAACAACAUGGUGCU
AGCACCAUGUUGUUUCAAA


 


1715
1716
1145-1163
UUGAAACAACAUGGUGCUG
CAGCACCAUGUUGUUUCAA


 


1717
1718
1146-1164
UGAAACAACAUGGUGCUGG
CCAGCACCAUGUUGUUUCA


 


1719
1720
1147-1165
GAAACAACAUGGUGCUGGG
CCCAGCACCAUGUUGUUUC


 


1721
1722
1148-1166
AAACAACAUGGUGCUGGGG
CCCCAGCACCAUGUUGUUU


 


1723
1724
1149-1167
AACAACAUGGUGCUGGGGC
GCCCCAGCACCAUGUUGUU


 


1725
1726
1150-1168
ACAACAUGGUGCUGGGGCA
UGCCCCAGCACCAUGUUGU


 


1727
1728
1151-1169
CAACAUGGUGCUGGGGCAG
CUGCCCCAGCACCAUGUUG


 


1729
1730
1152-1170
AACAUGGUGCUGGGGCAGG
CCUGCCCCAGCACCAUGUU


 


1731
1732
1153-1171
ACAUGGUGCUGGGGCAGGU
ACCUGCCCCAGCACCAUGU


 


1733
1734
1154-1172
CAUGGUGCUGGGGCAGGUG
CACCUGCCCCAGCACCAUG


 


1735
1736
1155-1173
AUGGUGCUGGGGCAGGUGG
CCACCUGCCCCAGCACCAU


 


1737
1738
1156-1174
UGGUGCUGGGGCAGGUGGU
ACCACCUGCCCCAGCACCA


 


1739
1740
1157-1175
GGUGCUGGGGCAGGUGGUA
UACCACCUGCCCCAGCACC


 


1741
1742
1158-1176
GUGCUGGGGCAGGUGGUAC
GUACCACCUGCCCCAGCAC


 


1743
1744
1159-1177
UGCUGGGGCAGGUGGUACU
AGUACCACCUGCCCCAGCA


 


1745
1746
1160-1178
GCUGGGGCAGGUGGUACUA
UAGUACCACCUGCCCCAGC


 


1747
1748
1161-1179
CUGGGGCAGGUGGUACUAG
CUAGUACCACCUGCCCCAG


 


1749
1750
1162-1180
UGGGGCAGGUGGUACUAGA
UCUAGUACCACCUGCCCCA


 


1751
1752
1166-1184
GCAGGUGGUACUAGAAAUA
UAUUUCUAGUACCACCUGC


 


1753
1754
1167-1185
CAGGUGGUACUAGAAAUAU
AUAUUUCUAGUACCACCUG


 


1755
1756
1168-1186
AGGUGGUACUAGAAAUAUU
AAUAUUUCUAGUACCACCU


 


1757
1758
1169-1187
GGUGGUACUAGAAAUAUUU
AAAUAUUUCUAGUACCACC


 


1759
1760
1170-1188
GUGGUACUAGAAAUAUUUC
GAAAUAUUUCUAGUACCAC


 


1761
1762
1171-1189
UGGUACUAGAAAUAUUUCU
AGAAAUAUUUCUAGUACCA


 


1763
1764
1172-1190
GGUACUAGAAAUAUUUCUG
CAGAAAUAUUUCUAGUACC


 


1765
1766
1173-1191
GUACUAGAAAUAUUUCUGG
CCAGAAAUAUUUCUAGUAC


 


1767
1768
1174-1192
UACUAGAAAUAUUUCUGGA
UCCAGAAAUAUUUCUAGUA


 


1769
1770
1175-1193
ACUAGAAAUAUUUCUGGAA
UUCCAGAAAUAUUUCUAGU


 


1771
1772
1176-1194
CUAGAAAUAUUUCUGGAAC
GUUCCAGAAAUAUUUCUAG


 


1773
1774
1177-1195
UAGAAAUAUUUCUGGAACU
AGUUCCAGAAAUAUUUCUA


 


1775
1776
1178-1196
AGAAAUAUUUCUGGAACUA
UAGUUCCAGAAAUAUUUCU


 


1777
1778
1179-1197
GAAAUAUUUCUGGAACUAG
CUAGUUCCAGAAAUAUUUC


 


1779
1780
1180-1198
AAAUAUUUCUGGAACUAGU
ACUAGUUCCAGAAAUAUUU


 


1781
1782
1181-1199
AAUAUUUCUGGAACUAGUA
UACUAGUUCCAGAAAUAUU


 


1783
1784
1183-1201
UAUUUCUGGAACUAGUAAA
UUUACUAGUUCCAGAAAUA


 


1785
1786
1186-1204
UUCUGGAACUAGUAAAUUC
GAAUUUACUAGUUCCAGAA


 


1787
1788
1187-1205
UCUGGAACUAGUAAAUUCC
GGAAUUUACUAGUUCCAGA


 


1789
1790
1189-1207
UGGAACUAGUAAAUUCCAU
AUGGAAUUUACUAGUUCCA


 


1791
1792
1190-1208
GGAACUAGUAAAUUCCAUG
CAUGGAAUUUACUAGUUCC


 


1793
1794
1192-1210
AACUAGUAAAUUCCAUGUG
CACAUGGAAUUUACUAGUU


 


1795
1796
1193-1211
ACUAGUAAAUUCCAUGUGG
CCACAUGGAAUUUACUAGU


 


1797
1798
1194-1212
CUAGUAAAUUCCAUGUGGA
UCCACAUGGAAUUUACUAG


 


1799
1800
1195-1213
UAGUAAAUUCCAUGUGGAC
GUCCACAUGGAAUUUACUA


 


1801
1802
1196-1214
AGUAAAUUCCAUGUGGACU
AGUCCACAUGGAAUUUACU


 


1803
1804
1197-1215
GUAAAUUCCAUGUGGACUU
AAGUCCACAUGGAAUUUAC


 


1805
1806
1198-1216
UAAAUUCCAUGUGGACUUA
UAAGUCCACAUGGAAUUUA


 


1807
1808
1199-1217
AAAUUCCAUGUGGACUUAG
CUAAGUCCACAUGGAAUUU


 


1809
1810
1200-1218
AAUUCCAUGUGGACUUAGA
UCUAAGUCCACAUGGAAUU


 


1811
1812
1201-1219
AUUCCAUGUGGACUUAGAG
CUCUAAGUCCACAUGGAAU


 


1813
1814
1202-1220
UUCCAUGUGGACUUAGAGC
GCUCUAAGUCCACAUGGAA


 


1815
1816
1222-1240
GGAGCUGGCAGACCUCCAU
AUGGAGGUCUGCCAGCUCC


 


1817
1818
1223-1241
GAGCUGGCAGACCUCCAUG
CAUGGAGGUCUGCCAGCUC


 


1819
1820
1224-1242
AGCUGGCAGACCUCCAUGG
CCAUGGAGGUCUGCCAGCU


 


1821
1822
1225-1243
GCUGGCAGACCUCCAUGGG
CCCAUGGAGGUCUGCCAGC


 


1823
1824
1226-1244
CUGGCAGACCUCCAUGGGA
UCCCAUGGAGGUCUGCCAG


 


1825
1826
1227-1245
UGGCAGACCUCCAUGGGAA
UUCCCAUGGAGGUCUGCCA


 


1827
1828
1228-1246
GGCAGACCUCCAUGGGAAA
UUUCCCAUGGAGGUCUGCC


 


1829
1830
1229-1247
GCAGACCUCCAUGGGAAAG
CUUUCCCAUGGAGGUCUGC


 


1831
1832
1230-1248
CAGACCUCCAUGGGAAAGA
UCUUUCCCAUGGAGGUCUG


 


1833
1834
1231-1249
AGACCUCCAUGGGAAAGAU
AUCUUUCCCAUGGAGGUCU


 


1835
1836
1232-1250
GACCUCCAUGGGAAAGAUG
CAUCUUUCCCAUGGAGGUC


 


1837
1838
1233-1251
ACCUCCAUGGGAAAGAUGC
GCAUCUUUCCCAUGGAGGU


 


1839
1840
1254-1272
CACUCUUGUUUUCCUCGUG
CACGAGGAAAACAAGAGUG


 


1841
1842
1255-1273
ACUCUUGUUUUCCUCGUGC
GCACGAGGAAAACAAGAGU


 


1843
1844
1256-1274
CUCUUGUUUUCCUCGUGCU
AGCACGAGGAAAACAAGAG


 


1845
1846
1257-1275
UCUUGUUUUCCUCGUGCUU
AAGCACGAGGAAAACAAGA


 


1847
1848
1259-1277
UUGUUUUCCUCGUGCUUUG
CAAAGCACGAGGAAAACAA


 


1849
1850
1260-1278
UGUUUUCCUCGUGCUUUGU
ACAAAGCACGAGGAAAACA


 


1851
1852
1261-1279
GUUUUCCUCGUGCUUUGUG
CACAAAGCACGAGGAAAAC


 


1853
1854
1262-1280
UUUUCCUCGUGCUUUGUGG
CCACAAAGCACGAGGAAAA


 


1855
1856
1263-1281
UUUCCUCGUGCUUUGUGGC
GCCACAAAGCACGAGGAAA


 


1857
1858
1264-1282
UUCCUCGUGCUUUGUGGCC
GGCCACAAAGCACGAGGAA


 


1859
1860
1265-1283
UCCUCGUGCUUUGUGGCCA
UGGCCACAAAGCACGAGGA


 


1861
1862
1266-1284
CCUCGUGCUUUGUGGCCAA
UUGGCCACAAAGCACGAGG


 


1863
1864
1267-1285
CUCGUGCUUUGUGGCCAAU
AUUGGCCACAAAGCACGAG


 


1865
1866
1268-1286
UCGUGCUUUGUGGCCAAUG
CAUUGGCCACAAAGCACGA


 


1867
1868
1269-1287
CGUGCUUUGUGGCCAAUGA
UCAUUGGCCACAAAGCACG


 


1869
1870
1270-1288
GUGCUUUGUGGCCAAUGAC
GUCAUUGGCCACAAAGCAC


 


1871
1872
1271-1289
UGCUUUGUGGCCAAUGACU
AGUCAUUGGCCACAAAGCA


 


1873
1874
1272-1290
GCUUUGUGGCCAAUGACUC
GAGUCAUUGGCCACAAAGC


 


1875
1876
1273-1291
CUUUGUGGCCAAUGACUCA
UGAGUCAUUGGCCACAAAG


 


1877
1878
1274-1292
UUUGUGGCCAAUGACUCAA
UUGAGUCAUUGGCCACAAA


 


1879
1880
1275-1293
UUGUGGCCAAUGACUCAAC
GUUGAGUCAUUGGCCACAA


 


1881
1882
1276-1294
UGUGGCCAAUGACUCAACC
GGUUGAGUCAUUGGCCACA


 


1883
1884
1277-1295
GUGGCCAAUGACUCAACCC
GGGUUGAGUCAUUGGCCAC


 


1885
1886
1278-1296
UGGCCAAUGACUCAACCCU
AGGGUUGAGUCAUUGGCCA


 


1887
1888
1279-1297
GGCCAAUGACUCAACCCUC
GAGGGUUGAGUCAUUGGCC


 


1889
1890
1280-1298
GCCAAUGACUCAACCCUCU
AGAGGGUUGAGUCAUUGGC


 


1891
1892
1281-1299
CCAAUGACUCAACCCUCUU
AAGAGGGUUGAGUCAUUGG


 


1893
1894
1282-1300
CAAUGACUCAACCCUCUUC
GAAGAGGGUUGAGUCAUUG


 


1895
1896
1283-1301
AAUGACUCAACCCUCUUCA
UGAAGAGGGUUGAGUCAUU


 


1897
1898
1284-1302
AUGACUCAACCCUCUUCAC
GUGAAGAGGGUUGAGUCAU


 


1899
1900
1285-1303
UGACUCAACCCUCUUCACC
GGUGAAGAGGGUUGAGUCA


 


1901
1902
1286-1304
GACUCAACCCUCUUCACCC
GGGUGAAGAGGGUUGAGUC


 


1903
1904
1287-1305
ACUCAACCCUCUUCACCCU
AGGGUGAAGAGGGUUGAGU


 


1905
1906
1288-1306
CUCAACCCUCUUCACCCUG
CAGGGUGAAGAGGGUUGAG


 


1907
1908
1289-1307
UCAACCCUCUUCACCCUGG
CCAGGGUGAAGAGGGUUGA


 


1909
1910
1290-1308
CAACCCUCUUCACCCUGGC
GCCAGGGUGAAGAGGGUUG


 


1911
1912
1291-1309
AACCCUCUUCACCCUGGCU
AGCCAGGGUGAAGAGGGUU


 


1913
1914
1292-1310
ACCCUCUUCACCCUGGCUA
UAGCCAGGGUGAAGAGGGU


 


1915
1916
1293-1311
CCCUCUUCACCCUGGCUAA
UUAGCCAGGGUGAAGAGGG


 


1917
1918
1294-1312
CCUCUUCACCCUGGCUAAG
CUUAGCCAGGGUGAAGAGG


 


1919
1920
1297-1315
CUUCACCCUGGCUAAGAUG
CAUCUUAGCCAGGGUGAAG


 


1921
1922
1298-1316
UUCACCCUGGCUAAGAUGA
UCAUCUUAGCCAGGGUGAA


 


1923
1924
1300-1318
CACCCUGGCUAAGAUGAUG
CAUCAUCUUAGCCAGGGUG


 


1925
1926
1301-1319
ACCCUGGCUAAGAUGAUGC
GCAUCAUCUUAGCCAGGGU


 


1927
1928
1302-1320
CCCUGGCUAAGAUGAUGCC
GGCAUCAUCUUAGCCAGGG


 


1929
1930
1303-1321
CCUGGCUAAGAUGAUGCCA
UGGCAUCAUCUUAGCCAGG


 


1931
1932
1304-1322
CUGGCUAAGAUGAUGCCAG
CUGGCAUCAUCUUAGCCAG


 


1933
1934
1305-1323
UGGCUAAGAUGAUGCCAGG
CCUGGCAUCAUCUUAGCCA


 


1935
1936
1306-1324
GGCUAAGAUGAUGCCAGGC
GCCUGGCAUCAUCUUAGCC


 


1937
1938
1307-1325
GCUAAGAUGAUGCCAGGCU
AGCCUGGCAUCAUCUUAGC


 


1939
1940
1308-1326
CUAAGAUGAUGCCAGGCUG
CAGCCUGGCAUCAUCUUAG


 


1941
1942
1309-1327
UAAGAUGAUGCCAGGCUGU
ACAGCCUGGCAUCAUCUUA


 


1943
1944
1310-1328
AAGAUGAUGCCAGGCUGUG
CACAGCCUGGCAUCAUCUU


 


1945
1946
1311-1329
AGAUGAUGCCAGGCUGUGA
UCACAGCCUGGCAUCAUCU


 


1947
1948
1312-1330
GAUGAUGCCAGGCUGUGAG
CUCACAGCCUGGCAUCAUC


 


1949
1950
1313-1331
AUGAUGCCAGGCUGUGAGA
UCUCACAGCCUGGCAUCAU


 


1951
1952
1314-1332
UGAUGCCAGGCUGUGAGAU
AUCUCACAGCCUGGCAUCA


 


1953
1954
1316-1334
AUGCCAGGCUGUGAGAUUU
AAAUCUCACAGCCUGGCAU


 


1955
1956
1317-1335
UGCCAGGCUGUGAGAUUUA
UAAAUCUCACAGCCUGGCA


 


1957
1958
1318-1336
GCCAGGCUGUGAGAUUUAC
GUAAAUCUCACAGCCUGGC


 


1959
1960
1319-1337
CCAGGCUGUGAGAUUUACU
AGUAAAUCUCACAGCCUGG


 


1961
1962
1320-1338
CAGGCUGUGAGAUUUACUC
GAGUAAAUCUCACAGCCUG


 


1963
1964
1321-1339
AGGCUGUGAGAUUUACUCU
AGAGUAAAUCUCACAGCCU


 


1965
1966
1322-1340
GGCUGUGAGAUUUACUCUG
CAGAGUAAAUCUCACAGCC


 


1967
1968
1323-1341
GCUGUGAGAUUUACUCUGA
UCAGAGUAAAUCUCACAGC


 


1969
1970
1326-1344
GUGAGAUUUACUCUGAUUC
GAAUCAGAGUAAAUCUCAC


 


1971
1972
1327-1345
UGAGAUUUACUCUGAUUCU
AGAAUCAGAGUAAAUCUCA


 


1973
1974
1328-1346
GAGAUUUACUCUGAUUCUG
CAGAAUCAGAGUAAAUCUC


 


1975
1976
1329-1347
AGAUUUACUCUGAUUCUGG
CCAGAAUCAGAGUAAAUCU


 


1977
1978
1330-1348
GAUUUACUCUGAUUCUGGG
CCCAGAAUCAGAGUAAAUC


 


1979
1980
1331-1349
AUUUACUCUGAUUCUGGGA
UCCCAGAAUCAGAGUAAAU


 


1981
1982
1332-1350
UUUACUCUGAUUCUGGGAA
UUCCCAGAAUCAGAGUAAA


 


1983
1984
1333-1351
UUACUCUGAUUCUGGGAAC
GUUCCCAGAAUCAGAGUAA


 


1985
1986
1334-1352
UACUCUGAUUCUGGGAACC
GGUUCCCAGAAUCAGAGUA


 


1987
1988
1335-1353
ACUCUGAUUCUGGGAACCA
UGGUUCCCAGAAUCAGAGU


 


1989
1990
1336-1354
CUCUGAUUCUGGGAACCAU
AUGGUUCCCAGAAUCAGAG


 


1991
1992
1337-1355
UCUGAUUCUGGGAACCAUG
CAUGGUUCCCAGAAUCAGA


 


1993
1994
1338-1356
CUGAUUCUGGGAACCAUGC
GCAUGGUUCCCAGAAUCAG


 


1995
1996
1339-1357
UGAUUCUGGGAACCAUGCC
GGCAUGGUUCCCAGAAUCA


 


1997
1998
1340-1358
GAUUCUGGGAACCAUGCCU
AGGCAUGGUUCCCAGAAUC


 


1999
2000
1341-1359
AUUCUGGGAACCAUGCCUC
GAGGCAUGGUUCCCAGAAU


 


2001
2002
1342-1360
UUCUGGGAACCAUGCCUCC
GGAGGCAUGGUUCCCAGAA


 


2003
2004
1343-1361
UCUGGGAACCAUGCCUCCA
UGGAGGCAUGGUUCCCAGA


 


2005
2006
1344-1362
CUGGGAACCAUGCCUCCAU
AUGGAGGCAUGGUUCCCAG


 


2007
2008
1345-1363
UGGGAACCAUGCCUCCAUG
CAUGGAGGCAUGGUUCCCA


 


2009
2010
1346-1364
GGGAACCAUGCCUCCAUGA
UCAUGGAGGCAUGGUUCCC


 


2011
2012
1348-1366
GAACCAUGCCUCCAUGAUC
GAUCAUGGAGGCAUGGUUC


 


2013
2014
1349-1367
AACCAUGCCUCCAUGAUCC
GGAUCAUGGAGGCAUGGUU


 


2015
2016
1350-1368
ACCAUGCCUCCAUGAUCCA
UGGAUCAUGGAGGCAUGGU


 


2017
2018
1351-1369
CCAUGCCUCCAUGAUCCAA
UUGGAUCAUGGAGGCAUGG


 


2019
2020
1352-1370
CAUGCCUCCAUGAUCCAAG
CUUGGAUCAUGGAGGCAUG


 


2021
2022
1353-1371
AUGCCUCCAUGAUCCAAGG
CCUUGGAUCAUGGAGGCAU


 


2023
2024
1354-1372
UGCCUCCAUGAUCCAAGGG
CCCUUGGAUCAUGGAGGCA


 


2025
2026
1358-1376
UCCAUGAUCCAAGGGAUUC
GAAUCCCUUGGAUCAUGGA


 


2027
2028
1359-1377
CCAUGAUCCAAGGGAUUCG
CGAAUCCCUUGGAUCAUGG


 


2029
2030
1360-1378
CAUGAUCCAAGGGAUUCGA
UCGAAUCCCUUGGAUCAUG


 


2031
2032
1361-1379
AUGAUCCAAGGGAUUCGAA
UUCGAAUCCCUUGGAUCAU


 


2033
2034
1362-1380
UGAUCCAAGGGAUUCGAAA
UUUCGAAUCCCUUGGAUCA


 


2035
2036
1363-1381
GAUCCAAGGGAUUCGAAAC
GUUUCGAAUCCCUUGGAUC


 


2037
2038
1365-1383
UCCAAGGGAUUCGAAACAG
CUGUUUCGAAUCCCUUGGA


 


2039
2040
1366-1384
CCAAGGGAUUCGAAACAGC
GCUGUUUCGAAUCCCUUGG


 


2041
2042
1367-1385
CAAGGGAUUCGAAACAGCC
GGCUGUUUCGAAUCCCUUG


 


2043
2044
1368-1386
AAGGGAUUCGAAACAGCCG
CGGCUGUUUCGAAUCCCUU


 


2045
2046
1369-1387
AGGGAUUCGAAACAGCCGA
UCGGCUGUUUCGAAUCCCU


 


2047
2048
1370-1388
GGGAUUCGAAACAGCCGAG
CUCGGCUGUUUCGAAUCCC


 


2049
2050
1371-1389
GGAUUCGAAACAGCCGAGU
ACUCGGCUGUUUCGAAUCC


 


2051
2052
1372-1390
GAUUCGAAACAGCCGAGUG
CACUCGGCUGUUUCGAAUC


 


2053
2054
1373-1391
AUUCGAAACAGCCGAGUGC
GCACUCGGCUGUUUCGAAU


 


2055
2056
1374-1392
UUCGAAACAGCCGAGUGCC
GGCACUCGGCUGUUUCGAA


 


2057
2058
1375-1393
UCGAAACAGCCGAGUGCCA
UGGCACUCGGCUGUUUCGA


 


2059
2060
1376-1394
CGAAACAGCCGAGUGCCAA
UUGGCACUCGGCUGUUUCG


 


2061
2062
1377-1395
GAAACAGCCGAGUGCCAAA
UUUGGCACUCGGCUGUUUC


 


2063
2064
1378-1396
AAACAGCCGAGUGCCAAAG
CUUUGGCACUCGGCUGUUU


 


2065
2066
1379-1397
AACAGCCGAGUGCCAAAGU
ACUUUGGCACUCGGCUGUU


 


2067
2068
1380-1398
ACAGCCGAGUGCCAAAGUA
UACUUUGGCACUCGGCUGU


 


2069
2070
1381-1399
CAGCCGAGUGCCAAAGUAC
GUACUUUGGCACUCGGCUG


 


2071
2072
1383-1401
GCCGAGUGCCAAAGUACAU
AUGUACUUUGGCACUCGGC


 


2073
2074
1384-1402
CCGAGUGCCAAAGUACAUC
GAUGUACUUUGGCACUCGG


 


2075
2076
1385-1403
CGAGUGCCAAAGUACAUCU
AGAUGUACUUUGGCACUCG


 


2077
2078
1386-1404
GAGUGCCAAAGUACAUCUU
AAGAUGUACUUUGGCACUC


 


2079
2080
1387-1405
AGUGCCAAAGUACAUCUUC
GAAGAUGUACUUUGGCACU


 


2081
2082
1388-1406
GUGCCAAAGUACAUCUUCC
GGAAGAUGUACUUUGGCAC


 


2083
2084
1389-1407
UGCCAAAGUACAUCUUCCG
CGGAAGAUGUACUUUGGCA


 


2085
2086
1390-1408
GCCAAAGUACAUCUUCCGC
GCGGAAGAUGUACUUUGGC


 


2087
2088
1391-1409
CCAAAGUACAUCUUCCGCC
GGCGGAAGAUGUACUUUGG


 


2089
2090
1392-1410
CAAAGUACAUCUUCCGCCA
UGGCGGAAGAUGUACUUUG


 


2091
2092
1393-1411
AAAGUACAUCUUCCGCCAC
GUGGCGGAAGAUGUACUUU


 


2093
2094
1394-1412
AAGUACAUCUUCCGCCACA
UGUGGCGGAAGAUGUACUU


 


2095
2096
1395-1413
AGUACAUCUUCCGCCACAA
UUGUGGCGGAAGAUGUACU


 


2097
2098
1396-1414
GUACAUCUUCCGCCACAAU
AUUGUGGCGGAAGAUGUAC


 


2099
2100
1397-1415
UACAUCUUCCGCCACAAUG
CAUUGUGGCGGAAGAUGUA


 


2101
2102
1398-1416
ACAUCUUCCGCCACAAUGA
UCAUUGUGGCGGAAGAUGU


 


2103
2104
1399-1417
CAUCUUCCGCCACAAUGAU
AUCAUUGUGGCGGAAGAUG


 


2105
2106
1400-1418
AUCUUCCGCCACAAUGAUG
CAUCAUUGUGGCGGAAGAU


 


2107
2108
1401-1419
UCUUCCGCCACAAUGAUGU
ACAUCAUUGUGGCGGAAGA


 


2109
2110
1402-1420
CUUCCGCCACAAUGAUGUC
GACAUCAUUGUGGCGGAAG


 


2111
2112
1403-1421
UUCCGCCACAAUGAUGUCA
UGACAUCAUUGUGGCGGAA


 


2113
2114
1404-1422
UCCGCCACAAUGAUGUCAG
CUGACAUCAUUGUGGCGGA


 


2115
2116
1405-1423
CCGCCACAAUGAUGUCAGC
GCUGACAUCAUUGUGGCGG


 


2117
2118
1406-1424
CGCCACAAUGAUGUCAGCC
GGCUGACAUCAUUGUGGCG


 


2119
2120
1407-1425
GCCACAAUGAUGUCAGCCA
UGGCUGACAUCAUUGUGGC


 


2121
2122
1427-1445
CUCAGAGAACUGCUGCAAA
UUUGCAGCAGUUCUCUGAG


 


2123
2124
1428-1446
UCAGAGAACUGCUGCAAAG
CUUUGCAGCAGUUCUCUGA


 


2125
2126
1429-1447
CAGAGAACUGCUGCAAAGA
UCUUUGCAGCAGUUCUCUG


 


2127
2128
1430-1448
AGAGAACUGCUGCAAAGAU
AUCUUUGCAGCAGUUCUCU


 


2129
2130
1431-1449
GAGAACUGCUGCAAAGAUC
GAUCUUUGCAGCAGUUCUC


 


2131
2132
1432-1450
AGAACUGCUGCAAAGAUCU
AGAUCUUUGCAGCAGUUCU


 


2133
2134
1433-1451
GAACUGCUGCAAAGAUCUG
CAGAUCUUUGCAGCAGUUC


 


2135
2136
1434-1452
AACUGCUGCAAAGAUCUGA
UCAGAUCUUUGCAGCAGUU


 


2137
2138
1435-1453
ACUGCUGCAAAGAUCUGAC
GUCAGAUCUUUGCAGCAGU


 


2139
2140
1436-1454
CUGCUGCAAAGAUCUGACC
GGUCAGAUCUUUGCAGCAG


 


2141
2142
1437-1455
UGCUGCAAAGAUCUGACCC
GGGUCAGAUCUUUGCAGCA


 


2143
2144
1457-1475
UCAGUCCCCAAGAUUGUGG
CCACAAUCUUGGGGACUGA


 


2145
2146
1458-1476
CAGUCCCCAAGAUUGUGGC
GCCACAAUCUUGGGGACUG


 


2147
2148
1459-1477
AGUCCCCAAGAUUGUGGCA
UGCCACAAUCUUGGGGACU


 


2149
2150
1461-1479
UCCCCAAGAUUGUGGCAUU
AAUGCCACAAUCUUGGGGA


 


2151
2152
1462-1480
CCCCAAGAUUGUGGCAUUU
AAAUGCCACAAUCUUGGGG


 


2153
2154
1463-1481
CCCAAGAUUGUGGCAUUUG
CAAAUGCCACAAUCUUGGG


 


2155
2156
1464-1482
CCAAGAUUGUGGCAUUUGA
UCAAAUGCCACAAUCUUGG


 


2157
2158
1465-1483
CAAGAUUGUGGCAUUUGAA
UUCAAAUGCCACAAUCUUG


 


2159
2160
1466-1484
AAGAUUGUGGCAUUUGAAA
UUUCAAAUGCCACAAUCUU


 


2161
2162
1467-1485
AGAUUGUGGCAUUUGAAAC
GUUUCAAAUGCCACAAUCU


 


2163
2164
1468-1486
GAUUGUGGCAUUUGAAACU
AGUUUCAAAUGCCACAAUC


 


2165
2166
1469-1487
AUUGUGGCAUUUGAAACUG
CAGUUUCAAAUGCCACAAU


 


2167
2168
1470-1488
UUGUGGCAUUUGAAACUGU
ACAGUUUCAAAUGCCACAA


 


2169
2170
1471-1489
UGUGGCAUUUGAAACUGUC
GACAGUUUCAAAUGCCACA


 


2171
2172
1472-1490
GUGGCAUUUGAAACUGUCC
GGACAGUUUCAAAUGCCAC


 


2173
2174
1473-1491
UGGCAUUUGAAACUGUCCA
UGGACAGUUUCAAAUGCCA


 


2175
2176
1474-1492
GGCAUUUGAAACUGUCCAU
AUGGACAGUUUCAAAUGCC


 


2177
2178
1475-1493
GCAUUUGAAACUGUCCAUU
AAUGGACAGUUUCAAAUGC


 


2179
2180
1476-1494
CAUUUGAAACUGUCCAUUC
GAAUGGACAGUUUCAAAUG


 


2181
2182
1477-1495
AUUUGAAACUGUCCAUUCA
UGAAUGGACAGUUUCAAAU


 


2183
2184
1479-1497
UUGAAACUGUCCAUUCAAU
AUUGAAUGGACAGUUUCAA


 


2185
2186
1480-1498
UGAAACUGUCCAUUCAAUG
CAUUGAAUGGACAGUUUCA


 


2187
2188
1481-1499
GAAACUGUCCAUUCAAUGG
CCAUUGAAUGGACAGUUUC


 


2189
2190
1482-1500
AAACUGUCCAUUCAAUGGA
UCCAUUGAAUGGACAGUUU


 


2191
2192
1483-1501
AACUGUCCAUUCAAUGGAU
AUCCAUUGAAUGGACAGUU


 


2193
2194
1484-1502
ACUGUCCAUUCAAUGGAUG
CAUCCAUUGAAUGGACAGU


 


2195
2196
1485-1503
CUGUCCAUUCAAUGGAUGG
CCAUCCAUUGAAUGGACAG


 


2197
2198
1486-1504
UGUCCAUUCAAUGGAUGGG
CCCAUCCAUUGAAUGGACA


 


2199
2200
1487-1505
GUCCAUUCAAUGGAUGGGG
CCCCAUCCAUUGAAUGGAC


 


2201
2202
1488-1506
UCCAUUCAAUGGAUGGGGC
GCCCCAUCCAUUGAAUGGA


 


2203
2204
1508-1526
GUGUGCCCACUGGAAGAGC
GCUCUUCCAGUGGGCACAC


 


2205
2206
1509-1527
UGUGCCCACUGGAAGAGCU
AGCUCUUCCAGUGGGCACA


 


2207
2208
1510-1528
GUGCCCACUGGAAGAGCUG
CAGCUCUUCCAGUGGGCAC


 


2209
2210
1511-1529
UGCCCACUGGAAGAGCUGU
ACAGCUCUUCCAGUGGGCA


 


2211
2212
1512-1530
GCCCACUGGAAGAGCUGUG
CACAGCUCUUCCAGUGGGC


 


2213
2214
1513-1531
CCCACUGGAAGAGCUGUGU
ACACAGCUCUUCCAGUGGG


 


2215
2216
1514-1532
CCACUGGAAGAGCUGUGUG
CACACAGCUCUUCCAGUGG


 


2217
2218
1515-1533
CACUGGAAGAGCUGUGUGA
UCACACAGCUCUUCCAGUG


 


2219
2220
1516-1534
ACUGGAAGAGCUGUGUGAU
AUCACACAGCUCUUCCAGU


 


2221
2222
1517-1535
CUGGAAGAGCUGUGUGAUG
CAUCACACAGCUCUUCCAG


 


2223
2224
1518-1536
UGGAAGAGCUGUGUGAUGU
ACAUCACACAGCUCUUCCA


 


2225
2226
1519-1537
GGAAGAGCUGUGUGAUGUG
CACAUCACACAGCUCUUCC


 


2227
2228
1520-1538
GAAGAGCUGUGUGAUGUGG
CCACAUCACACAGCUCUUC


 


2229
2230
1521-1539
AAGAGCUGUGUGAUGUGGC
GCCACAUCACACAGCUCUU


 


2231
2232
1522-1540
AGAGCUGUGUGAUGUGGCC
GGCCACAUCACACAGCUCU


 


2233
2234
1523-1541
GAGCUGUGUGAUGUGGCCC
GGGCCACAUCACACAGCUC


 


2235
2236
1524-1542
AGCUGUGUGAUGUGGCCCA
UGGGCCACAUCACACAGCU


 


2237
2238
1525-1543
GCUGUGUGAUGUGGCCCAU
AUGGGCCACAUCACACAGC


 


2239
2240
1526-1544
CUGUGUGAUGUGGCCCAUG
CAUGGGCCACAUCACACAG


 


2241
2242
1527-1545
UGUGUGAUGUGGCCCAUGA
UCAUGGGCCACAUCACACA


 


2243
2244
1528-1546
GUGUGAUGUGGCCCAUGAG
CUCAUGGGCCACAUCACAC


 


2245
2246
1529-1547
UGUGAUGUGGCCCAUGAGU
ACUCAUGGGCCACAUCACA


 


2247
2248
1532-1550
GAUGUGGCCCAUGAGUUUG
CAAACUCAUGGGCCACAUC


 


2249
2250
1533-1551
AUGUGGCCCAUGAGUUUGG
CCAAACUCAUGGGCCACAU


 


2251
2252
1534-1552
UGUGGCCCAUGAGUUUGGA
UCCAAACUCAUGGGCCACA


 


2253
2254
1535-1553
GUGGCCCAUGAGUUUGGAG
CUCCAAACUCAUGGGCCAC


 


2255
2256
1536-1554
UGGCCCAUGAGUUUGGAGC
GCUCCAAACUCAUGGGCCA


 


2257
2258
1537-1555
GGCCCAUGAGUUUGGAGCA
UGCUCCAAACUCAUGGGCC


 


2259
2260
1538-1556
GCCCAUGAGUUUGGAGCAA
UUGCUCCAAACUCAUGGGC


 


2261
2262
1539-1557
CCCAUGAGUUUGGAGCAAU
AUUGCUCCAAACUCAUGGG


 


2263
2264
1540-1558
CCAUGAGUUUGGAGCAAUC
GAUUGCUCCAAACUCAUGG


 


2265
2266
1542-1560
AUGAGUUUGGAGCAAUCAC
GUGAUUGCUCCAAACUCAU


 


2267
2268
1543-1561
UGAGUUUGGAGCAAUCACC
GGUGAUUGCUCCAAACUCA


 


2269
2270
1545-1563
AGUUUGGAGCAAUCACCUU
AAGGUGAUUGCUCCAAACU


 


2271
2272
1546-1564
GUUUGGAGCAAUCACCUUC
GAAGGUGAUUGCUCCAAAC


 


2273
2274
1547-1565
UUUGGAGCAAUCACCUUCG
CGAAGGUGAUUGCUCCAAA


 


2275
2276
1548-1566
UUGGAGCAAUCACCUUCGU
ACGAAGGUGAUUGCUCCAA


 


2277
2278
1549-1567
UGGAGCAAUCACCUUCGUG
CACGAAGGUGAUUGCUCCA


 


2279
2280
1550-1568
GGAGCAAUCACCUUCGUGG
CCACGAAGGUGAUUGCUCC


 


2281
2282
1551-1569
GAGCAAUCACCUUCGUGGA
UCCACGAAGGUGAUUGCUC


 


2283
2284
1552-1570
AGCAAUCACCUUCGUGGAU
AUCCACGAAGGUGAUUGCU


 


2285
2286
1553-1571
GCAAUCACCUUCGUGGAUG
CAUCCACGAAGGUGAUUGC


 


2287
2288
1554-1572
CAAUCACCUUCGUGGAUGA
UCAUCCACGAAGGUGAUUG


 


2289
2290
1555-1573
AAUCACCUUCGUGGAUGAG
CUCAUCCACGAAGGUGAUU


 


2291
2292
1556-1574
AUCACCUUCGUGGAUGAGG
CCUCAUCCACGAAGGUGAU


 


2293
2294
1557-1575
UCACCUUCGUGGAUGAGGU
ACCUCAUCCACGAAGGUGA


 


2295
2296
1558-1576
CACCUUCGUGGAUGAGGUC
GACCUCAUCCACGAAGGUG


 


2297
2298
1559-1577
ACCUUCGUGGAUGAGGUCC
GGACCUCAUCCACGAAGGU


 


2299
2300
1560-1578
CCUUCGUGGAUGAGGUCCA
UGGACCUCAUCCACGAAGG


 


2301
2302
1561-1579
CUUCGUGGAUGAGGUCCAC
GUGGACCUCAUCCACGAAG


 


2303
2304
1562-1580
UUCGUGGAUGAGGUCCACG
CGUGGACCUCAUCCACGAA


 


2305
2306
1563-1581
UCGUGGAUGAGGUCCACGC
GCGUGGACCUCAUCCACGA


 


2307
2308
1564-1582
CGUGGAUGAGGUCCACGCA
UGCGUGGACCUCAUCCACG


 


2309
2310
1565-1583
GUGGAUGAGGUCCACGCAG
CUGCGUGGACCUCAUCCAC


 


2311
2312
1566-1584
UGGAUGAGGUCCACGCAGU
ACUGCGUGGACCUCAUCCA


 


2313
2314
1567-1585
GGAUGAGGUCCACGCAGUG
CACUGCGUGGACCUCAUCC


 


2315
2316
1568-1586
GAUGAGGUCCACGCAGUGG
CCACUGCGUGGACCUCAUC


 


2317
2318
1569-1587
AUGAGGUCCACGCAGUGGG
CCCACUGCGUGGACCUCAU


 


2319
2320
1570-1588
UGAGGUCCACGCAGUGGGG
CCCCACUGCGUGGACCUCA


 


2321
2322
1571-1589
GAGGUCCACGCAGUGGGGC
GCCCCACUGCGUGGACCUC


 


2323
2324
1572-1590
AGGUCCACGCAGUGGGGCU
AGCCCCACUGCGUGGACCU


 


2325
2326
1595-1613
GGGGCUCGAGGCGGAGGGA
UCCCUCCGCCUCGAGCCCC


 


2327
2328
1596-1614
GGGCUCGAGGCGGAGGGAU
AUCCCUCCGCCUCGAGCCC


 


2329
2330
1597-1615
GGCUCGAGGCGGAGGGAUU
AAUCCCUCCGCCUCGAGCC


 


2331
2332
1598-1616
GCUCGAGGCGGAGGGAUUG
CAAUCCCUCCGCCUCGAGC


 


2333
2334
1599-1617
CUCGAGGCGGAGGGAUUGG
CCAAUCCCUCCGCCUCGAG


 


2335
2336
1600-1618
UCGAGGCGGAGGGAUUGGG
CCCAAUCCCUCCGCCUCGA


 


2337
2338
1601-1619
CGAGGCGGAGGGAUUGGGG
CCCCAAUCCCUCCGCCUCG


 


2339
2340
1602-1620
GAGGCGGAGGGAUUGGGGA
UCCCCAAUCCCUCCGCCUC


 


2341
2342
1603-1621
AGGCGGAGGGAUUGGGGAU
AUCCCCAAUCCCUCCGCCU


 


2343
2344
1604-1622
GGCGGAGGGAUUGGGGAUC
GAUCCCCAAUCCCUCCGCC


 


2345
2346
1605-1623
GCGGAGGGAUUGGGGAUCG
CGAUCCCCAAUCCCUCCGC


 


2347
2348
1606-1624
CGGAGGGAUUGGGGAUCGG
CCGAUCCCCAAUCCCUCCG


 


2349
2350
1607-1625
GGAGGGAUUGGGGAUCGGG
CCCGAUCCCCAAUCCCUCC


 


2351
2352
1608-1626
GAGGGAUUGGGGAUCGGGA
UCCCGAUCCCCAAUCCCUC


 


2353
2354
1609-1627
AGGGAUUGGGGAUCGGGAU
AUCCCGAUCCCCAAUCCCU


 


2355
2356
1610-1628
GGGAUUGGGGAUCGGGAUG
CAUCCCGAUCCCCAAUCCC


 


2357
2358
1611-1629
GGAUUGGGGAUCGGGAUGG
CCAUCCCGAUCCCCAAUCC


 


2359
2360
1612-1630
GAUUGGGGAUCGGGAUGGA
UCCAUCCCGAUCCCCAAUC


 


2361
2362
1613-1631
AUUGGGGAUCGGGAUGGAG
CUCCAUCCCGAUCCCCAAU


 


2363
2364
1614-1632
UUGGGGAUCGGGAUGGAGU
ACUCCAUCCCGAUCCCCAA


 


2365
2366
1615-1633
UGGGGAUCGGGAUGGAGUC
GACUCCAUCCCGAUCCCCA


 


2367
2368
1617-1635
GGGAUCGGGAUGGAGUCAU
AUGACUCCAUCCCGAUCCC


 


2369
2370
1618-1636
GGAUCGGGAUGGAGUCAUG
CAUGACUCCAUCCCGAUCC


 


2371
2372
1619-1637
GAUCGGGAUGGAGUCAUGC
GCAUGACUCCAUCCCGAUC


 


2373
2374
1620-1638
AUCGGGAUGGAGUCAUGCC
GGCAUGACUCCAUCCCGAU


 


2375
2376
1621-1639
UCGGGAUGGAGUCAUGCCA
UGGCAUGACUCCAUCCCGA


 


2377
2378
1622-1640
CGGGAUGGAGUCAUGCCAA
UUGGCAUGACUCCAUCCCG


 


2379
2380
1623-1641
GGGAUGGAGUCAUGCCAAA
UUUGGCAUGACUCCAUCCC


 


2381
2382
1624-1642
GGAUGGAGUCAUGCCAAAA
UUUUGGCAUGACUCCAUCC


 


2383
2384
1625-1643
GAUGGAGUCAUGCCAAAAA
UUUUUGGCAUGACUCCAUC


 


2385
2386
1626-1644
AUGGAGUCAUGCCAAAAAU
AUUUUUGGCAUGACUCCAU


 


2387
2388
1627-1645
UGGAGUCAUGCCAAAAAUG
CAUUUUUGGCAUGACUCCA


 


2389
2390
1628-1646
GGAGUCAUGCCAAAAAUGG
CCAUUUUUGGCAUGACUCC


 


2391
2392
1629-1647
GAGUCAUGCCAAAAAUGGA
UCCAUUUUUGGCAUGACUC


 


2393
2394
1630-1648
AGUCAUGCCAAAAAUGGAC
GUCCAUUUUUGGCAUGACU


 


2395
2396
1632-1650
UCAUGCCAAAAAUGGACAU
AUGUCCAUUUUUGGCAUGA


 


2397
2398
1633-1651
CAUGCCAAAAAUGGACAUC
GAUGUCCAUUUUUGGCAUG


 


2399
2400
1636-1654
GCCAAAAAUGGACAUCAUU
AAUGAUGUCCAUUUUUGGC


 


2401
2402
1638-1656
CAAAAAUGGACAUCAUUUC
GAAAUGAUGUCCAUUUUUG


 


2403
2404
1639-1657
AAAAAUGGACAUCAUUUCU
AGAAAUGAUGUCCAUUUUU


 


2405
2406
1640-1658
AAAAUGGACAUCAUUUCUG
CAGAAAUGAUGUCCAUUUU


 


2407
2408
1641-1659
AAAUGGACAUCAUUUCUGG
CCAGAAAUGAUGUCCAUUU


 


2409
2410
1642-1660
AAUGGACAUCAUUUCUGGA
UCCAGAAAUGAUGUCCAUU


 


2411
2412
1643-1661
AUGGACAUCAUUUCUGGAA
UUCCAGAAAUGAUGUCCAU


 


2413
2414
1644-1662
UGGACAUCAUUUCUGGAAC
GUUCCAGAAAUGAUGUCCA


 


2415
2416
1645-1663
GGACAUCAUUUCUGGAACA
UGUUCCAGAAAUGAUGUCC


 


2417
2418
1646-1664
GACAUCAUUUCUGGAACAC
GUGUUCCAGAAAUGAUGUC


 


2419
2420
1647-1665
ACAUCAUUUCUGGAACACU
AGUGUUCCAGAAAUGAUGU


 


2421
2422
1648-1666
CAUCAUUUCUGGAACACUU
AAGUGUUCCAGAAAUGAUG


 


2423
2424
1649-1667
AUCAUUUCUGGAACACUUG
CAAGUGUUCCAGAAAUGAU


 


2425
2426
1650-1668
UCAUUUCUGGAACACUUGG
CCAAGUGUUCCAGAAAUGA


 


2427
2428
1651-1669
CAUUUCUGGAACACUUGGC
GCCAAGUGUUCCAGAAAUG


 


2429
2430
1652-1670
AUUUCUGGAACACUUGGCA
UGCCAAGUGUUCCAGAAAU


 


2431
2432
1653-1671
UUUCUGGAACACUUGGCAA
UUGCCAAGUGUUCCAGAAA


 


2433
2434
1654-1672
UUCUGGAACACUUGGCAAA
UUUGCCAAGUGUUCCAGAA


 


2435
2436
1655-1673
UCUGGAACACUUGGCAAAG
CUUUGCCAAGUGUUCCAGA


 


2437
2438
1656-1674
CUGGAACACUUGGCAAAGC
GCUUUGCCAAGUGUUCCAG


 


2439
2440
1657-1675
UGGAACACUUGGCAAAGCC
GGCUUUGCCAAGUGUUCCA


 


2441
2442
1658-1676
GGAACACUUGGCAAAGCCU
AGGCUUUGCCAAGUGUUCC


 


2443
2444
1659-1677
GAACACUUGGCAAAGCCUU
AAGGCUUUGCCAAGUGUUC


 


2445
2446
1660-1678
AACACUUGGCAAAGCCUUU
AAAGGCUUUGCCAAGUGUU


 


2447
2448
1661-1679
ACACUUGGCAAAGCCUUUG
CAAAGGCUUUGCCAAGUGU


 


2449
2450
1662-1680
CACUUGGCAAAGCCUUUGG
CCAAAGGCUUUGCCAAGUG


 


2451
2452
1682-1700
UGUGUUGGAGGGUACAUCG
CGAUGUACCCUCCAACACA


 


2453
2454
1683-1701
GUGUUGGAGGGUACAUCGC
GCGAUGUACCCUCCAACAC


 


2455
2456
1684-1702
UGUUGGAGGGUACAUCGCC
GGCGAUGUACCCUCCAACA


 


2457
2458
1685-1703
GUUGGAGGGUACAUCGCCA
UGGCGAUGUACCCUCCAAC


 


2459
2460
1686-1704
UUGGAGGGUACAUCGCCAG
CUGGCGAUGUACCCUCCAA


 


2461
2462
1687-1705
UGGAGGGUACAUCGCCAGC
GCUGGCGAUGUACCCUCCA


 


2463
2464
1688-1706
GGAGGGUACAUCGCCAGCA
UGCUGGCGAUGUACCCUCC


 


2465
2466
1689-1707
GAGGGUACAUCGCCAGCAC
GUGCUGGCGAUGUACCCUC


 


2467
2468
1690-1708
AGGGUACAUCGCCAGCACG
CGUGCUGGCGAUGUACCCU


 


2469
2470
1691-1709
GGGUACAUCGCCAGCACGA
UCGUGCUGGCGAUGUACCC


 


2471
2472
1692-1710
GGUACAUCGCCAGCACGAG
CUCGUGCUGGCGAUGUACC


 


2473
2474
1693-1711
GUACAUCGCCAGCACGAGU
ACUCGUGCUGGCGAUGUAC


 


2475
2476
1694-1712
UACAUCGCCAGCACGAGUU
AACUCGUGCUGGCGAUGUA


 


2477
2478
1695-1713
ACAUCGCCAGCACGAGUUC
GAACUCGUGCUGGCGAUGU


 


2479
2480
1696-1714
CAUCGCCAGCACGAGUUCU
AGAACUCGUGCUGGCGAUG


 


2481
2482
1697-1715
AUCGCCAGCACGAGUUCUC
GAGAACUCGUGCUGGCGAU


 


2483
2484
1698-1716
UCGCCAGCACGAGUUCUCU
AGAGAACUCGUGCUGGCGA


 


2485
2486
1699-1717
CGCCAGCACGAGUUCUCUG
CAGAGAACUCGUGCUGGCG


 


2487
2488
1700-1718
GCCAGCACGAGUUCUCUGA
UCAGAGAACUCGUGCUGGC


 


2489
2490
1701-1719
CCAGCACGAGUUCUCUGAU
AUCAGAGAACUCGUGCUGG


 


2491
2492
1702-1720
CAGCACGAGUUCUCUGAUU
AAUCAGAGAACUCGUGCUG


 


2493
2494
1703-1721
AGCACGAGUUCUCUGAUUG
CAAUCAGAGAACUCGUGCU


 


2495
2496
1704-1722
GCACGAGUUCUCUGAUUGA
UCAAUCAGAGAACUCGUGC


 


2497
2498
1705-1723
CACGAGUUCUCUGAUUGAC
GUCAAUCAGAGAACUCGUG


 


2499
2500
1707-1725
CGAGUUCUCUGAUUGACAC
GUGUCAAUCAGAGAACUCG


 


2501
2502
1727-1745
GUACGGUCCUAUGCUGCUG
CAGCAGCAUAGGACCGUAC


 


2503
2504
1728-1746
UACGGUCCUAUGCUGCUGG
CCAGCAGCAUAGGACCGUA


 


2505
2506
1729-1747
ACGGUCCUAUGCUGCUGGC
GCCAGCAGCAUAGGACCGU


 


2507
2508
1730-1748
CGGUCCUAUGCUGCUGGCU
AGCCAGCAGCAUAGGACCG


 


2509
2510
1731-1749
GGUCCUAUGCUGCUGGCUU
AAGCCAGCAGCAUAGGACC


 


2511
2512
1732-1750
GUCCUAUGCUGCUGGCUUC
GAAGCCAGCAGCAUAGGAC


 


2513
2514
1733-1751
UCCUAUGCUGCUGGCUUCA
UGAAGCCAGCAGCAUAGGA


 


2515
2516
1734-1752
CCUAUGCUGCUGGCUUCAU
AUGAAGCCAGCAGCAUAGG


 


2517
2518
1735-1753
CUAUGCUGCUGGCUUCAUC
GAUGAAGCCAGCAGCAUAG


 


2519
2520
1736-1754
UAUGCUGCUGGCUUCAUCU
AGAUGAAGCCAGCAGCAUA


 


2521
2522
1737-1755
AUGCUGCUGGCUUCAUCUU
AAGAUGAAGCCAGCAGCAU


 


2523
2524
1738-1756
UGCUGCUGGCUUCAUCUUC
GAAGAUGAAGCCAGCAGCA


 


2525
2526
1739-1757
GCUGCUGGCUUCAUCUUCA
UGAAGAUGAAGCCAGCAGC


 


2527
2528
1740-1758
CUGCUGGCUUCAUCUUCAC
GUGAAGAUGAAGCCAGCAG


 


2529
2530
1741-1759
UGCUGGCUUCAUCUUCACC
GGUGAAGAUGAAGCCAGCA


 


2531
2532
1742-1760
GCUGGCUUCAUCUUCACCA
UGGUGAAGAUGAAGCCAGC


 


2533
2534
1743-1761
CUGGCUUCAUCUUCACCAC
GUGGUGAAGAUGAAGCCAG


 


2535
2536
1744-1762
UGGCUUCAUCUUCACCACC
GGUGGUGAAGAUGAAGCCA


 


2537
2538
1745-1763
GGCUUCAUCUUCACCACCU
AGGUGGUGAAGAUGAAGCC


 


2539
2540
1746-1764
GCUUCAUCUUCACCACCUC
GAGGUGGUGAAGAUGAAGC


 


2541
2542
1747-1765
CUUCAUCUUCACCACCUCU
AGAGGUGGUGAAGAUGAAG


 


2543
2544
1748-1766
UUCAUCUUCACCACCUCUC
GAGAGGUGGUGAAGAUGAA


 


2545
2546
1749-1767
UCAUCUUCACCACCUCUCU
AGAGAGGUGGUGAAGAUGA


 


2547
2548
1750-1768
CAUCUUCACCACCUCUCUG
CAGAGAGGUGGUGAAGAUG


 


2549
2550
1751-1769
AUCUUCACCACCUCUCUGC
GCAGAGAGGUGGUGAAGAU


 


2551
2552
1752-1770
UCUUCACCACCUCUCUGCC
GGCAGAGAGGUGGUGAAGA


 


2553
2554
1753-1771
CUUCACCACCUCUCUGCCA
UGGCAGAGAGGUGGUGAAG


 


2555
2556
1754-1772
UUCACCACCUCUCUGCCAC
GUGGCAGAGAGGUGGUGAA


 


2557
2558
1755-1773
UCACCACCUCUCUGCCACC
GGUGGCAGAGAGGUGGUGA


 


2559
2560
1756-1774
CACCACCUCUCUGCCACCC
GGGUGGCAGAGAGGUGGUG


 


2561
2562
1757-1775
ACCACCUCUCUGCCACCCA
UGGGUGGCAGAGAGGUGGU


 


2563
2564
1758-1776
CCACCUCUCUGCCACCCAU
AUGGGUGGCAGAGAGGUGG


 


2565
2566
1759-1777
CACCUCUCUGCCACCCAUG
CAUGGGUGGCAGAGAGGUG


 


2567
2568
1760-1778
ACCUCUCUGCCACCCAUGC
GCAUGGGUGGCAGAGAGGU


 


2569
2570
1761-1779
CCUCUCUGCCACCCAUGCU
AGCAUGGGUGGCAGAGAGG


 


2571
2572
1762-1780
CUCUCUGCCACCCAUGCUG
CAGCAUGGGUGGCAGAGAG


 


2573
2574
1763-1781
UCUCUGCCACCCAUGCUGC
GCAGCAUGGGUGGCAGAGA


 


2575
2576
1764-1782
CUCUGCCACCCAUGCUGCU
AGCAGCAUGGGUGGCAGAG


 


2577
2578
1765-1783
UCUGCCACCCAUGCUGCUG
CAGCAGCAUGGGUGGCAGA


 


2579
2580
1766-1784
CUGCCACCCAUGCUGCUGG
CCAGCAGCAUGGGUGGCAG


 


2581
2582
1767-1785
UGCCACCCAUGCUGCUGGC
GCCAGCAGCAUGGGUGGCA


 


2583
2584
1768-1786
GCCACCCAUGCUGCUGGCU
AGCCAGCAGCAUGGGUGGC


 


2585
2586
1769-1787
CCACCCAUGCUGCUGGCUG
CAGCCAGCAGCAUGGGUGG


 


2587
2588
1770-1788
CACCCAUGCUGCUGGCUGG
CCAGCCAGCAGCAUGGGUG


 


2589
2590
1771-1789
ACCCAUGCUGCUGGCUGGA
UCCAGCCAGCAGCAUGGGU


 


2591
2592
1772-1790
CCCAUGCUGCUGGCUGGAG
CUCCAGCCAGCAGCAUGGG


 


2593
2594
1773-1791
CCAUGCUGCUGGCUGGAGC
GCUCCAGCCAGCAGCAUGG


 


2595
2596
1774-1792
CAUGCUGCUGGCUGGAGCC
GGCUCCAGCCAGCAGCAUG


 


2597
2598
1775-1793
AUGCUGCUGGCUGGAGCCC
GGGCUCCAGCCAGCAGCAU


 


2599
2600
1776-1794
UGCUGCUGGCUGGAGCCCU
AGGGCUCCAGCCAGCAGCA


 


2601
2602
1777-1795
GCUGCUGGCUGGAGCCCUG
CAGGGCUCCAGCCAGCAGC


 


2603
2604
1778-1796
CUGCUGGCUGGAGCCCUGG
CCAGGGCUCCAGCCAGCAG


 


2605
2606
1779-1797
UGCUGGCUGGAGCCCUGGA
UCCAGGGCUCCAGCCAGCA


 


2607
2608
1780-1798
GCUGGCUGGAGCCCUGGAG
CUCCAGGGCUCCAGCCAGC


 


2609
2610
1781-1799
CUGGCUGGAGCCCUGGAGU
ACUCCAGGGCUCCAGCCAG


 


2611
2612
1782-1800
UGGCUGGAGCCCUGGAGUC
GACUCCAGGGCUCCAGCCA


 


2613
2614
1783-1801
GGCUGGAGCCCUGGAGUCU
AGACUCCAGGGCUCCAGCC


 


2615
2616
1784-1802
GCUGGAGCCCUGGAGUCUG
CAGACUCCAGGGCUCCAGC


 


2617
2618
1785-1803
CUGGAGCCCUGGAGUCUGU
ACAGACUCCAGGGCUCCAG


 


2619
2620
1786-1804
UGGAGCCCUGGAGUCUGUG
CACAGACUCCAGGGCUCCA


 


2621
2622
1787-1805
GGAGCCCUGGAGUCUGUGC
GCACAGACUCCAGGGCUCC


 


2623
2624
1788-1806
GAGCCCUGGAGUCUGUGCG
CGCACAGACUCCAGGGCUC


 


2625
2626
1789-1807
AGCCCUGGAGUCUGUGCGG
CCGCACAGACUCCAGGGCU


 


2627
2628
1790-1808
GCCCUGGAGUCUGUGCGGA
UCCGCACAGACUCCAGGGC


 


2629
2630
1792-1810
CCUGGAGUCUGUGCGGAUC
GAUCCGCACAGACUCCAGG


 


2631
2632
1793-1811
CUGGAGUCUGUGCGGAUCC
GGAUCCGCACAGACUCCAG


 


2633
2634
1795-1813
GGAGUCUGUGCGGAUCCUG
CAGGAUCCGCACAGACUCC


 


2635
2636
1796-1814
GAGUCUGUGCGGAUCCUGA
UCAGGAUCCGCACAGACUC


 


2637
2638
1797-1815
AGUCUGUGCGGAUCCUGAA
UUCAGGAUCCGCACAGACU


 


2639
2640
1798-1816
GUCUGUGCGGAUCCUGAAG
CUUCAGGAUCCGCACAGAC


 


2641
2642
1799-1817
UCUGUGCGGAUCCUGAAGA
UCUUCAGGAUCCGCACAGA


 


2643
2644
1800-1818
CUGUGCGGAUCCUGAAGAG
CUCUUCAGGAUCCGCACAG


 


2645
2646
1801-1819
UGUGCGGAUCCUGAAGAGC
GCUCUUCAGGAUCCGCACA


 


2647
2648
1802-1820
GUGCGGAUCCUGAAGAGCG
CGCUCUUCAGGAUCCGCAC


 


2649
2650
1803-1821
UGCGGAUCCUGAAGAGCGC
GCGCUCUUCAGGAUCCGCA


 


2651
2652
1804-1822
GCGGAUCCUGAAGAGCGCU
AGCGCUCUUCAGGAUCCGC


 


2653
2654
1805-1823
CGGAUCCUGAAGAGCGCUG
CAGCGCUCUUCAGGAUCCG


 


2655
2656
1806-1824
GGAUCCUGAAGAGCGCUGA
UCAGCGCUCUUCAGGAUCC


 


2657
2658
1807-1825
GAUCCUGAAGAGCGCUGAG
CUCAGCGCUCUUCAGGAUC


 


2659
2660
1808-1826
AUCCUGAAGAGCGCUGAGG
CCUCAGCGCUCUUCAGGAU


 


2661
2662
1809-1827
UCCUGAAGAGCGCUGAGGG
CCCUCAGCGCUCUUCAGGA


 


2663
2664
1810-1828
CCUGAAGAGCGCUGAGGGA
UCCCUCAGCGCUCUUCAGG


 


2665
2666
1811-1829
CUGAAGAGCGCUGAGGGAC
GUCCCUCAGCGCUCUUCAG


 


2667
2668
1812-1830
UGAAGAGCGCUGAGGGACG
CGUCCCUCAGCGCUCUUCA


 


2669
2670
1813-1831
GAAGAGCGCUGAGGGACGG
CCGUCCCUCAGCGCUCUUC


 


2671
2672
1814-1832
AAGAGCGCUGAGGGACGGG
CCCGUCCCUCAGCGCUCUU


 


2673
2674
1815-1833
AGAGCGCUGAGGGACGGGU
ACCCGUCCCUCAGCGCUCU


 


2675
2676
1816-1834
GAGCGCUGAGGGACGGGUG
CACCCGUCCCUCAGCGCUC


 


2677
2678
1817-1835
AGCGCUGAGGGACGGGUGC
GCACCCGUCCCUCAGCGCU


 


2679
2680
1818-1836
GCGCUGAGGGACGGGUGCU
AGCACCCGUCCCUCAGCGC


 


2681
2682
1819-1837
CGCUGAGGGACGGGUGCUU
AAGCACCCGUCCCUCAGCG


 


2683
2684
1820-1838
GCUGAGGGACGGGUGCUUC
GAAGCACCCGUCCCUCAGC


 


2685
2686
1821-1839
CUGAGGGACGGGUGCUUCG
CGAAGCACCCGUCCCUCAG


 


2687
2688
1822-1840
UGAGGGACGGGUGCUUCGC
GCGAAGCACCCGUCCCUCA


 


2689
2690
1823-1841
GAGGGACGGGUGCUUCGCC
GGCGAAGCACCCGUCCCUC


 


2691
2692
1824-1842
AGGGACGGGUGCUUCGCCG
CGGCGAAGCACCCGUCCCU


 


2693
2694
1825-1843
GGGACGGGUGCUUCGCCGC
GCGGCGAAGCACCCGUCCC


 


2695
2696
1826-1844
GGACGGGUGCUUCGCCGCC
GGCGGCGAAGCACCCGUCC


 


2697
2698
1827-1845
GACGGGUGCUUCGCCGCCA
UGGCGGCGAAGCACCCGUC


 


2699
2700
1828-1846
ACGGGUGCUUCGCCGCCAG
CUGGCGGCGAAGCACCCGU


 


2701
2702
1829-1847
CGGGUGCUUCGCCGCCAGC
GCUGGCGGCGAAGCACCCG


 


2703
2704
1830-1848
GGGUGCUUCGCCGCCAGCA
UGCUGGCGGCGAAGCACCC


 


2705
2706
1831-1849
GGUGCUUCGCCGCCAGCAC
GUGCUGGCGGCGAAGCACC


 


2707
2708
1832-1850
GUGCUUCGCCGCCAGCACC
GGUGCUGGCGGCGAAGCAC


 


2709
2710
1833-1851
UGCUUCGCCGCCAGCACCA
UGGUGCUGGCGGCGAAGCA


 


2711
2712
1834-1852
GCUUCGCCGCCAGCACCAG
CUGGUGCUGGCGGCGAAGC


 


2713
2714
1835-1853
CUUCGCCGCCAGCACCAGC
GCUGGUGCUGGCGGCGAAG


 


2715
2716
1836-1854
UUCGCCGCCAGCACCAGCG
CGCUGGUGCUGGCGGCGAA


 


2717
2718
1837-1855
UCGCCGCCAGCACCAGCGC
GCGCUGGUGCUGGCGGCGA


 


2719
2720
1838-1856
CGCCGCCAGCACCAGCGCA
UGCGCUGGUGCUGGCGGCG


 


2721
2722
1839-1857
GCCGCCAGCACCAGCGCAA
UUGCGCUGGUGCUGGCGGC


 


2723
2724
1840-1858
CCGCCAGCACCAGCGCAAC
GUUGCGCUGGUGCUGGCGG


 


2725
2726
1841-1859
CGCCAGCACCAGCGCAACG
CGUUGCGCUGGUGCUGGCG


 


2727
2728
1842-1860
GCCAGCACCAGCGCAACGU
ACGUUGCGCUGGUGCUGGC


 


2729
2730
1865-1883
CUCAUGAGACAGAUGCUAA
UUAGCAUCUGUCUCAUGAG


 


2731
2732
1866-1884
UCAUGAGACAGAUGCUAAU
AUUAGCAUCUGUCUCAUGA


 


2733
2734
1867-1885
CAUGAGACAGAUGCUAAUG
CAUUAGCAUCUGUCUCAUG


 


2735
2736
1868-1886
AUGAGACAGAUGCUAAUGG
CCAUUAGCAUCUGUCUCAU


 


2737
2738
1869-1887
UGAGACAGAUGCUAAUGGA
UCCAUUAGCAUCUGUCUCA


 


2739
2740
1871-1889
AGACAGAUGCUAAUGGAUG
CAUCCAUUAGCAUCUGUCU


 


2741
2742
1872-1890
GACAGAUGCUAAUGGAUGC
GCAUCCAUUAGCAUCUGUC


 


2743
2744
1873-1891
ACAGAUGCUAAUGGAUGCC
GGCAUCCAUUAGCAUCUGU


 


2745
2746
1874-1892
CAGAUGCUAAUGGAUGCCG
CGGCAUCCAUUAGCAUCUG


 


2747
2748
1875-1893
AGAUGCUAAUGGAUGCCGG
CCGGCAUCCAUUAGCAUCU


 


2749
2750
1876-1894
GAUGCUAAUGGAUGCCGGC
GCCGGCAUCCAUUAGCAUC


 


2751
2752
1877-1895
AUGCUAAUGGAUGCCGGCC
GGCCGGCAUCCAUUAGCAU


 


2753
2754
1878-1896
UGCUAAUGGAUGCCGGCCU
AGGCCGGCAUCCAUUAGCA


 


2755
2756
1879-1897
GCUAAUGGAUGCCGGCCUC
GAGGCCGGCAUCCAUUAGC


 


2757
2758
1880-1898
CUAAUGGAUGCCGGCCUCC
GGAGGCCGGCAUCCAUUAG


 


2759
2760
1881-1899
UAAUGGAUGCCGGCCUCCC
GGGAGGCCGGCAUCCAUUA


 


2761
2762
1882-1900
AAUGGAUGCCGGCCUCCCU
AGGGAGGCCGGCAUCCAUU


 


2763
2764
1883-1901
AUGGAUGCCGGCCUCCCUG
CAGGGAGGCCGGCAUCCAU


 


2765
2766
1884-1902
UGGAUGCCGGCCUCCCUGU
ACAGGGAGGCCGGCAUCCA


 


2767
2768
1885-1903
GGAUGCCGGCCUCCCUGUU
AACAGGGAGGCCGGCAUCC


 


2769
2770
1886-1904
GAUGCCGGCCUCCCUGUUG
CAACAGGGAGGCCGGCAUC


 


2771
2772
1887-1905
AUGCCGGCCUCCCUGUUGU
ACAACAGGGAGGCCGGCAU


 


2773
2774
1888-1906
UGCCGGCCUCCCUGUUGUC
GACAACAGGGAGGCCGGCA


 


2775
2776
1889-1907
GCCGGCCUCCCUGUUGUCC
GGACAACAGGGAGGCCGGC


 


2777
2778
1890-1908
CCGGCCUCCCUGUUGUCCA
UGGACAACAGGGAGGCCGG


 


2779
2780
1891-1909
CGGCCUCCCUGUUGUCCAC
GUGGACAACAGGGAGGCCG


 


2781
2782
1892-1910
GGCCUCCCUGUUGUCCACU
AGUGGACAACAGGGAGGCC


 


2783
2784
1893-1911
GCCUCCCUGUUGUCCACUG
CAGUGGACAACAGGGAGGC


 


2785
2786
1894-1912
CCUCCCUGUUGUCCACUGC
GCAGUGGACAACAGGGAGG


 


2787
2788
1895-1913
CUCCCUGUUGUCCACUGCC
GGCAGUGGACAACAGGGAG


 


2789
2790
1896-1914
UCCCUGUUGUCCACUGCCC
GGGCAGUGGACAACAGGGA


 


2791
2792
1897-1915
CCCUGUUGUCCACUGCCCC
GGGGCAGUGGACAACAGGG


 


2793
2794
1898-1916
CCUGUUGUCCACUGCCCCA
UGGGGCAGUGGACAACAGG


 


2795
2796
1899-1917
CUGUUGUCCACUGCCCCAG
CUGGGGCAGUGGACAACAG


 


2797
2798
1900-1918
UGUUGUCCACUGCCCCAGC
GCUGGGGCAGUGGACAACA


 


2799
2800
1901-1919
GUUGUCCACUGCCCCAGCC
GGCUGGGGCAGUGGACAAC


 


2801
2802
1902-1920
UUGUCCACUGCCCCAGCCA
UGGCUGGGGCAGUGGACAA


 


2803
2804
1903-1921
UGUCCACUGCCCCAGCCAC
GUGGCUGGGGCAGUGGACA


 


2805
2806
1904-1922
GUCCACUGCCCCAGCCACA
UGUGGCUGGGGCAGUGGAC


 


2807
2808
1905-1923
UCCACUGCCCCAGCCACAU
AUGUGGCUGGGGCAGUGGA


 


2809
2810
1906-1924
CCACUGCCCCAGCCACAUC
GAUGUGGCUGGGGCAGUGG


 


2811
2812
1907-1925
CACUGCCCCAGCCACAUCA
UGAUGUGGCUGGGGCAGUG


 


2813
2814
1908-1926
ACUGCCCCAGCCACAUCAU
AUGAUGUGGCUGGGGCAGU


 


2815
2816
1909-1927
CUGCCCCAGCCACAUCAUC
GAUGAUGUGGCUGGGGCAG


 


2817
2818
1910-1928
UGCCCCAGCCACAUCAUCC
GGAUGAUGUGGCUGGGGCA


 


2819
2820
1911-1929
GCCCCAGCCACAUCAUCCC
GGGAUGAUGUGGCUGGGGC


 


2821
2822
1912-1930
CCCCAGCCACAUCAUCCCU
AGGGAUGAUGUGGCUGGGG


 


2823
2824
1913-1931
CCCAGCCACAUCAUCCCUG
CAGGGAUGAUGUGGCUGGG


 


2825
2826
1914-1932
CCAGCCACAUCAUCCCUGU
ACAGGGAUGAUGUGGCUGG


 


2827
2828
1915-1933
CAGCCACAUCAUCCCUGUG
CACAGGGAUGAUGUGGCUG


 


2829
2830
1916-1934
AGCCACAUCAUCCCUGUGC
GCACAGGGAUGAUGUGGCU


 


2831
2832
1917-1935
GCCACAUCAUCCCUGUGCG
CGCACAGGGAUGAUGUGGC


 


2833
2834
1918-1936
CCACAUCAUCCCUGUGCGG
CCGCACAGGGAUGAUGUGG


 


2835
2836
1919-1937
CACAUCAUCCCUGUGCGGG
CCCGCACAGGGAUGAUGUG


 


2837
2838
1920-1938
ACAUCAUCCCUGUGCGGGU
ACCCGCACAGGGAUGAUGU


 


2839
2840
1922-1940
AUCAUCCCUGUGCGGGUUG
CAACCCGCACAGGGAUGAU


 


2841
2842
1923-1941
UCAUCCCUGUGCGGGUUGC
GCAACCCGCACAGGGAUGA


 


2843
2844
1924-1942
CAUCCCUGUGCGGGUUGCA
UGCAACCCGCACAGGGAUG


 


2845
2846
1925-1943
AUCCCUGUGCGGGUUGCAG
CUGCAACCCGCACAGGGAU


 


2847
2848
1926-1944
UCCCUGUGCGGGUUGCAGA
UCUGCAACCCGCACAGGGA


 


2849
2850
1928-1946
CCUGUGCGGGUUGCAGAUG
CAUCUGCAACCCGCACAGG


 


2851
2852
1929-1947
CUGUGCGGGUUGCAGAUGC
GCAUCUGCAACCCGCACAG


 


2853
2854
1930-1948
UGUGCGGGUUGCAGAUGCU
AGCAUCUGCAACCCGCACA


 


2855
2856
1931-1949
GUGCGGGUUGCAGAUGCUG
CAGCAUCUGCAACCCGCAC


 


2857
2858
1932-1950
UGCGGGUUGCAGAUGCUGC
GCAGCAUCUGCAACCCGCA


 


2859
2860
1933-1951
GCGGGUUGCAGAUGCUGCU
AGCAGCAUCUGCAACCCGC


 


2861
2862
1934-1952
CGGGUUGCAGAUGCUGCUA
UAGCAGCAUCUGCAACCCG


 


2863
2864
1935-1953
GGGUUGCAGAUGCUGCUAA
UUAGCAGCAUCUGCAACCC


 


2865
2866
1936-1954
GGUUGCAGAUGCUGCUAAA
UUUAGCAGCAUCUGCAACC


 


2867
2868
1937-1955
GUUGCAGAUGCUGCUAAAA
UUUUAGCAGCAUCUGCAAC


 


2869
2870
1938-1956
UUGCAGAUGCUGCUAAAAA
UUUUUAGCAGCAUCUGCAA


 


2871
2872
1939-1957
UGCAGAUGCUGCUAAAAAC
GUUUUUAGCAGCAUCUGCA


 


2873
2874
1940-1958
GCAGAUGCUGCUAAAAACA
UGUUUUUAGCAGCAUCUGC


 


2875
2876
1941-1959
CAGAUGCUGCUAAAAACAC
GUGUUUUUAGCAGCAUCUG


 


2877
2878
1961-1979
GAAGUCUGUGAUGAACUAA
UUAGUUCAUCACAGACUUC


 


2879
2880
1963-1981
AGUCUGUGAUGAACUAAUG
CAUUAGUUCAUCACAGACU


 


2881
2882
1965-1983
UCUGUGAUGAACUAAUGAG
CUCAUUAGUUCAUCACAGA


 


2883
2884
1966-1984
CUGUGAUGAACUAAUGAGC
GCUCAUUAGUUCAUCACAG


 


2885
2886
1968-1986
GUGAUGAACUAAUGAGCAG
CUGCUCAUUAGUUCAUCAC


 


2887
2888
1969-1987
UGAUGAACUAAUGAGCAGA
UCUGCUCAUUAGUUCAUCA


 


2889
2890
1970-1988
GAUGAACUAAUGAGCAGAC
GUCUGCUCAUUAGUUCAUC


 


2891
2892
1971-1989
AUGAACUAAUGAGCAGACA
UGUCUGCUCAUUAGUUCAU


 


2893
2894
1972-1990
UGAACUAAUGAGCAGACAU
AUGUCUGCUCAUUAGUUCA


 


2895
2896
1973-1991
GAACUAAUGAGCAGACAUA
UAUGUCUGCUCAUUAGUUC


 


2897
2898
1974-1992
AACUAAUGAGCAGACAUAA
UUAUGUCUGCUCAUUAGUU


 


2899
2900
1975-1993
ACUAAUGAGCAGACAUAAC
GUUAUGUCUGCUCAUUAGU


 


2901
2902
1978-1996
AAUGAGCAGACAUAACAUC
GAUGUUAUGUCUGCUCAUU


 


2903
2904
1979-1997
AUGAGCAGACAUAACAUCU
AGAUGUUAUGUCUGCUCAU


 


2905
2906
1980-1998
UGAGCAGACAUAACAUCUA
UAGAUGUUAUGUCUGCUCA


 


2907
2908
2000-2018
GUGCAAGCAAUCAAUUACC
GGUAAUUGAUUGCUUGCAC


 


2909
2910
2001-2019
UGCAAGCAAUCAAUUACCC
GGGUAAUUGAUUGCUUGCA


 


2911
2912
2002-2020
GCAAGCAAUCAAUUACCCU
AGGGUAAUUGAUUGCUUGC


 


2913
2914
2004-2022
AAGCAAUCAAUUACCCUAC
GUAGGGUAAUUGAUUGCUU


 


2915
2916
2024-2042
GUGCCCCGGGGAGAAGAGC
GCUCUUCUCCCCGGGGCAC


 


2917
2918
2025-2043
UGCCCCGGGGAGAAGAGCU
AGCUCUUCUCCCCGGGGCA


 


2919
2920
2026-2044
GCCCCGGGGAGAAGAGCUC
GAGCUCUUCUCCCCGGGGC


 


2921
2922
2027-2045
CCCCGGGGAGAAGAGCUCC
GGAGCUCUUCUCCCCGGGG


 


2923
2924
2028-2046
CCCGGGGAGAAGAGCUCCU
AGGAGCUCUUCUCCCCGGG


 


2925
2926
2029-2047
CCGGGGAGAAGAGCUCCUA
UAGGAGCUCUUCUCCCCGG


 


2927
2928
2030-2048
CGGGGAGAAGAGCUCCUAC
GUAGGAGCUCUUCUCCCCG


 


2929
2930
2031-2049
GGGGAGAAGAGCUCCUACG
CGUAGGAGCUCUUCUCCCC


 


2931
2932
2032-2050
GGGAGAAGAGCUCCUACGG
CCGUAGGAGCUCUUCUCCC


 


2933
2934
2033-2051
GGAGAAGAGCUCCUACGGA
UCCGUAGGAGCUCUUCUCC


 


2935
2936
2034-2052
GAGAAGAGCUCCUACGGAU
AUCCGUAGGAGCUCUUCUC


 


2937
2938
2060-2078
ACCCCUCACCACACACCCC
GGGGUGUGUGGUGAGGGGU


 


2939
2940
2061-2079
CCCCUCACCACACACCCCA
UGGGGUGUGUGGUGAGGGG


 


2941
2942
2062-2080
CCCUCACCACACACCCCAG
CUGGGGUGUGUGGUGAGGG


 


2943
2944
2063-2081
CCUCACCACACACCCCAGA
UCUGGGGUGUGUGGUGAGG


 


2945
2946
2064-2082
CUCACCACACACCCCAGAU
AUCUGGGGUGUGUGGUGAG


 


2947
2948
2065-2083
UCACCACACACCCCAGAUG
CAUCUGGGGUGUGUGGUGA


 


2949
2950
2066-2084
CACCACACACCCCAGAUGA
UCAUCUGGGGUGUGUGGUG


 


2951
2952
2067-2085
ACCACACACCCCAGAUGAU
AUCAUCUGGGGUGUGUGGU


 


2953
2954
2068-2086
CCACACACCCCAGAUGAUG
CAUCAUCUGGGGUGUGUGG


 


2955
2956
2069-2087
CACACACCCCAGAUGAUGA
UCAUCAUCUGGGGUGUGUG


 


2957
2958
2070-2088
ACACACCCCAGAUGAUGAA
UUCAUCAUCUGGGGUGUGU


 


2959
2960
2071-2089
CACACCCCAGAUGAUGAAC
GUUCAUCAUCUGGGGUGUG


 


2961
2962
2072-2090
ACACCCCAGAUGAUGAACU
AGUUCAUCAUCUGGGGUGU


 


2963
2964
2073-2091
CACCCCAGAUGAUGAACUA
UAGUUCAUCAUCUGGGGUG


 


2965
2966
2074-2092
ACCCCAGAUGAUGAACUAC
GUAGUUCAUCAUCUGGGGU


 


2967
2968
2076-2094
CCCAGAUGAUGAACUACUU
AAGUAGUUCAUCAUCUGGG


 


2969
2970
2077-2095
CCAGAUGAUGAACUACUUC
GAAGUAGUUCAUCAUCUGG


 


2971
2972
2078-2096
CAGAUGAUGAACUACUUCC
GGAAGUAGUUCAUCAUCUG


 


2973
2974
2079-2097
AGAUGAUGAACUACUUCCU
AGGAAGUAGUUCAUCAUCU


 


2975
2976
2080-2098
GAUGAUGAACUACUUCCUU
AAGGAAGUAGUUCAUCAUC


 


2977
2978
2081-2099
AUGAUGAACUACUUCCUUG
CAAGGAAGUAGUUCAUCAU


 


2979
2980
2082-2100
UGAUGAACUACUUCCUUGA
UCAAGGAAGUAGUUCAUCA


 


2981
2982
2083-2101
GAUGAACUACUUCCUUGAG
CUCAAGGAAGUAGUUCAUC


 


2983
2984
2084-2102
AUGAACUACUUCCUUGAGA
UCUCAAGGAAGUAGUUCAU


 


2985
2986
2085-2103
UGAACUACUUCCUUGAGAA
UUCUCAAGGAAGUAGUUCA


 


2987
2988
2086-2104
GAACUACUUCCUUGAGAAU
AUUCUCAAGGAAGUAGUUC


 


2989
2990
2087-2105
AACUACUUCCUUGAGAAUC
GAUUCUCAAGGAAGUAGUU


 


2991
2992
2088-2106
ACUACUUCCUUGAGAAUCU
AGAUUCUCAAGGAAGUAGU


 


2993
2994
2089-2107
CUACUUCCUUGAGAAUCUG
CAGAUUCUCAAGGAAGUAG


 


2995
2996
2090-2108
UACUUCCUUGAGAAUCUGC
GCAGAUUCUCAAGGAAGUA


 


2997
2998
2091-2109
ACUUCCUUGAGAAUCUGCU
AGCAGAUUCUCAAGGAAGU


 


2999
3000
2117-2135
UGGAAGCAAGUGGGGCUGG
CCAGCCCCACUUGCUUCCA


 


3001
3002
2118-2136
GGAAGCAAGUGGGGCUGGA
UCCAGCCCCACUUGCUUCC


 


3003
3004
2119-2137
GAAGCAAGUGGGGCUGGAA
UUCCAGCCCCACUUGCUUC


 


3005
3006
2120-2138
AAGCAAGUGGGGCUGGAAC
GUUCCAGCCCCACUUGCUU


 


3007
3008
2121-2139
AGCAAGUGGGGCUGGAACU
AGUUCCAGCCCCACUUGCU


 


3009
3010
2122-2140
GCAAGUGGGGCUGGAACUG
CAGUUCCAGCCCCACUUGC


 


3011
3012
2123-2141
CAAGUGGGGCUGGAACUGA
UCAGUUCCAGCCCCACUUG


 


3013
3014
2124-2142
AAGUGGGGCUGGAACUGAA
UUCAGUUCCAGCCCCACUU


 


3015
3016
2125-2143
AGUGGGGCUGGAACUGAAG
CUUCAGUUCCAGCCCCACU


 


3017
3018
2126-2144
GUGGGGCUGGAACUGAAGC
GCUUCAGUUCCAGCCCCAC


 


3019
3020
2127-2145
UGGGGCUGGAACUGAAGCC
GGCUUCAGUUCCAGCCCCA


 


3021
3022
2147-2165
CAUUCCUCAGCUGAGUGCA
UGCACUCAGCUGAGGAAUG


 


3023
3024
2148-2166
AUUCCUCAGCUGAGUGCAA
UUGCACUCAGCUGAGGAAU


 


3025
3026
2149-2167
UUCCUCAGCUGAGUGCAAC
GUUGCACUCAGCUGAGGAA


 


3027
3028
2150-2168
UCCUCAGCUGAGUGCAACU
AGUUGCACUCAGCUGAGGA


 


3029
3030
2151-2169
CCUCAGCUGAGUGCAACUU
AAGUUGCACUCAGCUGAGG


 


3031
3032
2152-2170
CUCAGCUGAGUGCAACUUC
GAAGUUGCACUCAGCUGAG


 


3033
3034
2153-2171
UCAGCUGAGUGCAACUUCU
AGAAGUUGCACUCAGCUGA


 


3035
3036
2154-2172
CAGCUGAGUGCAACUUCUG
CAGAAGUUGCACUCAGCUG


 


3037
3038
2155-2173
AGCUGAGUGCAACUUCUGC
GCAGAAGUUGCACUCAGCU


 


3039
3040
2156-2174
GCUGAGUGCAACUUCUGCA
UGCAGAAGUUGCACUCAGC


 


3041
3042
2157-2175
CUGAGUGCAACUUCUGCAG
CUGCAGAAGUUGCACUCAG


 


3043
3044
2158-2176
UGAGUGCAACUUCUGCAGG
CCUGCAGAAGUUGCACUCA


 


3045
3046
2159-2177
GAGUGCAACUUCUGCAGGA
UCCUGCAGAAGUUGCACUC


 


3047
3048
2160-2178
AGUGCAACUUCUGCAGGAG
CUCCUGCAGAAGUUGCACU


 


3049
3050
2161-2179
GUGCAACUUCUGCAGGAGG
CCUCCUGCAGAAGUUGCAC


 


3051
3052
2162-2180
UGCAACUUCUGCAGGAGGC
GCCUCCUGCAGAAGUUGCA


 


3053
3054
2163-2181
GCAACUUCUGCAGGAGGCC
GGCCUCCUGCAGAAGUUGC


 


3055
3056
2164-2182
CAACUUCUGCAGGAGGCCA
UGGCCUCCUGCAGAAGUUG


 


3057
3058
2165-2183
AACUUCUGCAGGAGGCCAC
GUGGCCUCCUGCAGAAGUU


 


3059
3060
2166-2184
ACUUCUGCAGGAGGCCACU
AGUGGCCUCCUGCAGAAGU


 


3061
3062
2167-2185
CUUCUGCAGGAGGCCACUG
CAGUGGCCUCCUGCAGAAG


 


3063
3064
2168-2186
UUCUGCAGGAGGCCACUGC
GCAGUGGCCUCCUGCAGAA


 


3065
3066
2169-2187
UCUGCAGGAGGCCACUGCA
UGCAGUGGCCUCCUGCAGA


 


3067
3068
2170-2188
CUGCAGGAGGCCACUGCAU
AUGCAGUGGCCUCCUGCAG


 


3069
3070
2171-2189
UGCAGGAGGCCACUGCAUU
AAUGCAGUGGCCUCCUGCA


 


3071
3072
2172-2190
GCAGGAGGCCACUGCAUUU
AAAUGCAGUGGCCUCCUGC


 


3073
3074
2173-2191
CAGGAGGCCACUGCAUUUU
AAAAUGCAGUGGCCUCCUG


 


3075
3076
2174-2192
AGGAGGCCACUGCAUUUUG
CAAAAUGCAGUGGCCUCCU


 


3077
3078
2175-2193
GGAGGCCACUGCAUUUUGA
UCAAAAUGCAGUGGCCUCC


 


3079
3080
2176-2194
GAGGCCACUGCAUUUUGAA
UUCAAAAUGCAGUGGCCUC


 


3081
3082
2177-2195
AGGCCACUGCAUUUUGAAG
CUUCAAAAUGCAGUGGCCU


 


3083
3084
2178-2196
GGCCACUGCAUUUUGAAGU
ACUUCAAAAUGCAGUGGCC


 


3085
3086
2179-2197
GCCACUGCAUUUUGAAGUG
CACUUCAAAAUGCAGUGGC


 


3087
3088
2180-2198
CCACUGCAUUUUGAAGUGA
UCACUUCAAAAUGCAGUGG


 


3089
3090
2181-2199
CACUGCAUUUUGAAGUGAU
AUCACUUCAAAAUGCAGUG


 


3091
3092
2182-2200
ACUGCAUUUUGAAGUGAUG
CAUCACUUCAAAAUGCAGU


 


3093
3094
2183-2201
CUGCAUUUUGAAGUGAUGA
UCAUCACUUCAAAAUGCAG


 


3095
3096
2184-2202
UGCAUUUUGAAGUGAUGAG
CUCAUCACUUCAAAAUGCA


 


3097
3098
2185-2203
GCAUUUUGAAGUGAUGAGU
ACUCAUCACUUCAAAAUGC


 


3099
3100
2186-2204
CAUUUUGAAGUGAUGAGUG
CACUCAUCACUUCAAAAUG


 


3101
3102
2187-2205
AUUUUGAAGUGAUGAGUGA
UCACUCAUCACUUCAAAAU


 


3103
3104
2188-2206
UUUUGAAGUGAUGAGUGAA
UUCACUCAUCACUUCAAAA


 


3105
3106
2190-2208
UUGAAGUGAUGAGUGAAAG
CUUUCACUCAUCACUUCAA


 


3107
3108
2191-2209
UGAAGUGAUGAGUGAAAGA
UCUUUCACUCAUCACUUCA


 


3109
3110
2192-2210
GAAGUGAUGAGUGAAAGAG
CUCUUUCACUCAUCACUUC


 


3111
3112
2193-2211
AAGUGAUGAGUGAAAGAGA
UCUCUUUCACUCAUCACUU


 


3113
3114
2194-2212
AGUGAUGAGUGAAAGAGAG
CUCUCUUUCACUCAUCACU


 


3115
3116
2195-2213
GUGAUGAGUGAAAGAGAGA
UCUCUCUUUCACUCAUCAC


 


3117
3118
2196-2214
UGAUGAGUGAAAGAGAGAA
UUCUCUCUUUCACUCAUCA


 


3119
3120
2197-2215
GAUGAGUGAAAGAGAGAAG
CUUCUCUCUUUCACUCAUC


 


3121
3122
2198-2216
AUGAGUGAAAGAGAGAAGU
ACUUCUCUCUUUCACUCAU


 


3123
3124
2199-2217
UGAGUGAAAGAGAGAAGUC
GACUUCUCUCUUUCACUCA


 


3125
3126
2200-2218
GAGUGAAAGAGAGAAGUCC
GGACUUCUCUCUUUCACUC


 


3127
3128
2201-2219
AGUGAAAGAGAGAAGUCCU
AGGACUUCUCUCUUUCACU


 


3129
3130
2202-2220
GUGAAAGAGAGAAGUCCUA
UAGGACUUCUCUCUUUCAC


 


3131
3132
2203-2221
UGAAAGAGAGAAGUCCUAU
AUAGGACUUCUCUCUUUCA


 


3133
3134
2204-2222
GAAAGAGAGAAGUCCUAUU
AAUAGGACUUCUCUCUUUC


 


3135
3136
2205-2223
AAAGAGAGAAGUCCUAUUU
AAAUAGGACUUCUCUCUUU


 


3137
3138
2206-2224
AAGAGAGAAGUCCUAUUUC
GAAAUAGGACUUCUCUCUU


 


3139
3140
2207-2225
AGAGAGAAGUCCUAUUUCU
AGAAAUAGGACUUCUCUCU


 


3141
3142
2208-2226
GAGAGAAGUCCUAUUUCUC
GAGAAAUAGGACUUCUCUC


 


3143
3144
2209-2227
AGAGAAGUCCUAUUUCUCA
UGAGAAAUAGGACUUCUCU


 


3145
3146
2210-2228
GAGAAGUCCUAUUUCUCAG
CUGAGAAAUAGGACUUCUC


 


3147
3148
2211-2229
AGAAGUCCUAUUUCUCAGG
CCUGAGAAAUAGGACUUCU


 


3149
3150
2212-2230
GAAGUCCUAUUUCUCAGGC
GCCUGAGAAAUAGGACUUC


 


3151
3152
2213-2231
AAGUCCUAUUUCUCAGGCU
AGCCUGAGAAAUAGGACUU


 


3153
3154
2214-2232
AGUCCUAUUUCUCAGGCUU
AAGCCUGAGAAAUAGGACU


 


3155
3156
2215-2233
GUCCUAUUUCUCAGGCUUG
CAAGCCUGAGAAAUAGGAC


 


3157
3158
2216-2234
UCCUAUUUCUCAGGCUUGA
UCAAGCCUGAGAAAUAGGA


 


3159
3160
2217-2235
CCUAUUUCUCAGGCUUGAG
CUCAAGCCUGAGAAAUAGG


 


3161
3162
2218-2236
CUAUUUCUCAGGCUUGAGC
GCUCAAGCCUGAGAAAUAG


 


3163
3164
2219-2237
UAUUUCUCAGGCUUGAGCA
UGCUCAAGCCUGAGAAAUA


 


3165
3166
2220-2238
AUUUCUCAGGCUUGAGCAA
UUGCUCAAGCCUGAGAAAU


 


3167
3168
2221-2239
UUUCUCAGGCUUGAGCAAG
CUUGCUCAAGCCUGAGAAA


 


3169
3170
2222-2240
UUCUCAGGCUUGAGCAAGU
ACUUGCUCAAGCCUGAGAA


 


3171
3172
2223-2241
UCUCAGGCUUGAGCAAGUU
AACUUGCUCAAGCCUGAGA


 


3173
3174
2224-2242
CUCAGGCUUGAGCAAGUUG
CAACUUGCUCAAGCCUGAG


 


3175
3176
2225-2243
UCAGGCUUGAGCAAGUUGG
CCAACUUGCUCAAGCCUGA


 


3177
3178
2226-2244
CAGGCUUGAGCAAGUUGGU
ACCAACUUGCUCAAGCCUG


 


3179
3180
2229-2247
GCUUGAGCAAGUUGGUAUC
GAUACCAACUUGCUCAAGC


 


3181
3182
2231-2249
UUGAGCAAGUUGGUAUCUG
CAGAUACCAACUUGCUCAA


 


3183
3184
2232-2250
UGAGCAAGUUGGUAUCUGC
GCAGAUACCAACUUGCUCA


 


3185
3186
2233-2251
GAGCAAGUUGGUAUCUGCU
AGCAGAUACCAACUUGCUC


 


3187
3188
2234-2252
AGCAAGUUGGUAUCUGCUC
GAGCAGAUACCAACUUGCU


 


3189
3190
2235-2253
GCAAGUUGGUAUCUGCUCA
UGAGCAGAUACCAACUUGC


 


3191
3192
2236-2254
CAAGUUGGUAUCUGCUCAG
CUGAGCAGAUACCAACUUG


 


3193
3194
2237-2255
AAGUUGGUAUCUGCUCAGG
CCUGAGCAGAUACCAACUU


 


3195
3196
2238-2256
AGUUGGUAUCUGCUCAGGC
GCCUGAGCAGAUACCAACU


 


3197
3198
2239-2257
GUUGGUAUCUGCUCAGGCC
GGCCUGAGCAGAUACCAAC


 


3199
3200
2240-2258
UUGGUAUCUGCUCAGGCCU
AGGCCUGAGCAGAUACCAA


 


3201
3202
2241-2259
UGGUAUCUGCUCAGGCCUG
CAGGCCUGAGCAGAUACCA


 


3203
3204
2242-2260
GGUAUCUGCUCAGGCCUGA
UCAGGCCUGAGCAGAUACC


 


3205
3206
2243-2261
GUAUCUGCUCAGGCCUGAG
CUCAGGCCUGAGCAGAUAC


 


3207
3208
2244-2262
UAUCUGCUCAGGCCUGAGC
GCUCAGGCCUGAGCAGAUA


 


3209
3210
2245-2263
AUCUGCUCAGGCCUGAGCA
UGCUCAGGCCUGAGCAGAU


 


3211
3212
2246-2264
UCUGCUCAGGCCUGAGCAU
AUGCUCAGGCCUGAGCAGA


 


3213
3214
2247-2265
CUGCUCAGGCCUGAGCAUG
CAUGCUCAGGCCUGAGCAG


 


3215
3216
2248-2266
UGCUCAGGCCUGAGCAUGA
UCAUGCUCAGGCCUGAGCA


 


3217
3218
2249-2267
GCUCAGGCCUGAGCAUGAC
GUCAUGCUCAGGCCUGAGC


 


3219
3220
2250-2268
CUCAGGCCUGAGCAUGACC
GGUCAUGCUCAGGCCUGAG


 


3221
3222
2251-2269
UCAGGCCUGAGCAUGACCU
AGGUCAUGCUCAGGCCUGA


 


3223
3224
2252-2270
CAGGCCUGAGCAUGACCUC
GAGGUCAUGCUCAGGCCUG


 


3225
3226
2253-2271
AGGCCUGAGCAUGACCUCA
UGAGGUCAUGCUCAGGCCU


 


3227
3228
2279-2297
CACUUAACCCCAGGCCAUU
AAUGGCCUGGGGUUAAGUG


 


3229
3230
2280-2298
ACUUAACCCCAGGCCAUUA
UAAUGGCCUGGGGUUAAGU


 


3231
3232
2281-2299
CUUAACCCCAGGCCAUUAU
AUAAUGGCCUGGGGUUAAG


 


3233
3234
2282-2300
UUAACCCCAGGCCAUUAUC
GAUAAUGGCCUGGGGUUAA


 


3235
3236
2283-2301
UAACCCCAGGCCAUUAUCA
UGAUAAUGGCCUGGGGUUA


 


3237
3238
2284-2302
AACCCCAGGCCAUUAUCAU
AUGAUAAUGGCCUGGGGUU


 


3239
3240
2285-2303
ACCCCAGGCCAUUAUCAUA
UAUGAUAAUGGCCUGGGGU


 


3241
3242
2287-2305
CCCAGGCCAUUAUCAUAUC
GAUAUGAUAAUGGCCUGGG


 


3243
3244
2288-2306
CCAGGCCAUUAUCAUAUCC
GGAUAUGAUAAUGGCCUGG


 


3245
3246
2289-2307
CAGGCCAUUAUCAUAUCCA
UGGAUAUGAUAAUGGCCUG


 


3247
3248
2290-2308
AGGCCAUUAUCAUAUCCAG
CUGGAUAUGAUAAUGGCCU


 


3249
3250
2291-2309
GGCCAUUAUCAUAUCCAGA
UCUGGAUAUGAUAAUGGCC


 


3251
3252
2292-2310
GCCAUUAUCAUAUCCAGAU
AUCUGGAUAUGAUAAUGGC


 


3253
3254
2314-2332
CUUCAGAGUUGUCUUUAUA
UAUAAAGACAACUCUGAAG


 


3255
3256
2315-2333
UUCAGAGUUGUCUUUAUAU
AUAUAAAGACAACUCUGAA


 


3257
3258
2316-2334
UCAGAGUUGUCUUUAUAUG
CAUAUAAAGACAACUCUGA


 


3259
3260
2318-2336
AGAGUUGUCUUUAUAUGUG
CACAUAUAAAGACAACUCU


 


3261
3262
2322-2340
UUGUCUUUAUAUGUGAAUU
AAUUCACAUAUAAAGACAA


 


3263
3264
2323-2341
UGUCUUUAUAUGUGAAUUA
UAAUUCACAUAUAAAGACA


 


3265
3266
2324-2342
GUCUUUAUAUGUGAAUUAA
UUAAUUCACAUAUAAAGAC


 


3267
3268
2325-2343
UCUUUAUAUGUGAAUUAAG
CUUAAUUCACAUAUAAAGA


 


3269
3270
2326-2344
CUUUAUAUGUGAAUUAAGU
ACUUAAUUCACAUAUAAAG


 


3271
3272
2327-2345
UUUAUAUGUGAAUUAAGUU
AACUUAAUUCACAUAUAAA


 


3273
3274
2328-2346
UUAUAUGUGAAUUAAGUUA
UAACUUAAUUCACAUAUAA


 


3275
3276
2329-2347
UAUAUGUGAAUUAAGUUAU
AUAACUUAAUUCACAUAUA


 


3277
3278
2330-2348
AUAUGUGAAUUAAGUUAUA
UAUAACUUAAUUCACAUAU


 


3279
3280
2331-2349
UAUGUGAAUUAAGUUAUAU
AUAUAACUUAAUUCACAUA


 


3281
3282
2332-2350
AUGUGAAUUAAGUUAUAUU
AAUAUAACUUAAUUCACAU


 


3283
3284
2333-2351
UGUGAAUUAAGUUAUAUUA
UAAUAUAACUUAAUUCACA


 


3285
3286
2334-2352
GUGAAUUAAGUUAUAUUAA
UUAAUAUAACUUAAUUCAC


 


3287
3288
2335-2353
UGAAUUAAGUUAUAUUAAA
UUUAAUAUAACUUAAUUCA


 


3289
3290
2336-2354
GAAUUAAGUUAUAUUAAAU
AUUUAAUAUAACUUAAUUC


 


3291
3292
2337-2355
AAUUAAGUUAUAUUAAAUU
AAUUUAAUAUAACUUAAUU


 


3293
3294
2338-2356
AUUAAGUUAUAUUAAAUUU
AAAUUUAAUAUAACUUAAU


 


3295
3296
2339-2357
UUAAGUUAUAUUAAAUUUU
AAAAUUUAAUAUAACUUAA


 


3297
3298
2340-2358
UAAGUUAUAUUAAAUUUUA
UAAAAUUUAAUAUAACUUA


 


3299
3300
2341-2359
AAGUUAUAUUAAAUUUUAA
UUAAAAUUUAAUAUAACUU


 


3301
3302
2342-2360
AGUUAUAUUAAAUUUUAAU
AUUAAAAUUUAAUAUAACU


 


3303
3304
2343-2361
GUUAUAUUAAAUUUUAAUC
GAUUAAAAUUUAAUAUAAC


 


3305
3306
2345-2363
UAUAUUAAAUUUUAAUCUA
UAGAUUAAAAUUUAAUAUA


 


3307
3308
2346-2364
AUAUUAAAUUUUAAUCUAU
AUAGAUUAAAAUUUAAUAU


 


3309
3310
2347-2365
UAUUAAAUUUUAAUCUAUA
UAUAGAUUAAAAUUUAAUA


 


3311
3312
2348-2366
AUUAAAUUUUAAUCUAUAG
CUAUAGAUUAAAAUUUAAU


 


3313
3314
2349-2367
UUAAAUUUUAAUCUAUAGU
ACUAUAGAUUAAAAUUUAA


 


3315
3316
2350-2368
UAAAUUUUAAUCUAUAGUA
UACUAUAGAUUAAAAUUUA


 


3317
3318
2351-2369
AAAUUUUAAUCUAUAGUAA
UUACUAUAGAUUAAAAUUU


 


3319
3320
2354-2372
UUUUAAUCUAUAGUAAAAA
UUUUUACUAUAGAUUAAAA


 


3321
3322
2355-2373
UUUAAUCUAUAGUAAAAAC
GUUUUUACUAUAGAUUAAA


 


3323
3324
2356-2374
UUAAUCUAUAGUAAAAACA
UGUUUUUACUAUAGAUUAA


 


3325
3326
2357-2375
UAAUCUAUAGUAAAAACAU
AUGUUUUUACUAUAGAUUA


 


3327
3328
2358-2376
AAUCUAUAGUAAAAACAUA
UAUGUUUUUACUAUAGAUU


 


3329
3330
2359-2377
AUCUAUAGUAAAAACAUAG
CUAUGUUUUUACUAUAGAU


 


3331
3332
2360-2378
UCUAUAGUAAAAACAUAGU
ACUAUGUUUUUACUAUAGA


 


3333
3334
2361-2379
CUAUAGUAAAAACAUAGUC
GACUAUGUUUUUACUAUAG


 


3335
3336
2362-2380
UAUAGUAAAAACAUAGUCC
GGACUAUGUUUUUACUAUA


 


3337
3338
2363-2381
AUAGUAAAAACAUAGUCCU
AGGACUAUGUUUUUACUAU


 


3339
3340
2364-2382
UAGUAAAAACAUAGUCCUG
CAGGACUAUGUUUUUACUA


 


3341
3342
2365-2383
AGUAAAAACAUAGUCCUGG
CCAGGACUAUGUUUUUACU


 


3343
3344
2366-2384
GUAAAAACAUAGUCCUGGA
UCCAGGACUAUGUUUUUAC


 


3345
3346
2367-2385
UAAAAACAUAGUCCUGGAA
UUCCAGGACUAUGUUUUUA


 


3347
3348
2368-2386
AAAAACAUAGUCCUGGAAA
UUUCCAGGACUAUGUUUUU


 


3349
3350
2369-2387
AAAACAUAGUCCUGGAAAU
AUUUCCAGGACUAUGUUUU


 


3351
3352
2370-2388
AAACAUAGUCCUGGAAAUA
UAUUUCCAGGACUAUGUUU


 


3353
3354
2371-2389
AACAUAGUCCUGGAAAUAA
UUAUUUCCAGGACUAUGUU


 


3355
3356
2372-2390
ACAUAGUCCUGGAAAUAAA
UUUAUUUCCAGGACUAUGU


 


3357
3358
2373-2391
CAUAGUCCUGGAAAUAAAU
AUUUAUUUCCAGGACUAUG


 


3359
3360
2374-2392
AUAGUCCUGGAAAUAAAUU
AAUUUAUUUCCAGGACUAU


 


3361
3362
2375-2393
UAGUCCUGGAAAUAAAUUC
GAAUUUAUUUCCAGGACUA


 


3363
3364
2377-2395
GUCCUGGAAAUAAAUUCUU
AAGAAUUUAUUUCCAGGAC


 


3365
3366
2378-2396
UCCUGGAAAUAAAUUCUUG
CAAGAAUUUAUUUCCAGGA









Example 9
Suppression of Porphyrin Precursors Using ALAS1 siRNA in an Acute Treatment Paradigm
The AIP mouse model (see Example 5) was used to investigate whether ALAS1 siRNA would work an acute treatment paradigm to lower already elevated levels of ALA and PBG, as would be present, for example, when a human porphyria patient suffers from an acute attack. Administration of the AD-53558 LNP11 formulation siRNA at a 1 mg/kg dose 12 hours after the last dose of phenobarbitol rapidly decreased the levels of both ALA and PBG in mouse plasma, whereas in Luc control treated animals the levels continued to rise (FIG. 14). These results indicate that ALAS siRNA is effective for treating an acute attack. The ALAS1 siRNA was effective to lower and prevent further increases in ALA and PBG levels.
Example 10
siRNAs that Target ALAS1
Further unmodified and modified siRNA sequences that target ALAS1 siRNA were designed and produced as described in Example 2. The in vitro activity of the modified duplexes was tested as described below.
Methods
Lipid Mediated Transfection
For Hep3B, PMH, and primary Cynomolgus hepatocytes, transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. catalog number 13778-150) to 5 μl of each siRNA duplex to an individual well in a 96-well plate. The mixture was then incubated at room temperature for 20 minutes. Eighty μl of complete growth media without antibiotic containing the appropriate cell number were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification.
Single dose experiments were performed at 1 uM, 500 nM, 20 nM, 10 nM and 0.2 nM final duplex concentration for GalNAc modified.
Free Uptake Transfection
Cryopreserved Primary Cynomolgus Hepatocytes (Celsis In Vitro Technologies, M003055-P) were thawed at 37° C. water bath immediately prior to usage and re-suspended at 0.26×106 cells/ml in InVitroGRO CP (plating) medium (Celsis In Vitro Technologies, catalog number Z99029). During transfections, cells were plated onto a BD BioCoat 96 well collagen plate (BD, 356407) at 25,000 cells per well and incubated at 37° C. in an atmosphere of 5% CO2. Free Uptake experiments were performed by adding 10 μl of siRNA duplexes in PBS per well into a 96 well (96 w) plate. Ninety μl of complete growth media containing appropriate cell number for the cell type was then added to the siRNA. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 1 uM, 500 nM, 20 nM and 10 nM final duplex.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using a magnetic stand and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, magnetic beads were washed 2 times with 150 Wash Buffer A and mixed for 1 minute. The beads were capturedagain and the supernatant was removed. The beads were then washed with 150 μl Wash Buffer B, captured and the supernatant was removed. The beads were next washed with 150 μl Elution Buffer, captured and the supernatant removed. Finally, the beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. The beads were captured on magnet for 5 minutes. Forty-five μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)
A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction as prepared. Equal volumes master mix and RNA were mixed for a final volume of 12 μl for in vitro screened or 20 μl for in vivo screened samples. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. for 10 minutes, 37° C. for 120 minutes, 85° C. for 5 seconds, and 4° C. hold.
Real Time PCR
Two μl of cDNA were added to a master mix containing 2 μl of H2O, 0.5 μl GAPDH TaqMan Probe (Life Technologies catalog number 4326317E for Hep3B cells, catalog number 352339E for primary mouse hepatocytes or custom probe for cynomolgus primary hepatocytes), 0.5 μl C5 TaqMan probe (Life Technologies catalog number Hs00167441_ml for Hep3B cells or Mm00457879_ml for Primary Mouse Hepatoctyes or custom probe for cynomolgus primary hepatocytes) and 5 μl Lightcycler 480 probe master mix (Roche catalog number 04887301001) per well in a 384 well (384 w) plates (Roche catalog number 04887301001). Real time PCR was performed in an Roche LC480 Real Time PCR system (Roche) using the ΔΔCt(RQ) assay. For in vitro screening, each duplex was tested with two biological replicates unless otherwise noted and each Real Time PCR was performed in duplicate technical replicates. For in vivo screening, each duplex was tested in one or more experiments (3 mice per group) and each Real Time PCR was run in duplicate technical replicates.
To calculate relative fold change in ALAS1 mRNA levels, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 over the same dose range, or to its own lowest dose.
The sense and antisense sequences of AD-1955 are:









SENSE:







(SEQ ID NO: 3682)









cuuAcGcuGAGuAcuucGAdTsdT



 



ANTISENSE:







(SEQ ID NO: 3683)









UCGAAGuACUcAGCGuAAGdTsdT.






The single strand and duplex sequences of the modified and unmodified siRNAs are provided in Table 14 and Table 15, respectively.







TABLE 14







Human ALAS1 Modified Single Strands and Duplex Sequences













SEQ



Target



ID



sites of


SEQ ID
NO:



antisense


NO:
(anti-
Duplex


sequence on


(sense)
sense)
Name
Sense Sequence (5′-3′)
Antisense Sequence (5′-3′)
NM_000688.4





3371
3372
AD-58848
CfsasUfgCfcAfaAfAfAfuGfgAfcAf
asUfsgAfuGfuCfcAfuuuUfuGfgCfaU
1635-1657





uCfaUfL96
fgsAfsc


 


3373
3374
AD-58849
AfsusUfuUfgAfaGfUfGfaUfgAfgU
usUfsuCfaCfuCfaUfcacUfuCfaAfaAf
2189-2211





fgAfaAfL96
usGfsc


 


3375
3376
AD-58850
AfsgsUfuAfuAfuUfAfAfaUfuUfuA
asGfsaUfuAfaAfaUfuuaAfuAfuAfaC
2344-2366





faUfcUfL96
fusUfsa


 


3377
3378
AD-58851
GfscsAfuUfuUfgAfAfGfuGfaUfgA
usCfsaCfuCfaUfcAfcuuCfaAfaAfuGf
2187-2209





fgUfgAfL96
csAfsg


 


3379
3380
AD-58852
GfsasAfcUfaAfuGfAfGfcAfgAfcAf
gsUfsuAfuGfuCfuGfcucAfuUfaGfuU
1975-1997





uAfaCfL96
fcsAfsu


 


3381
3382
AD-58853
AfsasUfgAfcCfaCfAfCfcUfaUfcGf
asAfscUfcGfaUfaGfgugUfgGfuCfaU
973-995





aGfuUfL96
fusCfsu


 


3383
3384
AD-58854
UfsasAfaUfuUfuAfAfUfcUfaUfaG
usUfsuAfcUfaUfaGfauuAfaAfaUfuU
2352-2374





fuAfaAfL96
fasAfsu


 


3385
3386
AD-58855
UfsusCfaGfuAfuGfAfUfcGfuUfuC
csAfsaAfgAfaAfcGfaucAfuAfcUfgAf
929-951





fuUfuGfL96
asAfsa


 


3387
3388
AD-58856
CfsasCfuUfuUfcAfGfUfaUfgAfuCf
asAfsaCfgAfuCfaUfacuGfaAfaAfgUf
924-946





gUfuUfL96
gsGfsa


 


3389
3390
AD-58857
AfsasAfuCfuGfuUfUfCfcAfcUfuUf
csUfsgAfaAfaGfuGfgaaAfcAfgAfuUf
913-935





uCfaGfL96
usUfsg


 


3391
3392
AD-58858
CfsasUfuUfgAfaAfCfUfgUfcCfaUf
usUfsgAfaUfgGfaCfaguUfuCfaAfaU
1478-1500





uCfaAfL96
fgsCfsc


 


3393
3394
AD-58859
CfscsUfaUfcGfaGfUfUfuUfuAfaA
csAfsgUfuUfuAfaAfaacUfcGfaUfaG
 983-1005





faCfuGfL96
fgsUfsg


 


3395
3396
AD-58861
GfsasCfcAfgAfaAfGfAfgUfgUfcUf
gsAfsuGfaGfaCfaCfucuUfuCfuGfgU
872-894





cAfuCfL96
fcsUfsu


 


3397
3398
AD-58862
AfscsCfaGfaAfaGfAfGfuGfuCfuCf
asGfsaUfgAfgAfcAfcucUfuUfcUfgGf
873-895





aUfcUfL96
usCfsu


 


3399
3400
AD-58863
AfscsUfaAfuGfaGfCfAfgAfcAfuAf
asUfsgUfuAfuGfuCfugcUfcAfuUfaG
1977-1999





aCfaUfL96
fusUfsc


 


3401
3402
AD-58864
UfsasGfuAfaAfaAfCfAfuAfgUfcCf
usCfscAfgGfaCfuAfuguUfuUfuAfcU
2366-2388





uGfgAfL96
fasUfsa


 


3403
3404
AD-58865
UfsasUfuUfcUfgGfAfAfcUfaGfuA
asAfsuUfuAfcUfaGfuucCfaGfaAfaU
1185-1207





faAfuUfL96
fasUfsu


 


3405
3406
AD-58867
UfsusCfuGfcAfaAfGfCfcAfgUfcUf
csUfscAfaGfaCfuGfgcuUfuGfcAfgAf
706-728





uGfaGfL96
asGfsa


 


3407
3408
AD-58868
GfsasGfgAfaAfgAfGfGfuUfgCfuG
gsUfsuUfcAfgCfaAfccuCfuUfuCfcUf
759-781





faAfaCfL96
csAfsc


 


3409
3410
AD-58869
GfsgsUfaCfuAfgAfAfAfuAfuUfuCf
usCfscAfgAfaAfuAfuuuCfuAfgUfaCf
1174-1196





uGfgAfL96
csAfsc


 


3411
3412
AD-58870
GfsasCfaUfcAfuGfCfAfaAfaGfcAf
usCfsuUfuGfcUfuUfugcAfuGfaUfgU
853-875





aAfgAfL96
fcsCfsu


 


3413
3414
AD-58871
AfsasAfuUfuUfaAfUfCfuAfuAfgU
usUfsuUfaCfuAfuAfgauUfaAfaAfuU
2353-2375





faAfaAfL96
fusAfsa


 


3415
3416
AD-58873
CfsasUfgAfuCfcAfAfGfgGfaUfuCf
usUfsuCfgAfaUfcCfcuuGfgAfuCfaUf
1362-1384





gAfaAfL96
gsGfsa


 


3417
3418
AD-58874
AfsgsAfcCfaGfaAfAfGfaGfuGfuCf
asUfsgAfgAfcAfcUfcuuUfcUfgGfuCf
871-893





uCfaUfL96
usUfsu


 


3419
3420
AD-58875
AfsusCfcUfgAfaGfAfGfcGfcUfgAf
usCfscCfuCfaGfcGfcucUfuCfaGfgAf
1810-1832





gGfgAfL96
usCfsc


 


3421
3422
AD-58876
GfsusCfuGfuGfaUfGfAfaCfuAfaU
gsCfsuCfaUfuAfgUfucaUfcAfcAfgAf
1966-1988





fgAfgCfL96
csUfsu


 


3423
3424
AD-58877
CfsasGfaAfaGfaGfUfGfuCfuCfaUf
gsAfsaGfaUfgAfgAfcacUfcUfuUfcUf
875-897





cUfuCfL96
gsGfsu


 


3425
3426
AD-58878
AfscsUfuUfuCfaGfUfAfuGfaUfcG
gsAfsaAfcGfaUfcAfuacUfgAfaAfaGf
925-947





fuUfuCfL96
usGfsg


 


3427
3428
AD-58879
UfscsAfuGfcCfaAfAfAfaUfgGfaCf
usGfsaUfgUfcCfaUfuuuUfgGfcAfuG
1634-1656





aUfcAfL96
fasCfsu


 


3429
3430
AD-58880
AfsasUfaUfuUfcUfGfGfaAfcUfaG
usUfsuAfcUfaGfuUfccaGfaAfaUfaU
1183-1205





fuAfaAfL96
fusUfsc


 


3431
3432
AD-58881
CfsusUfcUfuCfaAfGfAfuAfaCfuUf
usGfsgCfaAfgUfuAfucuUfgAfaGfaA
892-914





gCfcAfL96
fgsAfsu


 


3433
3434
AD-58882
UfsusUfcAfgUfaUfGfAfuCfgUfuU
asAfsaGfaAfaCfgAfucaUfaCfuGfaAf
928-950





fcUfuUfL96
asAfsg


 


3435
3436
AD-58883
CfscsCfaGfuGfuGfGfUfuAfgUfgU
usUfsuCfaCfaCfuAfaccAfcAfcUfgGf
790-812





fgAfaAfL96
gsGfsc


 


3437
3438
AD-58884
GfscsUfgUfgAfgAfUfUfuAfcUfcUf
asAfsuCfaGfaGfuAfaauCfuCfaCfaGf
1325-1347





gAfuUfL96
csCfsu


 


3439
3440
AD-58885
AfsgsGfcUfuGfaGfCfAfaGfuUfgG
gsAfsuAfcCfaAfcUfugcUfcAfaGfcCf
2229-2251





fuAfuCfL96
usGfsa


 


3441
3442
AD-58886
GfsasAfaGfaGfuGfUfCfuCfaUfcU
asAfsgAfaGfaUfgAfgacAfcUfcUfuUf
877-899





fuCfuUfL96
csUfsg


 


3443
3444
AD-58887
AfsusUfuCfuGfgAfAfCfuAfgUfaAf
gsAfsaUfuUfaCfuAfguuCfcAfgAfaAf
1186-1208





aUfuCfL96
usAfsu


 


3445
3446
AD-58888
UfsgsUfgAfuGfuGfGfCfcCfaUfgAf
asAfsaCfuCfaUfgGfgccAfcAfuCfaCf
1531-1553





gUfuUfL96
asCfsa


 


3447
3448
AD-58889
AfsasGfaGfaGfaAfGfUfcCfuAfuU
gsAfsgAfaAfuAfgGfacuUfcUfcUfcUf
2208-2230





fuCfuCfL96
usUfsc


 


3449
3450
AD-58890
UfsgsGfcAfgCfaCfAfGfaUfgAfaUf
usCfsuGfaUfuCfaUfcugUfgCfuGfcCf
671-693





cAfgAfL96
asGfsg


 


3451
3452
AD-58891
AfsusGfaUfcGfuUfUfCfuUfuGfaG
usUfsuUfcUfcAfaAfgaaAfcGfaUfcAf
935-957





faAfaAfL96
usAfsc


 


3453
3454
AD-58892
UfscsUfgGfaAfcUfAfGfuAfaAfuU
asUfsgGfaAfuUfuAfcuaGfuUfcCfaG
1189-1211





fcCfaUfL96
fasAfsa


 


3455
3456
AD-59095
GfscsCfcAfuUfcUfUfAfuCfcCfgAf
asCfsuCfgGfgAfuAfagaAfuGfgsgsc
360-382





gUfL96


 


3457
3458
AD-59096
GfsgsAfaCfcAfuGfCfCfuCfcAfuGf
asUfscAfuGfgAfgGfcauGfgUfuscsc
1347-1369





aUfL96


 


3459
3460
AD-59097
UfsgsGfaGfuCfuGfUfGfcGfgAfuC
asGfsgAfuCfcGfcAfcagAfcUfcscsa
1794-1816





fcUfL96


 


3461
3462
AD-59098
CfsasCfcCfaCfgGfGfUfgUfgUfgGf
usCfscCfaCfaCfaCfccgUfgGfgsusg
1112-1134





gAfL96


 


3463
3464
AD-59099
GfsgsAfgUfcUfgUfGfCfgGfaUfcCf
usAfsgGfaUfcCfgCfacaGfaCfuscsc
1795-1817





uAfL96


 


3465
3466
AD-59100
CfsasAfaAfcUfgCfCfCfcAfaGfaUf
usCfsaUfcUfuGfgGfgcaGfuUfususg
428-450





gAfL96


 


3467
3468
AD-59101
GfscsCfuCfcAfuGfAfUfcCfaAfgGf
usCfscCfuUfgGfaUfcauGfgAfgsgsc
1355-1377





gAfL96


 


3469
3470
AD-59102
CfsasUfcAfuCfcCfUfGfuGfcGfgGf
asAfscCfcGfcAfcAfgggAfuGfasusg
1921-1943





uUfL96


 


3471
3472
AD-59103
AfscsCfcAfcGfgGfUfGfuGfuGfgGf
usCfscCfcAfcAfcAfcccGfuGfgsgsu
1113-1135





gAfL96


 


3473
3474
AD-59104
CfsasCfaUfcAfuCfCfCfuGfuGfcGf
usCfscGfcAfcAfgGfgauGfaUfgsusg
1919-1941





gAfL96


 


3475
3476
AD-59105
CfsasGfaAfaGfaGfUfGfuCfuCfaUf
asGfsaUfgAfgAfcAfcucUfuUfcsusg
873-895





cUfL96


 


3477
3478
AD-59106
CfscsUfcCfaUfgAfUfCfcAfaGfgGf
asUfscCfcUfuGfgAfucaUfgGfasgsg
1356-1378





aUfL96


 


3479
3480
AD-59107
UfsgsCfcCfaUfuCfUfUfaUfcCfcGf
usUfscGfgGfaUfaAfgaaUfgGfgscsa
359-381





aAfL96


 


3481
3482
AD-59108
CfsusUfcAfcCfcUfGfGfcUfaAfgAf
usAfsuCfuUfaGfcCfaggGfuGfasasg
1297-1319





uAfL96


 


3483
3484
AD-59109
AfsusCfaUfcCfcUfGfUfgCfgGfgUf
usAfsaCfcCfgCfaCfaggGfaUfgsasu
1922-1944





uAfL96


 


3485
3486
AD-59110
AfsgsAfaAfgAfgUfGfUfcUfcAfuCf
asAfsgAfuGfaGfaCfacuCfuUfuscsu
874-896





uUfL96


 


3487
3488
AD-59111
CfsusCfcAfuGfaUfCfCfaAfgGfgAf
asAfsuCfcCfuUfgGfaucAfuGfgsasg
1357-1379





uUfL96


 


3489
3490
AD-59112
CfscsAfuUfcUfuAfUfCfcCfgAfgUf
usGfsaCfuCfgGfgAfuaaGfaAfusgsg
362-384





cAfL96


 


3491
3492
AD-59113
CfsasCfcCfuGfgCfUfAfaGfaUfgAf
usAfsuCfaUfcUfuAfgccAfgGfgsusg
1300-1322





uAfL96


 


3493
3494
AD-59114
UfscsAfuCfcCfuGfUfGfcGfgGfuUf
usCfsaAfcCfcGfcAfcagGfgAfusgsa
1923-1945





gAfL96


 


3495
3496
AD-59115
AfsasGfaGfuGfuCfUfCfaUfcUfuCf
asAfsgAfaGfaUfgAfgacAfcUfcsusu
877-899





uUfL96


 


3497
3498
AD-59116
GfsusCfaUfgCfcAfAfAfaAfuGfgAf
usGfsuCfcAfuUfuUfuggCfaUfgsasc
1631-1653





cAfL96


 


3499
3500
AD-59117
CfsasUfuCfuUfaUfCfCfcGfaGfuCf
usGfsgAfcUfcGfgGfauaAfgAfasusg
363-385





cAfL96


 


3501
3502
AD-59118
AfscsCfcUfgGfcUfAfAfgAfuGfaUf
usCfsaUfcAfuCfuUfagcCfaGfgsgsu
1301-1323





gAfL96


 


3503
3504
AD-59119
CfsusCfuUfcAfcCfCfUfgGfcUfaAf
usCfsuUfaGfcCfaGfgguGfaAfgsasg
1295-1317





gAfL96


 


3505
3506
AD-59120
AfsusGfcCfaAfaAfAfUfgGfaCfaUf
usGfsaUfgUfcCfaUfuuuUfgGfcsasu
1634-1656





cAfL96


 


3507
3508
AD-59121
UfsgsCfcCfcAfaGfAfUfgAfuGfgAf
asUfsuCfcAfuCfaUfcuuGfgGfgscsa
434-456





aUfL96


 


3509
3510
AD-59122
GfsasAfcCfaUfgCfCfUfcCfaUfgAf
usAfsuCfaUfgGfaGfgcaUfgGfususc
1348-1370





uAfL96


 


3511
3512
AD-59123
UfscsUfuCfaCfcCfUfGfgCfuAfaGf
asUfscUfuAfgCfcAfgggUfgAfasgsa
1296-1318





aUfL96


 


3513
3514
AD-59124
UfsgsCfcAfaAfaAfUfGfgAfcAfuCf
asUfsgAfuGfuCfcAfuuuUfuGfgscsa
1635-1657





aUfL96


 


3515
3516
AD-59125
CfscsAfgAfaAfgAfGfUfgUfcUfcAf
usAfsuGfaGfaCfaCfucuUfuCfusgsg
872-894





uAfL96


 


3517
3518
AD-59126
GfsasAfaCfuGfuCfCfAfuUfcAfaUf
usCfsaUfuGfaAfuGfgacAfgUfususc
1481-1503





gAfL96


 


3519
3520
AD-59127
UfscsAfcCfcUfgGfCfUfaAfgAfuGf
asUfscAfuCfuUfaGfccaGfgGfusgsa
1299-1321





aUfL96


 


3521
3522
AD-59128
CfscsCfuGfgAfgUfCfUfgUfgCfgGf
asUfscCfgCfaCfaGfacuCfcAfgsgsg
1791-1813





aUfL96


 


3523
3524
AD-59129
GfsasAfaGfaGfuGfUfCfuCfaUfcU
usAfsaGfaUfgAfgAfcacUfcUfususc
875-897





fuAfL96


 


3525
3526
AD-59130
UfsgsGfaGfcCfcUfGfGfaGfuCfuG
usAfscAfgAfcUfcCfaggGfcUfcscsa
1786-1808





fuAfL96
















TABLE 15







Human ALAS1 Unmodified Single Strands and Duplex Sequences













SEQ







ID



Target sites


SEQ ID
NO:



of antisense


NO:
(anti-
Duplex


sequence on


(sense)
sense)
Name
Sense Sequence (5′-3′)
Antisense Sequence (5′-3′)
NM_000688.4





3684
3527
AD-58848
CAUGCCAAAAAUGGACAUCAU
AUGAUGUCCAUUUUUGGCAUGAC
1635-1657


 


3528
3529
AD-58849
AUUUUGAAGUGAUGAGUGAAA
UUUCACUCAUCACUUCAAAAUGC
2189-2211


 


3530
3531
AD-58850
AGUUAUAUUAAAUUUUAAUCU
AGAUUAAAAUUUAAUAUAACUUA
2344-2366


 


3532
3533
AD-58851
GCAUUUUGAAGUGAUGAGUGA
UCACUCAUCACUUCAAAAUGCAG
2187-2209


 


3534
3535
AD-58852
GAACUAAUGAGCAGACAUAAC
GUUAUGUCUGCUCAUUAGUUCAU
1975-1997


 


3536
3537
AD-58853
AAUGACCACACCUAUCGAGUU
AACUCGAUAGGUGUGGUCAUUCU
973-995


 


3538
3539
AD-58854
UAAAUUUUAAUCUAUAGUAAA
UUUACUAUAGAUUAAAAUUUAAU
2352-2374


 


3540
3541
AD-58855
UUCAGUAUGAUCGUUUCUUUG
CAAAGAAACGAUCAUACUGAAAA
929-951


 


3542
3543
AD-58856
CACUUUUCAGUAUGAUCGUUU
AAACGAUCAUACUGAAAAGUGGA
924-946


 


3544
3545
AD-58857
AAAUCUGUUUCCACUUUUCAG
CUGAAAAGUGGAAACAGAUUUUG
913-935


 


3546
3547
AD-58858
CAUUUGAAACUGUCCAUUCAA
UUGAAUGGACAGUUUCAAAUGCC
1478-1500


 


3548
3549
AD-58859
CCUAUCGAGUUUUUAAAACUG
CAGUUUUAAAAACUCGAUAGGUG
 983-1005


 


3550
3551
AD-58861
GACCAGAAAGAGUGUCUCAUC
GAUGAGACACUCUUUCUGGUCUU
872-894


 


3552
3553
AD-58862
ACCAGAAAGAGUGUCUCAUCU
AGAUGAGACACUCUUUCUGGUCU
873-895


 


3554
3555
AD-58863
ACUAAUGAGCAGACAUAACAU
AUGUUAUGUCUGCUCAUUAGUUC
1977-1999


 


3556
3557
AD-58864
UAGUAAAAACAUAGUCCUGGA
UCCAGGACUAUGUUUUUACUAUA
2366-2388


 


3558
3559
AD-58865
UAUUUCUGGAACUAGUAAAUU
AAUUUACUAGUUCCAGAAAUAUU
1185-1207


 


3560
3561
AD-58867
UUCUGCAAAGCCAGUCUUGAG
CUCAAGACUGGCUUUGCAGAAGA
706-728


 


3562
3563
AD-58868
GAGGAAAGAGGUUGCUGAAAC
GUUUCAGCAACCUCUUUCCUCAC
759-781


 


3564
3565
AD-58869
GGUACUAGAAAUAUUUCUGGA
UCCAGAAAUAUUUCUAGUACCAC
1174-1196


 


3566
3567
AD-58870
GACAUCAUGCAAAAGCAAAGA
UCUUUGCUUUUGCAUGAUGUCCU
853-875


 


3568
3569
AD-58871
AAAUUUUAAUCUAUAGUAAAA
UUUUACUAUAGAUUAAAAUUUAA
2353-2375


 


3570
3571
AD-58873
CAUGAUCCAAGGGAUUCGAAA
UUUCGAAUCCCUUGGAUCAUGGA
1362-1384


 


3572
3573
AD-58874
AGACCAGAAAGAGUGUCUCAU
AUGAGACACUCUUUCUGGUCUUU
871-893


 


3574
3575
AD-58875
AUCCUGAAGAGCGCUGAGGGA
UCCCUCAGCGCUCUUCAGGAUCC
1810-1832


 


3576
3577
AD-58876
GUCUGUGAUGAACUAAUGAGC
GCUCAUUAGUUCAUCACAGACUU
1966-1988


 


3578
3579
AD-58877
CAGAAAGAGUGUCUCAUCUUC
GAAGAUGAGACACUCUUUCUGGU
875-897


 


3580
3581
AD-58878
ACUUUUCAGUAUGAUCGUUUC
GAAACGAUCAUACUGAAAAGUGG
925-947


 


3582
3583
AD-58879
UCAUGCCAAAAAUGGACAUCA
UGAUGUCCAUUUUUGGCAUGACU
1634-1656


 


3584
3585
AD-58880
AAUAUUUCUGGAACUAGUAAA
UUUACUAGUUCCAGAAAUAUUUC
1183-1205


 


3586
3587
AD-58881
CUUCUUCAAGAUAACUUGCCA
UGGCAAGUUAUCUUGAAGAAGAU
892-914


 


3588
3589
AD-58882
UUUCAGUAUGAUCGUUUCUUU
AAAGAAACGAUCAUACUGAAAAG
928-950


 


3590
3591
AD-58883
CCCAGUGUGGUUAGUGUGAAA
UUUCACACUAACCACACUGGGGC
790-812


 


3592
3593
AD-58884
GCUGUGAGAUUUACUCUGAUU
AAUCAGAGUAAAUCUCACAGCCU
1325-1347


 


3594
3595
AD-58885
AGGCUUGAGCAAGUUGGUAUC
GAUACCAACUUGCUCAAGCCUGA
2229-2251


 


3596
3597
AD-58886
GAAAGAGUGUCUCAUCUUCUU
AAGAAGAUGAGACACUCUUUCUG
877-899


 


3598
3599
AD-58887
AUUUCUGGAACUAGUAAAUUC
GAAUUUACUAGUUCCAGAAAUAU
1186-1208


 


3600
3601
AD-58888
UGUGAUGUGGCCCAUGAGUUU
AAACUCAUGGGCCACAUCACACA
1531-1553


 


3602
3603
AD-58889
AAGAGAGAAGUCCUAUUUCUC
GAGAAAUAGGACUUCUCUCUUUC
2208-2230


 


3604
3605
AD-58890
UGGCAGCACAGAUGAAUCAGA
UCUGAUUCAUCUGUGCUGCCAGG
671-693


 


3606
3607
AD-58891
AUGAUCGUUUCUUUGAGAAAA
UUUUCUCAAAGAAACGAUCAUAC
935-957


 


3608
3609
AD-58892
UCUGGAACUAGUAAAUUCCAU
AUGGAAUUUACUAGUUCCAGAAA
1189-1211


 


3610
3611
AD-59095
GCCCAUUCUUAUCCCGAGU
ACUCGGGAUAAGAAUGGGC
360-382


 


3612
3613
AD-59096
GGAACCAUGCCUCCAUGAU
AUCAUGGAGGCAUGGUUCC
1347-1369


 


3614
3615
AD-59097
UGGAGUCUGUGCGGAUCCU
AGGAUCCGCACAGACUCCA
1794-1816


 


3616
3617
AD-59098
CACCCACGGGUGUGUGGGA
UCCCACACACCCGUGGGUG
1112-1134


 


3618
3619
AD-59099
GGAGUCUGUGCGGAUCCUA
UAGGAUCCGCACAGACUCC
1795-1817


 


3620
3621
AD-59100
CAAAACUGCCCCAAGAUGA
UCAUCUUGGGGCAGUUUUG
428-450


 


3622
3623
AD-59101
GCCUCCAUGAUCCAAGGGA
UCCCUUGGAUCAUGGAGGC
1355-1377


 


3624
3625
AD-59102
CAUCAUCCCUGUGCGGGUU
AACCCGCACAGGGAUGAUG
1921-1943


 


3626
3627
AD-59103
ACCCACGGGUGUGUGGGGA
UCCCCACACACCCGUGGGU
1113-1135


 


3628
3629
AD-59104
CACAUCAUCCCUGUGCGGA
UCCGCACAGGGAUGAUGUG
1919-1941


 


3630
3631
AD-59105
CAGAAAGAGUGUCUCAUCU
AGAUGAGACACUCUUUCUG
873-895


 


3632
3633
AD-59106
CCUCCAUGAUCCAAGGGAU
AUCCCUUGGAUCAUGGAGG
1356-1378


 


3634
3635
AD-59107
UGCCCAUUCUUAUCCCGAA
UUCGGGAUAAGAAUGGGCA
359-381


 


3636
3637
AD-59108
CUUCACCCUGGCUAAGAUA
UAUCUUAGCCAGGGUGAAG
1297-1319


 


3638
3639
AD-59109
AUCAUCCCUGUGCGGGUUA
UAACCCGCACAGGGAUGAU
1922-1944


 


3640
3641
AD-59110
AGAAAGAGUGUCUCAUCUU
AAGAUGAGACACUCUUUCU
874-896


 


3642
3643
AD-59111
CUCCAUGAUCCAAGGGAUU
AAUCCCUUGGAUCAUGGAG
1357-1379


 


3644
3645
AD-59112
CCAUUCUUAUCCCGAGUCA
UGACUCGGGAUAAGAAUGG
362-384


 


3646
3647
AD-59113
CACCCUGGCUAAGAUGAUA
UAUCAUCUUAGCCAGGGUG
1300-1322


 


3648
3649
AD-59114
UCAUCCCUGUGCGGGUUGA
UCAACCCGCACAGGGAUGA
1923-1945


 


3650
3651
AD-59115
AAGAGUGUCUCAUCUUCUU
AAGAAGAUGAGACACUCUU
877-899


 


3652
3653
AD-59116
GUCAUGCCAAAAAUGGACA
UGUCCAUUUUUGGCAUGAC
1631-1653


 


3654
3655
AD-59117
CAUUCUUAUCCCGAGUCCA
UGGACUCGGGAUAAGAAUG
363-385


 


3656
3657
AD-59118
ACCCUGGCUAAGAUGAUGA
UCAUCAUCUUAGCCAGGGU
1301-1323


 


3658
3659
AD-59119
CUCUUCACCCUGGCUAAGA
UCUUAGCCAGGGUGAAGAG
1295-1317


 


3660
3661
AD-59120
AUGCCAAAAAUGGACAUCA
UGAUGUCCAUUUUUGGCAU
1634-1656


 


3662
3663
AD-59121
UGCCCCAAGAUGAUGGAAU
AUUCCAUCAUCUUGGGGCA
434-456


 


3664
3665
AD-59122
GAACCAUGCCUCCAUGAUA
UAUCAUGGAGGCAUGGUUC
1348-1370


 


3666
3667
AD-59123
UCUUCACCCUGGCUAAGAU
AUCUUAGCCAGGGUGAAGA
1296-1318


 


3668
3669
AD-59124
UGCCAAAAAUGGACAUCAU
AUGAUGUCCAUUUUUGGCA
1635-1657


 


3670
3671
AD-59125
CCAGAAAGAGUGUCUCAUA
UAUGAGACACUCUUUCUGG
872-894


 


3672
3673
AD-59126
GAAACUGUCCAUUCAAUGA
UCAUUGAAUGGACAGUUUC
1481-1503


 


3674
3675
AD-59127
UCACCCUGGCUAAGAUGAU
AUCAUCUUAGCCAGGGUGA
1299-1321


 


3676
3677
AD-59128
CCCUGGAGUCUGUGCGGAU
AUCCGCACAGACUCCAGGG
1791-1813


 


3678
3679
AD-59129
GAAAGAGUGUCUCAUCUUA
UAAGAUGAGACACUCUUUC
875-897


 


3680
3681
AD-59130
UGGAGCCCUGGAGUCUGUA
UACAGACUCCAGGGCUCCA
1786-1808









The results of the in vitro assays are provided in Table 16. Table 16 also notes the target species of each of the siRNAs.







TABLE 16







Results of Functional Assays











Cyno Free Uptake
Cyno Transfection
Hep3b Transfection


















Target

1 uM

20 nM

20 nM
0.2 nM
10 nM
0.1 nM


Duplex ID
Species
Type
Avg
500 nM
Avg
10 nM
Avg
Avg
Avg
Avg




















AD-58848
M/R/Rh/H
21/23
131.6
176.0
104.4
128.0
43.5
44.8
25.3
76.8


AD-58849
H/Rh
21/23
91.9
88.1
92.2
105.0
29.4
35.4
11.5
47.1


AD-58850
H/Rh
21/23
79.4
103.4
80.0
111.2
NA
62.2
31.3
72.0


AD-58851
H/Rh
21/23
99.7
74.7
94.8
104.7
NA
40.7
8.6
81.3


AD-58852
H/Rh
21/23
108.1
91.8
103.3
111.9
101.1
128.8
43.4
129.0


AD-58853
H/Rh
21/23
74.8
67.7
84.2
93.5
24.7
52.9
14.1
61.2


AD-58854
H/Rh
21/23
145.9
124.1
106.6
115.3
119.0
83.9
85.0
84.0


AD-58855
H/Rh
21/23
81.5
97.9
92.7
101.8
39.5
40.3
15.3
67.6


AD-58856
H/Rh
21/23
74.1
90.6
84.6
82.6
22.4
30.7
8.7
33.3


AD-58857
H/Rh
21/23
64.7
91.4
62.3
87.1
22.0
31.6
9.8
106.3


AD-58858
H/Rh
21/23
67.4
91.7
68.6
98.3
27.9
40.3
17.4
44.8


AD-58859
H/Rh
21/23
71.2
77.2
92.4
90.1
19.1
34.3
13.1
39.7


AD-58861
H/Rh
21/23
104.6
107.2
102.0
100.6
25.9
35.1
18.0
69.8


AD-58862
H/Rh
21/23
66.8
77.0
68.7
88.5
20.3
31.1
24.2
49.9


AD-58863
H/Rh
21/23
70.8
66.8
76.8
98.5
21.5
29.7
8.7
54.9


AD-58864
H/Rh
21/23
76.2
85.6
83.7
100.8
60.4
61.0
56.4
87.3


AD-58865
H/Rh
21/23
67.9
77.9
95.9
98.4
21.3
38.6
15.5
81.4


AD-58867
H/Rh
21/23
95.9
93.3
107.0
97.5
32.3
42.7
16.6
79.8


AD-58868
H/Rh
21/23
95.2
92.1
116.2
94.7
54.6
69.2
61.5
105.9


AD-58869
H/Rh
21/23
65.0
78.2
75.8
88.2
17.4
25.0
13.0
63.9


AD-58870
H/Rh
21/23
69.4
92.3
81.0
88.1
29.2
43.8
33.7
79.1


AD-58871
H/Rh
21/23
61.2
77.3
88.2
77.0
71.2
73.2
36.7
110.3


AD-58873
H/Rh
21/23
95.2
100.9
83.3
94.6
54.2
52.8
36.6
73.3


AD-58874
H/Rh
21/23
75.8
76.8
63.8
85.3
22.3
31.2
15.0
38.2


AD-58875
H/Rh
21/23
80.7
88.7
78.6
97.9
48.6
73.6
61.2
90.6


AD-58876
H/Rh
21/23
90.8
93.1
82.5
100.2
41.1
56.9
21.2
58.7


AD-58877
H/Rh
21/23
68.3
85.1
51.2
78.7
18.5
46.6
11.9
27.4


AD-58878
H/Rh
21/23
78.3
68.3
81.2
91.2
24.1
23.4
6.2
37.1


AD-58879
H/Rh
21/23
87.9
94.1
79.7
95.4
32.0
47.8
15.7
82.5


AD-58880
H/Rh
21/23
74.9
72.2
88.9
88.1
20.1
27.5
14.0
60.7


AD-58881
H/Rh
21/23
85.9
76.8
78.8
118.0
22.2
36.7
27.6
71.6


AD-58882
H/Rh
21/23
54.1
53.4
60.3
85.8
14.6
27.2
8.2
23.8


AD-58883
H/Rh
21/23
80.4
69.9
75.7
80.3
31.8
25.8
12.3
63.0


AD-58884
H/Rh
21/23
57.7
55.3
64.8
78.2
20.0
30.0
11.8
68.9


AD-58885
H/Rh
21/23
101.8
91.8
104.1
101.5
85.9
71.9
61.8
71.2


AD-58886
M/R/Rh/H
21/23
47.1
58.0
36.3
93.3
16.0
26.6
9.2
32.0


AD-58887
H/Rh
21/23
73.6
98.7
82.6
95.2
28.5
33.5
12.8
65.2


AD-58888
H/Rh
21/23
90.2
69.9
69.4
85.6
46.9
45.0
16.6
72.0


AD-58889
H/Rh
21/23
83.6
98.6
82.4
92.2
36.5
40.3
31.6
99.4


AD-58890
H/Rh
21/23
69.5
95.4
84.2
88.2
50.8
45.6
21.7
92.9


AD-58891
H/Rh
21/23
62.8
75.7
75.4
109.2
23.6
34.3
15.6
55.8


AD-58892
H/Rh
21/23
60.2
92.9
89.8
92.9
22.8
43.3
20.2
75.6


AD-59095
M/R/Rh/H
19mer
88.9
NA
132.8
NA
48.3
97.4
54.3
99.0


AD-59096
M/R/Rh/H
19mer
95.5
NA
90.5
NA
105.7
138.6
131.4
120.7


AD-59097
M/R/Rh/H
19mer
92.5
NA
84.2
NA
75.0
NA
94.7
108.5


AD-59098
M/R/Rh/H
19mer
84.0
NA
87.7
NA
109.3
NA
130.0
87.3


AD-59099
M/R/Rh/H
19mer
89.7
NA
90.0
NA
77.8
85.4
46.8
74.9


AD-59100
M/R/Rh/H
19mer
84.8
NA
144.3
NA
70.6
108.1
91.5
117.6


AD-59101
M/R/Rh/H
19mer
79.0
NA
103.8
NA
89.8
102.9
124.2
107.0


AD-59102
M/R/Rh/H
19mer
85.9
NA
100.6
NA
72.2
68.5
87.9
95.1


AD-59103
M/R/Rh/H
19mer
86.0
NA
91.1
NA
93.0
81.3
130.0
96.0


AD-59104
M/R/Rh/H
19mer
92.6
NA
96.9
NA
94.9
91.4
124.4
83.1


AD-59105
M/R/Rh/H
19mer
48.9
NA
101.7
NA
18.4
48.9
17.0
34.7


AD-59106
M/R/Rh/H
19mer
63.2
NA
76.7
NA
28.5
40.7
28.6
46.4


AD-59107
M/R/Rh/H
19mer
71.4
NA
68.7
NA
37.1
45.3
26.8
63.6


AD-59108
M/R/Rh/H
19mer
70.7
NA
85.1
NA
89.9
84.8
139.2
101.7


AD-59109
M/R/Rh/H
19mer
86.1
NA
83.4
NA
84.9
96.2
131.7
86.7


AD-59110
M/R/Rh/H
19mer
70.8
NA
119.7
NA
38.5
60.4
67.4
80.3


AD-59111
M/R/Rh/H
19mer
66.1
NA
76.5
NA
52.2
61.0
69.7
87.6


AD-59112
M/R/Rh/H
19mer
71.2
NA
80.2
NA
91.2
83.4
127.4
89.0


AD-59113
M/R/Rh/H
19mer
67.0
NA
77.8
NA
49.1
59.0
66.8
91.4


AD-59114
M/R/Rh/H
19mer
81.7
NA
79.3
NA
96.3
88.0
129.6
72.4


AD-59115
M/R/Rh/H
19mer
40.4
NA
69.6
NA
19.6
35.7
9.3
16.9


AD-59116
M/R/Rh/H
19mer
72.2
NA
78.3
NA
53.5
77.8
70.1
107.8


AD-59117
M/R/Rh/H
19mer
70.7
NA
75.6
NA
75.8
74.9
129.0
103.5


AD-59118
M/R/Rh/H
19mer
68.8
NA
75.9
NA
81.4
82.1
114.1
89.7


AD-59119
M/R/Rh/H
19mer
64.9
NA
86.5
NA
85.1
125.1
122.8
124.8


AD-59120
M/R/Rh/H
19mer
63.5
NA
75.1
NA
29.9
52.0
16.1
54.1


AD-59121
M/R/Rh/H
19mer
67.6
NA
72.0
NA
88.8
77.4
108.0
103.1


AD-59122
M/R/Rh/H
19mer
60.2
NA
62.3
NA
25.1
45.3
16.2
54.8


AD-59123
M/R/Rh/H
19mer
68.6
NA
108.2
NA
59.2
84.6
80.0
97.7


AD-59124
M/R/Rh/H
19mer
47.5
NA
56.5
NA
23.9
40.0
9.8
18.9


AD-59125
M/R/Rh/H
19mer
45.4
NA
47.2
NA
15.2
40.7
14.7
15.1


AD-59126
M/R/Rh/H
19mer
64.3
NA
74.6
NA
51.6
57.1
35.5
54.4


AD-59127
M/R/Rh/H
19mer
103.4
NA
105.8
NA
94.0
156.4
135.9
113.7


AD-59128
M/R/Rh/H
19mer
102.4
NA
81.4
NA
66.3
89.3
60.2
74.9


AD-59129
M/R/Rh/H
19mer
41.3
NA
38.8
NA
17.9
41.4
8.6
12.6


AD-59130
M/R/Rh/H
19mer
58.3
NA
80.8
NA
94.9
78.3
106.7
88.0









Table 17 illustrates the IC50s of select ALAS1 siRNA duplexes. The IC50s were determined from the knockdown of endogenously expressed ALAS1 in the Hep3B cell line, at 24 hours following transfection of each ALAS1 modified siRNA duplex (see Table 14). At least seven duplexes, including AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, and AD-59129, consistently demonstrated IC50s of less than 0.1 nm, indicating that these duplexes were particularly effective in suppressing ALAS1 expression.







TABLE 17







IC50s of select ALAS1 siRNA duplexes











Duplex ID
384w IC50 (nM)
96w IC50 (nM)















AD-58882
0.008
0.014



AD-58878
0.040
0.031



AD-58886
0.037
0.033



AD-58877
0.031
0.034



AD-59115
0.093
0.052



AD-58856
0.061
0.066



AD-59129
0.085
0.071



AD-59124
0.572
0.078



AD-58874
0.140
0.102



AD-59125
0.118
0.115



AD-59105
0.511
0.144



AD-59120
180.592 
0.498



AD-59122
36.646
0.646



AD-59106
7.906
0.847



AD-59126
n/a
1.014



AD-59107
n/a
1.971










EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

## Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, wherein said dsRNA comprises a sense strand and an antisense strand, wherein:
(i) the antisense strand is complementary to at least nucleotides 871-889 of SEQ ID NO:1 and comprises SEQ ID NO:1296,
(ii) the sense strand comprises at least 15 contiguous nucleotides from SEQ ID NO:1295,
(iii) a ligand and linker with a structure as shown below is attached to the 3′ end of the sense strand of the dsRNA





and
(iv) the dsRNA comprises one or more nucleotides with a modification chosen from a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, or both.

2. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, comprising:
(i) an antisense strand complementary to at least nucleotides 871-889 of SEQ ID NO:1;
(ii) a sense strand comprising at least 15 contiguous nucleotides from SEQ ID NO:1295;
(iii) one or more N-acetylgalactosamine (GalNAc) derivatives; and
(iv) one or more nucleotides with a modification chosen from a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, or both.

3. The dsRNA of claim 2, wherein the antisense strand comprises the sequence of SEQ ID NO:1296.

4. The dsRNA of claim 3, wherein the sense strand comprises the sequence of SEQ ID NO:1295.

5. The dsRNA of claim 3, wherein the dsRNA comprises at least five 2′-O-methyl modified nucleotides and at least five 2′-fluoro modified nucleotides.

6. The dsRNA of claim 5, wherein the dsRNA comprises one or more phosphorothioate linkages.

7. The dsRNA of claim 6, wherein the dsRNA comprises at least 20 modified nucleotides.

8. The dsRNA of claim 6, wherein the dsRNA comprises nucleotide modifications over the entire length of the sense and antisense strands.

9. The dsRNA of claim 3, which comprises at least one blunt-end.

10. The dsRNA of claim 3, wherein both ends of the dsRNA are blunt-ended.

11. The dsRNA of claim 3, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.

12. The dsRNA of claim 11, wherein the 3′ overhang is 2 nucleotides in length.

13. The dsRNA of claim 11, wherein the 3′ overhang is present on the 3′ end of either the antisense strand or the sense strand, or both 3′ ends of the antisense strand and the sense strand of the dsRNA.

14. The dsRNA of claim 11, wherein the 3′ overhang is in the antisense strand.

15. The dsRNA of claim 14, wherein one or more of the nucleotides in the overhang is a nucleoside thiophosphate.

16. The dsRNA of claim 3, comprising a duplex region of 15-30 base pairs in length.

17. The dsRNA of claim 16, wherein the duplex region is 19 to 23 nucleotides in length.

18. The dsRNA of claim 3, wherein each strand is no more than 30 nucleotides in length.

19. The dsRNA of claim 18, wherein each of the sense strand and the antisense strand are 15-30 nucleotides in length.

20. The dsRNA of claim 19, wherein each strand is 19 to 24 nucleotides in length.

21. The dsRNA of claim 3, wherein the one or more GalNAc derivatives is attached to the 3′ end of the sense strand.

22. The dsRNA of 3, wherein the one or more GalNAc derivatives is a biantennary or a triantennary GalNAc ligand.

23. The dsRNA of 22, wherein the GalNAc ligand is:

24. The dsRNA of 23, wherein the GalNAc ligand is attached to the 3′ end of the sense strand.

25. The dsRNA of 23, wherein the GalNAc ligand is attached to the 3′ end of the sense strand via linker.

26. The dsRNA of 22, wherein GalNAc ligand and linker have the structure of:

27. The dsRNA of claim 26, wherein the dsRNA inhibits the expression of ALAS1 mRNA by at least 50%.

28. The dsRNA of claim 3, wherein the antisense strand is fully complementary to at least nucleotides 871-889 of SEQ ID NO:1.

29. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, comprising:
(i) an antisense strand that comprises the sequence of SEQ ID NO:1296;
(ii) a sense strand comprising at least 15 contiguous nucleotides from SEQ ID NO:1295;
(iii) a duplex region of 15-30 base pairs in length;
(iv) a biantennary or a triantennary GalNAc ligand;
(v) 20 or more modified nucleotides chosen from 2′-O-methyl modified nucleotides and 2′-fluoro modified nucleotides; and
(vi) at least one strand comprising a 3′ overhang of at least 1 nucleotide.

30. The dsRNA of claim 29, wherein each of the sense strand and the antisense strand are 19-24 nucleotides in length.

31. The dsRNA of claim 30, wherein the antisense strand comprises the 3′-overhang.

32. The dsRNA of claim 31, wherein the 3′-overhang is 2 nucleotides in length.

33. The dsRNA of claim 32, which comprises a blunt-ended sense strand.

34. The dsRNA of claim 33, wherein the dsRNA comprises one or more phosphorothioate linkages.

35. The dsRNA of claim 34, comprising 2′-O-methyl modified nucleotides and 2′-fluoro modified nucleotides over the entire length of the sense and antisense strands.

36. The dsRNA of 33, wherein the GalNAc ligand is:

37. The dsRNA of 36, wherein the GalNAc ligand is attached to the 3′ end of the sense strand.

38. The dsRNA of 36, wherein the GalNAc ligand is attached to the 3′ end of the sense strand via linker.

39. The dsRNA of 38, wherein GalNAc ligand and linker have the structure of:

40. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of ALAS1, comprising:
(i) an antisense strand that is fully complementary to at least nucleotides 871-889 of SEQ ID NO:1;
(ii) a sense strand comprising at least 15 contiguous nucleotides from SEQ ID NO:1295, wherein each of the sense strand and the antisense strand are 19-24 nucleotides in length;
(iii) a ligand attached to the 3′ end of the sense strand and having the structure:




(iv) modified nucleotides over the entire length of the sense strand and the antisense strand consisting of 2′-O-methyl modified nucleotides, 2′-fluoro modified nucleotides, and phosphorothioate linkages;
(v) the antisense strand comprising a 3′-overhang of 2 nucleotides in length; and
(vi) a blunt-ended sense strand.

41. A pharmaceutical composition comprising the dsRNA of claim 3.

42. A pharmaceutical composition comprising the dsRNA of claim 29.

43. A pharmaceutical composition comprising the dsRNA of claim 40.

44. The pharmaceutical composition of claim 42, further comprising an unbuffered solution.

45. The pharmaceutical composition of claim 42, wherein said pharmaceutical composition is formulated for intravenous or subcutaneous administration.

46. An isolated cell containing the dsRNA of claim 3.

47. The cell of claim 46, which is a liver cell.

48. A method of treating a disorder related to ALAS1 expression comprising administering to a subject in need of such treatment the dsRNA of claim 29.

49. The method of claim 48, wherein the subject is at risk for developing, or is diagnosed with, a porphyria.

50. The method of claim 49, wherein the method decreases a level of a porphyrin or a porphyrin precursor in the subject.

51. The method of claim 49, wherein the method decreases the frequency of acute attacks of symptoms associated with the porphyria.

52. The method of claim 49, wherein the method decreases incidence of acute attacks of symptoms associated with a porphyria in the subject when the subject is exposed to a precipitating factor.

53. The method of claim 49, wherein the porphyria is a hepatic porphyria selected from the group consisting of acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), variegate porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic porphyria.

54. The method of claim 49, wherein the dsRNA or composition comprising the dsRNA is administered before, during, or after an acute attack of porphyria.

55. The method of claim 49, wherein the dsRNA or composition comprising the dsRNA is administered during a prodrome.

56. The method of claim 49, wherein the subject has an elevated level of ALA, PBG, or both ALA and PBG.

57. A method of treating a subject with an elevated level of ALA, PBG or both ALA and PBG, the method comprising administering to the subject the dsRNA of claim 29.

58. A method of treating a porphyria comprising administering to a subject in need of such treatment the dsRNA of claim 40.