# Patent

## Title
GNAQ targeted dsRNA compositions and methods for inhibiting expression

## Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) targeting a G-alpha q subunit (GNAQ) of a heterotrimeric G gene, and methods of using the dsRNA to inhibit expression of GNAQ.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 61/121,253, filed Dec. 10, 2008, and U.S. Provisional Application No. 61/185,543, filed Jun. 9, 2009, and U.S. Provisional Application No. 61/244,780, filed Sep. 22, 2009, which are hereby incorporated in their entirety by reference.


REFERENCE TO A SEQUENCE LISTING
This application includes a Sequence Listing submitted electronically as a text file named 16267US_sequencelisting.txt, created on Dec. 10, 2010, with a size of 560,474 bytes. The sequence listing is incorporated by reference.
FIELD OF THE INVENTION
The invention relates to a double-stranded ribonucleic acid (dsRNA) targeting a G-alpha q subunit (GNAQ) of a heterotrimeric G gene, and methods of using the dsRNA to inhibit expression of GNAQ.
BACKGROUND OF THE INVENTION
Guanine nucleotide-binding proteins (G proteins) are a family of heterotrimeric proteins that couple cell surface, 7-transmembrane domain receptors to intracellular signaling pathways. G proteins are composed of alpha, beta and gamma subunits. The G-alpha q subunit (GNAQ) is one of the G-alpha subunits. GNAQ mediates stimulation of phospholipase C-beta and hydrolysis of GTP.
Mice with GNAQ mutations leading to overexpression of GNAQ exhibit dermal hyperpigmentation. A point mutation in human GNAQ was reported in a melanoma sample (Bamford et al (2004) Br J Cancer, 91:355-358). In WO/2008/098208 (PCT/US2008/053484), the Applicant's described the presence of mutations that constitutively activate GNAQ in melanocytic neoplasms, e.g., uveal melanomas.
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.).
SUMMARY OF THE INVENTION
Disclosed herein are dsRNAs targeted to GNAQ for inhibiting expression of GNAQ in a cell. Also disclosed are methods of using the GNAQ dsRNA for siRNA inhibition of GNAQ expression and treatment of disease associated with expression and/or over expression of GNAQ, e.g., uveal melanoma.
Accordingly one aspect of the invention is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a G-alpha q subunit (GNAQ) of a heterotrimeric G gene, having a sense strand and an antisense strand having a region of complementarity complementary to an mRNA encoding GNAQ, wherein each strand is at least 15 nucleotides in length. In one embodiment the dsRNA is AD-20057, e.g., sense strand is SEQ ID NO:1579 and the antisense strand is SEQ ID NO:1580. In another embodiment, the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO:1421 or is complementary to at least the first 11 nucleotides of SEQ ID NO:1421. The sense strand can include 15 or more contiguous nucleotides of SEQ ID NO:1421 or SEQ ID NO:1579 and/or the antisense strand can include 15 or more contiguous nucleotides of SEQ ID NO:1422 or SEQ ID NO:1580. In some embodiments the sense strand nucleotide sequence includes SEQ ID NO:1421 and the antisense strand nucleotide sequence includes SEQ ID NO:1422.
In some embodiments the dsRNA of the invention results in the following: administration of 0.1 nM of the dsRNA to a A375 cell results in about 66% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of 1 nM of the dsRNA to a A375 cell results in about 61% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of 1 nM of the dsRNA to a A579 cell results in about 82% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of 10 nM of the dsRNA to a OMM1.3 cell results in about 42% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of the dsRNA to a UMEL202 cell results in about 81% inhibition of GNAQ mRNA expression as measured by a real time PCR assay.
In another embodiment, the dsRNA is AD-20051 and the sense strand is SEQ ID NO:1565 and the antisense strand is SEQ ID NO:1566. The dsRNA can be complementary to at least the first 11 nucleotides of SEQ ID NO:1407 and/or complementary to at least 15 contiguous nucleotides of SEQ ID NO:1407. In some embodiments the sense strand includes 15 or more contiguous nucleotides of SEQ ID NO: 1407 or SEQ ID NO:1565 and/or the antisense strand includes 15 or more contiguous nucleotides of SEQ ID NO:1408 or SEQ ID NO:1566. The sense strand nucleotide sequence can include SEQ ID NO:1407 and the antisense strand nucleotide sequence can include SEQ ID NO:1408.
In some embodiments the dsRNA of the invention results in the following: administration of 0.1 nM of the dsRNA to a A375 cell results in about 49% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of 1 nM of the dsRNA to a A375 cell results in about 55% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of 1 nM of the dsRNA to a A579 cell results in about 83% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of 10 nM of the dsRNA to a OMM1.3 cell results in about 42% inhibition of GNAQ mRNA expression as measured by a real time PCR assay.
In other embodiments the dsRNA is AD-20052 or AD-20069.
The antisense strand of the dsRNA is partially or completely complementary to an mRNA encoding a GNAQ, e.g., to a human GNAQ mRNA (e.g., NM—002072) or to a rat GNAQ mRNA (e.g., NM—031036). The region complementary is at least 15 nucleotides in length, e.g., between 19 and 21 nucleotides in length, e.g., 19 nucleotides in length. The region of complementarity can include at least 15 contiguous nucleotides of one of the antisense sequences listed in Tables 2a, 3a, or 4a. In other embodiments, the region of complementarity is one of the antisense sequences listed in Tables 2a, 3a, or 4a.
Additional exemplary dsRNA are provided in the tables herein. In some embodiments, the dsRNA of the invention includes a sense strand and antisense strand are selected from Tables 2b, 3b, 4b or Tables 2c, 3c, or 4c or Tables 2d, 3d, or 4d.
In one aspect, each strand of the dsRNA is no more than 30 nucleotides in length. At least one strand can include a 3′ overhang of at least 1 nucleotide, e.g., 2 nucleotides, e.g., dTdT.
In some embodiments, the dsRNA is modified. For example, the dsRNA can include a modification that causes the dsRNA to have increased stability in a biological sample. In one embodiment, the dsRNA includes at least one modified nucleotide, e.g., a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, or a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In other embodiments the modified nucleotide is 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, or a non-natural base comprising nucleotide. The dsRNA of the invention can include at least one 2′-O-methyl modified nucleotide and at least one 2′-deoxythymidine-3′-phosphate nucleotide comprising a 5′-phosphorothioate group.
Any of the dsRNA of the invention can be modified according to a set of rules, e.g., the sense strand includes all 2′-O-methyl modified pyrimidines and the antisense strand comprises 2′-O-methyl modified pyrimidines when the pyrimidine is adjacent to A and each strand comprises dTdT at the 3′ end or the sense strand comprises all 2′-O-methyl modified pyrimidines and the antisense strand comprises 2′-O-methyl modified pyrimidines when the pyrimidine is adjacent to A and each strand comprises dTsdT at the 3′ end or the sense strand comprises all 2′-O-methyl modified pyrimidines and the antisense strand comprises 2′-O-methyl modified pyrimidines when a) the pyrimidine is adjacent to A or b) the pyrimidine is a uracil adjacent to a U or a G, and each strand comprises dTsdT at the 3′ end.
In some embodiments the dsRNA include a ligand. The ligand can be conjugated to the 3′-end of the sense strand of the dsRNA.
Another aspect of the invention is a composition for inhibiting expression of a GNAQ gene including a dsRNA targeting GNAQ and a pharmaceutical formulation. In one embodiment, the pharmaceutical formulation is a lipid formulation. Exemplary formulations are described herein and include, for example, a LNP formulation, a LNP01 formulation, a XTC-SNALP formulation, a SNALP formulation, or a LNP11 formulation.
Also included herein is an isolated cell containing a dsRNA of the invention, a vector including the nucleotide sequence that encodes at least one strand of the dsRNA of the invention, and a cell including said vector.
A dsRNA of the invention, upon contact with a cell expressing said GNAQ, inhibits expression of said GNAQ gene by at least 40% compared to a cell not so contacted. In some embodiments, a dsRNA of the invention has a pM IC50, e.g., an IC50 of less than 10 pM.
Another aspect of the invention is method of inhibiting GNAQ expression in a cell, the method including introducing into the cell any of the dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of a GNAQ gene, thereby inhibiting expression of the GNAQ gene in the cell. In some embodiments, expression is inhibited by at least 20%, 40%, 60%, or at least 80%. Also included is a method of treating a disorder mediated by GNAQ expression by administering to a human in need of such treatment a therapeutically effective amount of any of the dsRNA of the invention. Examples of said disorders include uveal melanoma, cutaneous melanoma, Blue nevi, Nevi of Ota, a small lung tumor, or a neuroendocrine tumors. The method of treatment can include administering an addition composition, e.g., a second dsRNA.



DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing IFN-alpha cytokine induction in human PBMCs following transfection with a set of GNAQ targeted dsRNA.
FIG. 2 shows TNF-alpha cytokine induction in human PBMCs following transfection with a set of GNAQ targeted dsRNA.
FIG. 3 shows cell viability of OMM1.3 and MEL285 cells following transfection with 1 nM of dsRNAs. The Y-axis is viability normalized to control AD-1955.
FIG. 4 shows cell viability of MEL202 and MEL285 cells following transfection with 1 nM of dsRNAs. The Y-axis is viability normalized to control AD-1955.
FIG. 5 shows cell viability of OMM1.3 and MEL285 cells following transfection with 0.01 nM of dsRNAs. The Y-axis is viability normalized to control AD-1955.
FIG. 6 shows cell viability of MEL202 and MEL285 cells following transfection with 0.01 nM of dsRNAs. The Y-axis is viability normalized to control AD-1955.
FIG. 7 shows day 7 cell viability of OMM1.3, MEL202, and MEL285 cells following transfection with AD-20057 and AD-20051 dsRNAs
FIG. 8 shows day 7 cell viability of OMM1.3, MEL202, and MEL285 cells following transfection with AD-20069 and AD-20093 dsRNAs.



DETAILED DESCRIPTION OF THE INVENTION
The invention provides dsRNAs and methods of using the dsRNAs for inhibiting the expression of a G-alpha q subunit (GNAQ) of a heterotrimeric G gene in a cell or a mammal where the dsRNA targets a GNAQ gene. The invention also provides compositions and methods for treating pathological conditions and diseases, such as uveal melanoma in a mammal caused by the over-expression of a GNAQ gene. A dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).
The dsRNAs of the compositions featured herein include an antisense strand having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is complementary to at least part of an mRNA transcript of a GNAQ gene. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in pathologies associated with GNAQ expression in mammals. Very low dosages of GNAQ dsRNAs in particular can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a GNAQ gene. Using cell-based assays, the present inventors demonstrate that dsRNAs targeting GNAQ can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a GNAQ gene. Thus, methods and compositions including these dsRNAs are useful for treating pathological processes that can be mediated by down regulating GNAQ over-expression, such as, e.g., treatment of uveal melanoma.
The following detailed description discloses how to make and use the compositions containing dsRNAs to inhibit the expression of a GNAQ gene, as well as compositions (e.g., pharmaceutical compositions) and methods for treating diseases and disorders caused by the expression of this gene.
Accordingly, in some aspects, pharmaceutical compositions containing a GNAQ dsRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of a GNAQ gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of a GNAQ gene are featured in the invention.
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” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” or “deoxyribonucleotide” 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 the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
As used herein, “GNAQ” refers to a G-alpha q subunit (GNAQ) of a heterotrimeric G gene. GNAQ is also known as guanine nucleotide binding protein (G protein), q polypeptide and G-ALPHA-q, GAQ. The sequence of a human GNAQ mRNA transcript can be found at NM—002072.2. The sequence of rat GNAQ mRNA can be found at NM—031036.
A used herein “target” or “target gene” refers to a GNAQ gene.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a GNAQ gene, including mRNA that is a product of RNA processing of a primary transcription product.
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.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. 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 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. 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 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 a dsRNA 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., a target gene, e.g., an mRNA encoding GNAQ) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, a polynucleotide is complementary to at least a part of a GNAQ mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding GNAQ.
The term “double-stranded RNA” or “dsRNA,” as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims
The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. The term “siRNA” is also used herein to refer to a dsRNA as described above.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
The term “antisense strand” refers to the strand of 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 are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
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 agent or a plasmid from which an iRNA agent is transcribed. SNALP are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and U.S. Ser. No. 61/045,228 filed on Apr. 15, 2008. These applications are hereby incorporated by reference.
“Introducing into a cell,” when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA 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; a dsRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of” and the like, in as far as they refer to a target gene, herein refer to the at least partial suppression of the expression of a GNAQ gene, as manifested by a reduction of the amount of mRNA which may be isolated or detected from a first cell or group of cells in which a GNAQ gene is transcribed and which has or have been treated such that the expression of a GNAQ gene is inhibited, 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). The degree of inhibition is usually expressed in terms of




  
    
      
        
          
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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 GNAQ gene transcription, e.g., the amount of protein encoded by a GNAQ gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g., apoptosis. In principle, GNAQ gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a GNAQ 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 a GNAQ gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a GNAQ gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a GNAQ gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide featured in the invention.
As used herein in the context of GNAQ expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by GNAQ expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by GNAQ expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition, such as tumor reduction in uveal melanoma.
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 mediated by GNAQ expression or an overt symptom of pathological processes mediated by GNAQ 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 processes mediated by GNAQ expression, the patient's history and age, the stage of pathological processes mediated by GNAQ expression, and the administration of other anti-pathological processes mediated by GNAQ expression agents.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% 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 25% reduction in that parameter.
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.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
Double-stranded ribonucleic acid (dsRNA)
As described in more detail herein, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a GNAQ gene in a cell or mammal, where the dsRNA includes a sense strand having a first sequence and an antisense strand comprising a second sequence complementary to mRNA encoding GNAQ, wherein said first sequence is complementary to said second sequence at a region of complementarity and wherein each strand is 15 to 30 base pairs in length. In some embodiments, the dsRNA of the invention inhibits the expression of said GNAQ gene by at least 40% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of a GNAQ gene can be reduced by at least 30% when measured by an assay as described in the Examples below. For example, expression of a GNAQ gene in cell culture, such as in HepB3 cells, can be assayed by measuring GNAQ mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by ELISA assay.
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. The dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure.
One strand of the dsRNA (the antisense strand) includes a region of complementarity that is complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a target 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. The region of complementarity is generally at least 15 nucleotides in length, or between 19 and 21 nucleotides in length, or 19, 20, or 21 nucleotides in length. In some embodiments the region of complementarity includes at least 15 contiguous nucleotides of one of the antisense sequences listed in Tables 2a, 3a, or 4a. In other embodiments the region of complementarity includes one of the antisense sequences listed in Tables 2a, 3a, or 4a.
Generally, the duplex structure is between 15 and 30, or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different dsRNAs are used in combination, the duplex lengths can be identical or can differ.
Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is 25-30 nucleotides in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ.
The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, or 1, 2, 3, or 4 nucleotides. In another embodiment, the overhang include dTdT. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand.
A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In one embodiment, a GNAQ gene is a human GNAQ gene, e.g., the sequence identified by GenBank accession number NM—002072.2.
In specific embodiments, the sense strand of the dsRNA is one of the a sense sequences from Tables 2-4, and the antisense strand is one of the antisense sequences of Tables 2-4. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 2-4 can readily be determined using the target sequence and the flanking GNAQ sequence.
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 dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2-4, the dsRNAs featured in the invention can include at least one strand of a length described therein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Tables 2-4 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, 21, or 22, or more contiguous nucleotides from one of the sequences of Tables 2-4, and differing in their ability to inhibit the expression of a GNAQ gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired GNAQ target sequence can readily be made using the corresponding GNAQ antisense sequence and a complementary sense sequence.
In addition, the dsRNAs provided in Tables 2-4 identify a site in a GNAQ that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 2-4 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a GNAQ gene.
Additional dsRNA of the invention include those that cleave a target mRNA at the same location as a dsRNA described in any of the tables. In general, a RISC complex will cleave a target mRNA between the nucleotides complementary to nucleotides 10 and 11 of the antisense strand of a dsRNA, e.g., siRNA, of the invention. Cleavage e sites can be assayed using, e.g., a 5′ RACE assay.
For example, the duplex AD-20057 includes the sense and antisense strands below. Treatment of a cell with this duplex results in cleavage of human GNAQ mRNA at the nucleotides complementary to nucleotides 10 and 11 of the antisense strand, e.g., nucleotides 1211 and 1212. Therefore, also included in the invention are those dsRNA that cleave at that location.
The dsRNA featured in the invention can contain one or more mismatches to the target sequence. In one embodiment, the dsRNA featured in the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA 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 dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of a target gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a target gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a target gene is important, especially if the particular region of complementarity in a target gene is known to have polymorphic sequence variation within the population.
Modifications
In yet another embodiment, the dsRNA is chemically modified to enhance stability. 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. Specific examples of dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and 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 dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Modified dsRNA 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; and 5,625,050, each of which is herein incorporated by reference
Modified dsRNA 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 ore 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 suitable dsRNA mimetics, 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, a dsRNA 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 a dsRNA 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 in Nielsen et al., Science, 1991, 254, 1497-1500. Other embodiments of the invention are dsRNAs 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. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise 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. Particularly preferred are 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. Other preferred dsRNAs comprise 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 dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 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. A further preferred modification includes 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 preferred 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 dsRNA, 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. DsRNAs 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 in its entirety.
A dsRNA 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 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; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
Conjugates
Another modification of the dsRNAs featured in the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-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-3H-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).
Representative U.S. patents that teach the preparation of such dsRNA 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, 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 a dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which 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 dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA 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 dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs 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 dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (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-3H-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 dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs 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 dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.
Vector encoded dsRNAs
In another aspect, dsRNA molecules of the invention are 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). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. 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 strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the 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.
The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. 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 featured in the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors featured in the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
Viral vectors can be derived from AV and AAV. In one embodiment, the dsRNA featured in the invention is 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.
A suitable AV vector for expressing the dsRNA 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.
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. Nos. 5,252,479; 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.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector featured in the invention may be a eukaryotic RNA polymerase I (e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV early promoter or actin promoter or Ul snRNA promoter) or generally RNA polymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a 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 transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA 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.
dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKOTM). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single target gene or multiple target genes 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.
Target gene specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy 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.
Pharmaceutical compositions containing dsRNA
In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a GNAQ gene, such as pathological processes mediated by GNAQ expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.
The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of GNAQ genes. In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams siRNA 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 0.0059 mg/kg, 0.01 mg/kg, 0.0295 mg/kg, 0.05 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.628 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.
In one embodiment, the dosage is between 0.01 and 0.2 mg/kg. For example, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08 mg/kg 0.09 mg/kg, 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or 0.20 mg/kg.
In one embodiment, the dosage is between 0.005 mg/kg and 1.628 mg/kg. For example, the dsRNA can be administered at a dose of 0.0059 mg/kg, 0.0295 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, or 1.628 mg/kg.
In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg/kg. For example, the dsRNA can be administered at a dose of 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, or 1.5 mg/kg.
The dsRNA can be administered at a dose of 0.03 mg/kg.
The pharmaceutical composition may be administered once daily, or the dsRNA 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 dsRNA 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 dsRNA 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 could 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 GNAQ levels is 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, or at not more than 5, 6, 7, 8, 9, or 10 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 dsRNAs 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 mediated by GNAQ expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human GNAQ. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human GNAQ.
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.
The dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Administration
The present invention also includes pharmaceutical compositions and formulations which include the dsRNA 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, 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; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration.
The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
The present invention includes pharmaceutical compositions that can be delivered by injection directly into the brain. The injection can be by stereotactic injection into a particular region of the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus), or the dsRNA can be delivered into multiple regions of the central nervous system (e.g., into multiple regions of the brain, and/or into the spinal cord). The dsRNA can also be delivered into diffuse regions of the brain (e.g., diffuse delivery to the cortex of the brain).
In one embodiment, a dsRNA targeting GNAQ can be delivered by way of a cannula or other delivery device having one end implanted in a tissue, e.g., the brain, e.g., the substantia nigra, cortex, hippocampus, striatum, corpus callosum or globus pallidus of the brain. The cannula can be connected to a reservoir of the dsRNA composition. The flow or delivery can be mediated by a pump, e.g., an osmotic pump or minipump, such as an Alzet pump (Durect, Cupertino, Calif.). In one embodiment, a pump and reservoir are implanted in an area distant from the tissue, e.g., in the abdomen, and delivery is effected by a conduit leading from the pump or reservoir to the site of release. Infusion of the dsRNA composition into the brain can be over several hours or for several days, e.g., for 1, 2, 3, 5, or 7 days or more. Devices for delivery to the brain are described, for example, in U.S. Pat. Nos. 6,093,180, and 5,814,014.
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 dsRNAs 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, distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs 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 C1-10 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 cross 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 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, a GNAQ 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. In some embodiments the lipid to dsRNA ratio can be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1.
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.
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 (C12), a PEG-dimyristyloxypropyl (C14), a PEG-dipalmityloxypropyl (C16), or a PEG-distearyloxypropyl (C18). Other examples of PEG conjugates include PEG-cDMA (N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine), mPEG2000-DMG (mPEG-dimyrystylglycerol (with an average molecular weight of 2,000) and PEG-C-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 1.1., 1.2, 0.13, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 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 one 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.
For example, the lipid-siRNA particle can include 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.
In still another embodiment, the compound 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1) can be used to prepare lipid-siRNA particles. For example, the dsRNA can be formulated in a lipid formulation comprising Tech-G1, distearoyl phosphatidylcholine (DSPC), cholesterol and mPEG2000-DMG at a molar ratio of 50:10:38.5:1.5 at a total lipid to siRNA ratio of 7:1 (wt:wt).
LNP01
In one embodiment, the lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-siRNA nanoparticles (i.e., 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 siRNA (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-siRNA 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-siRNA formulations are as follows:
















cationic lipid/non-cationic





lipid/cholesterol/PEG-lipid conjugate



Cationic Lipid
Lipid:siRNA ratio
Process



















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


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


XTC
[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
Extrusion



[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
Extrusion



[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
In-line



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




lipid:siRNA ~6:1


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



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




lipid:siRNA ~11:1


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



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




Lipid:siRNA 10:1


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



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



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
In-line



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



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


LNP12
1,1′-(2-(4-(2-((2-(bis(2-
Tech G1/DSPC/Cholesterol/PEG-DMG
In-line



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



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



1-yl)ethylazanediyl)didodecan-2-ol



(Tech G1)









LNP09 formulations and XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, which is hereby incorporated by reference.
LNP11 formulations and MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, which is hereby incorporated by reference.
LNP12 formulations and TechG1 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009, which is hereby incorporated by reference.
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 siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA 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 siRNA 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” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA 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. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
The pharmaceutical formulations of 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 of 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 of the present invention 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.
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 (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 (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 (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 (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 (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 (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 dsRNAs 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 (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 (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 (M0310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750), 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 (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 (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 dsRNAs. 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 dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs 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 dsRNAs 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 dsRNAs, 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 (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 dsRNAs 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) (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, C.sub.1-10 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.) (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 (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) (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 dsRNAs 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 beta-diketones (enamines) (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 dsRNAs through the alimentary mucosa (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).
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 which 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 dsRNA compounds and (b) one or more anti-cytokine biologic agents which function by a non-RNAi mechanism. Examples of such biologics include, biologics that target IL1β (e.g., anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab, or certolizumab).
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 preferred.
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 dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by GNAQ expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Methods for treating diseases caused by expression of a GNAQ gene
The invention relates in particular to the use of a dsRNA targeting GNAQ and compositions containing at least one such dsRNA for the treatment of a GNAQ-mediated disorder or disease. For example, a dsRNA targeting a GNAQ gene can be useful for the treatment of cancers that have either an activating mutation of GNAQ and/or are the result of overexpression of GNAQ. Tumors to be targeted include uveal melanoma, cutaneous melanoma, Blue nevi, Nevi of Ota, and neuroendocrine tumors (including but not limited to carcinoid tumors, large cell lung cancer, and small cell lung cancer).
A dsRNA targeting a GNAQ gene is also used for treatment of symptoms of disorders, such as uveal melanoma. Symptoms associated include, e.g., melanoma progression, increasing eye pressure, pain in the eye, and impaired peripheral vision.
Owing to the inhibitory effects on GNAQ expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.
The invention further relates to the use of a dsRNA or a pharmaceutical composition thereof, e.g., for treating a GNAQ mediated disorder or disease, 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 these disorders. In one example, a dsRNA targeting GNAQ can be administered in combination with radiation therapy. In other examples, a dsRNA targeting GNAQ can be administered in combination with a pharmaceutical or therapeutic method for treating a symptom of a GNAQ disease, such as pain medication.
The dsRNA and an additional therapeutic agent can be administered in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.
The invention features a method of administering a dsRNA targeting GNAQ to a patient having a disease or disorder mediated by GNAQ expression, such as a uveal melanoma. Administration of the dsRNA can stabilize and improve vision, for example, in a patient with uveal melanoma. Patients can be administered a therapeutic amount of dsRNA, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dsRNA can be 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. The administration is repeated, for example, on a regular basis, such as 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. Administration of the dsRNA can reduce GNAQ levels in the blood or urine of the patient by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more.
Before administration of a full dose of the dsRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction.
Many GNAQ-associated diseases and disorders are hereditary. Therefore, a patient in need of a GNAQ dsRNA can be identified by taking a family history. A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a GNAQ dsRNA. A DNA test may also be performed on the patient to identify a mutation in the GNAQ gene, before a GNAQ dsRNA is administered to the patient.
Methods for inhibiting expression of a GNAQ gene
In yet another aspect, the invention provides a method for inhibiting the expression of a GNAQ gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target GNAQ gene is reduced or silenced.
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 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.
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 dsRNAs 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
Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
Other embodiments are, for example, in the claims.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).
Example 1
dsRNA 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.
Conjugates
For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3′), an appropriately modified solid support is used for RNA synthesis. The modified solid support is prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA




A 4.7 M aqueous solution of sodium hydroxide (50 mL) is added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) is added and the mixture is stirred at room temperature until completion of the reaction is ascertained by TLC. After 19 h the solution is partitioned with dichloromethane (3×100 mL). The organic layer is dried with anhydrous sodium sulfate, filtered and evaporated. The residue is distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB




Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) is dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) is added to the solution at 0° C. It is then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution is brought to room temperature and stirred further for 6 h. Completion of the reaction is ascertained by TLC. The reaction mixture is concentrated under vacuum and ethyl acetate is added to precipitate diisopropyl urea. The suspension is filtered. The filtrate is washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer is dried over sodium sulfate and concentrated to give the crude product which is purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC




3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) is dissolved in 20% piperidine in dimethylformamide at 0° C. The solution is continued stirring for 1 h. The reaction mixture is concentrated under vacuum, water is added to the residue, and the product is extracted with ethyl acetate. The crude product is purified by conversion into its hydrochloride salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD




The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) is taken up in dichloromethane. The suspension is cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) is added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) is added. The reaction mixture is stirred overnight. The reaction mixture is diluted with dichloromethane and washed with 10% hydrochloric acid. The product is purified by flash chromatography (10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a] phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE




Potassium t-butoxide (1.1 g, 9.8 mmol) is slurried in 30 mL of dry toluene. The mixture is cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD is added slowly with stirring within 20 mins. The temperature is kept below 5° C. during the addition. The stirring is continued for 30 mins at 0° C. and 1 mL of glacial acetic acid is added, immediately followed by 4 g of NaH2PO4.H2O in 40 mL of water The resultant mixture is extracted twice with 100 mL of dichloromethane each and the combined organic extracts are washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue is dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts are adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which are combined, dried and evaporated to dryness. The residue is purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF




Methanol (2 mL) is added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring is continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) is added, the mixture is extracted with ethylacetate (3×40 mL). The combined ethylacetate layer is dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which is purified by column chromatography (10% MeOH/CHCl3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG




Diol AF (1.25 gm 1.994 mmol) is dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) are added with stirring. The reaction is carried out at room temperature overnight. The reaction is quenched by the addition of methanol. The reaction mixture is concentrated under vacuum and to the residue dichloromethane (50 mL) is added. The organic layer is washed with 1M aqueous sodium bicarbonate. The organic layer is dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine is removed by evaporating with toluene. The crude product is purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).
Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH




Compound AG (1.0 g, 1.05 mmol) is mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture is dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) is added and the solution is stirred at room temperature under argon atmosphere for 16 h. It is then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase is dried over anhydrous sodium sulfate and concentrated to dryness. The residue is used as such for the next step.
Cholesterol Derivatised CPG AI




Succinate AH (0.254 g, 0.242 mmol) is dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) are added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) is added. The reaction mixture turned bright orange in color. The solution is agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) is added. The suspension is agitated for 2 h. The CPG is filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups are masked using acetic anhydride/pyridine. The achieved loading of the CPG is measured by taking UV measurement (37 mM/g).
The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) is performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step is performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.
Nucleic acid sequences are represented herein using standard nomenclature, and specifically the abbreviations of Table 1.







TABLE 1







Abbreviations of nucleoside 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
Nucleoside(s)







A
adenosine



C
cytidine



G
guanosine



U
uridine



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



a
2′-O-methyladenosine



c
2′-O-methylcytidine



g
2′-O-methylguanosine



u
2′-O-methyluridine



dT
2′-deoxythymidine



s
a phosphorothioate linkage










Example 2
siRNA Design and Synthesis
Transcripts
siRNA design was carried out to identify siRNAs targeting the G-alpha q subunit (GNAQ) of a heterotrimeric G gene. Three sets were designed, each specific for a different set of cross species: 1: human and monkey; 2) human, monkey and mouse; and 3) mouse and rat. GNAQ sequences were obtained from the NCBI Refseq collection on Nov. 24, 2008 as follows:














Species
GNAQ sequence ref









human
NM_002072.2



rat
NM_031036.1



monkey
AB170509.1



mouse
NM_008139.5










siRNA Design and Specificity Prediction
The predicted specificity of all possible 19mers was determined for each sequence. The GNAQ siRNAs were used in a comprehensive search against the human, cynomolgous monkey, mouse and rat transcriptomes (defined as the set of NM_ and XM_ records within the NCBI Refseq set for human, mouse and rat, and the ‘core’ sequences from the Unigene clusters for Macaca fascicularis) using the FASTA algorithm. The Python script ‘offtargetFasta.py’ was then used to parse the alignments and 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. The off-target score is calculated as follows: mismatches between the oligo and the transcript are given penalties. A mismatch in the seed region in positions 2-9 of the oligo is given a penalty of 2.8; mismatches in the putative cleavage sites 10 and 11 are given a penalty of 1.2, and all other mismatches a penalty of 1. The off-target score for each oligo-transcript pair is then calculated by summing the mismatch penalties. The lowest off-target score from all the oligo-transcript pairs is then determined and used in subsequent sorting of oligos. Both siRNA 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. In picking which oligos to synthesize, off-target score of the antisense strand was sorted from high to low.
Synthesis of dsRNA
The sense and antisense strands of the dsRNA duplexes were synthesized on a MerMade 192 synthesizer at 1 μmol scale. For each sense and antisense sequence listed in Tables 2a, 3a, and 4a, sequence were modified as follows and as listed in Tables 2d, 3d, and 4d:

    
    
        1. In the sense strand, all pyrimidines (U, C) were replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U); in the antisense strand, all pyrimidines (U, C) adjacent to A (UA, CA) were replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U); a 2 base dTdT extension at the 3′ end of both strands was introduced.
        2. In the sense strand, all pyrimidines (U, C) are replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U); in the antisense strand, all pyrimidines (U, C) adjacent to A (UA, CA) are replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U); a 2 base dTsdT (including a phosphorothioate) extension at the 3′ end of both strands was introduced.
        3. In the sense strand, all pyrimidines (U, C) are replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U); in the antisense strand, all pyrimidines (U, C) adjacent to A (UA, CA) and all U adjacent to another U (UU) or G (UG) were replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U); a 2 base dTsdT (including a phosphorothioate) extension at the 3′ end of both strands was introduced.
    
    


The synthesis of each strand of the dsRNA used solid supported oligonucleotide synthesis using phosphoramidite chemistry.
Synthesis was performed at 1 umole 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 an activator. The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and triethylamine.3HF in the second step. The crude sequences thus obtained were precipitated using acetone: ethanol mix and the pellet were re-suspended in 0.5M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS and the resulting mass data confirmed the identity of the sequences. A selected set of samples were also analyzed by IEX chromatography.
All sequences were purified on AKTA explorer purification system using Source 15Q column. A single peak corresponding to the full length sequence was collected in the eluent and was subsequently analyzed for purity by ion exchange chromatography.
The purified sequences were desalted on a Sephadex G25 column using AKTA purifier. The desalted sequences were analyzed for concentration and purity. For the preparation of duplexes, equimolar amounts of sense and antisense strand were heated in the required buffer (e.g. 1×PBS) at 95° C. for 2-5 minutes and slowly cooled to room temperature. Integrity of the duplex was confirmed by HPLC analysis.
Synthesis and Duplex Annealing for in vivo Studies
Step 1. Oligonucleotide Synthesis
Oligonucleotides for in vivo studies were synthesized on an AKTA oligopilot synthesizer or on an ABI 394 DNA/RNA synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500 Å, Prime Synthesis) or the in-house synthesized solid support cholesterol-CPG, AI were used for the synthesis. Other ligand conjugated solid supports amenable to the invention are described in U.S. patent application Ser. No. 10/946,873 filed Sep. 21, 2004, which is hereby incorporated by reference for all purposes. RNA phosphoramidites and 2′-β-methyl modified 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, 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N6-benzoyl-2′-O-methyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-O-methyl-cytidine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-O-methyl-guanosine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-2′-O-methyl-uridine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite and 5′-O-dimethoxytrityl-2′-deoxy-thymidine-3′-O-N,N′-diisopropyl-2-cyanoethylphosphoramidite) were obtained commercially (e.g. from Pierce Nucleic Acids Technologies and ChemGenes Research).
For the syntheses on AKTAoligopilot synthesizer, all phosphoramidites were used at a concentration of 0.2 M in CH3CN except for guanosine and 2′-O-methyl-uridine, which were used at 0.2 M concentration in 10% THF/CH3CN (v/v). Coupling/recycling time of 16 minutes was used for all phosphoramidite couplings. The activator was 5-ethyl-thio-tetrazole (0.75 M, American International Chemicals). For the PO-oxidation, 50 mM iodine in water/pyridine (10:90 v/v) was used and for the PS-oxidation 2% PADS (GL Synthesis) in 2,6-lutidine/CH3CN (1:1 v/v) was used. For the syntheses on ABI 394 DNA/RNA synthesizer, all phosphoramidites were used at a concentration of 0.15 M in CH3CN except for 2′-O-methyl-uridine, which was used at 0.15 M concentration in 10% THF/CH3CN (v/v). Coupling time of 10 minutes was used for all phosphoramidite couplings. The activator was 5-ethyl-thio-tetrazole (0.25 M, Glen Research). For the PO-oxidation, 20 mM iodine in water/pyridine (Glen Research) was used and for the PS-oxidation 0.1M DDTT (AM Chemicals) in pyridine was used.
Step 2. Deprotection of oligonucleotides
After completion of synthesis, the support was transferred to a 100 mL glass bottle (VWR). The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 40 mL of a 40% aq. methyl amine (Aldrich) 90 mins at 45° C. The bottle was cooled briefly on ice and then the methylamine was filtered into a new 500 mL bottle. The CPG was washed three times with 40 mL portions of DMSO. The mixture was then cooled on dry ice.
In order to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position, 60 mL triethylamine trihydrofluoride (Et3N—HF) was added to the above mixture. The mixture was heated at 40° C. for 60 minutes. The reaction was then quenched with 220 mL of 50 mM sodium acetate (pH 5.5) and stored in the freezer until purification.
Sequences synthesized on the ABI DNA/RNA synthesizer
After completion of synthesis, the support was transferred to a 15 mL tube (VWR). The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 7 mL of a 40% aq. methyl amine (Aldrich) 15 mins at 65° C. The bottle was cooled briefly on ice and then the methylamine solution was filtered into a 100 mL bottle (VWR). The CPG was washed three times with 7 mL portions of DMSO. The mixture was then cooled on dry ice.
In order to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position, 10.5 mL triethylamine trihydrofluoride (Et3N-HF) was added to the above mixture. The mixture was heated at 60° C. for 15 minutes. The reaction was then quenched with 38.5 mL of 50 mM sodium acetate (pH 5.5) and stored in the freezer until purification.
Step 3. Quantitation of Crude Oligonucleotides
For all samples, a 10 μL aliquot was diluted with 990 μL of deionised nuclease free water (1.0 mL) and the absorbance reading at 260 nm obtained.
Step 4. Purification of oligonucleotides
Unconjugated oligonucleotides
The unconjugated samples were purified by HPLC on a TSK-Gel SuperQ-5PW (20) column packed in house (17.3×5 cm) or on a commercially available TSK-Gel SuperQ-5PW column (15×0.215 cm) available from TOSOH Bioscience. The buffers were 20 mM phosphate in 10% CH3CN, pH 8.5 (buffer A) and 20 mM phosphate, 1.0 M NaBr in 10% CH3CN, pH 8.5 (buffer B). The flow rate was 50.0 mL/min for the in house packed column and 10.0 ml/min for the commercially obtained column. Wavelengths of 260 and 294 nm were monitored. The fractions containing the full-length oligonucleotides were pooled together, evaporated, and reconstituted to  ̃100 mL with deionised water.
Cholesterol-conjugated oligonucleotides
The cholesterol conjugated sequences were HPLC purified on RPC-Source15 reverse-phase columns packed in house (17.3×5 cm or 15×2 cm). The buffers were 20 mM NaOAc in 10% CH3CN (buffer A) and 20 mM NaOAc in 70% CH3CN (buffer B). The flow rate was 50.0 mL/min for the 17.3×5 cm column and 12.0 ml/min for the 15×2 cm column. Wavelengths of 260 and 284 nm were monitored. The fractions containing the full-length oligonucleotides were pooled, evaporated, and reconstituted to 100 mL with deionised water.
Step 5. Desalting of Purified Oligonucleotides
The purified oligonucleotides were desalted on either an AKTA Explorer or an AKTA Prime system (Amersham Biosciences) using a Sephadex G-25 column packed in house. First, the column was washed with water at a flow rate of 40 mL/min for 20-30 min. The sample was then applied in 40-60 mL fractions. The eluted salt-free fractions were combined, dried, and reconstituted in  ̃50 mL of RNase free water.
Step 6. Purity Analysis
Approximately 0.3 OD of each of the desalted oligonucleotides was diluted in water to 300 μL and were analyzed by CGE, ion exchange HPLC, and LC/MS.
Step 7. Duplex Formation
For the preparation of duplexes, equimolar amounts of sense and antisense strand were heated in the required buffer (e.g. 1×PBS) at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex was confirmed by HPLC analysis.
Tables of dsRNA sequences
Table 2 provides sequences used for design of dsRNA targeting human GNAQ that will cross react with monkey GNAQ. Table 3 provides sequences used for design of dsRNA targeting human GNAQ that will cross react with both monkey and rat GNAQ. Table 4 provides sequences used for design of dsRNA targeting rat GNAQ that will cross react with mouse GNAQ.
Tables 2a, 3a, and 4a following tables provide the sense and antisense strand of GNAQ target sequences. Tables 2b, 3b, and 4b provide exemplary sense and antisense dsRNA strands with a NN 2 base overhang. Tables 2c, 3c, and 4c provide exemplary sense and antisense dsRNA strands with dTdT 2 base overhang. Tables 2d, 3d, and 4d provide sequences of dsRNA that were synthesized, including the dTdT 2 base overhang and modified nucleotides.








TABLE 2a







GNAQ (human X monkey): target sequences



Numbering for target sequences is based on Human


GNAQ NM_002072.












Start of
SEQ

SEQ




target
ID
Target sequence, sense
ID
Target sequence, antisense


sequence
NO.
strand (5′-3′)
NO.
strand (5′-3′)















1217
1
CUAAUUUAUUGCCGUCCUG
74
CAGGACGGCAAUAAAUUAG



 


1213
2
AAUACUAAUUUAUUGCCGU
75
ACGGCAAUAAAUUAGUAUU


 


1810
3
CAGCCAUAGCUUGAUUGCU
76
AGCAAUCAAGCUAUGGCUG


 


1590
4
GUCAGGACACAUCGUUCGA
77
UCGAACGAUGUGUCCUGAC


 


1149
5
CUUCCCUGGUGGGCUAUUG
78
CAAUAGCCCACCAGGGAAG


 


1971
6
GACACUACAUUACCCUAAU
79
AUUAGGGUAAUGUAGUGUC


 


1237
7
ACUCUGUGUGAGCGUGUCC
80
GGACACGCUCACACAGAGU


 


1152
8
CCCUGGUGGGCUAUUGAAG
81
CUUCAAUAGCCCACCAGGG


 


1216
9
ACUAAUUUAUUGCCGUCCU
82
AGGACGGCAAUAAAUUAGU


 


1575
10
CUCUCAAAUGAUACAGUCA
83
UGACUGUAUCAUUUGAGAG


 


1105
11
AGUACAAUCUGGUCUAAUU
84
AAUUAGACCAGAUUGUACU


 


1407
12
CACAAAGAUAAGACUUGUU
85
AACAAGUCUUAUCUUUGUG


 


1108
13
ACAAUCUGGUCUAAUUGUG
86
CACAAUUAGACCAGAUUGU


 


1395
14
CAGUCAUGCACUCACAAAG
87
CUUUGUGAGUGCAUGACUG


 


1595
15
GACACAUCGUUCGAUUUAA
88
UUAAAUCGAACGAUGUGUC


 


1992
16
CUGCUACCCAGAACCUUUU
89
AAAAGGUUCUGGGUAGCAG


 


1809
17
UCAGCCAUAGCUUGAUUGC
90
GCAAUCAAGCUAUGGCUGA


 


1220
18
AUUUAUUGCCGUCCUGGAC
91
GUCCAGGACGGCAAUAAAU


 


1203
19
CAAUUUGCAUAAUACUAAU
92
AUUAGUAUUAUGCAAAUUG


 


1322
20
GUACAGUCCCAGCACAUUU
93
AAAUGUGCUGGGACUGUAC


 


1804
21
UACCUUCAGCCAUAGCUUG
94
CAAGCUAUGGCUGAAGGUA


 


1968
22
ACAGACACUACAUUACCCU
95
AGGGUAAUGUAGUGUCUGU


 


1214
23
AUACUAAUUUAUUGCCGUC
96
GACGGCAAUAAAUUAGUAU


 


1159
24
GGGCUAUUGAAGAUACACA
97
UGUGUAUCUUCAAUAGCCC


 


1603
25
GUUCGAUUUAAGCCAUCAU
98
AUGAUGGCUUAAAUCGAAC


 


1123
26
UGUGCCUCCUAGACACCCG
99
CGGGUGUCUAGGAGGCACA


 


1233
27
CUGGACUCUGUGUGAGCGU
100
ACGCUCACACAGAGUCCAG


 


1930
28
ACCCUCUCUUUCAAUUGCA
101
UGCAAUUGAAAGAGAGGGU


 


1969
29
CAGACACUACAUUACCCUA
102
UAGGGUAAUGUAGUGUCUG


 


1219
30
AAUUUAUUGCCGUCCUGGA
103
UCCAGGACGGCAAUAAAUU


 


1241
31
UGUGUGAGCGUGUCCACAG
104
CUGUGGACACGCUCACACA


 


1153
32
CCUGGUGGGCUAUUGAAGA
105
UCUUCAAUAGCCCACCAGG


 


1805
33
ACCUUCAGCCAUAGCUUGA
106
UCAAGCUAUGGCUGAAGGU


 


1312
34
GGAUGCUGAAGUACAGUCC
107
GGACUGUACUUCAGCAUCC


 


1546
35
AUCCUAGUUCCAUUCUUGG
108
CCAAGAAUGGAACUAGGAU


 


1547
36
UCCUAGUUCCAUUCUUGGU
109
ACCAAGAAUGGAACUAGGA


 


1103
37
GGAGUACAAUCUGGUCUAA
110
UUAGACCAGAUUGUACUCC


 


1334
38
CACAUUUCCUCUCUAUCUU
111
AAGAUAGAGAGGAAAUGUG


 


1255
39
CACAGAGUUUGUAGUAAAU
112
AUUUACUACAAACUCUGUG


 


1967
40
AACAGACACUACAUUACCC
113
GGGUAAUGUAGUGUCUGUU


 


1391
41
UUCUCAGUCAUGCACUCAC
114
GUGAGUGCAUGACUGAGAA


 


1124
42
GUGCCUCCUAGACACCCGC
115
GCGGGUGUCUAGGAGGCAC


 


1612
43
AAGCCAUCAUCAGCUUAAU
116
AUUAAGCUGAUGAUGGCUU


 


1933
44
CUCUCUUUCAAUUGCAGAU
117
AUCUGCAAUUGAAAGAGAG


 


1078
45
ACACCAUCCUCCAGUUGAA
118
UUCAACUGGAGGAUGGUGU


 


1545
46
UAUCCUAGUUCCAUUCUUG
119
CAAGAAUGGAACUAGGAUA


 


1109
47
CAAUCUGGUCUAAUUGUGC
120
GCACAAUUAGACCAGAUUG


 


1398
48
UCAUGCACUCACAAAGAUA
121
UAUCUUUGUGAGUGCAUGA


 


1970
49
AGACACUACAUUACCCUAA
122
UUAGGGUAAUGUAGUGUCU


 


1173
50
ACACAAGAGGGACUGUAUU
123
AAUACAGUCCCUCUUGUGU


 


1313
51
GAUGCUGAAGUACAGUCCC
124
GGGACUGUACUUCAGCAUC


 


1811
52
AGCCAUAGCUUGAUUGCUC
125
GAGCAAUCAAGCUAUGGCU


 


1862
53
CACAGGAGUCCUUUCUUUU
126
AAAAGAAAGGACUCCUGUG


 


1600
54
AUCGUUCGAUUUAAGCCAU
127
AUGGCUUAAAUCGAACGAU


 


1618
55
UCAUCAGCUUAAUUUAAGU
128
ACUUAAAUUAAGCUGAUGA


 


1332
56
AGCACAUUUCCUCUCUAUC
129
GAUAGAGAGGAAAUGUGCU


 


1157
57
GUGGGCUAUUGAAGAUACA
130
UGUAUCUUCAAUAGCCCAC


 


888
58
AUCAUGUAUUCCCAUCUAG
131
CUAGAUGGGAAUACAUGAU


 


1855
59
AAAGACACACAGGAGUCCU
132
AGGACUCCUGUGUGUCUUU


 


1579
60
CAAAUGAUACAGUCAGGAC
133
GUCCUGACUGUAUCAUUUG


 


805
61
UUAGAACAAUUAUCACAUA
134
UAUGUGAUAAUUGUUCUAA


 


1554
62
UCCAUUCUUGGUCAAGUUU
135
AAACUUGACCAAGAAUGGA


 


1113
63
CUGGUCUAAUUGUGCCUCC
136
GGAGGCACAAUUAGACCAG


 


1174
64
CACAAGAGGGACUGUAUUU
137
AAAUACAGUCCCUCUUGUG


 


1735
65
UCUUGUCUCACUUUGGACU
138
AGUCCAAAGUGAGACAAGA


 


1450
66
UUUUCUAUGGAGCAAAACA
139
UGUUUUGCUCCAUAGAAAA


 


1285
67
AUUUAAACUAUUCAGAGGA
140
UCCUCUGAAUAGUUUAAAU


 


804
68
UUUAGAACAAUUAUCACAU
141
AUGUGAUAAUUGUUCUAAA


 


1866
69
GGAGUCCUUUCUUUUGAAA
142
UUUCAAAAGAAAGGACUCC


 


1610
70
UUAAGCCAUCAUCAGCUUA
143
UAAGCUGAUGAUGGCUUAA


 


1117
71
UCUAAUUGUGCCUCCUAGA
144
UCUAGGAGGCACAAUUAGA


 


1320
72
AAGUACAGUCCCAGCACAU
145
AUGUGCUGGGACUGUACUU


 


1317
73
CUGAAGUACAGUCCCAGCA
146
UGCUGGGACUGUACUUCAG
















TABLE 2b







GNAQ (human and monkey): sense and


antisense sequences with 2 base overhangs;


Numbering for target sequences is based on


Human GNAQ NM_002072.













Start of





target


SEQ ID NO
SEQUENCE (5′-3′)
Strand
sequence













147
CUAAUUUAUUGCCGUCCUGNN
sense
1217


 


148
CAGGACGGCAAUAAAUUAGNN
antis
1217


 


149
AAUACUAAUUUAUUGCCGUNN
sense
1213


 


150
ACGGCAAUAAAUUAGUAUUNN
antis
1213


 


151
CAGCCAUAGCUUGAUUGCUNN
sense
1810


 


152
AGCAAUCAAGCUAUGGCUGNN
antis
1810


 


153
GUCAGGACACAUCGUUCGANN
sense
1590


 


154
UCGAACGAUGUGUCCUGACNN
antis
1590


 


155
CUUCCCUGGUGGGCUAUUGNN
sense
1149


 


156
CAAUAGCCCACCAGGGAAGNN
antis
1149


 


157
GACACUACAUUACCCUAAUNN
sense
1971


 


158
AUUAGGGUAAUGUAGUGUCNN
antis
1971


 


159
ACUCUGUGUGAGCGUGUCCNN
sense
1237


 


160
GGACACGCUCACACAGAGUNN
antis
1237


 


161
CCCUGGUGGGCUAUUGAAGNN
sense
1152


 


162
CUUCAAUAGCCCACCAGGGNN
antis
1152


 


163
ACUAAUUUAUUGCCGUCCUNN
sense
1216


 


164
AGGACGGCAAUAAAUUAGUNN
antis
1216


 


165
CUCUCAAAUGAUACAGUCANN
sense
1575


 


166
UGACUGUAUCAUUUGAGAGNN
antis
1575


 


167
AGUACAAUCUGGUCUAAUUNN
sense
1105


 


168
AAUUAGACCAGAUUGUACUNN
antis
1105


 


169
CACAAAGAUAAGACUUGUUNN
sense
1407


 


170
AACAAGUCUUAUCUUUGUGNN
antis
1407


 


171
ACAAUCUGGUCUAAUUGUGNN
sense
1108


 


172
CACAAUUAGACCAGAUUGUNN
antis
1108


 


173
CAGUCAUGCACUCACAAAGNN
sense
1395


 


174
CUUUGUGAGUGCAUGACUGNN
antis
1395


 


175
GACACAUCGUUCGAUUUAANN
sense
1595


 


176
UUAAAUCGAACGAUGUGUCNN
antis
1595


 


177
CUGCUACCCAGAACCUUUUNN
sense
1992


 


178
AAAAGGUUCUGGGUAGCAGNN
antis
1992


 


179
UCAGCCAUAGCUUGAUUGCNN
sense
1809


 


180
GCAAUCAAGCUAUGGCUGANN
antis
1809


 


181
AUUUAUUGCCGUCCUGGACNN
sense
1220


 


182
GUCCAGGACGGCAAUAAAUNN
antis
1220


 


183
CAAUUUGCAUAAUACUAAUNN
sense
1203


 


184
AUUAGUAUUAUGCAAAUUGNN
antis
1203


 


185
GUACAGUCCCAGCACAUUUNN
sense
1322


 


186
AAAUGUGCUGGGACUGUACNN
antis
1322


 


187
UACCUUCAGCCAUAGCUUGNN
sense
1804


 


188
CAAGCUAUGGCUGAAGGUANN
antis
1804


 


189
ACAGACACUACAUUACCCUNN
sense
1968


 


190
AGGGUAAUGUAGUGUCUGUNN
antis
1968


 


191
AUACUAAUUUAUUGCCGUCNN
sense
1214


 


192
GACGGCAAUAAAUUAGUAUNN
antis
1214


 


193
GGGCUAUUGAAGAUACACANN
sense
1159


 


194
UGUGUAUCUUCAAUAGCCCNN
antis
1159


 


195
GUUCGAUUUAAGCCAUCAUNN
sense
1603


 


196
AUGAUGGCUUAAAUCGAACNN
antis
1603


 


197
UGUGCCUCCUAGACACCCGNN
sense
1123


 


198
CGGGUGUCUAGGAGGCACANN
antis
1123


 


199
CUGGACUCUGUGUGAGCGUNN
sense
1233


 


200
ACGCUCACACAGAGUCCAGNN
antis
1233


 


201
ACCCUCUCUUUCAAUUGCANN
sense
1930


 


202
UGCAAUUGAAAGAGAGGGUNN
antis
1930


 


203
CAGACACUACAUUACCCUANN
sense
1969


 


204
UAGGGUAAUGUAGUGUCUGNN
antis
1969


 


205
AAUUUAUUGCCGUCCUGGANN
sense
1219


 


206
UCCAGGACGGCAAUAAAUUNN
antis
1219


 


207
UGUGUGAGCGUGUCCACAGNN
sense
1241


 


208
CUGUGGACACGCUCACACANN
antis
1241


 


209
CCUGGUGGGCUAUUGAAGANN
sense
1153


 


210
UCUUCAAUAGCCCACCAGGNN
antis
1153


 


211
ACCUUCAGCCAUAGCUUGANN
sense
1805


 


212
UCAAGCUAUGGCUGAAGGUNN
antis
1805


 


213
GGAUGCUGAAGUACAGUCCNN
sense
1312


 


214
GGACUGUACUUCAGCAUCCNN
antis
1312


 


215
AUCCUAGUUCCAUUCUUGGNN
sense
1546


 


216
CCAAGAAUGGAACUAGGAUNN
antis
1546


 


217
UCCUAGUUCCAUUCUUGGUNN
sense
1547


 


218
ACCAAGAAUGGAACUAGGANN
antis
1547


 


219
GGAGUACAAUCUGGUCUAANN
sense
1103


 


220
UUAGACCAGAUUGUACUCCNN
antis
1103


 


221
CACAUUUCCUCUCUAUCUUNN
sense
1334


 


222
AAGAUAGAGAGGAAAUGUGNN
antis
1334


 


223
CACAGAGUUUGUAGUAAAUNN
sense
1255


 


224
AUUUACUACAAACUCUGUGNN
antis
1255


 


225
AACAGACACUACAUUACCCNN
sense
1967


 


226
GGGUAAUGUAGUGUCUGUUNN
antis
1967


 


227
UUCUCAGUCAUGCACUCACNN
sense
1391


 


228
GUGAGUGCAUGACUGAGAANN
antis
1391


 


229
GUGCCUCCUAGACACCCGCNN
sense
1124


 


230
GCGGGUGUCUAGGAGGCACNN
antis
1124


 


231
AAGCCAUCAUCAGCUUAAUNN
sense
1612


 


232
AUUAAGCUGAUGAUGGCUUNN
antis
1612


 


233
CUCUCUUUCAAUUGCAGAUNN
sense
1933


 


234
AUCUGCAAUUGAAAGAGAGNN
antis
1933


 


235
ACACCAUCCUCCAGUUGAANN
sense
1078


 


236
UUCAACUGGAGGAUGGUGUNN
antis
1078


 


237
UAUCCUAGUUCCAUUCUUGNN
sense
1545


 


238
CAAGAAUGGAACUAGGAUANN
antis
1545


 


239
CAAUCUGGUCUAAUUGUGCNN
sense
1109


 


240
GCACAAUUAGACCAGAUUGNN
antis
1109


 


241
UCAUGCACUCACAAAGAUANN
sense
1398


 


242
UAUCUUUGUGAGUGCAUGANN
antis
1398


 


243
AGACACUACAUUACCCUAANN
sense
1970


 


244
UUAGGGUAAUGUAGUGUCUNN
antis
1970


 


245
ACACAAGAGGGACUGUAUUNN
sense
1173


 


246
AAUACAGUCCCUCUUGUGUNN
antis
1173


 


247
GAUGCUGAAGUACAGUCCCNN
sense
1313


 


248
GGGACUGUACUUCAGCAUCNN
antis
1313


 


249
AGCCAUAGCUUGAUUGCUCNN
sense
1811


 


250
GAGCAAUCAAGCUAUGGCUNN
antis
1811


 


251
CACAGGAGUCCUUUCUUUUNN
sense
1862


 


252
AAAAGAAAGGACUCCUGUGNN
antis
1862


 


253
AUCGUUCGAUUUAAGCCAUNN
sense
1600


 


254
AUGGCUUAAAUCGAACGAUNN
antis
1600


 


255
UCAUCAGCUUAAUUUAAGUNN
sense
1618


 


256
ACUUAAAUUAAGCUGAUGANN
antis
1618


 


257
AGCACAUUUCCUCUCUAUCNN
sense
1332


 


258
GAUAGAGAGGAAAUGUGCUNN
antis
1332


 


259
GUGGGCUAUUGAAGAUACANN
sense
1157


 


260
UGUAUCUUCAAUAGCCCACNN
antis
1157


 


261
AUCAUGUAUUCCCAUCUAGNN
sense
888


 


262
CUAGAUGGGAAUACAUGAUNN
antis
888


 


263
AAAGACACACAGGAGUCCUNN
sense
1855


 


264
AGGACUCCUGUGUGUCUUUNN
antis
1855


 


265
CAAAUGAUACAGUCAGGACNN
sense
1579


 


266
GUCCUGACUGUAUCAUUUGNN
antis
1579


 


267
UUAGAACAAUUAUCACAUANN
sense
805


 


268
UAUGUGAUAAUUGUUCUAANN
antis
805


 


269
UCCAUUCUUGGUCAAGUUUNN
sense
1554


 


270
AAACUUGACCAAGAAUGGANN
antis
1554


 


271
CUGGUCUAAUUGUGCCUCCNN
sense
1113


 


272
GGAGGCACAAUUAGACCAGNN
antis
1113


 


273
CACAAGAGGGACUGUAUUUNN
sense
1174


 


274
AAAUACAGUCCCUCUUGUGNN
antis
1174


 


275
UCUUGUCUCACUUUGGACUNN
sense
1735


 


276
AGUCCAAAGUGAGACAAGANN
antis
1735


 


277
UUUUCUAUGGAGCAAAACANN
sense
1450


 


278
UGUUUUGCUCCAUAGAAAANN
antis
1450


 


279
AUUUAAACUAUUCAGAGGANN
sense
1285


 


280
UCCUCUGAAUAGUUUAAAUNN
antis
1285


 


281
UUUAGAACAAUUAUCACAUNN
sense
804


 


282
AUGUGAUAAUUGUUCUAAANN
antis
804


 


283
GGAGUCCUUUCUUUUGAAANN
sense
1866


 


284
UUUCAAAAGAAAGGACUCCNN
antis
1866


 


285
UUAAGCCAUCAUCAGCUUANN
sense
1610


 


286
UAAGCUGAUGAUGGCUUAANN
antis
1610


 


287
UCUAAUUGUGCCUCCUAGANN
sense
1117


 


288
UCUAGGAGGCACAAUUAGANN
antis
1117


 


289
AAGUACAGUCCCAGCACAUNN
sense
1320


 


290
AUGUGCUGGGACUGUACUUNN
antis
1320


 


291
CUGAAGUACAGUCCCAGCANN
sense
1317


 


292
UGCUGGGACUGUACUUCAGNN
antis
1317
















TABLE 2c







GNAQ (human and monkey): sense and


antisense sequences with dTdT overhangs


Numbering for target sequences is based on


Human GNAQ NM_002072













Start of





target


SEQ ID NO
SEQUENCE (5′-3′)
Strand
sequence













293
CUAAUUUAUUGCCGUCCUGdTdT
sense
1217


 


294
CAGGACGGCAAUAAAUUAGdTdT
antis
1217


 


295
AAUACUAAUUUAUUGCCGUdTdT
sense
1213


 


296
ACGGCAAUAAAUUAGUAUUdTdT
antis
1213


 


297
CAGCCAUAGCUUGAUUGCUdTdT
sense
1810


 


298
AGCAAUCAAGCUAUGGCUGdTdT
antis
1810


 


299
GUCAGGACACAUCGUUCGAdTdT
sense
1590


 


300
UCGAACGAUGUGUCCUGACdTdT
antis
1590


 


301
CUUCCCUGGUGGGCUAUUGdTdT
sense
1149


 


302
CAAUAGCCCACCAGGGAAGdTdT
antis
1149


 


303
GACACUACAUUACCCUAAUdTdT
sense
1971


 


304
AUUAGGGUAAUGUAGUGUCdTdT
antis
1971


 


305
ACUCUGUGUGAGCGUGUCCdTdT
sense
1237


 


306
GGACACGCUCACACAGAGUdTdT
antis
1237


 


307
CCCUGGUGGGCUAUUGAAGdTdT
sense
1152


 


308
CUUCAAUAGCCCACCAGGGdTdT
antis
1152


 


309
ACUAAUUUAUUGCCGUCCUdTdT
sense
1216


 


310
AGGACGGCAAUAAAUUAGUdTdT
antis
1216


 


311
CUCUCAAAUGAUACAGUCAdTdT
sense
1575


 


312
UGACUGUAUCAUUUGAGAGdTdT
antis
1575


 


313
AGUACAAUCUGGUCUAAUUdTdT
sense
1105


 


314
AAUUAGACCAGAUUGUACUdTdT
antis
1105


 


315
CACAAAGAUAAGACUUGUUdTdT
sense
1407


 


316
AACAAGUCUUAUCUUUGUGdTdT
antis
1407


 


317
ACAAUCUGGUCUAAUUGUGdTdT
sense
1108


 


318
CACAAUUAGACCAGAUUGUdTdT
antis
1108


 


319
CAGUCAUGCACUCACAAAGdTdT
sense
1395


 


320
CUUUGUGAGUGCAUGACUGdTdT
antis
1395


 


321
GACACAUCGUUCGAUUUAAdTdT
sense
1595


 


322
UUAAAUCGAACGAUGUGUCdTdT
antis
1595


 


323
CUGCUACCCAGAACCUUUUdTdT
sense
1992


 


324
AAAAGGUUCUGGGUAGCAGdTdT
antis
1992


 


325
UCAGCCAUAGCUUGAUUGCdTdT
sense
1809


 


326
GCAAUCAAGCUAUGGCUGAdTdT
antis
1809


 


327
AUUUAUUGCCGUCCUGGACdTdT
sense
1220


 


328
GUCCAGGACGGCAAUAAAUdTdT
antis
1220


 


329
CAAUUUGCAUAAUACUAAUdTdT
sense
1203


 


330
AUUAGUAUUAUGCAAAUUGdTdT
antis
1203


 


331
GUACAGUCCCAGCACAUUUdTdT
sense
1322


 


332
AAAUGUGCUGGGACUGUACdTdT
antis
1322


 


333
UACCUUCAGCCAUAGCUUGdTdT
sense
1804


 


334
CAAGCUAUGGCUGAAGGUAdTdT
antis
1804


 


335
ACAGACACUACAUUACCCUdTdT
sense
1968


 


336
AGGGUAAUGUAGUGUCUGUdTdT
antis
1968


 


337
AUACUAAUUUAUUGCCGUCdTdT
sense
1214


 


338
GACGGCAAUAAAUUAGUAUdTdT
antis
1214


 


339
GGGCUAUUGAAGAUACACAdTdT
sense
1159


 


340
UGUGUAUCUUCAAUAGCCCdTdT
antis
1159


 


341
GUUCGAUUUAAGCCAUCAUdTdT
sense
1603


 


342
AUGAUGGCUUAAAUCGAACdTdT
antis
1603


 


343
UGUGCCUCCUAGACACCCGdTdT
sense
1123


 


344
CGGGUGUCUAGGAGGCACAdTdT
antis
1123


 


345
CUGGACUCUGUGUGAGCGUdTdT
sense
1233


 


346
ACGCUCACACAGAGUCCAGdTdT
antis
1233


 


347
ACCCUCUCUUUCAAUUGCAdTdT
sense
1930


 


348
UGCAAUUGAAAGAGAGGGUdTdT
antis
1930


 


349
CAGACACUACAUUACCCUAdTdT
sense
1969


 


350
UAGGGUAAUGUAGUGUCUGdTdT
antis
1969


 


351
AAUUUAUUGCCGUCCUGGAdTdT
sense
1219


 


352
UCCAGGACGGCAAUAAAUUdTdT
antis
1219


 


353
UGUGUGAGCGUGUCCACAGdTdT
sense
1241


 


354
CUGUGGACACGCUCACACAdTdT
antis
1241


 


355
CCUGGUGGGCUAUUGAAGAdTdT
sense
1153


 


356
UCUUCAAUAGCCCACCAGGdTdT
antis
1153


 


357
ACCUUCAGCCAUAGCUUGAdTdT
sense
1805


 


358
UCAAGCUAUGGCUGAAGGUdTdT
antis
1805


 


359
GGAUGCUGAAGUACAGUCCdTdT
sense
1312


 


360
GGACUGUACUUCAGCAUCCdTdT
antis
1312


 


361
AUCCUAGUUCCAUUCUUGGdTdT
sense
1546


 


362
CCAAGAAUGGAACUAGGAUdTdT
antis
1546


 


363
UCCUAGUUCCAUUCUUGGUdTdT
sense
1547


 


364
ACCAAGAAUGGAACUAGGAdTdT
antis
1547


 


365
GGAGUACAAUCUGGUCUAAdTdT
sense
1103


 


366
UUAGACCAGAUUGUACUCCdTdT
antis
1103


 


367
CACAUUUCCUCUCUAUCUUdTdT
sense
1334


 


368
AAGAUAGAGAGGAAAUGUGdTdT
antis
1334


 


369
CACAGAGUUUGUAGUAAAUdTdT
sense
1255


 


370
AUUUACUACAAACUCUGUGdTdT
antis
1255


 


371
AACAGACACUACAUUACCCdTdT
sense
1967


 


372
GGGUAAUGUAGUGUCUGUUdTdT
antis
1967


 


373
UUCUCAGUCAUGCACUCACdTdT
sense
1391


 


374
GUGAGUGCAUGACUGAGAAdTdT
antis
1391


 


375
GUGCCUCCUAGACACCCGCdTdT
sense
1124


 


376
GCGGGUGUCUAGGAGGCACdTdT
antis
1124


 


377
AAGCCAUCAUCAGCUUAAUdTdT
sense
1612


 


378
AUUAAGCUGAUGAUGGCUUdTdT
antis
1612


 


379
CUCUCUUUCAAUUGCAGAUdTdT
sense
1933


 


380
AUCUGCAAUUGAAAGAGAGdTdT
antis
1933


 


381
ACACCAUCCUCCAGUUGAAdTdT
sense
1078


 


382
UUCAACUGGAGGAUGGUGUdTdT
antis
1078


 


383
UAUCCUAGUUCCAUUCUUGdTdT
sense
1545


 


384
CAAGAAUGGAACUAGGAUAdTdT
antis
1545


 


385
CAAUCUGGUCUAAUUGUGCdTdT
sense
1109


 


386
GCACAAUUAGACCAGAUUGdTdT
antis
1109


 


387
UCAUGCACUCACAAAGAUAdTdT
sense
1398


 


388
UAUCUUUGUGAGUGCAUGAdTdT
antis
1398


 


389
AGACACUACAUUACCCUAAdTdT
sense
1970


 


390
UUAGGGUAAUGUAGUGUCUdTdT
antis
1970


 


391
ACACAAGAGGGACUGUAUUdTdT
sense
1173


 


392
AAUACAGUCCCUCUUGUGUdTdT
antis
1173


 


393
GAUGCUGAAGUACAGUCCCdTdT
sense
1313


 


394
GGGACUGUACUUCAGCAUCdTdT
antis
1313


 


395
AGCCAUAGCUUGAUUGCUCdTdT
sense
1811


 


396
GAGCAAUCAAGCUAUGGCUdTdT
antis
1811


 


397
CACAGGAGUCCUUUCUUUUdTdT
sense
1862


 


398
AAAAGAAAGGACUCCUGUGdTdT
antis
1862


 


399
AUCGUUCGAUUUAAGCCAUdTdT
sense
1600


 


400
AUGGCUUAAAUCGAACGAUdTdT
antis
1600


 


401
UCAUCAGCUUAAUUUAAGUdTdT
sense
1618


 


402
ACUUAAAUUAAGCUGAUGAdTdT
antis
1618


 


403
AGCACAUUUCCUCUCUAUCdTdT
sense
1332


 


404
GAUAGAGAGGAAAUGUGCUdTdT
antis
1332


 


405
GUGGGCUAUUGAAGAUACAdTdT
sense
1157


 


406
UGUAUCUUCAAUAGCCCACdTdT
antis
1157


 


407
AUCAUGUAUUCCCAUCUAGdTdT
sense
888


 


408
CUAGAUGGGAAUACAUGAUdTdT
antis
888


 


409
AAAGACACACAGGAGUCCUdTdT
sense
1855


 


410
AGGACUCCUGUGUGUCUUUdTdT
antis
1855


 


411
CAAAUGAUACAGUCAGGACdTdT
sense
1579


 


412
GUCCUGACUGUAUCAUUUGdTdT
antis
1579


 


413
UUAGAACAAUUAUCACAUAdTdT
sense
805


 


414
UAUGUGAUAAUUGUUCUAAdTdT
antis
805


 


415
UCCAUUCUUGGUCAAGUUUdTdT
sense
1554


 


416
AAACUUGACCAAGAAUGGAdTdT
antis
1554


 


417
CUGGUCUAAUUGUGCCUCCdTdT
sense
1113


 


418
GGAGGCACAAUUAGACCAGdTdT
antis
1113


 


419
CACAAGAGGGACUGUAUUUdTdT
sense
1174


 


420
AAAUACAGUCCCUCUUGUGdTdT
antis
1174


 


421
UCUUGUCUCACUUUGGACUdTdT
sense
1735


 


422
AGUCCAAAGUGAGACAAGAdTdT
antis
1735


 


423
UUUUCUAUGGAGCAAAACAdTdT
sense
1450


 


424
UGUUUUGCUCCAUAGAAAAdTdT
antis
1450


 


425
AUUUAAACUAUUCAGAGGAdTdT
sense
1285


 


426
UCCUCUGAAUAGUUUAAAUdTdT
antis
1285


 


427
UUUAGAACAAUUAUCACAUdTdT
sense
804


 


428
AUGUGAUAAUUGUUCUAAAdTdT
antis
804


 


429
GGAGUCCUUUCUUUUGAAAdTdT
sense
1866


 


430
UUUCAAAAGAAAGGACUCCdTdT
antis
1866


 


431
UUAAGCCAUCAUCAGCUUAdTdT
sense
1610


 


432
UAAGCUGAUGAUGGCUUAAdTdT
antis
1610


 


433
UCUAAUUGUGCCUCCUAGAdTdT
sense
1117


 


434
UCUAGGAGGCACAAUUAGAdTdT
antis
1117


 


435
AAGUACAGUCCCAGCACAUdTdT
sense
1320


 


436
AUGUGCUGGGACUGUACUUdTdT
antis
1320


 


437
CUGAAGUACAGUCCCAGCAdTdT
sense
1317


 


438
UGCUGGGACUGUACUUCAGdTdT
antis
1317
















TABLE 2d







GNAQ (human and monkey): modified sense


and antisense strands Numbering for target


sequences is based on Human


GNAQ NM_002072.












Start of





target


SEQUENCE (5′-3′)
Strand
sequence
SEQ ID NO:










Modifications: Sense strand - all pyrimidines


(U, C) are 2′OMe; antisense strand -


pyrimidines adjacent to A (UA, CA) are


2′Ome; 3′ end is dTdT










cuAAuuuAuuGccGuccuGdTdT
sense
1217
439


 


cAGGACGGcAAuAAAUuAGdTdT
antis
1217
440


 


AAuAcuAAuuuAuuGccGudTdT
sense
1213
441


 


ACGGcAAuAAAUuAGuAUUdTdT
antis
1213
442


 


cAGccAuAGcuuGAuuGcudTdT
sense
1810
443


 


AGcAAUcAAGCuAUGGCUGdTdT
antis
1810
444


 


GucAGGAcAcAucGuucGAdTdT
sense
1590
445


 


UCGAACGAUGUGUCCUGACdTdT
antis
1590
446


 


cuucccuGGuGGGcuAuuGdTdT
sense
1149
447


 


cAAuAGCCcACcAGGGAAGdTdT
antis
1149
448


 


GAcAcuAcAuuAcccuAAudTdT
sense
1971
449


 


AUuAGGGuAAUGuAGUGUCdTdT
antis
1971
450


 


AcucuGuGuGAGcGuGuccdTdT
sense
1237
451


 


GGAcACGCUcAcAcAGAGUdTdT
antis
1237
452


 


cccuGGuGGGcuAuuGAAGdTdT
sense
1152
453


 


CUUcAAuAGCCcACcAGGGdTdT
antis
1152
454


 


AcuAAuuuAuuGccGuccudTdT
sense
1216
455


 


AGGACGGcAAuAAAUuAGUdTdT
antis
1216
456


 


cucucAAAuGAuAcAGucAdTdT
sense
1575
457


 


UGACUGuAUcAUUUGAGAGdTdT
antis
1575
458


 


AGuAcAAucuGGucuAAuudTdT
sense
1105
459


 


AAUuAGACcAGAUUGuACUdTdT
antis
1105
460


 


cAcAAAGAuAAGAcuuGuudTdT
sense
1407
461


 


AAcAAGUCUuAUCUUUGUGdTdT
antis
1407
462


 


AcAAucuGGucuAAuuGuGdTdT
sense
1108
463


 


cAcAAUuAGACcAGAUUGUdTdT
antis
1108
464


 


cAGucAuGcAcucAcAAAGdTdT
sense
1395
465


 


CUUUGUGAGUGcAUGACUGdTdT
antis
1395
466


 


GAcAcAucGuucGAuuuAAdTdT
sense
1595
467


 


UuAAAUCGAACGAUGUGUCdTdT
antis
1595
468


 


cuGcuAcccAGAAccuuuudTdT
sense
1992
469


 


AAAAGGUUCUGGGuAGcAGdTdT
antis
1992
470


 


ucAGccAuAGcuuGAuuGcdTdT
sense
1809
471


 


GcAAUcAAGCuAUGGCUGAdTdT
antis
1809
472


 


AuuuAuuGccGuccuGGAcdTdT
sense
1220
473


 


GUCcAGGACGGcAAuAAAUdTdT
antis
1220
474


 


cAAuuuGcAuAAuAcuAAudTdT
sense
1203
475


 


AUuAGuAUuAUGcAAAUUGdTdT
antis
1203
476


 


GuAcAGucccAGcAcAuuudTdT
sense
1322
477


 


AAAUGUGCUGGGACUGuACdTdT
antis
1322
478


 


uAccuucAGccAuAGcuuGdTdT
sense
1804
479


 


cAAGCuAUGGCUGAAGGuAdTdT
antis
1804
480


 


AcAGAcAcuAcAuuAcccudTdT
sense
1968
481


 


AGGGuAAUGuAGUGUCUGUdTdT
antis
1968
482


 


AuAcuAAuuuAuuGccGucdTdT
sense
1214
483


 


GACGGcAAuAAAUuAGuAUdTdT
antis
1214
484


 


GGGcuAuuGAAGAuAcAcAdTdT
sense
1159
485


 


UGUGuAUCUUcAAuAGCCCdTdT
antis
1159
486


 


GuucGAuuuAAGccAucAudTdT
sense
1603
487


 


AUGAUGGCUuAAAUCGAACdTdT
antis
1603
488


 


uGuGccuccuAGAcAcccGdTdT
sense
1123
489


 


CGGGUGUCuAGGAGGcAcAdTdT
antis
1123
490


 


cuGGAcucuGuGuGAGcGudTdT
sense
1233
491


 


ACGCUcAcAcAGAGUCcAGdTdT
antis
1233
492


 


AcccucucuuucAAuuGcAdTdT
sense
1930
493


 


UGcAAUUGAAAGAGAGGGUdTdT
antis
1930
494


 


cAGAcAcuAcAuuAcccuAdTdT
sense
1969
495


 


uAGGGuAAUGuAGUGUCUGdTdT
antis
1969
496


 


AAuuuAuuGccGuccuGGAdTdT
sense
1219
497


 


UCcAGGACGGcAAuAAAUUdTdT
antis
1219
498


 


uGuGuGAGcGuGuccAcAGdTdT
sense
1241
499


 


CUGUGGAcACGCUcAcAcAdTdT
antis
1241
500


 


ccuGGuGGGcuAuuGAAGAdTdT
sense
1153
501


 


UCUUcAAuAGCCcACcAGGdTdT
antis
1153
502


 


AccuucAGccAuAGcuuGAdTdT
sense
1805
503


 


UcAAGCuAUGGCUGAAGGUdTdT
antis
1805
504


 


GGAuGcuGAAGuAcAGuccdTdT
sense
1312
505


 


GGACUGuACUUcAGcAUCCdTdT
antis
1312
506


 


AuccuAGuuccAuucuuGGdTdT
sense
1546
507


 


CcAAGAAUGGAACuAGGAUdTdT
antis
1546
508


 


uccuAGuuccAuucuuGGudTdT
sense
1547
509


 


ACcAAGAAUGGAACuAGGAdTdT
antis
1547
510


 


GGAGuAcAAucuGGucuAAdTdT
sense
1103
511


 


UuAGACcAGAUUGuACUCCdTdT
antis
1103
512


 


cAcAuuuccucucuAucuudTdT
sense
1334
513


 


AAGAuAGAGAGGAAAUGUGdTdT
antis
1334
514


 


cAcAGAGuuuGuAGuAAAudTdT
sense
1255
515


 


AUUuACuAcAAACUCUGUGdTdT
antis
1255
516


 


AAcAGAcAcuAcAuuAcccdTdT
sense
1967
517


 


GGGuAAUGuAGUGUCUGUUdTdT
antis
1967
518


 


uucucAGucAuGcAcucAcdTdT
sense
1391
519


 


GUGAGUGcAUGACUGAGAAdTdT
antis
1391
520


 


GuGccuccuAGAcAcccGcdTdT
sense
1124
521


 


GCGGGUGUCuAGGAGGcACdTdT
antis
1124
522


 


AAGccAucAucAGcuuAAudTdT
sense
1612
523


 


AUuAAGCUGAUGAUGGCUUdTdT
antis
1612
524


 


cucucuuucAAuuGcAGAudTdT
sense
1933
525


 


AUCUGcAAUUGAAAGAGAGdTdT
antis
1933
526


 


AcAccAuccuccAGuuGAAdTdT
sense
1078
527


 


UUcAACUGGAGGAUGGUGUdTdT
antis
1078
528


 


uAuccuAGuuccAuucuuGdTdT
sense
1545
529


 


cAAGAAUGGAACuAGGAuAdTdT
antis
1545
530


 


cAAucuGGucuAAuuGuGcdTdT
sense
1109
531


 


GcAcAAUuAGACcAGAUUGdTdT
antis
1109
532


 


ucAuGcAcucAcAAAGAuAdTdT
sense
1398
533


 


uAUCUUUGUGAGUGcAUGAdTdT
antis
1398
534


 


AGAcAcuAcAuuAcccuAAdTdT
sense
1970
535


 


UuAGGGuAAUGuAGUGUCUdTdT
antis
1970
536


 


AcAcAAGAGGGAcuGuAuudTdT
sense
1173
537


 


AAuAcAGUCCCUCUUGUGUdTdT
antis
1173
538


 


GAuGcuGAAGuAcAGucccdTdT
sense
1313
539


 


GGGACUGuACUUcAGcAUCdTdT
antis
1313
540


 


AGccAuAGcuuGAuuGcucdTdT
sense
1811
541


 


GAGcAAUcAAGCuAUGGCUdTdT
antis
1811
542


 


cAcAGGAGuccuuucuuuudTdT
sense
1862
543


 


AAAAGAAAGGACUCCUGUGdTdT
antis
1862
544


 


AucGuucGAuuuAAGccAudTdT
sense
1600
545


 


AUGGCUuAAAUCGAACGAUdTdT
antis
1600
546


 


ucAucAGcuuAAuuuAAGudTdT
sense
1618
547


 


ACUuAAAUuAAGCUGAUGAdTdT
antis
1618
548


 


AGcAcAuuuccucucuAucdTdT
sense
1332
549


 


GAuAGAGAGGAAAUGUGCUdTdT
antis
1332
550


 


GuGGGcuAuuGAAGAuAcAdTdT
sense
1157
551


 


UGuAUCUUcAAuAGCCcACdTdT
antis
1157
552


 


AucAuGuAuucccAucuAGdTdT
sense
888
553


 


CuAGAUGGGAAuAcAUGAUdTdT
antis
888
554


 


AAAGAcAcAcAGGAGuccudTdT
sense
1855
555


 


AGGACUCCUGUGUGUCUUUdTdT
antis
1855
556


 


cAAAuGAuAcAGucAGGAcdTdT
sense
1579
557


 


GUCCUGACUGuAUcAUUUGdTdT
antis
1579
558


 


uuAGAAcAAuuAucAcAuAdTdT
sense
805
559


 


uAUGUGAuAAUUGUUCuAAdTdT
antis
805
560


 


uccAuucuuGGucAAGuuudTdT
sense
1554
561


 


AAACUUGACcAAGAAUGGAdTdT
antis
1554
562


 


cuGGucuAAuuGuGccuccdTdT
sense
1113
563


 


GGAGGcAcAAUuAGACcAGdTdT
antis
1113
564


 


cAcAAGAGGGAcuGuAuuudTdT
sense
1174
565


 


AAAuAcAGUCCCUCUUGUGdTdT
antis
1174
566


 


ucuuGucucAcuuuGGAcudTdT
sense
1735
567


 


AGUCcAAAGUGAGAcAAGAdTdT
antis
1735
568


 


uuuucuAuGGAGcAAAAcAdTdT
sense
1450
569


 


UGUUUUGCUCcAuAGAAAAdTdT
antis
1450
570


 


AuuuAAAcuAuucAGAGGAdTdT
sense
1285
571


 


UCCUCUGAAuAGUUuAAAUdTdT
antis
1285
572


 


uuuAGAAcAAuuAucAcAudTdT
sense
804
573


 


AUGUGAuAAUUGUUCuAAAdTdT
antis
804
574


 


GGAGuccuuucuuuuGAAAdTdT
sense
1866
575


 


UUUcAAAAGAAAGGACUCCdTdT
antis
1866
576


 


uuAAGccAucAucAGcuuAdTdT
sense
1610
577


 


uAAGCUGAUGAUGGCUuAAdTdT
antis
1610
578


 


ucuAAuuGuGccuccuAGAdTdT
sense
1117
579


 


UCuAGGAGGcAcAAUuAGAdTdT
antis
1117
580


 


AAGuAcAGucccAGcAcAudTdT
sense
1320
581


 


AUGUGCUGGGACUGuACUUdTdT
antis
1320
582


 


cuGAAGuAcAGucccAGcAdTdT
sense
1317
583


 


UGCUGGGACUGuACUUcAGdTdT
antis
1317
584


 







Modifications: Sense strand - all pyrimidines


(U, C)are 2′OMe; antisense strand -


pyrimidines adjacent to A (UA, CA) are


2′Ome; 3′ end is thio (dTsdT)










cuAAuuuAuuGccGuccuGdTsdT
sense
1217
585


 


cAGGACGGcAAuAAAUuAGdTsdT
antis
1217
586


 


AAuAcuAAuuuAuuGccGudTsdT
sense
1213
587


 


ACGGcAAuAAAUuAGuAUUdTsdT
antis
1213
588


 


cAGccAuAGcuuGAuuGcudTsdT
sense
1810
589


 


AGcAAUcAAGCuAUGGCUGdTsdT
antis
1810
590


 


GucAGGAcAcAucGuucGAdTsdT
sense
1590
591


 


UCGAACGAUGUGUCCUGACdTsdT
antis
1590
592


 


cuucccuGGuGGGcuAuuGdTsdT
sense
1149
593


 


cAAuAGCCcACcAGGGAAGdTsdT
antis
1149
594


 


GAcAcuAcAuuAcccuAAudTsdT
sense
1971
595


 


AUuAGGGuAAUGuAGUGUCdTsdT
antis
1971
596


 


AcucuGuGuGAGcGuGuccdTsdT
sense
1237
597


 


GGAcACGCUcAcAcAGAGUdTsdT
antis
1237
598


 


cccuGGuGGGcuAuuGAAGdTsdT
sense
1152
599


 


CUUcAAuAGCCcACcAGGGdTsdT
antis
1152
600


 


AcuAAuuuAuuGccGuccudTsdT
sense
1216
601


 


AGGACGGcAAuAAAUuAGUdTsdT
antis
1216
602


 


cucucAAAuGAuAcAGucAdTsdT
sense
1575
603


 


UGACUGuAUcAUUUGAGAGdTsdT
antis
1575
604


 


AGuAcAAucuGGucuAAuudTsdT
sense
1105
605


 


AAUuAGACcAGAUUGuACUdTsdT
antis
1105
606


 


cAcAAAGAuAAGAcuuGuudTsdT
sense
1407
607


 


AAcAAGUCUuAUCUUUGUGdTsdT
antis
1407
608


 


AcAAucuGGucuAAuuGuGdTsdT
sense
1108
609


 


cAcAAUuAGACcAGAUUGUdTsdT
antis
1108
610


 


cAGucAuGcAcucAcAAAGdTsdT
sense
1395
611


 


CUUUGUGAGUGcAUGACUGdTsdT
antis
1395
612


 


GAcAcAucGuucGAuuuAAdTsdT
sense
1595
613


 


UuAAAUCGAACGAUGUGUCdTsdT
antis
1595
614


 


cuGcuAcccAGAAccuuuudTsdT
sense
1992
615


 


AAAAGGUUCUGGGuAGcAGdTsdT
antis
1992
616


 


ucAGccAuAGcuuGAuuGcdTsdT
sense
1809
617


 


GcAAUcAAGCuAUGGCUGAdTsdT
antis
1809
618


 


AuuuAuuGccGuccuGGAcdTsdT
sense
1220
619


 


GUCcAGGACGGcAAuAAAUdTsdT
antis
1220
620


 


cAAuuuGcAuAAuAcuAAudTsdT
sense
1203
621


 


AUuAGuAUuAUGcAAAUUGdTsdT
antis
1203
622


 


GuAcAGucccAGcAcAuuudTsdT
sense
1322
623


 


AAAUGUGCUGGGACUGuACdTsdT
antis
1322
624


 


uAccuucAGccAuAGcuuGdTsdT
sense
1804
625


 


cAAGCuAUGGCUGAAGGuAdTsdT
antis
1804
626


 


AcAGAcAcuAcAuuAcccudTsdT
sense
1968
627


 


AGGGuAAUGuAGUGUCUGUdTsdT
antis
1968
628


 


AuAcuAAuuuAuuGccGucdTsdT
sense
1214
629


 


GACGGcAAuAAAUuAGuAUdTsdT
antis
1214
630


 


GGGcuAuuGAAGAuAcAcAdTsdT
sense
1159
631


 


UGUGuAUCUUcAAuAGCCCdTsdT
antis
1159
632


 


GuucGAuuuAAGccAucAudTsdT
sense
1603
633


 


AUGAUGGCUuAAAUCGAACdTsdT
antis
1603
634


 


uGuGccuccuAGAcAcccGdTsdT
sense
1123
635


 


CGGGUGUCuAGGAGGcAcAdTsdT
antis
1123
636


 


cuGGAcucuGuGuGAGcGudTsdT
sense
1233
637


 


ACGCUcAcAcAGAGUCcAGdTsdT
antis
1233
638


 


AcccucucuuucAAuuGcAdTsdT
sense
1930
639


 


UGcAAUUGAAAGAGAGGGUdTsdT
antis
1930
640


 


cAGAcAcuAcAuuAcccuAdTsdT
sense
1969
641


 


uAGGGuAAUGuAGUGUCUGdTsdT
antis
1969
642


 


AAuuuAuuGccGuccuGGAdTsdT
sense
1219
643


 


UCcAGGACGGcAAuAAAUUdTsdT
antis
1219
644


 


uGuGuGAGcGuGuccAcAGdTsdT
sense
1241
645


 


CUGUGGAcACGCUcAcAcAdTsdT
antis
1241
646


 


ccuGGuGGGcuAuuGAAGAdTsdT
sense
1153
647


 


UCUUcAAuAGCCcACcAGGdTsdT
antis
1153
648


 


AccuucAGccAuAGcuuGAdTsdT
sense
1805
649


 


UcAAGCuAUGGCUGAAGGUdTsdT
antis
1805
650


 


GGAuGcuGAAGuAcAGuccdTsdT
sense
1312
651


 


GGACUGuACUUcAGcAUCCdTsdT
antis
1312
652


 


AuccuAGuuccAuucuuGGdTsdT
sense
1546
653


 


CcAAGAAUGGAACuAGGAUdTsdT
antis
1546
654


 


uccuAGuuccAuucuuGGudTsdT
sense
1547
655


 


ACcAAGAAUGGAACuAGGAdTsdT
antis
1547
656


 


GGAGuAcAAucuGGucuAAdTsdT
sense
1103
657


 


UuAGACcAGAUUGuACUCCdTsdT
antis
1103
658


 


cAcAuuuccucucuAucuudTsdT
sense
1334
659


 


AAGAuAGAGAGGAAAUGUGdTsdT
antis
1334
660


 


cAcAGAGuuuGuAGuAAAudTsdT
sense
1255
661


 


AUUuACuAcAAACUCUGUGdTsdT
antis
1255
662


 


AAcAGAcAcuAcAuuAcccdTsdT
sense
1967
663


 


GGGuAAUGuAGUGUCUGUUdTsdT
antis
1967
664


 


uucucAGucAuGcAcucAcdTsdT
sense
1391
665


 


GUGAGUGcAUGACUGAGAAdTsdT
antis
1391
666


 


GuGccuccuAGAcAcccGcdTsdT
sense
1124
667


 


GCGGGUGUCuAGGAGGcACdTsdT
antis
1124
668


 


AAGccAucAucAGcuuAAudTsdT
sense
1612
669


 


AUuAAGCUGAUGAUGGCUUdTsdT
antis
1612
670


 


cucucuuucAAuuGcAGAudTsdT
sense
1933
671


 


AUCUGcAAUUGAAAGAGAGdTsdT
antis
1933
672


 


AcAccAuccuccAGuuGAAdTsdT
sense
1078
673


 


UUcAACUGGAGGAUGGUGUdTsdT
antis
1078
674


 


uAuccuAGuuccAuucuuGdTsdT
sense
1545
675


 


cAAGAAUGGAACuAGGAuAdTsdT
antis
1545
676


 


cAAucuGGucuAAuuGuGcdTsdT
sense
1109
677


 


GcAcAAUuAGACcAGAUUGdTsdT
antis
1109
678


 


ucAuGcAcucAcAAAGAuAdTsdT
sense
1398
679


 


uAUCUUUGUGAGUGcAUGAdTsdT
antis
1398
680


 


AGAcAcuAcAuuAcccuAAdTsdT
sense
1970
681


 


UuAGGGuAAUGuAGUGUCUdTsdT
antis
1970
682


 


AcAcAAGAGGGAcuGuAuudTsdT
sense
1173
683


 


AAuAcAGUCCCUCUUGUGUdTsdT
antis
1173
684


 


GAuGcuGAAGuAcAGucccdTsdT
sense
1313
685


 


GGGACUGuACUUcAGcAUCdTsdT
antis
1313
686


 


AGccAuAGcuuGAuuGcucdTsdT
sense
1811
687


 


GAGcAAUcAAGCuAUGGCUdTsdT
antis
1811
688


 


cAcAGGAGuccuuucuuuudTsdT
sense
1862
689


 


AAAAGAAAGGACUCCUGUGdTsdT
antis
1862
690


 


AucGuucGAuuuAAGccAudTsdT
sense
1600
691


 


AUGGCUuAAAUCGAACGAUdTsdT
antis
1600
692


 


ucAucAGcuuAAuuuAAGudTsdT
sense
1618
693


 


ACUuAAAUuAAGCUGAUGAdTsdT
antis
1618
694


 


AGcAcAuuuccucucuAucdTsdT
sense
1332
695


 


GAuAGAGAGGAAAUGUGCUdTsdT
antis
1332
696


 


GuGGGcuAuuGAAGAuAcAdTsdT
sense
1157
697


 


UGuAUCUUcAAuAGCCcACdTsdT
antis
1157
698


 


AucAuGuAuucccAucuAGdTsdT
sense
888
699


 


CuAGAUGGGAAuAcAUGAUdTsdT
antis
888
700


 


AAAGAcAcAcAGGAGuccudTsdT
sense
1855
701


 


AGGACUCCUGUGUGUCUUUdTsdT
antis
1855
702


 


cAAAuGAuAcAGucAGGAcdTsdT
sense
1579
703


 


GUCCUGACUGuAUcAUUUGdTsdT
antis
1579
704


 


uuAGAAcAAuuAucAcAuAdTsdT
sense
805
705


 


uAUGUGAuAAUUGUUCuAAdTsdT
antis
805
706


 


uccAuucuuGGucAAGuuudTsdT
sense
1554
707


 


AAACUUGACcAAGAAUGGAdTsdT
antis
1554
708


 


cuGGucuAAuuGuGccuccdTsdT
sense
1113
709


 


GGAGGcAcAAUuAGACcAGdTsdT
antis
1113
710


 


cAcAAGAGGGAcuGuAuuudTsdT
sense
1174
711


 


AAAuAcAGUCCCUCUUGUGdTsdT
antis
1174
712


 


ucuuGucucAcuuuGGAcudTsdT
sense
1735
713


 


AGUCcAAAGUGAGAcAAGAdTsdT
antis
1735
714


 


uuuucuAuGGAGcAAAAcAdTsdT
sense
1450
715


 


UGUUUUGCUCcAuAGAAAAdTsdT
antis
1450
716


 


AuuuAAAcuAuucAGAGGAdTsdT
sense
1285
717


 


UCCUCUGAAuAGUUuAAAUdTsdT
antis
1285
718


 


uuuAGAAcAAuuAucAcAudTsdT
sense
804
719


 


AUGUGAuAAUUGUUCuAAAdTsdT
antis
804
720


 


GGAGuccuuucuuuuGAAAdTsdT
sense
1866
721


 


UUUcAAAAGAAAGGACUCCdTsdT
antis
1866
722


 


uuAAGccAucAucAGcuuAdTsdT
sense
1610
723


 


uAAGCUGAUGAUGGCUuAAdTsdT
antis
1610
724


 


ucuAAuuGuGccuccuAGAdTsdT
sense
1117
725


 


UCuAGGAGGcAcAAUuAGAdTsdT
antis
1117
726


 


AAGuAcAGucccAGcAcAudTsdT
sense
1320
727


 


AUGUGCUGGGACUGuACUUdTsdT
antis
1320
728


 


cuGAAGuAcAGucccAGcAdTsdT
sense
1317
729


 


UGCUGGGACUGuACUUcAGdTsdT
antis
1317
730


 







Modifications: Sense strand - all


pyrimidines are 2′OMe; antisense


strand - pyrimidines adjacent to A


(UA, CA) + U adjacent to another U


(UU) or G (UG) are 2′Ome;


3′ end is thio (dTsdT).










cuAAuuuAuuGccGuccuGdTsdT
sense
1217
731


 


cAGGACGGcAAuAAAUuAGdTsdT
antis
1217
732


 


AAuAcuAAuuuAuuGccGudTsdT
sense
1213
733


 


ACGGcAAuAAAUuAGuAuUdTsdT
antis
1213
734


 


cAGccAuAGcuuGAuuGcudTsdT
sense
1810
735


 


AGcAAUcAAGCuAuGGCuGdTsdT
antis
1810
736


 


GucAGGAcAcAucGuucGAdTsdT
sense
1590
737


 


UCGAACGAuGuGUCCuGACdTsdT
antis
1590
738


 


cuucccuGGuGGGcuAuuGdTsdT
sense
1149
739


 


cAAuAGCCcACcAGGGAAGdTsdT
antis
1149
740


 


GAcAcuAcAuuAcccuAAudTsdT
sense
1971
741


 


AUuAGGGuAAuGuAGuGUCdTsdT
antis
1971
742


 


AcucuGuGuGAGcGuGuccdTsdT
sense
1237
743


 


GGAcACGCUcAcAcAGAGUdTsdT
antis
1237
744


 


cccuGGuGGGcuAuuGAAGdTsdT
sense
1152
745


 


CuUcAAuAGCCcACcAGGGdTsdT
antis
1152
746


 


AcuAAuuuAuuGccGuccudTsdT
sense
1216
747


 


AGGACGGcAAuAAAUuAGUdTsdT
antis
1216
748


 


cucucAAAuGAuAcAGucAdTsdT
sense
1575
749


 


uGACuGuAUcAuUuGAGAGdTsdT
antis
1575
750


 


AGuAcAAucuGGucuAAuudTsdT
sense
1105
751


 


AAUuAGACcAGAuuGuACUdTsdT
antis
1105
752


 


cAcAAAGAuAAGAcuuGuudTsdT
sense
1407
753


 


AAcAAGUCUuAUCuUuGuGdTsdT
antis
1407
754


 


AcAAucuGGucuAAuuGuGdTsdT
sense
1108
755


 


cAcAAUuAGACcAGAuuGUdTsdT
antis
1108
756


 


cAGucAuGcAcucAcAAAGdTsdT
sense
1395
757


 


CuUuGuGAGuGcAuGACuGdTsdT
antis
1395
758


 


GAcAcAucGuucGAuuuAAdTsdT
sense
1595
759


 


UuAAAUCGAACGAuGuGUCdTsdT
antis
1595
760


 


cuGcuAcccAGAAccuuuudTsdT
sense
1992
761


 


AAAAGGuUCuGGGuAGcAGdTsdT
antis
1992
762


 


ucAGccAuAGcuuGAuuGcdTsdT
sense
1809
763


 


GcAAUcAAGCuAuGGCuGAdTsdT
antis
1809
764


 


AuuuAuuGccGuccuGGAcdTsdT
sense
1220
765


 


GUCcAGGACGGcAAuAAAUdTsdT
antis
1220
766


 


cAAuuuGcAuAAuAcuAAudTsdT
sense
1203
767


 


AUuAGuAUuAuGcAAAuuGdTsdT
antis
1203
768


 


GuAcAGucccAGcAcAuuudTsdT
sense
1322
769


 


AAAuGuGCuGGGACuGuACdTsdT
antis
1322
770


 


uAccuucAGccAuAGcuuGdTsdT
sense
1804
771


 


cAAGCuAuGGCuGAAGGuAdTsdT
antis
1804
772


 


AcAGAcAcuAcAuuAcccudTsdT
sense
1968
773


 


AGGGuAAuGuAGuGUCuGUdTsdT
antis
1968
774


 


AuAcuAAuuuAuuGccGucdTsdT
sense
1214
775


 


GACGGcAAuAAAUuAGuAUdTsdT
antis
1214
776


 


GGGcuAuuGAAGAuAcAcAdTsdT
sense
1159
777


 


uGuGuAUCuUcAAuAGCCCdTsdT
antis
1159
778


 


GuucGAuuuAAGccAucAudTsdT
sense
1603
779


 


AuGAuGGCUuAAAUCGAACdTsdT
antis
1603
780


 


uGuGccuccuAGAcAcccGdTsdT
sense
1123
781


 


CGGGuGUCuAGGAGGcAcAdTsdT
antis
1123
782


 


cuGGAcucuGuGuGAGcGudTsdT
sense
1233
783


 


ACGCUcAcAcAGAGUCcAGdTsdT
antis
1233
784


 


AcccucucuuucAAuuGcAdTsdT
sense
1930
785


 


uGcAAuuGAAAGAGAGGGUdTsdT
antis
1930
786


 


cAGAcAcuAcAuuAcccuAdTsdT
sense
1969
787


 


uAGGGuAAuGuAGuGUCuGdTsdT
antis
1969
788


 


AAuuuAuuGccGuccuGGAdTsdT
sense
1219
789


 


UCcAGGACGGcAAuAAAuUdTsdT
antis
1219
790


 


uGuGuGAGcGuGuccAcAGdTsdT
sense
1241
791


 


CuGuGGAcACGCUcAcAcAdTsdT
antis
1241
792


 


ccuGGuGGGcuAuuGAAGAdTsdT
sense
1153
793


 


UCuUcAAuAGCCcACcAGGdTsdT
antis
1153
794


 


AccuucAGccAuAGcuuGAdTsdT
sense
1805
795


 


UcAAGCuAuGGCuGAAGGUdTsdT
antis
1805
796


 


GGAuGcuGAAGuAcAGuccdTsdT
sense
1312
797


 


GGACuGuACuUcAGcAUCCdTsdT
antis
1312
798


 


AuccuAGuuccAuucuuGGdTsdT
sense
1546
799


 


CcAAGAAuGGAACuAGGAUdTsdT
antis
1546
800


 


uccuAGuuccAuucuuGGudTsdT
sense
1547
801


 


ACcAAGAAuGGAACuAGGAdTsdT
antis
1547
802


 


GGAGuAcAAucuGGucuAAdTsdT
sense
1103
803


 


UuAGACcAGAuuGuACUCCdTsdT
antis
1103
804


 


cAcAuuuccucucuAucuudTsdT
sense
1334
805


 


AAGAuAGAGAGGAAAuGuGdTsdT
antis
1334
806


 


cAcAGAGuuuGuAGuAAAudTsdT
sense
1255
807


 


AuUuACuAcAAACUCuGuGdTsdT
antis
1255
808


 


AAcAGAcAcuAcAuuAcccdTsdT
sense
1967
809


 


GGGuAAuGuAGuGUCuGuUdTsdT
antis
1967
810


 


uucucAGucAuGcAcucAcdTsdT
sense
1391
811


 


GuGAGuGcAuGACuGAGAAdTsdT
antis
1391
812


 


GuGccuccuAGAcAcccGcdTsdT
sense
1124
813


 


GCGGGuGUCuAGGAGGcACdTsdT
antis
1124
814


 


AAGccAucAucAGcuuAAudTsdT
sense
1612
815


 


AUuAAGCuGAuGAuGGCuUdTsdT
antis
1612
816


 


cucucuuucAAuuGcAGAudTsdT
sense
1933
817


 


AUCuGcAAuuGAAAGAGAGdTsdT
antis
1933
818


 


AcAccAuccuccAGuuGAAdTsdT
sense
1078
819


 


uUcAACuGGAGGAuGGuGUdTsdT
antis
1078
820


 


uAuccuAGuuccAuucuuGdTsdT
sense
1545
821


 


cAAGAAuGGAACuAGGAuAdTsdT
antis
1545
822


 


cAAucuGGucuAAuuGuGcdTsdT
sense
1109
823


 


GcAcAAUuAGACcAGAuuGdTsdT
antis
1109
824


 


ucAuGcAcucAcAAAGAuAdTsdT
sense
1398
825


 


uAUCuUuGuGAGuGcAuGAdTsdT
antis
1398
826


 


AGAcAcuAcAuuAcccuAAdTsdT
sense
1970
827


 


UuAGGGuAAuGuAGuGUCUdTsdT
antis
1970
828


 


AcAcAAGAGGGAcuGuAuudTsdT
sense
1173
829


 


AAuAcAGUCCCUCuuGuGUdTsdT
antis
1173
830


 


GAuGcuGAAGuAcAGucccdTsdT
sense
1313
831


 


GGGACuGuACuUcAGcAUCdTsdT
antis
1313
832


 


AGccAuAGcuuGAuuGcucdTsdT
sense
1811
833


 


GAGcAAUcAAGCuAuGGCUdTsdT
antis
1811
834


 


cAcAGGAGuccuuucuuuudTsdT
sense
1862
835


 


AAAAGAAAGGACUCCuGuGdTsdT
antis
1862
836


 


AucGuucGAuuuAAGccAudTsdT
sense
1600
837


 


AuGGCUuAAAUCGAACGAUdTsdT
antis
1600
838


 


ucAucAGcuuAAuuuAAGudTsdT
sense
1618
839


 


ACUuAAAUuAAGCuGAuGAdTsdT
antis
1618
840


 


AGcAcAuuuccucucuAucdTsdT
sense
1332
841


 


GAuAGAGAGGAAAuGuGCUdTsdT
antis
1332
842


 


GuGGGcuAuuGAAGAuAcAdTsdT
sense
1157
843


 


uGuAUCuUcAAuAGCCcACdTsdT
antis
1157
844


 


AucAuGuAuucccAucuAGdTsdT
sense
888
845


 


CuAGAuGGGAAuAcAuGAUdTsdT
antis
888
846


 


AAAGAcAcAcAGGAGuccudTsdT
sense
1855
847


 


AGGACUCCuGuGuGUCuUUdTsdT
antis
1855
848


 


cAAAuGAuAcAGucAGGAcdTsdT
sense
1579
849


 


GUCCuGACuGuAUcAuUuGdTsdT
antis
1579
850


 


uuAGAAcAAuuAucAcAuAdTsdT
sense
805
851


 


uAuGuGAuAAuuGuUCuAAdTsdT
antis
805
852


 


uccAuucuuGGucAAGuuudTsdT
sense
1554
853


 


AAACuuGACcAAGAAuGGAdTsdT
antis
1554
854


 


cuGGucuAAuuGuGccuccdTsdT
sense
1113
855


 


GGAGGcAcAAUuAGACcAGdTsdT
antis
1113
856


 


cAcAAGAGGGAcuGuAuuudTsdT
sense
1174
857


 


AAAuAcAGUCCCUCuuGuGdTsdT
antis
1174
858


 


ucuuGucucAcuuuGGAcudTsdT
sense
1735
859


 


AGUCcAAAGuGAGAcAAGAdTsdT
antis
1735
860


 


uuuucuAuGGAGcAAAAcAdTsdT
sense
1450
861


 


uGuUuuGCUCcAuAGAAAAdTsdT
antis
1450
862


 


AuuuAAAcuAuucAGAGGAdTsdT
sense
1285
863


 


UCCUCuGAAuAGuUuAAAUdTsdT
antis
1285
864


 


uuuAGAAcAAuuAucAcAudTsdT
sense
804
865


 


AuGuGAuAAuuGuUCuAAAdTsdT
antis
804
866


 


GGAGuccuuucuuuuGAAAdTsdT
sense
1866
867


 


uUUcAAAAGAAAGGACUCCdTsdT
antis
1866
868


 


uuAAGccAucAucAGcuuAdTsdT
sense
1610
869


 


uAAGCuGAuGAuGGCUuAAdTsdT
antis
1610
870


 


ucuAAuuGuGccuccuAGAdTsdT
sense
1117
871


 


UCuAGGAGGcAcAAUuAGAdTsdT
antis
1117
872


 


AAGuAcAGucccAGcAcAudTsdT
sense
1320
873


 


AuGuGCuGGGACuGuACuUdTsdT
antis
1320
874


 


cuGAAGuAcAGucccAGcAdTsdT
sense
1317
875


 


uGCuGGGACuGuACuUcAGdTsdT
antis
1317
876
















TABLE 3a







GNAQ (Human, monkey and mouse): target sequences


Numbering for target sequence is Human GNAQ NM_002072.











Start of
SEQ

SEQ



target
ID
Target sequence,
ID
Target sequence,


sequence
NO.
sense strand (5′-3′)
NO.
antisense strand (5′-3′)





1215
877
UACUAAUUUAUUGCCGUCC
888
GGACGGCAAUAAAUUAGUA


 


1217
878
CUAAUUUAUUGCCGUCCUG
889
CAGGACGGCAAUAAAUUAG


 


1216
879
ACUAAUUUAUUGCCGUCCU
890
AGGACGGCAAUAAAUUAGU


 


1322
880
GUACAGUCCCAGCACAUUU
891
AAAUGUGCUGGGACUGUAC


 


1220
881
AUUUAUUGCCGUCCUGGAC
892
GUCCAGGACGGCAAUAAAU


 


1265
882
GUAGUAAAUAUUAUGAUUU
893
AAAUCAUAAUAUUUACUAC


 


1218
883
UAAUUUAUUGCCGUCCUGG
894
CCAGGACGGCAAUAAAUUA


 


1175
884
ACAAGAGGGACUGUAUUUC
895
GAAAUACAGUCCCUCUUGU


 


1223
885
UAUUGCCGUCCUGGACUCU
896
AGAGUCCAGGACGGCAAUA


 


1319
886
GAAGUACAGUCCCAGCACA
897
UGUGCUGGGACUGUACUUC


 


1285
887
AUUUAAACUAUUCAGAGGA
898
UCCUCUGAAUAGUUUAAAU
















TABLE 3b







GNAQ (Human, monkey and mouse): sense and


antisense sequences with 2 base overhangs


Numbering for target sequence is


Human GNAQ NM_002072.










SEQ


Start of


ID


target


NO
SEQUENCE (5′-3′)
Strand
sequence





899
UACUAAUUUAUUGCCGUCCNN
sense
1215


 


900
GGACGGCAAUAAAUUAGUANN
antis
1215


 


901
CUAAUUUAUUGCCGUCCUGNN
sense
1217


 


902
CAGGACGGCAAUAAAUUAGNN
antis
1217


 


903
ACUAAUUUAUUGCCGUCCUNN
sense
1216


 


904
AGGACGGCAAUAAAUUAGUNN
antis
1216


 


905
GUACAGUCCCAGCACAUUUNN
sense
1322


 


906
AAAUGUGCUGGGACUGUACNN
antis
1322


 


907
AUUUAUUGCCGUCCUGGACNN
sense
1220


 


908
GUCCAGGACGGCAAUAAAUNN
antis
1220


 


909
GUAGUAAAUAUUAUGAUUUNN
sense
1265


 


910
AAAUCAUAAUAUUUACUACNN
antis
1265


 


911
UAAUUUAUUGCCGUCCUGGNN
sense
1218


 


912
CCAGGACGGCAAUAAAUUANN
antis
1218


 


913
ACAAGAGGGACUGUAUUUCNN
sense
1175


 


914
GAAAUACAGUCCCUCUUGUNN
antis
1175


 


915
UAUUGCCGUCCUGGACUCUNN
sense
1223


 


916
AGAGUCCAGGACGGCAAUANN
antis
1223


 


917
GAAGUACAGUCCCAGCACANN
sense
1319


 


918
UGUGCUGGGACUGUACUUCNN
antis
1319


 


919
AUUUAAACUAUUCAGAGGANN
sense
1285


 


920
UCCUCUGAAUAGUUUAAAUNN
antis
1285
















TABLE 3c







GNAQ (Human, monkey and mouse): sense and


antisense sequences with dTdT overhangs


Numbering for target sequence is Human


GNAQ NM_002072.










SEQ


Start of


ID


target


NO
SEQUENCE (5′-3′)
Strand
sequence





921
UACUAAUUUAUUGCCGUCCdTdT
sense
1215


 


922
GGACGGCAAUAAAUUAGUAdTdT
antis
1215


 


923
CUAAUUUAUUGCCGUCCUGdTdT
sense
1217


 


924
CAGGACGGCAAUAAAUUAGdTdT
antis
1217


 


925
ACUAAUUUAUUGCCGUCCUdTdT
sense
1216


 


926
AGGACGGCAAUAAAUUAGUdTdT
antis
1216


 


927
GUACAGUCCCAGCACAUUUdTdT
sense
1322


 


928
AAAUGUGCUGGGACUGUACdTdT
antis
1322


 


929
AUUUAUUGCCGUCCUGGACdTdT
sense
1220


 


930
GUCCAGGACGGCAAUAAAUdTdT
antis
1220


 


931
GUAGUAAAUAUUAUGAUUUdTdT
sense
1265


 


932
AAAUCAUAAUAUUUACUACdTdT
antis
1265


 


933
UAAUUUAUUGCCGUCCUGGdTdT
sense
1218


 


934
CCAGGACGGCAAUAAAUUAdTdT
antis
1218


 


935
ACAAGAGGGACUGUAUUUCdTdT
sense
1175


 


936
GAAAUACAGUCCCUCUUGUdTdT
antis
1175


 


937
UAUUGCCGUCCUGGACUCUdTdT
sense
1223


 


938
AGAGUCCAGGACGGCAAUAdTdT
antis
1223


 


939
GAAGUACAGUCCCAGCACAdTdT
sense
1319


 


940
UGUGCUGGGACUGUACUUCdTdT
antis
1319


 


941
AUUUAAACUAUUCAGAGGAdTdT
sense
1285


 


942
UCCUCUGAAUAGUUUAAAUdTdT
antis
1285
















TABLE 3d







GNAQ (Human, monkey and mouse): modified


sense and antisense strands Numbering


for target sequence is Human GNAQ NM_002072.












Start of





target



SEQUENCE (5′-3′)
Strand
sequence
SEQ ID NO










Modifications: Sense strand - all pyrimidines


(U, C)are 2′OMe; antisense strand - pyrimidines


adjacent to A (UA, CA) are 2′Ome;


3′ end is dTdT










uAcuAAuuuAuuGccGuccdTdT
sense
1215
943


 


GGACGGcAAuAAAUuAGuAdTdT
antis
1215
944


 


cuAAuuuAuuGccGuccuGdTdT
sense
1217
945


 


cAGGACGGcAAuAAAUuAGdTdT
antis
1217
946


 


AcuAAuuuAuuGccGuccudTdT
sense
1216
947


 


AGGACGGcAAuAAAUuAGUdTdT
antis
1216
948


 


GuAcAGucccAGcAcAuuudTdT
sense
1322
949


 


AAAUGUGCUGGGACUGuACdTdT
antis
1322
950


 


AuuuAuuGccGuccuGGAcdTdT
sense
1220
951


 


GUCcAGGACGGcAAuAAAUdTdT
antis
1220
952


 


GuAGuAAAuAuuAuGAuuudTdT
sense
1265
953


 


AAAUcAuAAuAUUuACuACdTdT
antis
1265
954


 


uAAuuuAuuGccGuccuGGdTdT
sense
1218
955


 


CcAGGACGGcAAuAAAUuAdTdT
antis
1218
956


 


AcAAGAGGGAcuGuAuuucdTdT
sense
1175
957


 


GAAAuAcAGUCCCUCUUGUdTdT
antis
1175
958


 


uAuuGccGuccuGGAcucudTdT
sense
1223
959


 


AGAGUCcAGGACGGcAAuAdTdT
antis
1223
960


 


GAAGuAcAGucccAGcAcAdTdT
sense
1319
961


 


UGUGCUGGGACUGuACUUCdTdT
antis
1319
962


 


AuuuAAAcuAuucAGAGGAdTdT
sense
1285
963


 


UCCUCUGAAuAGUUuAAAUdTdT
antis
1285
964


 







Modifications: Sense strand - all pyrimidines


(U, C) are 2′OMe; antisense strand -


pyrimidines adjacent to A (UA, CA) are 2′Ome;


3′ end is thio (dTsdT)










uAcuAAuuuAuuGccGuccdTsdT
sense
1215
965


 


GGACGGcAAuAAAUuAGuAdTsdT
antis
1215
966


 


cuAAuuuAuuGccGuccuGdTsdT
sense
1217
967


 


cAGGACGGcAAuAAAUuAGdTsdT
antis
1217
968


 


AcuAAuuuAuuGccGuccudTsdT
sense
1216
969


 


AGGACGGcAAuAAAUuAGUdTsdT
antis
1216
970


 


GuAcAGucccAGcAcAuuudTsdT
sense
1322
971


 


AAAUGUGCUGGGACUGuACdTsdT
antis
1322
972


 


AuuuAuuGccGuccuGGAcdTsdT
sense
1220
973


 


GUCcAGGACGGcAAuAAAUdTsdT
antis
1220
974


 


GuAGuAAAuAuuAuGAuuudTsdT
sense
1265
975


 


AAAUcAuAAuAUUuACuACdTsdT
antis
1265
976


 


uAAuuuAuuGccGuccuGGdTsdT
sense
1218
977


 


CcAGGACGGcAAuAAAUuAdTsdT
antis
1218
978


 


AcAAGAGGGAcuGuAuuucdTsdT
sense
1175
979


 


GAAAuAcAGUCCCUCUUGUdTsdT
antis
1175
980


 


uAuuGccGuccuGGAcucudTsdT
sense
1223
981


 


AGAGUCcAGGACGGcAAuAdTsdT
antis
1223
982


 


GAAGuAcAGucccAGcAcAdTsdT
sense
1319
983


 


UGUGCUGGGACUGuACUUCdTsdT
antis
1319
984


 


AuuuAAAcuAuucAGAGGAdTsdT
sense
1285
985


 


UCCUCUGAAuAGUUuAAAUdTsdT
antis
1285
986


 







Modifications: Sense strand - all pyrimidines


are 2′OMe; antisense strand - pyrimidines


adjacent to A (UA, CA) + U adjacent to


another U (UU) or G (UG) are 2′Ome;


3′ end is thio (dTsdT).










uAcuAAuuuAuuGccGuccdTsdT
sense
1215
987


 


GGACGGcAAuAAAUuAGuAdTsdT
antis
1215
988


 


cuAAuuuAuuGccGuccuGdTsdT
sense
1217
989


 


cAGGACGGcAAuAAAUuAGdTsdT
antis
1217
990


 


AcuAAuuuAuuGccGuccudTsdT
sense
1216
991


 


AGGACGGcAAuAAAUuAGUdTsdT
antis
1216
992


 


GuAcAGucccAGcAcAuuudTsdT
sense
1322
993


 


AAAuGuGCuGGGACuGuACdTsdT
antis
1322
994


 


AuuuAuuGccGuccuGGAcdTsdT
sense
1220
995


 


GUCcAGGACGGcAAuAAAUdTsdT
antis
1220
996


 


GuAGuAAAuAuuAuGAuuudTsdT
sense
1265
997


 


AAAUcAuAAuAuUuACuACdTsdT
antis
1265
998


 


uAAuuuAuuGccGuccuGGdTsdT
sense
1218
999


 


CcAGGACGGcAAuAAAUuAdTsdT
antis
1218
1000


 


AcAAGAGGGAcuGuAuuucdTsdT
sense
1175
1001


 


GAAAuAcAGUCCCUCuuGUdTsdT
antis
1175
1002


 


uAuuGccGuccuGGAcucudTsdT
sense
1223
1003


 


AGAGUCcAGGACGGcAAuAdTsdT
antis
1223
1004


 


GAAGuAcAGucccAGcAcAdTsdT
sense
1319
1005


 


uGuGCuGGGACuGuACuUCdTsdT
antis
1319
1006


 


AuuuAAAcuAuucAGAGGAdTsdT
sense
1285
1007


 


UCCUCuGAAuAGuUuAAAUdTsdT
antis
1285
1008
















TABLE 4a







GNAQ (rat and mouse): target sequences


Numbering for target sequences is Rat GNAQ NM_031036.











Start of


SEQ
Target sequence,


target
SEQ ID
Target sequence,
ID
antisense strand


sequence
NO.
sense strand (5′-3′)
NO.
(5′-3′)














853
1009
UAUUCCCACCUAGUCGACU
1039
AGUCGACUAGGUGGGAAUA


 


855
1010
UUCCCACCUAGUCGACUAC
1040
GUAGUCGACUAGGUGGGAA


 


367
1011
GCUUUUGAGAAUCCAUAUG
1041
CAUAUGGAUUCUCAAAAGC


 


55
1012
CGGAGGAUCAACGACGAGA
1042
UCUCGUCGUUGAUCCUCCG


 


459
1013
AUCUGACUCUACCAAAUAC
1043
GUAUUUGGUAGAGUCAGAU


 


312
1014
ACACAAUAAGGCUCAUGCA
1044
UGCAUGAGCCUUAUUGUGU


 


178
1015
AGGAUCAUCCACGGGUCGG
1045
CCGACCCGUGGAUGAUCCU


 


297
1016
CCCAUACAAGUAUGAACAC
1046
GUGUUCAUACUUGUAUGGG


 


315
1017
CAAUAAGGCUCAUGCACAA
1047
UUGUGCAUGAGCCUUAUUG


 


58
1018
AGGAUCAACGACGAGAUCG
1048
CGAUCUCGUCGUUGAUCCU


 


324
1019
UCAUGCACAAUUGGUUCGA
1049
UCGAACCAAUUGUGCAUGA


 


59
1020
GGAUCAACGACGAGAUCGA
1050
UCGAUCUCGUCGUUGAUCC


 


398
1021
AGAGCUUGUGGAAUGAUCC
1051
GGAUCAUUCCACAAGCUCU


 


57
1022
GAGGAUCAACGACGAGAUC
1052
GAUCUCGUCGUUGAUCCUC


 


56
1023
GGAGGAUCAACGACGAGAU
1053
AUCUCGUCGUUGAUCCUCC


 


369
1024
UUUUGAGAAUCCAUAUGUA
1054
UACAUAUGGAUUCUCAAAA


 


45
1025
CAAGGAAGCCCGGAGGAUC
1055
GAUCCUCCGGGCUUCCUUG


 


460
1026
UCUGACUCUACCAAAUACU
1056
AGUAUUUGGUAGAGUCAGA


 


97
1027
AAGCGCGACGCCCGCCGGG
1057
CCCGGCGGGCGUCGCGCUU


 


314
1028
ACAAUAAGGCUCAUGCACA
1058
UGUGCAUGAGCCUUAUUGU


 


318
1029
UAAGGCUCAUGCACAAUUG
1059
CAAUUGUGCAUGAGCCUUA


 


50
1030
AAGCCCGGAGGAUCAACGA
1060
UCGUUGAUCCUCCGGGCUU


 


323
1031
CUCAUGCACAAUUGGUUCG
1061
CGAACCAAUUGUGCAUGAG


 


327
1032
UGCACAAUUGGUUCGAGAG
1062
CUCUCGAACCAAUUGUGCA


 


329
1033
CACAAUUGGUUCGAGAGGU
1063
ACCUCUCGAACCAAUUGUG


 


862
1034
CUAGUCGACUACUUCCCAG
1064
CUGGGAAGUAGUCGACUAG


 


89
1035
GCAGGGACAAGCGCGACGC
1065
GCGUCGCGCUUGUCCCUGC


 


371
1036
UUGAGAAUCCAUAUGUAGA
1066
UCUACAUAUGGAUUCUCAA


 


868
1037
GACUACUUCCCAGAAUAUG
1067
CAUAUUCUGGGAAGUAGUC


 


62
1038
UCAACGACGAGAUCGAGCG
1068
CGCUCGAUCUCGUCGUUGA
















TABLE 4b







GNAQ (rat and mouse): sense and antisense


sequences with 2 base overhangs


Numbering for target sequences is


Rat GNAQ NM_031036.













Start of





target


SEQ ID NO
SEQUENCE (5′-3′)
Type
sequence













1069
UAUUCCCACCUAGUCGACUNN
sense
853


 


1070
AGUCGACUAGGUGGGAAUANN
antis
853


 


1071
UUCCCACCUAGUCGACUACNN
sense
855


 


1072
GUAGUCGACUAGGUGGGAANN
antis
855


 


1073
GCUUUUGAGAAUCCAUAUGNN
sense
367


 


1074
CAUAUGGAUUCUCAAAAGCNN
antis
367


 


1075
CGGAGGAUCAACGACGAGANN
sense
55


 


1076
UCUCGUCGUUGAUCCUCCGNN
antis
55


 


1077
AUCUGACUCUACCAAAUACNN
sense
459


 


1078
GUAUUUGGUAGAGUCAGAUNN
antis
459


 


1079
ACACAAUAAGGCUCAUGCANN
sense
312


 


1080
UGCAUGAGCCUUAUUGUGUNN
antis
312


 


1081
AGGAUCAUCCACGGGUCGGNN
sense
178


 


1082
CCGACCCGUGGAUGAUCCUNN
antis
178


 


1083
CCCAUACAAGUAUGAACACNN
sense
297


 


1084
GUGUUCAUACUUGUAUGGGNN
antis
297


 


1085
CAAUAAGGCUCAUGCACAANN
sense
315


 


1086
UUGUGCAUGAGCCUUAUUGNN
antis
315


 


1087
AGGAUCAACGACGAGAUCGNN
sense
58


 


1088
CGAUCUCGUCGUUGAUCCUNN
antis
58


 


1089
UCAUGCACAAUUGGUUCGANN
sense
324


 


1090
UCGAACCAAUUGUGCAUGANN
antis
324


 


1091
GGAUCAACGACGAGAUCGANN
sense
59


 


1092
UCGAUCUCGUCGUUGAUCCNN
antis
59


 


1093
AGAGCUUGUGGAAUGAUCCNN
sense
398


 


1094
GGAUCAUUCCACAAGCUCUNN
antis
398


 


1095
GAGGAUCAACGACGAGAUCNN
sense
57


 


1096
GAUCUCGUCGUUGAUCCUCNN
antis
57


 


1097
GGAGGAUCAACGACGAGAUNN
sense
56


 


1098
AUCUCGUCGUUGAUCCUCCNN
antis
56


 


1099
UUUUGAGAAUCCAUAUGUANN
sense
369


 


1100
UACAUAUGGAUUCUCAAAANN
antis
369


 


1101
CAAGGAAGCCCGGAGGAUCNN
sense
45


 


1102
GAUCCUCCGGGCUUCCUUGNN
antis
45


 


1103
UCUGACUCUACCAAAUACUNN
sense
460


 


1104
AGUAUUUGGUAGAGUCAGANN
antis
460


 


1105
AAGCGCGACGCCCGCCGGGNN
sense
97


 


1106
CCCGGCGGGCGUCGCGCUUNN
antis
97


 


1107
ACAAUAAGGCUCAUGCACANN
sense
314


 


1108
UGUGCAUGAGCCUUAUUGUNN
antis
314


 


1109
UAAGGCUCAUGCACAAUUGNN
sense
318


 


1110
CAAUUGUGCAUGAGCCUUANN
antis
318


 


1111
AAGCCCGGAGGAUCAACGANN
sense
50


 


1112
UCGUUGAUCCUCCGGGCUUNN
antis
50


 


1113
CUCAUGCACAAUUGGUUCGNN
sense
323


 


1114
CGAACCAAUUGUGCAUGAGNN
antis
323


 


1115
UGCACAAUUGGUUCGAGAGNN
sense
327


 


1116
CUCUCGAACCAAUUGUGCANN
antis
327


 


1117
CACAAUUGGUUCGAGAGGUNN
sense
329


 


1118
ACCUCUCGAACCAAUUGUGNN
antis
329


 


1119
CUAGUCGACUACUUCCCAGNN
sense
862


 


1120
CUGGGAAGUAGUCGACUAGNN
antis
862


 


1121
GCAGGGACAAGCGCGACGCNN
sense
89


 


1122
GCGUCGCGCUUGUCCCUGCNN
antis
89


 


1123
UUGAGAAUCCAUAUGUAGANN
sense
371


 


1124
UCUACAUAUGGAUUCUCAANN
antis
371


 


1125
GACUACUUCCCAGAAUAUGNN
sense
868


 


1126
CAUAUUCUGGGAAGUAGUCNN
antis
868


 


1127
UCAACGACGAGAUCGAGCGNN
sense
62


 


1128
CGCUCGAUCUCGUCGUUGANN
antis
62
















TABLE 4c







GNAQ (rat and mouse): sense and antisense


sequences with dTdT overhangs


Numbering for target sequences is


Rat GNAQ NM_031036.










SEQ


Start of


ID


target


NO
SEQUENCE (5′-3′)
Strand
sequence













1129
UAUUCCCACCUAGUCGACUdTdT
sense
853


 


1130
AGUCGACUAGGUGGGAAUAdTdT
antis
853


 


1131
UUCCCACCUAGUCGACUACdTdT
sense
855


 


1132
GUAGUCGACUAGGUGGGAAdTdT
antis
855


 


1133
GCUUUUGAGAAUCCAUAUGdTdT
sense
367


 


1134
CAUAUGGAUUCUCAAAAGCdTdT
antis
367


 


1135
CGGAGGAUCAACGACGAGAdTdT
sense
55


 


1136
UCUCGUCGUUGAUCCUCCGdTdT
antis
55


 


1137
AUCUGACUCUACCAAAUACdTdT
sense
459


 


1138
GUAUUUGGUAGAGUCAGAUdTdT
antis
459


 


1139
ACACAAUAAGGCUCAUGCAdTdT
sense
312


 


1140
UGCAUGAGCCUUAUUGUGUdTdT
antis
312


 


1141
AGGAUCAUCCACGGGUCGGdTdT
sense
178


 


1142
CCGACCCGUGGAUGAUCCUdTdT
antis
178


 


1143
CCCAUACAAGUAUGAACACdTdT
sense
297


 


1144
GUGUUCAUACUUGUAUGGGdTdT
antis
297


 


1145
CAAUAAGGCUCAUGCACAAdTdT
sense
315


 


1146
UUGUGCAUGAGCCUUAUUGdTdT
antis
315


 


1147
AGGAUCAACGACGAGAUCGdTdT
sense
58


 


1148
CGAUCUCGUCGUUGAUCCUdTdT
antis
58


 


1149
UCAUGCACAAUUGGUUCGAdTdT
sense
324


 


1150
UCGAACCAAUUGUGCAUGAdTdT
antis
324


 


1151
GGAUCAACGACGAGAUCGAdTdT
sense
59


 


1152
UCGAUCUCGUCGUUGAUCCdTdT
antis
59


 


1153
AGAGCUUGUGGAAUGAUCCdTdT
sense
398


 


1154
GGAUCAUUCCACAAGCUCUdTdT
antis
398


 


1155
GAGGAUCAACGACGAGAUCdTdT
sense
57


 


1156
GAUCUCGUCGUUGAUCCUCdTdT
antis
57


 


1157
GGAGGAUCAACGACGAGAUdTdT
sense
56


 


1158
AUCUCGUCGUUGAUCCUCCdTdT
antis
56


 


1159
UUUUGAGAAUCCAUAUGUAdTdT
sense
369


 


1160
UACAUAUGGAUUCUCAAAAdTdT
antis
369


 


1161
CAAGGAAGCCCGGAGGAUCdTdT
sense
45


 


1162
GAUCCUCCGGGCUUCCUUGdTdT
antis
45


 


1163
UCUGACUCUACCAAAUACUdTdT
sense
460


 


1164
AGUAUUUGGUAGAGUCAGAdTdT
antis
460


 


1165
AAGCGCGACGCCCGCCGGGdTdT
sense
97


 


1166
CCCGGCGGGCGUCGCGCUUdTdT
antis
97


 


1167
ACAAUAAGGCUCAUGCACAdTdT
sense
314


 


1168
UGUGCAUGAGCCUUAUUGUdTdT
antis
314


 


1169
UAAGGCUCAUGCACAAUUGdTdT
sense
318


 


1170
CAAUUGUGCAUGAGCCUUAdTdT
antis
318


 


1171
AAGCCCGGAGGAUCAACGAdTdT
sense
50


 


1172
UCGUUGAUCCUCCGGGCUUdTdT
antis
50


 


1173
CUCAUGCACAAUUGGUUCGdTdT
sense
323


 


1174
CGAACCAAUUGUGCAUGAGdTdT
antis
323


 


1175
UGCACAAUUGGUUCGAGAGdTdT
sense
327


 


1176
CUCUCGAACCAAUUGUGCAdTdT
antis
327


 


1177
CACAAUUGGUUCGAGAGGUdTdT
sense
329


 


1178
ACCUCUCGAACCAAUUGUGdTdT
antis
329


 


1179
CUAGUCGACUACUUCCCAGdTdT
sense
862


 


1180
CUGGGAAGUAGUCGACUAGdTdT
antis
862


 


1181
GCAGGGACAAGCGCGACGCdTdT
sense
89


 


1182
GCGUCGCGCUUGUCCCUGCdTdT
antis
89


 


1183
UUGAGAAUCCAUAUGUAGAdTdT
sense
371


 


1184
UCUACAUAUGGAUUCUCAAdTdT
antis
371


 


1185
GACUACUUCCCAGAAUAUGdTdT
sense
868


 


1186
CAUAUUCUGGGAAGUAGUCdTdT
antis
868


 


1187
UCAACGACGAGAUCGAGCGdTdT
sense
62


 


1188
CGCUCGAUCUCGUCGUUGAdTdT
antis
62
















TABLE 4d







GNAQ dsRNA (rat and mouse): modified sense


and antisense strands


Numbering for target sequences is


Rat GNAQ NM_031036.












Start of





target



SEQUENCE (5′-3′)
Strand
sequence
SEQ ID NO:










Modifications: Sense strand - all pyrimidines


(U, C)are 2′OMe; antisense strand -


pyrimidines adjacent to A (UA, CA) are


2′Ome; 3′ end is dTdT










uAuucccAccuAGucGAcudTdT
sense
853
1189


 


AGUCGACuAGGUGGGAAuAdTdT
antis
853
1190


 


uucccAccuAGucGAcuAcdTdT
sense
855
1191


 


GuAGUCGACuAGGUGGGAAdTdT
antis
855
1192


 


GcuuuuGAGAAuccAuAuGdTdT
sense
367
1193


 


cAuAUGGAUUCUcAAAAGCdTdT
antis
367
1194


 


cGGAGGAucAAcGAcGAGAdTdT
sense
55
1195


 


UCUCGUCGUUGAUCCUCCGdTdT
antis
55
1196


 


AucuGAcucuAccAAAuAcdTdT
sense
459
1197


 


GuAUUUGGuAGAGUcAGAUdTdT
antis
459
1198


 


AcAcAAuAAGGcucAuGcAdTdT
sense
312
1199


 


UGcAUGAGCCUuAUUGUGUdTdT
antis
312
1200


 


AGGAucAuccAcGGGucGGdTdT
sense
178
1201


 


CCGACCCGUGGAUGAUCCUdTdT
antis
178
1202


 


cccAuAcAAGuAuGAAcAcdTdT
sense
297
1203


 


GUGUUcAuACUUGuAUGGGdTdT
antis
297
1204


 


cAAuAAGGcucAuGcAcAAdTdT
sense
315
1205


 


UUGUGcAUGAGCCUuAUUGdTdT
antis
315
1206


 


AGGAucAAcGAcGAGAucGdTdT
sense
58
1207


 


CGAUCUCGUCGUUGAUCCUdTdT
antis
58
1208


 


ucAuGcAcAAuuGGuucGAdTdT
sense
324
1209


 


UCGAACcAAUUGUGcAUGAdTdT
antis
324
1210


 


GGAucAAcGAcGAGAucGAdTdT
sense
59
1211


 


UCGAUCUCGUCGUUGAUCCdTdT
antis
59
1212


 


AGAGcuuGuGGAAuGAuccdTdT
sense
398
1213


 


GGAUcAUUCcAcAAGCUCUdTdT
antis
398
1214


 


GAGGAucAAcGAcGAGAucdTdT
sense
57
1215


 


GAUCUCGUCGUUGAUCCUCdTdT
antis
57
1216


 


GGAGGAucAAcGAcGAGAudTdT
sense
56
1217


 


AUCUCGUCGUUGAUCCUCCdTdT
antis
56
1218


 


uuuuGAGAAuccAuAuGuAdTdT
sense
369
1219


 


uAcAuAUGGAUUCUcAAAAdTdT
antis
369
1220


 


cAAGGAAGcccGGAGGAucdTdT
sense
45
1221


 


GAUCCUCCGGGCUUCCUUGdTdT
antis
45
1222


 


ucuGAcucuAccAAAuAcudTdT
sense
460
1223


 


AGuAUUUGGuAGAGUcAGAdTdT
antis
460
1224


 


AAGcGcGAcGcccGccGGGdTdT
sense
97
1225


 


CCCGGCGGGCGUCGCGCUUdTdT
antis
97
1226


 


AcAAuAAGGcucAuGcAcAdTdT
sense
314
1227


 


UGUGcAUGAGCCUuAUUGUdTdT
antis
314
1228


 


uAAGGcucAuGcAcAAuuGdTdT
sense
318
1229


 


cAAUUGUGcAUGAGCCUuAdTdT
antis
318
1230


 


AAGcccGGAGGAucAAcGAdTdT
sense
50
1231


 


UCGUUGAUCCUCCGGGCUUdTdT
antis
50
1232


 


cucAuGcAcAAuuGGuucGdTdT
sense
323
1233


 


CGAACcAAUUGUGcAUGAGdTdT
antis
323
1234


 


uGcAcAAuuGGuucGAGAGdTdT
sense
327
1235


 


CUCUCGAACcAAUUGUGcAdTdT
antis
327
1236


 


cAcAAuuGGuucGAGAGGudTdT
sense
329
1237


 


ACCUCUCGAACcAAUUGUGdTdT
antis
329
1238


 


cuAGucGAcuAcuucccAGdTdT
sense
862
1239


 


CUGGGAAGuAGUCGACuAGdTdT
antis
862
1240


 


GcAGGGAcAAGcGcGAcGcdTdT
sense
89
1241


 


GCGUCGCGCUUGUCCCUGCdTdT
antis
89
1242


 


uuGAGAAuccAuAuGuAGAdTdT
sense
371
1243


 


UCuAcAuAUGGAUUCUcAAdTdT
antis
371
1244


 


GAcuAcuucccAGAAuAuGdTdT
sense
868
1245


 


cAuAUUCUGGGAAGuAGUCdTdT
antis
868
1246


 


ucAAcGAcGAGAucGAGcGdTdT
sense
62
1247


 


CGCUCGAUCUCGUCGUUGAdTdT
antis
62
1248


 







Modifications: Sense strand - all pyrimidines


(U, C) are 2′OMe; antisense strand -


pyrimidines adjacent to A (UA, CA) are


2′Ome; 3′ end is thio (dTsdT)










uAuucccAccuAGucGAcudTsdT
sense
853
1249


 


AGUCGACuAGGUGGGAAuAdTsdT
antis
853
1250


 


uucccAccuAGucGAcuAcdTsdT
sense
855
1251


 


GuAGUCGACuAGGUGGGAAdTsdT
antis
855
1252


 


GcuuuuGAGAAuccAuAuGdTsdT
sense
367
1253


 


cAuAUGGAUUCUcAAAAGCdTsdT
antis
367
1254


 


cGGAGGAucAAcGAcGAGAdTsdT
sense
55
1255


 


UCUCGUCGUUGAUCCUCCGdTsdT
antis
55
1256


 


AucuGAcucuAccAAAuAcdTsdT
sense
459
1257


 


GuAUUUGGuAGAGUcAGAUdTsdT
antis
459
1258


 


AcAcAAuAAGGcucAuGcAdTsdT
sense
312
1259


 


UGcAUGAGCCUuAUUGUGUdTsdT
antis
312
1260


 


AGGAucAuccAcGGGucGGdTsdT
sense
178
1261


 


CCGACCCGUGGAUGAUCCUdTsdT
antis
178
1262


 


cccAuAcAAGuAuGAAcAcdTsdT
sense
297
1263


 


GUGUUcAuACUUGuAUGGGdTsdT
antis
297
1264


 


cAAuAAGGcucAuGcAcAAdTsdT
sense
315
1265


 


UUGUGcAUGAGCCUuAUUGdTsdT
antis
315
1266


 


AGGAucAAcGAcGAGAucGdTsdT
sense
58
1267


 


CGAUCUCGUCGUUGAUCCUdTsdT
antis
58
1268


 


ucAuGcAcAAuuGGuucGAdTsdT
sense
324
1269


 


UCGAACcAAUUGUGcAUGAdTsdT
antis
324
1270


 


GGAucAAcGAcGAGAucGAdTsdT
sense
59
1271


 


UCGAUCUCGUCGUUGAUCCdTsdT
antis
59
1272


 


AGAGcuuGuGGAAuGAuccdTsdT
sense
398
1273


 


GGAUcAUUCcAcAAGCUCUdTsdT
antis
398
1274


 


GAGGAucAAcGAcGAGAucdTsdT
sense
57
1275


 


GAUCUCGUCGUUGAUCCUCdTsdT
antis
57
1276


 


GGAGGAucAAcGAcGAGAudTsdT
sense
56
1277


 


AUCUCGUCGUUGAUCCUCCdTsdT
antis
56
1278


 


uuuuGAGAAuccAuAuGuAdTsdT
sense
369
1279


 


uAcAuAUGGAUUCUcAAAAdTsdT
antis
369
1280


 


cAAGGAAGcccGGAGGAucdTsdT
sense
45
1281


 


GAUCCUCCGGGCUUCCUUGdTsdT
antis
45
1282


 


ucuGAcucuAccAAAuAcudTsdT
sense
460
1283


 


AGuAUUUGGuAGAGUcAGAdTsdT
antis
460
1284


 


AAGcGcGAcGcccGccGGGdTsdT
sense
97
1285


 


CCCGGCGGGCGUCGCGCUUdTsdT
antis
97
1286


 


AcAAuAAGGcucAuGcAcAdTsdT
sense
314
1287


 


UGUGcAUGAGCCUuAUUGUdTsdT
antis
314
1288


 


uAAGGcucAuGcAcAAuuGdTsdT
sense
318
1289


 


cAAUUGUGcAUGAGCCUuAdTsdT
antis
318
1290


 


AAGcccGGAGGAucAAcGAdTsdT
sense
50
1291


 


UCGUUGAUCCUCCGGGCUUdTsdT
antis
50
1292


 


cucAuGcAcAAuuGGuucGdTsdT
sense
323
1293


 


CGAACcAAUUGUGcAUGAGdTsdT
antis
323
1294


 


uGcAcAAuuGGuucGAGAGdTsdT
sense
327
1295


 


CUCUCGAACcAAUUGUGcAdTsdT
antis
327
1296


 


cAcAAuuGGuucGAGAGGudTsdT
sense
329
1297


 


ACCUCUCGAACcAAUUGUGdTsdT
antis
329
1298


 


cuAGucGAcuAcuucccAGdTsdT
sense
862
1299


 


CUGGGAAGuAGUCGACuAGdTsdT
antis
862
1300


 


GcAGGGAcAAGcGcGAcGcdTsdT
sense
89
1301


 


GCGUCGCGCUUGUCCCUGCdTsdT
antis
89
1302


 


uuGAGAAuccAuAuGuAGAdTsdT
sense
371
1303


 


UCuAcAuAUGGAUUCUcAAdTsdT
antis
371
1304


 


GAcuAcuucccAGAAuAuGdTsdT
sense
868
1305


 


cAuAUUCUGGGAAGuAGUCdTsdT
antis
868
1306


 


ucAAcGAcGAGAucGAGcGdTsdT
sense
62
1307


 


CGCUCGAUCUCGUCGUUGAdTsdT
antis
62
1308


 







Modifications: Sense strand - all pyrimidines


are 2′OMe; antisense strand -


pyrimidines adjacent to A (UA, CA) + U


adjacent to another U (UU) or G (UG) are


2′Ome; 3′ end is thio (dTsdT).


 










uAuucccAccuAGucGAcudTsdT
sense
853
1309


 


AGUCGACuAGGuGGGAAuAdTsdT
antis
853
1310


 


uucccAccuAGucGAcuAcdTsdT
sense
855
1311


 


GuAGUCGACuAGGuGGGAAdTsdT
antis
855
1312


 


GcuuuuGAGAAuccAuAuGdTsdT
sense
367
1313


 


cAuAuGGAuUCUcAAAAGCdTsdT
antis
367
1314


 


cGGAGGAucAAcGAcGAGAdTsdT
sense
55
1315


 


UCUCGUCGuuGAUCCUCCGdTsdT
antis
55
1316


 


AucuGAcucuAccAAAuAcdTsdT
sense
459
1317


 


GuAuUuGGuAGAGUcAGAUdTsdT
antis
459
1318


 


AcAcAAuAAGGcucAuGcAdTsdT
sense
312
1319


 


uGcAuGAGCCUuAuuGuGUdTsdT
antis
312
1320


 


AGGAucAuccAcGGGucGGdTsdT
sense
178
1321


 


CCGACCCGuGGAuGAUCCUdTsdT
antis
178
1322


 


cccAuAcAAGuAuGAAcAcdTsdT
sense
297
1323


 


GuGuUcAuACuuGuAuGGGdTsdT
antis
297
1324


 


cAAuAAGGcucAuGcAcAAdTsdT
sense
315
1325


 


uuGuGcAuGAGCCUuAuuGdTsdT
antis
315
1326


 


AGGAucAAcGAcGAGAucGdTsdT
sense
58
1327


 


CGAUCUCGUCGuuGAUCCUdTsdT
antis
58
1328


 


ucAuGcAcAAuuGGuucGAdTsdT
sense
324
1329


 


UCGAACcAAuuGuGcAuGAdTsdT
antis
324
1330


 


GGAucAAcGAcGAGAucGAdTsdT
sense
59
1331


 


UCGAUCUCGUCGuuGAUCCdTsdT
antis
59
1332


 


AGAGcuuGuGGAAuGAuccdTsdT
sense
398
1333


 


GGAUcAuUCcAcAAGCUCUdTsdT
antis
398
1334


 


GAGGAucAAcGAcGAGAucdTsdT
sense
57
1335


 


GAUCUCGUCGuuGAUCCUCdTsdT
antis
57
1336


 


GGAGGAucAAcGAcGAGAudTsdT
sense
56
1337


 


AUCUCGUCGuuGAUCCUCCdTsdT
antis
56
1338


 


uuuuGAGAAuccAuAuGuAdTsdT
sense
369
1339


 


uAcAuAuGGAuUCUcAAAAdTsdT
antis
369
1340


 


cAAGGAAGcccGGAGGAucdTsdT
sense
45
1341


 


GAUCCUCCGGGCuUCCuuGdTsdT
antis
45
1342


 


ucuGAcucuAccAAAuAcudTsdT
sense
460
1343


 


AGuAuUuGGuAGAGUcAGAdTsdT
antis
460
1344


 


AAGcGcGAcGcccGccGGGdTsdT
sense
97
1345


 


CCCGGCGGGCGUCGCGCuUdTsdT
antis
97
1346


 


AcAAuAAGGcucAuGcAcAdTsdT
sense
314
1347


 


uGuGcAuGAGCCUuAuuGUdTsdT
antis
314
1348


 


uAAGGcucAuGcAcAAuuGdTsdT
sense
318
1349


 


cAAuuGuGcAuGAGCCUuAdTsdT
antis
318
1350


 


AAGcccGGAGGAucAAcGAdTsdT
sense
50
1351


 


UCGuuGAUCCUCCGGGCuUdTsdT
antis
50
1352


 


cucAuGcAcAAuuGGuucGdTsdT
sense
323
1353


 


CGAACcAAuuGuGcAuGAGdTsdT
antis
323
1354


 


uGcAcAAuuGGuucGAGAGdTsdT
sense
327
1355


 


CUCUCGAACcAAuuGuGcAdTsdT
antis
327
1356


 


cAcAAuuGGuucGAGAGGudTsdT
sense
329
1357


 


ACCUCUCGAACcAAuuGuGdTsdT
antis
329
1358


 


cuAGucGAcuAcuucccAGdTsdT
sense
862
1359


 


CuGGGAAGuAGUCGACuAGdTsdT
antis
862
1360


 


GcAGGGAcAAGcGcGAcGcdTsdT
sense
89
1361


 


GCGUCGCGCuuGUCCCuGCdTsdT
antis
89
1362


 


uuGAGAAuccAuAuGuAGAdTsdT
sense
371
1363


 


UCuAcAuAuGGAuUCUcAAdTsdT
antis
371
1364


 


GAcuAcuucccAGAAuAuGdTsdT
sense
868
1365


 


cAuAuUCuGGGAAGuAGUCdTsdT
antis
868
1366


 


ucAAcGAcGAGAucGAGcGdTsdT
sense
62
1367


 


CGCUCGAUCUCGUCGuuGAdTsdT
antis
62
1368









Example 3
In vitro screening
For in vitro screening, cells expressing GNAQ were utilized. Some exemplary cell lines expressing GNAQ include, but are not limited to, human melanoma cell lines OMM1.3 and MEL 285, and Mel 202. OMM1.3 are liver metastisis cells that include a mutant GNAQ gene. MEL285 are primary uveal melanoma cells that include a WT GNAQ gene. MEL202 are also primary uveal melanoma but include a mutant GNAQ gene. A549 (lung carcinoma) and A375 (malignant melanoma) are cancer cell lines expressing WT GNAQ.
Cells expressing human GNAQ with the activating GNAQ mutation were obtained following the method outlined in PCT publication number WO2008/098208, which is incorporated herein in its entirety for all purposes.
The dsRNAs were screened for in vitro inhibition of the target gene. Tissue culture cells were transfected with the dsRNA. Target gene mRNA levels were assayed using qPCR (real time PCR).
Cell culture and transfections:
A549, A375, OMM1.3 and UMEL202 cells were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in specific medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transfection was carried out by adding 5 μl of Opti-MEM to 5 μl of siRNA duplexes (Tables 5-7) per well into a 96-well plate along with 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotics containing 2×104 cells were then added. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at either 0.1 nM, 1.0 nM, or and 10.0 nM final duplex concentration and dose response experiments were done with 10, 1.66, 0.27, 0.046, 0.0077, 0.0012, 0.00021, 0.000035 nM of selected duplexes.







TABLE 5







Duplex (dsRNA) names and corresponding sample names









Sample name
Duplex Name
ssRNA name












1
AD-20032
36864




36865


2
AD-20033
36866




36867


3
AD-20034
36868




36869


4
AD-20035
36870




36871


5
AD-20036
36872




36873


6
AD-20037
36874




36875


7
AD-20038
36876




36877


8
AD-20039
36878




36879


9
AD-20040
36880




36881


10
AD-20041
36882




36883


11
AD-20042
36884




36885


12
AD-20043
36886




36887


13
AD-20044
36888




36889


14
AD-20045
36890




36891


15
AD-20046
36892




36893


16
AD-20047
36894




36895


17
AD-20048
36896




36897


18
AD-20049
36898




36899


19
AD-20050
36900




36901


20
AD-20051
36902




36903


21
AD-20052
36904




36905


22
AD-20053
36906




36907


23
AD-20054
36910




36911


24
AD-20055
36912




36913


25
AD-20056
36914




36915


26
AD-20057
36916




36917


27
AD-20058
36918




36919


28
AD-20059
36920




36921


29
AD-20060
36922




36923


30
AD-20061
36924




36925


31
AD-20062
36926




36927


32
AD-20063
36928




36929


33
AD-20064
36930




36931


34
AD-20065
36932




36933


35
AD-20066
36934




36935


36
AD-20067
36936




36937


37
AD-20068
36938




36939


38
AD-20069
36940




36941


39
AD-20070
36942




36943


40
AD-20071
36946




36947


41
AD-20072
36948




36949


42
AD-20073
36950




36951


43
AD-20074
36954




36955


87
AD-20075
36956




36957


44
AD-20076
36958




36959


45
AD-20077
36960




36961


46
AD-20078
36962




36963


47
AD-20079
36964




36965


48
AD-20080
36966




36967


49
AD-20081
36968




36969


50
AD-20082
36970




36971


51
AD-20083
36972




36973


52
AD-20084
36974




36975


53
AD-20085
36976




36977


54
AD-20086
36978




36979


55
AD-20087
36980




36981


56
AD-20088
36982




36983


57
AD-20089
36984




36985


58
AD-20090
36986




36987


59
AD-20091
36988




36989


60
AD-20092
36990




36991


61
AD-20093
36992




36993


62
AD-20094
36994




36995


63
AD-20095
36996




36997


64
AD-20096
36998




36999


65
AD-20097
37000




37001


88
AD-20098
37002




37003


66
AD-20099
37004




37005


67
AD-20100
37006




37007


68
AD-20101
37008




37009


69
AD-20102
37010




37011


89
AD-20103
37012




37013


70
AD-20104
37014




37015


95
AD-20105
37016




37017


71
AD-20106
37018




37019


72
AD-20107
37022




37023


73
AD-20108
37024




37025


74
AD-20109
37026




37027


75
AD-20110
37032




37033


76
AD-20111
37034




37035


77
AD-20112
37036




37037


78
AD-20113
37038




37039


79
AD-20114
37040




37041


80
AD-20115
37042




37043


81
AD-20116
37044




37045


82
AD-20117
37046




37047


83
AD-20118
37048




37049


84
AD-20119
37050




37051


85
AD-20120
37052




37053


86
AD-20121
37054




37055


91
AD-20193
36908




36909


92
AD-20194
36945




36944


93
AD-20195
37020




37021


94
AD-20196
37028




37029


95
AD-20197
37030




37031
















TABLE 6







Sequences of dsRNA targeting Human GNAQ (NM_002072.2)


(target is position of 5′ base on transcript of NM_002072.2
















SEQ

SEQ



Duplex


ID
Unmodified sequence
ID
Modified sequence


name
Strand
Target
NO:
5′ to 3′
NO:
5′ to 3′
















AD-20032
S
1215
1369
UACUAAUUUAUUGCCGUCC
1527
uAcuAAuuuAuuGccGuccdTdT



A
1215
1370
GGACGGCAAUAAAUUAGUA
1528
GGACGGcAAuAAAUuAGuAdTdT


 


AD-20033
S
1217
1371
CUAAUUUAUUGCCGUCCUG
1529
cuAAuuuAuuGccGuccuGdTdT



A
1217
1372
CAGGACGGCAAUAAAUUAG
1530
cAGGACGGcAAuAAAUuAGdTdT


 


AD-20034
S
1216
1373
ACUAAUUUAUUGCCGUCCU
1531
AcuAAuuuAuuGccGuccudTdT



A
1216
1374
AGGACGGCAAUAAAUUAGU
1532
AGGACGGcAAuAAAUuAGUdTdT


 


AD-20035
S
1322
1375
GUACAGUCCCAGCACAUUU
1533
GuAcAGucccAGcAcAuuudTdT



A
1322
1376
AAAUGUGCUGGGACUGUAC
1534
AAAUGUGCUGGGACUGuACdTdT


 


AD-20036
S
1220
1377
AUUUAUUGCCGUCCUGGAC
1535
AuuuAuuGccGuccuGGAcdTdT



A
1220
1378
GUCCAGGACGGCAAUAAAU
1536
GUCcAGGACGGcAAuAAAUdTdT


 


AD-20037
S
1265
1379
GUAGUAAAUAUUAUGAUUU
1537
GuAGuAAAuAuuAuGAuuudTdT



A
1265
1380
AAAUCAUAAUAUUUACUAC
1538
AAAUcAuAAuAUUuACuACdTdT


 


AD-20038
S
1218
1381
UAAUUUAUUGCCGUCCUGG
1539
uAAuuuAuuGccGuccuGGdTdT



A
1218
1382
CCAGGACGGCAAUAAAUUA
1540
CcAGGACGGcAAuAAAUuAdTdT


 


AD-20039
S
1175
1383
ACAAGAGGGACUGUAUUUC
1541
AcAAGAGGGAcuGuAuuucdTdT



A
1175
1384
GAAAUACAGUCCCUCUUGU
1542
GAAAuAcAGUCCCUCUUGUdTdT


 


AD-20040
S
1223
1385
UAUUGCCGUCCUGGACUCU
1543
uAuuGccGuccuGGAcucudTdT



A
1223
1386
AGAGUCCAGGACGGCAAUA
1544
AGAGUCcAGGACGGcAAuAdTdT


 


AD-20041
S
1319
1387
GAAGUACAGUCCCAGCACA
1545
GAAGuAcAGucccAGcAcAdTdT



A
1319
1388
UGUGCUGGGACUGUACUUC
1546
UGUGCUGGGACUGuACUUCdTdT


 


AD-20042
S
1285
1389
AUUUAAACUAUUCAGAGGA
1547
AuuuAAAcuAuucAGAGGAdTdT



A
1285
1390
UCCUCUGAAUAGUUUAAAU
1548
UCCUCUGAAuAGUUuAAAUdTdT


 


AD-20043
S
1213
1391
AAUACUAAUUUAUUGCCGU
1549
AAuAcuAAuuuAuuGccGudTdT



A
1213
1392
ACGGCAAUAAAUUAGUAUU
1550
ACGGcAAuAAAUuAGuAUUdTdT


 


AD-20044
S
1810
1393
CAGCCAUAGCUUGAUUGCU
1551
cAGccAuAGcuuGAuuGcudTdT



A
1810
1394
AGCAAUCAAGCUAUGGCUG
1552
AGcAAUcAAGCuAUGGCUGdTdT


 


AD-20045
S
1590
1395
GUCAGGACACAUCGUUCGA
1553
GucAGGAcAcAucGuucGAdTdT



A
1590
1396
UCGAACGAUGUGUCCUGAC
1554
UCGAACGAUGUGUCCUGACdTdT


 


AD-20046
S
1149
1397
CUUCCCUGGUGGGCUAUUG
1555
cuucccuGGuGGGcuAuuGdTdT



A
1149
1398
CAAUAGCCCACCAGGGAAG
1556
cAAuAGCCcACcAGGGAAGdTdT


 


AD-20047
S
1971
1399
GACACUACAUUACCCUAAU
1557
GAcAcuAcAuuAcccuAAudTdT



A
1971
1400
AUUAGGGUAAUGUAGUGUC
1558
AUuAGGGuAAUGuAGUGUCdTdT


 


AD-20048
S
1237
1401
ACUCUGUGUGAGCGUGUCC
1559
AcucuGuGuGAGcGuGuccdTdT



A
1237
1402
GGACACGCUCACACAGAGU
1560
GGAcACGCUcAcAcAGAGUdTdT


 


AD-20049
S
1152
1403
CCCUGGUGGGCUAUUGAAG
1561
cccuGGuGGGcuAuuGAAGdTdT



A
1152
1404
CUUCAAUAGCCCACCAGGG
1562
CUUcAAuAGCCcACcAGGGdTdT


 


AD-20050
S
1575
1405
CUCUCAAAUGAUACAGUCA
1563
cucucAAAuGAuAcAGucAdTdT



A
1575
1406
UGACUGUAUCAUUUGAGAG
1564
UGACUGuAUcAUUUGAGAGdTdT


 


AD-20051
S
1105
1407
AGUACAAUCUGGUCUAAUU
1565
AGuAcAAucuGGucuAAuudTdT



A
1105
1408
AAUUAGACCAGAUUGUACU
1566
AAUuAGACcAGAUUGuACUdTdT


 


AD-20052
S
1407
1409
CACAAAGAUAAGACUUGUU
1567
cAcAAAGAuAAGAcuuGuudTdT



A
1407
1410
AACAAGUCUUAUCUUUGUG
1568
AAcAAGUCUuAUCUUUGUGdTdT


 


AD-20053
S
1108
1411
ACAAUCUGGUCUAAUUGUG
1569
AcAAucuGGucuAAuuGuGdTdT



A
1108
1412
CACAAUUAGACCAGAUUGU
1570
cAcAAUuAGACcAGAUUGUdTdT


 


AD-20193
S
1395
1413
CAGUCAUGCACUCACAAAG
1571
cAGucAuGcAcucAcAAAGdTdT



A
1395
1414
CUUUGUGAGUGCAUGACUG
1572
CUUUGUGAGUGcAUGACUGdTdT


 


AD-20054
S
1595
1415
GACACAUCGUUCGAUUUAA
1573
GAcAcAucGuucGAuuuAAdTdT



A
1595
1416
UUAAAUCGAACGAUGUGUC
1574
UuAAAUCGAACGAUGUGUCdTdT


 


AD-20055
S
1992
1417
CUGCUACCCAGAACCUUUU
1575
cuGcuAcccAGAAccuuuudTdT



A
1992
1418
AAAAGGUUCUGGGUAGCAG
1576
AAAAGGUUCUGGGuAGcAGdTdT


 


AD-20056
S
1809
1419
UCAGCCAUAGCUUGAUUGC
1577
ucAGccAuAGcuuGAuuGcdTdT



A
1809
1420
GCAAUCAAGCUAUGGCUGA
1578
GcAAUcAAGCuAUGGCUGAdTdT


 


AD-20057
S
1203
1421
CAAUUUGCAUAAUACUAAU
1579
cAAuuuGcAuAAuAcuAAudTdT



A
1203
1422
AUUAGUAUUAUGCAAAUUG
1580
AUuAGuAUuAUGcAAAUUGdTdT


 


AD-20058
S
1804
1423
UACCUUCAGCCAUAGCUUG
1581
uAccuucAGccAuAGcuuGdTdT



A
1804
1424
CAAGCUAUGGCUGAAGGUA
1582
cAAGCuAUGGCUGAAGGuAdTdT


 


AD-20059
S
1968
1425
ACAGACACUACAUUACCCU
1583
AcAGAcAcuAcAuuAcccudTdT



A
1968
1426
AGGGUAAUGUAGUGUCUGU
1584
AGGGuAAUGuAGUGUCUGUdTdT


 


AD-20060
S
1214
1427
AUACUAAUUUAUUGCCGUC
1585
AuAcuAAuuuAuuGccGucdTdT



A
1214
1428
GACGGCAAUAAAUUAGUAU
1586
GACGGcAAuAAAUuAGuAUdTdT


 


AD-20061
S
1159
1429
GGGCUAUUGAAGAUACACA
1587
GGGcuAuuGAAGAuAcAcAdTdT



A
1159
1430
UGUGUAUCUUCAAUAGCCC
1588
UGUGuAUCUUcAAuAGCCCdTdT


 


AD-20062
S
1603
1431
GUUCGAUUUAAGCCAUCAU
1589
GuucGAuuuAAGccAucAudTdT



A
1603
1432
AUGAUGGCUUAAAUCGAAC
1590
AUGAUGGCUuAAAUCGAACdTdT


 


AD-20063
S
1123
1433
UGUGCCUCCUAGACACCCG
1591
uGuGccuccuAGAcAcccGdTdT



A
1123
1434
CGGGUGUCUAGGAGGCACA
1592
CGGGUGUCuAGGAGGcAcAdTdT


 


AD-20064
S
1233
1435
CUGGACUCUGUGUGAGCGU
1593
cuGGAcucuGuGuGAGcGudTdT



A
1233
1436
ACGCUCACACAGAGUCCAG
1594
ACGCUcAcAcAGAGUCcAGdTdT


 


AD-20065
S
1930
1437
ACCCUCUCUUUCAAUUGCA
1595
AcccucucuuucAAuuGcAdTdT



A
1930
1438
UGCAAUUGAAAGAGAGGGU
1596
UGcAAUUGAAAGAGAGGGUdTdT


 


AD-20066
S
1969
1439
CAGACACUACAUUACCCUA
1597
cAGAcAcuAcAuuAcccuAdTdT



A
1969
1440
UAGGGUAAUGUAGUGUCUG
1598
uAGGGuAAUGuAGUGUCUGdTdT


 


AD-20067
S
1219
1441
AAUUUAUUGCCGUCCUGGA
1599
AAuuuAuuGccGuccuGGAdTdT



A
1219
1442
UCCAGGACGGCAAUAAAUU
1600
UCcAGGACGGcAAuAAAUUdTdT


 


AD-20068
S
1241
1443
UGUGUGAGCGUGUCCACAG
1601
uGuGuGAGcGuGuccAcAGdTdT



A
1241
1444
CUGUGGACACGCUCACACA
1602
CUGUGGAcACGCUcAcAcAdTdT


 


AD-20069
S
1153
1445
CCUGGUGGGCUAUUGAAGA
1603
ccuGGuGGGcuAuuGAAGAdTdT



A
1153
1446
UCUUCAAUAGCCCACCAGG
1604
UCUUcAAuAGCCcACcAGGdTdT


 


AD-20070
S
1805
1447
ACCUUCAGCCAUAGCUUGA
1605
AccuucAGccAuAGcuuGAdTdT



A
1805
1448
UCAAGCUAUGGCUGAAGGU
1606
UcAAGCuAUGGCUGAAGGUdTdT


 


AD-20194
S
1312
1449
GGAUGCUGAAGUACAGUCC
1607
GGAuGcuGAAGuAcAGuccdTdT



A
1312
1450
GGACUGUACUUCAGCAUCC
1608
GGACUGuACUUcAGcAUCCdTdT


 


AD-20071
S
1546
1451
AUCCUAGUUCCAUUCUUGG
1609
AuccuAGuuccAuucuuGGdTdT



A
1546
1452
CCAAGAAUGGAACUAGGAU
1610
CcAAGAAUGGAACuAGGAUdTdT


 


AD-20072
S
1547
1453
UCCUAGUUCCAUUCUUGGU
1611
uccuAGuuccAuucuuGGudTdT



A
1547
1454
ACCAAGAAUGGAACUAGGA
1612
ACcAAGAAUGGAACuAGGAdTdT


 


AD-20073
S
1103
1455
GGAGUACAAUCUGGUCUAA
1613
GGAGuAcAAucuGGucuAAdTdT



A
1103
1456
UUAGACCAGAUUGUACUCC
1614
UuAGACcAGAUUGuACUCCdTdT



A
1334
1457
CACAUUUCCUCUCUAUCUU
1615
cAcAuuuccucucuAucuudTdT



A
1334
1458
AAGAUAGAGAGGAAAUGUG
1616
AAGAuAGAGAGGAAAUGUGdTdT


 


AD-20074
S
1255
1459
CACAGAGUUUGUAGUAAAU
1617
cAcAGAGuuuGuAGuAAAudTdT



A
1255
1460
AUUUACUACAAACUCUGUG
1618
AUUuACuAcAAACUCUGUGdTdT


 


AD-20075
S
1967
1461
AACAGACACUACAUUACCC
1619
AAcAGAcAcuAcAuuAcccdTdT



A
1967
1462
GGGUAAUGUAGUGUCUGUU
1620
GGGuAAUGuAGUGUCUGUUdTdT


 


AD-20076
S
1391
1463
UUCUCAGUCAUGCACUCAC
1621
uucucAGucAuGcAcucAcdTdT



A
1391
1464
GUGAGUGCAUGACUGAGAA
1622
GUGAGUGcAUGACUGAGAAdTdT


 


AD-20077
S
1124
1465
GUGCCUCCUAGACACCCGC
1623
GuGccuccuAGAcAcccGcdTdT



A
1124
1466
GCGGGUGUCUAGGAGGCAC
1624
GCGGGUGUCuAGGAGGcACdTdT


 


AD-20078
S
1612
1467
AAGCCAUCAUCAGCUUAAU
1625
AAGccAucAucAGcuuAAudTdT



A
1612
1468
AUUAAGCUGAUGAUGGCUU
1626
AUuAAGCUGAUGAUGGCUUdTdT


 


AD-20079
S
1933
1469
CUCUCUUUCAAUUGCAGAU
1627
cucucuuucAAuuGcAGAudTdT



A
1933
1470
AUCUGCAAUUGAAAGAGAG
1628
AUCUGcAAUUGAAAGAGAGdTdT


 


AD-20080
S
1078
1471
ACACCAUCCUCCAGUUGAA
1629
AcAccAuccuccAGuuGAAdTdT



A
1078
1472
UUCAACUGGAGGAUGGUGU
1630
UUcAACUGGAGGAUGGUGUdTdT


 


AD-20081
S
1545
1473
UAUCCUAGUUCCAUUCUUG
1631
uAuccuAGuuccAuucuuGdTdT



A
1545
1474
CAAGAAUGGAACUAGGAUA
1632
cAAGAAUGGAACuAGGAuAdTdT


 


AD-20082
S
1109
1475
CAAUCUGGUCUAAUUGUGC
1633
cAAucuGGucuAAuuGuGcdTdT



A
1109
1476
GCACAAUUAGACCAGAUUG
1634
GcAcAAUuAGACcAGAUUGdTdT


 


AD-20083
S
1398
1477
UCAUGCACUCACAAAGAUA
1635
ucAuGcAcucAcAAAGAuAdTdT



A
1398
1478
UAUCUUUGUGAGUGCAUGA
1636
uAUCUUUGUGAGUGcAUGAdTdT


 


AD-20084
S
1970
1479
AGACACUACAUUACCCUAA
1637
AGAcAcuAcAuuAcccuAAdTdT



A
1970
1480
UUAGGGUAAUGUAGUGUCU
1638
UuAGGGuAAUGuAGUGUCUdTdT


 


AD-20085
S
1173
1481
ACACAAGAGGGACUGUAUU
1639
AcAcAAGAGGGAcuGuAuudTdT



A
1173
1482
AAUACAGUCCCUCUUGUGU
1640
AAuAcAGUCCCUCUUGUGUdTdT


 


AD-20086
S
1313
1483
GAUGCUGAAGUACAGUCCC
1641
GAuGcuGAAGuAcAGucccdTdT



A
1313
1484
GGGACUGUACUUCAGCAUC
1642
GGGACUGuACUUcAGcAUCdTdT


 


AD-20087
S
1811
1485
AGCCAUAGCUUGAUUGCUC
1643
AGccAuAGcuuGAuuGcucdTdT



A
1811
1486
GAGCAAUCAAGCUAUGGCU
1644
GAGcAAUcAAGCuAUGGCUdTdT


 


AD-20088
S
1862
1487
CACAGGAGUCCUUUCUUUU
1645
cAcAGGAGuccuuucuuuudTdT



A
1862
1488
AAAAGAAAGGACUCCUGUG
1646
AAAAGAAAGGACUCCUGUGdTdT


 


AD-20089
S
1600
1489
AUCGUUCGAUUUAAGCCAU
1647
AucGuucGAuuuAAGccAudTdT



A
1600
1490
AUGGCUUAAAUCGAACGAU
1648
AUGGCUuAAAUCGAACGAUdTdT


 


AD-20090
S
1618
1491
UCAUCAGCUUAAUUUAAGU
1649
ucAucAGcuuAAuuuAAGudTdT



A
1618
1492
ACUUAAAUUAAGCUGAUGA
1650
ACUuAAAUuAAGCUGAUGAdTdT


 


AD-20091
S
1332
1493
AGCACAUUUCCUCUCUAUC
1651
AGcAcAuuuccucucuAucdTdT



A
1332
1494
GAUAGAGAGGAAAUGUGCU
1652
GAuAGAGAGGAAAUGUGCUdTdT


 


AD-20092
S
1157
1495
GUGGGCUAUUGAAGAUACA
1653
GuGGGcuAuuGAAGAuAcAdTdT



A
1157
1496
UGUAUCUUCAAUAGCCCAC
1654
UGuAUCUUcAAuAGCCcACdTdT


 


AD-20093
S
888
1497
AUCAUGUAUUCCCAUCUAG
1655
AucAuGuAuucccAucuAGdTdT



A
888
1498
CUAGAUGGGAAUACAUGAU
1656
CuAGAUGGGAAuAcAUGAUdTdT


 


AD-20094
S
1855
1499
AAAGACACACAGGAGUCCU
1657
AAAGAcAcAcAGGAGuccudTdT



A
1855
1500
AGGACUCCUGUGUGUCUUU
1658
AGGACUCCUGUGUGUCUUUdTdT


 


AD-20095
S
1579
1501
CAAAUGAUACAGUCAGGAC
1659
cAAAuGAuAcAGucAGGAcdTdT



A
1579
1502
GUCCUGACUGUAUCAUUUG
1660
GUCCUGACUGuAUcAUUUGdTdT


 


AD-20096
S
805
1503
UUAGAACAAUUAUCACAUA
1661
uuAGAAcAAuuAucAcAuAdTdT



A
805
1504
UAUGUGAUAAUUGUUCUAA
1662
uAUGUGAuAAUUGUUCuAAdTdT


 


AD-20097
S
1554
1505
UCCAUUCUUGGUCAAGUUU
1663
uccAuucuuGGucAAGuuudTdT



A
1554
1506
AAACUUGACCAAGAAUGGA
1664
AAACUUGACcAAGAAUGGAdTdT


 


AD-20098
S
1113
1507
CUGGUCUAAUUGUGCCUCC
1665
cuGGucuAAuuGuGccuccdTdT



A
1113
1508
GGAGGCACAAUUAGACCAG
1666
GGAGGcAcAAUuAGACcAGdTdT


 


AD-20099
S
1174
1509
CACAAGAGGGACUGUAUUU
1667
cAcAAGAGGGAcuGuAuuudTdT



A
1174
1510
AAAUACAGUCCCUCUUGUG
1668
AAAuAcAGUCCCUCUUGUGdTdT


 


AD-20100
S
1735
1511
UCUUGUCUCACUUUGGACU
1669
ucuuGucucAcuuuGGAcudTdT



A
1735
1512
AGUCCAAAGUGAGACAAGA
1670
AGUCcAAAGUGAGAcAAGAdTdT


 


AD-20101
S
1450
1513
UUUUCUAUGGAGCAAAACA
1671
uuuucuAuGGAGcAAAAcAdTdT



A
1450
1514
UGUUUUGCUCCAUAGAAAA
1672
UGUUUUGCUCcAuAGAAAAdTdT


 


AD-20102
S
804
1515
UUUAGAACAAUUAUCACAU
1673
uuuAGAAcAAuuAucAcAudTdT



A
804
1516
AUGUGAUAAUUGUUCUAAA
1674
AUGUGAuAAUUGUUCuAAAdTdT


 


AD-20103
S
1866
1517
GGAGUCCUUUCUUUUGAAA
1675
GGAGuccuuucuuuuGAAAdTdT



A
1866
1518
UUUCAAAAGAAAGGACUCC
1676
UUUcAAAAGAAAGGACUCCdTdT


 


AD-20104
S
1610
1519
UUAAGCCAUCAUCAGCUUA
1677
uuAAGccAucAucAGcuuAdTdT



A
1610
1520
UAAGCUGAUGAUGGCUUAA
1678
uAAGCUGAUGAUGGCUuAAdTdT


 


AD-20105
S
1117
1521
UCUAAUUGUGCCUCCUAGA
1679
ucuAAuuGuGccuccuAGAdTdT



A
1117
1522
UCUAGGAGGCACAAUUAGA
1680
UCuAGGAGGcAcAAUuAGAdTdT


 


AD-20106
S
1320
1523
AAGUACAGUCCCAGCACAU
1681
AAGuAcAGucccAGcAcAudTdT



A
1320
1524
AUGUGCUGGGACUGUACUU
1682
AUGUGCUGGGACUGuACUUdTdT


 


AD-20195
S
1317
1525
CUGAAGUACAGUCCCAGCA
1683
cuGAAGuAcAGucccAGcAdTdT



A
1317
1526
UGCUGGGACUGUACUUCAG
1684
UGCUGGGACUGuACUUcAGdTdT
















TABLE 7a







Sequences of dsRNA targeting Mouse GNAQ (NM_031036)


(target is position of 5′ base on transcript of NM_031036
















SEQ

SEQ



Duplex


ID
Unmodified sequence
ID
Modified sequence


Name
Strand
Target
NO:
5′ to 3′
NO:
5′ to 3′
















AD-20107
S
853
1685
UAUUCCCACCUAGUCGACU
1719
uAuucccAccuAGucGAcudTdT



A
853
1686
AGUCGACUAGGUGGGAAUA
1720
AGUCGACuAGGUGGGAAuAdTdT


 


AD-20108
S
855
1687
UUCCCACCUAGUCGACUAC
1721
uucccAccuAGucGAcuAcdTdT



A
855
1688
GUAGUCGACUAGGUGGGAA
1722
GuAGUCGACuAGGUGGGAAdTdT


 


AD-20109
S
367
1689
GCUUUUGAGAAUCCAUAUG
1723
GcuuuuGAGAAuccAuAuGdTdT



A
367
1690
CAUAUGGAUUCUCAAAAGC
1724
cAuAUGGAUUCUcAAAAGCdTdT


 


AD-20196
S
55
1691
CGGAGGAUCAACGACGAGA
1725
cGGAGGAucAAcGAcGAGAdTdT



A
55
1692
UCUCGUCGUUGAUCCUCCG
1726
UCUCGUCGUUGAUCCUCCGdTdT


 


AD-20197
S
459
1693
AUCUGACUCUACCAAAUAC
1727
AucuGAcucuAccAAAuAcdTdT



A
459
1694
GUAUUUGGUAGAGUCAGAU
1728
GuAUUUGGuAGAGUcAGAUdTdT


 


AD-20110
S
312
1695
ACACAAUAAGGCUCAUGCA
1729
AcAcAAuAAGGcucAuGcAdTdT



A
312
1696
UGCAUGAGCCUUAUUGUGU
1730
UGcAUGAGCCUuAUUGUGUdTdT


 


AD-20111
S
178
1697
AGGAUCAUCCACGGGUCGG
1731
AGGAucAuccAcGGGucGGdTdT



A
178
1698
CCGACCCGUGGAUGAUCCU
1732
CCGACCCGUGGAUGAUCCUdTdT


 


AD-20112
S
297
1699
CCCAUACAAGUAUGAACAC
1733
cccAuAcAAGuAuGAAcAcdTdT



A
297
1700
GUGUUCAUACUUGUAUGGG
1734
GUGUUcAuACUUGuAUGGGdTdT


 


AD-20113
S
315
1701
CAAUAAGGCUCAUGCACAA
1735
cAAuAAGGcucAuGcAcAAdTdT



A
315
1702
UUGUGCAUGAGCCUUAUUG
1736
UUGUGcAUGAGCCUuAUUGdTdT


 


AD-20114
S
58
1703
AGGAUCAACGACGAGAUCG
1737
AGGAucAAcGAcGAGAucGdTdT



A
58
1704
CGAUCUCGUCGUUGAUCCU
1738
CGAUCUCGUCGUUGAUCCUdTdT


 


AD-20115
S
324
1705
UCAUGCACAAUUGGUUCGA
1739
ucAuGcAcAAuuGGuucGAdTdT



A
324
1706
UCGAACCAAUUGUGCAUGA
1740
UCGAACcAAUUGUGcAUGAdTdT


 


AD-20116
S
59
1707
GGAUCAACGACGAGAUCGA
1741
GGAucAAcGAcGAGAucGAdTdT



A
59
1708
UCGAUCUCGUCGUUGAUCC
1742
UCGAUCUCGUCGUUGAUCCdTdT


 


AD-20117
S
398
1709
AGAGCUUGUGGAAUGAUCC
1743
AGAGcuuGuGGAAuGAuccdTdT



A
398
1710
GGAUCAUUCCACAAGCUCU
1744
GGAUcAUUCcAcAAGCUCUdTdT


 


AD-20118
S
57
1711
GAGGAUCAACGACGAGAUC
1745
GAGGAucAAcGAcGAGAucdTdT



A
57
1712
GAUCUCGUCGUUGAUCCUC
1746
GAUCUCGUCGUUGAUCCUCdTdT


 


AD-20119
S
56
1713
GGAGGAUCAACGACGAGAU
1747
GGAGGAucAAcGAcGAGAudTdT



A
56
1714
AUCUCGUCGUUGAUCCUCC
1748
AUCUCGUCGUUGAUCCUCCdTdT


 


AD-20120
S
369
1715
UUUUGAGAAUCCAUAUGUA
1749
uuuuGAGAAuccAuAuGuAdTdT



A
369
1716
UACAUAUGGAUUCUCAAAA
1750
uAcAuAUGGAUUCUcAAAAdTdT


 


AD-20121
S
45
1717
CAAGGAAGCCCGGAGGAUC
1751
cAAGGAAGcccGGAGGAucdTdT



A
45
1718
GAUCCUCCGGGCUUCCUUG
1752
GAUCCUCCGGGCUUCCUUGdTdT
















TABLE 7b







Sequences of dsRNA targeting GNAQ (AD-20196 and AD-20197 only)














SEQ

SEQ



Duplex

ID
Unmodified sequence
ID
Modified sequence


Name
Strand
NO:
5′ to 3′
NO:
5′ to 3′





AD-20196
S
1753
CGGAGGAUCAACGACGAGA
1757
cGGAGGAucAAcGAcGAGAdTdT



A
1754
UCUCGUCGUUGAUCCUCCG
1758
UCUCGUCGUUGAUCCUCCGdTdT


 


AD-20197
S
1755
AUCUGACUCUACCAAAUAC
1759
AucuGAcucuAccAAAuAcdTdT



A
1756
GUAUUUGGUAGAGUCAGAU
1760
GuAUUUGGuAGAGUcAGAUdTdT









Total RNA isolation using MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Foster City Calif., Part #: AM1830):
Cells were harvested and lysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads were added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Solution 2 (Ethanol added), captured and supernatant was removed. 50 ul of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μl of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μl Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA it was eluted with 50 μl of water.
Total RNA Isolation Using RNAqueous®-96 Well Plate Procedure (Applied Biosystem, Foster City Calif., Part #: 1812):
Cells were lysed for 5 minutes in 200 μl of Lysis/Binding Solution. 100 μl of 100% ethanol was added into each cell lysate and the total 300 μl lysates were transferred into one wells of “filter plate”. Filter plate was centrifuged at RCF of 10,000-15,000 g for 2 minutes. 300 μl Wash Solution was then added into each well and the plate was centrifuged at RCF of 10,000-15,000g for 2 minutes. For DNase treatment, 20 μl of DNase mixture was added on top of each filter and the plate was incubated for 15 minutes at room temperature. RNA rebinding was performed by washing filters with 200 μL of Rebinding Mix and 1 minute later samples were centrifuged at RCF of 10,000-15,000g for 2 minutes. Filter was washed then twice with 200 μl of Wash Solution and centrifuged at RCF of 10,000-15,000g for 2 minutes. A third centrifugation of 2 minutes was then applied after the reservoir unit was emptied and elution of the RNA was done into a clean culture plate by adding into the filters 50 μL of preheated (80° C.) Nuclease-free Water.
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 was added to a master mix of 1 μl GAPDH TaqMan Probe (Human GAPD Endogenous Control VIC/MGB Probe, Primer Limited Applied Biosystems Cat #4326317E), 1 μl GNAQ TaqMan probe (Applied Biosystems cat #HS00387073_M1) and 10 μl TaqMan Universal PCR Master Mix (Applied Biosystems Cat #4324018) per well in a MicroAmp Optical 96 well plate (Applied Biosystems cat #4326659). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. All reactions were done in triplicate.
Real time data were analyzed using the ΔΔCt method and normalized to assays performed from cells transfected with 10 nM BlockIT fluorescent Oligo (Invitrogen Cat #2013) or 10 nM AD-1955 a duplex that targets luciferase to calculate fold change.
Results
A total of 94 chemically modified siRNAs were screened. Single dose screens were performed in A549 (lung carcinoma), A375 (malignant melanoma) and uveal melanoma cell lines GNAQmut, OMM1.3, and MEL202. Tables 8-14 show the results of the single-dose in vitro siRNA screen.







TABLE 8







A375 cells (0.1 nM) GNAQ dsRNA single dose in vitro screen


A375 cells (0.1 nM conc.)










Sample

% Target
St. Dev


Name
Duplex Name
Remaining
error













26
AD-20057
44.52
4.74


21
AD-20052
49.83
4.55


20
AD-20051
51.94
6.86


38
AD-20069
53.68
5.80


60
AD-20092
54.34
5.94


66
AD-20099
56.06
5.86


14
AD-20045
56.35
5.74


91
AD-20193
57.53
3.82


68
AD-20101
58.44
4.72


23
AD-20054
60.08
6.13


89
AD-20103
60.82
5.02


16
AD-20047
61.66
5.59


53
AD-20085
61.99
8.52


56
AD-20088
62.09
7.48


81
AD-20116
63.84
7.76


19
AD-20050
64.39
8.36


78
AD-20113
64.84
7.95


8
AD-20039
65.29
9.23


51
AD-20083
70.34
8.09


65
AD-20097
71.57
5.92


11
AD-20042
74.74
8.67


43
AD-20074
74.87
6.70


47
AD-20079
75.39
6.12


24
AD-20055
77.24
36.15


58
AD-20090
77.65
9.17


57
AD-20089
78.32
9.94


44
AD-20076
78.59
5.98


46
AD-20078
79.00
5.54


64
AD-20096
80.39
5.96


48
AD-20080
80.66
10.31


84
AD-20119
80.94
5.22


3
AD-20034
81.37
7.63


10
AD-20041
81.65
6.39


12
AD-20043
81.65
11.97


6
AD-20037
81.79
11.99


59
AD-20091
81.79
9.24


13
AD-20044
81.79
6.42


30
AD-20061
85.41
8.80


63
AD-20095
85.71
8.69


18
AD-20049
85.71
10.26


75
AD-20110
86.60
13.52


52
AD-20084
87.81
11.94


69
AD-20102
87.81
6.48


94
AD-20196
88.58
6.22


71
AD-20106
88.73
9.38


70
AD-20104
89.35
14.88


35
AD-20066
89.81
7.73


54
AD-20086
89.97
12.64


45
AD-20077
90.28
10.05


72
AD-20107
90.59
5.99


83
AD-20118
90.75
8.80


34
AD-20065
91.54
12.41


62
AD-20094
92.02
10.71


74
AD-20109
92.82
11.79


79
AD-20114
92.82
11.48


73
AD-20108
93.14
8.59


80
AD-20115
93.47
9.77


93
AD-20195
93.63
7.82


55
AD-20087
93.95
15.29


76
AD-20111
93.95
14.29


92
AD-20194
94.44
6.29


82
AD-20117
94.61
12.80


15
AD-20046
94.61
10.42


22
AD-20053
94.93
16.04


77
AD-20112
95.10
12.23


29
AD-20060
95.10
11.08


67
AD-20100
95.26
11.16


28
AD-20059
95.43
11.09


32
AD-20063
95.93
14.32


25
AD-20056
96.09
12.23


90

96.26
9.50


95
AD-20105
96.76
10.01


9
AD-20040
97.10
7.88


17
AD-20048
97.10
11.44


88
AD-20098
97.27
6.97


61
AD-20093
97.27
12.46


39
AD-20070
97.43
9.70


7
AD-20038
97.60
11.22


87

97.94
8.37


49
AD-20081
98.45
9.22


31
AD-20062
98.62
13.40


86
AD-20121
98.62
10.71


50
AD-20082
98.79
12.83


41
AD-20072
98.97
9.54


42
AD-20073
99.48
9.92


85
AD-20120
99.65
7.42


27
AD-20058
99.83
16.38


33
AD-20064
100.35
10.82


1
AD-20032
101.40
9.56


37
AD-20068
101.57
9.44


4
AD-20035
102.99
15.49


2
AD-20033
103.71
13.63


40
AD-20071
104.25
10.82


5
AD-20036
106.25
23.63
















TABLE 9







A375 cells (1.0 nM) single dose GNAQ in vitro screen


A375 cells (1 nM conc.)










Sample

% Target
St. Dev


Name
Duplex Name
Remaining
error













26
AD-20057
39.55
7.92


21
AD-20052
41.23
9.20


38
AD-20069
44.42
6.19


68
AD-20101
45.04
6.93


20
AD-20051
45.11
8.89


14
AD-20045
45.98
7.80


19
AD-20050
47.11
11.07


53
AD-20085
47.52
9.93


56
AD-20088
47.60
9.91


16
AD-20047
48.35
8.35


66
AD-20099
48.44
8.52


78
AD-20113
48.69
8.81


81
AD-20116
49.20
9.77


23
AD-20054
49.71
8.20


89
AD-20103
49.80
7.27


91
AD-20193
51.29
8.73


65
AD-20097
52.27
9.60


60
AD-20092
52.46
6.04


51
AD-20083
55.64
9.34


58
AD-20090
57.30
9.63


8
AD-20039
57.70
15.80


11
AD-20042
58.51
9.24


43
AD-20074
59.43
9.18


24
AD-20055
59.53
13.24


47
AD-20079
59.74
8.98


57
AD-20089
59.94
11.78


46
AD-20078
61.10
12.31


18
AD-20049
61.31
8.08


30
AD-20061
63.14
11.19


6
AD-20037
63.91
10.65


10
AD-20041
64.25
12.25


59
AD-20091
64.36
10.87


3
AD-20034
65.26
12.38


13
AD-20044
65.26
10.78


64
AD-20096
66.51
10.16


44
AD-20076
66.86
8.87


93
AD-20195
67.44
8.49


12
AD-20043
68.74
12.02


94
AD-20196
68.98
12.81


35
AD-20066
69.70
10.38


54
AD-20086
70.79
13.09


45
AD-20077
71.04
10.55


84
AD-20119
71.28
8.50


52
AD-20084
71.53
14.24


34
AD-20065
72.15
12.77


29
AD-20060
74.44
11.33


48
AD-20080
74.83
9.32


63
AD-20095
75.09
12.36


75
AD-20110
75.35
15.56


92
AD-20194
76.40
12.95


70
AD-20104
76.67
10.72


28
AD-20059
78.41
12.71


74
AD-20109
78.55
15.25


15
AD-20046
78.69
12.82


55
AD-20087
79.37
12.92


69
AD-20102
80.90
11.23


31
AD-20062
80.90
15.87


4
AD-20035
81.18
19.43


83
AD-20118
82.45
19.97


49
AD-20081
82.60
15.95


67
AD-20100
82.88
13.22


42
AD-20073
83.32
14.05


25
AD-20056
84.19
16.11


62
AD-20094
84.48
13.94


41
AD-20072
84.92
12.80


9
AD-20040
85.21
15.48


71
AD-20106
85.51
16.45


90

85.81
15.45


7
AD-20038
86.85
16.09


79
AD-20114
87.76
16.56


33
AD-20064
88.07
20.64


80
AD-20115
88.07
17.42


2
AD-20033
88.68
16.03


61
AD-20093
89.76
13.56


32
AD-20063
90.07
14.83


36
AD-20067
90.23
9.73


77
AD-20112
90.54
15.45


86
AD-20121
91.49
20.81


95
AD-20105
91.65
17.40


22
AD-20053
91.97
20.15


5
AD-20036
92.13
23.89


37
AD-20068
92.77
14.46


39
AD-20070
93.09
16.90


27
AD-20058
93.09
17.29


17
AD-20048
93.25
14.32


88
AD-20098
93.25
14.60


82
AD-20117
93.41
17.84


40
AD-20071
94.39
15.66


50
AD-20082
94.88
17.58


87

95.71
15.99


1
AD-20032
96.71
14.37


73
AD-20108
96.71
17.36


85
AD-20120
97.04
11.67


72
AD-20107
108.05
12.36
















TABLE 10







A549 cells (1.0 nM) single dose GNAQ in vitro screen


A549 cells (1 nM conc.)










Sample Name
Duplex Name
% Target Remaining
St. Dev error













78
AD-20113
13.33
2.98


53
AD-20085
15.79
3.53


81
AD-20116
16.44
3.68


21
AD-20052
16.90
3.78


20
AD-20051
17.31
3.87


38
AD-20069
17.71
3.96


66
AD-20099
17.77
3.98


19
AD-20050
18.11
4.05


64
AD-20096
18.17
4.07


26
AD-20057
18.75
4.20


89
AD-20103
19.11
4.28


43
AD-20074
19.28
4.31


51
AD-20083
19.41
4.34


68
AD-20101
19.61
4.39


14
AD-20045
20.06
4.49


8
AD-20039
20.20
4.52


11
AD-20042
20.41
4.57


65
AD-20097
20.99
4.70


60
AD-20092
21.02
4.70


56
AD-20088
22.53
5.04


44
AD-20076
22.57
5.05


58
AD-20090
23.29
5.21


57
AD-20089
23.29
5.21


47
AD-20079
23.69
5.30


74
AD-20109
23.86
5.34


16
AD-20047
24.02
5.38


63
AD-20095
24.36
5.45


59
AD-20091
25.04
5.60


23
AD-20054
25.17
5.63


45
AD-20077
25.61
5.73


48
AD-20080
25.84
5.78


91
AD-20193
28.92
6.47


13
AD-20044
29.83
6.68


6
AD-20037
30.89
6.91


46
AD-20078
31.10
6.96


24
AD-20055
31.64
7.08


85
AD-20120
31.70
7.09


18
AD-20049
33.74
7.55


84
AD-20119
34.75
7.77


3
AD-20034
35.85
8.02


35
AD-20066
36.73
8.22


70
AD-20104
36.92
8.26


12
AD-20043
38.62
8.64


54
AD-20086
38.96
8.72


15
AD-20046
39.98
8.95


34
AD-20065
40.19
8.99


93
AD-20195
41.18
9.21


75
AD-20110
41.18
9.21


69
AD-20102
41.68
9.33


52
AD-20084
42.19
9.44


30
AD-20061
44.29
9.91


94
AD-20196
48.13
10.77


40
AD-20071
48.21
10.79


49
AD-20081
48.72
10.90


10
AD-20041
48.80
10.92


36
AD-20067
48.97
10.96


29
AD-20060
50.79
11.36


31
AD-20062
51.05
11.42


90

52.12
11.66


55
AD-20087
52.30
11.70


61
AD-20093
52.85
11.83


2
AD-20033
53.50
11.97


25
AD-20056
55.77
12.48


4
AD-20035
56.25
12.59


1
AD-20032
57.43
12.85


92
AD-20194
60.19
13.47


42
AD-20073
61.03
13.65


5
AD-20036
61.45
13.75


28
AD-20059
61.99
13.87


50
AD-20082
62.09
13.89


67
AD-20100
63.29
14.16


83
AD-20118
64.06
14.33


62
AD-20094
64.17
14.36


27
AD-20058
64.95
14.53


7
AD-20038
69.26
15.50


79
AD-20114
71.45
15.99


39
AD-20070
72.07
16.13


41
AD-20072
74.61
16.69


86
AD-20121
74.61
16.69


33
AD-20064
75.39
16.87


9
AD-20040
80.11
17.92


72
AD-20107
82.22
18.40


95
AD-20105
86.90
19.45


73
AD-20108
87.96
19.68


17
AD-20048
89.04
19.92


88
AD-20098
90.13
20.17


77
AD-20112
90.44
20.24


80
AD-20115
91.07
20.38


22
AD-20053
91.86
20.55


37
AD-20068
92.50
20.70


32
AD-20063
92.66
20.73


76
AD-20111
92.82
20.77


71
AD-20106
92.98
20.80


82
AD-20117
109.81
24.57


87

110.19
24.65
















TABLE 11







OMM1.3 cells (10 nM) single dose GNAQ in vitro screen


OMM1.3 cells (10 nM conc.)










Sample

% Target



Name
Duplex Name
Remaining
St. Dev error













85
AD-20120
51.12
7.27


58
AD-20090
51.83
11.83


89
AD-20103
53.57
6.93


68
AD-20101
54.50
10.88


64
AD-20096
54.60
9.30


57
AD-20089
54.98
10.87


53
AD-20085
55.07
11.92


38
AD-20069
55.55
10.05


59
AD-20091
55.94
13.82


51
AD-20083
56.42
13.08


60
AD-20092
56.72
11.64


65
AD-20097
57.61
8.03


45
AD-20077
57.81
11.18


63
AD-20095
57.81
10.19


43
AD-20074
58.01
11.58


91
AD-20193
58.11
10.38


26
AD-20057
58.21
10.36


20
AD-20051
58.41
7.60


24
AD-20055
58.92
9.65


66
AD-20099
59.74
10.83


44
AD-20076
59.74
12.63


23
AD-20054
59.95
8.25


47
AD-20079
60.06
11.09


56
AD-20088
60.06
12.78


61
AD-20093
60.06
13.48


41
AD-20072
60.37
12.49


13
AD-20044
61.11
12.23


35
AD-20066
61.32
11.53


90

61.53
10.72


19
AD-20050
61.64
10.53


14
AD-20045
61.85
7.21


15
AD-20046
61.96
10.96


21
AD-20052
62.07
7.36


34
AD-20065
62.61
8.87


29
AD-20060
62.71
11.52


16
AD-20047
62.93
8.91


93
AD-20195
63.26
10.94


69
AD-20102
63.59
7.49


54
AD-20086
64.25
16.58


50
AD-20082
64.59
16.58


94
AD-20196
64.70
9.71


48
AD-20080
64.70
12.16


30
AD-20061
64.81
9.02


A2

65.26
13.18


70
AD-20104
65.83
8.26


A3

66.41
11.43


18
AD-20049
68.27
11.57


49
AD-20081
68.75
15.03


55
AD-20087
69.35
14.25


31
AD-20062
69.71
10.58


52
AD-20084
71.42
17.10


A4

72.29
8.52


67
AD-20100
73.68
15.34


27
AD-20058
74.19
12.01


36
AD-20067
74.32
17.93


33
AD-20064
75.23
14.71


72
AD-20107
75.88
10.61


28
AD-20059
76.94
13.68


A1

76.94
14.61


71
AD-20106
77.08
12.79


25
AD-20056
79.11
12.52


 8
AD-20039
80.21
10.01


39
AD-20070
80.49
14.56


88
AD-20098
80.63
11.15


40
AD-20071
80.77
16.38


62
AD-20094
81.75
16.23


86
AD-20121
84.49
9.13


17
AD-20048
84.64
16.94


12
AD-20043
86.87
14.40


22
AD-20053
87.93
14.30


11
AD-20042
88.23
13.27


37
AD-20068
91.66
17.18


32
AD-20063
91.98
14.78


87

94.56
10.00


 9
AD-20040
96.89
12.28


 6
AD-20037
97.90
16.58


 2
AD-20033
100.48
17.62


 3
AD-20034
100.83
12.65


 1
AD-20032
105.84
19.01


 7
AD-20038
114.62
16.88


 5
AD-20036
115.42
14.21


 4
AD-20035
123.49
11.58


10
AD-20041
135.05
65.85
















TABLE 12







OMM1.3 cells (10 nM) single dose GNAQ in vitro screen


OMM1.3 (10 nM conc.)










Sample Name
Duplex Name
% Target Remaining
St. Dev error













38
AD-20069
50.04
6.45


68
AD-20101
50.30
7.35


53
AD-20085
51.09
11.53


66
AD-20099
51.45
8.97


64
AD-20096
51.72
8.35


43
AD-20074
53.17
6.93


21
AD-20052
53.54
8.56


51
AD-20083
53.54
10.85


58
AD-20090
53.82
9.62


45
AD-20077
54.29
8.36


26
AD-20057
54.76
12.81


56
AD-20088
54.86
12.18


65
AD-20097
54.86
7.64


89
AD-20103
55.24
9.10


63
AD-20095
55.33
9.42


23
AD-20054
55.53
7.94


19
AD-20050
55.53
8.95


57
AD-20089
55.82
10.84


91
AD-20193
56.01
10.65


16
AD-20047
56.20
8.85


20
AD-20051
56.50
9.35


47
AD-20079
56.50
7.15


15
AD-20046
56.69
7.92


44
AD-20076
57.09
8.01


59
AD-20091
57.09
9.56


 8
AD-20039
57.29
7.18


61
AD-20093
57.58
10.14


14
AD-20045
57.78
10.19


85
AD-20120
57.78
9.98


54
AD-20086
57.88
9.77


11
AD-20042
58.90
11.84


13
AD-20044
59.41
11.72


48
AD-20080
60.55
9.45


41
AD-20072
60.87
6.66


A2

61.08
9.29


12
AD-20043
61.72
13.93


35
AD-20066
61.72
11.27


 6
AD-20037
61.72
9.69


69
AD-20102
61.93
10.90


34
AD-20065
62.15
12.75


60
AD-20092
62.25
10.76


50
AD-20082
62.80
11.11


 3
AD-20034
63.12
7.93


10
AD-20041
63.89
9.55


18
AD-20049
64.00
9.43


30
AD-20061
64.12
10.78


29
AD-20060
64.23
12.25


70
AD-20104
65.35
10.65


52
AD-20084
67.54
14.15


55
AD-20087
67.77
12.89


90

67.77
10.53


24
AD-20055
68.36
10.68


 9
AD-20040
68.60
11.16


 5
AD-20036
69.08
10.37


93
AD-20195
69.44
10.54


 2
AD-20033
70.04
12.33


31
AD-20062
71.02
11.73


49
AD-20081
71.02
11.45


39
AD-20070
71.51
9.18


27
AD-20058
71.88
11.58


67
AD-20100
72.01
9.95


94
AD-20196
72.26
15.50


A1

72.89
8.79


33
AD-20064
73.39
13.32


A4

73.65
12.42


A3

74.55
14.37


25
AD-20056
74.81
12.70


28
AD-20059
74.94
14.91


40
AD-20071
75.20
10.84


36
AD-20067
76.64
12.54


71
AD-20106
76.64
10.67


 4
AD-20035
76.91
10.25


22
AD-20053
78.80
15.37


86
AD-20121
79.48
10.26


 7
AD-20038
79.62
10.32


17
AD-20048
80.59
13.99


88
AD-20098
81.01
12.34


72
AD-20107
82.00
16.19


62
AD-20094
82.43
14.56


32
AD-20063
84.60
12.39


37
AD-20068
93.22
16.05


87

94.52
14.29


 1
AD-20032
115.87
15.00
















TABLE 13







UMEL 202 cells (10 nM) single dose GNAQ in vitro screen


UMEL 202 cells (10 nm Conc.










Sample Name
Duplex Name
% Target Remaining
St. Dev error













51
AD-20083
17.87
3.17


85
AD-20120
18.37
4.48


45
AD-20077
18.76
5.42


68
AD-20101
18.82
3.16


26
AD-20057
19.42
3.43


64
AD-20096
19.66
3.25


15
AD-20046
19.83
4.71


58
AD-20090
19.90
4.30


57
AD-20089
20.74
4.31


53
AD-20085
21.55
4.68


89
AD-20103
22.15
4.48


63
AD-20095
22.31
2.70


21
AD-20052
22.46
4.02


11
AD-20042
22.66
2.36


59
AD-20091
22.78
4.06


20
AD-20051
22.86
3.46


38
AD-20069
23.34
5.47


16
AD-20047
23.58
3.90


43
AD-20074
23.62
5.71


19
AD-20050
23.87
4.41


 8
AD-20039
23.91
2.96


14
AD-20045
24.33
4.08


47
AD-20079
25.10
5.85


50
AD-20082
25.27
4.51


 3
AD-20034
25.49
4.73


61
AD-20093
25.54
4.75


60
AD-20092
25.76
4.91


56
AD-20088
25.94
3.59


66
AD-20099
26.03
4.28


65
AD-20097
26.30
4.82


41
AD-20072
27.09
6.80


13
AD-20044
27.61
5.71


 2
AD-20033
27.70
3.68


91
AD-20193
27.90
4.64


29
AD-20060
27.99
5.33


44
AD-20076
28.04
7.95


A2

28.29
6.73


54
AD-20086
28.78
5.43


69
AD-20102
29.18
4.79


48
AD-20080
29.28
8.11


 5
AD-20036
30.90
4.82


A3

30.95
6.03


18
AD-20049
31.06
4.57


 6
AD-20037
31.17
3.46


30
AD-20061
31.49
6.96


35
AD-20066
31.71
39.01


34
AD-20065
34.05
7.29


90

34.11
5.07


94
AD-20196
34.17
6.48


23
AD-20054
34.46
5.09


12
AD-20043
34.70
2.93


10
AD-20041
34.76
6.00


55
AD-20087
36.55
8.21


31
AD-20062
36.81
7.00


49
AD-20081
37.06
7.58


25
AD-20056
39.04
9.55


70
AD-20104
39.59
6.12


52
AD-20084
39.93
6.61


A4

40.42
7.46


93
AD-20195
41.99
7.10


40
AD-20071
42.28
8.86


27
AD-20058
43.40
9.27


 4
AD-20035
47.00
6.36


24
AD-20055
47.08
6.37


A1

48.65
10.76


 9
AD-20040
50.46
8.04


28
AD-20059
50.63
12.97


39
AD-20070
51.43
9.23


36
AD-20067
52.42
10.11


33
AD-20064
52.78
10.02


17
AD-20048
54.36
8.74


88
AD-20098
55.50
9.72


86
AD-20121
57.16
8.59


67
AD-20100
58.87
8.34


22
AD-20053
65.32
10.91


62
AD-20094
68.10
10.87


 7
AD-20038
72.48
9.86


37
AD-20068
74.00
17.25


71
AD-20106
82.39
11.32


32
AD-20063
83.11
17.34


72
AD-20107
89.39
11.20


87

99.18
18.11


 1
AD-20032
119.33
18.54
















TABLE 14







MEL 202 cells (10 nM) single dose GNAQ in vitro screen


UMEL202 cells (10 nM)










Sample Name
Duplex Name
% Target Remaining
St. Dev error













85
AD-20120
16.28
1.84


26
AD-20057
18.41
3.50


68
AD-20101
18.73
3.64


45
AD-20077
19.09
4.41


64
AD-20096
19.33
4.19


21
AD-20052
21.08
3.11


51
AD-20083
21.22
4.27


58
AD-20090
22.36
4.62


63
AD-20095
22.55
3.04


20
AD-20051
23.22
2.94


53
AD-20085
23.43
4.97


57
AD-20089
23.43
4.55


 8
AD-20039
24.00
3.73


89
AD-20103
24.25
5.69


15
AD-20046
24.30
3.82


38
AD-20069
25.02
5.88


19
AD-20050
25.11
3.28


11
AD-20042
25.20
3.93


16
AD-20047
25.20
3.98


59
AD-20091
25.41
5.04


43
AD-20074
25.50
6.00


61
AD-20093
25.50
4.07


66
AD-20099
25.68
3.88


65
AD-20097
25.90
2.90


56
AD-20088
25.95
4.67


47
AD-20079
26.31
5.00


41
AD-20072
26.96
5.21


69
AD-20102
27.19
3.81


14
AD-20045
27.72
5.20


13
AD-20044
28.10
5.06


50
AD-20082
28.25
5.67


54
AD-20086
28.35
4.75


60
AD-20092
28.84
4.72


29
AD-20060
29.04
5.29


 2
AD-20033
29.24
4.55


91
AD-20193
29.30
7.31


35
AD-20066
29.40
6.42


 3
AD-20034
29.45
5.51


A2

30.70
5.81


48
AD-20080
30.86
7.14


44
AD-20076
31.07
7.63


12
AD-20043
31.29
7.00


30
AD-20061
31.40
5.57


94
AD-20196
32.22
8.75


A3

32.73
7.68


18
AD-20049
33.36
6.21


 5
AD-20036
34.12
4.63


34
AD-20065
34.60
4.89


 6
AD-20037
34.66
5.71


70
AD-20104
35.32
5.17


23
AD-20054
35.39
5.08


90

36.19
8.71


10
AD-20041
36.82
5.22


A4

36.89
11.72


93
AD-20195
37.73
9.95


31
AD-20062
37.93
7.86


25
AD-20056
40.51
8.37


55
AD-20087
40.65
10.00


52
AD-20084
41.72
7.24


24
AD-20055
43.26
6.08


49
AD-20081
43.34
13.40


27
AD-20058
45.57
7.25


A1

45.89
8.52


 4
AD-20035
46.13
8.13


28
AD-20059
48.25
8.52


36
AD-20067
48.84
13.01


40
AD-20071
48.93
9.64


88
AD-20098
50.30
12.25


33
AD-20064
50.48
7.61


 9
AD-20040
50.74
6.96


67
AD-20100
50.92
9.41


39
AD-20070
53.36
14.44


17
AD-20048
53.45
6.78


22
AD-20053
66.61
12.90


86
AD-20121
66.84
16.28


62
AD-20094
67.89
11.19


 7
AD-20038
70.53
8.81


71
AD-20106
81.44
14.31


32
AD-20063
83.29
12.02


72
AD-20107
85.04
14.05


87

100.26
29.22


37
AD-20068
108.58
54.53


 1
AD-20032
124.62
15.51









Duplexes with desirable levels of GNAQ inhibition were selected for further analysis of IC50 in A549 (lung carcinoma) MEL202 (GNAQmut uveal melanoma), and OMM1.3 cells (GNAQmut liver metastisis). Tables 15-17 show the results of the IC50 experiments in A549, MEL202, and OMM1.3 cells. Dose response screen identified pM IC50s in lung carcinoma cell line and GNAQmut uveal melanoma MEL202 and OMM1.3, including duplexes AD-20057 and AD-20051.







TABLE 15







IC50 in A549 cells










Rank
Duplex Name
IC50 in [nM]
IC50 in [pM]













1
AD-20057
0.0002
0.2


2
AD-20069
0.0026
2.6


3
AD-20051
0.0031
3.1


4
AD-20052
0.0032
3.2


5
AD-20099
0.0033
3.3


6
AD-20045
0.0052
5.2


7
AD-20193
0.0064
6.4


8
AD-20092
0.0094
9.4


9
AD-20116
0.0098
9.8


10
AD-20039
0.0137
13.7


11
AD-20042
0.0172
17.2
















TABLE 16







IC50 in MEL 202 cells









Rank
Duplex Name/(Sample Name)
IC50 in [nM]












1
AD-20057 (26)
0.001


2
AD-20069 (38)
0.002


3
AD-20051 (20)
0.002


4
AD-20052 (21)
0.003


5
AD-20045 (14)
0.003


6
AD-20193 (91)
0.003


7
AD-20092 (60)
0.003


8
AD-20099 (66)
0.004


9
AD-20101 (68)
0.005


10
AD-20116 (81)
0.006


11
AD-20039 (8)
0.006


12
AD-20103 (89)
0.007


13
AD-20085 (53)
0.008


14
AD-20113 (78)
0.010


15
AD-20083 (51)
0.010


16
AD-20096 (64)
0.010


17
AD-20042 (11)
0.011


18
AD-20090 (58)
0.023


19
AD-20119 (84)
0.024


20
AD-20120 (85)
0.037


21
AD-20109 (74)
0.047


22
AD-20077 (45)
0.084
















TABLE 17







IC50 in OMM1.3 cells









Rank
Duplex Name (Sample)
IC50 in [nM]












1
AD-20057 (26)
0.0043


2
AD-20069 (38)
0.0115


3
AD-20052 (21)
0.0183


4
AD-20051 (20)
0.0197


5
AD-20099 (66)
0.0270


6
AD-20092 (60)
0.0280


7
AD-20193 (91)
0.0335


8
AD-20101 (68)
0.0531


9
AD-20045 (14)
0.0538


10
AD-20113 (78)
0.0625


11
AD-20039 (8)
0.0693


12
AD-20103 (89)
0.0820


13
AD-20085 (53)
0.0842


14
AD-20116 (81)
0.1280


15
AD-20083 (51)
0.1653


16
AD-20042 (11)
0.2470


17
AD-20090 (58)
0.2593


18
AD-20096 (64)
0.3006


19
AD-20120 (85)
0.6189


20
AD-20119 (84)
1.2276


21
AD-20109 (74)
1.2558


22
AD-20077 (45)
2.0044









Example 4
In Vitro Dose Response
For in vitro dose response experiments, cells expressing GNAQ were utilized. Some exemplary cell lines expressing GNAQ include, but are not limited to, human melanoma cell lines OMM1.3 and Mel 202 and MEL-285.
The dsRNAs were screened for in vitro inhibition of the target gene at 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM. Tissue culture cells were transfected with the dsRNA. Target gene mRNA levels were assayed using qPCR (real time PCR).
Cell Culture and Transfection
For knockdown, OMM-1.3, MEL-202 and MEL-285 were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in RPMI (Invitrogen) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transfection was carried out by adding 5 μl of Opti-MEM to 5 μl of siRNA duplexes per well into a 96-well plate along with 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing 2.0×104 OMM-1.3, MEL-202 or MEL-285 cells were then added. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 1, 0.1, 0.01 and 0.001 nM final duplex concentration.
Total RNA Isolation Using MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Foster City Calif., Part #: AM1830):
Cells were harvested and lysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads and Lysis/Binding Enhancer mixture were added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Solution 2 (Ethanol added), captured and supernatant was removed. 50 μl of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μl of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μl Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA was eluted with 50 μl of water.
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 GNAQ TaqMan probe (Applied Biosystems cat #HS00387073_m1) and 5 μl Roche Probes Master Mix (Roche Cat #04887301001) per well in a LightCycler 480 384 well plate (Roche cat #0472974001). Real time PCR was done in a LightCycler 480 Real Time PCR machine (Roche). Each duplex was tested in two independent transfections and each transfections was assayed in duplicate.
Real time data were analyzed using the ΔΔCt method. Each sample was normalized to GAPDH expression and knockdown was assessed relative to cells transfected with the non-targeting duplex AD-1955.
The data are presented in Table 18a. Data are expressed as the fraction of message remaining relative to cells targeted with AD-1955. The calculated IC50s are presented in Table 18b.







TABLE 18a







In vitro dose response in 3 cell lines













Duplex







name
1 nM
0.1 nM
0.01 nM
0.001 nM










OMM-1.3













AD-20039
0.38
0.46
0.74
0.73



AD-20045
0.42
0.52
0.60
0.79



AD-20051
0.34
0.46
0.63
1.18



AD-20052
0.36
0.37
0.53
0.61



AD-20057
0.32
0.36
0.43
0.59



AD-20063
0.63
0.69
0.99
0.74



AD-20069
0.37
0.35
0.43
0.69



AD-20092
0.42
0.51
0.71
0.75



AD-20099
0.35
0.46
0.52
0.63



AD-20101
0.39
0.57
0.60
0.69



AD-20111
0.64
0.68
0.65
0.70



AD-20113
0.37
0.51
0.71
0.92



AD-20116
0.56
0.58
0.66
0.75



AD-20193
0.45
0.50
0.64
0.75



AD-1955
1.12
1.17
0.83
0.92







MEL-202













AD-20039
0.35
0.44
0.63
0.83



AD-20045
0.30
0.36
0.53
0.55



AD-20051
0.22
0.37
0.67
0.88



AD-20052
0.33
0.39
0.66
0.85



AD-20057
0.28
0.29
0.39
0.77



AD-20063
0.93
0.87
0.95
0.97



AD-20069
0.35
0.39
0.39
0.75



AD-20092
0.37
0.49
0.93
0.98



AD-20099
0.28
0.33
0.61
0.96



AD-20101
0.38
0.46
0.83
0.92



AD-20111
0.67
0.81
0.91
0.98



AD-20113
0.31
0.48
0.82
0.99



AD-20116
0.33
0.34
0.72
0.92



AD-20193
0.32
0.44
0.65
0.87



AD-1955
1.11
0.85
1.11
0.95







MEL-285













AD-20039
0.29
0.47
0.95
1.09



AD-20045
0.39
0.42
0.69
0.86



AD-20051
0.34
0.34
0.73
0.90



AD-20052
0.30
0.53
1.17
1.22



AD-20057
0.37
0.34
0.54
0.86



AD-20063
0.99
1.05
1.52
1.37



AD-20069
0.27
0.33
0.55
0.80



AD-20092
0.39
0.58
0.78
0.82



AD-20099
0.28
0.40
0.92
1.10



AD-20101
0.35
0.57
0.82
1.05



AD-20111
0.75
0.79
0.78
0.73



AD-20113
0.32
0.53
0.92
1.18



AD-20116
0.55
0.51
1.17
0.91



AD-20193
0.42
0.47
0.79
0.95



AD-1955
0.93
1.01
0.93
1.15
















TABLE 18b







IC50 (pM) in 3 cell lines












duplex number
MEL202
OMM1.3
A549















AD-20057
0.7
4.3
0.2



AD-20069
1.8
11.5
2.6



AD-20051
2.5
19.7
3.1



AD-20052
2.6
18.3
3.2



AD-20045
2.8
53.8
5.2



AD-20193
3.2
33.5
6.4



AD-20092
3.5
28
9.4



AD-20099
3.6
27
3.3



AD-20101
4.9
53.1




AD-20116
5.5
128
9.8



AD-20113
9.5
62.5




AD-20039
6.1
69.3
13.7









Example 5
Immunostimulatory Assays: Screening siRNA Sequences for Immunostimulatory Ability
Twelve siRNA candidates were tested for induction of cytokines associated with immunostimulation (TNF-alpha and IFN-alpha).
Human PBMC were isolated from whole blood from healthy donors (Research Blood Components, Inc., Boston, Mass.) by a standard Ficoll-Hypaque density gradient centrifugation technique. PBMC (1×105/well/100 μL) were seeded in 96-well flat bottom plates and cultured in RPMI 1640 GlutaMax-1 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Omega Scientific) and 1% antibiotic/antimycotic (Invitrogen).
GNAC siRNAs was transfected into PBMC using N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP; Roche). The DOTAP was first diluted in Opti-MEM Reduced Serum medium (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing the siRNA. siRNA/DOTAP complexes were incubated for 10-15 minutes at room temperature and subsequently added to PBMC (50 μL/well) which were then cultured at 37° C. 5% CO2. siRNAs were used at a final concentration of 133 nM. The ratio of RNA to transfection reagent was 16.5 pmoles per μL of DOTAP. Transfections were conducted in quadruplicate in all experiments and were performed within two hours of cell plating. Culture supernatants were collected after 20-24 h and assayed for IFN-α and TNF-α by ELISA.
Cytokines were detected and quantified in culture supernatants with a commercially available ELISA kit for IFN-α (BMS216INST) and TNF-α (BMS223INST) from Bender MedSystems (Vienna, Austria).
Results
The data in Table 19 are presented as a percentage to a AD-5048 stimulated cytokine response. AD-5048 (positive control) corresponds to a sequence that targets human Apolipoprotein B (Soutschek et al., 2004) and elicits both an IFN-α and TNF-α. FIG. 1 and FIG. 2 shows the cytokine induction following transfection with siRNAs.
None of the siRNAs tested demonstrated significant expression of IFN-α and TNF-α in Human PBMCs compared to AD-5048. In particular, AD-20051 and AD-20057 were found to be non immunostimulatory in HuPBMC assay.







TABLE 19







Immunostimulatory activity











Duplex name
% IFN-α/AD-5048
% TNF-α/AD-5048






AD-20039
0
0



AD-20045
0
0



AD-20051
0
0



AD-20052
0
0



AD-20057
0
0



AD-20069
0
0



AD-20092
0
0



AD-20099
0
0



AD-20101
0
0



AD-20113
0
0



AD-20116
0
0



AD-20193
0
0









Example 6
In Vitro Cell Viability
A set of dsRNAs were screened for effects on in vitro cell viability. Tissue culture cells were transfected with the dsRNA and viability was assayed by staining with CellTiterBLue and microscopic evaluation.
Cell Culture and Transfection
For viability, OMM-1.3, MEL-202 and MEL-285 cells were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in RPMI, (Invitrogen) supplemented with 10% FBS, Penn/streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transfection was carried out by adding 5 μl of Opti-MEM to 5 μl of siRNA duplexes per well into a 96-well plate along with 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing 1.0×103 OMM-1.3, MEL-202 or MEL-285 cells were then added. Cells were incubated for 3, 5 or 7 days prior to viability assays. Experiments were performed at 1, 0.1, 0.01 and 0.001 nM final duplex concentration. All transfections were done in triplicate. The siRNAs PLK, and AD-19200 were included as positive controls (result in loss of viability) and AD-1955 was included as a negative control and was used for data normalization.
Cell Viability Assay
For viability assays, 20 μl of CellTiterBlue (Promega, Cat #G8080) was added and mixed into each well of the culture plate 3, 5 or 7 days after transfection with an siRNAs at 1, 0.1, 0.01 or 0.001 nM final concentration. The plates, containing transfected, cultured cells, media and CellTiterBlue were incubated for 1.5 hours and then read on a SpectraMax M5 plate reader (Molecular Devices) at 560 nm (excitation) and 590 nm (emission).
To measure viability, three replicate wells were averaged and subtracted from background (wells containing media and CellTiterBlue, but no cells). Viability is expressed as a normalized value in which cells transfected with GNAQ specific siRNAs or other controls are compared to cells transfected with AD-1955, a non-targeting duplex, cultured under the same conditions.
Results
The results are shown in Table 20. Graphical summaries of the results comparing viability at 3, 5, and 7 days in a single cell line after treatment with each of the duplexes at a single concentration are shown in FIG. 3, FIG. 4, FIG. 5, and FIG. 6.
The results show decreased cell viability in vitro following GNAQ knockdown that was specific for GNAQ mutant cell lines (e.g., OMM1.3, MEL202), but not GNAQ wild-type (e.g., MEL285) cell lines. In particular these results were shown for duplexes AD-20057, AD-20051, AD-20069, and AD-20093 as illustrated by the graphs in FIG. 7 and FIG. 8.







TABLE 20







Cell viability after treatment with siRNA











Day 3
Day 5
Day 7









Conc. (in nM)























0.001



0.001



0.001



1 nM
0.1 nM
0.01 nM
nM
1 nM
0.1 nM
0.01 nM
nM
1 nM
0.1 nM
0.01 nM
nM
























OMM-1.3
AD-20039
0.95
0.96
1.18
1.27
0.46
0.48
0.79
0.94
0.25
0.52
1.18
1.32



AD-20045
0.99
0.94
1.07
1.20
0.53
0.53
0.70
1.03
0.42
0.44
0.71
1.16



AD-20051
0.78
0.90
1.01
0.63
0.35
0.42
0.65
0.94
0.23
0.35
0.74
1.15



AD-20052
0.82
0.90
1.17
1.38
0.41
0.47
0.79
1.02
0.31
0.45
1.06
1.24



AD-20057
0.86
0.88
0.90
1.31
0.36
0.39
0.49
0.83
0.22
0.31
0.55
1.03



AD-20063
1.26
1.26
1.10
0.53
1.27
1.06
1.04
0.93
1.11
0.95
1.00
1.06



AD-20069
0.79
0.72
0.96
1.16
0.35
0.39
0.46
0.86
0.17
0.21
0.58
0.89



AD-20092
0.68
0.93
1.11
1.15
0.36
0.58
0.85
0.91
0.27
0.63
1.08
0.96



AD-20099
0.51
0.72
0.95
1.07
0.18
0.37
0.58
0.88
0.08
0.22
0.56
0.86



AD-20101
0.72
0.76
1.34
1.53
0.33
0.40
0.78
1.00
0.18
0.39
0.98
0.95



AD-20111
1.25
1.15
1.30
1.32
0.89
1.10
0.87
1.00
0.93
0.95
0.98
0.95



AD-20113
0.56
0.73
1.02
1.03
0.22
0.44
0.75
0.80
0.12
0.35
0.88
0.82



AD-20116
0.82
1.23
1.64
1.88
0.41
0.70
0.92
0.98
0.17
0.51
0.75
0.73



AD-20193
1.22
0.84
1.31
1.67
0.46
0.53
0.66
0.80
0.16
0.30
0.48
0.74



AD-12115
0.60
0.65
1.03
1.00
0.19
0.26
0.62
0.93
0.08
0.26
0.63
0.61



PLK
0.47
0.80
0.65
1.67
0.12
0.54
0.74
1.00
0.06
0.64
0.88
0.78



AD-19200
0.62
0.85
0.72
1.55
0.64
0.78
0.92
0.81
0.64
0.83
0.81
0.87



AD-1955
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00


MEL-202
AD-20039
1.21
0.98
1.02
0.93
0.72
0.56
0.79
1.00
0.78
0.66
0.75
0.88



AD-20045
0.95
0.90
0.95
0.85
0.47
0.38
0.61
0.92
0.57
0.36
0.47
0.80



AD-20051
0.70
0.78
0.80
0.38
0.62
0.36
0.77
0.82
0.82
0.42
0.62
0.80



AD-20052
0.98
1.02
1.06
0.93
0.46
0.42
0.67
1.02
0.33
0.55
0.95
1.03



AD-20057
0.61
0.91
0.86
0.85
0.30
0.27
0.47
0.86
0.31
0.34
0.53
0.88



AD-20063
0.91
1.00
1.02
0.37
0.74
0.81
1.01
0.91
1.54
1.32
1.08
1.04



AD-20069
0.77
1.03
0.91
1.00
0.28
0.44
0.43
0.64
0.34
0.37
0.49
0.79



AD-20092
0.87
0.88
0.95
0.87
0.25
0.44
0.70
0.80
0.26
0.73
0.97
1.11



AD-20099
0.80
0.69
0.75
0.41
0.24
0.36
0.53
0.68
0.13
0.26
0.66
1.16



AD-20101
0.72
0.92
0.80
0.87
0.16
0.48
0.57
0.73
0.17
0.51
1.21
0.92



AD-20111
1.18
0.90
0.75
0.84
0.67
0.83
0.74
0.80
1.30
1.37
1.25
1.03



AD-20113
0.63
0.55
0.74
0.36
0.18
0.31
0.66
0.68
0.15
0.37
1.00
1.05



AD-20116
0.42
0.59
0.62
0.93
0.41
0.51
0.73
0.90
0.29
0.38
0.71
0.77



AD-20193
0.39
0.53
0.53
0.94
0.36
0.49
0.70
0.76
0.29
0.32
0.59
0.80



AD-12115
0.22
0.22
0.30
0.50
0.09
0.12
0.42
0.78
0.03
0.04
0.08
0.68



PLK
0.23
0.27
0.37
0.63
0.10
0.16
0.56
0.65
0.03
0.07
0.97
1.19



AD-19200
0.37
0.52
0.49
0.56
0.29
0.75
0.76
0.74
0.44
1.15
1.04
0.85



AD-1955
1
1
1
1
1
1
1
1
1
1
1
1


MEL-285
AD-20039
0.58
1.37
1.23
1.23
1.07
1.25
1.13
1.09
0.82
1.06
0.96
0.93



AD-20045
1.16
1.31
1.15
1.05
1.10
1.10
1.12
1.24
0.84
0.85
0.97
0.90



AD-20051
1.14
1.20
0.97
0.98
1.27
1.16
1.06
1.03
0.84
0.99
0.89
0.97



AD-20052
0.63
1.40
1.26
1.04
1.03
1.40
1.42
1.21
0.92
1.02
1.13
0.98



AD-20057
1.14
1.17
1.20
1.04
1.00
1.04
1.26
1.35
0.98
0.98
1.03
1.00



AD-20063
1.10
1.14
0.88
0.89
1.33
1.14
0.94
0.95
1.16
1.05
0.87
0.91



AD-20069
0.46
1.09
1.08
0.96
1.17
1.20
1.03
1.04
1.29
1.17
1.01
0.99



AD-20092
1.02
1.14
1.15
0.96
1.02
1.11
1.03
1.05
1.06
1.11
1.04
1.02



AD-20099
0.89
1.10
0.95
0.95
0.48
0.92
0.96
1.00
0.54
0.91
0.89
1.05



AD-20101
0.70
1.16
1.12
1.04
0.47
1.12
1.41
1.42
0.66
1.01
1.22
1.03



AD-20111
1.12
1.05
1.13
1.01
1.21
1.49
1.30
1.29
1.04
1.18
1.04
1.03



AD-20113
0.81
0.97
1.02
1.03
0.41
0.85
0.81
0.97
0.31
0.76
0.85
1.01



AD-20116
0.50
0.86
1.07
1.01
1.03
0.98
1.03
1.01
0.99
0.91
1.01
0.94



AD-20193
0.91
0.88
1.03
0.94
0.58
0.86
1.25
1.24
0.72
0.80
1.09
1.03



AD-12115
0.34
0.35
0.81
0.43
0.10
0.12
0.54
1.02
0.07
0.12
0.82
1.00



PLK
0.23
0.65
0.46
0.97
0.31
0.54
1.40
1.31
0.18
0.72
1.38
1.17



AD-19200
0.53
0.81
0.68
0.94
0.53
0.77
1.22
1.32
0.46
0.97
1.22
1.15



AD-1955
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00









Example 7
In Vivo Efficacy Studies
The dsRNAs are screened for in vivo inhibition of the target gene in mice. Mice are injected with varying amounts of the dsRNA. Target gene protein levels are assayed using, e.g., mouse plasma and an ELISA with a target gene specific antibody. Target gene mRNA levels are assayed using, e.g., mouse liver and branched DNA assays. The lead candidates are dsRNA that reduce levels of the target gene protein and/or mRNA in a dose-dependent manner.
Regimen for Treatment of Mice with dsRNA
A single-dose IV bolus efficacy study is designed for each dsRNA to be tested: dose level, dosing days, formulation, and number of animals. Mice are intravenously (i.v.) administered target gene specific dsRNA, control dsRNA) or PBS systemically and/or subcutaneously in a range of concentrations, e.g., 1.0 mg/kg, 3.0 mg/kg, or 6.0 mg/kg.
Mice are observed for forty-hours then anesthetized with 200 μl of ketamine, and are exsanguinated by severing the right caudal artery. Whole blood is isolated and placed into EDTA plasma separator tubes and centrifuged at 3000 rpm for 10 minutes. Plasma is isolated and stored at 80° C. until assaying. Liver tissue is collected, flash-frozen and stored at −80° C. until processing.
Efficacy of treatment is evaluated by methods including (i) measurement of protein in plasma at prebleed and at 48 hours post-dose, (ii) measurement of mRNA in liver at 48 hours post-dose, and (iii) efficacy in modulation of target gene specific phenotype, e.g., anti-tumor activity.
Assay of Target Gene Protein in Mouse Plasma
Target plasma levels are assayed by ELISA utilizing the commercially available anti GNAQ antibodies, for example G alpha q (K-17) or G alpha q (E-17) (Santa Cruz Biotechnology Inc. Santa Cruz, Calif., USA, cat #SC-26791 and cat #SC-393), according to manufacturer's guidelines.
Assay of Target Gene mRNA Levels in Mouse Liver
Target gene mRNA levels are assayed utilizing the Branched DNA assays Quantigene 2.0 (Panomics cat #: QS0011). Briefly, mouse liver samples are ground and tissue lysates are prepared. Liver lysis Mixture (a mixture of 1 volume of lysis mixture, 2 volume of nuclease-free water and 10 μl of Proteinase-K/ml for a final concentration of 20 mg/ml.) is incubated at 65° C. for 35 minutes. 20 μl of Working Probe Set (target probe for detection of target gene and GAPDH probe for endogenous control) and 80 ul of tissue-lysate are then added into the Capture Plate. Capture Plates are incubated at 55° C.±1° C. (aprx. 16-20 hrs). The next day, the Capture Plate are washed 3 times with 1× Wash Buffer (nuclease-free water, Buffer Component 1 and Wash Buffer Component 2), then dried by centrifuging for 1 minute at 240 g. 100 ul of pre-Amplifer Working Reagent is added into the Capture Plate, which is sealed with aluminum foil and incubated for 1 hour at 55° C.±1° C. Following 1 hour incubation, the wash step is repeated, then 100 μl of Amplifier Working Reagent is added. After 1 hour, the wash and dry steps are repeated, and 100 μl of Label Probe is added. Capture plates are incubated 50° C.±1° C. for 1 hour. The plate is then washed with 1× Wash Buffer, dried and 100 μl Substrate is added into the Capture Plate. Capture Plates are read using the SpectraMax Luminometer following a 5 to 15 minute incubation. bDNA data are analyzed by subtracting the average background from each triplicate sample, averaging the triplicate GAPDH (control probe) and target gene probe (experimental probe) then taking the ratio: (experimental probe-background)/(control probe-background).
GNAQ Materials and Methods
The GNAQ specific dsRNA are formulated in lipid particles (SNALP) as describe herein and administered systemically or subcutaneously to mice with GNAQ-mutant human uveal melanoma cell tumors implanted in the liver to assess in vivo target knockdown and antitumor activity. The dsRNA duplexes with positive results are selected for further studies to develop a Phase I/II trial in patients with GNAQ-mutant uveal melanoma metastatic to liver.
Example 8
Inhibition of GNAQ in Humans
A human subject is treated with a dsRNA targeted to a GNAQ gene to inhibit expression of the GNAQ gene to treat a condition.
A subject in need of treatment is selected or identified. The subject can have uveal melanoma, cutaneous melanoma, Blue nevi, Nevi of Ota, a neuroendocrine tumor, or a small lung tumor.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an anti-GNAQ siRNA is administered to the subject. The dsRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated, e.g., by measuring tumor growth. This measurement can be accompanied by a measurement of GNAQ expression in said subject, and/or the products of the successful siRNA-targeting of GNAQ mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
After treatment, the subject's tumor growth rate is lowered relative to the rate existing prior to the treatment, or relative to the rate measured in a similarly afflicted but untreated subject.
Example 9
GNAQ mRNA Sequences








(SEQ ID NO: 1761)



Human GNAQ mRNA NM_002072.2










1
agggggtgcc ggcggggctg cagcggaggc actttggaag aatgactctg gagtccatca



 


61
tggcgtgctg cctgagcgag gaggccaagg aagcccggcg gatcaacgac gagatcgagc


 


121
ggcagctccg cagggacaag cgggacgccc gccgggagct caagctgctg ctgctcggga


 


181
caggagagag tggcaagagt acgtttatca agcagatgag aatcatccat gggtcaggat


 


241
actctgatga agataaaagg ggcttcacca agctggtgta tcagaacatc ttcacggcca


 


301
tgcaggccat gatcagagcc atggacacac tcaagatccc atacaagtat gagcacaata


 


361
aggctcatgc acaattagtt cgagaagttg atgtggagaa ggtgtctgct tttgagaatc


 


421
catatgtaga tgcaataaag agtttatgga atgatcctgg aatccaggaa tgctatgata


 


481
gacgacgaga atatcaatta tctgactcta ccaaatacta tcttaatgac ttggaccgcg


 


541
tagctgaccc tgcctacctg cctacgcaac aagatgtgct tagagttcga gtccccacca


 


601
cagggatcat cgaatacccc tttgacttac aaagtgtcat tttcagaatg gtcgatgtag


 


661
ggggccaaag gtcagagaga agaaaatgga tacactgctt tgaaaatgtc acctctatca


 


721
tgtttctagt agcgcttagt gaatatgatc aagttctcgt ggagtcagac aatgagaacc


 


781
gaatggagga aagcaaggct ctctttagaa caattatcac atacccctgg ttccagaact


 


841
cctcggttat tctgttctta aacaagaaag atcttctaga ggagaaaatc atgtattccc


 


901
atctagtcga ctacttccca gaatatgatg gaccccagag agatgcccag gcagcccgag


 


961
aattcattct gaagatgttc gtggacctga acccagacag tgacaaaatt atctactccc


 


1021
acttcacgtg cgccacagac accgagaata tccgctttgt ctttgctgcc gtcaaggaca


 


1081
ccatcctcca gttgaacctg aaggagtaca atctggtcta attgtgcctc ctagacaccc


 


1141
gccctgccct tccctggtgg gctattgaag atacacaaga gggactgtat ttctgtggaa


 


1201
aacaatttgc ataatactaa tttattgccg tcctggactc tgtgtgagcg tgtccacaga


 


1261
gtttgtagta aatattatga ttttatttaa actattcaga ggaaaaacag aggatgctga


 


1321
agtacagtcc cagcacattt cctctctatc ttttttttag gcaaaacctt gtgactcagt


 


1381
gtattttaaa ttctcagtca tgcactcaca aagataagac ttgtttcttt ctgtctctct


 


1441
ctctttttct tttctatgga gcaaaacaaa gctgatttcc cttttttctt cccccgctaa


 


1501
ttcatacctc cctcctgatg tttttcccag gttacaatgg cctttatcct agttccattc


 


1561
ttggtcaagt ttttctctca aatgatacag tcaggacaca tcgttcgatt taagccatca


 


1621
tcagcttaat ttaagtttgt agtttttgct gaaggattat atgtattaat acttacggtt


 


1681
ttaaatgtgt tgctttggat acacacatag tttctttttt aatagaatat actgtcttgt


 


1741
ctcactttgg actgggacag tggatgccca tctaaaagtt aagtgtcatt tcttttagat


 


1801
gtttaccttc agccatagct tgattgctca gagaaatatg cagaaggcag gatcaaagac


 


1861
acacaggagt cctttctttt gaaatgccac gtgccattgt ctttcctccc ttctttgctt


 


1921
ctttttctta ccctctcttt caattgcaga tgccaaaaaa gatgccaaca gacactacat


 


1981
taccctaatg gctgctaccc agaacctttt tataggttgt tcttaatttt tttgttgttg


 


2041
ttgttcaagc ttttcctttc ttttttttct tagtgtttgg gccacgattt taaaatgact


 


2101
tttattatgg gtatgtgttg ccaaagctgg ctttttgtca aataaaatga atacgaactt


 


2161
aaaaaataaa aaaaaaaaaa aaaaaaaa


 








(SEQ ID NO: 1762)



Rat GNAQ mRNA NM_031036










1
atgactctgg agtccatcat ggcgtgctgc ctgagcgagg aggccaagga agcccggagg



 


61
atcaacgacg agatcgagcg gcagctgcgc agggacaagc gcgacgcccg ccgggagctc


 


121
aagctgctgc tgctggggac aggggagagt ggcaagagta ccttcattaa gcagatgagg


 


181
atcatccacg ggtcggggta ctctgatgaa gacaagaggg gctttaccaa actggtgtat


 


241
cagaacatct ttacagccat gcaggccatg gtcagagcta tggacactct caagatccca


 


301
tacaagtatg aacacaataa ggctcatgca caattggttc gagaggttga tgtggagaag


 


361
gtgtctgctt ttgagaatcc atatgtagac gcaataaaga gcttgtggaa tgatcctgga


 


421
atccaggaat gctacgatag acggcgagaa tatcagctat ctgactctac caaatactat


 


481
ctgaacgact tggaccgtgt ggctgaccct tcctatctgc ctacacaaca agatgtgctt


 


541
agagttcgag tccccaccac agggatcatt gagtacccct tcgacttaca gagtgtcatc


 


601
ttcagaatgg tcgatgtagg aggccaaagg tcagagagaa gaaaatggat acactgcttt


 


661
gaaaacgtca cctcgatcat gtttctggta gcgcttagcg aatacgatca agttcttgtg


 


721
gagtcagaca atgagaaccg aatggaggag agcaaagcac tctttagaac cattatcaca


 


781
tatccctggt tccagaactc ctctgttatt ctgttcttaa acaagaaaga tcttctagag


 


841
gagaaaatta tgtattccca cctagtcgac tacttcccag aatatgatgg accccagaga


 


901
gatgcccagg cagcacgaga attcatcctg aagatgttcg tggacctgaa ccccgacagt


 


961
gacaaaatca tctactcgca cttcacgtgt gccacagaca cggagaacat ccgcttcgtg


 


1021
tttgctgctg tcaaggacac catcctgcag ctgaacctga aggagtacaa tctggtctaa

## Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a G-alpha q subunit (GNAQ) of a heterotrimeric G gene, comprising a sense strand and an antisense strand complementary to at least the first 11 nucleotides of SEQ ID NO:1421, wherein each strand is at least 15 nucleotides in length.

2. The dsRNA of claim 1, wherein the sense strand consists of SEQ ID NO:1579 (cAAuuuGcAuAAuAcuAAudTdT) and the antisense strand consists of SEQ ID NO:1580 (AUuAGuAUuAUGcAAAUUGdTdT) wherein each strand includes 2′-O-methyl ribonucleotides as indicated by a lower case letter “c” and “u”.

3. The dsRNA of claim 1, wherein the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO:1421.

4. The dsRNA of claim 1, wherein the sense strand comprises 15 or more contiguous nucleotides of SEQ ID NO:1421 or SEQ ID NO:1579.

5. The dsRNA of claim 1, wherein the antisense strand comprises 15 or more contiguous nucleotides of SEQ ID NO:1422 or SEQ ID NO:1580.

6. The dsRNA of claim 1, wherein the sense strand sequence comprises SEQ ID NO:1421 and the antisense strand sequence comprises of SEQ ID NO:1422.

7. The dsRNA of claim 1, wherein administration of 0.1 nM of the dsRNA to a A375 cell results in about 66% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of 1 nM of the dsRNA to a A375 cell results in about 61% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of 1 nM of the dsRNA to a A579 cell results in about 82% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of 10 nM of the dsRNA to a OMM1.3 cell results in about 42% inhibition of GNAQ mRNA expression as measured by a real time PCR assay or administration of the dsRNA to a UMEL202 cell results in about 81% inhibition of GNAQ mRNA expression as measured by a real time PCR assay.

8. The dsRNA of claim 1, wherein the antisense strand is complementary to 16, 17, or 18 nucleotides of SEQ ID NO:1421.

9. The dsRNA of claim 1, wherein the antisense strand is complementary to 19 nucleotides of SEQ ID NO:1421.

10. The dsRNA of claim 1, wherein each strand is no more than 30 nucleotides in length.

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

12. The dsRNA of claim 1, wherein each strand comprises a 3′ overhang consisting of 2 nucleotides.

13. The dsRNA of claim 1, wherein each strand comprises a 3′ overhang consisting of dTdT.

14. The dsRNA of claim 1 or claim 6, comprising a modification that causes the dsRNA to have increased stability in a biological sample.

15. The dsRNA of claim 1 or claim 6, wherein said dsRNA comprises at least one modified nucleotide.

16. The dsRNA of claim 14, wherein said modified nucleotide is selected from the group 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.

17. The dsRNA of claim 14, wherein said modified nucleotide is 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.

18. The dsRNA of claim 14, comprising at least one 2′-O-methyl modified nucleotide and at least one 2′-deoxythymidine-3′-phosphate nucleotide comprising a 5′-phosphorothioate group.

19. The dsRNA of claim 14, wherein the sense strand comprises all 2′-O-methyl modified pyrimidines and the antisense strand comprises 2′-O-methyl modified pyrimidines when the pyrimidine is adjacent to A and each strand comprises dTdT at the 3′ end.

20. The dsRNA of claim 14, wherein the sense strand comprises all 2′-O-methyl modified pyrimidines and the antisense strand comprises 2′-O-methyl modified pyrimidines when the pyrimidine is adjacent to A and each strand comprises dTsdT at the 3′ end.

21. The dsRNA of claim 14, wherein the sense strand comprises all 2′-O-methyl modified pyrimidines and the antisense strand comprises 2′-O-methyl modified pyrimidines when a) the pyrimidine is adjacent to A or b) the pyrimidine is a uracil adjacent to a U or a G, and each strand comprises dTsdT at the 3′ end.

22. The dsRNA of claim 1 or claim 2 or claim 6, further comprising a ligand.

23. The dsRNA of claim 22, wherein the ligand is conjugated to the 3′-end of the sense strand of the dsRNA.

24. A composition for inhibiting expression of a GNAQ gene comprising the dsRNA of claim 1 or claim 2 or claim 6 and pharmaceutical formulation.

25. The composition of claim 24, wherein the pharmaceutical formulation is a lipid formulation.

26. The composition of claim 24, wherein the pharmaceutical formulation is a a (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3) comprising formulation, or a LNP11 formulation.

27. An isolated cell containing the dsRNA of claim 1 or claim 2 or claim 6.

28. A vector comprising a nucleotide sequence that encodes at least one strand of the dsRNA of claim 1 or claim 6.

29. An isolated cell comprising the vector of claim 28.

30. The dsRNA of claim 1, wherein said dsRNA, upon contact with a cell expressing said GNAQ, inhibits expression of said GNAQ gene by at least 40% compared to a cell not so contacted.

31. The dsRNA of claim 1, wherein the dsRNA has an IC50 of less than 10 pM.

32. A method of inhibiting GNAQ expression in a cell, the method comprising:
(a) introducing into the cell the dsRNA of claim 1 or claim 2 or claim 6; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a GNAQ gene, thereby inhibiting expression of the GNAQ gene in the cell.

33. A method of treating a disorder mediated by GNAQ expression comprising administering to a human in need of such treatment a therapeutically effective amount of the dsRNA of claim 1 or claim 2 or claim 6.

34. The method of claim 33, wherein the human has uveal melanoma, cutaneous melanoma, Blue nevi, Nevi of Ota, a small lung tumor, or a neuroendocrine tumors.

35. The composition of claim 24, wherein the pharmaceutical formulation is a (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3) comprising formulation.

36. The composition of claim 24, wherein the pharmaceutical formulation is a LNP11 formulation.