# Patent

## Title
Reduced size self-delivering RNAi compounds

## Abstract

The present invention relates to RNAi constructs with minimal double-stranded regions, and their use in gene silencing. RNAi constructs associated with the invention include a double stranded region of 8-14 nucleotides and a variety of chemical modifications, and are highly effective in gene silencing.

## Background
RELATED APPLICATIONS
This application is a national stage filing under 35 U.S.C. §371 of international application PCT/US2009/005247, filed Sep. 22, 2009, which was published under PCT Article 21(20) in English, and claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application serial number U.S. 61/192,954, entitled “Chemically Modified Nucleotides and Methods of Using the Same,” filed on Sep. 22, 2008, U.S. 61/149,946, entitled “Minimum Length Triggers of RNA Interference,” filed on Feb. 4, 2009, and U.S. 61/224,031, entitled “Minimum Length Triggers of RNA Interference,” filed on Jul. 8, 2009, the disclosure of each of which is incorporated by reference herein in its entirety.


FIELD OF INVENTION
The invention pertains to the field of RNA interference (RNAi). The invention more specifically relates to nucleic acid molecules with improved in vivo delivery properties without the use of a delivering agent and their use in efficient gene silencing.
BACKGROUND OF INVENTION
Complementary oligonucleotide sequences are promising therapeutic agents and useful research tools in elucidating gene functions. However, prior art oligonucleotide molecules suffer from several problems that may impede their clinical development, and frequently make it difficult to achieve intended efficient inhibition of gene expression (including protein synthesis) using such compositions in vivo.
A major problem has been the delivery of these compounds to cells and tissues. Conventional double-stranded RNAi compounds, 19-29 bases long, form a highly negatively-charged rigid helix of approximately 1.5 by 10-15 nm in size. This rod type molecule cannot get through the cell-membrane and as a result has very limited efficacy both in vitro and in vivo. As a result, all conventional RNAi compounds require some kind of a delivery vehicle to promote their tissue distribution and cellular uptake. This is considered to be a major limitation of the RNAi technology.
There have been previous attempts to apply chemical modifications to oligonucleotides to improve their cellular uptake properties. One such modification was the attachment of a cholesterol molecule to the oligonucleotide. A first report on this approach was by Letsinger et al., in 1989. Subsequently, ISIS Pharmaceuticals, Inc. (Carlsbad, Calif.) reported on more advanced techniques in attaching the cholesterol molecule to the oligonucleotide (Manoharan, 1992).
With the discovery of siRNAs in the late nineties, similar types of modifications were attempted on these molecules to enhance their delivery profiles. Cholesterol molecules conjugated to slightly modified (Soutschek, 2004) and heavily modified (Wolfrum, 2007) siRNAs appeared in the literature. Yamada et al., 2008 also reported on the use of advanced linker chemistries which further improved cholesterol mediated uptake of siRNAs. In spite of all this effort, the uptake of these types of compounds appears to be inhibited in the presence of biological fluids resulting in highly limited efficacy in gene silencing in vivo, limiting the applicability of these compounds in a clinical setting.
Therefore, it would be of great benefit to improve upon the prior art oligonucleotides by designing oligonucleotides that have improved delivery properties in vivo and are clinically meaningful.
SUMMARY OF INVENTION
Described herein are asymmetric chemically modified nucleic acid molecules with minimal double stranded regions, and the use of such molecules in gene silencing. RNAi molecules associated with the invention contain single stranded regions and double stranded regions, and can contain a variety of chemical modifications within both the single stranded and double stranded regions of the molecule. Additionally, the RNAi molecules can be attached to a hydrophobic conjugate such as a conventional and advanced sterol-type molecule. This new class of RNAi molecules has superior efficacy both in vitro and in vivo than previously described RNAi molecules.
Aspects of the invention relate to asymmetric nucleic acid molecules including a guide strand, with a minimal length of 16 nucleotides, and a passenger strand forming a double stranded nucleic acid, having a double stranded region and a single stranded region, the double stranded region having 8-15 nucleotides in length, the single stranded region having 5-12 nucleotides in length, wherein the passenger strand is linked to a lipophilic group, wherein at least 40% of the nucleotides of the double stranded nucleic acid are modified, and wherein the single stranded region has at least 2 phosphorothioate modifications. In some embodiments position 1 of the guide strand is 5′ phosphorylated. In certain embodiments, position 1 of the guide strand is 2′O-methyl modified and 5′ phosphorylated.
Aspects of the invention relate to isolated double stranded nucleic acid molecules including a longer strand of 15-21 nucleotides in length that has complementarily to a miRNA sequence, a shorter strand of 8-15 nucleotides in length linked at the 3′ end to a lipophilic group, wherein the longer strand and the passenger strand form the double stranded nucleic acid molecule having a double stranded region and a single stranded region, wherein the longer strand has a 3′ single stranded region of 2-13 nucleotides in length, comprising at least two phosphorothioate modification, and at least 50% nucleotides are modified.
Further aspects of the invention relate to isolated double stranded nucleic acid molecules including a guide strand of 17-21 nucleotides in length that has complementarity to a target gene, a passenger strand of 8-16 nucleotides in length linked at the 3′ end to a lipophilic group, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule having a double stranded region and a single stranded region, wherein the guide strand has a 3′ single stranded region of 2-13 nucleotides in length, each nucleotide within the single stranded region having a phosphorothioate modification, wherein the guide strand has a 5′ phosphate modification and wherein at least 50% of C and U nucleotides in the double stranded region include at least one 2′ O-methyl modification or 2′-fluoro modification.
In another aspect, the invention is an isolated double stranded nucleic acid molecule having a guide strand of 17-21 nucleotides in length that has complementarity to a target gene, a passenger strand of 10-16 nucleotides in length linked at the 3′ end to a lipophilic group, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule having a double stranded region and a single stranded region, wherein the guide strand has a 3′ single stranded region of 5-11 nucleotides in length, at least two nucleotide within the single stranded region having a phosphorothioate modification, wherein the guide strand has a 5′ phosphate modification and wherein at least 50% of C and U nucleotides in the double stranded region are 2′ O-methyl modification or 2′-fluoro modified.
The invention in another aspect is an isolated double stranded nucleic acid molecule having a guide strand of 17-21 nucleotides in length that has complementarity to a target gene, a passenger strand of 8-16 nucleotides in length linked at the 3′ end to a lipophilic group, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule having a double stranded region and a single stranded region, wherein the guide strand has a 3′ single stranded region of 6-8 nucleotides in length, each nucleotide within the single stranded region having a phosphorothioate modification, wherein the guide strand has a 5′ phosphate modification, wherein the passenger strand includes at least two phosphorothioate modifications, wherein at least 50% of C and U nucleotides in the double stranded region include a 2′ O-methyl modification or 2′-fluoro modification, and wherein the double stranded nucleic acid molecule has one end that is blunt or includes a one-two nucleotide overhang.
An isolated double stranded nucleic acid molecule having a guide strand of 17-21 nucleotides in length that has complementarity to a target gene, a passenger strand of 8-16 nucleotides in length linked at the 3′ end to a lipophilic group, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule having a double stranded region and a single stranded region, wherein the guide strand has a 3′ single stranded region, each nucleotide within the single stranded region having a phosphorothioate modification, wherein the guide strand has a 5′ phosphate modification, wherein every C and U nucleotide in position 11-18 of the guide strand has a 2′ O-methyl modification, wherein every nucleotide of the passenger strand is 2′ O-methyl modified, and wherein the double stranded nucleic acid molecule has one end that is blunt or includes a one-two nucleotide overhang is provided in other aspects of the invention.
In another aspect the invention is an isolated double stranded nucleic acid molecule having a guide strand of 17-21 nucleotides in length that has complementarity to a target gene, a passenger strand of 8-15 nucleotides in length linked at the 3′ end to a lipophilic group, wherein the lipophilic group is selected from the group consisting of cholesterol and a sterol type molecule with C17 polycarbon chain of 5-7 or 9-18 carbons in length, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule having a double stranded region and a single stranded region, wherein the guide strand has a 3′ single stranded region, each nucleotide within the single stranded region having a phosphorothioate modification, wherein the guide strand has a 5′ phosphate modification, wherein every C and U nucleotide in position 11-18 of the guide strand has a 2′ O-methyl modification, wherein every C and U nucleotide in position 2-10 of the guide strand has a 2′F modification, wherein every nucleotide of the passenger strand is 2′ O-methyl modified, and wherein the double stranded nucleic acid molecule has one end that is blunt or includes a one-two nucleotide overhang.
In yet another aspect the invention is an isolated nucleic acid molecule having a guide sequence that has complementarity to a target gene, a passenger sequence linked at the 3′ end to a lipophilic group, wherein the guide sequence and the passenger sequence form a nucleic acid molecule having a double stranded region and a single stranded region, wherein the guide sequence has a 3′ single stranded region of 2-13 nucleotides in length, each nucleotide within the single stranded region having a phosphorothioate modification, wherein the guide sequence has a 5′ phosphate modification, wherein at to least 50% of C and U nucleotides in the double stranded region include at least one 2′ O-methyl modification or 2′-fluoro modification, and wherein the double stranded nucleic acid molecule has one end that is blunt or includes a one-two nucleotide overhang.
An isolated double stranded nucleic acid molecule having a guide strand and a passenger strand, wherein the region of the molecule that is double stranded is from 8-14 nucleotides long, wherein the guide strand contains a single stranded region that is 4-12 nucleotides long, and wherein the single stranded region of the guide strand contains 2-12 phosphorothioate modifications is provided in other aspects of the invention.
In some embodiments the guide strand contains 6-8 phosphorothioate modifications. In other embodiments the single stranded region of the guide strand is 6 nucleotides long.
In yet other embodiments the double stranded region is 13 nucleotides long. Optionally the double stranded nucleic acid molecule has one end that is blunt or includes a one-two nucleotide overhang.
In another aspect the invention is an isolated double stranded nucleic acid molecule having a guide strand, wherein the guide strand is 16-28 nucleotides long and has complementarity to a target gene, wherein the 3′ terminal 10 nucleotides of the guide strand include at least two phosphate modifications, and wherein the guide strand has a 5′ phosphate modification and includes at least one 2′ O-methyl modification or 2′-fluoro modification, and a passenger strand, wherein the passenger strand is 8-14 nucleotides long and has complementarity to the guide strand, wherein the passenger strand is linked to a lipophilic group, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule.
In some embodiments the nucleotide in position one of the guide strand or sequence has a 2′-O-methyl modification. In other embodiments at least one C or U nucleotide in positions 2-10 of the guide strand or sequence has a 2′-fluoro modification. In yet other embodiments every C and U nucleotide in positions 2-10 of the guide strand or sequence has a 2′-fluoro modification. At least one C or U nucleotide in positions 11-18 of the guide strand or sequence may have a 2′-O-methyl modification. In some embodiments every C and U nucleotide in positions 11-18 of the guide strand or sequence has a 2′-O-methyl modification.
In yet other embodiments the 3′ terminal 10 nucleotides of the guide strand include at least four phosphate modifications. Optionally the 3′ terminal 10 nucleotides of the guide strand include at least eight phosphate modifications. In some embodiments the guide strand includes 4-14 phosphate modifications. In other embodiments the guide strand includes 4-10 phosphate modifications. In yet other embodiments the 3′ terminal 6 nucleotides of the guide strand all include phosphate modifications. The phosphate modifications may be phosphorothioate modifications.
In some embodiments every C and U nucleotide on the passenger strand has a 2′-O-methyl modification. In other embodiments every nucleotide on the passenger strand has a 2′-O-methyl modification. In an embodiment at least one nucleotide on the passenger strand is phosphorothioate modified. At least two nucleotides on the passenger strand are phosphorothioate modified in other embodiments.
The lipophilic molecule may be a sterol, such as cholesterol.
In some embodiments the guide strand is 18-19 nucleotides long. In other embodiments the passenger strand is 11-13 nucleotides long.
The double stranded nucleic acid molecule has one end that is blunt or includes a one-two nucleotide overhang in other embodiments.
In other aspects the invention is an isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-14 nucleotides long and has complementarity to the guide strand, and wherein the guide stand has at least two chemical modifications. In some embodiments the at least two chemical modifications include at least two phosphorothioate modifications. In some embodiments the double stranded nucleic acid molecule has one end that is blunt or includes a one-two nucleotide overhang.
In some aspects the invention is an isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-14 nucleotides long and has complementarity to the guide strand, and wherein the guide stand has a single stranded 3′ region that is 5 nucleotides or longer and a 5′ region that is 1 nucleotide or less. The single stranded region may contain at least 2 phosphorothioate modifications.
An isolated double stranded nucleic acid molecule having a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-16 nucleotides long and has complementarity to the guide strand, and wherein the guide stand has a single stranded 3′ region that is 5 nucleotides or longer and a passenger strand has a sterol type molecule with C17 attached chain longer than 9 is provided in other aspects of the invention.
A duplex polynucleotide is provided in other aspects of the invention. The polynucleotide has a first polynucleotide wherein said first polynucleotide is complementary to a second polynucleotide and a target gene; and a second polynucleotide wherein said second polynucleotide is at least 6 nucleotides shorter than said first polynucleotide, wherein said first polynucleotide includes a single stranded region containing modifications selected from the group consisting of 40-90% hydrophobic base modifications, 40-90% phosphorothioates, and 40-90% modifications of the ribose moiety, or any combination thereof.
In other aspects the invention is a duplex polynucleotide having a first polynucleotide wherein said first polynucleotide is complementary to a second polynucleotide and a target gene; and a second polynucleotide wherein said second polynucleotide is at least 6 nucleotides shorter than said first polynucleotide, wherein the duplex polynucleotide includes a mismatch between nucleotides 9, 11, 12, 13 or 14 on the first polynucleotide and the opposite nucleotide on the second polynucleotide.
In other aspects the invention is a method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with an isolated double stranded nucleic acid molecule or a duplex polynucleotide.
A method of inducing RNAi in a subject is provided in other aspects of the invention. The method involves administering to a subject an effective amount for inducing RNAi of an mRNA of a target gene, an isolated double stranded nucleic acid molecule or a duplex polynucleotide. In other embodiment the subject is a human. In other embodiments the target gene is PPIB, MAP4K4, or SOD1.
In other aspects an isolated hydrophobic modified polynucleotide having a polynucleotide, wherein the polynucleotide is double stranded RNA, attached to a hydrophobic molecule, wherein the hydrophobic molecule is attached to a base, a ribose or a backbone of a non-terminal nucleotide and wherein the isolated double stranded nucleic acid molecule comprises a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-14 nucleotides long and has complementarity to the guide strand is provided.
In one embodiment the hydrophobic molecule is attached to the guide strand of the double stranded RNA. In another embodiment the 3′ terminal 10 nucleotides of the guide strand include at least two phosphate modifications, and wherein the guide strand has a 5′ phosphate modification and includes at least one 2′ O-methyl modification or 2′-fluoro modification. In yet another embodiment the hydrophobic molecule is attached to the passenger strand of the double stranded RNA.
The invention provides an isolated hydrophobic modified polynucleotide having a polynucleotide non-covalently complexed to a hydrophobic molecule, wherein the hydrophobic molecule is a polycationic molecule. In some embodiments the polycationic molecule is selected from the group consisting of protamine, arginine rich peptides, and spermine.
In other aspects the invention an isolated hydrophobic modified polynucleotide having a polynucleotide, wherein the polynucleotide is double stranded RNA, directly complexed to a hydrophobic molecule without a linker, wherein the hydrophobic molecule is not cholesterol.
A composition having a hydrophobic modified polynucleotide, wherein the polynucleotide is double stranded RNA, attached to a hydrophobic molecule, wherein the double stranded nucleic acid molecule comprises a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-14 nucleotides long and has complementarity to the guide strand, wherein position 1 of the guide strand is 5′ phosphorylated or has a 2′ O-methyl modification, wherein at least 40% of the nucleotides of the double stranded nucleic acid are modified, and wherein the double stranded nucleic acid molecule has one end that is blunt or includes a one-two nucleotide overhang; a neutral fatty mixture; and optionally a cargo molecule, wherein the hydrophobic modified polynucleotide and the neutral fatty mixture forms a micelle is provided in other aspects of the invention.
In some embodiments the 3′ end of the passenger strand is linked to the hydrophobic molecule. In other embodiments the composition is sterile. In yet other embodiments the neutral fatty mixture comprises a DOPC (dioleoylphosphatidylcholine). In further embodiments the neutral fatty mixture comprises a DSPC (distearoylphosphatidylcholine). The neutral fatty mixture further comprises a sterol such as cholesterol in other embodiments.
In yet other embodiments the composition includes at least 20% DOPC and at least 20% cholesterol. The hydrophobic portion of the hydrophobic modified polynucleotide is a sterol in other embodiments. The sterol may be a cholesterol, a cholesteryl or modified cholesteryl residue. In other embodiments the hydrophobic portion of the hydrophobic modified polynucleotide is selected from the group consisting of bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, vitamins, saturated fatty acids, unsaturated fatty acids, fatty acid esters, triglycerides, pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, and ibuprofen.
In yet other embodiments the hydrophobic portion of the hydrophobic modified polynucleotide is a polycationic molecule, such as, for instance, protamine, arginine rich peptides, and/or spermine.
The composition optionally includes a cargo molecule such as a lipid, a peptide, vitamin, and/or a small molecule. In some embodiments the cargo molecule is a commercially available fat emulsions available for a variety of purposes selected from the group consisting of parenteral feeding. In some embodiments the commercially available fat emulsion is an intralipid or a nutralipid. In other embodiments the cargo molecule is a fatty acid mixture containing more then 74% of linoleic acid, a fatty acid mixture containing at least 6% of cardiolipin, or a fatty acid mixture containing at least 74% of linoleic acid and at least 6% of cardiolipin. In another embodiment the cargo molecule is a fusogenic lipid, such as for example, DOPE, and preferably is at least 10% fusogenic lipid
In some embodiments the polynucleotide includes chemical modifications. For instance it may be at least 40% modified.
A method of inducing RNAi in a subject is provided in another aspect of the invention. The method involves administering to a subject an effective amount for inducing RNAi of mRNA of a target gene, an isolated double stranded nucleic acid molecule or a duplex polynucleotide or a composition of the invention, wherein the polynucleotide has at least a region of sequence correspondence to the target gene, wherein the step of administering is systemic, intravenous, intraperitoneal, intradermal, topical, intranasal, inhalation, oral, intramucosal, local injection, subcutaneous, oral tracheal, or intraocular.
In other embodiment the subject is a human. In other embodiments the target gene is PPIB, MAP4K4, or SOD1.
In some aspects the invention is a single-stranded RNA of less than 35 nucleotides in length that forms a hairpin structure, said hairpin includes a double-stranded stem and a single-stranded loop, said double-stranded stem having a 5′-stem sequence having a 5′-end, and a 3′-stem sequence having a 3′-end; and said 5′-stem sequence and at least a portion of said loop form a guide sequence complementary to a transcript of a target gene, wherein said polynucleotide mediates sequence-dependent gene silencing of expression of said target gene, wherein each nucleotide within the single-stranded loop region has a phosphorothioate modification, and wherein at least 50% of C and U nucleotides in the double stranded region include a 2′ O-methyl modification or 2′-fluoro modification. In one embodiment every C and U nucleotide in position 11-18 of the guide sequence has a 2′ O-methyl modification.
A polynucleotide construct is provided in other aspects, the polynucleotide having two identical single-stranded polynucleotides, each of said single-stranded polynucleotide comprising a 5′-stem sequence having a 5′-end, a 3′-stem sequence having a 3′-end, and a linker sequence linking the 5′-stem sequence and the 3′-stem sequence, wherein: (1) the 5′-stem sequence of a first single-stranded polynucleotide hybridizes with the 3′-stem sequence of a second single-stranded polynucleotide to form a first double-stranded stem region; (2) the 5′-stem sequence of the second single-stranded polynucleotide hybridize with the 3′-stem sequence of the first single-stranded polynucleotide to form a second double-stranded stem region; and, (3) the linker sequences of the first and the second single-stranded polynucleotides form a loop or bulge connecting said first and said second double-stranded stem regions, wherein the 5′-stem sequence and at least a portion of the linker sequence form a guide sequence complementary to a transcript of a target gene, wherein said polynucleotide construct mediates sequence-dependent gene silencing of expression of said target gene, wherein each nucleotide within the single-stranded loop region has a phosphorothioate modification, and wherein at least 50% of C and U nucleotides in the double stranded regions include a 2′ O-methyl modification or 2′-fluoro modification.
In one embodiment every C and U nucleotide in position 11-18 of the guide sequence has a 2′ O-methyl modification.
In some embodiments, the guide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides long. In some embodiments, the passenger strand is 8, 9, 10, 11, 12, 13 or 14 nucleotides long. In some embodiments, the nucleic acid molecule has a thermodynamic stability (ΔG) of less than −20 kkal/mol.
Aspects of the invention relate to nucleic acid molecules that are chemically modified. In some embodiments, the chemical modification is selected from the group consisting of 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU), C-5 propynyl-C (pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine), 5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5 propynyl-fU (pfU), 5-methyl IC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid), MGB (minor groove binder) and other base modifications which increase base hydrophobicity. More than one chemical modification may be present in the same molecule. In some embodiments, chemical modification increases stability and/or improves thermodynamic stability (ΔG). In some embodiments, at least 90% of CU residues on a nucleic acid molecule are modified.
In some embodiments, the nucleotide in position one of the guide strand has a 2′-O-methyl modification and/or a 5′ Phosphate modification. In some embodiments, at least one C or U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In certain embodiments, every C and U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In some embodiments, at least one C or U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In certain embodiments, every C and U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In some embodiments, every C and U nucleotide on the passenger strand has a 2′-O-methyl modification. In certain embodiments, every nucleotide on the passenger strand has a 2′-O-methyl modification.
In some embodiments, nucleic acid molecules associated with the invention contain a stretch of at least 4 nucleotides that are phosphorothioate modified. In certain embodiments, the stretch of nucleotides that are phosphorothioate modified is at least 12 nucleotides long. In some embodiments, the stretch of nucleotides that are phosphorothioate modified is not fully single stranded.
Nucleic acid molecules associated with the invention may be attached to a conjugate. In some embodiments, the conjugate is attached to the guide strand, while in other embodiments the conjugate is attached to the passenger strand. In some embodiments, the conjugate is hydrophobic. In some embodiments, the conjugate is a sterol such as cholesterol. In some embodiments, nucleic acid molecules associated with the invention are blunt-ended.
Aspects of the invention relate to double stranded nucleic acid molecule including a guide strand and a passenger strand, wherein the region of the molecule that is double stranded is from 8-14 nucleotides long, and wherein the molecule has a thermodynamic stability (ΔG) of less than −13 kkal/mol.
In some embodiments, the region of the molecule that is double stranded is 8, 9, 10, 11, 12, 13, or 14 nucleotides long. In some embodiments, the molecule has a thermodynamic stability (ΔG) of less than −20 kkal/mol. The nucleic acid molecules, in some embodiments are chemically modified. In certain embodiments, the chemical modification is selected from the group consisting of 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU), C-5 propynyl-C (pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine), 5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5 propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid), MGB (minor groove binder) and other base modifications which increase base hydrophobicity. More than one chemical modification may be present in the same molecule. In some embodiments, chemical modification increases stability and/or improves thermodynamic stability (ΔG). In some embodiments, at least 90% of CU residues on a nucleic acid molecule are modified.
In some embodiments, the nucleotide in position one of the guide strand has a 2′-O-methyl modification and/or a 5′ Phosphate modification. In some embodiments, at least one C or U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In certain embodiments, every C and U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In some embodiments, at least one C or U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In certain embodiments, every C and U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In some embodiments, every C and U nucleotide on the passenger strand has a 2′-O-methyl modification. In certain embodiments, every nucleotide on the passenger strand has a 2′-O-methyl modification.
The nucleic acid molecules associated with the invention may contain a stretch of at least 4 nucleotides that are phosphorothioate modified. In certain embodiments, the stretch of nucleotides that are phosphorothioate modified is at least 12 nucleotides long. In some embodiments, the stretch of nucleotides that are phosphorothioate modified is not fully single stranded. In some embodiments, the nucleic acid molecules are attached to a conjugate. In some embodiments, the conjugate is attached to the guide strand, while in other embodiments the conjugate is attached to the passenger strand. In some embodiments, the conjugate is hydrophobic. In some embodiments, the conjugate is a sterol such as cholesterol. In some embodiments, nucleic acid molecules associated with the invention are blunt-ended. In some embodiments, the nucleic acid molecules are blunt ended at the 5′ end. In certain embodiments, the nucleic acid molecules are blunt ended at the 5′ end where the region of complementarity between the two strands of the molecule begins.
Aspects of the invention relate to methods for inhibiting the expression of a target gene in a mammalian cell. Methods include contacting the mammalian cell with an isolated double stranded nucleic acid molecule including a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and has complementarity to a target gene, wherein the passenger strand is from 8-14 nucleotides long and has complementarity to the guide strand, and wherein the double stranded nucleic acid molecule has a thermodynamic stability (ΔG) of less than −13 kkal/mol.
The cell may be contacted in vivo or in vitro. In some embodiments, the guide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides long. In some embodiments, the passenger strand is 8, 9, 10, 11, 12, 13 or 14 nucleotides long. In some embodiments, the nucleic acid molecule has a thermodynamic stability (ΔG) of less than −20 kkal/mol.
The nucleic acid molecules associated with methods described herein may be chemically modified. In some embodiments, the chemical modification is selected from the group consisting of 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU), C-5 propynyl-C (pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine), 5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5 propynyl-fU (pfU), 5-methyl IC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid), MGB (minor groove binder) and other base modifications which increase base hydrophobicity. More than one chemical modification may be present in the same molecule. In some embodiments, chemical modification increases stability and/or improves thermodynamic stability (ΔG). In some embodiments, at least 90% of CU residues on a nucleic acid molecule are modified.
In some embodiments, the nucleotide in position one of the guide strand has a 2′-O-methyl modification and/or a 5′ Phosphate modification. In some embodiments, at least one C or U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In certain embodiments, every C and U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In some embodiments, at least one C or U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In certain embodiments, every C and U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In some embodiments, every C and U nucleotide on the passenger strand has a 2′-O-methyl modification. In certain embodiments, every nucleotide on the passenger strand has a 2′-O-methyl modification.
In some embodiments, nucleic acid molecules associated with the invention contain a stretch of at least 4 nucleotides that are phosphorothioate modified. In certain embodiments, the stretch of nucleotides that are phosphorothioate modified is at least 12 nucleotides long. In some embodiments, the stretch of nucleotides that are phosphorothioate modified is not fully single stranded.
Nucleic acid molecules associated with the invention may be attached to a conjugate. In some embodiments, the conjugate is attached to the guide strand, while in other embodiments the conjugate is attached to the passenger strand. In some embodiments, the conjugate is hydrophobic. In some embodiments, the conjugate is a sterol such as cholesterol. In some embodiments, nucleic acid molecules associated with the invention are blunt-ended.
Methods for inhibiting the expression of a target gene in a mammalian cell described herein include contacting the mammalian cell with an isolated double stranded nucleic acid molecule including a guide strand and a passenger strand, wherein the region of the molecule that is double stranded is from 8-14 nucleotides long, and wherein the molecule has a thermodynamic stability (ΔG) of less than −13 kkal/mol.
In some embodiments, the region of the molecule that is double stranded is 8, 9, 10, 11, 12, 13, or 14 nucleotides long. In some embodiments, the molecule has a thermodynamic stability (ΔG) of less than −20 kkal/mol. The nucleic acid molecules, in some embodiments are chemically modified. In certain embodiments, the chemical modification is selected from the group consisting of 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU), C-5 propynyl-C (pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine), 5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5 propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid), MGB (minor groove binder) and other base modifications which increase base hydrophobicity. More than one chemical modification may be present in the same molecule. In some embodiments, chemical modification increases stability and/or improves thermodynamic stability (ΔG). In some embodiments, at least 90% of CU residues on a nucleic acid molecule are modified.
In some embodiments, the nucleotide in position one of the guide strand has a 2′-O-methyl modification and/or a 5′ Phosphate modification. In some embodiments, at least one C or U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In certain embodiments, every C and U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In some embodiments, at least one C or U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In certain embodiments, every C and U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In some embodiments, every C and U nucleotide on the passenger strand has a 2′-O-methyl modification. In certain embodiments, every nucleotide on the passenger strand has a 2′-O-methyl modification.
The nucleic acid molecules associated with the invention may contain a stretch of at least 4 nucleotides that are phosphorothioate modified. In certain embodiments, the stretch of nucleotides that are phosphorothioate modified is at least 12 nucleotides long. In some embodiments, the stretch of nucleotides that are phosphorothioate modified is not fully single stranded. In some embodiments, the nucleic acid molecules are attached to a conjugate. In some embodiments, the conjugate is attached to the guide strand, while in other embodiments the conjugate is attached to the passenger strand. In some embodiments, the conjugate is hydrophobic. In some embodiments, the conjugate is a sterol such as cholesterol. In some embodiments, nucleic acid molecules associated with the invention are blunt-ended.
In another embodiment, the invention provides a method for selecting an siRNA for gene silencing by (a) selecting a target gene, wherein the target gene comprises a target sequence; (b) selecting a candidate siRNA, wherein said candidate siRNA comprises a guide strand of 16-29 nucleotide base pairs and a passenger strand of 8-14 nucleotide base pairs that form a duplex comprised of an antisense region and a sense region and said antisense region of said candidate siRNA is at least 80% complementary to a region of said target sequence; (c) determining a thermodynamic stability (ΔG) of the candidate siRNA; and (e) selecting said candidate siRNA as an siRNA for gene silencing, if said thermodynamic stability is less than −13 kkal/mol.
Aspects of the invention relate to isolated double stranded nucleic acid molecules including a guide strand and a passenger strand, wherein the guide strand is 18-19 nucleotides long and has complementarity to a target gene, wherein the passenger strand is 11-13 nucleotides long and has complementarity to the guide strand, and wherein the double stranded nucleic acid molecule has a thermodynamic stability (ΔG) of less than −13 kkal/mol.
In some embodiments, the nucleotide in position one of the guide strand has a 2′-O-methyl modification and/or a 5′ Phosphate modification. In some embodiments, at least one C or U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In certain embodiments, every C and U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In some embodiments, at least one C or U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In certain embodiments, every C and U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification.
In some embodiments, the guide strand contains a stretch of at least 4 nucleotides that are phosphorothioate modified. In certain embodiments, the guide strand contains a stretch of at least 8 nucleotides that are phosphorothioate modified. In some embodiments, every C and U nucleotide on the passenger strand has a 2′-O-methyl modification. In certain embodiments, every nucleotide on the passenger strand has a 2′-O-methyl modification. In some embodiments, at least one, or at least two nucleotides on the passenger strand is phosphorothioate modified. The nucleic acid molecule can be attached to a conjugate on either the guide or passenger strand. In some embodiments, the conjugate is a sterol such as cholesterol.
Aspects of the invention relate to isolated double stranded nucleic acid molecules including a guide strand, wherein the guide strand is 16-28 nucleotides long and has complementarity to a target gene, wherein the 3′ terminal 10 nucleotides of the guide strand include at least two phosphate modifications, and wherein the guide strand includes at least one 2′ O-methyl modification or 2′-fluoro modification, and a passenger strand, wherein the passenger strand is 8-28 nucleotides long and has complementarity to the guide strand, wherein the passenger strand is linked to a lipophilic group, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule.
In some embodiments, the nucleotide in position one of the guide strand has a 2′-O-methyl modification and/or a 5′ Phosphate modification. In some embodiments, at least one C or U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In certain embodiments, every C and U nucleotide in positions 2-10 of the guide strand has a 2′-fluoro modification. In some embodiments, at least one C or U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification. In certain embodiments, every C and U nucleotide in positions 11-18 of the guide strand has a 2′-O-methyl modification.
In some embodiments, the 3′ terminal 10 nucleotides of the guide strand include at least four, or at least eight phosphate modifications. In certain embodiments, the guide strand includes 2-14 or 4-10 phosphate modifications. In some embodiments, the 3′ terminal 6 nucleotides of the guide strand all include phosphate modifications. In certain embodiments, the phosphate modifications are phosphorothioate modifications.
In some embodiments, every C and U nucleotide on the passenger strand has a 2′-O-methyl modification. In certain embodiments, every nucleotide on the passenger strand has a 2′-O-methyl modification. In some embodiments, at least one, or at least two nucleotides on the passenger strand is phosphorothioate modified. In some embodiments, the lipophilic molecule is a sterol such as cholesterol. In some embodiments, the guide strand is 18-19 nucleotides long and the passenger strand is 11-13 nucleotides long.
Aspects of the invention relate to isolated double stranded nucleic acid molecules including a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-14 nucleotides long and has complementarity to the guide strand, and wherein the guide stand has at least two chemical modifications. In some embodiments, the two chemical modifications are phosphorothioate modifications.
Further aspects of the invention relate to isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-14 nucleotides long and has complementarity to the guide strand, and wherein the guide stand has a single stranded 3′ region that is 5 nucleotides or longer. In some embodiments, the single stranded region contains at least 2 phosphorothioate modifications.
Further aspects of the invention relate to isolated double stranded nucleic acid molecules including a guide strand and a passenger strand, wherein the guide strand is from 18-21 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 11-14 nucleotides long and has complementarity to the guide strand, and wherein position one of the guide stand has 2-OMe and 5′ phosphate modifications, every C and U in positions 2 to 11 of the guide strand are 2-F modified, every C and U in positions 12-18 of the guide strand are 2′OMe modified, and 80% of Cs and Us on the passenger strand are 2′OMe modified
Another aspect of the invention relates to isolated double stranded nucleic acid molecules including a guide strand and a passenger strand, wherein the guide strand is from 18-21 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 11-14 nucleotides long and has complementarity to the guide strand, and wherein the guide stand has 2-OMe and 5′ phosphate modifications at position 1, every C and U in positions 2 to 11 of the guide strand are 2-F modified, every C and U in positions 12-18 of the guide strand are 2′OMe modified, 80% of Cs and Us on the passenger strand are 2′OMe and the 3′ end of the passenger strand is attached to a conjugate. In some embodiments the conjugate is selected from sterols, sterol-type molecules, hydrophobic vitamins or fatty acids.
Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.



BRIEF DESCRIPTION OF DRAWINGS
The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:
FIG. 1 is a schematic depicting proposed structures of asymmetric double stranded RNA molecules (adsRNA). Bold lines represent sequences carrying modification patterns compatible with RISC loading. Striped lines represent polynucleotides carrying modifications compatible with passenger strands. Plain lines represent a single stranded polynucleotide with modification patterns optimized for cell interaction and uptake. FIG. 1A depicts adsRNA with extended guide or passenger strands; FIG. 1B depicts adsRNA with length variations of a cell penetrating polynucleotide; FIG. 1C depicts adsRNA with 3′ and 5′ conjugates; FIG. 1D depicts adsRNAs with mismatches.
FIG. 2 is a schematic depicting asymmetric dsRNA molecules with different chemical modification patterns. Several examples of chemical modifications that might be used to increase hydrophobicity are shown including 4-pyridyl, 2-pyridyl, isobutyl and indolyl based position 5 uridine modifications.
FIG. 3 is a schematic depicting the use of dsRNA binding domains, protamine (or other Arg rich peptides), spermidine or similar chemical structures to block duplex charge to facilitate cellular entry.
FIG. 4 is a schematic depicting positively charged chemicals that might be used for polynucleotide charge blockage.
FIG. 5 is a schematic depicting examples of structural and chemical compositions of single stranded RISC entering polynucleotides. The combination of one or more modifications including 2′ d, 2′Ome, 2′F, hydrophobic and phosphorothioate modifications can be used to optimize single strand entry into the RISC.
FIG. 6 is a schematic depicting examples of structural and chemical composition of RISC substrate inhibitors. Combinations of one or more chemical modifications can be used to mediate efficient uptake and efficient binding to preloaded RISC complex.
FIG. 7 is a schematic depicting structures of polynucleotides with sterol type molecules attached, where R represent a polycarbonic tail of 9 carbons or longer. FIG. 7A depicts an adsRNA molecule; FIG. 7B depicts an siRNA molecule of approximately 17-30 bp long; FIG. 7C depicts a RISC entering strand; FIG. 7D depicts a substrate analog strand. Chemical modification patterns, as depicted in FIG. 7, can be optimized to promote desired function.
FIG. 8 is a schematic depicting examples of naturally occurring phytosterols with a polycarbon chain that is longer than 8, attached at position 17. More than 250 different types of phytosterols are known.
FIG. 9 is a schematic depicting examples of sterol-like structures, with variations in the size of the polycarbon chains attached at position 17.
FIG. 10 presents schematics and graphs demonstrating that the percentage of liver uptake and plasma clearance of lipid emulsions containing sterol type molecules is directly affected by the size of the polycarbon chain attached at position 17. This figure is adapted from Martins et al, Journal of Lipid Research (1998).
FIG. 11 is a schematic depicting micelle formation. FIG. 11A depicts a polynucleotide with a hydrophobic conjugate; FIG. 11B depicts linoleic acid; FIG. 11C depicts a micelle formed from a mixture of polynucleotides containing hydrophobic conjugates combined with fatty acids.
FIG. 12 is a schematic depicting how alteration in lipid composition can affect pharmacokinetic behavior and tissue distribution of hydrophobically modified and/or hydrophobically conjugated polynucleotides. In particular, use of lipid mixtures enriched in linoleic acid and cardiolipin results in preferential uptake by cardiomyocites.
FIG. 13 is a schematic showing examples of RNAi constructs and controls used to target MAP4K4 expression. RNAi construct 12083 corresponds to SEQ ID NOs:597 and 598. RNAi construct 12089 corresponds to SEQ ID NO:599.
FIG. 14 is a graph showing MAP4K4 expression following transfection with RNAi constructs associated with the invention. RNAi constructs tested were: 12083 (Nicked), 12085 (13 nt Duplex), 12089 (No Stem Pairing) and 12134 (13 nt miniRNA). Results of transfection were compared to an untransfected control sample. RNAi construct 12083 corresponds to SEQ ID NOs:597 and 598. RNAi construct 12085 corresponds to SEQ ID NOs:600 and 601. RNAi construct 12089 corresponds to SEQ ID NO:599. RNAi construct 12134 corresponds to SEQ ID NOs:602 and 603.
FIG. 15 is a graph showing expression of MAP4K4 24 hours post-transfection with RNAi constructs associated with the invention. RNAi constructs tested were: 11546 (MAP4K4 rxRNA), 12083 (MAP4K4 Nicked Construct), 12134 (12 bp soloRNA) and 12241 (14/3/14 soloRNA). Results of transfection were compared to a filler control sample. RNAi construct 11546 corresponds to SEQ ID NOs:604 and 605. RNAi construct 12083 corresponds to SEQ ID NOs:597 and 598. RNAi construct 12134 corresponds to SEQ ID NOs:602 and 603. RNAi construct 12241 corresponds to SEQ ID NOs:606 and 607.
FIG. 16 presents a graph and several tables comparing parameters associated with silencing of MAP4K4 expression following transfection with RNAi constructs associated with the invention. The rxRNA construct corresponds to SEQ ID NOs:604 and 605. The 14-3-14 soloRNA construct corresponds to SEQ ID NOs:606 and 607. The 13/19 duplex (nicked construct) corresponds to SEQ ID NOs:597 and 598. The 12-bp soloRNA construct corresponds to SEQ ID NOs:602 and 603.
FIG. 17 is a schematic showing examples of RNAi constructs and controls used to target SOD1 expression. The 12084 RNAi construct corresponds to SEQ ID NOs:612 and 613.
FIG. 18 is a graph showing SOD1 expression following transfection with RNAi constructs associated with the invention. RNAi constructs tested were: 12084 (Nicked), 12086 (13 nt Duplex), 12090 (No Stem Pairing) and 12035 (13 nt MiniRNA). Results of transfection were compared to an untransfected control sample. The 12084 RNAi construct corresponds to SEQ ID NOs:612 and 613. The 12086 RNAi construct corresponds to SEQ ID NOs:608 and 609. The 12035 RNAi construct corresponds to SEQ ID NOs:610 and 611.
FIG. 19 is a graph showing expression of SOD1 24 hours post-transfection with RNAi constructs associated with the invention. RNAi constructs tested were: 10015 (SOD1 rxRNA) and 12084 (SOD 1 Nicked Construct). Results of transfection were compared to a filler control sample. The 10015 RNAi construct corresponds to SEQ ID NOs:614 and 615. The 12084 RNAi construct corresponds to SEQ ID NOs:612 and 613.
FIG. 20 is a schematic indicating that RNA molecules with double stranded regions that are less than 10 nucleotides are not cleaved by Dicer.
FIG. 21 is a schematic revealing a hypothetical RNAi model for RNA induced gene silencing.
FIG. 22 is a graph showing chemical optimization of asymmetric RNAi compounds. The presence of chemical modifications, in particular 2′F UC, phosphorothioate modifications on the guide strand, and complete CU 2′OMe modification of the passenger strands results in development of functional compounds. Silencing of MAP4K4 following lipid-mediated transfection is shown using RNAi molecules with specific modifications. RNAi molecules tested had sense strands that were 13 nucleotides long and contained the following modifications: unmodified; C and U 2′OMe; C and U 2′OMe and 3′ Chl; rxRNA 2′OMe pattern; or full 2′OMe, except base 1. Additionally, the guide (anti-sense) strands of the RNAi molecules tested contained the following modifications: unmodified; unmodified with 5′P; C and U 2′F; C and U 2′F with 8 PS 3′ end; and unmodified (17 nt length). Results for rxRNA 12/10 Duplex and negative controls are also shown.
FIG. 23 demonstrates that the chemical modifications described herein significantly increase in vitro efficacy in un-assisted delivery of RNAi molecules in HeLa cells. The structure and sequence of the compounds were not altered; only the chemical modification patterns of the molecules were modified. Compounds lacking 2′ F, 2′O-me, phosphorothioate modification, or cholesterol conjugates were completely inactive in passive uptake. A combination of all 4 of these types of modifications produced the highest levels of activity (compound 12386).
FIG. 24 is a graph showing MAP4K4 expression in Hela cells following passive uptake transfection of: NT Accell modified siRNA, MAP4K4 Accell siRNA, Non-Chl nanoRNA (12379) and sd-nanoRNA (12386).
FIG. 25 is a graph showing expression of MAP4K4 in HeLa cells following passive uptake transfection of various concentrations of RNA molecules containing the following parameters: Nano Lead with no 3′Chl; Nano Lead; Accell MAP4K4; 21mer GS with 8 PS tail; 21mer GS with 12 PS tail; and 25mer GS with 12 PS tail.
FIG. 26 is a graph demonstrating that reduction in oligonucleotide content increases the efficacy of unassisted uptake. Similar chemical modifications were applied to assymetric compounds, traditional siRNA compounds and 25 mer RNAi compounds. The assymetric small compounds demonstrated the most significant efficacy.
FIG. 27 is a graph demonstrating the importance of phosphorothioate content for un-assisted delivery. FIG. 27A demonstrates the results of a systematic screen that revealed that the presence of at least 2-12 phosphorothioates in the guide strand significantly improves uptake; in some embodiments, 4-8 phosphorothioate modifications were found to be preferred. FIG. 27 B reveals that the presence or absence of phosphorothioate modifications in the sense strand did not alter efficacy.
FIG. 28 is a graph showing expression of MAP4K4 in primary mouse hepatocytes following passive uptake transfection of: Accell Media-Ctrl-UTC; MM APOB Alnylam; Active APOB Alnylam; nanoRNA without chl; nanoRNA MAP4K4; Mouse MAP4K4 Accell Smartpool; DY547 Accell Control; Luc Ctrl rxRNA with Dy547; MAP4K4 rxRNA with DY547; and AS Strand Alone (nano).
FIG. 29 is a graph showing expression of ApoB in mouse primary hepatocytes following passive uptake transfection of: Accell Media-Ctrl-UTC; MM APOB Alnylam; Active APOB Alnylam; nanoRNA without chl; nanoRNA MAP4K4; Mouse MAP4K4 Accell Smartpool; DY547 Accell Control; Luc Ctrl rxRNA with Dy547; MAP4K4 rxRNA with DY547; and AS Strand Alone (nano).
FIG. 30 is a graph showing expression of MAP4K4 in primary human hepatocytes following passive uptake transfection of: 11550 MAP4K4 rxRNA; 12544 mM MAP4K4 nanoRNA; 12539 Active MAP4K4 nanoRNA; Accell Media; and UTC.
FIG. 31 is a graph showing ApoB expression in primary human hepatoctyes following passive uptake transfection of: 12505 Active ApoB chol-siRNA; 12506 mM ApoB chol-siRNA; Accell Media; and UTC.
FIG. 32 is an image depicting localization of sd-rxRNAnano localization.
FIG. 33 is an image depicting localization of Chol-siRNA (Alnylam).
FIG. 34 is a schematic of 1st generation (G1) sd-rxRNAnano molecules associated with the invention indicating regions that are targeted for modification, and functions associated with different regions of the molecules.
FIG. 35 depicts modification patterns that were screened for optimization of sd-rxRNAnano (G1). The modifications that were screened included, on the guide strand, lengths of 19, 21 and 25 nucleotides, phosphorothioate modifications of 0-18 nucleotides, and replacement of 2′F modifications with 2′OMe, 5 Methyl C and/or ribo Thymidine modifications. Modifications on the sense strand that were screened included nucleotide lengths of 11, 13 and 19 nucleotides, phosphorothiote modifications of 0-4 nucleotides and 2′OMe modifications.
FIG. 36 is a schematic depicting modifications of sd-rxRNAnano that were screened for optimization.
FIG. 37 is a graph showing percent MAP4K4 expression in Hek293 cells following transfection of: Risc Free siRNA; rxRNA; Nano (unmodified); GS alone; Nano Lead (no Chl); Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 8 PS, 19 nt); Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 8 PS, 21 nt); Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 12 PS, 21 nt); and Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 12 PS, 25 nt);
FIG. 38 is a graph showing percent MAP4K4 expression in HeLa cells following passive uptake transfection of GS alone; Nano Lead; Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 8 PS, 19 nt); Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 8 PS, 21 nt); Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 12 PS, 21 nt); Nano (GS: (3) 2′OMe at positions 1, 18, and 19, 12 PS, 25 nt).
FIG. 39 is a graph showing percent MAP4K4 expression in Hek293 cells following lipid mediated transfection of: Guide Strand alone (GS: 8 PS, 19 nt); Guide Strand alone (GS: 18 PS, 19 nt); Nano (GS: no PS, 19 nt); Nano (GS: 2 PS, 19 nt); Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt); Nano Lead (GS: 8 PS, 19 nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS, 19 nt); and Nano (GS: 18 PS, 19 nt).
FIG. 40 is a graph showing percent MAP4K4 expression in Hek293 cells following lipid mediated transfection of: Guide Strand alone (GS: 8 PS, 19 nt); Guide Strand alone (GS: 18 PS, 19 nt); Nano (GS: no PS, 19 nt); Nano (GS: 2 PS, 19 nt); Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt); Nano Lead (GS: 8 PS, 19 nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS, 19 nt); and Nano (GS: 18 PS, 19 nt).
FIG. 41 is a graph showing percent MAP4K4 expression in HeLa cells following passive uptake transfection of Nano Lead (no Chl); Guide Strand alone (18 PS); Nano (GS: 0 PS, 19 nt); Nano (GS: 2 PS, 19 nt); Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt); Nano Lead (GS: 8 PS, 19 nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS, 19 nt); and Nano (GS: 18 PS, 19 nt).
FIG. 42 is a graph showing percent MAP4K4 expression in HeLa cells following passive uptake transfection of: Nano Lead (no Chl); Guide Strand alone (18 PS); Nano (GS: 0 PS, 19 nt); Nano (GS: 2 PS, 19 nt); Nano (GS: 4 PS, 19 nt); Nano (GS: 6 PS, 19 nt); Nano Lead (GS: 8 PS, 19 nt); Nano (GS: 10 PS, 19 nt); Nano (GS: 12 PS, 19 nt); and Nano (GS: 18 PS, 19 nt).
FIG. 43 is a schematic depicting guide strand chemical modifications that were screened for optimization.
FIG. 44 is a graph showing percent MAP4K4 expression in Hek293 cells following reverse transfection of: RISC free siRNA; GS only (2′F C and Us); GS only (2′OMe C and Us); Nano Lead (2′F C and Us); nano (GS: (3) 2′OMe, positions 16-18); nano (GS: (3) 2′OMe, positions 16, 17 and 19); nano (GS: (4) 2′OMe, positions 11, 16-18); nano (GS: (10) 2′OMe, C and Us); nano (GS: (6) 2′OMe, positions 1 and 5-9); nano (GS: (3) 2′OMe, positions 1, 18 and 19); and nano (GS: (5) 2′OMe Cs).
FIG. 45 is a graph demonstrating efficacy of various chemical modification patterns. In particular, 2-OMe modification in positions 1 and 11-18 was well tolerated. 2′OMe modifications in the seed area resulted in a slight reduction of efficacy (but were still highly efficient). Ribo-modifications in the seed were well tolerated. This data enabled the generation of self delivering compounds with reduced or no 2′F modifications. This is significant because 2′F modifications may be associated with toxicity in vivo.
FIG. 46 is a schematic depicting sense strand modifications.
FIG. 47 is a graph demonstrating sense strand length optimization. A sense strand length between 10-15 bases was found to be optimal in this assay. Increasing sense strand length resulted in a reduction of passive uptake of these compounds but may be tolerated for other compounds. Sense strands containing LNA modification demonstrated similar efficacy to non-LNA containing compounds. In some embodiments, the addition of LNA or other thermodynamically stabilizing compounds can be beneficial, resulting in converting non-functional sequences into functional sequences.
FIG. 48 is a graph showing percent MAP4K4 expression in HeLa cells following passive uptake transfection of: Guide Strand Alone (2′F C and U); Nano Lead; Nano Lead (No Chl); Nano (SS: 11 nt 2′OMe C and Us, Chl); Nano (SS: 1 int, complete 2′OMe, Chl); Nano (SS: 19 nt, 2′OMe C and Us, Chl); Nano (SS: 19 nt, 2′OMe C and Us, no Chl).
FIG. 49 is a graph showing percent MAP4K4 expression in HeLa cells following passive uptake transfection of: Nano Lead (No Chl); Nano (SS no PS); Nano Lead (SS:2 PS); Nano (SS:4 PS).
FIG. 50 is a schematic depicting a sd-rxRNAnano second generation (GII) lead molecule.
FIG. 51 presents a graph indicating EC50 values for MAP4K4 silencing in the presence of sd-rxRNA, and images depicting localization of DY547-labeled rxRNAnano and DY547-labeled sd-rxRNA.
FIG. 52 is a graph showing percent MAP4K4 expression in HeLa cells in the presence of optimized sd-rxRNA molecules.
FIG. 53 is a graph depicting the relevance of chemistry content in optimization of sd-rxRNA efficacy.
FIG. 54 presents schematics of sterol-type molecules and a graph revealing that sd-rxRNA compounds are fully functional with a variety of linker chemistries. GII asymmetric compounds were synthesized with steroltype molecules attached through TEG and amino caproic acid linkers. Both linkers showed identical potency. This functionality independent of linker chemistry indicates a significant difference between the molecules described herein and previously described molecules, and offers significant advantages for the molecules described herein in terms of scale up and synthesis.
FIG. 55 demonstrates the stability of chemically modified sd-rxRNA compounds in human serum in comparison to non modified RNA. The oligonucleotides were incubated in 75% serum at 37° C. for the number of hours indicated. The level of degradation was determined by running the samples on non-denaturing gels and staining with SYBGR.
FIG. 56 is a graph depicting optimization of cellular uptake of sd-rxRNA through minimizing oligonucleotide content.
FIG. 57 is a graph showing percent MAP4K4 expression after spontaneous cellular uptake of sd-rxRNA in mouse PEC-derived macrophages, and phase and fluorescent images showing localization of sd-rxRNA.
FIG. 58 is a graph showing percent MAP4K4 expression after spontaneous cellular uptake of sd-rxRNA (targeting) and sd-rxRNA (mismatch) in mouse primary hepatocytes, and phase and fluorescent images showing localization of sd-rxRNA.
FIG. 59 presents images depicting localization of DY547-labeled sd-rxRNA delivered to RPE cells with no formulation.
FIG. 60 is a graph showing silencing of MAP4K4 expression in RPE cells treated with sd-rxRNAnano without formulation.
FIG. 61 presents a graph and schematics of RNAi compounds showing the chemical/structural composition of highly effective sd-rxRNA compounds. Highly effective compounds were found to have the following characteristics: antisense strands of 17-21 nucleotides, sense strands of 10-15 nucleotides, single-stranded regions that contained 2-12 phosphorothioate modifications, preferentially 6-8 phosphorothioate modifications, and sense strands in which the majority of nucleotides were 2′OMe modified, with or without phosphorothioate modification. Any linker chemistry can be used to attach these molecules to hydrophobic moieties such as cholesterol at the 3′ end of the sense strand. Version GIIa-b of these RNA compounds demonstrate that elimination of 2′F content has no impact on efficacy.
FIG. 62 presents a graph and schematics of RNAi compounds demonstrating the superior performance of sd-rxRNA compounds compared to compounds published by Wolfrum et. al. Nature Biotech, 2007. Both generation I and II compounds (GI and GIIa) developed herein show great efficacy. By contrast, when the chemistry described in Wolfrum et al. (all oligos contain cholesterol conjugated to the 3′ end of the sense strand) was applied to the same sequence in a context of conventional siRNA (19 bp duplex with two overhang) the compound was practically inactive. These data emphasize the significance of the combination of chemical modifications and assymetrical molecules described herein, producing highly effective RNA compounds.
FIG. 63 presents images showing that sd-rxRNA accumulates inside cells while other less effective conjugate RNAs accumulate on the surface of cells.
FIG. 64 presents images showing that sd-rxRNA molecules, but not other molecules, are internalized into cells within minutes.
FIG. 65 presents images demonstrating that sd-rxRNA compounds have drastically better cellular and tissue uptake characteristics when compared to conventional cholesterol conjugated siRNAs (such as those published by Soucheck et al). FIG. 65A,B compare uptake in RPE cells, FIG. 65C,D compare uptake upon local administration to skin and FIG. 65E,F compare uptake by the liver upon systemic administration. The level of uptake is at least an order of magnitude higher for the sd-rxRNA compounds relative to the regular siRNA-cholesterol compounds.
FIG. 66 presents images depicting localization of rxRNAori and sd-rxRNA following local delivery.
FIG. 67 presents images depicting localization of sd-rxRNA and other conjugate RNAs following local delivery.
FIG. 68 presents a graph revealing the results of a screen performed with sd-rxRNAGII chemistry to identify functional compounds targeting the SPP1 gene. Multiple effective compounds were identified, with 14131 being the most effective. The compounds were added to A-549 cells and the level of the ratio of SPP1/PPIB was determined by B-DNA after 48 hours.
FIG. 69 presents a graph and several images demonstrating efficient cellular uptake of sd-rxRNA within minutes of exposure. This is a unique characteristics of the sd-rxRNA compounds described herein, not observed with any other RNAi compounds. The Soutschek et al. compound was used as a negative control.
FIG. 70 presents a graph and several images demonstrating efficient uptake and silencing of sd-rxRNA compounds in multiple cell types with multiple sequences. In each case silencing was confirmed by looking at target gene expression using a Branched DNA assay.
FIG. 71 presents a graph revealing that sd-rxRNA is active in the presence and absence of serum. A slight reduction in efficacy (2-5 fold) was observed in the presence of serum. This minimal reduction in efficacy in the presence of serum differentiates the sd-rxRNA compounds described herein from previously described RNAi compounds, which had a greater reduction in efficacy, and thus creates a foundation for in vivo efficacy of the sd-rxRNA molecules described herein.
FIG. 72 presents images demonstrating efficient tissue penetration and cellular uptake upon single intradermal injection of sd-rxRNA compounds described herein. This represents a model for local delivery of sd-rxRNA compounds as well as an effective demonstration of delivery of sd-rxRNA compounds and silencing of genes in dermatological applications.
FIG. 73 presents images and a graph demonstrating efficient cellular uptake and in vivo silencing with sd-rxRNA following intradermal injection.
FIG. 74 presents graphs demonstrating that sd-rxRNA compounds have improved blood clearance and induce effective gene silencing in vivo in the liver upon systemic administration.
FIG. 75 presents a graph demonstrating that the presence of 5-Methyl C in an RNAi compound resulted in an increase in potency of lipid mediated transfection, demonstrating that hydrophobic modification of Cs and Us in the content of RNAi compounds can be beneficial. In some embodiments, these types of modifications can be used in the context of 2′ ribose modified bases to insure optimal stability and efficacy.
FIG. 76 presents a graph showing percent MAP4K4 expression in HeLa cells following passive uptake transfection of: Guide strand alone; Nano Lead; Nano Lead (No cholesterol); Guide Strand w/5MeC and 2′F Us Alone; Nano Lead w/GS 5MeC and 2′F Us; Nano Lead w/GS riboT and 5 Methyl Cs; and Nano Lead w/Guide dT and 5 Methyl Cs.
FIG. 77 presents images comparing localization of sd-rxRNA and other RNA conjugates following systemic delivery to the liver.
FIG. 78 presents schematics demonstrating 5-uridyl modifications with improved hydrophobicity characteristics. Incorporation of such modifications into sd-rxRNA compounds can increase cellular and tissue uptake properties. FIG. 78B presents a new type of RNAi compound modification which can be applied to compounds to improve cellular uptake and pharmacokinetic behavior. This type of modification, when applied to sd-rxRNA compounds, may contribute to making such compounds orally available.
FIG. 79 presents schematics revealing the structures of synthesized modified sterol type molecules, where the length and structure of the C17 attached tail is modified. Without wishing to be bound by any theory, the length of the C17 attached tail may contribute to improving in vitro and in vivo efficacy of sd-rxRNA compounds.
FIG. 80 presents a schematic demonstrating the lithocholic acid route to long side chain cholesterols.
FIG. 81 presents a schematic demonstrating a route to 5-uridyl phosphoramidite synthesis.
FIG. 82 presents a schematic demonstrating synthesis of tri-functional hydroxyprolinol linker for 3′-cholesterol attachment.
FIG. 83 presents a schematic demonstrating synthesis of solid support for the manufacture of a shorter asymmetric RNAi compound strand.
FIG. 84 demonstrates SPPI sd-rxRNA compound selection. Sd-rxRNA compounds targeting SPP1 were added to A549 cells (using passive transfection) and the level of SPPI expression was evaluated after 48 hours. Several novel compounds effective in SPPI silencing were identified, the most potent of which was compound 14131.
FIG. 85 demonstrates independent validation of sd-rxRNA compounds 14116, 14121, 14131, 14134, 14139, 14149, and 14152 efficacy in SPP1 silencing.
FIG. 86 demonstrates results of sd-rxRNA compound screens to identify sd-rxRNA compounds functional in CTGF knockdown.
FIG. 87 demonstrates results of sd-rxRNA compound screens to identify sd-rxRNA functional in CTGF knockdown.
FIG. 88 demonstrates a systematic screen identifying the minimal length of the asymmetric compounds. The passenger strand of 10-19 bases was hybridized to a guide strand of 17-25 bases. In this assay, compounds with duplex regions as short as 10 bases were found to be effective in inducing.
FIG. 89 demonstrates that positioning of the sense strand relative to the guide strand is critical for RNAi Activity. In this assay, a blunt end was found to be optimal, a 3′ overhang was tolerated, and a 5′ overhang resulted in complete loss of functionality.
FIG. 90 demonstrates that the guide strand, which has homology to the target only at nucleotides 2-17, resulted in effective RNAi when hybridized with sense strands of different lengths. The compounds were introduced into HeLa cells via lipid mediated transfection.
FIG. 91 is a schematic depicting a panel of sterol-type molecules which can be used as a hydrophobic entity in place of cholesterol. In some instances, the use of sterol-type molecules comprising longer chains results in generation of sd-rxRNA compounds with significantly better cellular uptake and tissue distribution properties.
FIG. 92 presents schematics depicting a panel of hydrophobic molecules which might be used as a hydrophobic entity in place of cholesterol. These list just provides representative examples; any small molecule with substantial hydrophobicity can be used.



DETAILED DESCRIPTION
Aspects of the invention relate to methods and compositions involved in gene silencing. The invention is based at least in part on the surprising discovery that asymmetric nucleic acid molecules with a double stranded region of a minimal length such as 8-14 nucleotides, are effective in silencing gene expression. Molecules with such a short double stranded region have not previously been demonstrated to be effective in mediating RNA interference. It had previously been assumed that that there must be a double stranded region of 19 nucleotides or greater. The molecules described herein are optimized through chemical modification, and in some instances through attachment of hydrophobic conjugates.
The invention is based at least in part on another surprising discovery that asymmetric nucleic acid molecules with reduced double stranded regions are much more effectively taken up by cells compared to conventional siRNAs. These molecules are highly efficient in silencing of target gene expression and offer significant advantages over previously described RNAi molecules including high activity in the presence of serum, efficient self delivery, compatibility with a wide variety of linkers, and reduced presence or complete absence of chemical modifications that are associated with toxicity.
In contrast to single-stranded polynucleotides, duplex polynucleotides have been difficult to deliver to a cell as they have rigid structures and a large number of negative charges which makes membrane transfer difficult. Unexpectedly, it was found that the polynucleotides of the present invention, although partially double-stranded, are recognized in vivo as single-stranded and, as such, are capable of efficiently being delivered across cell membranes. As a result the polynucleotides of the invention are capable in many instances of self delivery. Thus, the polynucleotides of the invention may be formulated in a manner similar to conventional RNAi agents or they may be delivered to the cell or subject alone (or with non-delivery type carriers) and allowed to self deliver. In one embodiment of the present invention, self delivering asymmetric double-stranded RNA molecules are provided in which one portion of the molecule resembles a conventional RNA duplex and a second portion of the molecule is single stranded.
The polynucleotides of the invention are referred to herein as isolated double stranded or duplex nucleic acids, oligonucleotides or polynucleotides, nano molecules, nano RNA, sd-rxRNAnano, sd-rxRNA or RNA molecules of the invention.
The oligonucleotides of the invention in some aspects have a combination of asymmetric structures including a double stranded region and a single stranded region of 5 nucleotides or longer, specific chemical modification patterns and are conjugated to lipophilic or hydrophobic molecules. This new class of RNAi like compounds have superior efficacy in vitro and in vivo. Based on the data described herein it is believed that the reduction in the size of the rigid duplex region in combination with phosphorothioate modifications applied to a single stranded region are new and important for achieving the observed superior efficacy. Thus, the RNA molecules described herein are different in both structure and composition as well as in vitro and in vivo activity.
In a preferred embodiment the RNAi compounds of the invention comprise an asymmetric compound comprising a duplex region (required for efficient RISC entry of 10-15 bases long) and single stranded region of 4-12 nucleotides long; with a 13 nucleotide duplex. A 6 nucleotide single stranded region is preferred in some embodiments. The single stranded region of the new RNAi compounds also comprises 2-12 phosphorothioate internucleotide linkages (referred to as phosphorothioate modifications). 6-8 phosphorothioate internucleotide linkages are preferred in some embodiments. Additionally, the RNAi compounds of the invention also include a unique chemical modification pattern, which provides stability and is compatible with RISC entry. The combination of these elements has resulted in unexpected properties which are highly useful for delivery of RNAi reagents in vitro and in vivo.
The chemically modification pattern, which provides stability and is compatible with RISC entry includes modifications to the sense, or passenger, strand as well as the antisense, or guide, strand. For instance the passenger strand can be modified with any chemical entities which confirm stability and do not interfere with activity. Such modifications include 2′ ribo modifications (O-methyl, 2′ F, 2 deoxy and others) and backbone modification like phosphorothioate modifications. A preferred chemical modification pattern in the passenger strand includes Omethyl modification of C and U nucleotides within the passenger strand or alternatively the passenger strand may be completely Omethyl modified.
The guide strand, for example, may also be modified by any chemical modification which confirms stability without interfering with RISC entry. A preferred chemical modification pattern in the guide strand includes the majority of C and U nucleotides being 2′ F modified and the 5′ end being phosphorylated. Another preferred chemical modification pattern in the guide strand includes 2′Omethyl modification of position 1 and C/U in positions 11-18 and 5′ end chemical phosphorylation. Yet another preferred chemical modification pattern in the guide strand includes 2′Omethyl modification of position 1 and C/U in positions 11-18 and 5′ end chemical phosphorylation and 2′F modification of C/U in positions 2-10.
It was surprisingly discovered according to the invention that the above-described chemical modification patterns of the oligonucleotides of the invention are well tolerated and actually improved efficacy of asymmetric RNAi compounds. See, for instance, FIG. 22.
It was also demonstrated experimentally herein that the combination of modifications to RNAi when used together in a polynucleotide results in the achievement of optimal efficacy in passive uptake of the RNAi. Elimination of any of the described components (Guide strand stabilization, phosphorothioate stretch, sense strand stabilization and hydrophobic conjugate) or increase in size results in sub-optimal efficacy and in some instances complete lost of efficacy. The combination of elements results in development of compound, which is fully active following passive delivery to cells such as HeLa cells. (FIG. 23). The degree to which the combination of elements results in efficient self delivery of RNAi molecules was completely unexpected.
The data shown in FIGS. 26, 27 and 43 demonstrated the importance of the various modifications to the RNAi in achieving stabilization and activity. For instance, FIG. 26 demonstrates that use off asymmetric configuration is important in getting efficacy in passive uptake. When the same chemical composition is applied to compounds of traditional configurations (19-21 bases duplex and 25 mer duplex) the efficacy was drastically decreased in a length dependent manner. FIG. 27 demonstrated a systematic screen of the impact of phosphorothioate chemical modifications on activity. The sequence, structure, stabilization chemical modifications, hydrophobic conjugate were kept constant and compound phosphorothioate content was varied (from 0 to 18 PS bond). Both compounds having no phosphorothioate linkages and having 18 phosphorothioate linkages were completely inactive in passive uptake. Compounds having 2-16 phosphorothioate linkages were active, with compounds having 4-10 phosphorothioate being the most active compounds.
The data in the Examples presented below demonstrates high efficacy of the oligonucleotides of the invention both in vitro in variety of cell types (supporting data) and in vivo upon local and systemic administration. For instance, the data compares the ability of several competitive RNAi molecules having different chemistries to silence a gene. Comparison of sd-rxRNA (oligonucleotides of the invention) with RNAs described in Soucheck et al. and Wolfrum at al., as applied to the same targeting region, demonstrated that only sd-rxRNA chemistry showed a significant functionality in passive uptake. The composition of the invention achieved EC50 values of 10-50 pM. This level of efficacy is un-attainable with conventional chemistries like those described in Sauthceck at and Accell. Similar comparisons were made in other systems, such as in vitro (RPE cell line), in vivo upon local administration (wounded skin) and systemic (50 mg/kg) as well as other genes (FIGS. 65 and 68). In each case the oligonucleotides of the invention achieved better results. FIG. 64 includes data demonstrating efficient cellular uptake and resulting silencing by sd-rxRNA compounds only after 1 minute of exposure. Such an efficacy is unique to this composition and have not been seen with other types of molecules in this class. FIG. 70 demonstrates efficient uptake and silencing of sd-rxRNA compounds in multiple cell types with multiple sequences. The sd-rxRNA compounds are also active in cells in presence and absence of serum and other biological liquids. FIG. 71 demonstrates only a slight reduction in activity in the presence of serum. This ability to function in biologically aggressive environment effectively further differentiates sd-rxRNA compounds from other compounds described previously in this group, like Accell and Soucheck et al, in which uptake is drastically inhibited in a presence of serum.
Significant amounts of data also demonstrate the in vivo efficacy of the compounds of the invention. For instance FIGS. 72-74 involve multiple routes of in vivo delivery of the compounds of the invention resulting in significant activity. FIG. 72, for example, demonstrates efficient tissue penetration and cellular uptake upon single intradermal injection. This is a model for local delivery of sd-rxRNA compounds as well as an effective delivery mode for sd-rxRNA compounds and silencing genes in any dermatology applications. FIG. 73 demonstrated efficient tissue penetration, cellular uptake and silencing upon local in vivo intradermal injection of sd-rxRNA compounds. The data of FIG. 74 demonstrate that sd-rxRNA compounds result in highly effective liver uptake upon IV administration. Comparison to Souicheck at al molecule showed that the level of liver uptake at identical dose level was quite surprisingly, at least 50 fold higher with the sd-rxRNA compound than the Souicheck at al molecule.
The sd-rxRNA can be further improved in some instances by improving the hydrophobicity of compounds using of novel types of chemistries. For example one chemistry is related to use of hydrophobic base modifications. Any base in any position might be modified, as long as modification results in an increase of the partition coefficient of the base. The preferred locations for modification chemistries are positions 4 and 5 of the pyrimidines. The major advantage of these positions is (a) ease of synthesis and (b) lack of interference with base-pairing and A form helix formation, which are essential for RISC complex loading and target recognition. Examples of these chemistries is shown in FIGS. 75-83. A version of sd-rxRNA compounds where multiple deoxy Uridines are present without interfering with overall compound efficacy was used. In addition major improvement in tissue distribution and cellular uptake might be obtained by optimizing the structure of the hydrophobic conjugate. In some of the preferred embodiment the structure of sterol is modified to alter (increase/decrease) C17 attached chain. This type of modification results in significant increase in cellular uptake and improvement of tissue uptake prosperities in vivo.
This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
Thus, aspects of the invention relate to isolated double stranded nucleic acid molecules comprising a guide (antisense) strand and a passenger (sense) strand. As used herein, the term “double-stranded” refers to one or more nucleic acid molecules in which at least a portion of the nucleomonomers are complementary and hydrogen bond to form a double-stranded region. In some embodiments, the length of the guide strand ranges from 16-29 nucleotides long. In certain embodiments, the guide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides long. The guide strand has complementarity to a target gene. Complementarity between the guide strand and the target gene may exist over any portion of the guide strand. Complementarity as used herein may be perfect complementarity or less than perfect complementarity as long as the guide strand is sufficiently complementary to the target that it mediates RNAi. In some embodiments complementarity refers to less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% mismatch between the guide strand and the target. Perfect complementarity refers to 100% complementarity. Thus the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Moreover, not all positions of a siRNA contribute equally to target recognition. Mismatches in the center of the siRNA are most critical and essentially abolish target RNA cleavage. Mismatches upstream of the center or upstream of the cleavage site referencing the antisense strand are tolerated but significantly reduce target RNA cleavage. Mismatches downstream of the center or cleavage site referencing the antisense strand, preferably located near the 3′ end of the antisense strand, e.g. 1, 2, 3, 4, 5 or 6 nucleotides from the 3′ end of the antisense strand, are tolerated and reduce target RNA cleavage only slightly.
While not wishing to be bound by any particular theory, in some embodiments, the guide strand is at least 16 nucleotides in length and anchors the Argonaute protein in RISC. In some embodiments, when the guide strand loads into RISC it has a defined seed region and target mRNA cleavage takes place across from position 10-11 of the guide strand. In some embodiments, the 5′ end of the guide strand is or is able to be phosphorylated. The nucleic acid molecules described herein may be referred to as minimum trigger RNA.
In some embodiments, the length of the passenger strand ranges from 8-14 nucleotides long. In certain embodiments, the passenger strand is 8, 9, 10, 11, 12, 13 or 14 nucleotides long. The passenger strand has complementarity to the guide strand. Complementarity between the passenger strand and the guide strand can exist over any portion of the passenger or guide strand. In some embodiments, there is 100% complementarity between the guide and passenger strands within the double stranded region of the molecule.
Aspects of the invention relate to double stranded nucleic acid molecules with minimal double stranded regions. In some embodiments the region of the molecule that is double stranded ranges from 8-14 nucleotides long. In certain embodiments, the region of the molecule that is double stranded is 8, 9, 10, 11, 12, 13 or 14 nucleotides long. In certain embodiments the double stranded region is 13 nucleotides long. There can be 100% complementarity between the guide and passenger strands, or there may be one or more mismatches between the guide and passenger strands. In some embodiments, on one end of the double stranded molecule, the molecule is either blunt-ended or has a one-nucleotide overhang. The single stranded region of the molecule is in some embodiments between 4-12 nucleotides long. For example the single stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides long. However, in certain embodiments, the single stranded region can also be less than 4 or greater than 12 nucleotides long. In certain embodiments, the single stranded region is 6 nucleotides long.
RNAi constructs associated with the invention can have a thermodynamic stability (ΔG) of less than −13 kkal/mol. In some embodiments, the thermodynamic stability (ΔG) is less than −20 kkal/mol. In some embodiments there is a loss of efficacy when (ΔG) goes below −21 kkal/mol. In some embodiments a (ΔG) value higher than −13 kkal/mol is compatible with aspects of the invention. Without wishing to be bound by any theory, in some embodiments a molecule with a relatively higher (ΔG) value may become active at a relatively higher concentration, while a molecule with a relatively lower (ΔG) value may become active at a relatively lower concentration. In some embodiments, the (ΔG) value may be higher than −9 kkcal/mol. The gene silencing effects mediated by the RNAi constructs associated with the invention, containing minimal double stranded regions, are unexpected because molecules of almost identical design but lower thermodynamic stability have been demonstrated to be inactive (Rana et al 2004).
Without wishing to be bound by any theory, results described herein suggest that a stretch of 8-10 bp of dsRNA or dsDNA will be structurally recognized by protein components of RISC or co-factors of RISC. Additionally, there is a free energy requirement for the triggering compound that it may be either sensed by the protein components and/or stable enough to interact with such components so that it may be loaded into the Argonaute protein. If optimal thermodynamics are present and there is a double stranded portion that is preferably at least 8 nucleotides then the duplex will be recognized and loaded into the RNAi machinery.
In some embodiments, thermodynamic stability is increased through the use of LNA bases. In some embodiments, additional chemical modifications are introduced. Several non-limiting examples of chemical modifications include: 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, ribothymidine, C-5 propynyl-dC (pdC) and C-5 propynyl-dU (pdU); C-5 propynyl-C (pC) and C-5 propynyl-U (pU); 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine), 5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine and MGB (minor groove binder). It should be appreciated that more than one chemical modification can be combined within the same molecule.
Molecules associated with the invention are optimized for increased potency and/or reduced toxicity. For example, nucleotide length of the guide and/or passenger strand, and/or the number of phosphorothioate modifications in the guide and/or passenger strand, can in some aspects influence potency of the RNA molecule, while replacing 2′-fluoro (2′F) modifications with 2′-O-methyl (2′OMe) modifications can in some aspects influence toxicity of the molecule. Specifically, reduction in 2′F content of a molecule is predicted to reduce toxicity of the molecule. The Examples section presents molecules in which 2′F modifications have been eliminated, offering an advantage over previously described RNAi compounds due to a predicted reduction in toxicity. Furthermore, the number of phosphorothioate modifications in an RNA molecule can influence the uptake of the molecule into a cell, for example the efficiency of passive uptake of the molecule into a cell. Preferred embodiments of molecules described herein have no 2′F modification and yet are characterized by equal efficacy in cellular uptake and tissue penetration. Such molecules represent a significant improvement over prior art, such as molecules described by Accell and Wolfrum, which are heavily modified with extensive use of 2′F.
In some embodiments, a guide strand is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate modifications. For example, a guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more than 14 nucleotides that are phosphate-modified. The guide strand may contain one or more modifications that confer increased stability without interfering with RISC entry. The phosphate modified nucleotides, such as phosphorothioate modified nucleotides, can be at the 3′ end, 5′ end or spread throughout the guide strand. In some embodiments, the 3′ terminal 10 nucleotides of the guide strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides. The guide strand can also contain 2′F and/or 2′OMe modifications, which can be located throughout the molecule. In some embodiments, the nucleotide in position one of the guide strand (the nucleotide in the most 5′ position of the guide strand) is 2′OMe modified and/or phosphorylated. C and U nucleotides within the guide strand can be 2′F modified. For example, C and U nucleotides in positions 2-10 of a 19 nt guide strand (or corresponding positions in a guide strand of a different length) can be 2′F modified. C and U nucleotides within the guide strand can also be 2′OMe modified. For example, C and U nucleotides in positions 11-18 of a 19 nt guide strand (or corresponding positions in a guide strand of a different length) can be 2′OMe modified. In some embodiments, the nucleotide at the most 3′ end of the guide strand is unmodified. In certain embodiments, the majority of Cs and Us within the guide strand are 2′F modified and the 5′ end of the guide strand is phosphorylated. In other embodiments, position 1 and the Cs or Us in positions 11-18 are 2′OMe modified and the 5′ end of the guide strand is phosphorylated. In other embodiments, position 1 and the Cs or Us in positions 11-18 are 2′OMe modified, the 5′ end of the guide strand is phosphorylated, and the Cs or Us in position 2-10 are 2′F modified.
In some aspects, an optimal passenger strand is approximately 11-14 nucleotides in length. The passenger strand may contain modifications that confer increased stability. One or more nucleotides in the passenger strand can be 2′OMe modified. In some embodiments, one or more of the C and/or U nucleotides in the passenger strand is 2′OMe modified, or all of the C and U nucleotides in the passenger strand are 2′OMe modified. In certain embodiments, all of the nucleotides in the passenger strand are 2′OMe modified. One or more of the nucleotides on the passenger strand can also be phosphate-modified such as phosphorothioate modified. The passenger strand can also contain 2′ ribo, 2′F and 2 deoxy modifications or any combination of the above. As demonstrated in the Examples, chemical modification patterns on both the guide and passenger strand are well tolerated and a combination of chemical modifications is shown herein to lead to increased efficacy and self-delivery of RNA molecules.
Aspects of the invention relate to RNAi constructs that have extended single-stranded regions relative to double stranded regions, as compared to molecules that have been used previously for RNAi. The single stranded region of the molecules may be modified to promote cellular uptake or gene silencing. In some embodiments, phosphorothioate modification of the single stranded region influences cellular uptake and/or gene silencing. The region of the guide strand that is phosphorothioate modified can include nucleotides within both the single stranded and double stranded regions of the molecule. In some embodiments, the single stranded region includes 2-12 phosphorothioate modifications. For example, the single stranded region can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphorothioate modifications. In some instances, the single stranded region contains 6-8 phosphorothioate modifications.
Molecules associated with the invention are also optimized for cellular uptake. In RNA molecules described herein, the guide and/or passenger strands can be attached to a conjugate. In certain embodiments the conjugate is hydrophobic. The hydrophobic conjugate can be a small molecule with a partition coefficient that is higher than 10. The conjugate can be a sterol-type molecule such as cholesterol, or a molecule with an increased length polycarbon chain attached to C17, and the presence of a conjugate can influence the ability of an RNA molecule to be taken into a cell with or without a lipid transfection reagent. The conjugate can be attached to the passenger or guide strand through a hydrophobic linker. In some embodiments, a hydrophobic linker is 5-12C in length, and/or is hydroxypyrrolidine-based. In some embodiments, a hydrophobic conjugate is attached to the passenger strand and the CU residues of either the passenger and/or guide strand are modified. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the CU residues on the passenger strand and/or the guide strand are modified. In some aspects, molecules associated with the invention are self-delivering (sd). As used herein, “self-delivery” refers to the ability of a molecule to be delivered into a cell without the need for an additional delivery vehicle such as a transfection reagent.
Aspects of the invention relate to selecting molecules for use in RNAi. Based on the data described herein, molecules that have a double stranded region of 8-14 nucleotides can be selected for use in RNAi. In some embodiments, molecules are selected based on their thermodynamic stability (ΔG). In some embodiments, molecules will be selected that have a (ΔG) of less than −13 kkal/mol. For example, the (ΔG) value may be −13, −14, −15, −16, −17, −18, −19, −21, −22 or less than −22 kkal/mol. In other embodiments, the (ΔG) value may be higher than −13 kkal/mol. For example, the (ΔG) value may be −12, −11, −10, −9, −8, −7 or more than −7 kkal/mol. It should be appreciated that ΔG can be calculated using any method known in the art. In some embodiments ΔG is calculated using Mfold, available through the Mfold internet site (http://mfold.bioinfospi.edu/cgi-bin/rna-form1.cgi). Methods for calculating ΔG are described in, and are incorporated by reference from, the following references: Zuker, M. (2003) Nucleic Acids Res., 31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M. and Turner, D. H. (1999) J. Mol. Biol. 288:911-940; Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl. Acad. Sci. 101:7287-7292; Duan, S., Mathews, D. H., and Turner, D. H. (2006) Biochemistry 45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I. L., and Schuster, P. (1999) Biopolymers 49:145-165.
Aspects of the invention relate to using nucleic acid molecules described herein, with minimal double stranded regions and/or with a (ΔG) of less than −13 kkal/mol, for gene silencing. RNAi molecules can be administered in vivo or in vitro, and gene silencing effects can be achieved in vivo or in vitro.
In certain embodiments, the polynucleotide contains 5′- and/or 3′-end overhangs. The number and/or sequence of nucleotides overhang on one end of the polynucleotide may be the same or different from the other end of the polynucleotide. In certain embodiments, one or more of the overhang nucleotides may contain chemical modification(s), such as phosphorothioate or 2′-OMe modification.
In certain embodiments, the polynucleotide is unmodified. In other embodiments, at least one nucleotide is modified. In further embodiments, the modification includes a 2′-H or 2′-modified ribose sugar at the 2nd nucleotide from the 5′-end of the guide sequence. The “2nd nucleotide” is defined as the second nucleotide from the 5′-end of the polynucleotide.
As used herein, “2′-modified ribose sugar” includes those ribose sugars that do not have a 2′-OH group. “2′-modified ribose sugar” does not include 2′-deoxyribose (found in unmodified canonical DNA nucleotides). For example, the 2′-modified ribose sugar may be 2′-O-alkyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, or combination thereof.
In certain embodiments, the 2′-modified nucleotides are pyrimidine nucleotides (e.g., C/U). Examples of 2′-O-alkyl nucleotides include 2′-O-methyl nucleotides, or 2′-O-allyl nucleotides.
In certain embodiments, the miniRNA polynucleotide of the invention with the above-referenced 5′-end modification exhibits significantly (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) less “off-target” gene silencing when compared to similar constructs without the specified 5′-end modification, thus greatly improving the overall specificity of the RNAi reagent or therapeutics.
As used herein, “off-target” gene silencing refers to unintended gene silencing due to, for example, spurious sequence homology between the antisense (guide) sequence and the unintended target mRNA sequence.
According to this aspect of the invention, certain guide strand modifications further increase nuclease stability, and/or lower interferon induction, without significantly decreasing RNAi activity (or no decrease in RNAi activity at all).
In some embodiments, the 5′-stem sequence may comprise a 2′-modified ribose sugar, such as 2′-O-methyl modified nucleotide, at the 2nd nucleotide on the 5′-end of the polynucleotide and, in some embodiments, no other modified nucleotides. The hairpin structure having such modification may have enhanced target specificity or reduced off-target silencing compared to a similar construct without the 2′-O-methyl modification at said position.
Certain combinations of specific 5′-stem sequence and 3′-stem sequence modifications may result in further unexpected advantages, as partly manifested by enhanced ability to inhibit target gene expression, enhanced serum stability, and/or increased target specificity, etc.
In certain embodiments, the guide strand comprises a 2′-O-methyl modified nucleotide at the 2nd nucleotide on the 5′-end of the guide strand and no other modified nucleotides.
In other aspects, the miniRNA structures of the present invention mediates sequence-dependent gene silencing by a microRNA mechanism. As used herein, the term “microRNA” (“miRNA”), also referred to in the art as “small temporal RNAs” (“stRNAs”), refers to a small (10-50 nucleotide) RNA which are genetically encoded (e.g., by viral, mammalian, or plant genomes) and are capable of directing or mediating RNA silencing. An “miRNA disorder” shall refer to a disease or disorder characterized by an aberrant expression or activity of an miRNA.
microRNAs are involved in down-regulating target genes in critical pathways, such as development and cancer, in mice, worms and mammals. Gene silencing through a microRNA mechanism is achieved by specific yet imperfect base-pairing of the miRNA and its target messenger RNA (mRNA). Various mechanisms may be used in microRNA-mediated down-regulation of target mRNA expression.
miRNAs are noncoding RNAs of approximately 22 nucleotides which can regulate gene expression at the post transcriptional or translational level during plant and animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNase III-type enzyme, or a homolog thereof. Naturally-occurring miRNAs are expressed by endogenous genes in vivo and are processed from a hairpin or stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or other RNAses. miRNAs can exist transiently in vivo as a double-stranded duplex but only one strand is taken up by the RISC complex to direct gene silencing.
In some embodiments a version of sd-rxRNA compounds, which are effective in cellular uptake and inhibiting of miRNA activity are described. Essentially the compounds are similar to RISC entering version but large strand chemical modification patterns are optimized in the way to block cleavage and act as an effective inhibitor of the RISC action. For example, the compound might be completely or mostly Omethyl modified with the PS content described previously. For these types of compounds the 5′ phosphorilation is not necessary. The presence of double stranded region is preferred as it is promotes cellular uptake and efficient RISC loading.
Another pathway that uses small RNAs as sequence-specific regulators is the RNA interference (RNAi) pathway, which is an evolutionarily conserved response to the presence of double-stranded RNA (dsRNA) in the cell. The dsRNAs are cleaved into  ̃20-base pair (bp) duplexes of small-interfering RNAs (siRNAs) by Dicer. These small RNAs get assembled into multiprotein effector complexes called RNA-induced silencing complexes (RISCs). The siRNAs then guide the cleavage of target mRNAs with perfect complementarity.
Some aspects of biogenesis, protein complexes, and function are shared between the siRNA pathway and the miRNA pathway. The subject single-stranded polynucleotides may mimic the dsRNA in the siRNA mechanism, or the microRNA in the miRNA mechanism.
In certain embodiments, the modified RNAi constructs may have improved stability in serum and/or cerebral spinal fluid compared to an unmodified RNAi constructs having the same sequence.
In certain embodiments, the structure of the RNAi construct does not induce interferon response in primary cells, such as mammalian primary cells, including primary cells from human, mouse and other rodents, and other non-human mammals. In certain embodiments, the RNAi construct may also be used to inhibit expression of a target gene in an invertebrate organism.
To further increase the stability of the subject constructs in vivo, the 3′-end of the hairpin structure may be blocked by protective group(s). For example, protective groups such as inverted nucleotides, inverted abasic moieties, or amino-end modified nucleotides may be used. Inverted nucleotides may comprise an inverted deoxynucleotide. Inverted abasic moieties may comprise an inverted deoxyabasic moiety, such as a 3′,3′-linked or 5′,5′-linked deoxyabasic moiety.
The RNAi constructs of the invention are capable of inhibiting the synthesis of any target protein encoded by target gene(s). The invention includes methods to inhibit expression of a target gene either in a cell in vitro, or in vivo. As such, the RNAi constructs of the invention are useful for treating a patient with a disease characterized by the overexpression of a target gene.
The target gene can be endogenous or exogenous (e.g., introduced into a cell by a virus or using recombinant DNA technology) to a cell. Such methods may include introduction of RNA into a cell in an amount sufficient to inhibit expression of the target gene. By way of example, such an RNA molecule may have a guide strand that is complementary to the nucleotide sequence of the target gene, such that the composition inhibits expression of the target gene.
The invention also relates to vectors expressing the nucleic acids of the invention, and cells comprising such vectors or the nucleic acids. The cell may be a mammalian cell in vivo or in culture, such as a human cell.
The invention further relates to compositions comprising the subject RNAi constructs, and a pharmaceutically acceptable carrier or diluent.
Another aspect of the invention provides a method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with any of the subject RNAi constructs.
The method may be carried out in vitro, ex vivo, or in vivo, in, for example, mammalian cells in culture, such as a human cell in culture.
The target cells (e.g., mammalian cell) may be contacted in the presence of a delivery reagent, such as a lipid (e.g., a cationic lipid) or a liposome.
Another aspect of the invention provides a method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with a vector expressing the subject RNAi constructs.
In one aspect of the invention, a longer duplex polynucleotide is provided, including a first polynucleotide that ranges in size from about 16 to about 30 nucleotides; a second polynucleotide that ranges in size from about 26 to about 46 nucleotides, wherein the first polynucleotide (the antisense strand) is complementary to both the second polynucleotide (the sense strand) and a target gene, and wherein both polynucleotides form a duplex and wherein the first polynucleotide contains a single stranded region longer than 6 bases in length and is modified with alternative chemical modification pattern, and/or includes a conjugate moiety that facilitates cellular delivery. In this embodiment, between about 40% to about 90% of the nucleotides of the passenger strand between about 40% to about 90% of the nucleotides of the guide strand, and between about 40% to about 90% of the nucleotides of the single stranded region of the first polynucleotide are chemically modified nucleotides.
In an embodiment, the chemically modified nucleotide in the polynucleotide duplex may be any chemically modified nucleotide known in the art, such as those discussed in detail above. In a particular embodiment, the chemically modified nucleotide is selected from the group consisting of 2′ F modified nucleotides, 2′-O-methyl modified and 2′ deoxy nucleotides. In another particular embodiment, the chemically modified nucleotides results from “hydrophobic modifications” of the nucleotide base. In another particular embodiment, the chemically modified nucleotides are phosphorothioates. In an additional particular embodiment, chemically modified nucleotides are combination of phosphorothioates, 2′-O-methyl, 2′ deoxy, hydrophobic modifications and phosphorothioates. As these groups of modifications refer to modification of the ribose ring, back bone and nucleotide, it is feasible that some modified nucleotides will carry a combination of all three modification types.
In another embodiment, the chemical modification is not the same across the various regions of the duplex. In a particular embodiment, the first polynucleotide (the passenger strand), has a large number of diverse chemical modifications in various positions. For this polynucleotide up to 90% of nucleotides might be chemically modified and/or have mismatches introduced.
In another embodiment, chemical modifications of the first or second polynucleotide include, but not limited to, 5′ position modification of Uridine and Cytosine (4-pyridyl, 2-pyridyl, indolyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl; naphthyl, etc), where the chemical modification might alter base pairing capabilities of a nucleotide. For the guide strand an important feature of this aspect of the invention is the position of the chemical modification relative to the 5′ end of the antisense and sequence. For example, chemical phosphorylation of the 5′ end of the guide strand is usually beneficial for efficacy. O-methyl modifications in the seed region of the sense strand (position 2-7 relative to the 5′ end) are not generally well tolerated, whereas 2′F and deoxy are well tolerated. The mid part of the guide strand and the 3′ end of the guide strand are more permissive in a type of chemical modifications applied. Deoxy modifications are not tolerated at the 3′ end of the guide strand.
A unique feature of this aspect of the invention involves the use of hydrophobic modification on the bases. In one embodiment, the hydrophobic modifications are preferably positioned near the 5′ end of the guide strand, in other embodiments, they localized in the middle of the guides strand, in other embodiment they localized at the 3′ end of the guide strand and yet in another embodiment they are distributed thought the whole length of the polynucleotide. The same type of patterns is applicable to the passenger strand of the duplex.
The other part of the molecule is a single stranded region. The single stranded region is expected to range from 7 to 40 nucleotides.
In one embodiment, the single stranded region of the first polynucleotide contains modifications selected from the group consisting of between 40% and 90% hydrophobic base modifications, between 40%-90% phosphorothioates, between 40%-90% modification of the ribose moiety, and any combination of the preceding.
Efficiency of guide strand (first polynucleotide) loading into the RISC complex might be altered for heavily modified polynucleotides, so in one embodiment, the duplex polynucleotide includes a mismatch between nucleotide 9, 11, 12, 13, or 14 on the guide strand (first polynucleotide) and the opposite nucleotide on the sense strand (second polynucleotide) to promote efficient guide strand loading.
More detailed aspects of the invention are described in the sections below.
Duplex Characteristics
Double-stranded oligonucleotides of the invention may be formed by two separate complementary nucleic acid strands. Duplex formation can occur either inside or outside the cell containing the target gene.
As used herein, the term “duplex” includes the region of the double-stranded nucleic acid molecule(s) that is (are) hydrogen bonded to a complementary sequence. Double-stranded oligonucleotides of the invention may comprise a nucleotide sequence that is sense to a target gene and a complementary sequence that is antisense to the target gene. The sense and antisense nucleotide sequences correspond to the target gene sequence, e.g., are identical or are sufficiently identical to effect target gene inhibition (e.g., are about at least about 98% identical, 96% identical, 94%, 90% identical, 85% identical, or 80% identical) to the target gene sequence.
In certain embodiments, the double-stranded oligonucleotide of the invention is double-stranded over its entire length, i.e., with no overhanging single-stranded sequence at either end of the molecule, i.e., is blunt-ended. In other embodiments, the individual nucleic acid molecules can be of different lengths. In other words, a double-stranded oligonucleotide of the invention is not double-stranded over its entire length. For instance, when two separate nucleic acid molecules are used, one of the molecules, e.g., the first molecule comprising an antisense sequence, can be longer than the second molecule hybridizing thereto (leaving a portion of the molecule single-stranded). Likewise, when a single nucleic acid molecule is used a portion of the molecule at either end can remain single-stranded.
In one embodiment, a double-stranded oligonucleotide of the invention contains mismatches and/or loops or bulges, but is double-stranded over at least about 70% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 80% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 90%-95% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 96%-98% of the length of the oligonucleotide. In certain embodiments, the double-stranded oligonucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.
Modifications
The nucleotides of the invention may be modified at various locations, including the sugar moiety, the phosphodiester linkage, and/or the base.
Sugar moieties include natural, unmodified sugars, e.g., monosaccharide (such as pentose, e.g., ribose, deoxyribose), modified sugars and sugar analogs. In general, possible modifications of nucleomonomers, particularly of a sugar moiety, include, for example, replacement of one or more of the hydroxyl groups with a halogen, a heteroatom, an aliphatic group, or the functionalization of the hydroxyl group as an ether, an amine, a thiol, or the like.
One particularly useful group of modified nucleomonomers are 2′-O-methyl nucleotides. Such 2′-O-methyl nucleotides may be referred to as “methylated,” and the corresponding nucleotides may be made from unmethylated nucleotides followed by alkylation or directly from methylated nucleotide reagents. Modified nucleomonomers may be used in combination with unmodified nucleomonomers. For example, an oligonucleotide of the invention may contain both methylated and unmethylated nucleomonomers.
Some exemplary modified nucleomonomers include sugar- or backbone-modified ribonucleotides. Modified ribonucleotides may contain a non-naturally occurring base (instead of a naturally occurring base), such as uridines or cytidines modified at the 5′-position, e.g., 5′-(2-amino)propyl uridine and 5′-bromo uridine; adenosines and guanosines modified at the 8-position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; and N-alkylated nucleotides, e.g., N6-methyl adenosine. Also, sugar-modified ribonucleotides may have the 2′-OH group replaced by a H, alxoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as NH2, NHR, NR2,), or CN group, wherein R is lower alkyl, alkenyl, or alkynyl.
Modified ribonucleotides may also have the phosphodiester group connecting to adjacent ribonucleotides replaced by a modified group, e.g., of phosphorothioate group. More generally, the various nucleotide modifications may be combined.
Although the antisense (guide) strand may be substantially identical to at least a portion of the target gene (or genes), at least with respect to the base pairing properties, the sequence need not be perfectly identical to be useful, e.g., to inhibit expression of a target gene's phenotype. Generally, higher homology can be used to compensate for the use of a shorter antisense gene. In some cases, the antisense strand generally will be substantially identical (although in antisense orientation) to the target gene.
The use of 2′-O-methyl modified RNA may also be beneficial in circumstances in which it is desirable to minimize cellular stress responses. RNA having 2′-O-methyl nucleomonomers may not be recognized by cellular machinery that is thought to recognize unmodified RNA. The use of 2′-O-methylated or partially 2′-O-methylated RNA may avoid the interferon response to double-stranded nucleic acids, while maintaining target RNA inhibition. This may be useful, for example, for avoiding the interferon or other cellular stress responses, both in short RNAi (e.g., siRNA) sequences that induce the interferon response, and in longer RNAi sequences that may induce the interferon response.
Overall, modified sugars may include D-ribose, 2′-O-alkyl (including 2′-β-methyl and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino, 2′-S-alkyl, 2′-halo (including 2′-fluoro), 2′-methoxyethoxy, 2′-allyloxy (—OCH2CH═CH2), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, and cyano and the like. In one embodiment, the sugar moiety can be a hexose and incorporated into an oligonucleotide as described (Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992)). Exemplary nucleomonomers can be found, e.g., in U.S. Pat. No. 5,849,902, incorporated by reference herein.
The term “alkyl” includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and more preferably 4 or fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C1-C6 includes alkyl groups containing 1 to 6 carbon atoms.
Moreover, unless otherwise specified, the term alkyl includes both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyls can be further substituted, e.g., with the substituents described above. An “alkylaryl” or an “arylalkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term “alkyl” also includes the side chains of natural and unnatural amino acids. The term “n-alkyl” means a straight chain (i.e., unbranched) unsubstituted alkyl group.
The term “alkenyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term “alkenyl” includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. In certain embodiments, a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C2-C6 includes alkenyl groups containing 2 to 6 carbon atoms.
Moreover, unless otherwise specified, the term alkenyl includes both “unsubstituted alkenyls” and “substituted alkenyls,” the latter of which refers to alkenyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term “alkynyl” includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, the term “alkynyl” includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term C2-C6 includes alkynyl groups containing 2 to 6 carbon atoms.
Moreover, unless otherwise specified, the term alkynyl includes both “unsubstituted alkynyls” and “substituted alkynyls,” the latter of which refers to alkynyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to five carbon atoms in its backbone structure. “Lower alkenyl” and “lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.
The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with independently selected groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulffiydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.
The term “heteroatom” includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O− (with an appropriate counterion).
The term “halogen” includes fluorine, bromine, chlorine, iodine, etc. The term “perhalogenated” generally refers to a moiety wherein all hydrogens are replaced by halogen atoms.
The term “substituted” includes independently selected substituents which can be placed on the moiety and which allow the molecule to perform its intended function. Examples of substituents include alkyl, alkenyl, alkynyl, aryl, (CR′R′′)0-3NR′R′′, (CR′R′′)0-3CN, NO2, halogen, (CR′R′′)0-3C(halogen)3, (CR′R′′)0-3CH(halogen)2, (CR′R′′)0-3CH2(halogen), (CR′R′′)0-3CONR′R′′, (CR′R′′)0-3S(O)1-2NR′R′′, (CR′R′′)0-3CHO, (CR′R′′)0-3O(CR′R′′)0-3H, (CR′R′′)0-3S(O)0-2R′, (CR′R′′)0-3O(CR′R′′)0-3H, (CR′R′′)0-3COR′, (CR′R′′)0-3CO2R′, or (CR′R′′)0-30R′ groups; wherein each R′ and R′′ are each independently hydrogen, a C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, or aryl group, or R′ and R′′ taken together are a benzylidene group or a —(CH2)2—O—(CH2)2— group.
The term “amine” or “amino” includes compounds or moieties in which a nitrogen atom is covalently bonded to at least one carbon or heteroatom. The term “alkyl amino” includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group. The term “dialkyl amino” includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups.
The term “ether” includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms. For example, the term includes “alkoxyalkyl,” which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.
The term “base” includes the known purine and pyrimidine heterocyclic bases, deazapurines, and analogs (including heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomers thereof. Examples of purines include adenine, guanine, inosine, diaminopurine, and xanthine and analogs (e.g., 8-oxo-N6-methyladenine or 7-diazaxanthine) and derivatives thereof. Pyrimidines include, for example, thymine, uracil, and cytosine, and their analogs (e.g., 5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and 4,4-ethanocytosine). Other examples of suitable bases include non-purinyl and non-pyrimidinyl bases such as 2-aminopyridine and triazines.
In a preferred embodiment, the nucleomonomers of an oligonucleotide of the invention are RNA nucleotides. In another preferred embodiment, the nucleomonomers of an oligonucleotide of the invention are modified RNA nucleotides. Thus, the oligonucleotides contain modified RNA nucleotides.
The term “nucleoside” includes bases which are covalently attached to a sugar moiety, preferably ribose or deoxyribose. Examples of preferred nucleosides include ribonucleosides and deoxyribonucleosides. Nucleosides also include bases linked to amino acids or amino acid analogs which may comprise free carboxyl groups, free amino groups, or protecting groups. Suitable protecting groups are well known in the art (see P. G. M. Wuts and T. W. Greene, “Protective Groups in Organic Synthesis”, 2nd Ed., Wiley-Interscience, New York, 1999).
The term “nucleotide” includes nucleosides which further comprise a phosphate group or a phosphate analog.
As used herein, the term “linkage” includes a naturally occurring, unmodified phosphodiester moiety (—O—(PO2−)—O—) that covalently couples adjacent nucleomonomers. As used herein, the term “substitute linkage” includes any analog or derivative of the native phosphodiester group that covalently couples adjacent nucleomonomers. Substitute linkages include phosphodiester analogs, e.g., phosphorothioate, phosphorodithioate, and P-ethyoxyphosphodiester, P-ethoxyphosphodiester, P-alkyloxyphosphotriester, methylphosphonate, and nonphosphorus containing linkages, e.g., acetals and amides. Such substitute linkages are known in the art (e.g., Bjergarde et al. 1991. Nucleic Acids Res. 19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47). In certain embodiments, non-hydrolizable linkages are preferred, such as phosphorothiate linkages.
In certain embodiments, oligonucleotides of the invention comprise hydrophobicly modified nucleotides or “hydrophobic modifications.” As used herein “hydrophobic modifications” refers to bases that are modified such that (1) overall hydrophobicity of the base is significantly increased, and/or (2) the base is still capable of forming close to regular Watson-Crick interaction. Several non-limiting examples of base modifications include 5-position uridine and cytidine modifications such as phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), Isobutyl, butyl, aminobenzyl; phenyl; and naphthyl.
In certain embodiments, oligonucleotides of the invention comprise 3′ and 5′ termini (except for circular oligonucleotides). In one embodiment, the 3′ and 5′ termini of an oligonucleotide can be substantially protected from nucleases e.g., by modifying the 3′ or 5′ linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For example, oligonucleotides can be made resistant by the inclusion of a “blocking group.” The term “blocking group” as used herein refers to substituents (e.g., other than OH groups) that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (e.g., FITC, propyl (CH2—CH2—CH3), glycol (—O—CH2—CH2—O—) phosphate (PO32−), hydrogen phosphonate, or phosphoramidite). “Blocking groups” also include “end blocking groups” or “exonuclease blocking groups” which protect the 5′ and 3′ termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.
Exemplary end-blocking groups include cap structures (e.g., a 7-methylguanosine cap), inverted nucleomonomers, e.g., with 3′-3′ or 5′-5′ end inversions (see, e.g., to Ortiagao et al. 1992. Antisense Res. Dev. 2:129), methylphosphonate, phosphoramidite, non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers, conjugates) and the like. The 3′ terminal nucleomonomer can comprise a modified sugar moiety. The 3′ terminal nucleomonomer comprises a 3′-O that can optionally be substituted by a blocking group that prevents 3′-exonuclease degradation of the oligonucleotide. For example, the 3′-hydroxyl can be esterified to a nucleotide through a 3′→3′ internucleotide linkage. For example, the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, and preferably, ethoxy. Optionally, the 3′→3′ linked nucleotide at the 3′ terminus can be linked by a substitute linkage. To reduce nuclease degradation, the 5′ most 3′→5′ linkage can be a modified linkage, e.g., a phosphorothioate or a P-alkyloxyphosphotriester linkage. Preferably, the two 5′ most 3′→5′ linkages are modified linkages. Optionally, the 5′ terminal hydroxy moiety can be esterified with a phosphorus containing moiety, e.g., phosphate, phosphorothioate, or P-ethoxyphosphate.
Another type of conjugates that can be attached to the end (3′ or 5′ end), the loop region, or any other parts of the miniRNA might include a sterol, sterol type molecule, peptide, small molecule, protein, etc. In some embodiments, a miniRNA may contain more than one conjugates (same or different chemical nature). In some embodiments, the conjugate is cholesterol.
Another way to increase target gene specificity, or to reduce off-target silencing effect, is to introduce a 2′-modification (such as the 2′-O methyl modification) at a position corresponding to the second 5′-end nucleotide of the guide sequence. This allows the positioning of this 2′-modification in the Dicer-resistant hairpin structure, thus enabling one to design better RNAi constructs with less or no off-target silencing.
In one embodiment, a hairpin polynucleotide of the invention can comprise one nucleic acid portion which is DNA and one nucleic acid portion which is RNA. Antisense (guide) sequences of the invention can be “chimeric oligonucleotides” which comprise an RNA-like and a DNA-like region.
The language “RNase H activating region” includes a region of an oligonucleotide, e.g., a chimeric oligonucleotide, that is capable of recruiting RNase H to cleave the target RNA strand to which the oligonucleotide binds. Typically, the RNase activating region contains a minimal core (of at least about 3-5, typically between about 3-12, more typically, between about 5-12, and more preferably between about 5-10 to contiguous nucleomonomers) of DNA or DNA-like nucleomonomers. (See, e.g., U.S. Pat. No. 5,849,902). Preferably, the RNase H activating region comprises about nine contiguous deoxyribose containing nucleomonomers.
The language “non-activating region” includes a region of an antisense sequence, e.g., a chimeric oligonucleotide, that does not recruit or activate RNase H. Preferably, a non-activating region does not comprise phosphorothioate DNA. The oligonucleotides of the invention comprise at least one non-activating region. In one embodiment, the non-activating region can be stabilized against nucleases or can provide specificity for the target by being complementary to the target and forming hydrogen bonds with the target nucleic acid molecule, which is to be bound by the oligonucleotide.
In one embodiment, at least a portion of the contiguous polynucleotides are linked by a substitute linkage, e.g., a phosphorothioate linkage.
In certain embodiments, most or all of the nucleotides beyond the guide sequence (2′-modified or not) are linked by phosphorothioate linkages. Such constructs tend to have improved pharmacokinetics due to their higher affinity for serum proteins. The phosphorothioate linkages in the non-guide sequence portion of the polynucleotide generally do not interfere with guide strand activity, once the latter is loaded into RISC.
Antisense (guide) sequences of the present invention may include “morpholino oligonucleotides.” Morpholino oligonucleotides are non-ionic and function by an RNase H-independent mechanism. Each of the 4 genetic bases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholino oligonucleotides is linked to a 6-membered morpholine ring. Morpholino oligonucleotides are made by joining the 4 different subunit types by, e.g., non-ionic phosphorodiamidate inter-subunit linkages. Morpholino oligonucleotides have many advantages including: complete resistance to nucleases (Antisense & Nucl. Acid Drug Dev. 1996. 6:267); predictable targeting (Biochemica Biophysica Acta. 1999. 1489:141); reliable activity in cells (Antisense & Nucl. Acid Drug Dev. 1997. 7:63); excellent sequence specificity (Antisense & Nucl. Acid Drug Dev. 1997. 7:151); minimal non-antisense activity (Biochemica Biophysica Acta. 1999. 1489:141); and simple osmotic or scrape delivery (Antisense & Nucl. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides are also preferred because of their non-toxicity at high doses. A discussion of the preparation of morpholino oligonucleotides can be found in Antisense & Nucl. Acid Drug Dev. 1997. 7:187.
The chemical modifications described herein are believed, based on the data described herein, to promote single stranded polynucleotide loading into the RISC. Single stranded polynucleotides have been shown to be active in loading into RISC and inducing gene silencing. However, the level of activity for single stranded polynucleotides appears to be 2 to 4 orders of magnitude lower when compared to a duplex polynucleotide.
The present invention provides a description of the chemical modification patterns, which may (a) significantly increase stability of the single stranded polynucleotide (b) promote efficient loading of the polynucleotide into the RISC complex and (c) improve uptake of the single stranded nucleotide by the cell. FIG. 5 provides some non-limiting examples of the chemical modification patterns which may be beneficial for achieving single stranded polynucleotide efficacy inside the cell. The chemical modification patterns may include combination of ribose, backbone, hydrophobic nucleoside and conjugate type of modifications. In addition, in some of the embodiments, the 5′ end of the single polynucleotide may be chemically phosphorylated.
In yet another embodiment, the present invention provides a description of the chemical modifications patterns, which improve functionality of RISC inhibiting polynucleotides. Single stranded polynucleotides have been shown to inhibit activity of a preloaded RISC complex through the substrate competition mechanism. For these types of molecules, conventionally called antagomers, the activity usually requires high concentration and in vivo delivery is not very effective. The present invention provides a description of the chemical modification patterns, which may (a) significantly increase stability of the single stranded polynucleotide (b) promote efficient recognition of the polynucleotide by the RISC as a substrate and/or (c) improve uptake of the single stranded nucleotide by the cell. FIG. 6 provides some non-limiting examples of the chemical modification patterns that may be beneficial for achieving single stranded polynucleotide efficacy inside the cell. The chemical modification patterns may include combination of ribose, backbone, hydrophobic nucleoside and conjugate type of modifications.
The modifications provided by the present invention are applicable to all polynucleotides. This includes single stranded RISC entering polynucleotides, single stranded RISC inhibiting polynucleotides, conventional duplexed polynucleotides of variable length (15-40 bp), asymmetric duplexed polynucleotides, and the like. Polynucleotides may be modified with wide variety of chemical modification patterns, including 5′ end, ribose, backbone and hydrophobic nucleoside modifications.
Synthesis
Oligonucleotides of the invention can be synthesized by any method known in the art, e.g., using enzymatic synthesis and/or chemical synthesis. The oligonucleotides can be synthesized in vitro (e.g., using enzymatic synthesis and chemical synthesis) or in vivo (using recombinant DNA technology well known in the art).
In a preferred embodiment, chemical synthesis is used for modified polynucleotides. Chemical synthesis of linear oligonucleotides is well known in the art and can be achieved by solution or solid phase techniques. Preferably, synthesis is by solid phase methods. Oligonucleotides can be made by any of several different synthetic procedures including the phosphoramidite, phosphite triester, H-phosphonate, and phosphotriester methods, typically by automated synthesis methods.
Oligonucleotide synthesis protocols are well known in the art and can be found, e.g., in U.S. Pat. No. 5,830,653; WO 98/13526; Stec et al. 1984. J. Am. Chem. Soc. 106:6077; Stec et al. 1985. J. Org. Chem. 50:3908; Stec et al. J. Chromatog. 1985. 326:263; LaPlanche et al. 1986. Nucl. Acid. Res. 1986. 14:9081; Fasman G. D., 1989. Practical Handbook of Biochemistry and Molecular Biology. 1989. CRC Press, Boca Raton, Fla.; Lamone. 1993. Biochem. Soc. Trans. 21:1; U.S. Pat. No. 5,013,830; U.S. Pat. No. 5,214,135; U.S. Pat. No. 5,525,719; Kawasaki et al. 1993. J. Med. Chem. 36:831; WO 92/03568; U.S. Pat. No. 5,276,019; and U.S. Pat. No. 5,264,423.
The synthesis method selected can depend on the length of the desired oligonucleotide and such choice is within the skill of the ordinary artisan. For example, the phosphoramidite and phosphite triester method can produce oligonucleotides having 175 or more nucleotides, while the H-phosphonate method works well for oligonucleotides of less than 100 nucleotides. If modified bases are incorporated into the oligonucleotide, and particularly if modified phosphodiester linkages are used, then the synthetic procedures are altered as needed according to known procedures. In this regard, Uhlmann et al. (1990, Chemical Reviews 90:543-584) provide references and outline procedures for making oligonucleotides with modified bases and modified phosphodiester linkages. Other exemplary methods for making oligonucleotides are taught in Sonveaux. 1994. “Protecting Groups in Oligonucleotide Synthesis”; Agrawal. Methods in Molecular Biology 26:1. Exemplary synthesis methods are also taught in “Oligonucleotide Synthesis—A Practical Approach” (Gait, M. J. IRL Press at Oxford University Press. 1984). Moreover, linear oligonucleotides of defined sequence, including some sequences with modified nucleotides, are readily available from several commercial sources.
The oligonucleotides may be purified by polyacrylamide gel electrophoresis, or by any of a number of chromatographic methods, including gel chromatography and high pressure liquid chromatography. To confirm a nucleotide sequence, especially unmodified nucleotide sequences, oligonucleotides may be subjected to DNA sequencing by any of the known procedures, including Maxam and Gilbert sequencing, Sanger sequencing, capillary electrophoresis sequencing, the wandering spot sequencing procedure or by using selective chemical degradation of oligonucleotides bound to Hybond paper. Sequences of short oligonucleotides can also be analyzed by laser desorption mass spectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am. Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass Spectrom. 14:83; Grotjahn et al., 1982, Nuc. Acid Res. 10:4671). Sequencing methods are also available for RNA oligonucleotides.
The quality of oligonucleotides synthesized can be verified by testing the oligonucleotide by capillary electrophoresis and denaturing strong anion HPLC (SAX-HPLC) using, e.g., the method of Bergot and Egan. 1992. J. Chrom. 599:35.
Other exemplary synthesis techniques are well known in the art (see, e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (DN Glover Ed. 1985); Oligonucleotide Synthesis (M J Gait Ed, 1984; Nucleic Acid Hybridisation (B D Hames and S J Higgins eds. 1984); A Practical Guide to Molecular Cloning (1984); or the series, Methods in Enzymology (Academic Press, Inc.)).
In certain embodiments, the subject RNAi constructs or at least portions thereof are transcribed from expression vectors encoding the subject constructs. Any art recognized vectors may be use for this purpose. The transcribed RNAi constructs may be isolated and purified, before desired modifications (such as replacing an unmodified sense strand with a modified one, etc.) are carried out.
Delivery/Carrier
Uptake of Oligonucleotides by Cells
Oligonucleotides and oligonucleotide compositions are contacted with (i.e., brought into contact with, also referred to herein as administered or delivered to) and taken up by one or more cells or a cell lysate. The term “cells” includes prokaryotic and eukaryotic cells, preferably vertebrate cells, and, more preferably, mammalian cells. In a preferred embodiment, the oligonucleotide compositions of the invention are contacted with human cells.
Oligonucleotide compositions of the invention can be contacted with cells in vitro, e.g., in a test tube or culture dish, (and may or may not be introduced into a subject) or in vivo, e.g., in a subject such as a mammalian subject. Oligonucleotides are taken up by cells at a slow rate by endocytosis, but endocytosed oligonucleotides are generally sequestered and not available, e.g., for hybridization to a target nucleic acid molecule. In one embodiment, cellular uptake can be facilitated by electroporation or calcium phosphate precipitation. However, these procedures are only useful for in vitro or ex vivo embodiments, are not convenient and, in some cases, are associated with cell toxicity.
In another embodiment, delivery of oligonucleotides into cells can be enhanced by suitable art recognized methods including calcium phosphate, DMSO, glycerol or dextran, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes using methods known in the art (see e.g., WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No. 4,897,355; Bergan et al. 1993. Nucleic Acids Research. 21:3567). Enhanced delivery of oligonucleotides can also be mediated by the use of vectors (See e.g., Shi, Y. 2003. Trends Genet 2003 Jan. 19:9; Reichhart J M et al. Genesis. 2002. 34(1-2):1604, Yu et al. 2002. Proc. Natl. Acad Sci. USA 99:6047; Sui et al. 2002. Proc. Natl. Acad Sci. USA 99:5515) viruses, polyamine or polycation conjugates using compounds such as polylysine, protamine, or Ni, N12-bis(ethyl) spermine (see, e.g., Bartzatt, R. et al. 1989. Biotechnol. Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl. Acad. Sci. 88:4255).
In certain embodiments, the miniRNA of the invention may be delivered by using various beta-glucan containing particles, such as those described in US 2005/0281781 A1, WO 2006/007372, and WO 2007/050643 (all incorporated herein by reference). In certain embodiments, the beta-glucan particle is derived from yeast. In to certain embodiments, the payload trapping molecule is a polymer, such as those with a molecular weight of at least about 1000 Da, 10,000 Da, 50,000 Da, 100 kDa, 500 kDa, etc. Preferred polymers include (without limitation) cationic polymers, chitosans, or PEI (polyethylenimine), etc.
Such beta-glucan based delivery system may be formulated for oral delivery, where the orally delivered beta-glucan/miniRNA constructs may be engulfed by macrophages or other related phagocytic cells, which may in turn release the miniRNA constructs in selected in vivo sites. Alternatively or in addition, the miniRNA may changes the expression of certain macrophage target genes.
The optimal protocol for uptake of oligonucleotides will depend upon a number of factors, the most crucial being the type of cells that are being used. Other factors that are important in uptake include, but are not limited to, the nature and concentration of the oligonucleotide, the confluence of the cells, the type of culture the cells are in (e.g., a suspension culture or plated) and the type of media in which the cells are grown.
Encapsulating Agents
Encapsulating agents entrap oligonucleotides within vesicles. In another embodiment of the invention, an oligonucleotide may be associated with a carrier or vehicle, e.g., liposomes or micelles, although other carriers could be used, as would be appreciated by one skilled in the art. Liposomes are vesicles made of a lipid bilayer having a structure similar to biological membranes. Such carriers are used to facilitate the cellular uptake or targeting of the oligonucleotide, or improve the oligonucleotide's pharmacokinetic or toxicologic properties.
For example, the oligonucleotides of the present invention may also be administered encapsulated in liposomes, pharmaceutical compositions wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The oligonucleotides, depending upon solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phopholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, or other materials of a hydrophobic nature. The diameters of the liposomes generally range from about 15 nm to about 5 microns.
The use of liposomes as drug delivery vehicles offers several advantages. Liposomes increase intracellular stability, increase uptake efficiency and improve biological activity. Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. They have an internal aqueous space for entrapping water soluble compounds and range in size from 0.05 to several microns in diameter. Several studies have shown that liposomes can deliver nucleic acids to cells and that the nucleic acids remain biologically active. For example, a lipid delivery vehicle originally designed as a research tool, such as Lipofectin or LIPOFECTAMINETM 2000, can deliver intact nucleic acid molecules to cells.
Specific advantages of using liposomes include the following: they are non-toxic and biodegradable in composition; they display long circulation half-lives; and recognition molecules can be readily attached to their surface for targeting to tissues. Finally, cost-effective manufacture of liposome-based pharmaceuticals, either in a liquid suspension or lyophilized product, has demonstrated the viability of this technology as an acceptable drug delivery system.
In some aspects, formulations associated with the invention might be selected for a class of naturally occurring or chemically synthesized or modified saturated and unsaturated fatty acid residues. Fatty acids might exist in a form of triglycerides, diglycerides or individual fatty acids. In another embodiment, the use of well-validated mixtures of fatty acids and/or fat emulsions currently used in pharmacology for parenteral nutrition may be utilized.
Liposome based formulations are widely used for oligonucleotide delivery. However, most of commercially available lipid or liposome formulations contain at least one positively charged lipid (cationic lipids). The presence of this positively charged lipid is believed to be essential for obtaining a high degree of oligonucleotide loading and for enhancing liposome fusogenic properties. Several methods have been performed and published to identify optimal positively charged lipid chemistries. However, the commercially available liposome formulations containing cationic lipids are characterized by a high level of toxicity. In vivo limited therapeutic indexes have revealed that liposome formulations containing positive charged lipids are associated with toxicity (i.e. elevation in liver enzymes) at concentrations only slightly higher than concentration required to achieve RNA silencing.
New liposome formulations, lacking the toxicity of the prior art liposomes have been developed according to the invention. These new liposome formulations are neutral fat-based formulations for the efficient delivery of oligonucleotides, and in particular for the delivery of the RNA molecules of the invention. The compositions are referred to as neutral nanotransporters because they enable quantitative oligonucleotide incorporation into non-charged lipids mixtures. The lack of toxic levels of cationic lipids in the neutral nanotransporter compositions of the invention is an important feature.
The neutral nanotransporters compositions enable efficient loading of oligonucleotide into neutral fat formulation. The composition includes an oligonucleotide that is modified in a manner such that the hydrophobicity of the molecule is increased (for example a hydrophobic molecule is attached (covalently or no-covalently) to a hydrophobic molecule on the oligonucleotide terminus or a non-terminal nucleotide, base, sugar, or backbone), the modified oligonucleotide being mixed with a neutral fat formulation (for example containing at least 25% of cholesterol and 25% of DOPC or analogs thereof). A cargo molecule, such as another lipid can also be included in the composition. This composition, where part of the formulation is build into the oligonucleotide itself, enables efficient encapsulation of oligonucleotide in neutral lipid particles.
One of several unexpected observations associated with the invention was that the oligonucleotides of the invention could effectively be incorporated in a lipid mixture that was free of cationic lipids and that such a composition could effectively deliver the therapeutic oligonucleotide to a cell in a manner that it is functional. Another unexpected observation was the high level of activity observed when the fatty mixture is composed of a phosphatidylcholine base fatty acid and a sterol such as a cholesterol. For instance, one preferred formulation of neutral fatty mixture is composed of at least 20% of DOPC or DSPC and at least 20% of sterol such as cholesterol. Even as low as 1:5 lipid to oligonucleotide ratio was shown to be sufficient to get complete encapsulation of the oligonucleotide in a non charged formulation. The prior art demonstrated only a 1-5% oligonucleotide encapsulation with non-charged formulations, which is not sufficient to get to a desired amount of in vivo efficacy. Compared to the prior art using neutral lipids the level of oligonucleotide delivery to a cell was quite unexpected.
Stable particles ranging in size from 50 to 140 nm were formed upon complexing of hydrophobic oligonucleotides with preferred formulations. It is interesting to mention that the formulation by itself typically does not form small particles, but rather, forms agglomerates, which are transformed into stable 50-120 nm particles upon addition of the hydrophobic modified oligonucleotide.
The neutral nanotransporter compositions of the invention include a hydrophobic modified polynucleotide, a neutral fatty mixture, and optionally a cargo molecule. A “hydrophobic modified polynucleotide” as used herein is a polynucleotide of the invention (i.e. sd-rxRNA) that has at least one modification that renders the polynucleotide more hydrophobic than the polynucleotide was prior to modification. The modification may be achieved by attaching (covalently or non-covalently) a hydrophobic molecule to the polynucleotide. In some instances the hydrophobic molecule is or includes a lipophilic group.
The term “lipophilic group” means a group that has a higher affinity for lipids than its affinity for water. Examples of lipophilic groups include, but are not limited to, cholesterol, a cholesteryl or modified cholesteryl residue, adamantine, dihydrotesterone, long chain alkyl, long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic, oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, such as steroids, vitamins, such as vitamin E, fatty acids either saturated or unsaturated, fatty acid esters, such as triglycerides, pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. The cholesterol moiety may be reduced (e.g. as in cholestan) or may be substituted (e.g. by halogen). A combination of different lipophilic groups in one molecule is also possible.
The hydrophobic molecule may be attached at various positions of the polynucleotide. As described above, the hydrophobic molecule may be linked to the terminal residue of the polynucleotide such as the 3′ of 5′-end of the polynucleotide. Alternatively, it may be linked to an internal nucleotide or a nucleotide on a branch of the polynucleotide. The hydrophobic molecule may be attached, for instance to a 2′-position of the nucleotide. The hydrophobic molecule may also be linked to the heterocyclic base, the sugar or the backbone of a nucleotide of the polynucleotide.
The hydrophobic molecule may be connected to the polynucleotide by a linker moiety. Optionally the linker moiety is a non-nucleotidic linker moiety. Non-nucleotidic linkers are e.g. abasic residues (dSpacer), oligoethyleneglycol, such as triethyleneglycol (spacer 9) or hexaethylenegylcol (spacer 18), or alkane-diol, such as butanediol. The spacer units are preferably linked by phosphodiester or phosphorothioate bonds. The linker units may appear just once in the molecule or may be incorporated several times, e.g. via phosphodiester, phosphorothioate, methylphosphonate, or amide linkages.
Typical conjugation protocols involve the synthesis of polynucleotides bearing an aminolinker at one or more positions of the sequence, however, a linker is not required. 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 polynucleotide still bound to a solid support or following cleavage of the polynucleotide in solution phase. Purification of the modified polynucleotide by HPLC typically results in a pure material.
In some embodiments the hydrophobic molecule is a sterol type conjugate, a PhytoSterol conjugate, cholesterol conjugate, sterol type conjugate with altered side chain length, fatty acid conjugate, any other hydrophobic group conjugate, and/or hydrophobic modifications of the internal nucleoside, which provide sufficient hydrophobicity to be incorporated into micelles.
For purposes of the present invention, the term “sterols”, refers or steroid alcohols are a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring. They are amphipathic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway. The overall molecule is quite flat. The hydroxyl group on the A ring is polar. The rest of the aliphatic chain is non-polar. Usually sterols are considered to have an 8 carbon chain at position 17.
For purposes of the present invention, the term “sterol type molecules”, refers to steroid alcohols, which are similar in structure to sterols. The main difference is the structure of the ring and number of carbons in a position 21 attached side chain.
For purposes of the present invention, the term “PhytoSterols” (also called plant sterols) are a group of steroid alcohols, phytochemicals naturally occurring in plants. There are more then 200 different known PhytoSterols.
For purposes of the present invention, the term “Sterol side chain” refers to a chemical composition of a side chain attached at the position 17 of sterol-type molecule. In a standard definition sterols are limited to a 4 ring structure carrying a 8 carbon chain at position 17. In this invention, the sterol type molecules with side chain longer and shorter than conventional are described. The side chain may branched or contain double back bones.
Thus, sterols useful in the invention, for example, include cholesterols, as well as unique sterols in which position 17 has attached side chain of 2-7 or longer then 9 carbons. In a particular embodiment, the length of the polycarbon tail is varied between 5 and 9 carbons. FIG. 9 demonstrates that there is a correlation between plasma clearance, liver uptake and the length of the polycarbon chain. Such conjugates may have significantly better in vivo efficacy, in particular delivery to liver. These types of molecules are expected to work at concentrations 5 to 9 fold lower then oligonucleotides conjugated to conventional cholesterols.
Alternatively the polynucleotide may be bound to a protein, peptide or positively charged chemical that functions as the hydrophobic molecule. The proteins may be selected from the group consisting of protamine, dsRNA binding domain, and arginine rich peptides. Exemplary positively charged chemicals include spermine, spermidine, cadaverine, and putrescine.
In another embodiment hydrophobic molecule conjugates may demonstrate even higher efficacy when it is combined with optimal chemical modification patterns of the polynucleotide (as described herein in detail), containing but not limited to hydrophobic modifications, phosphorothioate modifications, and 2′ ribo modifications.
In another embodiment the sterol type molecule may be a naturally occurring PhytoSterols such as those shown in FIG. 8. The polycarbon chain may be longer than 9 and may be linear, branched and/or contain double bonds. Some PhytoSterol containing polynucleotide conjugates may be significantly more potent and active in delivery of polynucleotides to various tissues. Some PhytoSterols may demonstrate tissue preference and thus be used as a way to delivery RNAi specifically to particular tissues.
The hydrophobic modified polynucleotide is mixed with a neutral fatty mixture to form a micelle. The neutral fatty acid mixture is a mixture of fats that has a net neutral or slightly net negative charge at or around physiological pH that can form a micelle with the hydrophobic modified polynucleotide. For purposes of the present invention, the term “micelle” refers to a small nanoparticle formed by a mixture of non charged fatty acids and phospholipids. The neutral fatty mixture may include cationic lipids as long as they are present in an amount that does not cause toxicity. In preferred embodiments the neutral fatty mixture is free of cationic lipids. A mixture that is free of cationic lipids is one that has less than 1% and preferably 0% of the total lipid being cationic lipid. The term “cationic lipid” includes lipids and synthetic lipids having a net positive charge at or around physiological pH. The term “anionic lipid” includes lipids and synthetic lipids having a net negative charge at or around physiological pH.
The neutral fats bind to the oligonucleotides of the invention by a strong but non-covalent attraction (e.g., an electrostatic, van der Waals, pi-stacking, etc. interaction).
The neutral fat mixture may include formulations selected from a class of naturally occurring or chemically synthesized or modified saturated and unsaturated fatty acid residues. Fatty acids might exist in a form of triglycerides, diglycerides or individual fatty acids. In another embodiment the use of well-validated mixtures of fatty acids and/or fat emulsions currently used in pharmacology for parenteral nutrition may be utilized.
The neutral fatty mixture is preferably a mixture of a choline based fatty acid and a sterol. Choline based fatty acids include for instance, synthetic phosphocholine derivatives such as DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, and DEPC. DOPC (chemical registry number 4235-95-4) is dioleoylphosphatidylcholine (also known as dielaidoylphosphatidylcholine, dioleoyl-PC, dioleoylphosphocholine, dioleoyl-sn-glycero-3-phosphocholine, dioleylphosphatidylcholine). DSPC (chemical registry number 816-94-4) is distearoylphosphatidylcholine (also known as 1,2-Distearoyl-sn-Glycero-3-phosphocholine).
The sterol in the neutral fatty mixture may be for instance cholesterol. The neutral fatty mixture may be made up completely of a choline based fatty acid and a sterol or it may optionally include a cargo molecule. For instance, the neutral fatty mixture may have at least 20% or 25% fatty acid and 20% or 25% sterol.
For purposes of the present invention, the term “Fatty acids” relates to conventional description of fatty acid. They may exist as individual entities or in a form of two- and triglycerides. For purposes of the present invention, the term “fat emulsions” refers to safe fat formulations given intravenously to subjects who are unable to get enough fat in their diet. It is an emulsion of soy bean oil (or other naturally occurring oils) and egg phospholipids. Fat emulsions are being used for formulation of some insoluble anesthetics. In this disclosure, fat emulsions might be part of commercially available preparations like Intralipid, Liposyn, Nutrilipid, modified commercial preparations, where they are enriched with particular fatty acids or fully de novo-formulated combinations of fatty acids and phospholipids.
In one embodiment, the cells to be contacted with an oligonucleotide composition of the invention are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 12 hours to about 24 hours. In another embodiment, the cells to be contacted with an oligonucleotide composition are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 1 and about five days. In one embodiment, the cells are contacted with a mixture comprising a lipid and the oligonucleotide for between about three days to as long as about 30 days. In another embodiment, a mixture comprising a lipid is left in contact with the cells for at least about five to about 20 days. In another embodiment, a mixture comprising a lipid is left in contact with the cells for at least about seven to about 15 days.
50%-60% of the formulation can optionally be any other lipid or molecule. Such a lipid or molecule is referred to herein as a cargo lipid or cargo molecule. Cargo molecules include but are not limited to intralipid, small molecules, fusogenic peptides or lipids or other small molecules might be added to alter cellular uptake, endosomal release or tissue distribution properties. The ability to tolerate cargo molecules is important for modulation of properties of these particles, if such properties are desirable. For instance the presence of some tissue specific metabolites might drastically alter tissue distribution profiles. For example use of Intralipid type formulation enriched in shorter or longer fatty chains with various degrees of saturation affects tissue distribution profiles of these type of formulations (and their loads).
An example of a cargo lipid useful according to the invention is a fusogenic lipid. For instance, the zwiterionic lipid DOPE (chemical registry number 4004-5-1,1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine) is a preferred cargo lipid.
Intralipid may be comprised of the following composition: 1 000 mL contain: purified soybean oil 90 g, purified egg phospholipids 12 g, glycerol anhydrous 22 g, water for injection q.s. ad 1 000 mL. pH is adjusted with sodium hydroxide to pH approximately 8. Energy content/L: 4.6 MJ (190 kcal). Osmolality (approx.): 300 mOsm/kg water. In another embodiment fat emulsion is Liposyn that contains 5% safflower oil, 5% soybean oil, up to 1.2% egg phosphatides added as an emulsifier and 2.5% glycerin in water for injection. It may also contain sodium hydroxide for pH adjustment. pH 8.0 (6.0-9.0). Liposyn has an osmolarity of 276 m Osmol/liter (actual).
Variation in the identity, amounts and ratios of cargo lipids affects the cellular uptake and tissue distribution characteristics of these compounds. For example, the length of lipid tails and level of saturability will affect differential uptake to liver, lung, fat and cardiomyocytes. Addition of special hydrophobic molecules like vitamins or different forms of sterols can favor distribution to special tissues which are involved in the metabolism of particular compounds. Complexes are formed at different oligonucleotide concentrations, with higher concentrations favoring more efficient complex formation (FIGS. 21-22).
In another embodiment, the fat emulsion is based on a mixture of lipids. Such lipids may include natural compounds, chemically synthesized compounds, purified fatty acids or any other lipids. In yet another embodiment the composition of fat emulsion is entirely artificial. In a particular embodiment, the fat emulsion is more then 70% linoleic acid. In yet another particular embodiment the fat emulsion is at least 1% of cardiolipin. Linoleic acid (LA) is an unsaturated omega-6 fatty acid. It is a colorless liquid made of a carboxylic acid with an 18-carbon chain and two cis double bonds.
In yet another embodiment of the present invention, the alteration of the composition of the fat emulsion is used as a way to alter tissue distribution of hydrophobicly modified polynucleotides. This methodology provides for the specific delivery of the polynucleotides to particular tissues (FIG. 12).
In another embodiment the fat emulsions of the cargo molecule contain more then 70% of Linoleic acid (C18H32O2) and/or cardiolipin are used for specifically delivering RNAi to heart muscle.
Fat emulsions, like intralipid have been used before as a delivery formulation for some non-water soluble drugs (such as Propofol, re-formulated as Diprivan). Unique features of the present invention include (a) the concept of combining modified polynucleotides with the hydrophobic compound(s), so it can be incorporated in the fat micelles and (b) mixing it with the fat emulsions to provide a reversible carrier. After injection into a blood stream, micelles usually bind to serum proteins, including albumin, HDL, LDL and other. This binding is reversible and eventually the fat is absorbed by cells. The polynucleotide, incorporated as a part of the micelle will then be delivered closely to the surface of the cells. After that cellular uptake might be happening though variable mechanisms, including but not limited to sterol type delivery.
Complexing Agents
Complexing agents bind to the oligonucleotides of the invention by a strong but non-covalent attraction (e.g., an electrostatic, van der Waals, pi-stacking, etc. interaction). In one embodiment, oligonucleotides of the invention can be complexed with a complexing agent to increase cellular uptake of oligonucleotides. An example of a complexing agent includes cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells. However, as discussed above, formulations free in cationic lipids are preferred in some embodiments.
The term “cationic lipid” includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells. In general cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof. Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g., from 1 to about 25 carbon atoms. Preferred straight chain or branched alkyl or alkene groups have six or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., Cl−, Br−, I−, F−, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.
Examples of cationic lipids include polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINETM (e.g., LIPOFECTAMINETM 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3β-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). The cationic lipid N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), for example, was found to increase 1000-fold the antisense effect of a phosphorothioate oligonucleotide. (Vlassov et al., 1994, Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides can also be complexed with, e.g., poly (L-lysine) or avidin and lipids may, or may not, be included in this mixture, e.g., steryl-poly (L-lysine).
Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998. Molecular Membrane Biology 15:1). Other lipid compositions which can be used to facilitate uptake of the instant oligonucleotides can be used in connection with the claimed methods. In addition to those listed supra, other lipid compositions are also known in the art and include, e.g., those taught in U.S. Pat. No. 4,235,871; U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.
In one embodiment lipid compositions can further comprise agents, e.g., viral proteins to enhance lipid-mediated transfections of oligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536). In another embodiment, oligonucleotides are contacted with cells as part of a composition comprising an oligonucleotide, a peptide, and a lipid as taught, e.g., in U.S. Pat. No. 5,736,392. Improved lipids have also been described which are serum resistant (Lewis, et al., 1996. Proc. Natl. Acad. Sci. 93:3176). Cationic lipids and other complexing agents act to increase the number of oligonucleotides carried into the cell through endocytosis.
In another embodiment N-substituted glycine oligonucleotides (peptoids) can be used to optimize uptake of oligonucleotides. Peptoids have been used to create cationic lipid-like compounds for transfection (Murphy, et al., 1998. Proc. Natl. Acad. Sci. 95:1517). Peptoids can be synthesized using standard methods (e.g., Zuckermann, R. N., et al. 1992. J. Am. Chem. Soc. 114:10646; Zuckermann, R. N., et al. 1992. Int. J. Peptide Protein Res. 40:497). Combinations of cationic lipids and peptoids, liptoids, can also be used to optimize uptake of the subject oligonucleotides (Hunag, et al., 1998. Chemistry and Biology. 5:345). Liptoids can be synthesized by elaborating peptoid oligonucleotides and coupling the amino terminal submonomer to a lipid via its amino group (Hunag, et al., 1998. Chemistry and Biology. 5:345).
It is known in the art that positively charged amino acids can be used for creating highly active cationic lipids (Lewis et al. 1996. Proc. Natl. Acad. Sci. USA. 93:3176). In one embodiment, a composition for delivering oligonucleotides of the invention comprises a number of arginine, lysine, histidine or ornithine residues linked to a lipophilic moiety (see e.g., U.S. Pat. No. 5,777,153).
In another embodiment, a composition for delivering oligonucleotides of the invention comprises a peptide having from between about one to about four basic residues. These basic residues can be located, e.g., on the amino terminal, C-terminal, or internal region of the peptide. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine (can also be considered non-polar), asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Apart from the basic amino acids, a majority or all of the other residues of the peptide can be selected from the non-basic amino acids, e.g., amino acids other than lysine, arginine, or histidine. Preferably a preponderance of neutral amino acids with long neutral side chains are used.
In one embodiment, a composition for delivering oligonucleotides of the invention comprises a natural or synthetic polypeptide having one or more gamma carboxyglutamic acid residues, or γ-Gla residues. These gamma carboxyglutamic acid residues may enable the polypeptide to bind to each other and to membrane surfaces. In other words, a polypeptide having a series of γ-Gla may be used as a general delivery modality that helps an RNAi construct to stick to whatever membrane to which it comes in contact. This may at least slow RNAi constructs from being cleared from the blood stream and enhance their chance of homing to the target.
The gamma carboxyglutamic acid residues may exist in natural proteins (for example, prothrombin has 10 γ-Gla residues). Alternatively, they can be introduced into the purified, recombinantly produced, or chemically synthesized polypeptides by carboxylation using, for example, a vitamin K-dependent carboxylase. The gamma carboxyglutamic acid residues may be consecutive or non-consecutive, and the total number and location of such gamma carboxyglutamic acid residues in the polypeptide can be regulated/fine tuned to achieve different levels of “stickiness” of the polypeptide.
In one embodiment, the cells to be contacted with an oligonucleotide composition of the invention are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 12 hours to about 24 hours. In another embodiment, the cells to be contacted with an oligonucleotide composition are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 1 and about five days. In one embodiment, the cells are contacted with a mixture comprising a lipid and the oligonucleotide for between about three days to as long as about 30 days. In another embodiment, a mixture comprising a lipid is left in contact with the cells for at least about five to about 20 days. In another embodiment, a mixture comprising a lipid is left in contact with the cells for at least about seven to about 15 days.
For example, in one embodiment, an oligonucleotide composition can be contacted with cells in the presence of a lipid such as cytofectin CS or GSV (available from Glen Research; Sterling, Va.), GS3815, GS2888 for prolonged incubation periods as described herein.
In one embodiment, the incubation of the cells with the mixture comprising a lipid and an oligonucleotide composition does not reduce the viability of the cells. Preferably, after the transfection period the cells are substantially viable. In one embodiment, after transfection, the cells are between at least about 70% and at least about 100% viable. In another embodiment, the cells are between at least about 80% and at least about 95% viable. In yet another embodiment, the cells are between at least about 85% and at least about 90% viable.
In one embodiment, oligonucleotides are modified by attaching a peptide sequence that transports the oligonucleotide into a cell, referred to herein as a “transporting peptide.” In one embodiment, the composition includes an oligonucleotide which is complementary to a target nucleic acid molecule encoding the protein, and a covalently attached transporting peptide.
The language “transporting peptide” includes an amino acid sequence that facilitates the transport of an oligonucleotide into a cell. Exemplary peptides which facilitate the transport of the moieties to which they are linked into cells are known in the art, and include, e.g., HIV TAT transcription factor, lactoferrin, Herpes VP22 protein, and fibroblast growth factor 2 (Pooga et al. 1998. Nature Biotechnology. 16:857; and Derossi et al. 1998. Trends in Cell Biology. 8:84; Elliott and O'Hare. 1997. Cell 88:223).
Oligonucleotides can be attached to the transporting peptide using known techniques, e.g., (Prochiantz, A. 1996. Curr. Opin. Neurobiol. 6:629; Derossi et al. 1998. Trends Cell Biol. 8:84; Troy et al. 1996. J. Neurosci. 16:253), Vives et al. 1997. J. Biol. Chem. 272:16010). For example, in one embodiment, oligonucleotides bearing an activated thiol group are linked via that thiol group to a cysteine present in a transport peptide (e.g., to the cysteine present in the J3 turn between the second and the third helix of the antennapedia homeodomain as taught, e.g., in Derossi et al. 1998. Trends Cell Biol. 8:84; Prochiantz. 1996. Current Opinion in Neurobiol. 6:629; Allinquant et al. 1995. J Cell Biol. 128:919). In another embodiment, a Boc-Cys-(Npys)OH group can be coupled to the transport peptide as the last (N-terminal) amino acid and an oligonucleotide bearing an SH group can be coupled to the peptide (Troy et al. 1996. J. Neurosci. 16:253).
In one embodiment, a linking group can be attached to a nucleomonomer and the transporting peptide can be covalently attached to the linker. In one embodiment, a linker can function as both an attachment site for a transporting peptide and can provide stability against nucleases. Examples of suitable linkers include substituted or unsubstituted C1-C20 alkyl chains, C2-C20 alkenyl chains, C2-C20 alkynyl chains, peptides, and heteroatoms (e.g., S, O, NH, etc.). Other exemplary linkers include bifunctional crosslinking agents such as sulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smith et al. Biochem J 1991.276: 417-2).
In one embodiment, oligonucleotides of the invention are synthesized as molecular conjugates which utilize receptor-mediated endocytotic mechanisms for delivering genes into cells (see, e.g., Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559, and the references cited therein).
Targeting Agents
The delivery of oligonucleotides can also be improved by targeting the oligonucleotides to a cellular receptor. The targeting moieties can be conjugated to the oligonucleotides or attached to a carrier group (i.e., poly(L-lysine) or liposomes) linked to the oligonucleotides. This method is well suited to cells that display specific receptor-mediated endocytosis.
For instance, oligonucleotide conjugates to 6-phosphomannosylated proteins are internalized 20-fold more efficiently by cells expressing mannose 6-phosphate specific receptors than free oligonucleotides. The oligonucleotides may also be coupled to a ligand for a cellular receptor using a biodegradable linker. In another example, the delivery construct is mannosylated streptavidin which forms a tight complex with biotinylated oligonucleotides. Mannosylated streptavidin was found to increase 20-fold the internalization of biotinylated oligonucleotides. (Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).
In addition specific ligands can be conjugated to the polylysine component of polylysine-based delivery systems. For example, transferrin-polylysine, adenovirus-polylysine, and influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides-polylysine conjugates greatly enhance receptor-mediated DNA delivery in eucaryotic cells. Mannosylated glycoprotein conjugated to poly(L-lysine) in aveolar macrophages has been employed to enhance the cellular uptake of oligonucleotides. Liang et al. 1999. Pharmazie 54:559-566.
Because malignant cells have an increased need for essential nutrients such as folic acid and transferrin, these nutrients can be used to target oligonucleotides to cancerous cells. For example, when folic acid is linked to poly(L-lysine) enhanced oligonucleotide uptake is seen in promyelocytic leukaemia (HL-60) cells and human melanoma (M-14) cells. Ginobbi et al. 1997. Anticancer Res. 17:29. In another example, liposomes coated with maleylated bovine serum albumin, folic acid, or ferric protoporphyrin IX, show enhanced cellular uptake of oligonucleotides in murine macrophages, KB cells, and 2.2.15 human hepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.
Liposomes naturally accumulate in the liver, spleen, and reticuloendothelial system (so-called, passive targeting). By coupling liposomes to various ligands such as antibodies are protein A, they can be actively targeted to specific cell populations. For example, protein A-bearing liposomes may be pretreated with H-2K specific antibodies which are targeted to the mouse major histocompatibility complex-encoded H-2K protein expressed on L cells. (Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).
Other in vitro and/or in vivo delivery of RNAi reagents are known in the art, and can be used to deliver the subject RNAi constructs. See, for example, U.S. patent application publications 20080152661, 20080112916, 20080107694, 20080038296, 20070231392, 20060240093, 20060178327, 20060008910, 20050265957, 20050064595, 20050042227, 20050037496, 20050026286, 20040162235, 20040072785, 20040063654, 20030157030, WO 2008/036825, WO04/065601, and AU2004206255B2, just to name a few (all incorporated by reference).
Administration
The optimal course of administration or delivery of the oligonucleotides may vary depending upon the desired result and/or on the subject to be treated. As used herein “administration” refers to contacting cells with oligonucleotides and can be performed in vitro or in vivo. The dosage of oligonucleotides may be adjusted to optimally reduce expression of a protein translated from a target nucleic acid molecule, e.g., as measured by a readout of RNA stability or by a therapeutic response, without undue experimentation.
For example, expression of the protein encoded by the nucleic acid target can be measured to determine whether or not the dosage regimen needs to be adjusted accordingly. In addition, an increase or decrease in RNA or protein levels in a cell or produced by a cell can be measured using any art recognized technique. By determining whether transcription has been the effectiveness of the oligonucleotide in inducing the cleavage of a target RNA can be determined.
Any of the above-described oligonucleotide compositions can be used alone or in conjunction with a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes appropriate solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, it can be used in the therapeutic compositions. Supplementary active ingredients can also be incorporated into the compositions.
Oligonucleotides may be incorporated into liposomes or liposomes modified with polyethylene glycol or admixed with cationic lipids for parenteral administration. Incorporation of additional substances into the liposome, for example, antibodies reactive against membrane proteins found on specific target cells, can help target the oligonucleotides to specific cell types.
Moreover, the present invention provides for administering the subject oligonucleotides with an osmotic pump providing continuous infusion of such oligonucleotides, for example, as described in Rataiczak et al. (1992 Proc. Natl. Acad. Sci. USA 89:11823-11827). Such osmotic pumps are commercially available, e.g., from Alzet Inc. (Palo Alto, Calif.). Topical administration and parenteral administration in a cationic lipid carrier are preferred.
With respect to in vivo applications, the formulations of the present invention can be administered to a patient in a variety of forms adapted to the chosen route of administration, e.g., parenterally, orally, or intraperitoneally. Parenteral administration, which is preferred, includes administration by the following routes: intravenous; intramuscular; interstitially; intraarterially; subcutaneous; intra ocular; intrasynovial; trans epithelial, including transdermal; pulmonary via inhalation; ophthalmic; sublingual and buccal; topically, including ophthalmic; dermal; ocular; rectal; and nasal inhalation via insufflation.
Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, or dextran, optionally, the suspension may also contain stabilizers. The oligonucleotides of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligonucleotides may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included in the invention.
Pharmaceutical preparations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, sprays, suppositories, liquids and powders. In addition, conventional pharmaceutical carriers, aqueous, powder or oily bases, or thickeners may be used in pharmaceutical preparations for topical administration.
Pharmaceutical preparations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. In addition, thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be used in pharmaceutical preparations for oral administration.
For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, and detergents. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligonucleotides are formulated into conventional oral administration forms such as capsules, tablets, and tonics. For topical administration, the oligonucleotides of the invention are formulated into ointments, salves, gels, or creams as known in the art.
Drug delivery vehicles can be chosen e.g., for in vitro, for systemic, or for topical administration. These vehicles can be designed to serve as a slow release reservoir or to deliver their contents directly to the target cell. An advantage of using some direct delivery drug vehicles is that multiple molecules are delivered per uptake. Such vehicles have been shown to increase the circulation half-life of drugs that would otherwise be rapidly cleared from the blood stream. Some examples of such specialized drug delivery vehicles which fall into this category are liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
The described oligonucleotides may be administered systemically to a subject. Systemic absorption refers to the entry of drugs into the blood stream followed by distribution throughout the entire body. Administration routes which lead to systemic absorption include: intravenous, subcutaneous, intraperitoneal, and intranasal. Each of these administration routes delivers the oligonucleotide to accessible diseased cells. Following subcutaneous administration, the therapeutic agent drains into local lymph nodes and proceeds through the lymphatic network into the circulation. The rate of entry into the circulation has been shown to be a function of molecular weight or size. The use of a liposome or other drug carrier localizes the oligonucleotide at the lymph node. The oligonucleotide can be modified to diffuse into the cell, or the liposome can directly participate in the delivery of either the unmodified or modified oligonucleotide into the cell.
The chosen method of delivery will result in entry into cells. Preferred delivery methods include liposomes (10-400 nm), hydrogels, controlled-release polymers, and other pharmaceutically applicable vehicles, and microinjection or electroporation (for ex vivo treatments).
The pharmaceutical preparations of the present invention may be prepared and formulated as emulsions. Emulsions are usually heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. The emulsions of the present invention may contain excipients such as emulsifiers, stabilizers, dyes, fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and anti-oxidants may also be present in emulsions as needed. These excipients may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.
Examples of naturally occurring emulsifiers that may be used in emulsion formulations of the present invention include lanolin, beeswax, phosphatides, lecithin and acacia. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. Examples of finely divided solids that may be used as emulsifiers include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montrnorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
Examples of preservatives that may be included in the emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Examples of antioxidants that may be included in the emulsion formulations include 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.
In one embodiment, the compositions of oligonucleotides are formulated as microemulsions. A microemulsion is a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution. Typically microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a 4th component, generally an intermediate chain-length alcohol to form a transparent system.
Surfactants that may be 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 (MO750), 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 oil/water and water/oil) have been proposed to enhance the oral bioavailability of drugs.
Microemulsions offer 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). 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 oligonucleotides from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
In an embodiment, the present invention employs various penetration enhancers to affect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to increasing the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also act to enhance the permeability of lipophilic drugs.
Five categories of penetration enhancers that may be used in the present invention include: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Other agents may be utilized to enhance the penetration of the administered oligonucleotides include: glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-15 pyrrol, azones, and terpenes such as limonene, and menthone.
The oligonucleotides, especially in lipid formulations, can also be administered by coating a medical device, for example, a catheter, such as an angioplasty balloon catheter, with a cationic lipid formulation. Coating may be achieved, for example, by dipping the medical device into a lipid formulation or a mixture of a lipid formulation and a suitable solvent, for example, an aqueous-based buffer, an aqueous solvent, ethanol, methylene chloride, chloroform and the like. An amount of the formulation will naturally adhere to the surface of the device which is subsequently administered to a patient, as appropriate. Alternatively, a lyophilized mixture of a lipid formulation may be specifically bound to the surface of the device. Such binding techniques are described, for example, in K. Ishihara et al., Journal of Biomedical Materials Research, Vol. 27, pp. 1309-1314 (1993), the disclosures of which are incorporated herein by reference in their entirety.
The useful dosage to be administered and the particular mode of administration will vary depending upon such factors as the cell type, or for in vivo use, the age, weight and the particular animal and region thereof to be treated, the particular oligonucleotide and delivery method used, the therapeutic or diagnostic use contemplated, and the form of the formulation, for example, suspension, emulsion, micelle or liposome, as will be readily apparent to those skilled in the art. Typically, dosage is administered at lower levels and increased until the desired effect is achieved. When lipids are used to deliver the oligonucleotides, the amount of lipid compound that is administered can vary and generally depends upon the amount of oligonucleotide agent being administered. For example, the weight ratio of lipid compound to oligonucleotide agent is preferably from about 1:1 to about 15:1, with a weight ratio of about 5:1 to about 10:1 being more preferred. Generally, the amount of cationic lipid compound which is administered will vary from between about 0.1 milligram (mg) to about 1 gram (g). By way of general guidance, typically between about 0.1 mg and about 10 mg of the particular oligonucleotide agent, and about 1 mg to about 100 mg of the lipid compositions, each per kilogram of patient body weight, is administered, although higher and lower amounts can be used.
The agents of the invention are administered to subjects or contacted with cells in a biologically compatible form suitable for pharmaceutical administration. By “biologically compatible form suitable for administration” is meant that the oligonucleotide is administered in a form in which any toxic effects are outweighed by the therapeutic effects of the oligonucleotide. In one embodiment, oligonucleotides can be administered to subjects. Examples of subjects include mammals, e.g., humans and other primates; cows, pigs, horses, and farming (agricultural) animals; dogs, cats, and other domesticated pets; mice, rats, and transgenic non-human animals.
Administration of an active amount of an oligonucleotide of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, an active amount of an oligonucleotide may vary according to factors such as the type of cell, the oligonucleotide used, and for in vivo uses the disease state, age, sex, and weight of the individual, and the ability of the oligonucleotide to elicit a desired response in the individual. Establishment of therapeutic levels of oligonucleotides within the cell is dependent upon the rates of uptake and efflux or degradation. Decreasing the degree of degradation prolongs the to intracellular half-life of the oligonucleotide. Thus, chemically-modified oligonucleotides, e.g., with modification of the phosphate backbone, may require different dosing.
The exact dosage of an oligonucleotide and number of doses administered will depend upon the data generated experimentally and in clinical trials. Several factors such as the desired effect, the delivery vehicle, disease indication, and the route of administration, will affect the dosage. Dosages can be readily determined by one of ordinary skill in the art and formulated into the subject pharmaceutical compositions. Preferably, the duration of treatment will extend at least through the course of the disease symptoms.
Dosage regim may be adjusted to provide the optimum therapeutic response. For example, the oligonucleotide may be repeatedly administered, e.g., several doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. One of ordinary skill in the art will readily be able to determine appropriate doses and schedules of administration of the subject oligonucleotides, whether the oligonucleotides are to be administered to cells or to subjects.
Physical methods of introducing nucleic acids include injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. A viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of nucleic acid encoded by the expression construct. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, such as calcium phosphate, and the like. Thus the nucleic acid may be introduced along with components that perform one or more of the following activities: enhance nucleic acid uptake by the cell, inhibit annealing of single strands, stabilize the single strands, or other-wise increase inhibition of the target gene.
Nucleic acid may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally or by inhalation, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.
The cell with the target gene may be derived from or contained in any organism. The organism may a plant, animal, protozoan, bacterium, virus, or fungus. The plant may be a monocot, dicot or gymnosperm; the animal may be a vertebrate or invertebrate. Preferred microbes are those used in agriculture or by industry, and those that are pathogenic for plants or animals.
Alternatively, vectors, e.g., transgenes encoding a siRNA of the invention can be engineered into a host cell or transgenic animal using art recognized techniques.
Another use for the nucleic acids of the present invention (or vectors or transgenes encoding same) is a functional analysis to be carried out in eukaryotic cells, or eukaryotic non-human organisms, preferably mammalian cells or organisms and most preferably human cells, e.g. cell lines such as HeLa or 293 or rodents, e.g. rats and mice. By administering a suitable nucleic acid of the invention which is sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference, a specific knockout or knockdown phenotype can be obtained in a target cell, e.g. in cell culture or in a target organism.
Thus, a further subject matter of the invention is a eukaryotic cell or a eukaryotic non-human organism exhibiting a target gene-specific knockout or knockdown phenotype comprising a fully or at least partially deficient expression of at least one endogenous target gene wherein said cell or organism is transfected with at least one vector comprising DNA encoding an RNAi agent capable of inhibiting the expression of the target gene. It should be noted that the present invention allows a target-specific knockout or knockdown of several different endogenous genes due to the specificity of the RNAi agent.
Gene-specific knockout or knockdown phenotypes of cells or non-human organisms, particularly of human cells or non-human mammals may be used in analytic to procedures, e.g. in the functional and/or phenotypical analysis of complex physiological processes such as analysis of gene expression profiles and/or proteomes. Preferably the analysis is carried out by high throughput methods using oligonucleotide based chips.
Assays of Oligonucleotide Stability
In some embodiments, the oligonucleotides of the invention are stabilized, i.e., substantially resistant to endonuclease and exonuclease degradation. An oligonucleotide is defined as being substantially resistant to nucleases when it is at least about 3-fold more resistant to attack by an endogenous cellular nuclease, and is highly nuclease resistant when it is at least about 6-fold more resistant than a corresponding oligonucleotide. This can be demonstrated by showing that the oligonucleotides of the invention are substantially resistant to nucleases using techniques which are known in the art.
One way in which substantial stability can be demonstrated is by showing that the oligonucleotides of the invention function when delivered to a cell, e.g., that they reduce transcription or translation of target nucleic acid molecules, e.g., by measuring protein levels or by measuring cleavage of mRNA. Assays which measure the stability of target RNA can be performed at about 24 hours post-transfection (e.g., using Northern blot techniques, RNase Protection Assays, or QC-PCR assays as known in the art). Alternatively, levels of the target protein can be measured. Preferably, in addition to testing the RNA or protein levels of interest, the RNA or protein levels of a control, non-targeted gene will be measured (e.g., actin, or preferably a control with sequence similarity to the target) as a specificity control. RNA or protein measurements can be made using any art-recognized technique. Preferably, measurements will be made beginning at about 16-24 hours post transfection. (M. Y. Chiang, et al. 1991. J Biol Chem. 266:18162-71; T. Fisher, et al. 1993. Nucleic Acids Research. 21 3857).
The ability of an oligonucleotide composition of the invention to inhibit protein synthesis can be measured using techniques which are known in the art, for example, by detecting an inhibition in gene transcription or protein synthesis. For example, Nuclease S1 mapping can be performed. In another example, Northern blot analysis can be used to measure the presence of RNA encoding a particular protein. For example, total RNA can be prepared over a cesium chloride cushion (see, e.g., Ausebel et al., 1987. Current Protocols in Molecular Biology (Greene & Wiley, New York)). Northern blots can then be made using the RNA and probed (see, e.g., Id.). In another example, the level of the specific mRNA produced by the target protein can be measured, e.g., using PCR. In yet another example, Western blots can be used to measure the amount of target protein present. In still another embodiment, a phenotype influenced by the amount of the protein can be detected. Techniques for performing Western blots are well known in the art, see, e.g., Chen et al. J. Biol. Chem. 271:28259.
In another example, the promoter sequence of a target gene can be linked to a reporter gene and reporter gene transcription (e.g., as described in more detail below) can be monitored. Alternatively, oligonucleotide compositions that do not target a promoter can be identified by fusing a portion of the target nucleic acid molecule with a reporter gene so that the reporter gene is transcribed. By monitoring a change in the expression of the reporter gene in the presence of the oligonucleotide composition, it is possible to determine the effectiveness of the oligonucleotide composition in inhibiting the expression of the reporter gene. For example, in one embodiment, an effective oligonucleotide composition will reduce the expression of the reporter gene.
A “reporter gene” is a nucleic acid that expresses a detectable gene product, which may be RNA or protein. Detection of mRNA expression may be accomplished by Northern blotting and detection of protein may be accomplished by staining with antibodies specific to the protein. Preferred reporter genes produce a readily detectable product. A reporter gene may be operably linked with a regulatory DNA sequence such that detection of the reporter gene product provides a measure of the transcriptional activity of the regulatory sequence. In preferred embodiments, the gene product of the reporter gene is detected by an intrinsic activity associated with that product. For instance, the reporter gene may encode a gene product that, by enzymatic activity, gives rise to a detectable signal based on color, fluorescence, or luminescence. Examples of reporter genes include, but are not limited to, those coding for chloramphenicol acetyl transferase (CAT), luciferase, beta-galactosidase, and alkaline phosphatase.
One skilled in the art would readily recognize numerous reporter genes suitable for use in the present invention. These include, but are not limited to, chloramphenicol acetyltransferase (CAT), luciferase, human growth hormone (hGH), and beta-galactosidase. Examples of such reporter genes can be found in F. A. Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley & Sons, New York, (1989). Any gene that encodes a detectable product, e.g., any product having detectable enzymatic activity or against which a specific antibody can be raised, can be used as a reporter gene in the present methods.
One reporter gene system is the firefly luciferase reporter system. (Gould, S. J., and Subramani, S. 1988. Anal. Biochem., 7:404-408 incorporated herein by reference). The luciferase assay is fast and sensitive. In this assay, a lysate of the test cell is prepared and combined with ATP and the substrate luciferin. The encoded enzyme luciferase catalyzes a rapid, ATP dependent oxidation of the substrate to generate a light-emitting product. The total light output is measured and is proportional to the amount of luciferase present over a wide range of enzyme concentrations.
CAT is another frequently used reporter gene system; a major advantage of this system is that it has been an extensively validated and is widely accepted as a measure of promoter activity. (Gorman C. M., Moffat, L. F., and Howard, B. H. 1982. Mol. Cell. Biol., 2:1044-1051). In this system, test cells are transfected with CAT expression vectors and incubated with the candidate substance within 2-3 days of the initial transfection. Thereafter, cell extracts are prepared. The extracts are incubated with acetyl CoA and radioactive chloramphenicol. Following the incubation, acetylated chloramphenicol is separated from nonacetylated form by thin layer chromatography. In this assay, the degree of acetylation reflects the CAT gene activity with the particular promoter.
Another suitable reporter gene system is based on immunologic detection of hGH. This system is also quick and easy to use. (Selden, R., Burke-Howie, K. Rowe, M. E., Goodman, H. M., and Moore, D. D. (1986), Mol. Cell, Biol., 6:3173-3179 incorporated herein by reference). The hGH system is advantageous in that the expressed hGH polypeptide is assayed in the media, rather than in a cell extract. Thus, this system does not require the destruction of the test cells. It will be appreciated that the principle of this reporter gene system is not limited to hGH but rather adapted for use with any polypeptide for which an antibody of acceptable specificity is available or can be prepared.
In one embodiment, nuclease stability of a double-stranded oligonucleotide of the invention is measured and compared to a control, e.g., an RNAi molecule typically used in the art (e.g., a duplex oligonucleotide of less than 25 nucleotides in length and comprising 2 nucleotide base overhangs) or an unmodified RNA duplex with blunt ends.
The target RNA cleavage reaction achieved using the siRNAs of the invention is highly sequence specific. Sequence identity may determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). A preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. Additionally, numerous commercial entities, such as Dharmacon, and Invitrogen provide access to algorithms on their website. The Whitehead Institute also offers a free siRNA Selection Program. Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the siRNA and the portion of the target gene is preferred. Alternatively, the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the target gene transcript. Examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
Therapeutic Use
By inhibiting the expression of a gene, the oligonucleotide compositions of the present invention can be used to treat any disease involving the expression of a protein. Examples of diseases that can be treated by oligonucleotide compositions, just to illustrate, include: cancer, retinopathies, autoimmune diseases, inflammatory diseases (i.e., ICAM-1 related disorders, Psoriasis, Ulcerative Colitus, Crohn's disease), viral diseases (i.e., HIV, Hepatitis C), miRNA disorders, and cardiovascular diseases.
In one embodiment, in vitro treatment of cells with oligonucleotides can be used for ex vivo therapy of cells removed from a subject (e.g., for treatment of leukemia or viral infection) or for treatment of cells which did not originate in the subject, but are to be administered to the subject (e.g., to eliminate transplantation antigen expression on cells to be transplanted into a subject). In addition, in vitro treatment of cells can be used in non-therapeutic settings, e.g., to evaluate gene function, to study gene regulation and protein synthesis or to evaluate improvements made to oligonucleotides designed to modulate gene expression or protein synthesis. In vivo treatment of cells can be useful in certain clinical settings where it is desirable to inhibit the expression of a protein. There are numerous medical conditions for which antisense therapy is reported to be suitable (see, e.g., U.S. Pat. No. 5,830,653) as well as respiratory syncytial virus infection (WO 95/22,553) influenza virus (WO 94/23,028), and malignancies (WO 94/08,003). Other examples of clinical uses of antisense sequences are reviewed, e.g., in Glaser. 1996. Genetic Engineering News 16:1. Exemplary targets for cleavage by oligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf kinase, p53, c-myb, and the bcr/abl fusion gene found in chronic myelogenous leukemia.
The subject nucleic acids can be used in RNAi-based therapy in any animal having RNAi pathway, such as human, non-human primate, non-human mammal, non-human vertebrates, rodents (mice, rats, hamsters, rabbits, etc.), domestic livestock animals, pets (cats, dogs, etc.), Xenopus, fish, insects (Drosophila, etc.), and worms (C. elegans), etc.
The invention provides methods for inhibiting or preventing in a subject, a disease or condition associated with an aberrant or unwanted target gene expression or activity, by administering to the subject a nucleic acid of the invention. If appropriate, subjects are first treated with a priming agent so as to be more responsive to the subsequent RNAi therapy. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted target gene expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays known in the art. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the target gene aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of target gene aberrancy, for example, a target gene, target gene agonist or target gene antagonist agent can be used for treating the subject.
In another aspect, the invention pertains to methods of modulating target gene expression, protein expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the methods of the invention involve contacting a cell capable of expressing target gene with a nucleic acid of the invention that is specific for the target gene or protein (e.g., is specific for the mRNA encoded by said gene or specifying the amino acid sequence of said protein) such that expression or one or more of the activities of target protein is modulated. These methods can be performed in vitro (e.g., by culturing the cell with the agent), in vivo (e.g., by administering the agent to a subject), or ex vivo. The subjects may be first treated with a priming agent so as to be more responsive to the subsequent RNAi therapy if desired. As such, the present invention provides methods of treating a subject afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a target gene polypeptide or nucleic acid molecule. Inhibition of target gene activity is desirable in situations in which target gene is abnormally unregulated and/or in which decreased target gene activity is likely to have a beneficial effect.
Thus the therapeutic agents of the invention can be administered to subjects to treat (prophylactically or therapeutically) disorders associated with aberrant or unwanted target gene activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a therapeutic agent as well as tailoring the dosage and/or therapeutic regimen of treatment with a therapeutic agent. Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons.
For the purposes of the invention, ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
Moreover, for the purposes of the present invention, the term “a” or “an” entity refers to one or more of that entity; for example, “a protein” or “a nucleic acid molecule” refers to one or more of those compounds or at least one compound. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably. Furthermore, a compound “selected from the group consisting of” refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.
The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.
EXAMPLES
Example 1
Inhibition of Gene Expression Using Minimum Length Trigger RNAs
Transfection of Minimum Length Trigger (mlt) RNA
mltRNA constructs were chemically synthesized (Integrated DNA Technologies, Coralville, Iowa) and transfected into HEK293 cells (ATCC, Manassas, Va.) using the Lipofectamine RNAiMAX (Invitrogen, Carlsbad, Calif.) reagent according to manufacturer's instructions. In brief, RNA was diluted to a 12× concentration and then combined with a 12× concentration of Lipofectamine RNAiMAX to complex. The RNA and transfection reagent were allowed to complex at room temperature for 20 minutes and make a 6× concentration. While complexing, HEK293 cells were washed, trypsinized and counted. The cells were diluted to a concentration recommended by the manufacturer and previously described conditions which was at 1×105 cells/ml. When RNA had completed complexing with the RNAiMAX transfection reagent, 20 ul of the complexes were added to the appropriate well of the 96-well plate in triplicate. Cells were added to each well (100 ul volume) to make the final cell count per well at 1×104 cells/well. The volume of cells diluted the 6× concentration of complex to IX which was equal to a concentration noted (between 10-0.05 nM). Cells were incubated for 24 or 48 hours under normal growth conditions.
After 24 or 48 hour incubation cells were lysed and gene silencing activity was measured using the QuantiGene assay (Panomics, Freemont, Calif.) which employs bDNA hybridization technology. The assay was carried out according to manufacturer's instructions.
ΔG Calculation
ΔG was calculated using Mfold, available through the Mfold interne site (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi). Methods for calculating ΔG are described in, and are incorporated by reference from, the following references: Zuker, M. (2003) Nucleic Acids Res., 31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M. and Turner, D. H. (1999) J. Mol. Biol. 288:911-940; Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl. Acad. Sci. 101:7287-7292; Duan, S., Mathews, D. H., and Turner, D. H. (2006) Biochemistry 45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I. L., and Schuster, P. (1999) Biopolymers 49:145-165.
Example 2
Optimization of sd-rxRNAnano Molecules for Gene Silencing
Asymmetric double stranded RNAi molecules, with minimal double stranded regions, were developed herein and are highly effective at gene silencing. These molecules can contain a variety of chemical modifications on the sense and/or anti-sense strands, and can be conjugated to sterol-like compounds such as cholesterol.
FIGS. 1-3 present schematics of RNAi molecules associated with the invention. In the asymmetric molecules, which contain a sense and anti-sense strand, either of the strands can be the longer strand. Either strand can also contain a single-stranded region. There can also be mismatches between the sense and anti-sense strand, as indicated in FIG. 1D. Preferably, one end of the double-stranded molecule is either blunt-ended or contains a short overhang such as an overhang of one nucleotide. FIG. 2 indicates types of chemical modifications applied to the sense and anti-sense strands including 2′F, 2′OMe, hydrophobic modifications and phosphorothioate modifications. Preferably, the single stranded region of the molecule contains multiple phosphorothioate modifications. Hydrophobicity of molecules can be increased using such compounds as 4-pyridyl at 5-U, 2-pyridyl at 5-U, isobutyl at 5-U and indolyl at 5-U (FIG. 2). Proteins or peptides such as protamine (or other Arg rich peptides), spermidine or other similar chemical structures can also be used to block duplex charge and facilitate cellular entry (FIG. 3). Increased hydrophobicity can be achieved through either covalent or non-covalent modifications. Several positively charged chemicals, which might be used for polynucleotide charge blockage are depicted in FIG. 4.
Chemical modifications of polynucleotides, such as the guide strand in a duplex molecule, can facilitate RISC entry. FIG. 5 depicts single stranded polynucleotides, representing a guide strand in a duplex molecule, with a variety of chemical modifications including 2′ d, 2′OMe, 2′F, hydrophobic modifications, phosphorothioate modifications, and attachment of conjugates such as “X” in FIG. 5, where X can be a small molecule with high affinity to a PAZ domain, or sterol-type entity. Similarly, FIG. 6 depicts single stranded polynucleotides, representing a passenger strand in a duplex molecule, with proposed structural and chemical compositions of RISC substrate inhibitors. Combinations of chemical modifications can ensure efficient uptake and efficient binding to preloaded RISC complexes.
FIG. 7 depicts structures of polynucleotides with sterol-type molecules attached, where R represents a polycarbonic tail of 9 carbons or longer. FIG. 8 presents examples of naturally occurring phytosterols with a polycarbon chain longer than 8 attached at position 17. More than 250 different types of phytosterols are known. FIG. 9 presents examples of sterol-like structures with variations in the sizes of the polycarbon chains attached at position 17. FIG. 91 presents further examples of sterol-type molecules that can be used as a hydrophobic entity in place of cholesterol. FIG. 92 presents further examples of hydrophobic molecules that might be used as hydrophobic entities in place of cholestesterol. Optimization of such characteristics can improve uptake properties of the RNAi molecules. FIG. 10 presents data adapted from Martins et al. (J Lipid Research), showing that the percentage of liver uptake and plasma clearance of lipid emulsions containing sterol-type molecules is directly affected by the size of the attached polycarbon chain at position 17. FIG. 11 depicts a micelle formed from a mixture of polynucleotides attached to hydrophobic conjugates and fatty acids. FIG. 12 describes how alteration in lipid composition can affect pharmacokinetic behavior and tissue distribution of hydrophobically modified and/or hydrophobically conjugated polynucleotides. In particular, the use of lipid mixtures that are enriched in linoleic acid and cardiolipin results in preferential uptake by cardiomyocites.
FIG. 13 depicts examples of RNAi constructs and controls designed to target MAP4K4 expression. FIGS. 14 and 15 reveal that RNAi constructs with minimal duplex regions (such as duplex regions of approximately 13 nucleotides) are effective in mediating RNA silencing in cell culture. Parameters associated with these RNA molecules are shown in FIG. 16. FIG. 17 depicts examples of RNAi constructs and controls designed to target SOD1 expression. FIGS. 18 and 19 reveal the results of gene silencing experiments using these RNAi molecules to target SOD1 in cells. FIG. 20 presents a schematic indicating that RNA molecules with double stranded regions that are less than 10 nucleotides are not cleaved by Dicer, and FIG. 21 presents a schematic of a hypothetical RNAi model for RNA induced gene silencing.
The RNA molecules described herein were subject to a variety of chemical modifications on the sense and antisense strands, and the effects of such modifications were observed. RNAi molecules were synthesized and optimized through testing of a variety of modifications. In first generation optimization, the sense (passenger) and anti-sense (guide) strands of the sd-rxRNAnano molecules were modified for example through incorporation of C and U 2′OMe modifications, 2′F modifications, phosphorothioate modifications, phosphorylation, and conjugation of cholesterol. Molecules were tested for inhibition of MAP4K4 expression in cells including HeLa, primary mouse hepatocytes and primary human hepatocytes through both lipid-mediated and passive uptake transfection.
FIG. 22 reveals that chemical modifications can enhance gene silencing. In particular, modifying the guide strand with 2′F UC modifications, and with a stretch of phosphorothioate modifications, combined with complete CU O′Me modification of the passenger strands, resulted in molecules that were highly effective in gene silencing. The effect of chemical modification on in vitro efficacy in un-assisted delivery in HeLa cells was also examined. FIG. 23 reveals that compounds lacking any of 2′F, 2′OMe, a stretch of phosphorothioate modifications, or cholesterol conjugates, were completely inactive in passive uptake. A combination of all 4 types of chemical modifications, for example in compound 12386, was found to be highly effective in gene silencing. FIG. 24 also shows the effectiveness of compound 12386 in gene silencing.
Optimization of the length of the oligonucleotide was also investigated. FIGS. 25 and 26 reveal that oligonucleotides with a length of 21 nucleotides were more effective than oligonucleotides with a length of 25 nucleotides, indicating that reduction in the size of an RNA molecule can improve efficiency, potentially by assisting in its uptake. Screening was also conducted to optimize the size of the duplex region of double stranded RNA molecules. FIG. 88 reveals that compounds with duplexes of 10 nucleotides were effective in inducing gene silencing. Positioning of the sense strand relative to the guide strand can also be critical for silencing gene expression (FIG. 89). In this assay, a blunt end was found to be most effective. 3′ overhangs were tolerated, but 5′ overhangs resulted in a complete loss of functionality. The guide strand can be effective in gene silencing when hybridized to a sense strand of varying lengths (FIG. 90). In this assay presented in FIG. 90, the compounds were introduced into HeLa cells via lipid mediated transfection.
The importance of phosphorothioate content of the RNA molecule for unassisted delivery was also investigated. FIG. 27 presents the results of a systematic screen that identified that the presence of at least 2-12 phosphorothioates in the guide strand as being highly advantageous for achieving uptake, with 4-8 being the preferred number. FIG. 27 also shows that presence or absence of phosphorothioate modifications in the sense strand did not alter efficacy.
FIGS. 28-29 reveal the effects of passive uptake of RNA compounds on gene silencing in primary mouse hepatocytes. nanoRNA molecules were found to be highly effective, especially at a concentration of 1 μM (FIG. 28). FIGS. 30 and 31 reveal that the RNA compounds associated with the invention were also effective in gene silencing following passive uptake in primary human hepatocytes. The cellular localization of the RNA molecules associated with the invention was examined and compared to the localization of Chol-siRNA (Alnylam) molecules, as shown in FIGS. 32 and 33.
A summary of 1st generation sd-rxRNA molecules is presented in FIG. 21. Chemical modifications were introduced into the RNA molecules, at least in part, to increase potency, such as through optimization of nucleotide length and phosphorothioate content, to reduce toxicity, such as through replacing 2′F modifications on the guide strand with other modifications, to improve delivery such as by adding or conjugating the RNA molecules to linker and sterol modalities, and to improve the ease of manufacturing the RNA molecules. FIG. 35 presents schematic depictions of some of the chemical modifications that were screened in 1st generation molecules. Parameters that were optimized for the guide strand included nucleotide length (e.g., 19, 21 and 25 nucleotides), phosphorothioate content (e.g., 0-18 phosphorothioate linkages) and replacement of 2′F groups with 2′OMe and 5 Me C or riboThymidine. Parameters that were optimized for the sense strand included nucleotide length (e.g., 11, 13 and 19 nucleotides), phosphorothioate content (e.g., 0-4 phosphorothioate linkages), and 2′OMe modifications. FIG. 36 summarizes parameters that were screened. For example, the nucleotide length and the phosphorothioate tail length were modified and screened for optimization, as were the additions of 2′OMe C and U modifications. Guide strand length and the length of the phosphorothioate modified stretch of nucleotides were found to influence efficacy (FIGS. 37-38). Phosphorothioate modifications were tolerated in the guide strand and were found to influence passive uptake (FIGS. 39-42).
FIG. 43 presents a schematic revealing guide strand chemical modifications that were screened. FIGS. 44 and 45 reveal that 2′OMe modifications were tolerated in the 3′ end of the guide strand. In particular, 2′OMe modifications in positions 1 and 11-18 were well tolerated. The 2′OMe modifications in the seed area were tolerated but resulted in slight reduction of efficacy. Ribo-modifications in the seed were also well tolerated. These data indicate that the molecules associated with the invention offer the significant advantage of having reduced or no 2′F modification content. This is advantageous because 2′F modifications are thought to generate toxicity in vivo. In some instances, a complete substitution of 2′F modifications with 2′OMe was found to lead to some reduction in potency. However, the 2′ OMe substituted molecules were still very active. A molecule with 50% reduction in 2′F content (including at positions 11, 16-18 which were changed to 2′OMe modifications), was found to have comparable efficacy to a compound with complete 2′F C and U modification. 2′OMe modification in position was found in some instances to reduce efficacy, although this can be at least partially compensated by 2′OMe modification in position 1 (with chemical phosphate). In some instances, 5 Me C and/or ribothymidine substitution for 2′F modifications led to a reduction in passive uptake efficacy, but increased potency in lipid mediated transfections compared to 2′F modifications. Optimization results for lipid mediated transfection were not necessarily the same as for passive uptake.
Modifications to the sense strand were also developed and tested, as depicted in FIG. 46. FIG. 47 reveals that in some instances, a sense strand length between 10-15 bases was found to be optimal. For the molecules tested in FIG. 47, an increase in the sense strand length resulted in reduction of passive uptake, however an increase in sense strand length may be tolerated for some compounds. FIG. 47 also reveals that LNA modification of the sense strand demonstrated similar efficacy to non-LNA containing compounds. In general, the addition of LNA or other thermodynamically stabilizing compounds has been found to be beneficial, in some instances resulting in converting non-functional sequences to functional sequences. FIG. 48 also presents data on sense strand length optimization, while FIG. 49 shows that phosphorothioate modification of the sense strand is not required for passive uptake.
Based on the above-described optimization experiments, 2nd generation RNA molecules were developed. As shown in FIG. 50, these molecules contained reduced phosphorothioate modification content and reduced 2′F modification content, relative to 1st generation RNA molecules. Significantly, these RNA molecules exhibit spontaneous cellular uptake and efficacy without a delivery vehicle (FIG. 51). These molecules can achieve self-delivery (i.e., with no transfection reagent) and following self-delivery can exhibit nanomolar activity in cell culture. These molecules can also be delivered using lipid-mediated transfection, and exhibit picomolar activity levels following transfection. Significantly, these molecules exhibit highly efficient uptake, 95% by most cells in cell culture, and are stable for more than three days in the presence of 100% human serum. These molecules are also highly specific and exhibit little or no immune induction. FIGS. 52 and 53 reveal the significance of chemical modifications and the configurations of such modifications in influencing the properties of the RNA molecules associated with the invention.
Linker chemistry was also tested in conjunction with the RNA molecules associated with the invention. As depicted in FIG. 54, 2nd generation RNA molecules were synthesized with sterol-type molecules attached through TEG and amino caproic acid linkers. Both linkers showed identical potency. This functionality of the RNA molecules, independent of linker chemistry offers additional advantages in terms of scale up and synthesis and demonstrates that the mechanism of function of these RNA molecules is very different from other previously described RNA molecules.
Stability of the chemically modified sd-rxRNA molecules described herein in human serum is shown in FIG. 55 in comparison to unmodified RNA. The duplex molecules were incubated in 75% serum at 37° C. for the indicated periods of time. The level of degradation was determined by running the samples on non-denaturing gels and staining with SYBGR.
FIGS. 56 and 57 present data on cellular uptake of the sd-rxRNA molecules. FIG. 56 shows that minimizing the length of the RNA molecule is importance for cellular uptake, while FIG. 57 presents data showing target gene silencing after spontaneous cellular uptake in mouse PEC-derived macrophages. FIG. 58 demonstrates spontaneous uptake and target gene silencing in primary cells. FIG. 59 shows the results of delivery of sd-rxRNA molecules associated with the invention to RPE cells with no formulation. Imaging with Hoechst and DY547 reveals the clear presence of a signal representing the RNA molecule in the sd-rxRNA sample, while no signal is detectable in the other samples including the samples competing a competing conjugate, an rxRNA, and an untransfected control. FIG. 60 reveals silencing of target gene expression in RPE cells treated with sd-rxRNA molecules associated with the invention following 24-48 hours without any transfection formulation.
FIG. 61 shows further optimization of the chemical/structural composition of sd-rxRNA compounds. In some instances, preferred properties included an antisense strand that was 17-21 nucleotides long, a sense strand that was 10-15 nucleotides long, phosphorothioate modification of 2-12 nucleotides within the single stranded region of the molecule, preferentially phosphorothioate modification of 6-8 nucleotides within the single stranded region, and 2′OMe modification at the majority of positions within the sense strand, with or without phosphorothioate modification. Any linker chemistry can be used to attach the hydrophobic moiety, such as cholesterol, to the 3′ end of the sense strand. Version GIIb molecules, as shown in FIG. 61, have no 2′F modifications. Significantly, there is was no impact on efficacy in these molecules.
FIG. 62 demonstrates the superior performance of sd-rxRNA compounds compared to compounds published by Wolfrum et. al. Nature Biotech, 2007. Both generation I and II compounds (GI and GIIa) developed herein show great efficacy in reducing target gene expression. By contrast, when the chemistry described in Wolfrum et al. (all oligos contain cholesterol conjugated to the 3′ end of the sense strand) was applied to the same sequence in a context of conventional siRNA (19 bp duplex with two overhang) the compound was practically inactive. These data emphasize the significance of the combination of chemical modifications and assymetrical molecules described herein, producing highly effective RNA compounds.
FIG. 63 shows localization of sd-rxRNA molecules developed herein compared to localization of other RNA molecules such as those described in Soutschek et al. (2004) Nature, 432:173. sd-rxRNA molecules accumulate inside the cells whereas competing conjugate RNAs accumulate on the surface of cells. Significantly, FIG. 64 shows that sd-rxRNA molecules, but not competitor molecules such as those described in Soutschek et al. are internalized within minutes. FIG. 65 compares localization of sd-rxRNA molecules compared to regular siRNA-cholesterol, as described in Soutschek et al. A signal representing the RNA molecule is clearly detected for the sd-rxRNA molecule in tissue culture RPE cells, following local delivery to compromised skin, and following systemic delivery where uptake to the liver is seen. In each case, no signal is detected for the regular siRNA-cholesterol molecule. The sd-rxRNA molecule thus has drastically better cellular and tissue uptake characteristics when compared to conventional cholesterol conjugated siRNAs such as those described in Soutschek et al. The level of uptake is at least order of magnitude higher and is due at least in part to the unique combination of chemistries and conjugated structure. Superior delivery of sd-rxRNA relative to previously described RNA molecules is also demonstrated in FIGS. 66 and 67.
Based on the analysis of 2nd generation RNA molecules associated with the invention, a screen was performed to identify functional molecules for targeting the SPP1/PPIB gene. As revealed in FIG. 68, several effective molecules were identified, with 14131 being the most effective. The compounds were added to A-549 cells and then the level of SPP1/PPIB ratio was determined by B-DNA after 48 hours.
FIG. 69 reveals efficient cellular uptake of sd-rxRNA within minutes of exposure. This is a unique characteristics of these molecules, not observed with any other RNAi compounds. Compounds described in Soutschek et al. were used as negative controls. FIG. 70 reveals that the uptake and gene silencing of the sd-rxRNA is effective in multiple different cell types including SH-SY5Y neuroblastoma derived cells, ARPE-19 (retinal pigment epithelium) cells, primary hepatocytes, and primary macrophages. In each case silencing was confirmed by looking at target gene expression by a Branched DNA assay.
FIG. 70 reveals that sd-rxRNA is active in the presence or absence of serum. While a slight reduction in efficacy (2-5 fold) was observed in the presence of serum, this small reduction in efficacy in the presence of serum differentiate the sd-rxRNA molecules from previously described molecules which exhibited a larger reduction in efficacy in the presence of serum. This demonstrated level of efficacy in the presence of serum creates a foundation for in vivo efficacy.
FIG. 72 reveals efficient tissue penetration and cellular uptake upon single intradermal injection. This data indicates the potential of the sd-rxRNA compounds described herein for silencing genes in any dermatology applications, and also represents a model for local delivery of sd-rxRNA compounds. FIG. 73 also demonstrates efficient cellular uptake and in vivo silencing with sd-rxRNA following intradermal injection. Silencing is determined as the level of MAP4K4 knockdown in several individual biopsies taken from the site of injection as compared to biopsies taken from a site injected with a negative control. FIG. 74 reveals that sd-rxRNA compounds has improved blood clearance and induced effective gene silencing in vivo in the liver upon systemic administration. In comparison to the RNA molecules described by Soutschek et al., the level of liver uptake at identical dose level is at least 50 fold higher with the sd-rxRNA molecules. The uptake results in productive silencing. sd-rxRNA compounds are also characterized by improved blood clearance kinetics.
The effect of 5-Methyl C modifications was also examined. FIG. 75 demonstrates that the presence of 5-Methyl C in an RNAi molecule resulted in increased potency in lipid mediated transfection. This suggests that hydrophobic modification of Cs and Us in an RNAi molecule can be beneficial. These types of modifications can also be used in the context 2′ ribose modified bases to ensure optimal stability and efficacy. FIG. 76 presents data showing that incorporation of 5-Methyl C and/or ribothymidine in the guide strand can in some instances reduce efficacy.
FIG. 77 reveals that sd-rxRNA molecules are more effective than competitor molecules such as molecules described in Soutschek et al., in systemic delivery to the liver. A signal representing the RNA molecule is clearly visible in the sample containing sd-rxRNA, while no signal representing the RNA molecule is visible in the sample containing the competitor RNA molecule.
The addition of hydrophobic conjugates to the sd-rxRNA molecules was also explored (FIGS. 78-83). FIG. 78 presents schematics demonstrating 5-uridyl modifications with improved hydrophobicity characteristics. Incorporation of such modifications into sd-rxRNA compounds can increase cellular and tissue uptake properties. FIG. 78B presents a new type of RNAi compound modification which can be applied to compounds to improve cellular uptake and pharmacokinetic behavior. Significantly, this type of modification, when applied to sd-rxRNA compounds, may contribute to making such compounds orally available. FIG. 79 presents schematics revealing the structures of synthesized modified sterol-type molecules, where the length and structure of the C17 attached tail is modified. Without wishing to be bound by any theory, the length of the C17 attached tail may contribute to improving in vitro and in vivo efficacy of sd-rxRNA compounds.
FIG. 80 presents a schematic demonstrating the lithocholic acid route to long side chain cholesterols. FIG. 81 presents a schematic demonstrating a route to 5-uridyl phosphoramidite synthesis. FIG. 82 presents a schematic demonstrating synthesis of tri-functional hydroxyprolinol linker for 3′-cholesterol attachment. FIG. 83 presents a schematic demonstrating synthesis of solid support for the manufacture of a shorter asymmetric RNAi compound strand.
A screen was conducted to identify compounds that could effectively silence expression of SPP1 (Osteopontin). Compounds targeting SPP1 were added to A549 cells (using passive transfection), and the level of SPPI expression was evaluated at 48 hours. Several novel compounds effective in SPPI silencing were identified. Compounds that were effective in silencing of SPPI included 14116, 14121, 14131, 14134, 14139, 14149, and 14152 (FIGS. 84-86). The most potent compound in this assay was 14131 (FIG. 84). The efficacy of these sd-rxRNA compounds in silencing SPP1 expression was independently validated (FIG. 85).
A similar screen was conducted to identify compounds that could effectively silence expression of CTGF (FIGS. 86-87). Compounds that were effective in silencing of CTGF included 14017, 14013, 14016, 14022, 14025, 14027.
Methods
Transfection of sd-rxRNAnano 
Lipid Mediated Transfection
sd-rxRNAnano constructs were chemically synthesized (Dharmacon, Lafayette, Colo.) and transfected into HEK293 cells (ATCC, Manassas, Va.) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. In brief, RNA was diluted to a 12× concentration in Opti-MEM® 1 Reduced Serum Media (Invitrogen, Carlsbad, Calif.) and then combined with a 12× concentration of Lipofectamine RNAiMAX. The RNA and transfection reagent were allowed to complex at room temperature for 20 minutes and make a 6× concentration. While complexing, HEK293 cells were washed, trypsinized and counted. The cells were diluted to a concentration recommended by the manufacturer and previously described of 1×105 cells/ml. When RNA had completed complexing with the RNAiMAX transfection reagent, 20 ul of the complexes were added to the appropriate well of the 96-well plate in triplicate. Cells were added to each well (100 ul volume) to make the final cell count per well 1×104 cells/well. The volume of cells diluted the 6× concentration of complex to 1× (between 10-0.05 nM). Cells were incubated for 24 or 48 hours under normal growth conditions. After 24 or 48 hour incubation, cells were lysed and gene silencing activity was measured using the QuantiGene assay (Panomics, Freemont, Calif.) which employs bDNA hybridization technology. The assay was carried out according to manufacturer's instructions.
Passive Uptake Transfection
sd-rxRNAnano constructs were chemically synthesized (Dharmacon, Lafayette, Colo.). 24 hours prior to transfection, HeLa cells (ATCC, Manassas, Va.) were plated at 1×104 cells/well in a 96 well plate under normal growth conditions (DMEM, 10% FBS and 1% Penicillin and Streptomycin). Prior to transfection of HeLa cells, sd-rxRNAnano were diluted to a final concentration of 0.01 uM to 1 uM in Accell siRNA Delivery Media (Dharmacon, Lafayette, Colo.). Normal growth media was aspirated off cells and 100 uL of Accell Delivery media containing the appropriate concentration of sd-rxRNAnano was applied to the cells. 48 hours post transfection, delivery media was aspirated off the cells and normal growth media was applied to cells for an additional 24 hours.
After 48 or 72 hour incubation, cells were lysed and gene silencing activity was measured using the QuantiGene assay (Panomics, Freemont, Calif.) according to manufacturer's instructions.











TABLE 1






Oligo
Accession

Gene


ID Number
Number
number
Gene Name
Symbol



















APOB-10167-
12138
NM_000384
Apolipoprotein B (including Ag(x)
APOB


20-12138


antigen)


APOB-10167-
12139
NM_000384
Apolipoprotein B (including Ag(x)
APOB


20-12139


antigen)


MAP4K4-
12266
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-13-


Kinase Kinase Kinase 4 (MAP4K4),


12266


transcript variant 1


MAP4K4-
12293
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12293


transcript variant 1


MAP4K4-
12383
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12383


transcript variant 1


MAP4K4-
12384
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12384


transcript variant 1


MAP4K4-
12385
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12385


transcript variant 1


MAP4K4-
12386
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12386


transcript variant 1


MAP4K4-
12387
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12387


transcript variant 1


MAP4K4-
12388
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-15-


Kinase Kinase Kinase 4 (MAP4K4),


12388


transcript variant 1


MAP4K4-
12432
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-13-


Kinase Kinase Kinase 4 (MAP4K4),


12432


transcript variant 1


MAP4K4-
12266.2
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-13-


Kinase Kinase Kinase 4 (MAP4K4),


12266.2


transcript variant 1


APOB--21-
12434
NM_000384
Apolipoprotein B (including Ag(x)
APOB


12434


antigen)


APOB--21-
12435
NM_000384
Apolipoprotein B (including Ag(x)
APOB


12435


antigen)


MAP4K4-
12451
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12451


transcript variant 1


MAP4K4-
12452
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12452


transcript variant 1


MAP4K4-
12453
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12453


transcript variant 1


MAP4K4-
12454
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-17-


Kinase Kinase Kinase 4 (MAP4K4),


12454


transcript variant 1


MAP4K4-
12455
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-17-


Kinase Kinase Kinase 4 (MAP4K4),


12455


transcript variant 1


MAP4K4-
12456
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-19-


Kinase Kinase Kinase 4 (MAP4K4),


12456


transcript variant 1


--27-12480
12480


--27-12481
12481


APOB-10167-
12505
NM_000384
Apolipoprotein B (including Ag(x)
APOB


21-12505


antigen)


APOB-10167-
12506
NM_000384
Apolipoprotein B (including Ag(x)
APOB


21-12506


antigen)


MAP4K4-
12539
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12539


transcript variant 1


APOB-10167-
12505.2
NM_000384
Apolipoprotein B (including Ag(x)
APOB


21-12505.2


antigen)


APOB-10167-
12506.2
NM_000384
Apolipoprotein B (including Ag(x)
APOB


21-12506.2


antigen)


MAP4K4--13-
12565


MAP4K4


12565


MAP4K4-
12386.2
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-16-


Kinase Kinase Kinase 4 (MAP4K4),


12386.2


transcript variant 1


MAP4K4-
12815
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-13-


Kinase Kinase Kinase 4 (MAP4K4),


12815


transcript variant 1


APOB--13-
12957
NM_000384
Apolipoprotein B (including Ag(x)
APOB


12957


antigen)


MAP4K4--16-
12983

Mitogen-Activated Protein Kinase
MAP4K4


12983


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12984

Mitogen-Activated Protein Kinase
MAP4K4


12984


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12985

Mitogen-Activated Protein Kinase
MAP4K4


12985


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12986

Mitogen-Activated Protein Kinase
MAP4K4


12986


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12987

Mitogen-Activated Protein Kinase
MAP4K4


12987


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12988

Mitogen-Activated Protein Kinase
MAP4K4


12988


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12989

Mitogen-Activated Protein Kinase
MAP4K4


12989


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12990

Mitogen-Activated Protein Kinase
MAP4K4


12990


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12991

Mitogen-Activated Protein Kinase
MAP4K4


12991


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12992

Mitogen-Activated Protein Kinase
MAP4K4


12992


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12993

Mitogen-Activated Protein Kinase
MAP4K4


12993


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12994

Mitogen-Activated Protein Kinase
MAP4K4


12994


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4--16-
12995

Mitogen-Activated Protein Kinase
MAP4K4


12995


Kinase Kinase Kinase 4 (MAP4K4),





transcript variant 1


MAP4K4-
13012
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-19-


Kinase Kinase Kinase 4 (MAP4K4),


13012


transcript variant 1


MAP4K4-
13016
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-19-


Kinase Kinase Kinase 4 (MAP4K4),


13016


transcript variant 1


PPIB--13-
13021
NM_000942
Peptidylprolyl Isomerase B
PPIB


13021


(cyclophilin B)


pGL3-1172-
13038
U47296
Cloning vector pGL3-Control
pGL3


13-13038


pGL3-1172-
13040
U47296
Cloning vector pGL3-Control
pGL3


13-13040


--16-13047
13047


SOD1-530-
13090
NM_000454
Superoxide Dismutase 1, soluble
SOD1


13-13090


(amyotrophic lateral sclerosis 1





(adult))


SOD1-523-
13091
NM_000454
Superoxide Dismutase 1, soluble
SOD1


13-13091


(amyotrophic lateral sclerosis 1





(adult))


SOD1-535-
13092
NM_000454
Superoxide Dismutase 1, soluble
SOD1


13-13092


(amyotrophic lateral sclerosis 1





(adult))


SOD1-536-
13093
NM_000454
Superoxide Dismutase 1, soluble
SOD1


13-13093


(amyotrophic lateral sclerosis 1





(adult))


SOD1-396-
13094
NM_000454
Superoxide Dismutase 1, soluble
SOD1


13-13094


(amyotrophic lateral sclerosis 1





(adult))


SOD1-385-
13095
NM_000454
Superoxide Dismutase 1, soluble
SOD1


13-13095


(amyotrophic lateral sclerosis 1





(adult))


SOD1-195-
13096
NM_000454
Superoxide Dismutase 1, soluble
SOD1


13-13096


(amyotrophic lateral sclerosis 1





(adult))


APOB-4314-
13115
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13-13115


antigen)


APOB-3384-
13116
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13-13116


antigen)


APOB-3547-
13117
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13-13117


antigen)


APOB-4318-
13118
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13-13118


antigen)


APOB-3741-
13119
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13-13119


antigen)


PPIB--16-
13136
NM_000942
Peptidylprolyl Isomerase B
PPIB


13136


(cyclophilin B)


APOB-4314-
13154
NM_000384
Apolipoprotein B (including Ag(x)
APOB


15-13154


antigen)


APOB-3547-
13155
NM_000384
Apolipoprotein B (including Ag(x)
APOB


15-13155


antigen)


APOB-4318-
13157
NM_000384
Apolipoprotein B (including Ag(x)
APOB


15-13157


antigen)


APOB-3741-
13158
NM_000384
Apolipoprotein B (including Ag(x)
APOB


15-13158


antigen)


APOB--13-
13159
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13159


antigen)


APOB--15-
13160
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13160


antigen)


SOD1-530-
13163
NM_000454
Superoxide Dismutase 1, soluble
SOD1


16-13163


(amyotrophic lateral sclerosis 1





(adult))


SOD1-523-
13164
NM_000454
Superoxide Dismutase 1, soluble
SOD1


16-13164


(amyotrophic lateral sclerosis 1





(adult))


SOD1-535-
13165
NM_000454
Superoxide Dismutase 1, soluble
SOD1


16-13165


(amyotrophic lateral sclerosis 1





(adult))


SOD1-536-
13166
NM_000454
Superoxide Dismutase 1, soluble
SOD1


16-13166


(amyotrophic lateral sclerosis 1





(adult))


SOD1-396-
13167
NM_000454
Superoxide Dismutase 1, soluble
SOD1


16-13167


(amyotrophic lateral sclerosis 1





(adult))


SOD1-385-
13168
NM_000454
Superoxide Dismutase 1, soluble
SOD1


16-13168


(amyotrophic lateral sclerosis 1





(adult))


SOD1-195-
13169
NM_000454
Superoxide Dismutase 1, soluble
SOD1


16-13169


(amyotrophic lateral sclerosis 1





(adult))


pGL3-1172-
13170
U47296
Cloning vector pGL3-Control
pGL3


16-13170


pGL3-1172-
13171
U47296
Cloning vector pGL3-Control
pGL3


16-13171


MAP4k4-
13189
NM_004834
Mitogen-Activated Protein Kinase
MAP4k4


2931-19-


Kinase Kinase Kinase 4 (MAP4K4),


13189


transcript variant 1


CTGF-1222-
13190
NM_001901.2
connective tissue growth factor
CTGF


13-13190


CTGF-813-
13192
NM_001901.2
connective tissue growth factor
CTGF


13-13192


CTGF-747-
13194
NM_001901.2
connective tissue growth factor
CTGF


13-13194


CTGF-817-
13196
NM_001901.2
connective tissue growth factor
CTGF


13-13196


CTGF-1174-
13198
NM_001901.2
connective tissue growth factor
CTGF


13-13198


CTGF-1005-
13200
NM_001901.2
connective tissue growth factor
CTGF


13-13200


CTGF-814-
13202
NM_001901.2
connective tissue growth factor
CTGF


13-13202


CTGF-816-
13204
NM_001901.2
connective tissue growth factor
CTGF


13-13204


CTGF-1001-
13206
NM_001901.2
connective tissue growth factor
CTGF


13-13206


CTGF-1173-
13208
NM_001901.2
connective tissue growth factor
CTGF


13-13208


CTGF-749-
13210
NM_001901.2
connective tissue growth factor
CTGF


13-13210


CTGF-792-
13212
NM_001901.2
connective tissue growth factor
CTGF


13-13212


CTGF-1162-
13214
NM_001901.2
connective tissue growth factor
CTGF


13-13214


CTGF-811-
13216
NM_001901.2
connective tissue growth factor
CTGF


13-13216


CTGF-797-
13218
NM_001901.2
connective tissue growth factor
CTGF


13-13218


CTGF-1175-
13220
NM_001901.2
connective tissue growth factor
CTGF


13-13220


CTGF-1172-
13222
NM_001901.2
connective tissue growth factor
CTGF


13-13222


CTGF-1177-
13224
NM_001901.2
connective tissue growth factor
CTGF


13-13224


CTGF-1176-
13226
NM_001901.2
connective tissue growth factor
CTGF


13-13226


CTGF-812-
13228
NM_001901.2
connective tissue growth factor
CTGF


13-13228


CTGF-745-
13230
NM_001901.2
connective tissue growth factor
CTGF


13-13230


CTGF-1230-
13232
NM_001901.2
connective tissue growth factor
CTGF


13-13232


CTGF-920-
13234
NM_001901.2
connective tissue growth factor
CTGF


13-13234


CTGF-679-
13236
NM_001901.2
connective tissue growth factor
CTGF


13-13236


CTGF-992-
13238
NM_001901.2
connective tissue growth factor
CTGF


13-13238


CTGF-1045-
13240
NM_001901.2
connective tissue growth factor
CTGF


13-13240


CTGF-1231-
13242
NM_001901.2
connective tissue growth factor
CTGF


13-13242


CTGF-991-
13244
NM_001901.2
connective tissue growth factor
CTGF


13-13244


CTGF-998-
13246
NM_001901.2
connective tissue growth factor
CTGF


13-13246


CTGF-1049-
13248
NM_001901.2
connective tissue growth factor
CTGF


13-13248


CTGF-1044-
13250
NM_001901.2
connective tissue growth factor
CTGF


13-13250


CTGF-1327-
13252
NM_001901.2
connective tissue growth factor
CTGF


13-13252


CTGF-1196-
13254
NM_001901.2
connective tissue growth factor
CTGF


13-13254


CTGF-562-
13256
NM_001901.2
connective tissue growth factor
CTGF


13-13256


CTGF-752-
13258
NM_001901.2
connective tissue growth factor
CTGF


13-13258


CTGF-994-
13260
NM_001901.2
connective tissue growth factor
CTGF


13-13260


CTGF-1040-
13262
NM_001901.2
connective tissue growth factor
CTGF


13-13262


CTGF-1984-
13264
NM_001901.2
connective tissue growth factor
CTGF


13-13264


CTGF-2195-
13266
NM_001901.2
connective tissue growth factor
CTGF


13-13266


CTGF-2043-
13268
NM_001901.2
connective tissue growth factor
CTGF


13-13268


CTGF-1892-
13270
NM_001901.2
connective tissue growth factor
CTGF


13-13270


CTGF-1567-
13272
NM_001901.2
connective tissue growth factor
CTGF


13-13272


CTGF-1780-
13274
NM_001901.2
connective tissue growth factor
CTGF


13-13274


CTGF-2162-
13276
NM_001901.2
connective tissue growth factor
CTGF


13-13276


CTGF-1034-
13278
NM_001901.2
connective tissue growth factor
CTGF


13-13278


CTGF-2264-
13280
NM_001901.2
connective tissue growth factor
CTGF


13-13280


CTGF-1032-
13282
NM_001901.2
connective tissue growth factor
CTGF


13-13282


CTGF-1535-
13284
NM_001901.2
connective tissue growth factor
CTGF


13-13284


CTGF-1694-
13286
NM_001901.2
connective tissue growth factor
CTGF


13-13286


CTGF-1588-
13288
NM_001901.2
connective tissue growth factor
CTGF


13-13288


CTGF-928-
13290
NM_001901.2
connective tissue growth factor
CTGF


13-13290


CTGF-1133-
13292
NM_001901.2
connective tissue growth factor
CTGF


13-13292


CTGF-912-
13294
NM_001901.2
connective tissue growth factor
CTGF


13-13294


CTGF-753-
13296
NM_001901.2
connective tissue growth factor
CTGF


13-13296


CTGF-918-
13298
NM_001901.2
connective tissue growth factor
CTGF


13-13298


CTGF-744-
13300
NM_001901.2
connective tissue growth factor
CTGF


13-13300


CTGF-466-
13302
NM_001901.2
connective tissue growth factor
CTGF


13-13302


CTGF-917-
13304
NM_001901.2
connective tissue growth factor
CTGF


13-13304


CTGF-1038-
13306
NM_001901.2
connective tissue growth factor
CTGF


13-13306


CTGF-1048-
13308
NM_001901.2
connective tissue growth factor
CTGF


13-13308


CTGF-1235-
13310
NM_001901.2
connective tissue growth factor
CTGF


13-13310


CTGF-868-
13312
NM_001901.2
connective tissue growth factor
CTGF


13-13312


CTGF-1131-
13314
NM_001901.2
connective tissue growth factor
CTGF


13-13314


CTGF-1043-
13316
NM_001901.2
connective tissue growth factor
CTGF


13-13316


CTGF-751-
13318
NM_001901.2
connective tissue growth factor
CTGF


13-13318


CTGF-1227-
13320
NM_001901.2
connective tissue growth factor
CTGF


13-13320


CTGF-867-
13322
NM_001901.2
connective tissue growth factor
CTGF


13-13322


CTGF-1128-
13324
NM_001901.2
connective tissue growth factor
CTGF


13-13324


CTGF-756-
13326
NM_001901.2
connective tissue growth factor
CTGF


13-13326


CTGF-1234-
13328
NM_001901.2
connective tissue growth factor
CTGF


13-13328


CTGF-916-
13330
NM_001901.2
connective tissue growth factor
CTGF


13-13330


CTGF-925-
13332
NM_001901.2
connective tissue growth factor
CTGF


13-13332


CTGF-1225-
13334
NM_001901.2
connective tissue growth factor
CTGF


13-13334


CTGF-445-
13336
NM_001901.2
connective tissue growth factor
CTGF


13-13336


CTGF-446-
13338
NM_001901.2
connective tissue growth factor
CTGF


13-13338


CTGF-913-
13340
NM_001901.2
connective tissue growth factor
CTGF


13-13340


CTGF-997-
13342
NM_001901.2
connective tissue growth factor
CTGF


13-13342


CTGF-277-
13344
NM_001901.2
connective tissue growth factor
CTGF


13-13344


CTGF-1052-
13346
NM_001901.2
connective tissue growth factor
CTGF


13-13346


CTGF-887-
13348
NM_001901.2
connective tissue growth factor
CTGF


13-13348


CTGF-914-
13350
NM_001901.2
connective tissue growth factor
CTGF


13-13350


CTGF-1039-
13352
NM_001901.2
connective tissue growth factor
CTGF


13-13352


CTGF-754-
13354
NM_001901.2
connective tissue growth factor
CTGF


13-13354


CTGF-1130-
13356
NM_001901.2
connective tissue growth factor
CTGF


13-13356


CTGF-919-
13358
NM_001901.2
connective tissue growth factor
CTGF


13-13358


CTGF-922-
13360
NM_001901.2
connective tissue growth factor
CTGF


13-13360


CTGF-746-
13362
NM_001901.2
connective tissue growth factor
CTGF


13-13362


CTGF-993-
13364
NM_001901.2
connective tissue growth factor
CTGF


13-13364


CTGF-825-
13366
NM_001901.2
connective tissue growth factor
CTGF


13-13366


CTGF-926-
13368
NM_001901.2
connective tissue growth factor
CTGF


13-13368


CTGF-923-
13370
NM_001901.2
connective tissue growth factor
CTGF


13-13370


CTGF-866-
13372
NM_001901.2
connective tissue growth factor
CTGF


13-13372


CTGF-563-
13374
NM_001901.2
connective tissue growth factor
CTGF


13-13374


CTGF-823-
13376
NM_001901.2
connective tissue growth factor
CTGF


13-13376


CTGF-1233-
13378
NM_001901.2
connective tissue growth factor
CTGF


13-13378


CTGF-924-
13380
NM_001901.2
connective tissue growth factor
CTGF


13-13380


CTGF-921-
13382
NM_001901.2
connective tissue growth factor
CTGF


13-13382


CTGF-443-
13384
NM_001901.2
connective tissue growth factor
CTGF


13-13384


CTGF-1041-
13386
NM_001901.2
connective tissue growth factor
CTGF


13-13386


CTGF-1042-
13388
NM_001901.2
connective tissue growth factor
CTGF


13-13388


CTGF-755-
13390
NM_001901.2
connective tissue growth factor
CTGF


13-13390


CTGF-467-
13392
NM_001901.2
connective tissue growth factor
CTGF


13-13392


CTGF-995-
13394
NM_001901.2
connective tissue growth factor
CTGF


13-13394


CTGF-927-
13396
NM_001901.2
connective tissue growth factor
CTGF


13-13396


SPP1-1025-
13398
NM_000582.2
Osteopontin
SPP1


13-13398


SPP1-1049-
13400
NM_000582.2
Osteopontin
SPP1


13-13400


SPP1-1051-
13402
NM_000582.2
Osteopontin
SPP1


13-13402


SPP1-1048-
13404
NM_000582.2
Osteopontin
SPP1


13-13404


SPP1-1050-
13406
NM_000582.2
Osteopontin
SPP1


13-13406


SPP1-1047-
13408
NM_000582.2
Osteopontin
SPP1


13-13408


SPP1-800-
13410
NM_000582.2
Osteopontin
SPP1


13-13410


SPP1-492-
13412
NM_000582.2
Osteopontin
SPP1


13-13412


SPP1-612-
13414
NM_000582.2
Osteopontin
SPP1


13-13414


SPP1-481-
13416
NM_000582.2
Osteopontin
SPP1


13-13416


SPP1-614-
13418
NM_000582.2
Osteopontin
SPP1


13-13418


SPP1-951-
13420
NM_000582.2
Osteopontin
SPP1


13-13420


SPP1-482-
13422
NM_000582.2
Osteopontin
SPP1


13-13422


SPP1-856-
13424
NM_000582.2
Osteopontin
SPP1


13-13424


SPP1-857-
13426
NM_000582.2
Osteopontin
SPP1


13-13426


SPP1-365-
13428
NM_000582.2
Osteopontin
SPP1


13-13428


SPP1-359-
13430
NM_000582.2
Osteopontin
SPP1


13-13430


SPP1-357-
13432
NM_000582.2
Osteopontin
SPP1


13-13432


SPP1-858-
13434
NM_000582.2
Osteopontin
SPP1


13-13434


SPP1-1012-
13436
NM_000582.2
Osteopontin
SPP1


13-13436


SPP1-1014-
13438
NM_000582.2
Osteopontin
SPP1


13-13438


SPP1-356-
13440
NM_000582.2
Osteopontin
SPP1


13-13440


SPP1-368-
13442
NM_000582.2
Osteopontin
SPP1


13-13442


SPP1-1011-
13444
NM_000582.2
Osteopontin
SPP1


13-13444


SPP1-754-
13446
NM_000582.2
Osteopontin
SPP1


13-13446


SPP1-1021-
13448
NM_000582.2
Osteopontin
SPP1


13-13448


SPP1-1330-
13450
NM_000582.2
Osteopontin
SPP1


13-13450


SPP1-346-
13452
NM_000582.2
Osteopontin
SPP1


13-13452


SPP1-869-
13454
NM_000582.2
Osteopontin
SPP1


13-13454


SPP1-701-
13456
NM_000582.2
Osteopontin
SPP1


13-13456


SPP1-896-
13458
NM_000582.2
Osteopontin
SPP1


13-13458


SPP1-1035-
13460
NM_000582.2
Osteopontin
SPP1


13-13460


SPP1-1170-
13462
NM_000582.2
Osteopontin
SPP1


13-13462


SPP1-1282-
13464
NM_000582.2
Osteopontin
SPP1


13-13464


SPP1-1537-
13466
NM_000582.2
Osteopontin
SPP1


13-13466


SPP1-692-
13468
NM_000582.2
Osteopontin
SPP1


13-13468


SPP1-840-
13470
NM_000582.2
Osteopontin
SPP1


13-13470


SPP1-1163-
13472
NM_000582.2
Osteopontin
SPP1


13-13472


SPP1-789-
13474
NM_000582.2
Osteopontin
SPP1


13-13474


SPP1-841-
13476
NM_000582.2
Osteopontin
SPP1


13-13476


SPP1-852-
13478
NM_000582.2
Osteopontin
SPP1


13-13478


SPP1-209-
13480
NM_000582.2
Osteopontin
SPP1


13-13480


SPP1-1276-
13482
NM_000582.2
Osteopontin
SPP1


13-13482


SPP1-137-
13484
NM_000582.2
Osteopontin
SPP1


13-13484


SPP1-711-
13486
NM_000582.2
Osteopontin
SPP1


13-13486


SPP1-582-
13488
NM_000582.2
Osteopontin
SPP1


13-13488


SPP1-839-
13490
NM_000582.2
Osteopontin
SPP1


13-13490


SPP1-1091-
13492
NM_000582.2
Osteopontin
SPP1


13-13492


SPP1-884-
13494
NM_000582.2
Osteopontin
SPP1


13-13494


SPP1-903-
13496
NM_000582.2
Osteopontin
SPP1


13-13496


SPP1-1090-
13498
NM_000582.2
Osteopontin
SPP1


13-13498


SPP1-474-
13500
NM_000582.2
Osteopontin
SPP1


13-13500


SPP1-575-
13502
NM_000582.2
Osteopontin
SPP1


13-13502


SPP1-671-
13504
NM_000582.2
Osteopontin
SPP1


13-13504


SPP1-924-
13506
NM_000582.2
Osteopontin
SPP1


13-13506


SPP1-1185-
13508
NM_000582.2
Osteopontin
SPP1


13-13508


SPP1-1221-
13510
NM_000582.2
Osteopontin
SPP1


13-13510


SPP1-347-
13512
NM_000582.2
Osteopontin
SPP1


13-13512


SPP1-634-
13514
NM_000582.2
Osteopontin
SPP1


13-13514


SPP1-877-
13516
NM_000582.2
Osteopontin
SPP1


13-13516


SPP1-1033-
13518
NM_000582.2
Osteopontin
SPP1


13-13518


SPP1-714-
13520
NM_000582.2
Osteopontin
SPP1


13-13520


SPP1-791-
13522
NM_000582.2
Osteopontin
SPP1


13-13522


SPP1-813-
13524
NM_000582.2
Osteopontin
SPP1


13-13524


SPP1-939-
13526
NM_000582.2
Osteopontin
SPP1


13-13526


SPP1-1161-
13528
NM_000582.2
Osteopontin
SPP1


13-13528


SPP1-1164-
13530
NM_000582.2
Osteopontin
SPP1


13-13530


SPP1-1190-
13532
NM_000582.2
Osteopontin
SPP1


13-13532


SPP1-1333-
13534
NM_000582.2
Osteopontin
SPP1


13-13534


SPP1-537-
13536
NM_000582.2
Osteopontin
SPP1


13-13536


SPP1-684-
13538
NM_000582.2
Osteopontin
SPP1


13-13538


SPP1-707-
13540
NM_000582.2
Osteopontin
SPP1


13-13540


SPP1-799-
13542
NM_000582.2
Osteopontin
SPP1


13-13542


SPP1-853-
13544
NM_000582.2
Osteopontin
SPP1


13-13544


SPP1-888-
13546
NM_000582.2
Osteopontin
SPP1


13-13546


SPP1-1194-
13548
NM_000582.2
Osteopontin
SPP1


13-13548


SPP1-1279-
13550
NM_000582.2
Osteopontin
SPP1


13-13550


SPP1-1300-
13552
NM_000582.2
Osteopontin
SPP1


13-13552


SPP1-1510-
13554
NM_000582.2
Osteopontin
SPP1


13-13554


SPP1-1543-
13556
NM_000582.2
Osteopontin
SPP1


13-13556


SPP1-434-
13558
NM_000582.2
Osteopontin
SPP1


13-13558


SPP1-600-
13560
NM_000582.2
Osteopontin
SPP1


13-13560


SPP1-863-
13562
NM_000582.2
Osteopontin
SPP1


13-13562


SPP1-902-
13564
NM_000582.2
Osteopontin
SPP1


13-13564


SPP1-921-
13566
NM_000582.2
Osteopontin
SPP1


13-13566


SPP1-154-
13568
NM_000582.2
Osteopontin
SPP1


13-13568


SPP1-217-
13570
NM_000582.2
Osteopontin
SPP1


13-13570


SPP1-816-
13572
NM_000582.2
Osteopontin
SPP1


13-13572


SPP1-882-
13574
NM_000582.2
Osteopontin
SPP1


13-13574


SPP1-932-
13576
NM_000582.2
Osteopontin
SPP1


13-13576


SPP1-1509-
13578
NM_000582.2
Osteopontin
SPP1


13-13578


SPP1-157-
13580
NM_000582.2
Osteopontin
SPP1


13-13580


SPP1-350-
13582
NM_000582.2
Osteopontin
SPP1


13-13582


SPP1-511-
13584
NM_000582.2
Osteopontin
SPP1


13-13584


SPP1-605-
13586
NM_000582.2
Osteopontin
SPP1


13-13586


SPP1-811-
13588
NM_000582.2
Osteopontin
SPP1


13-13588


SPP1-892-
13590
NM_000582.2
Osteopontin
SPP1


13-13590


SPP1-922-
13592
NM_000582.2
Osteopontin
SPP1


13-13592


SPP1-1169-
13594
NM_000582.2
Osteopontin
SPP1


13-13594


SPP1-1182-
13596
NM_000582.2
Osteopontin
SPP1


13-13596


SPP1-1539-
13598
NM_000582.2
Osteopontin
SPP1


13-13598


SPP1-1541-
13600
NM_000582.2
Osteopontin
SPP1


13-13600


SPP1-427-
13602
NM_000582.2
Osteopontin
SPP1


13-13602


SPP1-533-
13604
NM_000582.2
Osteopontin
SPP1


13-13604


APOB--13-
13763
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13763


antigen)


APOB--13-
13764
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13764


antigen)


MAP4K4--16-
13766


MAP4K4


13766


PPIB--13-
13767
NM_000942
peptidylprolyl isomerase B
PPIB


13767


(cyclophilin B)


PPIB--15-
13768
NM_000942
peptidylprolyl isomerase B
PPIB


13768


(cyclophilin B)


PPIB--17-
13769
NM_000942
peptidylprolyl isomerase B
PPIB


13769


(cyclophilin B)


MAP4K4--16-
13939


MAP4K4


13939


APOB-4314-
13940
NM_000384
Apolipoprotein B (including Ag(x)
APOB


16-13940


antigen)


APOB-4314-
13941
NM_000384
Apolipoprotein B (including Ag(x)
APOB


17-13941


antigen)


APOB--16-
13942
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13942


antigen)


APOB--18-
13943
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13943


antigen)


APOB--17-
13944
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13944


antigen)


APOB--19-
13945
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13945


antigen)


APOB-4314-
13946
NM_000384
Apolipoprotein B (including Ag(x)
APOB


16-13946


antigen)


APOB-4314-
13947
NM_000384
Apolipoprotein B (including Ag(x)
APOB


17-13947


antigen)


APOB--16-
13948
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13948


antigen)


APOB--17-
13949
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13949


antigen)


APOB--16-
13950
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13950


antigen)


APOB--18-
13951
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13951


antigen)


APOB--17-
13952
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13952


antigen)


APOB--19-
13953
NM_000384
Apolipoprotein B (including Ag(x)
APOB


13953


antigen)


MAP4K4--16-
13766.2


MAP4K4


13766.2


CTGF-1222-
13980
NM_001901.2
connective tissue growth factor
CTGF


16-13980


CTGF-813-
13981
NM_001901.2
connective tissue growth factor
CTGF


16-13981


CTGF-747-
13982
NM_001901.2
connective tissue growth factor
CTGF


16-13982


CTGF-817-
13983
NM_001901.2
connective tissue growth factor
CTGF


16-13983


CTGF-1174-
13984
NM_001901.2
connective tissue growth factor
CTGF


16-13984


CTGF-1005-
13985
NM_001901.2
connective tissue growth factor
CTGF


16-13985


CTGF-814-
13986
NM_001901.2
connective tissue growth factor
CTGF


16-13986


CTGF-816-
13987
NM_001901.2
connective tissue growth factor
CTGF


16-13987


CTGF-1001-
13988
NM_001901.2
connective tissue growth factor
CTGF


16-13988


CTGF-1173-
13989
NM_001901.2
connective tissue growth factor
CTGF


16-13989


CTGF-749-
13990
NM_001901.2
connective tissue growth factor
CTGF


16-13990


CTGF-792-
13991
NM_001901.2
connective tissue growth factor
CTGF


16-13991


CTGF-1162-
13992
NM_001901.2
connective tissue growth factor
CTGF


16-13992


CTGF-811-
13993
NM_001901.2
connective tissue growth factor
CTGF


16-13993


CTGF-797-
13994
NM_001901.2
connective tissue growth factor
CTGF


16-13994


CTGF-1175-
13995
NM_001901.2
connective tissue growth factor
CTGF


16-13995


CTGF-1172-
13996
NM_001901.2
connective tissue growth factor
CTGF


16-13996


CTGF-1177-
13997
NM_001901.2
connective tissue growth factor
CTGF


16-13997


CTGF-1176-
13998
NM_001901.2
connective tissue growth factor
CTGF


16-13998


CTGF-812-
13999
NM_001901.2
connective tissue growth factor
CTGF


16-13999


CTGF-745-
14000
NM_001901.2
connective tissue growth factor
CTGF


16-14000


CTGF-1230-
14001
NM_001901.2
connective tissue growth factor
CTGF


16-14001


CTGF-920-
14002
NM_001901.2
connective tissue growth factor
CTGF


16-14002


CTGF-679-
14003
NM_001901.2
connective tissue growth factor
CTGF


16-14003


CTGF-992-
14004
NM_001901.2
connective tissue growth factor
CTGF


16-14004


CTGF-1045-
14005
NM_001901.2
connective tissue growth factor
CTGF


16-14005


CTGF-1231-
14006
NM_001901.2
connective tissue growth factor
CTGF


16-14006


CTGF-991-
14007
NM_001901.2
connective tissue growth factor
CTGF


16-14007


CTGF-998-
14008
NM_001901.2
connective tissue growth factor
CTGF


16-14008


CTGF-1049-
14009
NM_001901.2
connective tissue growth factor
CTGF


16-14009


CTGF-1044-
14010
NM_001901.2
connective tissue growth factor
CTGF


16-14010


CTGF-1327-
14011
NM_001901.2
connective tissue growth factor
CTGF


16-14011


CTGF-1196-
14012
NM_001901.2
connective tissue growth factor
CTGF


16-14012


CTGF-562-
14013
NM_001901.2
connective tissue growth factor
CTGF


16-14013


CTGF-752-
14014
NM_001901.2
connective tissue growth factor
CTGF


16-14014


CTGF-994-
14015
NM_001901.2
connective tissue growth factor
CTGF


16-14015


CTGF-1040-
14016
NM_001901.2
connective tissue growth factor
CTGF


16-14016


CTGF-1984-
14017
NM_001901.2
connective tissue growth factor
CTGF


16-14017


CTGF-2195-
14018
NM_001901.2
connective tissue growth factor
CTGF


16-14018


CTGF-2043-
14019
NM_001901.2
connective tissue growth factor
CTGF


16-14019


CTGF-1892-
14020
NM_001901.2
connective tissue growth factor
CTGF


16-14020


CTGF-1567-
14021
NM_001901.2
connective tissue growth factor
CTGF


16-14021


CTGF-1780-
14022
NM_001901.2
connective tissue growth factor
CTGF


16-14022


CTGF-2162-
14023
NM_001901.2
connective tissue growth factor
CTGF


16-14023


CTGF-1034-
14024
NM_001901.2
connective tissue growth factor
CTGF


16-14024


CTGF-2264-
14025
NM_001901.2
connective tissue growth factor
CTGF


16-14025


CTGF-1032-
14026
NM_001901.2
connective tissue growth factor
CTGF


16-14026


CTGF-1535-
14027
NM_001901.2
connective tissue growth factor
CTGF


16-14027


CTGF-1694-
14028
NM_001901.2
connective tissue growth factor
CTGF


16-14028


CTGF-1588-
14029
NM_001901.2
connective tissue growth factor
CTGF


16-14029


CTGF-928-
14030
NM_001901.2
connective tissue growth factor
CTGF


16-14030


CTGF-1133-
14031
NM_001901.2
connective tissue growth factor
CTGF


16-14031


CTGF-912-
14032
NM_001901.2
connective tissue growth factor
CTGF


16-14032


CTGF-753-
14033
NM_001901.2
connective tissue growth factor
CTGF


16-14033


CTGF-918-
14034
NM_001901.2
connective tissue growth factor
CTGF


16-14034


CTGF-744-
14035
NM_001901.2
connective tissue growth factor
CTGF


16-14035


CTGF-466-
14036
NM_001901.2
connective tissue growth factor
CTGF


16-14036


CTGF-917-
14037
NM_001901.2
connective tissue growth factor
CTGF


16-14037


CTGF-1038-
14038
NM_001901.2
connective tissue growth factor
CTGF


16-14038


CTGF-1048-
14039
NM_001901.2
connective tissue growth factor
CTGF


16-14039


CTGF-1235-
14040
NM_001901.2
connective tissue growth factor
CTGF


16-14040


CTGF-868-
14041
NM_001901.2
connective tissue growth factor
CTGF


16-14041


CTGF-1131-
14042
NM_001901.2
connective tissue growth factor
CTGF


16-14042


CTGF-1043-
14043
NM_001901.2
connective tissue growth factor
CTGF


16-14043


CTGF-751-
14044
NM_001901.2
connective tissue growth factor
CTGF


16-14044


CTGF-1227-
14045
NM_001901.2
connective tissue growth factor
CTGF


16-14045


CTGF-867-
14046
NM_001901.2
connective tissue growth factor
CTGF


16-14046


CTGF-1128-
14047
NM_001901.2
connective tissue growth factor
CTGF


16-14047


CTGF-756-
14048
NM_001901.2
connective tissue growth factor
CTGF


16-14048


CTGF-1234-
14049
NM_001901.2
connective tissue growth factor
CTGF


16-14049


CTGF-916-
14050
NM_001901.2
connective tissue growth factor
CTGF


16-14050


CTGF-925-
14051
NM_001901.2
connective tissue growth factor
CTGF


16-14051


CTGF-1225-
14052
NM_001901.2
connective tissue growth factor
CTGF


16-14052


CTGF-445-
14053
NM_001901.2
connective tissue growth factor
CTGF


16-14053


CTGF-446-
14054
NM_001901.2
connective tissue growth factor
CTGF


16-14054


CTGF-913-
14055
NM_001901.2
connective tissue growth factor
CTGF


16-14055


CTGF-997-
14056
NM_001901.2
connective tissue growth factor
CTGF


16-14056


CTGF-277-
14057
NM_001901.2
connective tissue growth factor
CTGF


16-14057


CTGF-1052-
14058
NM_001901.2
connective tissue growth factor
CTGF


16-14058


CTGF-887-
14059
NM_001901.2
connective tissue growth factor
CTGF


16-14059


CTGF-914-
14060
NM_001901.2
connective tissue growth factor
CTGF


16-14060


CTGF-1039-
14061
NM_001901.2
connective tissue growth factor
CTGF


16-14061


CTGF-754-
14062
NM_001901.2
connective tissue growth factor
CTGF


16-14062


CTGF-1130-
14063
NM_001901.2
connective tissue growth factor
CTGF


16-14063


CTGF-919-
14064
NM_001901.2
connective tissue growth factor
CTGF


16-14064


CTGF-922-
14065
NM_001901.2
connective tissue growth factor
CTGF


16-14065


CTGF-746-
14066
NM_001901.2
connective tissue growth factor
CTGF


16-14066


CTGF-993-
14067
NM_001901.2
connective tissue growth factor
CTGF


16-14067


CTGF-825-
14068
NM_001901.2
connective tissue growth factor
CTGF


16-14068


CTGF-926-
14069
NM_001901.2
connective tissue growth factor
CTGF


16-14069


CTGF-923-
14070
NM_001901.2
connective tissue growth factor
CTGF


16-14070


CTGF-866-
14071
NM_001901.2
connective tissue growth factor
CTGF


16-14071


CTGF-563-
14072
NM_001901.2
connective tissue growth factor
CTGF


16-14072


CTGF-823-
14073
NM_001901.2
connective tissue growth factor
CTGF


16-14073


CTGF-1233-
14074
NM_001901.2
connective tissue growth factor
CTGF


16-14074


CTGF-924-
14075
NM_001901.2
connective tissue growth factor
CTGF


16-14075


CTGF-921-
14076
NM_001901.2
connective tissue growth factor
CTGF


16-14076


CTGF-443-
14077
NM_001901.2
connective tissue growth factor
CTGF


16-14077


CTGF-1041-
14078
NM_001901.2
connective tissue growth factor
CTGF


16-14078


CTGF-1042-
14079
NM_001901.2
connective tissue growth factor
CTGF


16-14079


CTGF-755-
14080
NM_001901.2
connective tissue growth factor
CTGF


16-14080


CTGF-467-
14081
NM_001901.2
connective tissue growth factor
CTGF


16-14081


CTGF-995-
14082
NM_001901.2
connective tissue growth factor
CTGF


16-14082


CTGF-927-
14083
NM_001901.2
connective tissue growth factor
CTGF


16-14083


SPP1-1091-
14131
NM_000582.2
Osteopontin
SPP1


16-14131


PPIB--16-
14188
NM_000942
peptidylprolyl isomerase B
PPIB


14188


(cyclophilin B)


PPIB--17-
14189
NM_000942
peptidylprolyl isomerase B
PPIB


14189


(cyclophilin B)


PPIB--18-
14190
NM_000942
peptidylprolyl isomerase B
PPIB


14190


(cyclophilin B)


pGL3-1172-
14386
U47296
Cloning vector pGL3-Control
pGL3


16-14386


pGL3-1172-
14387
U47296
Cloning vector pGL3-Control
pGL3


16-14387


MAP4K4-
14390
NM_004834
Mitogen-Activated Protein Kinase
MAP4K4


2931-25-


Kinase Kinase Kinase 4 (MAP4K4),


14390


transcript variant 1


miR-122--
14391


miR-


23-14391



122



14084
NM_000582.2
Osteopontin
SPP1



14085
NM_000582.2
Osteopontin
SPP1



14086
NM_000582.2
Osteopontin
SPP1



14087
NM_000582.2
Osteopontin
SPP1



14088
NM_000582.2
Osteopontin
SPP1



14089
NM_000582.2
Osteopontin
SPP1



14090
NM_000582.2
Osteopontin
SPP1



14091
NM_000582.2
Osteopontin
SPP1



14092
NM_000582.2
Osteopontin
SPP1



14093
NM_000582.2
Osteopontin
SPP1



14094
NM_000582.2
Osteopontin
SPP1



14095
NM_000582.2
Osteopontin
SPP1



14096
NM_000582.2
Osteopontin
SPP1



14097
NM_000582.2
Osteopontin
SPP1



14098
NM_000582.2
Osteopontin
SPP1



14099
NM_000582.2
Osteopontin
SPP1



14100
NM_000582.2
Osteopontin
SPP1



14101
NM_000582.2
Osteopontin
SPP1



14102
NM_000582.2
Osteopontin
SPP1



14103
NM_000582.2
Osteopontin
SPP1



14104
NM_000582.2
Osteopontin
SPP1



14105
NM_000582.2
Osteopontin
SPP1



14106
NM_000582.2
Osteopontin
SPP1



14107
NM_000582.2
Osteopontin
SPP1



14108
NM_000582.2
Osteopontin
SPP1



14109
NM_000582.2
Osteopontin
SPP1



14110
NM_000582.2
Osteopontin
SPP1



14111
NM_000582.2
Osteopontin
SPP1



14112
NM_000582.2
Osteopontin
SPP1



14113
NM_000582.2
Osteopontin
SPP1



14114
NM_000582.2
Osteopontin
SPP1



14115
NM_000582.2
Osteopontin
SPP1



14116
NM_000582.2
Osteopontin
SPP1



14117
NM_000582.2
Osteopontin
SPP1



14118
NM_000582.2
Osteopontin
SPP1



14119
NM_000582.2
Osteopontin
SPP1



14120
NM_000582.2
Osteopontin
SPP1



14121
NM_000582.2
Osteopontin
SPP1



14122
NM_000582.2
Osteopontin
SPP1



14123
NM_000582.2
Osteopontin
SPP1



14124
NM_000582.2
Osteopontin
SPP1



14125
NM_000582.2
Osteopontin
SPP1



14126
NM_000582.2
Osteopontin
SPP1



14127
NM_000582.2
Osteopontin
SPP1



14128
NM_000582.2
Osteopontin
SPP1



14129
NM_000582.2
Osteopontin
SPP1



14130
NM_000582.2
Osteopontin
SPP1



14132
NM_000582.2
Osteopontin
SPP1



14133
NM_000582.2
Osteopontin
SPP1



14134
NM_000582.2
Osteopontin
SPP1



14135
NM_000582.2
Osteopontin
SPP1



14136
NM_000582.2
Osteopontin
SPP1



14137
NM_000582.2
Osteopontin
SPP1



14138
NM_000582.2
Osteopontin
SPP1



14139
NM_000582.2
Osteopontin
SPP1



14140
NM_000582.2
Osteopontin
SPP1



14141
NM_000582.2
Osteopontin
SPP1



14142
NM_000582.2
Osteopontin
SPP1



14143
NM_000582.2
Osteopontin
SPP1



14144
NM_000582.2
Osteopontin
SPP1



14145
NM_000582.2
Osteopontin
SPP1



14146
NM_000582.2
Osteopontin
SPP1



14147
NM_000582.2
Osteopontin
SPP1



14148
NM_000582.2
Osteopontin
SPP1



14149
NM_000582.2
Osteopontin
SPP1



14150
NM_000582.2
Osteopontin
SPP1



14151
NM_000582.2
Osteopontin
SPP1



14152
NM_000582.2
Osteopontin
SPP1



14153
NM_000582.2
Osteopontin
SPP1



14154
NM_000582.2
Osteopontin
SPP1



14155
NM_000582.2
Osteopontin
SPP1



14156
NM_000582.2
Osteopontin
SPP1



14157
NM_000582.2
Osteopontin
SPP1



14158
NM_000582.2
Osteopontin
SPP1



14159
NM_000582.2
Osteopontin
SPP1



14160
NM_000582.2
Osteopontin
SPP1



14161
NM_000582.2
Osteopontin
SPP1



14162
NM_000582.2
Osteopontin
SPP1



14163
NM_000582.2
Osteopontin
SPP1



14164
NM_000582.2
Osteopontin
SPP1



14165
NM_000582.2
Osteopontin
SPP1



14166
NM_000582.2
Osteopontin
SPP1



14167
NM_000582.2
Osteopontin
SPP1



14168
NM_000582.2
Osteopontin
SPP1



14169
NM_000582.2
Osteopontin
SPP1



14170
NM_000582.2
Osteopontin
SPP1



14171
NM_000582.2
Osteopontin
SPP1



14172
NM_000582.2
Osteopontin
SPP1



14173
NM_000582.2
Osteopontin
SPP1



14174
NM_000582.2
Osteopontin
SPP1



14175
NM_000582.2
Osteopontin
SPP1



14176
NM_000582.2
Osteopontin
SPP1



14177
NM_000582.2
Osteopontin
SPP1



14178
NM_000582.2
Osteopontin
SPP1



14179
NM_000582.2
Osteopontin
SPP1



14180
NM_000582.2
Osteopontin
SPP1



14181
NM_000582.2
Osteopontin
SPP1



14182
NM_000582.2
Osteopontin
SPP1



14183
NM_000582.2
Osteopontin
SPP1



14184
NM_000582.2
Osteopontin
SPP1



14185
NM_000582.2
Osteopontin
SPP1



14186
NM_000582.2
Osteopontin
SPP1



14187
NM_000582.2
Osteopontin
SPP1
















TABLE 2







Antisense backbone, chemistry, and sequence information.













ID
Oligo
AntiSense
AntiSense
AntiSense
SEQ ID



Number
Number
Backbone
Chemistry
Sequence
NO:
















APOB-
12138
oooooooooooooooo
00000000000000
AUUGGUAUUCAGUGUGA
1



10167-

ooo
000000m
UG


20-12138


 


APOB-
12139
oooooooooooooooo
00000000000000
AUUCGUAUUGAGUCUGA
2


10167-

ooo
000000m
UC


20-12139


 


MAP4K4-
12266


2931-13-


12266


 


MAP4K4-
12293
oooooooooooooooo
Pf000fffff0f00
UAGACUUCCACAGAACU
3


2931-16-

ooo
00fff0
CU


12293


 


MAP4K4-
12383
oooooooooooooooo
00000000000000
UAGACUUCCACAGAACU
4


2931-16-

ooo
00000
CU


12383


 


MAP4K4-
12384
oooooooooooooooo
P0000000000000
UAGACUUCCACAGAACU
5


2931-16-

ooo
000000
CU


12384


 


MAP4K4-
12385
oooooooooooooooo
Pf000fffff0f00
UAGACUUCCACAGAACU
6


2931-16-

ooo
00fff0
CU


12385


 


MAP4K4-
12386
oooooooooossssss
Pf000fffff0f00
UAGACUUCCACAGAACU
7


2931-16-

sso
00fff0
CU


12386


 


MAP4K4-
12387
oooooooooossssss
P0000000000000
UAGACUUCCACAGAACU
8


2931-16-

sso
000000
CU


12387


 


MAP4K4-
12388
ooooooooooooooooo
00000000000000
UAGACUUCCACAGAACU
9


2931-15-


000


12388


 


MAP4K4-
12432


2931-13-


12432


 


MAP4K4-
12266.2


2931-13-


12266.2


 


APOB--
12434
oooooooooooooooo
00000000000000
AUUGGUAUUCAGUGUGA
10


21-12434

ooooo
000000m
UGAC


 


APOB--
12435
oooooooooooooooo
00000000000000
AUUCGUAUUGAGUCUGA
11


21-12435

ooooo
000000m
UCAC


 


MAP4K4-
12451
oooooooooossssss
Pf000fffff0f00
UAGACUUCCACAGAACU
12


2931-16-

sso
00ffmm
CU


12451


 


MAP4K4-
12452
oooooooooossssss
Pm000fffff0f00
UAGACUUCCACAGAACU
13


2931-16-

sso
00ffmm
CU


12452


 


MAP4K4-
12453
oooooossssssssss
Pm000fffff0f00
UAGACUUCCACAGAACU
14


2931-16-

sso
00ffmm
CU


12453


 


MAP4K4-
12454
oooooooooooossss
Pm000fffff0f00
UAGACUUCCACAGAACU
15


2931-17-

sssso
00ffffmm
CUUC


12454


 


MAP4K4-
12455
oooooooossssssss
Pm000fffff0f00
UAGACUUCCACAGAACU
16


2931-17-

sssso
00ffffmm
CUUC


12455


 


MAP4K4-
12456
oooooooooooossss
Pm000fffff0f00
UAGACUUCCACAGAACU
17


2931-19-

sssssssso
00ffffff00mm
CUUCAAAG


12456


 


--27-
12480


12480


 


--27-
12481


12481


 


APOB-
12505
oooooooooooooooo
00000000000000
AUUGGUAUUCAGUGUGA
18


10167-

oooos
000000m
UGAC


21-12505


 


APOB-
12506
oooooooooooooooo
00000000000000
AUUCGUAUUGAGUCUGA
19


10167-

oooos
000000m
UCAC


21-12506


 


MAP4K4-
12539
ooooooooooosssss
Pf000fffff0f00
UAGACUUCCACAGAACU
20


2931-16-

sss
00fff0
CU


12539


 


APOB-
12505.2
oooooooooooooooo
00000000000000
AUUGGUAUUCAGUGUGA
21


10167-

ooooo
000000m
UGAC


21-


12505.2


 


APOB-
12506.2
oooooooooooooooo
00000000000000
AUUCGUAUUGAGUCUGA
22


10167-

ooooo
000000m
UCAC


21-


12506.2


 


MAP4K4--
12565


13-12565


 


MAP4K4-
12386.2
oooooooooossssss
Pf000fffff0f00
UAGACUUCCACAGAACU
23


2931-16-

sso
00fff0
CU


12386.2


 


MAP4K4-
12815


2931-13-


12815


 


APOB--
12957


13-12957


 


MAP4K4--
12983
oooooooooooossss
Pm000fffff0m00
uagacuuccacagaacu
24


16-12983

sso
00mmm0
cu


 


MAP4K4--
12984
oooooooooooossss
Pm000fffff0m00
uagacuuccacagaacu
25


16-12984

ss
00mmm0
cu


 


MAP4K4--
12985
oooooooooooossss
Pm000fffff0m00
uagacuuccacagaacu
26


16-12985

sso
00mmm0
cu


 


MAP4K4--
12986
oooooooooossssss
Pf000fffff0f00
UAGACUUCCACAGAACU
27


16-12986

sso
00fff0
CU


 


MAP4K4--
12987
ooooooooooooosss
P0000f00ff0m00
UagacUUccacagaacU
28


16-12987

sss
00m0m0
cU


 


MAP4K4--
12988
ooooooooooooosss
P0000f00ff0m00
UagacUUccacagaacU
29


16-12988

sss
00m0m0
cu


 


MAP4K4--
12989
ooooooooooooosss
P0000ff0ff0m00
UagacuUccacagaacU
30


16-12989

sss
00m0m0
cu


 


MAP4K4--
12990
ooooooooooooosss
Pf0000ff000000
uagaCuuCCaCagaaCu
31


16-12990

sss
000m00
Cu


 


MAP4K4--
12991
ooooooooooooosss
Pf0000fff00m00
uagaCuucCacagaaCu
32


16-12991

sss
000mm0
cu


 


MAP4K4--
12992
ooooooooooooosss
Pf000fffff0000
uagacuuccaCagaaCu
33


16-12992

sss
000m00
Cu


 


MAP4K4--
12993
ooooooooooooosss
P0000000000000
UagaCUUCCaCagaaCU
34


16-12993

sss
000000
CU


 


MAP4K4--
12994
ooooooooooooosss
P0000f0f0f0000
UagacUuCcaCagaaCu
35


16-12994

sss
000m00
Cu


 


MAP4K4--
12995
oooooooooooossss
Pf000fffff0000
uagacuuccaCagaaCU
36


16-12995

sso
000000
CU


 


MAP4K4-
13012


2931-19-


13012


 


MAP4K4-
13016


2931-19-


13016


 


PPIB--
13021


13-13021


 


pGL3-
13038


1172-13-


13038


 


pGL3-
13040


1172-13-


13040


 


--16-
13047
oooooooooooossss
Pm000000000m00
UAGACUUCCACAGAACU
37


13047

ss
00mmm0
CU


 


SOD1-
13090


530-13-


13090


 


SOD1-
13091


523-13-


13091


 


SOD1-
13092


535-13-


13092


 


SOD1-
13093


536-13-


13093


 


SOD1-
13094


396-13-


13094


 


SOD1-
13095


385-13-


13095


 


SOD1-
13096


195-13-


13096


 


APOB-
13115


4314-13-


13115


 


APOB-
13116


3384-13-


13116


 


APOB-
13117


3547-13-


13117


 


APOB-
13118


4318-13-


13118


 


APOB-
13119


3741-13-


13119


 


PPIB--
13136
oooooooooooossss
Pm0fffff0f00mm
UGUUUUUGUAGCCAAAU
38


16-13136

ss
000mm0
CC


 


APOB-
13154


4314-15-


13154


 


APOB-
13155


3547-15-


13155


 


APOB-
13157


4318-15-


13157


 


APOB-
13158


3741-15-


13158


 


APOB--
13159


13-13159


 


APOB--
13160


15-13160


 


SOD1-
13163
oooooooooooossss
Pm0ffffffff0mm
UACUUUCUUCAUUUCCA
39


530-16-

sso
mmm0m0
CC


13163


 


SOD1-
13164
oooooooooooossss
Pmff0fffff0fmm
UUCAUUUCCACCUUUGC
40


523-16-

sso
mm0mm0
CC


13164


 


SOD1-
13165
oooooooooooossss
Pmfff0f0ffffmm
CUUUGUACUUUCUUCAU
41


535-16-

sso
mm0mm0
UU


13165


 


SOD1-
13166
oooooooooooossss
Pmffff0f0fffmm
UCUUUGUACUUUCUUCA
42


536-16-

sso
mmm0m0
UU


13166


 


SOD1-
13167
oooooooooooossss
Pmf00f00ff0f0m
UCAGCAGUCACAUUGCC
43


396-16-

sso
m0mmm0
CA


13167


 


SOD1-
13168
oooooooooooossss
Pmff0fff000fmm
AUUGCCCAAGUCUCCAA
44


385-16-

sso
mm00m0
CA


13168


 


SOD1-
13169
oooooooooooossss
Pmfff0fff0000m
UUCUGCUCGAAAUUGAU
45


195-16-

sso
m00m00
GA


13169


 


pGL3-
13170
oooooooooooossss
Pm00ff0f0ffm0f
AAAUCGUAUUUGUCAAU
46


1172-16-

sso
f00mm0
CA


13170


 


pGL3-
13171
ooooooooooooosss
Pm00ff0f0ffm0f
AAAUCGUAUUUGUCAAU
47


1172-16-

sss
f00mm0
CA


13171


 


MAP4k4-
13189
oooooooooooooooo
00000000000000
UAGACUUCCACAGAACU
48


2931-19-

ooo
00000
CU


13189


 


CTGF-
13190


1222-13-


13190


 


CTGF-
13192


813-13-


13192


 


CTGF-
13194


747-13-


13194


 


CTGF-
13196


817-13-


13196


 


CTGF-
13198


1174-13-


13198


 


CTGF-
13200


1005-13-


13200


 


CTGF-
13202


814-13-


13202


 


CTGF-
13204


816-13-


13204


 


CTGF-
13206


1001-13-


13206


 


CTGF-
13208


1173-13-


13208


 


CTGF-
13210


749-13-


13210


 


CTGF-
13212


792-13-


13212


 


CTGF-
13214


1162-13-


13214


 


CTGF-
13216


811-13-


13216


 


CTGF-
13218


797-13-


13218


 


CTGF-
13220


1175-13-


13220


 


CTGF-
13222


1172-13-


13222


 


CTGF-
13224


1177-13-


13224


 


CTGF-
13226


1176-13-


13226


 


CTGF-
13228


812-13-


13228


 


CTGF-
13230


745-13-


13230


 


CTGF-
13232


1230-13-


13232


 


CTGF-
13234


920-13-


13234


 


CTGF-
13236


679-13-


13236


 


CTGF-
13238


992-13-


13238


 


CTGF-
13240


1045-13-


13240


 


CTGF-
13242


1231-13-


13242


 


CTGF-
13244


991-13-


13244


 


CTGF-
13246


998-13-


13246


 


CTGF-
13248


1049-13-


13248


 


CTGF-
13250


1044-13-


13250


 


CTGF-
13252


1327-13-


13252


 


CTGF-
13254


1196-13-


13254


 


CTGF-
13256


562-13-


13256


 


CTGF-
13258


752-13-


13258


 


CTGF-
13260


994-13-


13260


 


CTGF-
13262


1040-13-


13262


 


CTGF-
13264


1984-13-


13264


 


CTGF-
13266


2195-13-


13266


 


CTGF-
13268


2043-13-


13268


 


CTGF-
13270


1892-13-


13270


 


CTGF-
13272


1567-13-


13272


 


CTGF-
13274


1780-13-


13274


 


CTGF-
13276


2162-13-


13276


 


CTGF-
13278


1034-13-


13278


 


CTGF-
13280


2264-13-


13280


 


CTGF-
13282


1032-13-


13282


 


CTGF-
13284


1535-13-


13284


 


CTGF-
13286


1694-13-


13286


 


CTGF-
13288


1588-13-


13288


 


CTGF-
13290


928-13-


13290


 


CTGF-
13292


1133-13-


13292


 


CTGF-
13294


912-13-


13294


 


CTGF-
13296


753-13-


13296


 


CTGF-
13298


918-13-


13298


 


CTGF-
13300


744-13-


13300


 


CTGF-
13302


466-13-


13302


 


CTGF-
13304


917-13-


13304


 


CTGF-
13306


1038-13-


13306


 


CTGF-
13308


1048-13-


13308


 


CTGF-
13310


1235-13-


13310


 


CTGF-
13312


868-13-


13312


 


CTGF-
13314


1131-13-


13314


 


CTGF-
13316


1043-13-


13316


 


CTGF-
13318


751-13-


13318


 


CTGF-
13320


1227-13-


13320


 


CTGF-
13322


867-13-


13322


 


CTGF-
13324


1128-13-


13324


 


CTGF-
13326


756-13-


13326


 


CTGF-
13328


1234-13-


13328


 


CTGF-
13330


916-13-


13330


 


CTGF-
13332


925-13-


13332


 


CTGF-
13334


1225-13-


13334


 


CTGF-
13336


445-13-


13336


 


CTGF-
13338


446-13-


13338


 


CTGF-
13340


913-13-


13340


 


CTGF-
13342


997-13-


13342


 


CTGF-
13344


277-13-


13344


 


CTGF-
13346


1052-13-


13346


 


CTGF-
13348


887-13-


13348


 


CTGF-
13350


914-13-


13350


 


CTGF-
13352


1039-13-


13352


 


CTGF-
13354


754-13-


13354


 


CTGF-
13356


1130-13-


13356


 


CTGF-
13358


919-13-


13358


 


CTGF-
13360


922-13-


13360


 


CTGF-
13362


746-13-


13362


 


CTGF-
13364


993-13-


13364


 


CTGF-
13366


825-13-


13366


 


CTGF-
13368


926-13-


13368


 


CTGF-
13370


923-13-


13370


 


CTGF-
13372


866-13-


13372


 


CTGF-
13374


563-13-


13374


 


CTGF-
13376


823-13-


13376


 


CTGF-
13378


1233-13-


13378


 


CTGF-
13380


924-13-


13380


 


CTGF-
13382


921-13-


13382


 


CTGF-
13384


443-13-


13384


 


CTGF-
13386


1041-13-


13386


 


CTGF-
13388


1042-13-


13388


 


CTGF-
13390


755-13-


13390


 


CTGF-
13392


467-13-


13392


 


CTGF-
13394


995-13-


13394


 


CTGF-
13396


927-13-


13396


 


SPP1-
13398


1025-13-


13398


 


SPP1-
13400


1049-13-


13400


 


SPP1-
13402


1051-13-


13402


 


SPP1-
13404


1048-13-


13404


 


SPP1-
13406


1050-13-


13406


 


SPP1-
13408


1047-13-


13408


 


SPP1-
13410


800-13-


13410


 


SPP1-
13412


492-13-


13412


 


SPP1-
13414


612-13-


13414


 


SPP1-
13416


481-13-


13416


 


SPP1-
13418


614-13-


13418


 


SPP1-
13420


951-13-


13420


 


SPP1-
13422


482-13-


13422


 


SPP1-
13424


856-13-


13424


 


SPP1-
13426


857-13-


13426


 


SPP1-
13428


365-13-


13428


 


SPP1-
13430


359-13-


13430


 


SPP1-
13432


357-13-


13432


 


SPP1-
13434


858-13-


13434


 


SPP1-
13436


1012-13-


13436


 


SPP1-
13438


1014-13-


13438


 


SPP1-
13440


356-13-


13440


 


SPP1-
13442


368-13-


13442


 


SPP1-
13444


1011-13-


13444


 


SPP1-
13446


754-13-


13446


 


SPP1-
13448


1021-13-


13448


 


SPP1-
13450


1330-13-


13450


 


SPP1-
13452


346-13-


13452


 


SPP1-
13454


869-13-


13454


 


SPP1-
13456


701-13-


13456


 


SPP1-
13458


896-13-


13458


 


SPP1-
13460


1035-13-


13460


 


SPP1-
13462


1170-13-


13462


 


SPP1-
13464


1282-13-


13464


 


SPP1-
13466


1537-13-


13466


 


SPP1-
13468


692-13-


13468


 


SPP1-
13470


840-13-


13470


 


SPP1-
13472


1163-13-


13472


 


SPP1-
13474


789-13-


13474


 


SPP1-
13476


841-13-


13476


 


SPP1-
13478


852-13-


13478


 


SPP1-
13480


209-13-


13480


 


SPP1-
13482


1276-13-


13482


 


SPP1-
13484


137-13-


13484


 


SPP1-
13486


711-13-


13486


 


SPP1-
13488


582-13-


13488


 


SPP1-
13490


839-13-


13490


 


SPP1-
13492


1091-13-


13492


 


SPP1-
13494


884-13-


13494


 


SPP1-
13496


903-13-


13496


 


SPP1-
13498


1090-13-


13498


 


SPP1-
13500


474-13-


13500


 


SPP1-
13502


575-13-


13502


 


SPP1-
13504


671-13-


13504


 


SPP1-
13506


924-13-


13506


 


SPP1-
13508


1185-13-


13508


 


SPP1-
13510


1221-13-


13510


 


SPP1-
13512


347-13-


13512


 


SPP1-
13514


634-13-


13514


 


SPP1-
13516


877-13-


13516


 


SPP1-
13518


1033-13-


13518


 


SPP1-
13520


714-13-


13520


 


SPP1-
13522


791-13-


13522


 


SPP1-
13524


813-13-


13524


 


SPP1-
13526


939-13-


13526


 


SPP1-
13528


1161-13-


13528


 


SPP1-
13530


1164-13-


13530


 


SPP1-
13532


1190-13-


13532


 


SPP1-
13534


1333-13-


13534


 


SPP1-
13536


537-13-


13536


 


SPP1-
13538


684-13-


13538


 


SPP1-
13540


707-13-


13540


 


SPP1-
13542


799-13-


13542


 


SPP1-
13544


853-13-


13544


 


SPP1-
13546


888-13-


13546


 


SPP1-
13548


1194-13-


13548


 


SPP1-
13550


1279-13-


13550


 


SPP1-
13552


1300-13-


13552


 


SPP1-
13554


1510-13-


13554


 


SPP1-
13556


1543-13-


13556


 


SPP1-
13558


434-13-


13558


 


SPP1-
13560


600-13-


13560


 


SPP1-
13562


863-13-


13562


 


SPP1-
13564


902-13-


13564


 


SPP1-
13566


921-13-


13566


 


SPP1-
13568


154-13-


13568


 


SPP1-
13570


217-13-


13570


 


SPP1-
13572


816-13-


13572


 


SPP1-
13574


882-13-


13574


 


SPP1-
13576


932-13-


13576


 


SPP1-
13578


1509-13-


13578


 


SPP1-
13580


157-13-


13580


 


SPP1-
13582


350-13-


13582


 


SPP1-
13584


511-13-


13584


 


SPP1-
13586


605-13-


13586


 


SPP1-
13588


811-13-


13588


 


SPP1-
13590


892-13-


13590


 


SPP1-
13592


922-13-


13592


 


SPP1-
13594


1169-13-


13594


 


SPP1-
13596


1182-13-


13596


 


SPP1-
13598


1539-13-


13598


 


SPP1-
13600


1541-13-


13600


 


SPP1-
13602


427-13-


13602


 


SPP1-
13604


533-13-


13604


 


APOB--
13763


13-13763


 


APOB--
13764


13-13764


 


MAP4K4--
13766
oooooooooooossss
Pm000fffff0m00
UAGACUUCCACAGAACU
49


16-13766

sso
00mmm0
CU


 


PPIB--
13767


13-13767


 


PPIB--
13768


15-13768


 


PPIB--
13769


17-13769


 


MAP4K4--
13939
oooooooooooossss
m000f0ffff0m0m
UAGACAUCCUACACAGC
50


16-13939

sso
00m0m
AC


 


APOB-
13940
oooooooooooossss
Pm0fffffff000m
UGUUUCUCCAGAUCCUU
51


4314-16-

sso
mmmm00
GC


13940


 


APOB-
13941
oooooooooooossss
Pm0fffffff000m
UGUUUCUCCAGAUCCUU
52


4314-17-

sso
mmmm00
GC


13941


 


APOB--
13942
oooooooooooossss
Pm00f000f000mm
UAGCAGAUGAGUCCAUU
53


16-13942

sso
m0mmm0
UG


 


APOB--
13943
oooooooooooooooo
Pm00f000f000mm
UAGCAGAUGAGUCCAUU
54


18-13943

sssssso
m0mmm00000
UGGAGA


 


APOB--
13944
oooooooooooossss
Pm00f000f000mm
UAGCAGAUGAGUCCAUU
55


17-13944

sso
m0mmm0
UG


 


APOB--
13945
oooooooooooooooo
Pm00f000f000mm
UAGCAGAUGAGUCCAUU
56


19-13945

sssssso
m0mmm00000
UGGAGA


 


APOB-
13946
oooooooooooossss
Pmf0ff0ffffmmm
AUGUUGUUUCUCCAGAU
57


4314-16-

sso
000mm0
CC


13946


 


APOB-
13947
oooooooooooossss
Pmf0ff0ffffmmm
AUGUUGUUUCUCCAGAU
58


4314-17-

sso
000mm0
CC


13947


 


APOB--
13948
oooooooooooossss
Pm0fff000000mm
UGUUUGAGGGACUCUGU
59


16-13948

sso
mm0m00
GA


 


APOB--
13949
oooooooooooossss
Pm0fff000000mm
UGUUUGAGGGACUCUGU
60


17-13949

sso
mm0m00
GA


 


APOB--
13950
oooooooooooossss
Pmff00f0fff00m
AUUGGUAUUCAGUGUGA
61


16-13950

sso
0m00m0
UG


 


APOB--
13951
oooooooooooooooo
Pmff00f0fff00m
AUUGGUAUUCAGUGUGA
62


18-13951

sssssso
0m00m00m00
UGACAC


 


APOB--
13952
oooooooooooossss
Pmff00f0fff00m
AUUGGUAUUCAGUGUGA
63


17-13952

sso
0m00m0
UG


 


APOB--
13953
oooooooooooooooo
Pmff00f0fff00m
AUUGGUAUUCAGUGUGA
64


19-13953

sssssso
0m00m00m00
UGACAC


 


MAP4K4--
13766.2
oooooooooooossss
Pm000fffff0m00
UAGACUUCCACAGAACU
65


16-

sso
00mmm0
CU


13766.2


 


CTGF-
13980
oooooooooooossss
Pm0f0ffffffm0m
UACAUCUUCCUGUAGUA
66


1222-16-

sso
00m0m0
CA


13980


 


CTGF-
13981
oooooooooooossss
Pm0f0ffff0mmmm
AGGCGCUCCACUCUGUG
67


813-16-

sso
0m000
GU


13981


 


CTGF-
13982
oooooooooooossss
Pm0ffffff00mm0
UGUCUUCCAGUCGGUAA
68


747-16-

sso
m0000
GC


13982


 


CTGF-
13983
oooooooooooossss
Pm00f000f0fmmm
GAACAGGCGCUCCACUC
69


817-16-

sso
0mmmm0
UG


13983


 


CTGF-
13984
oooooooooooossss
Pm00ff0f00f00m
CAGUUGUAAUGGCAGGC
70


1174-16-

sso
000m00
AC


13984


 


CTGF-
13985
oooooooooooossss
Pmff000000mmm0
AGCCAGAAAGCUCAAAC
71


1005-16-

sso
00mm0
UU


13985


 


CTGF-
13986
oooooooooooossss
Pm000f0ffff0mm
CAGGCGCUCCACUCUGU
72


814-16-

sso
mm0m00
GG


13986


 


CTGF-
13987
oooooooooooossss
Pm0f000f0ffmm0
AACAGGCGCUCCACUCU
73


816-16-

sso
mmmm00
GU


13987


 


CTGF-
13988
oooooooooooossss
Pm0000fff000mm
AGAAAGCUCAAACUUGA
74


1001-16-

sso
m00m0
UA


13988


 


CTGF-
13989
oooooooooooossss
Pmff0f00f00m00
AGUUGUAAUGGCAGGCA
75


1173-16-

sso
0m0m0
CA


13989


 


CTGF-
13990
oooooooooooossss
Pmf0ffffff00mm
CGUGUCUUCCAGUCGGU
76


749-16-

sso
00m00
AA


13990


 


CTGF-
13991
oooooooooooossss
Pm00ff000f00mm
GGACCAGGCAGUUGGCU
77


792-16-

sso
00mmm0
CU


13991


 


CTGF-
13992
oooooooooooossss
Pm000f0f000mmm
CAGGCACAGGUCUUGAU
78


1162-16-

sso
m00m00
GA


13992


 


CTGF-
13993
oooooooooooossss
Pmf0ffff0ffmm0
GCGCUCCACUCUGUGGU
79


811-16-

sso
m00mm0
CU


13993


 


CTGF-
13994
oooooooooooossss
Pm0fff000ff000
GGUCUGGACCAGGCAGU
80


797-16-

sso
m00mm0
UG


13994


 


CTGF-
13995
oooooooooooossss
Pmf00ff0f00m00
ACAGUUGUAAUGGCAGG
81


1175-16-

sso
m000m0
CA


13995


 


CTGF-
13996
oooooooooooossss
Pmff0f00f00m00
GUUGUAAUGGCAGGCAC
82


1172-16-

sso
0m0m00
AG


13996


 


CTGF-
13997
oooooooooooossss
Pm00f00ff0f00m
GGACAGUUGUAAUGGCA
83


1177-16-

sso
00m000
GG


13997


 


CTGF-
13998
oooooooooooossss
Pm0f00ff0f00m0
GACAGUUGUAAUGGCAG
84


1176-16-

sso
0m0000
GC


13998


 


CTGF-
13999
oooooooooooossss
Pm0f0ffff0fmmm
GGCGCUCCACUCUGUGG
85


812-16-

sso
0m00m0
UC


13999


 


CTGF-
14000
oooooooooooossss
Pmfffff00ff00m
UCUUCCAGUCGGUAAGC
86


745-16-

sso
000mm0
CG


14000


 


CTGF-
14001
oooooooooooossss
Pm0fffff0f0m0m
UGUCUCCGUACAUCUUC
87


1230-16-

sso
mmmmm0
CU


14001


 


CTGF-
14002
oooooooooooossss
Pmffff0f0000mm
AGCUUCGCAAGGCCUGA
88


920-16-

sso
m00m0
CC


14002


 


CTGF-
14003
oooooooooooossss
Pm0ffffff0f00m
CACUCCUCGCAGCAUUU
89


679-16-

sso
0mmmm0
CC


14003


 


CTGF-
14004
oooooooooooossss
Pm00fff00f000m
AAACUUGAUAGGCUUGG
90


992-16-

sso
mm0000
AG


14004


 


CTGF-
14005
oooooooooooossss
Pmffff0f0000mm
ACUCCACAGAAUUUAGC
91


1045-16-

sso
m00mm0
UC


14005


 


CTGF-
14006
oooooooooooossss
Pmf0fffff0f0m0
AUGUCUCCGUACAUCUU
92


1231-16-

sso
mmmmm0
CC


14006


 


CTGF-
14007
oooooooooooossss
Pm0fff00f000mm
AACUUGAUAGGCUUGGA
93


991-16-

sso
m00000
GA


14007


 


CTGF-
14008
oooooooooooossss
Pm00fff000fmm0
AAGCUCAAACUUGAUAG
94


998-16-

sso
0m0000
GC


14008


 


CTGF-
14009
oooooooooooossss
Pmf0f0ffff0m00
ACAUACUCCACAGAAUU
95


1049-16-

sso
00mmm0
UA


14009


 


CTGF-
14010
oooooooooooossss
Pmfff0f0000mmm
CUCCACAGAAUUUAGCU
96


1044-16-

sso
00mmm0
CG


14010


 


CTGF-
14011
oooooooooooossss
Pm0f0ff0ff0000
UGUGCUACUGAAAUCAU
97


1327-16-

sso
mm0mm0
UU


14011


 


CTGF-
14012
oooooooooooossss
Pm0000f0ff0mm0
AAAGAUGUCAUUGUCUC
98


1196-16-

sso
mmmmm0
CG


14012


 


CTGF-
14013
oooooooooooossss
Pmf0f0ff00f0mm
GUGCACUGGUACUUGCA
99


562-16-

sso
m0m000
GC


14013


 


CTGF-
14014
oooooooooooossss
Pm00f0f0fffmmm
AAACGUGUCUUCCAGUC
100


752-16-

sso
00mm00
GG


14014


 


CTGF-
14015
oooooooooooossss
Pmf000fff00m00
UCAAACUUGAUAGGCUU
101


994-16-

sso
0mmm00
GG


14015


 


CTGF-
14016
oooooooooooossss
Pmf0000fff00mm
ACAGAAUUUAGCUCGGU
102


1040-16-

sso
m00m00
AU


14016


 


CTGF-
14017
oooooooooooossss
Pmf0f0ffff0mmm
UUACAUUCUACCUAUGG
103


1984-16-

sso
0m00m0
UG


14017


 


CTGF-
14018
oooooooooooossss
Pm00ff00ff00mm
AAACUGAUCAGCUAUAU
104


2195-16-

sso
0m0m00
AG


14018


 


CTGF-
14019
oooooooooooossss
Pm0fff000f0000
UAUCUGAGCAGAAUUUC
105


2043-16-

sso
mmmmm0
CA


14019


 


CTGF-
14020
oooooooooooossss
Pmf00fff000m00
UUAACUUAGAUAACUGU
106


1892-16-

sso
mm0m00
AC


14020


 


CTGF-
14021
oooooooooooossss
Pm0ff0fff0f0m0
UAUUACUCGUAUAAGAU
107


1567-16-

sso
000m00
GC


14021


 


CTGF-
14022
oooooooooooossss
Pm00ff0fff00mm
AAGCUGUCCAGUCUAAU
108


1780-16-

sso
m00mm0
CG


14022


 


CTGF-
14023
oooooooooooossss
Pm00f00000fm0m
UAAUAAAGGCCAUUUGU
109


2162-16-

sso
mm0mm0
UC


14023


 


CTGF-
14024
oooooooooooossss
Pmff00fff00m0m
UUUAGCUCGGUAUGUCU
110


1034-16-

sso
0mmmm0
UC


14024


 


CTGF-
14025
oooooooooooossss
Pmf0fffff00m00
ACACUCUCAACAAAUAA
111


2264-16-

sso
0m0000
AC


14025


 


CTGF-
14026
oooooooooooossss
Pm00fff00f0m0m
UAGCUCGGUAUGUCUUC
112


1032-16-

sso
mmmm00
AU


14026


 


CTGF-
14027
oooooooooooossss
Pm00fffffff0mm
UAACCUUUCUGCUGGUA
113


1535-16-

sso
00m0m0
CC


14027


 


CTGF-
14028
oooooooooooossss
Pmf000000f00mm
UUAAGGAACAACUUGAC
114


1694-16-

sso
m00mm0
UC


14028


 


CTGF-
14029
oooooooooooossss
Pmf0f0ffff000m
UUACACUUCAAAUAGCA
115


1588-16-

sso
00m000
GG


14029


 


CTGF-
14030
oooooooooooossss
Pmff000ff00mmm
UCCAGGUCAGCUUCGCA
116


928-16-

sso
m0m000
AG


14030


 


CTGF-
14031
oooooooooooossss
Pmffffff0f00mm
CUUCUUCAUGACCUCGC
117


1133-16-

sso
mm0mm0
CG


14031


 


CTGF-
14032
oooooooooooossss
Pm000fff00fm0m
AAGGCCUGACCAUGCAC
118


912-16-

sso
0m0m00
AG


14032


 


CTGF-
14033
oooooooooooossss
Pm000f0f0ffmmm
CAAACGUGUCUUCCAGU
119


753-16-

sso
m00mm0
CG


14033


 


CTGF-
14034
oooooooooooossss
Pmfff0f0000mmm
CUUCGCAAGGCCUGACC
120


918-16-

sso
00mm00
AU


14034


 


CTGF-
14035
oooooooooooossss
Pmffff00ff00m0
CUUCCAGUCGGUAAGCC
121


744-16-

sso
00mm00
GC


14035


 


CTGF-
14036
oooooooooooossss
Pmf00ffff0f00m
CCGAUCUUGCGGUUGGC
122


466-16-

sso
m00mm0
CG


14036


 


CTGF-
14037
oooooooooooossss
Pmff0f0000fmm0
UUCGCAAGGCCUGACCA
123


917-16-

sso
0mm0m0
UG


14037


 


CTGF-
14038
oooooooooooossss
Pm00fff00fmm0m
AGAAUUUAGCUCGGUAU
124


1038-16-

sso
0m00
GU


14038


 


CTGF-
14039
oooooooooooossss
Pm0f0ffff0f000
CAUACUCCACAGAAUUU
125


1048-16-

sso
0mmm00
AG


14039


 


CTGF-
14040
oooooooooooossss
Pm0ff0f0fffmmm
UGCCAUGUCUCCGUACA
126


1235-16-

sso
0m0m0
UC


14040


 


CTGF-
14041
oooooooooooossss
Pm000f0ff0fm0m
GAGGCGUUGUCAUUGGU
127


868-16-

sso
m00m00
AA


14041


 


CTGF-
14042
oooooooooooossss
Pmffff0f00fmmm
UCUUCAUGACCUCGCCG
128


1131-16-

sso
0mm0m0
UC


14042


 


CTGF-
14043
oooooooooooossss
Pmff0f0000fmm0
UCCACAGAAUUUAGCUC
129


1043-16-

sso
0mmm00
GG


14043


 


CTGF-
14044
oooooooooooossss
Pm0f0f0ffffmm0
AACGUGUCUUCCAGUCG
130


751-16-

sso
0mm000
GU


14044


 


CTGF-
14045
oooooooooooossss
Pmfff0f0f0fmmm
CUCCGUACAUCUUCCUG
131


1227-16-

sso
mmm0m0
UA


14045


 


CTGF-
14046
oooooooooooossss
Pm0f0ff0ff0mm0
AGGCGUUGUCAUUGGUA
132


867-16-

sso
0m000
AC


14046


 


CTGF-
14047
oooooooooooossss
Pmf0f00ffff0mm
UCAUGACCUCGCCGUCA
133


1128-16-

sso
0mm000
GG


14047


 


CTGF-
14048
oooooooooooossss
Pm0ff000f0f0mm
GGCCAAACGUGUCUUCC
134


756-16-

sso
mmmm00
AG


14048


 


CTGF-
14049
oooooooooooossss
Pmff0f0ffffmm0
GCCAUGUCUCCGUACAU
135


1234-16-

sso
m0mm0
CU


14049


 


CTGF-
14050
oooooooooooossss
Pmf0f0000ffm00
UCGCAAGGCCUGACCAU
136


916-16-

sso
mm0m00
GC


14050


 


CTGF-
14051
oooooooooooossss
Pm0ff00fffmm00
AGGUCAGCUUCGCAAGG
137


925-16-

sso
00m0
CC


14051


 


CTGF-
14052
oooooooooooossss
Pmf0f0f0fffmmm
CCGUACAUCUUCCUGUA
138


1225-16-

sso
m0m000
GU


14052


 


CTGF-
14053
oooooooooooossss
Pm00ff0000fm0m
GAGCCGAAGUCACAGAA
139


445-16-

sso
000000
GA


14053


 


CTGF-
14054
oooooooooooossss
Pm000ff0000mm0
GGAGCCGAAGUCACAGA
140


446-16-

sso
m00000
AG


14054


 


CTGF-
14055
oooooooooooossss
Pm0000fff00mm0
CAAGGCCUGACCAUGCA
141


913-16-

sso
m0m0m0
CA


14055


 


CTGF-
14056
oooooooooooossss
Pmfff000ffm00m
AGCUCAAACUUGAUAGG
142


997-16-

sso
000m0
CU


14056


 


CTGF-
14057
oooooooooooossss
Pmf0f00ffff00m
CUGCAGUUCUGGCCGAC
143


277-16-

sso
m00m00
GG


14057


 


CTGF-
14058
oooooooooooossss
Pm0f0f0f0ffmm0
GGUACAUACUCCACAGA
144


1052-16-

sso
m00000
AU


14058


 


CTGF-
14059
oooooooooooossss
Pmf0fffffff00m
CUGCUUCUCUAGCCUGC
145


887-16-

sso
mm0m00
AG


14059


 


CTGF-
14060
oooooooooooossss
Pmf0000fff00mm
GCAAGGCCUGACCAUGC
146


914-16-

sso
0m0m00
AC


14060


 


CTGF-
14061
oooooooooooossss
Pm0000fff00mmm
CAGAAUUUAGCUCGGUA
147


1039-16-

sso
00m0m0
UG


14061


 


CTGF-
14062
oooooooooooossss
Pmf000f0f0fmmm
CCAAACGUGUCUUCCAG
148


754-16-

sso
mm00m0
UC


14062


 


CTGF-
14063
oooooooooooossss
Pmfff0f00ffmmm
CUUCAUGACCUCGCCGU
149


1130-16-

sso
m0mm0
CA


14063


 


CTGF-
14064
oooooooooooossss
Pmffff0f0000mm
GCUUCGCAAGGCCUGAC
150


919-16-

sso
m00mm0
CA


14064


 


CTGF-
14065
oooooooooooossss
Pmf00ffff0f000
UCAGCUUCGCAAGGCCU
151


922-16-

sso
0mmm00
GA


14065


 


CTGF-
14066
oooooooooooossss
Pmffffff00fm0m
GUCUUCCAGUCGGUAAG
152


746-16-

sso
000m0
CC


14066


 


CTGF-
14067
oooooooooooossss
Pm000fff00f000
CAAACUUGAUAGGCUUG
153


993-16-

sso
mmm000
GA


14067


 


CTGF-
14068
oooooooooooossss
Pm0ffff0000m00
AGGUCUUGGAACAGGCG
154


825-16-

sso
0m0m0
CU


14068


 


CTGF-
14069
oooooooooooossss
Pm000ff00ffmmm
CAGGUCAGCUUCGCAAG
155


926-16-

sso
00000
GC


14069


 


CTGF-
14070
oooooooooooossss
Pmff00ffff0m00
GUCAGCUUCGCAAGGCC
156


923-16-

sso
00mmm0
UG


14070


 


CTGF-
14071
oooooooooooossss
Pm0f0ff0ff0mm0
GGCGUUGUCAUUGGUAA
157


866-16-

sso
0m00m0
CC


14071


 


CTGF-
14072
oooooooooooossss
Pmf0f0ff00m0mm
CGUGCACUGGUACUUGC
158


563-16-

sso
m0m00
AG


14072


 


CTGF-
14073
oooooooooooossss
Pmffff0000f000
GUCUUGGAACAGGCGCU
159


823-16-

sso
m0mmm0
CC


14073


 


CTGF-
14074
oooooooooooossss
Pmf0f0fffff0m0
CCAUGUCUCCGUACAUC
160


1233-16-

sso
m0mmm0
UU


14074


 


CTGF-
14075
oooooooooooossss
Pm0ff00ffff0m0
GGUCAGCUUCGCAAGGC
161


924-16-

sso
000mm0
CU


14075


 


CTGF-
14076
oooooooooooossss
Pm00ffff0f0000
CAGCUUCGCAAGGCCUG
162


921-16-

sso
mmm000
AC


14076


 


CTGF-
14077
oooooooooooossss
Pmff0000ff0m00
GCCGAAGUCACAGAAGA
163


443-16-

sso
000000
GG


14077


 


CTGF-
14078
oooooooooooossss
Pm0f0000fff00m
CACAGAAUUUAGCUCGG
164


1041-16-

sso
mm00m0
UA


14078


 


CTGF-
14079
oooooooooooossss
Pmf0f0000ffm00
CCACAGAAUUUAGCUCG
165


1042-16-

sso
mmm000
GU


14079


 


CTGF-
14080
oooooooooooossss
Pmff000f0f0mmm
GCCAAACGUGUCUUCCA
166


755-16-

sso
mmm000
GU


14080


 


CTGF-
14081
oooooooooooossss
Pmf0f00ffff0m0
GCCGAUCUUGCGGUUGG
167


467-16-

sso
mm00m0
CC


14081


 


CTGF-
14082
oooooooooooossss
Pmff000fff00m0
CUCAAACUUGAUAGGCU
168


995-16-

sso
00mmm0
UG


14082


 


CTGF-
14083
oooooooooooossss
Pmf000ff00fmmm
CCAGGUCAGCUUCGCAA
169


927-16-

sso
0m0000
GG


14083


 


SPP1-
14131
oooooooooooossss
Pmff00ff000m0m
UUUGACUAAAUGCAAAG
170


1091-16-

sso
0000m0
UG


14131


 


PPIB--
14188
ooooooooooooosss
Pm0fffff0f00mm
UGUUUUUGUAGCCAAAU
171


16-14188

sss
000mm0
CC


 


PPIB--
14189
ooooooooooooosss
Pm0fffff0f00mm
UGUUUUUGUAGCCAAAU
172


17-14189

sss
000mm0
CC


 


PPIB--
14190
ooooooooooooosss
Pm0fffff0f00mm
UGUUUUUGUAGCCAAAU
173


18-14190

sss
000mm0
CC


 


pGL3-
14386
oooooooooooossss
Pm00ff0f0ffm0m
AAAUCGUAUUUGUCAAU
174


1172-16-

sso
m00mm0
CA


14386


 


pGL3-
14387
oooooooooooossss
Pm00ff0f0ffm0m
AAAUCGUAUUUGUCAAU
175


1172-16-

sso
m00mm0
CA


14387


 


MAP4K4-
14390


2931-25-


14390


 


miR-122--


23-
14391


14391


 



14084
oooooooooooossss
Pmff00fff0f000
UCUAAUUCAUGAGAAAU
616




sso
000m00
AC


 



14085
oooooooooooossss
Pm00ff00fffm00
UAAUUGACCUCAGAAGA
617




sso
0000m0
UG


 



14086
oooooooooooossss
Pmff00ff00fmmm
UUUAAUUGACCUCAGAA
618




sso
000000
GA


 



14087
oooooooooooossss
Pm0ff00ffff000
AAUUGACCUCAGAAGAU
619




sso
000m00
GC


 



14088
oooooooooooossss
Pmf00ff00ffmm0
UUAAUUGACCUCAGAAG
620




sso
000000
AU


 



14089
oooooooooooossss
Pmff00ffff0000
AUUGACCUCAGAAGAUG
621




sso
00m0m0
CA


 



14090
oooooooooooossss
Pmf0fff00ff00m
UCAUCCAGCUGACUCGU
622




sso
mm0mm0
UU


 



14091
oooooooooooossss
Pm0fff0ff0000m
AGAUUCAUCAGAAUGGU
623




sso
00m00
GA


 



14092
oooooooooooossss
Pm00ffff00fmm0
UGACCUCAGUCCAUAAA
624




sso
m000m0
CC


 



14093
oooooooooooossss
Pm0f00f0000mmm
AAUGGUGAGACUCAUCA
625




sso
0mm000
GA


 



14094
oooooooooooossss
Pmff00ffff00mm
UUUGACCUCAGUCCAUA
626




sso
m0m000
AA


 



14095
oooooooooooossss
Pmff0f00ff0m00
UUCAUGGCUGUGAAAUU
627




sso
00mmm0
CA


 



14096
oooooooooooossss
Pm00f00f0000mm
GAAUGGUGAGACUCAUC
628




sso
m0mm00
AG


 



14097
oooooooooooossss
Pm00ffffff0mmm
UGGCUUUCCGCUUAUAU
629




sso
0m0m00
AA


 



14098
oooooooooooossss
Pmf00ffffff0mm
UUGGCUUUCCGCUUAUA
630




sso
m0m0m0
UA


 



14099
oooooooooooossss
Pmf0fff0f0f00m
UCAUCCAUGUGGUCAUG
631




sso
m0m000
GC


 



14100
oooooooooooossss
Pmf0f00ff0f00m
AUGUGGUCAUGGCUUUC
632




sso
mmmm00
GU


 



14101
oooooooooooossss
Pmf00ff0f00mmm
GUGGUCAUGGCUUUCGU
633




sso
mm0mm0
UG


 



14102
oooooooooooossss
Pmff00fffffmmm
AUUGGCUUUCCGCUUAU
634




sso
m0m00
AU


 



14103
oooooooooooossss
Pm00f0f0000mmm
AAAUACGAAAUUUCAGG
635




sso
m000m0
UG


 



14104
oooooooooooossss
Pm000f0f0000mm
AGAAAUACGAAAUUUCA
636




sso
mm000
GG


 



14105
oooooooooooossss
Pm00ff0f00fmmm
UGGUCAUGGCUUUCGUU
637




sso
m0mm00
GG


 



14106
oooooooooooossss
Pmf0ff0fff0m0m
AUAUCAUCCAUGUGGUC
638




sso
00mm00
AU


 



14107
oooooooooooossss
Pm0f0f0000fmmm
AAUACGAAAUUUCAGGU
639




sso
000m00
GU


 



14108
oooooooooooossss
Pm0ff000000mm0
AAUCAGAAGGCGCGUUC
640




sso
mmm00
AG


 



14109
oooooooooooossss
Pmfff0f000000m
AUUCAUGAGAAAUACGA
641




sso
0m0000
AA


 



14110
oooooooooooossss
Pmf0fff0f00000
CUAUUCAUGAGAGAAUA
642




sso
00m000
AC


 



14111
oooooooooooossss
Pmfff0ff000mmm
UUUCGUUGGACUUACUU
643




sso
0mmm00
GG


 



14112
oooooooooooossss
Pmf0fffff0fm0m
UUGCUCUCAUCAUUGGC
644




sso
m00mm0
UU


 



14113
oooooooooooossss
Pmff00fffffmmm
UUCAACUCCUCGCUUUC
645




sso
mmmm0
CA


 



14114
oooooooooooossss
Pm00ff0ff00mm0
UGACUAUCAAUCACAUC
646




sso
m0mm00
GG


 



14115
oooooooooooossss
Pm0f0f0ff0mmm0
AGAUGCACUAUCUAAUU
647




sso
0mmm0
CA


 



14116
oooooooooooossss
Pm0f000f0f0m0m
AAUAGAUACACAUUCAA
648




sso
mm00m0
CC


 



14117
oooooooooooossss
Pmffffff0f0000
UUCUUCUAUAGAAUGAA
649




sso
m000m0
CA


 



14118
oooooooooooossss
Pm0ff0ff000m00
AAUUGCUGGACAACCGU
650




sso
mm0m00
GG


 



14119
oooooooooooossss
Pmf0ffffff0m0m
UCGCUUUCCAUGUGUGA
651




sso
0m0000
GG


 



14120
oooooooooooossss
Pm00fff000fm0m
UAAUCUGGACUGCUUGU
652




sso
mm0m00
GG


 



14121
oooooooooooossss
Pmf0f0fff00mm0
ACACAUUCAACCAAUAA
653




sso
0m0000
AC


 



14122
oooooooooooossss
Pmfff0ffff0m00
ACUCGUUUCAUAACUGU
654




sso
mm0mm0
CC


 



14123
oooooooooooossss
Pmf00fff000mm0
AUAAUCUGGACUGCUUG
655




sso
mmm0m0
UG


 



14124
oooooooooooossss
Pmffff0fff0m0m
UUUCCGCUUAUAUAAUC
656




sso
00mmm0
UG


 



14125
oooooooooooossss
Pm0fff00ff00m0
UGUUUAACUGGUAUGGC
657




sso
m00m00
AC


 



14126
oooooooooooossss
Pm0f0000f000m0
UAUAGAAUGAACAUAGA
658




sso
m000m0
CA


 



14127
oooooooooooossss
Pmffffff00fm0m
UUUCCUUGGUCGGCGUU
659




sso
0mmm0
UG


 



14128
oooooooooooossss
Pmf0f0f0ff0mmm
GUAUGCACCAUUCAACU
660




sso
00mmm0
CC


 



14129
oooooooooooossss
Pmf00ff0ff0m0m
UCGGCCAUCAUAUGUGU
661




sso
0m0mm0
CU


 



14130
oooooooooooossss
Pm0fff000ff0mm
AAUCUGGACUGCUUGUG
662




sso
m0m000
GC


 



14132
oooooooooooossss
Pmf0ff0000f0mm
ACAUCGGAAUGCUCAUU
663




sso
m0mm00
GC


 



14133
oooooooooooossss
Pm00fffff00mm0
AAGUUCCUGACUAUCAA
664




sso
mm00m0
UC


 



14134
oooooooooooossss
Pmf00ff000f0m0
UUGACUAAAUGCAAAGU
665




sso
000m00
GA


 



14135
oooooooooooossss
Pm0fff0ff000mm
AGACUCAUCAGACUGGU
666




sso
00m00
GA


 



14136
oooooooooooossss
Pmf0f0f0f0fmm0
UCAUAUGUGUCUACUGU
667




sso
mm0m00
GG


 



14137
oooooooooooossss
Pmf0fffff0fmm0
AUGUCCUCGUCUGUAGC
668




sso
m00m00
AU


 



14138
oooooooooooossss
Pm00fff0f00mm0
GAAUUCACGGCUGACUU
669




sso
0mmmm0
UG


 



14139
oooooooooooossss
Pmf0fffff000mm
UUAUUUCCAGACUCAAA
670




sso
m000m0
UA


 



14140
oooooooooooossss
Pm000ff0f000mm
GAAGCCACAAACUAAAC
671




sso
000mm0
UA


 



14141
oooooooooooossss
Pmffff0ff000mm
CUUUCGUUGGACUUACU
672




sso
m0mmm0
UG


 



14142
oooooooooooossss
Pmfff0f0000mmm
GUCUGCGAAACUUCUUA
673




sso
mmm000
GA


 



14143
oooooooooooossss
Pm0f0fff0ff0mm
AAUGCUCAUUGCUCUCA
674




sso
mmm0m0
UC


 



14144
oooooooooooossss
Pmf0f0ff0ffm00
AUGCACUAUCUAAUUCA
675




sso
mmm0m0
UG


 



14145
oooooooooooossss
Pmff0f0f0f0mm0
CUUGUAUGCACCAUUCA
676




sso
mmm000
AC


 



14146
oooooooooooossss
Pm00fff0fffm0m
UGACUCGUUUCAUAACU
677




sso
00mm00
GU


 



14147
oooooooooooossss
Pmff00f0fffm00
UUCAGCACUCUGGUCAU
678




sso
mm0mm0
CC


 



14148
oooooooooooossss
Pm00fff0f00mm0
AAAUUCAUGGCUGUGGA
679




sso
m00000
AU


 



14149
oooooooooooossss
Pmf0fff00ff00m
ACAUUCAACCAAUAAAC
680




sso
000mm0
UG


 



14150
oooooooooooossss
Pm0f0f0fff00mm
UACACAUUCAACCAAUA
681




sso
00m000
AA


 



14151
oooooooooooossss
Pmff00ff0ffmmm
AUUAGUUAUUUCCAGAC
682




sso
000mm0
UC


 



14152
oooooooooooossss
Pmffff0fff0m00
UUUCUAUUCAUGAGAGA
683




sso
000000
AU


 



14153
oooooooooooossss
Pmff00ff0ff00m
UUCGGUUGCUGGCAGGU
684




sso
000mm0
CC


 



14154
oooooooooooossss
Pm0f0f0f0000m0
CAUGUGUGAGGUGAUGU
685




sso
0m0mm0
CC


 



14155
oooooooooooossss
Pmf0ff0fff00mm
GCACCAUUCAACUCCUC
686




sso
mmmm00
GC


 



14156
oooooooooooossss
Pm0fff00ff00mm
CAUCCAGCUGACUCGUU
687




sso
m0mmm0
UC


 



14157
oooooooooooossss
Pmfffff0fff0m0
CUUUCCGCUUAUAUAAU
688




sso
m00mm0
CU


 



14158
oooooooooooossss
Pm0ff0f0ff0000
AAUCACAUCGGAAUGCU
689




sso
m0mmm0
CA


 



14159
oooooooooooossss
Pmf0f0ff00fm0m
ACACAUUAGUUAUUUCC
690




sso
mmmm00
AG


 



14160
oooooooooooossss
Pmfff0f0000m00
UUCUAUAGAAUGAACAU
691




sso
0m0m00
AG


 



14161
oooooooooooossss
Pm0f00f00f00mm
UACAGUGAUAGUUUGCA
692




sso
m0m0m0
UU


 



14162
oooooooooooossss
Pmf000f00ff00m
AUAAGCAAUUGACACCA
693




sso
0mm0m0
CC


 



14163
oooooooooooossss
Pmff0ff00ff0mm
UUUAUUAAUUGCUGGAC
694




sso
000m00
AA


 



14164
oooooooooooossss
Pmf0ff0000fmmm
UCAUCAGAGUCGUUCGA
695




sso
m0000
GU


 



14165
oooooooooooossss
Pmf000ff0f0mm0
AUAAACCACACUAUCAC
696




sso
mm0mm0
CU


 



14166
oooooooooooossss
Pmf0ff0ff00mmm
UCAUCAUUGGCUUUCCG
697




sso
mmm0m0
CU


 



14167
oooooooooooossss
Pmfffff00fm0mm
AGUUCCUGACUAUCAAU
698




sso
00mm0
CA


 



14168
oooooooooooossss
Pmff0f00ff00mm
UUCACGGCUGACUUUGG
699




sso
mm0000
AA


 



14169
oooooooooooossss
Pmffff0f00f00m
UUCUCAUGGUAGUGAGU
700




sso
000mm0
UU


 



14170
oooooooooooossss
Pm0ff00fff0mmm
AAUCAGCCUGUUUAACU
701




sso
00mm00
GG


 



14171
oooooooooooossss
Pm0ffff00f0mmm
GGUUUCAGCACUCUGGU
702




sso
m00mm0
CA


 



14172
oooooooooooossss
Pmff0000f0fmm0
AUCGGAAUGCUCAUUGC
703




sso
mm0mm0
UC


 



14173
oooooooooooossss
Pm00ff0f0000mm
UGGCUGUGGAAUUCACG
704




sso
m0m000
GC


 



14174
oooooooooooossss
Pm000f00ff00m0
UAAGCAAUUGACACCAC
705




sso
mm0mm0
CA


 



14175
oooooooooooossss
Pm00fffff0f00m
CAAUUCUCAUGGUAGUG
706




sso
00m000
AG


 



14176
oooooooooooossss
Pm00fffff0fm00
UGGCUUUCGUUGGACUU
707




sso
0mmm00
AC


 



14177
oooooooooooossss
Pm0ff00f00fm00
AAUCAGUGACCAGUUCA
708




sso
mmm0m0
UC


 



14178
oooooooooooossss
Pmfff0f000mm0m
AGUCCAUAAACCACACU
709




sso
0mm00
AU


 



14179
oooooooooooossss
Pm00f0ffff00mm
CAGCACUCUGGUCAUCC
710




sso
0mmm00
AG


 



14180
oooooooooooossss
Pm0ff00ff0f0mm
UAUCAAUCACAUCGGAA
711




sso
0000m0
UG


 



14181
oooooooooooossss
Pmfff0f00ff00m
AUUCACGGCUGACUUUG
712




sso
mmm000
GA


 



14182
oooooooooooossss
Pmf000f0f0f0mm
AUAGAUACACAUUCAAC
713




sso
m00mm0
CA


 



14183
oooooooooooossss
Pmffff000ffm00
UUUCCAGACUCAAAUAG
714




sso
0m0000
AU


 



14184
oooooooooooossss
Pmf00ff0ff000m
UUAAUUGCUGGACAACC
715




sso
00mm00
GU


 



14185
oooooooooooossss
Pm0ff00ff0fm00
UAUUAAUUGCUGGACAA
716




sso
0m00m0
CC


 



14186
oooooooooooossss
Pmff0fff000mm0
AGUCGUUCGAGUCAAUG
717




sso
0m000
GA


 



14187
oooooooooooossss
Pmff0ff00f000m
GUUGCUGGCAGGUCCGU
718




sso
mm0m00
GG





o: phosphodiester;


s: phosphorothioate;


P: 5′ phosphorylation;


0: 2′-OH;


F: 2′-fluoro;


m: 2′ O-methyl;


+: LNA modification.


Capital letters in the sequence signify riobonucleotides, lower case letters


signify deoxyribonucleotides.













TABLE 3







Sense backbone, chemistry, and sequence information.
















OHang



SEQ




Oligo
Sense
Sense
Sense
Sense
ID


ID Number
Number
Chem.
Backbone
Chemistry
Sequence
NO:

















APOB-10167-
12138
chl
oooooooooooo
0000000000000
GUCAUCACACUGA
176



20-12138


oooooooso
0000000
AUACCAAU


 


APOB-10167-
12139
chl
oooooooooooo
0000000000000
GUGAUCAGACUCA
177


20-12139


oooooooso
0000000
AUACGAAU


 


MAP4K4-
12266
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
178


2931-13-


12266


 


MAP4K4-
12293
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
179


2931-16-


12293


 


MAP4K4-
12383
chl
ooooooooooooo
mm0m00000mmm0
CUGUGGAAGUCUA
180


2931-16-


12383


 


MAP4K4-
12384
chl
ooooooooooooo
mm0m00000mmm0
CUGUGGAAGUCUA
181


2931-16-


12384


 


MAP4K4-
12385
chl
ooooooooooooo
mm0m00000mmm0
CUGUGGAAGUCUA
182


2931-16-


12385


 


MAP4K4-
12386
chl
oooooooooosso
0mm0m00000mmm0
CUGUGGAAGUCUA
183


2931-16-


12386


 


MAP4K4-
12387
chl
ooooooooooooo
mm0m00000mmm0
CUGUGGAAGUCUA
184


2931-16-


12387


 


MAP4K4-
12388
chl
ooooooooooooo
mm0m00000mmm0
CUGUGGAAGUCUA
185


2931-15-


12388


 


MAP4K4-
12432
chl
ooooooooooooo
DY547mm0m0000
CUGUGGAAGUCUA
186


2931-13-



0mmm0


12432


 


MAP4K4-
12266.2
chl
oooooooooooss
mm0m00000mmm0
CUGUGGAAGUCUA
187


2931-13-


12266.2


 


APOB--21-
12434
chl
oooooooooooo
0000000000000
GUCAUCACACUGA
188


12434


oooooooso
0000000
AUACCAAU


 


APOB--21-
12435
chl
oooooooooooo
DY54700000000
GUGAUCAGACUCA
189


12435


oooooooso
000000000000
AUACGAAU


 


MAP4K4-
12451
chl
oooooooooooss
0mm0m00000mmm
CUGUGGAAGUCUA
190


2931-16-


12451


 


MAP4K4-
12452
chl
oooooooooooss
mm0m00000mmm0
CUGUGGAAGUCUA
191


2931-16-


12452


 


MAP4K4-
12453
chl
oooooooooooss
mm0m00000mmm0
CUGUGGAAGUCUA
192


2931-16-


12453


 


MAP4K4-
12454
chl
oooooooooooss
0mm0m00000mmm0
CUGUGGAAGUCUA
193


2931-17-


12454


 


MAP4K4-
12455
chl
oooooooooooss
mm0m00000mmm0
CUGUGGAAGUCUA
194


2931-17-


12455


 


MAP4K4-
12456
chl
oooooooooooss
mm0m00000mmm0
CUGUGGAAGUCUA
195


2931-19-


12456


 


--27-12480
12480
chl
oooooooooooo
DY547mm0f000f
UCAUAGGUAACCU
196





oooooooooooo
0055f5f00mm00
CUGGUUGAAAGUGA





sso
000m000


 


--27-12481
12481
chl
oooooooooooo
DY547mm05f050
CGGCUACAGGUGC
197





oooooooooooo
00f05ff0m0000
UUAUGAAGAAAGUA





sso
0000m00


 


APOB-10167-
12505
chl
oooooooooooo
0000000000000
GUCAUCACACUGA
198


21-12505


oooooooos
00000000
AUACCAAU


 


APOB-10167-
12506
chl
oooooooooooo
0000000000000
GUGAUCAGACUCA
199


21-12506


oooooooos
00000000
AUACGAAU


 


MAP4K4-
12539
chl
ooooooooooss
DY547mm0m0000
CUGUGGAAGUCUA
200


2931-16-



0mmm0


12539


 


APOB-10167-
12505.2
chl
oooooooooooo
0000000000000
GUCAUCACACUGA
201


21-12505.2


oooooooso
0000000
AUACCAAU


 


APOB-10167-
12506.2
chl
oooooooooooo
0000000000000
GUGAUCAGACUCA
202


21-12506.2


oooooooso
0000000
AUACGAAU


 


MAP4K4--13-
12565
Chl
ooooooooooooo
m0m0000m0mmm0
UGUAGGAUGUCUA
203


12565


 


MAP4K4-
12386.2
chl
ooooooooooooo
0mm0m00000mmm0
CUGUGGAAGUCUA
204


2931-16-


12386.2


 


MAP4K4-
12815
chl
ooooooooooooo
m0m0m0m0m0m0m
CUGUGGAAGUCUA
205


2931-13-



0m0m0m0m0m0m0


12815


 


APOB--13-
12957
Chl
oooooooooooss
0mmmmmmmmmmmmm
ACUGAAUACCAAU
206


12957

TEG


 


MAP4K4--16-
12983
chl
oooooooooooss
mm0m00000mmm0
CUGUGGAAGUCUA
207


12983


 


MAP4K4--16-
12984
Chl
oooooooooooo
mm0m00000mmm0
CUGUGGAAGUCUA
208


12984


oo


 


MAP4K4--16-
12985
chl
oooooooooosso
mmmmmmmmmmmmm
CUGUGGAAGUCUA
209


12985


 


MAP4K4--16-
12986
chl
oooooooooosso
mmmmmmmmmmmmm
CUGUGGAAGUCUA
210


12986


 


MAP4K4--16-
12987
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
211


12987


 


MAP4K4--16-
12988
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
212


12988


 


MAP4K4--16-
12989
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
213


12989


 


MAP4K4--16-
12990
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
214


12990


 


MAP4K4--16-
12991
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
215


12991


 


MAP4K4--16-
12992
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
216


12992


 


MAP4K4--16-
12993
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
217


12993


 


MAP4K4--16-
12994
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
218


12994


 


MAP4K4--16-
12995
chl
oooooooooosso
mm0m00000mmm0
CUGUGGAAGUCUA
219


12995


 


MAP4K4-
13012
chl
oooooooooooo
0000000000000
AGAGUUCUGUGGA
220


2931-19-


ooooooo
00000000
AGUCUA


13012


 


MAP4K4-
13016
chl
oooooooooooo
DY54700000000
AGAGUUCUGUGGA
221


2931-19-


ooooooo
0000000000000
AGUCUA


13016


 


PPIB--13-
13021
Chl
ooooooooooooo
0mmm00mm0m000
AUUUGGCUACAAA
222


13021


 


pGL3-1172-
13038
chl
ooooooooooooo
00m000m0m00mmm
ACAAAUACGAUUU
223


13-13038


 


pGL3-1172-
13040
chl
ooooooooooooo
DY5470m000m0m
ACAAAUACGAUUU
224


13-13040



00mmm


 


--16-13047
13047
Chl
oooooooooooo
mm0m00000mmm0
CUGUGGAAGUCUA
225





oo


 


SOD1-530-
13090
chl
ooooooooooooo
00m00000000m0
AAUGAAGAAAGUA
226


13-13090


 


SOD1-523-
13091
chl
ooooooooooooo
000m00000m000
AGGUGGAAAUGAA
227


13-13091


 


SOD1-535-
13092
chl
ooooooooooooo
000000m0m0000
AGAAAGUACAAAG
228


13-13092


 


SOD1-536-
13093
chl
ooooooooooooo
00000m0m00000
GAAAGUACAAAGA
229


13-13093


 


SOD1-396-
13094
chl
ooooooooooooo
0m0m00mm0mm00
AUGUGACUGCUGA
230


13-13094


 


SOD1-385-
13095
chl
ooooooooooooo
000mmm000m00m
AGACUUGGGCAAU
231


13-13095


 


SOD1-195-
13096
chl
ooooooooooooo
0mmmm000m0000
AUUUCGAGCAGAA
232


13-13096


 


APOB-4314-
13115
Chl
ooooooooooooo
0mmm0000000m0
AUCUGGAGAAACA
233


13-13115


 


APOB-3384-
13116
Chl
ooooooooooooo
mm0000m000000
UCAGAACAAGAAA
234


13-13116


 


APOB-3547-
13117
Chl
ooooooooooooo
00mmm0mmm0mm0
GACUCAUCUGCUA
235


13-13117


 


APOB-4318-
13118
Chl
ooooooooooooo
0000000m00m0m
GGAGAAACAACAU
236


13-13118


 


APOB-3741-
13119
Chl
ooooooooooooo
00mmmmmm000m0
AGUCCCUCAAACA
237


13-13119


 


PPIB--16-
13136
Chl
oooooooooooo
00mm0m00000m0
GGCUACAAAAACA
238


13136


oo


 


APOB-4314-
13154
chl
oooooooooooo
000mmm0000000
AGAUCUGGAGAAA
239


15-13154


oo
m0
CA


 


APOB-3547-
13155
chl
oooooooooooo
m000mmm0mmm0m
UGGACUCAUCUGC
240


15-13155


oo
m0
UA


 


APOB-4318-
13157
chl
oooooooooooo
mm0000000m00m
CUGGAGAAACAAC
241


15-13157


oo
0m
AU


 


APOB-3741-
13158
chl
oooooooooooo
0000mmmmmm000
AGAGUCCCUCAAA
242


15-13158


oo
m0
CA


 


APOB--13-
13159
chl
oooooooooooo
0mm000m0mm00m
ACUGAAUACCAAU
243


13159


 


APOB--15-
13160
chl
oooooooooooo
0m0mm000m0mm0
ACACUGAAUACCA
244


13160


oo
0m
AU


 


SOD1-530-
13163
chl
ooooooooooooo
00m00000000m0
AAUGAAGAAAGUA
245


16-13163


 


SOD1-523-
13164
chl
ooooooooooooo
000m00000m000
AGGUGGAAAUGAA
246


16-13164


 


SOD1-535-
13165
chl
ooooooooooooo
000000m0m0000
AGAAAGUACAAAG
247


16-13165


 


SOD1-536-
13166
chl
ooooooooooooo
00000m0m00000
GAAAGUACAAAGA
248


16-13166


 


SOD1-396-
13167
chl
ooooooooooooo
0m0m00mm0mm00
AUGUGACUGCUGA
249


16-13167


 


SOD1-385-
13168
chl
ooooooooooooo
000mmm000m00m
AGACUUGGGCAAU
250


16-13168


 


SOD1-195-
13169
chl
ooooooooooooo
0mmmm000m0000
AUUUCGAGCAGAA
251


16-13169


 


pGL3-1172-
13170
chl
ooooooooooooo
0m000m0m00mmm
ACAAAUACGAUUU
252


16-13170


 


pGL3-1172-
13171
chl
ooooooooooooo
DY5470m000m0m
ACAAAUACGAUUU
253


16-13171



00mmm


 


MAP4k4-
13189
chl
oooooooooooo
0000000000000
AGAGUUCUGUGGA
254


2931-19-


ooooooo
00000000
AGUCUA


13189


 


CTGF-1222-
13190
Chl
ooooooooooooo
0m0000000m0m0
ACAGGAAGAUGUA
255


13-13190


 


CTGF-813-
13192
Chl
ooooooooooooo
000m0000m0mmm
GAGUGGAGCGCCU
256


13-13192


 


CTGF-747-
13194
Chl
ooooooooooooo
m00mm000000m0
CGACUGGAAGACA
257


13-13194


 


CTGF-817-
13196
Chl
ooooooooooooo
0000m0mmm0mmm
GGAGCGCCUGUUC
258


13-13196


 


CTGF-1174-
13198
Chl
ooooooooooooo
0mm0mm0m00mm0
GCCAUUACAACUG
259


13-13198


 


CTGF-1005-
13200
Chl
ooooooooooooo
000mmmmmm00mm
GAGCUUUCUGGCU
260


13-13200


 


CTGF-814-
13202
Chl
ooooooooooooo
00m0000m0mmm0
AGUGGAGCGCCUG
261


13-13202


 


CTGF-816-
13204
Chl
ooooooooooooo
m0000m0mmm0mm
UGGAGCGCCUGUU
262


13-13204


 


CTGF-1001-
13206
Chl
ooooooooooooo
0mmm000mmmmmm
GUUUGAGCUUUCU
263


13-13206


 


CTGF-1173-
13208
Chl
ooooooooooooo
m0mm0mm0m00mm
UGCCAUUACAACU
264


13-13208


 


CTGF-749-
13210
Chl
ooooooooooooo
0mm000000m0m0
ACUGGAAGACACG
265


13-13210


 


CTGF-792-
13212
Chl
ooooooooooooo
00mm0mmm00mmm
AACUGCCUGGUCC
266


13-13212


 


CTGF-1162-
13214
Chl
ooooooooooooo
000mmm0m0mmm0
AGACCUGUGCCUG
267


13-13214


 


CTGF-811-
13216
Chl
ooooooooooooo
m0000m0000m0m
CAGAGUGGAGCGC
268


13-13216


 


CTGF-797-
13218
Chl
ooooooooooooo
mmm00mmm000mm
CCUGGUCCAGACC
269


13-13218


 


CTGF-1175-
13220
Chl
ooooooooooooo
mm0mm0m00mm0m
CCAUUACAACUGU
270


13-13220


 


CTGF-1172-
13222
Chl
ooooooooooooo
mm0mm0mm0m00m
CUGCCAUUACAAC
271


13-13222


 


CTGF-1177-
13224
Chl
ooooooooooooo
0mm0m00mm0mmm
AUUACAACUGUCC
272


13-13224


 


CTGF-1176-
13226
Chl
ooooooooooooo
m0mm0m00mm0mm
CAUUACAACUGUC
273


13-13226


 


CTGF-812-
13228
Chl
ooooooooooooo
0000m0000m0mm
AGAGUGGAGCGCC
274


13-13228


 


CTGF-745-
13230
Chl
ooooooooooooo
0mm00mm000000
ACCGACUGGAAGA
275


13-13230


 


CTGF-1230-
13232
Chl
ooooooooooooo
0m0m0m00000m0
AUGUACGGAGACA
276


13-13232


 


CTGF-920-
13234
Chl
ooooooooooooo
0mmmm0m0000mm
GCCUUGCGAAGCU
277


13-13234


 


CTGF-679-
13236
Chl
ooooooooooooo
0mm0m000000m0
GCUGCGAGGAGUG
278


13-13236


 


CTGF-992-
13238
Chl
ooooooooooooo
0mmm0mm000mmm
GCCUAUCAAGUUU
279


13-13238


 


CTGF-1045-
13240
Chl
ooooooooooooo
00mmmm0m0000m
AAUUCUGUGGAGU
280


13-13240


 


CTGF-1231-
13242
Chl
ooooooooooooo
m0m0m00000m0m
UGUACGGAGACAU
281


13-13242


 


CTGF-991-
13244
Chl
ooooooooooooo
00mmm0mm000mm
AGCCUAUCAAGUU
282


13-13244


 


CTGF-998-
13246
Chl
ooooooooooooo
m000mmm000mmm
CAAGUUUGAGCUU
283


13-13246


 


CTGF-1049-
13248
Chl
ooooooooooooo
mm0m0000m0m0m
CUGUGGAGUAUGU
284


13-13248


 


CTGF-1044-
13250
Chl
ooooooooooooo
000mmmm0m0000
AAAUUCUGUGGAG
285


13-13250


 


CTGF-1327-
13252
Chl
ooooooooooooo
mmmm00m00m0m0
UUUCAGUAGCACA
286


13-13252


 


CTGF-1196-
13254
Chl
ooooooooooooo
m00m00m0mmmmm
CAAUGACAUCUUU
287


13-13254


 


CTGF-562-
13256
Chl
ooooooooooooo
00m0mm00m0m0m
AGUACCAGUGCAC
288


13-13256


 


CTGF-752-
13258
Chl
ooooooooooooo
000000m0m0mmm
GGAAGACACGUUU
289


13-13258


 


CTGF-994-
13260
Chl
ooooooooooooo
mm0mm000mmm00
CUAUCAAGUUUGA
290


13-13260


 


CTGF-1040-
13262
Chl
ooooooooooooo
00mm000mmmm0m
AGCUAAAUUCUGU
291


13-13262


 


CTGF-1984-
13264
Chl
ooooooooooooo
000m0000m0m00
AGGUAGAAUGUAA
292


13-13264


 


CTGF-2195-
13266
Chl
ooooooooooooo
00mm00mm00mmm
AGCUGAUCAGUUU
293


13-13266


 


CTGF-2043-
13268
Chl
ooooooooooooo
mmmm0mmm000m0
UUCUGCUCAGAUA
294


13-13268


 


CTGF-1892-
13270
Chl
ooooooooooooo
mm0mmm000mm00
UUAUCUAAGUUAA
295


13-13270


 


CTGF-1567-
13272
Chl
ooooooooooooo
m0m0m000m00m0
UAUACGAGUAAUA
296


13-13272


 


CTGF-1780-
13274
Chl
ooooooooooooo
00mm000m00mmm
GACUGGACAGCUU
297


13-13274


 


CTGF-2162-
13276
Chl
ooooooooooooo
0m00mmmmm0mm0
AUGGCCUUUAUUA
298


13-13276


 


CTGF-1034-
13278
Chl
ooooooooooooo
0m0mm000mm000
AUACCGAGCUAAA
299


13-13278


 


CTGF-2264-
13280
Chl
ooooooooooooo
mm0mm00000m0m
UUGUUGAGAGUGU
300


13-13280


 


CTGF-1032-
13282
Chl
ooooooooooooo
0m0m0mm000mm0
ACAUACCGAGCUA
301


13-13282


 


CTGF-1535-
13284
Chl
ooooooooooooo
00m0000000mm0
AGCAGAAAGGUUA
302


13-13284


 


CTGF-1694-
13286
Chl
ooooooooooooo
00mm0mmmmmm00
AGUUGUUCCUUAA
303


13-13286


 


CTGF-1588-
13288
Chl
ooooooooooooo
0mmm0000m0m00
AUUUGAAGUGUAA
304


13-13288


 


CTGF-928-
13290
Chl
ooooooooooooo
000mm00mmm000
AAGCUGACCUGGA
305


13-13290


 


CTGF-1133-
13292
Chl
ooooooooooooo
00mm0m0000000
GGUCAUGAAGAAG
306


13-13292


 


CTGF-912-
13294
Chl
ooooooooooooo
0m00mm000mmmm
AUGGUCAGGCCUU
307


13-13294


 


CTGF-753-
13296
Chl
ooooooooooooo
00000m0m0mmm0
GAAGACACGUUUG
308


13-13296


 


CTGF-918-
13298
Chl
ooooooooooooo
000mmmm0m0000
AGGCCUUGCGAAG
309


13-13298


 


CTGF-744-
13300
Chl
ooooooooooooo
m0mm0mm00000
UACCGACUGGAAG
310


13-13300


 


CTGF-466-
13302
Chl
ooooooooooooo
0mm0m0000mm0
ACCGCAAGAUCGG
311


13-13302


 


CTGF-917-
13304
Chl
ooooooooooooo
m000mmmm0m000
CAGGCCUUGCGAA
312


13-13304


 


CTGF-1038-
13306
Chl
ooooooooooooo
m000mm000mmmm
CGAGCUAAAUUCU
313


13-13306


 


CTGF-1048-
13308
Chl
ooooooooooooo
mmm0m0000m0m0
UCUGUGGAGUAUG
314


13-13308


 


CTGF-1235-
13310
Chl
ooooooooooooo
m00000m0m00m0
CGGAGACAUGGCA
315


13-13310


 


CTGF-868-
13312
Chl
ooooooooooooo
0m00m00m0mmmm
AUGACAACGCCUC
316


13-13312


 


CTGF-1131-
13314
Chl
ooooooooooooo
0000mm0m00000
GAGGUCAUGAAGA
317


13-13314


 


CTGF-1043-
13316
Chl
ooooooooooooo
m000mmmm0m000
UAAAUUCUGUGGA
318


13-13316


 


CTGF-751-
13318
Chl
ooooooooooooo
m000000m0m0mm
UGGAAGACACGUU
319


13-13318


 


CTGF-1227-
13320
Chl
ooooooooooooo
0000m0m0m0000
AAGAUGUACGGAG
320


13-13320


 


CTGF-867-
13322
Chl
ooooooooooooo
00m00m00m0mmm
AAUGACAACGCCU
321


13-13322


 


CTGF-1128-
13324
Chl
ooooooooooooo
00m0000mm0m00
GGCGAGGUCAUGA
322


13-13324


 


CTGF-756-
13326
Chl
ooooooooooooo
00m0m0mmm00mm
GACACGUUUGGCC
323


13-13326


 


CTGF-1234-
13328
Chl
ooooooooooooo
0m00000m0m00m
ACGGAGACAUGGC
324


13-13328


 


CTGF-916-
13330
Chl
ooooooooooooo
mm000mmmm0m00
UCAGGCCUUGCGA
325


13-13330


 


CTGF-925-
13332
Chl
ooooooooooooo
0m0000mm00mmm
GCGAAGCUGACCU
326


13-13332


 


CTGF-1225-
13334
Chl
ooooooooooooo
000000m0m0m00
GGAAGAUGUACGG
327


13-13334


 


CTGF-445-
13336
Chl
ooooooooooooo
0m00mmmm00mmm
GUGACUUCGGCUC
328


13-13336


 


CTGF-446-
13338
Chl
ooooooooooooo
m00mmmm00mmmm
UGACUUCGGCUCC
329


13-13338


 


CTGF-913-
13340
Chl
ooooooooooooo
m00mm000mmmm0
UGGUCAGGCCUUG
330


13-13340


 


CTGF-997-
13342
Chl
ooooooooooooo
mm000mmm000mm
UCAAGUUUGAGCU
331


13-13342


 


CTGF-277-
13344
Chl
ooooooooooooo
0mm0000mm0m00
GCCAGAACUGCAG
332


13-13344


 


CTGF-1052-
13346
Chl
ooooooooooooo
m0000m0m0m0mm
UGGAGUAUGUACC
333


13-13346


 


CTGF-887-
13348
Chl
ooooooooooooo
0mm0000000m00
GCUAGAGAAGCAG
334


13-13348


 


CTGF-914-
13350
Chl
ooooooooooooo
00mm000mmmm0m
GGUCAGGCCUUGC
335


13-13350


 


CTGF-1039-
13352
Chl
ooooooooooooo
000mm000mmmm0
GAGCUAAAUUCUG
336


13-13352


 


CTGF-754-
13354
Chl
ooooooooooooo
0000m0m0mmm00
AAGACACGUUUGG
337


13-13354


 


CTGF-1130-
13356
Chl
ooooooooooooo
m0000mm0m0000
CGAGGUCAUGAAG
338


13-13356


 


CTGF-919-
13358
Chl
ooooooooooooo
00mmmm0m0000m
GGCCUUGCGAAGC
339


13-13358


 


CTGF-922-
13360
Chl
ooooooooooooo
mmm0m0000mm00
CUUGCGAAGCUGA
340


13-13360


 


CTGF-746-
13362
Chl
ooooooooooooo
mm00mm000000m
CCGACUGGAAGAC
341


13-13362


 


CTGF-993-
13364
Chl
ooooooooooooo
mmm0mm000mmm0
CCUAUCAAGUUUG
342


13-13364


 


CTGF-825-
13366
Chl
ooooooooooooo
m0mmmm0000mmm
UGUUCCAAGACCU
343


13-13366


 


CTGF-926-
13368
Chl
ooooooooooooo
m0000mm00mmm0
CGAAGCUGACCUG
344


13-13368


 


CTGF-923-
13370
Chl
ooooooooooooo
mm0m0000mm00m
UUGCGAAGCUGAC
345


13-13370


 


CTGF-866-
13372
Chl
ooooooooooooo
m00m00m00m0mm
CAAUGACAACGCC
346


13-13372


 


CTGF-563-
13374
Chl
ooooooooooooo
0m0mm00m0m0m0
GUACCAGUGCACG
347


13-13374


 


CTGF-823-
13376
Chl
ooooooooooooo
mmm0mmmm0000m
CCUGUUCCAAGAC
348


13-13376


 


CTGF-1233-
13378
Chl
ooooooooooooo
m0m00000m0m00
UACGGAGACAUGG
349


13-13378


 


CTGF-924-
13380
Chl
ooooooooooooo
m0m0000mm00mm
UGCGAAGCUGACC
350


13-13380


 


CTGF-921-
13382
Chl
ooooooooooooo
mmmm0m0000mm0
CCUUGCGAAGCUG
351


13-13382


 


CTGF-443-
13384
Chl
oooooooooooo
mm0m00mmmm00m
CUGUGACUUCGGC
352


13-13384


 


CTGF-1041-
13386
Chl
ooooooooooooo
0mm000mmmm0m0
GCUAAAUUCUGUG
353


13-13386


 


CTGF-1042-
13388
Chl
ooooooooooooo
mm000mmmm0m00
CUAAAUUCUGUGG
354


13-13388


 


CTGF-755-
13390
Chl
ooooooooooooo
000m0m0mmm00m
AGACACGUUUGGC
355


13-13390


 


CTGF-467-
13392
Chl
ooooooooooooo
mm0m0000mm00m
CCGCAAGAUCGGC
356


13-13392


 


CTGF-995-
13394
Chl
ooooooooooooo
m0mm000mmm000
UAUCAAGUUUGAG
357


13-13394


 


CTGF-927-
13396
Chl
ooooooooooooo
0000mm00mmm00
GAAGCUGACCUGG
358


13-13396


 


SPP1-1025-
13398
Chl
ooooooooooooo
mmm0m000mm000
CUCAUGAAUUAGA
359


13-13398


 


SPP1-1049-
13400
Chl
ooooooooooooo
mm0000mm00mm0
CUGAGGUCAAUUA
360


13-13400


 


SPP1-1051-
13402
Chl
ooooooooooooo
0000mm00mm000
GAGGUCAAUUAAA
361


13-13402


 


SPP1-1048-
13404
Chl
ooooooooooooo
mmm0000mm00mm
UCUGAGGUCAAUU
362


13-13404


 


SPP1-1050-
13406
Chl
ooooooooooooo
m0000mm00mm00
UGAGGUCAAUUAA
363


13-13406


 


SPP1-1047-
13408
Chl
ooooooooooooo
mmmm0000mm00m
UUCUGAGGUCAAU
364


13-13408


 


SPP1-800-
13410
Chl
ooooooooooooo
0mm00mm000m00
GUCAGCUGGAUGA
365


13-13410


 


SPP1-492-
13412
Chl
ooooooooooooo
mmmm00m000mmm
UUCUGAUGAAUCU
366


13-13412


 


SPP1-612-
13414
Chl
ooooooooooooo
m000mm0000mm0
UGGACUGAGGUCA
367


13-13414


 


SPP1-481-
13416
Chl
ooooooooooooo
000mmmm0mm0mm
GAGUCUCACCAUU
368


13-13416


 


SPP1-614-
13418
Chl
ooooooooooooo
00mm0000mm000
GACUGAGGUCAAA
369


13-13418


 


SPP1-951-
13420
Chl
ooooooooooooo
mm0m00mm0m000
UCACAGCCAUGAA
370


13-13420


 


SPP1-482-
13422
Chl
ooooooooooooo
00mmmm0mm0mmm
AGUCUCACCAUUC
371


13-13422


 


SPP1-856-
13424
Chl
ooooooooooooo
000m000000mm0
AAGCGGAAAGCCA
372


13-13424


 


SPP1-857-
13426
Chl
ooooooooooooo
00m000000mm00
AGCGGAAAGCCAA
373


13-13426


 


SPP1-365-
13428
Chl
ooooooooooooo
0mm0m0m000m00
ACCACAUGGAUGA
374


13-13428


 


SPP1-359-
13430
Chl
ooooooooooooo
0mm0m00mm0m0m
GCCAUGACCACAU
375


13-13430


 


SPP1-357-
13432
Chl
ooooooooooooo
000mm0m00mm0m
AAGCCAUGACCAC
376


13-13432


 


SPP1-858-
13434
Chl
ooooooooooooo
0m000000mm00m
GCGGAAAGCCAAU
377


13-13434


 


SPP1-1012-
13436
Chl
ooooooooooooo
000mmmm0m0mmm
AAAUUUCGUAUUU
378


13-13436


 


SPP1-1014-
13438
Chl
ooooooooooooo
0mmmm0m0mmmmm
AUUUCGUAUUUCU
379


13-13438


 


SPP1-356-
13440
Chl
ooooooooooooo
0000mm0m00mm0
AAAGCCAUGACCA
380


13-13440


 


SPP1-368-
13442
Chl
ooooooooooooo
0m0m000m00m0m
ACAUGGAUGAUAU
381


13-13442


 


SPP1-1011-
13444
Chl
ooooooooooooo
0000mmmm0m0mm
GAAAUUUCGUAUU
382


13-13444


 


SPP1-754-
13446
Chl
ooooooooooooo
0m0mmmmmm00mm
GCGCCUUCUGAUU
383


13-13446


 


SPP1-1021-
13448
Chl
ooooooooooooo
0mmmmmm0m000m
AUUUCUCAUGAAU
384


13-13448


 


SPP1-1330-
13450
Chl
ooooooooooooo
mmmmm0m000m00
CUCUCAUGAAUAG
385


13-13450


 


SPP1-346-
13452
Chl
ooooooooooooo
000mmm00m0000
AAGUCCAACGAAA
386


13-13452


 


SPP1-869-
13454
Chl
ooooooooooooo
0m00m00000m00
AUGAUGAGAGCAA
387


13-13454


 


SPP1-701-
13456
Chl
ooooooooooooo
0m000000mm000
GCGAGGAGUUGAA
388


13-13456


 


SPP1-896-
13458
Chl
ooooooooooooo
m00mm00m00mm0
UGAUUGAUAGUCA
389


13-13458


 


SPP1-1035-
13460
Chl
ooooooooooooo
000m00m0m0mmm
AGAUAGUGCAUCU
390


13-13460


 


SPP1-1170-
13462
Chl
ooooooooooooo
0m0m0m0mmm0mm
AUGUGUAUCUAUU
391


13-13462


 


SPP1-1282-
13464
Chl
ooooooooooooo
mmmm0m0000000
UUCUAUAGAAGAA
392


13-13464


 


SPP1-1537-
13466
Chl
ooooooooooooo
mm0mmm00m00mm
UUGUCCAGCAAUU
393


13-13466


 


SPP1-692-
13468
Chl
ooooooooooooo
0m0m000000m00
ACAUGGAAAGCGA
394


13-13468


 


SPP1-840-
13470
Chl
ooooooooooooo
0m00mmm000mm0
GCAGUCCAGAUUA
395


13-13470


 


SPP1-1163-
13472
Chl
ooooooooooooo
m00mm000m0m0m
UGGUUGAAUGUGU
396


13-13472


 


SPP1-789-
13474
Chl
ooooooooooooo
mm0m0000m000m
UUAUGAAACGAGU
397


13-13474


 


SPP1-841-
13476
Chl
ooooooooooooo
m00mmm000mm0m
CAGUCCAGAUUAU
398


13-13476


 


SPP1-852-
13478
Chl
ooooooooooooo
0m0m000m00000
AUAUAAGCGGAAA
399


13-13478


 


SPP1-209-
13480
Chl
ooooooooooooo
m0mm00mm000m0
UACCAGUUAAACA
400


13-13480


 


SPP1-1276-
13482
Chl
ooooooooooooo
m0mmm0mmmm0m0
UGUUCAUUCUAUA
401


13-13482


 


SPP1-137-
13484
Chl
ooooooooooooo
mm00mm0000000
CCGACCAAGGAAA
402


13-13484


 


SPP1-711-
13486
Chl
ooooooooooooo
000m00m0m0m0m
GAAUGGUGCAUAC
403


13-13486


 


SPP1-582-
13488
Chl
ooooooooooooo
0m0m00m00mm00
AUAUGAUGGCCGA
404


13-13488


 


SPP1-839-
13490
Chl
ooooooooooooo
00m00mmm000mm
AGCAGUCCAGAUU
405


13-13490


 


SPP1-1091-
13492
Chl
ooooooooooooo
0m0mmm00mm000
GCAUUUAGUCAAA
406


13-13492


 


SPP1-884-
13494
Chl
ooooooooooooo
00m0mmmm00m0m
AGCAUUCCGAUGU
407


13-13494


 


SPP1-903-
13496
Chl
ooooooooooooo
m00mm00000mmm
UAGUCAGGAACUU
408


13-13496


 


SPP1-1090-
13498
Chl
ooooooooooooo
m0m0mmm00mm00
UGCAUUUAGUCAA
409


13-13498


 


SPP1-474-
13500
Chl
ooooooooooooo
0mmm00m000mmm
GUCUGAUGAGUCU
410


13-13500


 


SPP1-575-
13502
Chl
ooooooooooooo
m000m0m0m0m00
UAGACACAUAUGA
411


13-13502


 


SPP1-671-
13504
Chl
ooooooooooooo
m000m00000m0m
CAGACGAGGACAU
412


13-13504


 


SPP1-924-
13506
Chl
ooooooooooooo
m00mm0m000mmm
CAGCCGUGAAUUC
413


13-13506


 


SPP1-1185-
13508
Chl
ooooooooooooo
00mmm00000m00
AGUCUGGAAAUAA
414


13-13508


 


SPP1-1221-
13510
Chl
ooooooooooooo
00mmm0m00mmmm
AGUUUGUGGCUUC
415


13-13510


 


SPP1-347-
13512
Chl
ooooooooooooo
00mmm00m00000
AGUCCAACGAAAG
416


13-13512


 


SPP1-634-
13514
Chl
ooooooooooooo
000mmmm0m000m
AAGUUUCGCAGAC
417


13-13514


 


SPP1-877-
13516
Chl
ooooooooooooo
00m00m000m0mm
AGCAAUGAGCAUU
418


13-13516


 


SPP1-1033-
13518
Chl
ooooooooooooo
mm000m00m0m0m
UUAGAUAGUGCAU
419


13-13518


 


SPP1-714-
13520
Chl
ooooooooooooo
m00m0m0m0m000
UGGUGCAUACAAG
420


13-13520


 


SPP1-791-
13522
Chl
ooooooooooooo
0m0000m000mm0
AUGAAACGAGUCA
421


13-13522


 


SPP1-813-
13524
Chl
ooooooooooooo
mm0000m0mm000
CCAGAGUGCUGAA
422


13-13524


 


SPP1-939-
13526
Chl
ooooooooooooo
m00mm0m000mmm
CAGCCAUGAAUUU
423


13-13526


 


SPP1-1161-
13528
Chl
ooooooooooooo
0mm00mm000m0m
AUUGGUUGAAUGU
424


13-13528


 


SPP1-1164-
13530
Chl
ooooooooooooo
00mm000m0m0m0
GGUUGAAUGUGUA
425


13-13530


 


SPP1-1190-
13532
Chl
ooooooooooooo
00000m00mm00m
GGAAAUAACUAAU
426


13-13532


 


SPP1-1333-
13534
Chl
ooooooooooooo
mm0m000m00000
UCAUGAAUAGAAA
427


13-13534


 


SPP1-537-
13536
Chl
ooooooooooooo
0mm00m00mm000
GCCAGCAACCGAA
428


13-13536


 


SPP1-684-
13538
Chl
ooooooooooooo
m0mmmm0m0m0m0
CACCUCACACAUG
429


13-13538


 


SPP1-707-
13540
Chl
ooooooooooooo
00mm000m00m0m
AGUUGAAUGGUGC
430


13-13540


 


SPP1-799-
13542
Chl
ooooooooooooo
00mm00mm000m0
AGUCAGCUGGAUG
431


13-13542


 


SPP1-853-
13544
Chl
ooooooooooooo
m0m000m000000
UAUAAGCGGAAAG
432


13-13544


 


SPP1-888-
13546
Chl
ooooooooooooo
mmmm00m0m00mm
UUCCGAUGUGAUU
433


13-13546


 


SPP1-1194-
13548
Chl
ooooooooooooo
0m00mm00m0m0m
AUAACUAAUGUGU
434


13-13548


 


SPP1-1279-
13550
Chl
ooooooooooooo
mm0mmmm0m0000
UCAUUCUAUAGAA
435


13-13550


 


SPP1-1300-
13552
Chl
ooooooooooooo
00mm0mm0mm0m0
AACUAUCACUGUA
436


13-13552


 


SPP1-1510-
13554
Chl
ooooooooooooo
0mm00mm0mmm0m
GUCAAUUGCUUAU
437


13-13554


 


SPP1-1543-
13556
Chl
ooooooooooooo
00m00mm00m000
AGCAAUUAAUAAA
438


13-13556


 


SPP1-434-
13558
Chl
ooooooooooooo
0m00mmmm00m00
ACGACUCUGAUGA
439


13-13558


 


SPP1-600-
13560
Chl
ooooooooooooo
m00m0m00mmm0m
UAGUGUGGUUUAU
440


13-13560


 


SPP1-863-
13562
Chl
ooooooooooooo
000mm00m00m00
AAGCCAAUGAUGA
441


13-13562


 


SPP1-902-
13564
Chl
ooooooooooooo
0m00mm00000mm
AUAGUCAGGAACU
442


13-13564


 


SPP1-921-
13566
Chl
ooooooooooooo
00mm00mm0m000
AGUCAGCCGUGAA
443


13-13566


 


SPP1-154-
13568
Chl
ooooooooooooo
0mm0mm0m00000
ACUACCAUGAGAA
444


13-13568


 


SPP1-217-
13570
Chl
ooooooooooooo
000m000mm00mm
AAACAGGCUGAUU
445


13-13570


 


SPP1-816-
13572
Chl
ooooooooooooo
000m0mm0000mm
GAGUGCUGAAACC
446


13-13572


 


SPP1-882-
13574
Chl
ooooooooooooo
m000m0mmmm00m
UGAGCAUUCCGAU
447


13-13574


 


SPP1-932-
13576
Chl
ooooooooooooo
00mmmm0m00mm0
AAUUCCACAGCCA
448


13-13576


 


SPP1-1509-
13578
Chl
ooooooooooooo
m0mm00mm0mmm0
UGUCAAUUGCUUA
449


13-13578


 


SPP1-157-
13580
Chl
ooooooooooooo
0mm0m00000mm0
ACCAUGAGAAUUG
450


13-13580


 


SPP1-350-
13582
Chl
ooooooooooooo
mm00m00000mm0
CCAACGAAAGCCA
451


13-13582


 


SPP1-511-
13584
Chl
ooooooooooooo
mm00mm0mm00mm
CUGGUCACUGAUU
452


13-13584


 


SPP1-605-
13586
Chl
ooooooooooooo
m00mmm0m000mm
UGGUUUAUGGACU
453


13-13586


 


SPP1-811-
13588
Chl
ooooooooooooo
00mm0000m0mm0
GACCAGAGUGCUG
454


13-13588


 


SPP1-892-
13590
Chl
ooooooooooooo
00m0m00mm00m0
GAUGUGAUUGAUA
455


13-13590


 


SPP1-922-
13592
Chl
ooooooooooooo
0mm00mm0m000m
GUCAGCCGUGAAU
456


13-13592


 


SPP1-1169-
13594
Chl
ooooooooooooo
00m0m0m0mmm0m
AAUGUGUAUCUAU
457


13-13594


 


SPP1-1182-
13596
Chl
ooooooooooooo
mm000mmm00000
UUGAGUCUGGAAA
458


13-13596


 


SPP1-1539-
13598
Chl
ooooooooooooo
0mmm00m00mm00
GUCCAGCAAUUAA
459


13-13598


 


SPP1-1541-
13600
Chl
ooooooooooooo
mm00m00mm00m0
CCAGCAAUUAAUA
460


13-13600


 


SPP1-427-
13602
Chl
ooooooooooooo
00mmm000m00mm
GACUCGAACGACU
461


13-13602


 


SPP1-533-
13604
Chl
ooooooooooooo
0mmm0mm00m00m
ACCUGCCAGCAAC
462


13-13604


 


APOB--13-
13763
Chl
ooooooooooooo
0m+00+m0+m0+m
ACtGAaUAcCAaU
463


13763

TEG


 


APOB--13-
13764
Chl
ooooooooooooo
0mm000m0mm00m
ACUGAAUACCAAU
464


13764

TEG


 


MAP4K4--16-
13766
Chl
ooooooooooooo
DY547mm0m0000
CUGUGGAAGUCUA
465


13766



0mmm0


 


PPIB--13-
13767
Chl
ooooooooooooo
mmmmmmmmmmmmm
GGCUACAAAAACA
466


13767


 


PPIB--15-
13768
Chl
oooooooooooo
mm00mm0m00000
UUGGCUACAAAAA
467


13768


ooo
m0
CA


 


PPIB--17-
13769
Chl
oooooooooooo
0mmm00mm0m000
AUUUGGCUACAAA
468


13769


ooooo
00m0
AACA


 


MAP4K4--16-
13939
Chl
ooooooooooooo
m0m0000m0mmm0
UGUAGGAUGUCUA
469


13939


 


APOB-4314-
13940
Chl
ooooooooooooo
0mmm0000000m0
AUCUGGAGAAACA
470


16-13940


 


APOB-4314-
13941
Chl
oooooooooooo
000mmm0000000
AGAUCUGGAGAAA
471


17-13941


ooo
m0
CA


 


APOB--16-
13942
Chl
ooooooooooooo
00mmm0mmm0mm0
GACUCAUCUGCUA
472


13942


 


APOB--18-
13943
Chl
ooooooooooooo
00mmm0mmm0mm0
GACUCAUCUGCUA
473


13943


 


APOB--17-
13944
Chl
oooooooooooo
m000mmm0mmm0m
UGGACUCAUCUGC
474


13944


ooo
m0
UA


 


APOB--19-
13945
Chl
oooooooooooo
m000mmm0mmm0m
UGGACUCAUCUGC
475


13945


ooo
m0
UA


 


APOB-4314-
13946
Chl
ooooooooooooo
0000000m00m0m
GGAGAAACAACAU
476


16-13946


 


APOB-4314-
13947
Chl
oooooooooooo
mm0000000m00m
CUGGAGAAACAAC
477


17-13947


ooo
0m
AU


 


APOB--16-
13948
Chl
ooooooooooooo
00mmmmmm000m0
AGUCCCUCAAACA
478


13948


 


APOB--17-
13949
Chl
oooooooooooo
0000mmmmmm000
AGAGUCCCUCAAA
479


13949


ooo
m0
CA


 


APOB--16-
13950
Chl
ooooooooooooo
0mm000m0mm00m
ACUGAAUACCAAU
480


13950


 


APOB--18-
13951
Chl
ooooooooooooo
0mm000m0mm00m
ACUGAAUACCAAU
481


13951


 


APOB--17-
13952
Chl
oooooooooooo
0m0mm000m0mm0
ACACUGAAUACCA
482


13952


ooo
0m
AU


 


APOB--19-
13953
Chl
oooooooooooo
0m0mm000m0mm0
ACACUGAAUACCA
483


13953


ooo
0m
AU


 


MAP4K4--16-
13766.2
Chl
ooooooooooooo
DY547mm0m0000
CUGUGGAAGUCUA
484


13766.2



0mmm0


 


CTGF-1222-
13980
Chl
ooooooooooooo
0m0000000m0m0
ACAGGAAGAUGUA
485


16-13980


 


CTGF-813-
13981
Chl
ooooooooooooo
000m0000mmmm
GAGUGGAGCGCCU
486


16-13981


 


CTGF-747-
13982
Chl
ooooooooooooo
m0mm000000m0
CGACUGGAAGACA
487


16-13982


 


CTGF-817-
13983
Chl
ooooooooooooo
0000mmmm0mmm
GGAGCGCCUGUUC
488


16-13983


 


CTGF-1174-
13984
Chl
ooooooooooooo
0mm0mm0m00mm0
GCCAUUACAACUG
489


16-13984


 


CTGF-1005-
13985
Chl
ooooooooooooo
000mmmmmm00mm
GAGCUUUCUGGCU
490


16-13985


 


CTGF-814-
13986
Chl
ooooooooooooo
00m0000mmmm0
AGUGGAGCGCCUG
491


16-13986


 


CTGF-816-
13987
Chl
ooooooooooooo
m0000mmmm0mm
UGGAGCGCCUGUU
492


16-13987


 


CTGF-1001-
13988
Chl
ooooooooooooo
0mmm000mmmmmm
GUUUGAGCUUUCU
493


16-13988


 


CTGF-1173-
13989
Chl
ooooooooooooo
m0mm0mm0m00mm
UGCCAUUACAACU
494


16-13989


 


CTGF-749-
13990
Chl
ooooooooooooo
0mm000000m0m
ACUGGAAGACACG
495


16-13990


 


CTGF-792-
13991
Chl
ooooooooooooo
00mm0mmm00mmm
AACUGCCUGGUCC
496


16-13991


 


CTGF-1162-
13992
Chl
ooooooooooooo
000mmm0m0mmm0
AGACCUGUGCCUG
497


16-13992


 


CTGF-811-
13993
Chl
ooooooooooooo
m0000m0000mm
CAGAGUGGAGCGC
498


16-13993


 


CTGF-797-
13994
Chl
ooooooooooooo
mmm00mmm000mm
CCUGGUCCAGACC
499


16-13994


 


CTGF-1175-
13995
Chl
ooooooooooooo
mm0mm0m00mm0m
CCAUUACAACUGU
500


16-13995


 


CTGF-1172-
13996
Chl
ooooooooooooo
mm0mm0mm0m00m
CUGCCAUUACAAC
501


16-13996


 


CTGF-1177-
13997
Chl
ooooooooooooo
0mm0m00mm0mmm
AUUACAACUGUCC
502


16-13997


 


CTGF-1176-
13998
Chl
ooooooooooooo
m0mm0m00mm0mm
CAUUACAACUGUC
503


16-13998


 


CTGF-812-
13999
Chl
ooooooooooooo
0000m0000mmm
AGAGUGGAGCGCC
504


16-13999


 


CTGF-745-
14000
Chl
ooooooooooooo
0mm0mm000000
ACCGACUGGAAGA
505


16-14000


 


CTGF-1230-
14001
Chl
ooooooooooooo
0m0m0m0000m0
AUGUACGGAGACA
506


16-14001


 


CTGF-920-
14002
Chl
ooooooooooooo
0mmmm0m000mm
GCCUUGCGAAGCU
507


16-14002


 


CTGF-679-
14003
Chl
ooooooooooooo
0mm0m00000m0
GCUGCGAGGAGUG
508


16-14003


 


CTGF-992-
14004
Chl
ooooooooooooo
0mmm0mm000mmm
GCCUAUCAAGUUU
509


16-14004


 


CTGF-1045-
14005
Chl
ooooooooooooo
00mmmm0m0000m
AAUUCUGUGGAGU
510


16-14005


 


CTGF-1231-
14006
Chl
ooooooooooooo
m0m0m0000m0m
UGUACGGAGACAU
511


16-14006


 


CTGF-991-
14007
Chl
ooooooooooooo
00mmm0mm000mm
AGCCUAUCAAGUU
512


16-14007


 


CTGF-998-
14008
Chl
ooooooooooooo
m000mmm000mmm
CAAGUUUGAGCUU
513


16-14008


 


CTGF-1049-
14009
Chl
ooooooooooooo
mm0m0000m0m0m
CUGUGGAGUAUGU
514


16-14009


 


CTGF-1044-
14010
Chl
ooooooooooooo
000mmmm0m0000
AAAUUCUGUGGAG
515


16-14010


 


CTGF-1327-
14011
Chl
ooooooooooooo
mmmm00m00m0m0
UUUCAGUAGCACA
516


16-14011


 


CTGF-1196-
14012
Chl
ooooooooooooo
m00m00m0mmmmm
CAAUGACAUCUUU
517


16-14012


 


CTGF-562-
14013
Chl
ooooooooooooo
00m0mm00m0m0m
AGUACCAGUGCAC
518


16-14013


 


CTGF-752-
14014
Chl
ooooooooooooo
000000m0mmmm
GGAAGACACGUUU
519


16-14014


 


CTGF-994-
14015
Chl
ooooooooooooo
mm0mm000mmm00
CUAUCAAGUUUGA
520


16-14015


 


CTGF-1040-
14016
Chl
ooooooooooooo
00mm000mmmm0m
AGCUAAAUUCUGU
521


16-14016


 


CTGF-1984-
14017
Chl
ooooooooooooo
000m0000m0m00
AGGUAGAAUGUAA
522


16-14017


 


CTGF-2195-
14018
Chl
ooooooooooooo
00mm00mm00mmm
AGCUGAUCAGUUU
523


16-14018


 


CTGF-2043-
14019
Chl
ooooooooooooo
mmmm0mmm000m0
UUCUGCUCAGAUA
524


16-14019


 


CTGF-1892-
14020
Chl
ooooooooooooo
mm0mmm000mm00
UUAUCUAAGUUAA
525


16-14020


 


CTGF-1567-
14021
Chl
ooooooooooooo
m0m0m00m00m0
UAUACGAGUAAUA
526


16-14021


 


CTGF-1780-
14022
Chl
ooooooooooooo
00mm000m00mmm
GACUGGACAGCUU
527


16-14022


 


CTGF-2162-
14023
Chl
ooooooooooooo
0m00mmmmm0mm0
AUGGCCUUUAUUA
528


16-14023


 


CTGF-1034-
14024
Chl
ooooooooooooo
0m0mm00mm000
AUACCGAGCUAAA
529


16-14024


 


CTGF-2264-
14025
Chl
ooooooooooooo
mm0mm00000m0m
UUGUUGAGAGUGU
530


16-14025


 


CTGF-1032-
14026
Chl
ooooooooooooo
0m0m0mm00mm0
ACAUACCGAGCUA
531


16-14026


 


CTGF-1535-
14027
Chl
ooooooooooooo
00m0000000mm0
AGCAGAAAGGUUA
532


16-14027


 


CTGF-1694-
14028
Chl
ooooooooooooo
00mm0mmmmmm00
AGUUGUUCCUUAA
533


16-14028


 


CTGF-1588-
14029
Chl
ooooooooooooo
0mmm0000m0m00
AUUUGAAGUGUAA
534


16-14029


 


CTGF-928-
14030
Chl
ooooooooooooo
000mm00mmm000
AAGCUGACCUGGA
535


16-14030


 


CTGF-1133-
14031
Chl
ooooooooooooo
00mm0m0000000
GGUCAUGAAGAAG
536


16-14031


 


CTGF-912-
14032
Chl
ooooooooooooo
0m00mm000mmmm
AUGGUCAGGCCUU
537


16-14032


 


CTGF-753-
14033
Chl
ooooooooooooo
00000m0mmmm0
GAAGACACGUUUG
538


16-14033


 


CTGF-918-
14034
Chl
ooooooooooooo
000mmmm0m000
AGGCCUUGCGAAG
539


16-14034


 


CTGF-744-
14035
Chl
ooooooooooooo
m0mm0mm00000
UACCGACUGGAAG
540


16-14035


 


CTGF-466-
14036
Chl
ooooooooooooo
0mmm0000mm0
ACCGCAAGAUCGG
541


16-14036


 


CTGF-917-
14037
Chl
ooooooooooooo
m000mmmm0m00
CAGGCCUUGCGAA
542


16-14037


 


CTGF-1038-
14038
Chl
ooooooooooooo
m00mm000mmmm
CGAGCUAAAUUCU
543


16-14038


 


CTGF-1048-
14039
Chl
ooooooooooooo
mmm0m0000m0m0
UCUGUGGAGUAUG
544


16-14039


 


CTGF-1235-
14040
Chl
ooooooooooooo
m0000m0m00m0
CGGAGACAUGGCA
545


16-14040


 


CTGF-868
14041
Chl
ooooooooooooo
0m00m00mmmmm
AUGACAACGCCUC
546


16-14041


 


CTGF-1131-
14042
Chl
ooooooooooooo
0000mm0m00000
GAGGUCAUGAAGA
547


16-14042


 


CTGF-1043-
14043
Chl
ooooooooooooo
m000mmmm0m000
UAAAUUCUGUGGA
548


16-14043


 


CTGF-751-
14044
Chl
ooooooooooooo
m000000m0mmm
UGGAAGACACGUU
549


16-14044


 


CTGF-1227-
14045
Chl
ooooooooooooo
0000m0m0m000
AAGAUGUACGGAG
550


16-14045


 


CTGF-867-
14046
Chl
ooooooooooooo
00m00m00mmmm
AAUGACAACGCCU
551


16-14046


 


CTGF-1128-
14047
Chl
ooooooooooooo
00m000mm0m00
GGCGAGGUCAUGA
552


16-14047


 


CTGF-756-
14048
Chl
ooooooooooooo
00m0m0mmm00mm
GACACGUUUGGCC
553


16-14048


 


CTGF-1234-
14049
Chl
ooooooooooooo
0m00000m0m00m
ACGGAGACAUGGC
554


16-14049


 


CTGF-916-
14050
Chl
ooooooooooooo
mm000mmmm0m00
UCAGGCCUUGCGA
555


16-14050


 


CTGF-925-
14051
Chl
ooooooooooooo
0m0000mm00mmm
GCGAAGCUGACCU
556


16-14051


 


CTGF-1225-
14052
Chl
ooooooooooooo
000000m0m0m00
GGAAGAUGUACGG
557


16-14052


 


CTGF-445-
14053
Chl
ooooooooooooo
0m00mmmm00mmm
GUGACUUCGGCUC
558


16-14053


 


CTGF-446-
14054
Chl
ooooooooooooo
m00mmmm00mmmm
UGACUUCGGCUCC
559


16-14054


 


CTGF-913-
14055
Chl
ooooooooooooo
m00mm000mmmm0
UGGUCAGGCCUUG
560


16-14055


 


CTGF-997-
14056
Chl
ooooooooooooo
mm000mmm000mm
UCAAGUUUGAGCU
561


16-14056


 


CTGF-277-
14057
Chl
ooooooooooooo
0mm0000mm0m00
GCCAGAACUGCAG
562


16-14057


 


CTGF-1052-
14058
Chl
ooooooooooooo
m0000m0m0m0mm
UGGAGUAUGUACC
563


16-14058


 


CTGF-887-
14059
Chl
ooooooooooooo
0mm0000000m00
GCUAGAGAAGCAG
564


16-14059


 


CTGF-914-
14060
Chl
ooooooooooooo
00mm000mmmm0m
GGUCAGGCCUUGC
565


16-14060


 


CTGF-1039-
14061
Chl
ooooooooooooo
000mm000mmmm0
GAGCUAAAUUCUG
566


16-14061


 


CTGF-754-
14062
Chl
ooooooooooooo
0000m0m0mmm00
AAGACACGUUUGG
567


16-14062


 


CTGF-1130-
14063
Chl
ooooooooooooo
m0000mm0m0000
CGAGGUCAUGAAG
568


16-14063


 


CTGF-919-
14064
Chl
ooooooooooooo
00mmmm0m0000m
GGCCUUGCGAAGC
569


16-14064


 


CTGF-922-
14065
Chl
ooooooooooooo
mmm0m0000mm00
CUUGCGAAGCUGA
570


16-14065


 


CTGF-746-
14066
Chl
ooooooooooooo
mm00mm000000m
CCGACUGGAAGAC
571


16-14066


 


CTGF-993-
14067
Chl
ooooooooooooo
mmm0mm000mmm0
CCUAUCAAGUUUG
572


16-14067


 


CTGF-825-
14068
Chl
ooooooooooooo
m0mmmm0000mmm
UGUUCCAAGACCU
573


16-14068


 


CTGF-926-
14069
Chl
ooooooooooooo
m0000mm00mmm0
CGAAGCUGACCUG
574


16-14069


 


CTGF-923-
14070
Chl
ooooooooooooo
mm0m0000mm00m
UUGCGAAGCUGAC
575


16-14070


 


CTGF-866-
14071
Chl
ooooooooooooo
m00m00m00m0mm
CAAUGACAACGCC
576


16-14071


 


CTGF-563-
14072
Chl
ooooooooooooo
0m0mm00m0m0m0
GUACCAGUGCACG
577


16-14072


 


CTGF-823-
14073
Chl
ooooooooooooo
mmm0mmmm0000m
CCUGUUCCAAGAC
578


16-14073


 


CTGF-1233-
14074
Chl
ooooooooooooo
m0m00000m0m00
UACGGAGACAUGG
579


16-14074


 


CTGF-924-
14075
Chl
ooooooooooooo
m0m0000mm00mm
UGCGAAGCUGACC
580


16-14075


 


CTGF-921-
14076
Chl
ooooooooooooo
mmmm0m0000mm0
CCUUGCGAAGCUG
581


16-14076


 


CTGF-443-
14077
Chl
ooooooooooooo
mm0m00mmmm00m
CUGUGACUUCGGC
582


16-14077


 


CTGF-1041-
14078
Chl
ooooooooooooo
0mm000mmmm0m0
GCUAAAUUCUGUG
583


16-14078


 


CTGF-1042-
14079
Chl
ooooooooooooo
mm000mmmm0m00
CUAAAUUCUGUGG
584


16-14079


 


CTGF-755-
14080
Chl
ooooooooooooo
000m0m0mmm00m
AGACACGUUUGGC
585


16-14080


 


CTGF-467-
14081
Chl
ooooooooooooo
mm0m0000mm00m
CCGCAAGAUCGGC
586


16-14081


 


CTGF-995-
14082
Chl
ooooooooooooo
m0mm000mmm000
UAUCAAGUUUGAG
587


16-14082


 


CTGF-927-
14083
Chl
ooooooooooooo
0000mm00mmm00
GAAGCUGACCUGG
588


16-14083


 


SPP1-1091-
14131
Chl
ooooooooooooo
0m0mmm00mm000
GCAUUUAGUCAAA
589


16-14131


 


PPIB--16-
14188
Chl
ooooooooooooo
mmmmmmmmmmmmm
GGCUACAAAAACA
590


14188


 


PPIB--17-
14189
Chl
oooooooooooo
mm00mm0m00000
UUGGCUACAAAAA
591


14189


ooo
m0
CA


 


PPIB--18-
14190
Chl
oooooooooooo
0mmm00mm0m000
AUUUGGCUACAAA
592


14190


ooooo
00m0
AACA


 


pGL3-1172-
14386
chl
ooooooooooooo
0m000m0m00mmm
ACAAAUACGAUUU
593


16-14386


 


pGL3-1172-
14387
chl
ooooooooooooo
DY5470m000m0m
ACAAAUACGAUUU
594


16-14387



00mmm


 


MAP4K4-
14390
Chl
oooooooooooo
Pmmmmmmmmmmmm
CUUUGAAGAGUUC
595


2931-25-


ooooooooooooo
000mmmmmmmmmm
UGUGGAAGUCUA


14390


 


miR-122--
14391
Chl
ssoooooooooo
mmmmmmmmmmmmm
ACAAACACCAUUG
596


23-14391


ooooooossss
mmmmmmmmmm
UCACACUCCA


 



14084
Chl
ooooooooooooo
mmm0m000mm000
CUCAUGAAUUAGA
719


 



14085
Chl
ooooooooooooo
mm0000mm00mm0
CUGAGGUCAAUUA
720


 



14086
Chl
ooooooooooooo
0000mm00mm000
GAGGUCAAUUAAA
721


 



14087
Chl
ooooooooooooo
mmm0000mm00mm
UCUGAGGUCAAUU
722


 



14088
Chl
ooooooooooooo
m0000mm00mm00
UGAGGUCAAUUAA
723


 



14089
Chl
ooooooooooooo
mmmm0000mm00m
UUCUGAGGUCAAU
724


 



14090
Chl
ooooooooooooo
0mm00mm000m00
GUCAGCUGGAUGA
725


 



14091
Chl
ooooooooooooo
mmmm00m000mmm
UUCUGAUGAAUCU
726


 



14092
Chl
ooooooooooooo
m000mm0000mm0
UGGACUGAGGUCA
727


 



14093
Chl
ooooooooooooo
000mmmm0mm0mm
GAGUCUCACCAUU
728


 



14094
Chl
ooooooooooooo
00mm0000mm000
GACUGAGGUCAAA
729


 



14095
Chl
ooooooooooooo
mm0m00mm0m000
UCACAGCCAUGAA
730


 



14096
Chl
ooooooooooooo
00mmmm0mm0mmm
AGUCUCACCAUUC
731


 



14097
Chl
ooooooooooooo
000m00000mm0
AAGCGGAAAGCCA
732


 



14098
Chl
ooooooooooooo
00m00000mm00
AGCGGAAAGCCAA
733


 



14099
Chl
ooooooooooooo
0mm0m0m000m00
ACCACAUGGAUGA
734


 



14100
Chl
ooooooooooooo
0mm0m00mm0m0m
GCCAUGACCACAU
735


 



14101
Chl
ooooooooooooo
000mm0m00mm0m
AAGCCAUGACCAC
736


 



14102
Chl
ooooooooooooo
0m00000mm00m
GCGGAAAGCCAAU
737


 



14103
Chl
ooooooooooooo
000mmmmm0mmm
AAAUUUCGUAUUU
738


 



14104
Chl
ooooooooooooo
0mmmmm0mmmmm
AUUUCGUAUUUCU
739


 



14105
Chl
ooooooooooooo
0000mm0m00mm0
AAAGCCAUGACCA
740


 



14106
Chl
ooooooooooooo
0m0m000m00m0m
ACAUGGAUGAUAU
741


 



14107
Chl
ooooooooooooo
0000mmmmm0mm
GAAAUUUCGUAUU
742


 



14108
Chl
ooooooooooooo
0mmmmmmm00mm
GCGCCUUCUGAUU
743


 



14109
Chl
ooooooooooooo
0mmmmmm0m000m
AUUUCUCAUGAAU
744


 



14110
Chl
ooooooooooooo
mmmmm0m000m00
CUCUCAUGAAUAG
745


 



14111
Chl
ooooooooooooo
000mmm00m000
AAGUCCAACGAAA
746


 



14112
Chl
ooooooooooooo
0m00m00000m00
AUGAUGAGAGCAA
747


 



14113
Chl
ooooooooooooo
0m00000mm000
GCGAGGAGUUGAA
748


 



14114
Chl
ooooooooooooo
m00mm00m00mm0
UGAUUGAUAGUCA
749


 



14115
Chl
ooooooooooooo
000m00m0m0mmm
AGAUAGUGCAUCU
750


 



14116
Chl
ooooooooooooo
0m0m0m0mmm0mm
AUGUGUAUCUAUU
751


 



14117
Chl
ooooooooooooo
mmmm0m0000000
UUCUAUAGAAGAA
752


 



14118
Chl
ooooooooooooo
mm0mmm00m00mm
UUGUCCAGCAAUU
753


 



14119
Chl
ooooooooooooo
0m0m000000m0
ACAUGGAAAGCGA
754


 



14120
Chl
ooooooooooooo
0m00mmm000mm0
GCAGUCCAGAUUA
755


 



14121
Chl
ooooooooooooo
m00mm000m0m0m
UGGUUGAAUGUGU
756


 



14122
Chl
ooooooooooooo
mm0m0000m00m
UUAUGAAACGAGU
757


 



14123
Chl
ooooooooooooo
m00mmm000mm0m
CAGUCCAGAUUAU
758


 



14124
Chl
ooooooooooooo
0m0m000m0000
AUAUAAGCGGAAA
759


 



14125
Chl
ooooooooooooo
m0mm00mm000m0
UACCAGUUAAACA
760


 



14126
Chl
ooooooooooooo
m0mmm0mmmm0m0
UGUUCAUUCUAUA
761


 



14127
Chl
ooooooooooooo
mm0mm0000000
CCGACCAAGGAAA
762


 



14128
Chl
ooooooooooooo
000m00m0m0m0m
GAAUGGUGCAUAC
763


 



14129
Chl
ooooooooooooo
0m0m00m00mm0
AUAUGAUGGCCGA
764


 



14130
Chl
ooooooooooooo
00m00mmm000mm
AGCAGUCCAGAUU
765


 



14132
Chl
ooooooooooooo
00m0mmmm0m0m
AGCAUUCCGAUGU
766


 



14133
Chl
ooooooooooooo
m00mm00000mmm
UAGUCAGGAACUU
767


 



14134
Chl
ooooooooooooo
m0m0mmm00mm00
UGCAUUUAGUCAA
768


 



14135
Chl
ooooooooooooo
0mmm00m000mmm
GUCUGAUGAGUCU
769


 



14136
Chl
ooooooooooooo
m000m0m0m0m00
UAGACACAUAUGA
770


 



14137
Chl
ooooooooooooo
m000m0000m0m
CAGACGAGGACAU
771


 



14138
Chl
ooooooooooooo
m00mmm000mmm
CAGCCGUGAAUUC
772


 



14139
Chl
ooooooooooooo
00mmm00000m00
AGUCUGGAAAUAA
773


 



14140
Chl
ooooooooooooo
00mmm0m00mmmm
AGUUUGUGGCUUC
774


 



14141
Chl
ooooooooooooo
00mmm00m0000
AGUCCAACGAAAG
775


 



14142
Chl
ooooooooooooo
000mmmmm000m
AAGUUUCGCAGAC
776


 



14143
Chl
ooooooooooooo
00m00m000m0mm
AGCAAUGAGCAUU
777


 



14144
Chl
ooooooooooooo
mm000m00m0m0m
UUAGAUAGUGCAU
778


 



14145
Chl
ooooooooooooo
m00m0m0m0m000
UGGUGCAUACAAG
779


 



14146
Chl
ooooooooooooo
0m0000m00mm0
AUGAAACGAGUCA
780


 



14147
Chl
ooooooooooooo
mm0000m0mm000
CCAGAGUGCUGAA
781


 



14148
Chl
ooooooooooooo
m00mm0m000mmm
CAGCCAUGAAUUU
782


 



14149
Chl
ooooooooooooo
0mm00mm000m0m
AUUGGUUGAAUGU
783


 



14150
Chl
ooooooooooooo
00mm000m0m0m0
GGUUGAAUGUGUA
784


 



14151
Chl
ooooooooooooo
00000m00mm00m
GGAAAUAACUAAU
785


 



14152
Chl
ooooooooooooo
mm0m000m00000
UCAUGAAUAGAAA
786


 



14153
Chl
ooooooooooooo
0mm00m00mm00
GCCAGCAACCGAA
787


 



14154
Chl
ooooooooooooo
m0mmmm0m0m0m0
CACCUCACACAUG
788


 



14155
Chl
ooooooooooooo
00mm000m00m0m
AGUUGAAUGGUGC
789


 



14156
Chl
ooooooooooooo
00mm00mm000m0
AGUCAGCUGGAUG
790


 



14157
Chl
ooooooooooooo
m0m000m00000
UAUAAGCGGAAAG
791


 



14158
Chl
ooooooooooooo
mmmm0m0m00mm
UUCCGAUGUGAUU
792


 



14159
Chl
ooooooooooooo
0m00mm00m0m0m
AUAACUAAUGUGU
793


 



14160
Chl
ooooooooooooo
mm0mmmm0m0000
UCAUUCUAUAGAA
794


 



14161
Chl
ooooooooooooo
00mm0mm0mm0m0
AACUAUCACUGUA
795


 



14162
Chl
ooooooooooooo
0mm00mm0mmm0m
GUCAAUUGCUUAU
796


 



14163
Chl
ooooooooooooo
00m00mm00m000
AGCAAUUAAUAAA
797


 



14164
Chl
ooooooooooooo
0m0mmmm00m00
ACGACUCUGAUGA
798


 



14165
Chl
ooooooooooooo
m00m0m00mmm0m
UAGUGUGGUUUAU
799


 



14166
Chl
ooooooooooooo
000mm00m00m00
AAGCCAAUGAUGA
800


 



14167
Chl
ooooooooooooo
0m00mm00000mm
AUAGUCAGGAACU
801


 



14168
Chl
ooooooooooooo
00mm00mmm000
AGUCAGCCGUGAA
802


 



14169
Chl
ooooooooooooo
0mm0mm0m00000
ACUACCAUGAGAA
803


 



14170
Chl
ooooooooooooo
000m000mm00mm
AAACAGGCUGAUU
804


 



14171
Chl
ooooooooooooo
000m0mm0000mm
GAGUGCUGAAACC
805


 



14172
Chl
ooooooooooooo
m000m0mmmm0m
UGAGCAUUCCGAU
806


 



14173
Chl
ooooooooooooo
00mmmm0m00mm0
AAUUCCACAGCCA
807


 



14174
Chl
ooooooooooooo
m0mm00mm0mmm0
UGUCAAUUGCUUA
808


 



14175
Chl
ooooooooooooo
0mm0m00000mm0
ACCAUGAGAAUUG
809


 



14176
Chl
ooooooooooooo
mm00m0000mm0
CCAACGAAAGCCA
810


 



14177
Chl
ooooooooooooo
mm00mm0mm00mm
CUGGUCACUGAUU
811


 



14178
Chl
ooooooooooooo
m00mmm0m000mm
UGGUUUAUGGACU
812


 



14179
Chl
ooooooooooooo
00mm0000m0mm0
GACCAGAGUGCUG
813


 



14180
Chl
ooooooooooooo
00m0m00mm00m0
GAUGUGAUUGAUA
814


 



14181
Chl
ooooooooooooo
0mm00mmm000m
GUCAGCCGUGAAU
815


 



14182
Chl
ooooooooooooo
00m0m0m0mmm0m
AAUGUGUAUCUAU
816


 



14183
Chl
ooooooooooooo
mm000mmm00000
UUGAGUCUGGAAA
817


 



14184
Chl
ooooooooooooo
0mmm00m00mm00
GUCCAGCAAUUAA
818


 



14185
Chl
ooooooooooooo
mm00m00mm00m0
CCAGCAAUUAAUA
819


 



14186
Chl
ooooooooooooo
00mmm00m0mm
GACUCGAACGACU
820


 



14187
Chl
ooooooooooooo
0mmm0mm00m00m
ACCUGCCAGCAAC
821





o: phosphodiester;


s: phosphorothioate;


P: 5′ phosphorylation;


0: 2′-OH;


F: 2′-fluoro;


m: 2′ O-methyl;


+: LNA modification.


Capital letters in the sequence signify ribonucleotides, lower case letters signify deoxyribonucleotides.






Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
All references, including patent documents, disclosed herein are incorporated by reference in their entirety. This application incorporates by reference the entire contents, including all the drawings and all parts of the specification (including sequence listing or amino acid/polynucleotide sequences) of the co-pending U.S. Provisional Application No. 61/135,855, filed on Jul. 24, 2008, entitled “SHORT HAIRPIN RNAI CONSTRUCTS AND USES THEREOF,” and U.S. Provisional Application No. 61/197,768, filed on Oct. 30, 2008, entitled “MINIRNA CONSTRUCTS AND USES THEREOF.”

## Claims

1. An isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand,
wherein the isolated double stranded nucleic acid molecule includes a double stranded region and a single stranded region, wherein the double stranded region is from 8-15 nucleotides long, wherein the single stranded region is at the 3′ end of the guide strand and is 4-12 nucleotides long, wherein the single stranded region contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, wherein at least 40% of the nucleotides of the isolated double stranded nucleic acid molecule are modified, wherein a hydrophobic conjugate is attached to the isolated double stranded nucleic acid molecule, and wherein the isolated double stranded nucleic acid molecule does not form a hairpin.

2. The isolated double stranded nucleic acid molecule of claim 1, wherein the double stranded region is 11, 12, 13, or 14 nucleotides long and/or wherein the single stranded region is at least 6 or at least 7 nucleotides long.

3. The isolated double stranded nucleic acid molecule of claim 1, wherein each nucleotide within the single stranded region has a phosphorothioate modification.

4. The isolated double stranded nucleic acid molecule of claim 1, wherein at least one of the nucleotides of the isolated double stranded nucleic acid molecule that is modified comprises a 2′ O-methyl or a 2′-fluoro modification and/or wherein at least one of the nucleotides of the isolated double stranded nucleic acid molecule that is modified comprises a hydrophobic modification.

5. The isolated double stranded nucleic acid molecule of claim 1, wherein the guide strand of the double stranded nucleic acid molecule exhibits complementarity to a gene encoding for Osteopontin (SPP1), SOD1 or MAP4K4, optionally wherein the guide strand comprises SEQ ID NO:170, SEQ ID NO:40 or SEQ ID NO:25.

6. An isolated asymmetric nucleic acid molecule comprising:
a first polynucleotide wherein the first polynucleotide is complementary to a second polynucleotide and a target gene; and
a second polynucleotide,
wherein the second polynucleotide is at least 6 nucleotides shorter than the first polynucleotide, wherein the first polynucleotide includes a single stranded region of 6, 7, 8, 9, 10, 11 or 12 nucleotides, wherein the single stranded region of the first polynucleotide contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, wherein the asymmetric nucleic acid molecule also includes a double stranded region of 8-15 nucleotides long, wherein at least 50% of C and U nucleotides in the double stranded region are 2′ O-methyl modified or 2′-fluoro modified, and wherein a hydrophobic conjugate is attached to the asymmetric nucleic acid molecule.

7. The isolated asymmetric nucleic acid molecule of claim 6, wherein the single stranded region is 6 or 7 nucleotides long and/or wherein each nucleotide within the single stranded region has a phosphorothioate modification.

8. The isolated asymmetric nucleic acid molecule of claim 6, wherein each of the first polynucleotide and the second polynucleotide has a 5′ end and a 3′ end, wherein the hydrophobic conjugate is attached to:
i) the 3′ end of the second polynucleotide;
ii) the 3′ end of the first polynucleotide; or
iii) the 5′ end of the second polynucleotide.

9. An isolated double stranded nucleic acid molecule comprising:
a guide strand of 17-21 nucleotides in length that has complementarity to a target gene, and a passenger strand of 8-16 nucleotides in length,
wherein the isolated double stranded nucleic acid molecule includes a double stranded region of 8-15 nucleotides long and a single stranded region, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule having the double stranded region and the single stranded region, wherein the single stranded region is at the 3′ end of the guide strand and is 4-12 nucleotides in length, wherein the single stranded region comprises 2-12 phosphorothioate modifications, wherein at least 40% of the nucleotides of the isolated double stranded nucleic acid molecule are modified, and wherein the double stranded nucleic acid molecule is linked to a hydrophobic conjugate, and wherein the isolated double stranded nucleic acid molecule does not form a hairpin.

10. The isolated double stranded nucleic acid molecule of claim 9, wherein the isolated double stranded nucleic acid molecule contains at least one hydrophobic base modification and wherein the hydrophobic base modification comprises a hydrophobic modification of a pyrimidine base, optionally at position 4 or 5, optionally wherein the hydrophobic base modification is selected from the group consisting of a phenyl, 4-pyridyl, 2-pyridyl, indolyl, isobutyl, tryptophanyl (C8H6N)CH2CH(NH2)CO), methyl, butyl, aminobenzyl, and naphthyl modification of a uridine or cytidine.

11. The isolated double stranded nucleic acid molecule of claim 9, wherein the hydrophobic conjugate is a small molecule, optionally wherein the small molecule is a sterol-type molecule, optionally wherein the sterol-type molecule is cholesterol.

12. The isolated double stranded nucleic acid molecule of claim 9, wherein the hydrophobic conjugate is attached to the double stranded nucleic acid molecule through a linker, optionally wherein the linker is a TEG linker.

13. A method for inhibiting the expression of a target gene in a mammalian cell, comprising contacting the mammalian cell with an isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand,
wherein the isolated double stranded nucleic acid molecule includes a double stranded region and a single stranded region, wherein the double stranded region is from 8-15 nucleotides long, wherein the single stranded region is at the 3′ end of the guide strand and is 4-12 nucleotides long, wherein the single stranded region of the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, wherein at least 40% of the nucleotides of the isolated double stranded nucleic acid molecule are modified, and wherein a hydrophobic conjugate is attached to the isolated double stranded nucleic acid molecule, and wherein the isolated double stranded nucleic acid molecule does not form a hairpin.

14. The method of claim 13, wherein the double stranded region is 11, 12, 13, or 14 nucleotides long and/or wherein the single stranded region is at least 6 or at least 7 nucleotides long.

15. The method of claim 13, wherein each nucleotide within the single stranded region has a phosphorothioate modification.

16. The method of claim 13, wherein at least one of the nucleotides of the isolated double stranded nucleic acid molecule that is modified comprises a 2′ O-methyl or a 2′-fluoro modification and/or wherein at least one of the nucleotides of the isolated double stranded nucleic acid molecule that is modified comprises a hydrophobic modification.

17. The method of claim 13, wherein the double stranded nucleic acid molecule exhibits complementarity to a gene encoding for Osteopontin (SPP1), SOD1 or MAP4K4, optionally wherein the guide strand comprises SEQ ID NO:170, SEQ ID NO:40 or SEQ ID NO:25.

18. The isolated double stranded nucleic acid molecule of claim 1, wherein each of the guide strand and the passenger strand has a 5′ end and a 3′ end, and wherein the hydrophobic conjugate is attached to:
i) the 3′ end of the passenger strand;
ii) the 3′ end of the guide strand; or
iii) the 5′ end of the passenger strand.

19. The method of claim 13, wherein each of the guide strand and the passenger strand has a 5′ end and a 3′ end, and wherein the hydrophobic conjugate is attached to:
i) the 3′ end of the passenger strand;
ii) the 3′ end of the guide strand; or
iii) the 5′ end of the passenger strand.