US20210261968A1 - Rna interference in dermal and fibrotic indications - Google Patents

Abstract

The present invention relates to RNAi constructs with improved tissue and cellular uptake characteristics and methods of use of these compounds in dermal and fibrotic applications.

Description

RELATED APPLICATIONS
• This application is a National Stage Application of PCT/US2011/029867 which claims priority under 35 U.S.C. § 119(e) to U.S. 61/317,252, entitled “RNA INTERFERENCE IN SKIN INDICATIONS.” filed on Mar. 24, 2010, and U.S. Provisional Application Serial No. U.S. 61/317,633, entitled “RNA INTERFERENCE IN SKIN INDICATIONS,” filed on Mar. 25, 2010, each of which are herein incorporated by reference in their 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 and their use for dermal and fibrotic indications.
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.
SUMMARY OF INVENTION
• Described herein is the efficient in vivo delivery of sd-rxRNA molecules to the skin and the use of such molecules for gene silencing. This class of RNAi molecules has superior efficacy both in vitro and in vivo than previously described RNAi molecules. Molecules associated with the invention have widespread potential as therapeutics for disorders or conditions associated with compromised skin and fibrosis.
• Aspects of the invention relate to double-stranded ribonucleic acids (dsRNAs) including a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23, and wherein the dsRNA is an sd-rxRNA.
• Further aspects of the invention relate to double-stranded ribonucleic acids (dsRNAs) comprising a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27, and wherein the dsRNA is an sd-rxRNA.
• Further aspects of the invention relate to double-stranded ribonucleic acids (dsRNAs) comprising a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23, and wherein the dsRNA is an rxRNAori.
• Further aspects of the invention relate to double-stranded ribonucleic acids (dsRNAs) comprising a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27, and wherein the dsRNA is an rxRNAori.
• In some embodiments, the dsRNA is directed against CTGF. In some embodiments, the antisense strand of the dsRNA is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 11, 12 and 15. In some embodiments, the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 10, 11, 12, 15, 20 and 24.
• In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 2463, 3429, 2443, 3445, 2459, 3493, 2465 and 3469. In some embodiments, the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: 2464, 3430, 4203, 3446, 2460, 3494, 2466 and 3470.
• In certain embodiments, the sense strand comprises SEQ ID NO:2463 and the antisense strand comprises SEQ ID NO:2464. In certain embodiments, the sense strand comprises SEQ ID NO:3429 and the antisense strand comprises SEQ ID NO:3430.
• In certain embodiments, the sense strand comprises SEQ ID NO:2443 and the antisense strand comprises SEQ ID NO:4203. In certain embodiments, the sense strand comprises SEQ ID NO:3445 and the antisense strand comprises SEQ ID NO:3446.
• In certain embodiments, the sense strand comprises SEQ ID NO:2459 and the antisense strand comprises SEQ ID NO:2460. In certain embodiments, the sense strand comprises SEQ ID NO:3493 and the antisense strand comprises SEQ ID NO:3494.
• In certain embodiments, the sense strand comprises SEQ ID NO:2465 and the antisense strand comprises SEQ ID NO:2466. In certain embodiments, the sense strand comprises SEQ ID NO:3469 and the antisense strand comprises SEQ ID NO:3470.
• In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849. In certain embodiments, the sense strand comprises a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849.
• In some embodiments, the dsRNA is hydrophobically modified. In certain embodiments, the dsRNA is linked to a hydrophobic conjugate.
• Aspects of the invention relate to compositions comprising the dsRNA described herein. In some embodiments, the composition comprises dsRNA directed against genes encoding for more than one protein.
• In some embodiments, the composition is formulated for delivery to the skin. In certain embodiments, the composition is in a neutral formulation. In some embodiments, the composition is formulated for topical delivery or for intradermal injection.
• Aspects of the invention relate to methods comprising delivering any of the dsRNA described herein or a composition comprising any of the dsRNA described herein to the skin of a subject in need thereof.
• Aspects of the invention relate to methods comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23, and wherein the dsRNA is an sd-rxRNA.
• Further aspects of the invention relate to methods comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27, and wherein the dsRNA is an sd-rxRNA.
• Further aspects of the invention relate to methods comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23, and wherein the dsRNA is an rxRNAori.
• Further aspects of the invention relate to methods comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27, and wherein the dsRNA is an rxRNAori.
• In some embodiments, the method is a method for treating compromised skin. In some embodiments, the method is a method for treating or preventing a fibrotic disorder.
• In some embodiments, the dsRNA is administered via intradermal injection. In some embodiments, the dsRNA is administered locally to the skin. In some embodiments, two or more nucleic acid molecules are administered simultaneously or sequentially.
• In some embodiments, one or more of the dsRNAs is hydrophobically modified. In certain embodiments, one or more of the dsRNAs is linked to a hydrophobic conjugate.
• In some embodiments, the dsRNA is directed against CTGF. In certain embodiments, the antisense strand of the dsRNA is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 11, 12 and 15. In some embodiments, the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 10, 11, 12, 15, 20 and 24.
• In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 2463, 3429, 2443, 3445, 2459, 3493, 2465 and 3469. In certain embodiments, the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: 2464, 3430, 4203, 3446, 2460, 3494, 2466 and 3470.
• In certain embodiments, the sense strand comprises SEQ ID NO:2463 and the antisense strand comprises SEQ ID NO:2464. In certain embodiments, the sense strand comprises SEQ ID NO:3429 and the antisense strand comprises SEQ ID NO:3430.
• In certain embodiments, the sense strand comprises SEQ ID NO:2443 and the antisense strand comprises SEQ ID NO:4203. In certain embodiments, the sense strand comprises SEQ ID NO:3445 and the antisense strand comprises SEQ ID NO:3446.
• In certain embodiments, the sense strand comprises SEQ ID NO:2459 and the antisense strand comprises SEQ ID NO:2460. In certain embodiments, the sense strand comprises SEQ ID NO:3493 and the antisense strand comprises SEQ ID NO:3494.
• In certain embodiments, the sense strand comprises SEQ ID NO:2465 and the antisense strand comprises SEQ ID NO:2466. In certain embodiments, the sense strand comprises SEQ ID NO:3469 and the antisense strand comprises SEQ ID NO:3470.
• In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849. In some embodiments, the sense strand comprises a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849.
• Aspects of the invention relate to treating or preventing a fibrotic disorder. In some embodiments, the fibrotic disorder is selected from the group consisting of pulmonary fibrosis, liver cirrhosis, scleroderma and glomerulonephritis, lung fibrosis, liver fibrosis, skin fibrosis, muscle fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy, restenosis and uterine fibrosis, and trabeculectomy failure due to scarring.
• In some embodiments, the dsRNA are administered via intradermal injection, while in other embodiments, the one or more dsRNA are administered subcutaneously or epicutaneously.
• The one or more dsRNA can be administered prior to, during and/or after a medical procedure. In some embodiments, administration occurs within 8 days prior to or within 8 days after the medical procedure. In some embodiments, the medical procedure is surgery. In certain embodiments, the surgery is elective. In some embodiments, the surgery comprises epithelial grafting or skin grafting. In some embodiments, the one or more double stranded nucleic acid molecules are administered to a graft donor site and/or a graft recipient site.
• Aspects of the invention relate to methods for administering one or more dsRNA prior to, during and/or after an injury. In some embodiments, the subject has a wound such as a chronic wound. In certain embodiments, the wound is a result of elective surgery. The wound can be external or internal. In some embodiments, the dsRNA is administered after burn injury.
• Methods described herein include methods for promoting wound healing and methods for preventing scarring.
• In some embodiments, one or more of the dsRNA administered to a subject is directed against a gene selected from the group consisting of TGFB1, TGFB2, hTGFB1, hTGFB2, PTGS2, SPP1, hSPP1, CTGF or hCTGF. In some embodiments, the one or more dsRNA are administered on the skin of the subject. In certain embodiments, the one or more dsRNA molecules are in the form of a cream or ointment. In some embodiments, two or more or three or more nucleic acids are administered. Two or more nucleic acid molecules can be administered simultaneously or sequentially.
• Aspects of the invention related to nucleic acids that are optimized. In some embodiments, one or more double stranded nucleic acid molecules are hydrophobically modified. In certain embodiments, the one or more double stranded nucleic acid molecules are linked to a hydrophobic conjugate or multiple hydrophobic conjugates. In some embodiments, the one or more double stranded nucleic acid molecule are linked to a lipophilic group. In certain embodiments, the lipophilic group is linked to the passenger strand of the one or more double stranded nucleic acid molecules. In some embodiment, the one or more double stranded nucleic acid molecules are linked to cholesterol, a long chain alkyl cholesterol analog, vitamin A or vitamin E. In some embodiments, the one or more double stranded nucleic acid molecules is attached to chloroformate.
• Aspects of the invention related to nucleic acids that are optimized through modifications. In some embodiments, the one or more double stranded nucleic acid molecules includes at least one 2′ O methyl or 2′ fluoro modification and/or at least one 5 methyl C or U modification. In some embodiments, the one or more double stranded nucleic acid molecules has a guide strand of 16-28 nucleotides in length. In certain embodiments, at least 40% of the nucleotides of the one or more double stranded nucleic acid molecules are modified. Double stranded nucleic acid molecules described herein can also be attached to linkers. In some embodiments, the linker is protonatable.
• Aspects of the invention relate to double stranded nucleic acid molecules that contain at least two single stranded regions. In some embodiments, the single stranded regions contain phosphorothioate modifications. In certain embodiments, the single stranded regions are located at the 3′ end of the guide strand and the 5′ end of the passenger strand.
• Aspects of the invention relate to methods for delivering a nucleic acid to a subject, involving administering to a subject within 8 days prior to a medical procedure a therapeutically effective amount for treating compromised skin of one or more sd-rxRNAs.
• 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 demonstrates the expression profiles for non-limiting examples of target genes including MAP4K4, SPP1, CTGF, PTGS2 and TGFB1. As expected, target gene expression is elevated early and returns to normal byday 10.
• FIG. 2 presents schematics depicting an experimental approach to visualizing tissue after intradermal injection.
• FIG. 3 demonstrates silencing of MAP4K4 following intradermal injection of sd-rxRNA targeting MAP4K4.
• FIG. 4 demonstrates silencing of MAP4K4, PPIB and CTGF following intradermal injection of sd-rxRNA molecules targeting each gene.
• FIG. 5 demonstrates silencing of MAP4K4 following intradermal injection of sd-rxRNA targeting MAP4K4. Normalized expression of MAP4K4 relative to controls is demonstrated.
• FIG. 6 demonstrates silencing of PPIB following intradermal injection of sd-rxRNA targeting PPIB. Normalized expression of PPIB relative to controls is demonstrated.
• FIG. 7 demonstrates the duration of PPIB silencing following intradermal injection of sd-rxRNA targeting PPIB.
• FIG. 8 demonstrates the duration of MAP4K4 silencing following intradermal injection of sd-rxRNA targeting MAP4K4.
• FIG. 9 demonstrates equivalent silencing achieved using two different dosing regimens.
• FIG. 10 demonstrates examples of sd-rxRNA molecules targeting CTGF that are efficacious for gene silencing.
• FIG. 1 demonstrates examples of sd-rxRNA molecules targeting CTGF that are efficacious for gene silencing.
• FIG. 12 demonstrates a dose response for sd-rxRNA molecules targeting CTGF.
• FIG. 13 demonstrates a sample of an original sd-rxRNA screen.
• FIG. 14 presents data on a hit from the original sd-rxRNA screen.
• FIG. 15 demonstrates gene expression of PTGS2 following administration of sd-rxRNA targeting PTGS2.
• FIG. 16 demonstrates gene expression of hTGFB1 following administration of sd-rxRNA targeting hTGFB1.
• FIG. 17 demonstrates gene expression of hTGFB1 following administration of sd-rxRNA targeting hTGFB1.
• FIG. 18 demonstrates results of TGFB1 sd-rxRNA screening.
• FIG. 19 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.
• FIG. 20 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.
• FIG. 21 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.
• FIG. 22 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.
• FIG. 23 demonstrates gene expression of TGFB2 following administration of sd-rxRNA targeting TGFB2.
• FIG. 24 demonstrates results of TGFB2 sd-rxRNA screening.
• FIG. 25 demonstrates identification of potent hSPP1 sd-rxRNAs.
• FIG. 26 demonstrates identification of potent hSPP1 sd-rxRNAs.
• FIG. 27 demonstrates identification of potent hSPP1 sd-rxRNAs.
• FIG. 28 demonstrates SPP1 sd-rxRNA compound selection.
• FIG. 29 demonstrates that variation of linker chemistry does not influence silencing activity of sd-rxRNAs in vitro. Two different linker chemistries were evaluated, a hydroxyproline linker and ribo linker, on multiple sd-rxRNAs (targeting Map4k4 or PPIB) in passive uptake assays to determine linkers which favor self delivery. HeLa cells were transfected in the absence of a delivery vehicle (passive transfection) with sd-rxRNAs at 1 uM, 0.1 uM or 0.01 uM for 48 hrs. Use of either linker results in an efficacious delivery of sd-rxRNA.
• FIG. 30 depicts CTGF as a central factor in the pathway to fibrosis.
• FIG. 31 depicts the phases of wound healing.
• FIG. 32 depicts the chemical optimization of sd-rxRNA leads.
• FIG. 33 demonstrates that chemically optimized CTGF L1 sd-rxRNAs are active.
• FIG. 34 demonstrates in vitro efficacy of chemically optimized CTGF L1 sd-rxRNAs.
• FIG. 35 demonstrates in vitro stability of chemically optimized CTGF L1 sd-rxRNAs.
• FIG. 36 demonstrates that chemically optimized CTGF L2 sd-rxRNAs are active.
• FIG. 37 demonstrates in vitro efficacy of chemically optimized CTGF L2 sd-rxRNAs.
• FIG. 38 demonstrates in vitro stability of chemically optimized CTGF L2 sd-rxRNAs.
• FIG. 39 provides a summary of compounds that are active in vivo.
• FIG. 40 demonstrates that treatment with CTGF L1B target sequence resulted in mRNA silencing.
• FIG. 41 demonstrates that treatment with CTGF L2 target sequence resulted in mRNA silencing.
• FIG. 42 demonstrates CTGF silencing after two intradermal injections of RXi-109.
• FIG. 43 demonstrates the duration of CTGF silencing in skin after intradermal injection of the sd-rxRNA in SD rats. Eight millimeter skin biopsies were harvested, and mRNA levels were quantified by QPCR and normalized to a housekeeping gene. Shown is percent (%) silencing vs. Non Targeting Control (NTC); PBS at each time point is one experimental group; * p≤0.04; ** p≤0.002.
• FIG. 44 demonstrates that chemically optimized CTGF L3 sd-rxRNAs are active.
• FIG. 45 demonstrates absolute luminescence of CTGF L4 sd-rxRNAs.
• FIG. 46 demonstrates that chemically optimized CTGF L4 sd-rxRNAs are active.
• FIG. 47 demonstrates changes in mRNA expression levels of CTGF, α-SM actin, collagen 1A2, andcollagen 3A 1 after intradermal injection of CTFG sd-rxRNA in SD rats. mRNA levels were quantified by qPCR.
• FIG. 48 demonstrates that there is no apparent delay in wound healing with treatment of CTGF-targeting sd-rxRNA. Some changes was observed with treatment of a combination of CTGF- and COX2-targeting sd-rxRNAs.
• FIG. 49 demonstrates that administration of sd-rxRNAs decreases wound width over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats ondays 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.
• FIG. 50 demonstrates that administration of sd-rxRNAs decreases wound area over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats ondays 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.
• FIG. 51 demonstrates that administration of sd-rxRNAs increase the percentage of wound re-epithelialization over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats ondays 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.
• FIG. 52 demonstrates that administration of sd-rxRNAs increases the average granulation tissue maturity scores over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats ondays 3, 6, and 9 post-wounding (5=mature, 1=immature). Each group represents 5 rats.
• FIG. 53 demonstrates CD68 labeling inday 9 wounds (0=no labeling, 3=substantial labeling). Each group represents 5 rats.
• FIG. 54 demonstrates that CTGF leads have different toxicity levels in vitro.
• FIG. 55 shows percentage (%) of cell viability afterRXI 109 dose escalation (oligos formulated in PBS).
• FIG. 56 is a schematic ofPhases 1 and 2 clinical trial design.
• FIG. 57 is a schematic ofPhases 1 and 2 clinical trial design.
• FIG. 58 demonstrates a percent (%) decrease in PPIB expression in the liver relative to PBS control. Lipoid formulated rxRNAs (10 mg/kg) were delivery systemically to Balb/c mice (n=5) by single tail vein injections. Liver tissue was harvested at 24 hours after injection and expression was analyzed by qPCR (normalized to β-actin). Map4K4 rxRNAori also showed significant silencing (˜83%, p<0.001) although Map4K4 sd-rxRNA did not significantly reduce target gene expression (˜17%, p=0.019). TD.035.2278, Published lipidoid delivery reagent, 98N12-5(1), from Akine, 2009.
• FIG. 59 demonstrates that chemically optimized PTGS2 L1 sd-rxRNAs are active.
• FIG. 60 demonstrates that chemically optimized PTGS2 L2 sd-rxRNAs are active.
• FIG. 61 demonstrates that chemically optimized hTGFB1 L1 sd-rxRNAs are active.
• FIG. 62 demonstrates that chemically optimized hTGFB1 L1 sd-rxRNAs are active.
• FIG. 63 demonstrates that chemically optimized hTGFB2 L1 sd-rxRNAs are active.
• FIG. 64 demonstrates that chemically optimized hTGFB2 sd-rxRNAs are active.
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 administration of sd-rxRNA molecules to the skin, such as through intradermal injection or subcutaneous administration, results in efficient silencing of gene expression in the skin. Highly potent sd-rxRNA molecules that target genes including SPP1, CTGF, PTGS2, TGFB1 and TGFB2 were also identified herein through cell-based screening. sd-rxRNAs represent a new class of therapeutic RNAi molecules with significant potential in treatment of compromised skin.
• sd-rxRNA Molecules
• Aspects of the invention relate to sd-rxRNA molecules. As used herein, an “sd-rxRNA” or an “sd-rxRNA molecule” refers to a self-delivering RNA molecule such as those described in, and incorporated by reference from, PCT Publication No. WO2010/033247 (Application No. PCT/US2009/005247), filed on Sep. 22, 2009, and entitled “REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS,” and PCT application PCT/US2009/005246, filed on Sep. 22, 2009, and entitled “RNA INTERFERENCE IN SKIN INDICATIONS.” Briefly, an sd-rxRNA, (also referred to as an sd-rxRNAn^nano) is an isolated asymmetric double stranded nucleic acid molecule comprising a guide strand, with a minimal length of 16 nucleotides, and a passenger strand of 8-18 nucleotides in length, wherein the double stranded nucleic acid molecule has a double stranded region and a single stranded region, the single stranded region having 4-12 nucleotides in length and having at least three nucleotide backbone modifications. In preferred embodiments, the double stranded nucleic acid molecule has one end that is blunt or includes a one or two nucleotide overhang. sd-rxRNA molecules can be optimized through chemical modification, and in some instances through attachment of hydrophobic conjugates.
• In some embodiments, an sd-rxRNA comprises an isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the region of the molecule that is double stranded is from 8-15 nucleotides long, wherein the guide strand contains a single stranded region that 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, and wherein at least 40% of the nucleotides of the double stranded nucleic acid are modified.
• 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-rxRNA^nano, sd-rxRNA or RNA molecules of the invention.
• sd-rxRNAs 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 traditionally been difficult to deliver to a cell as they have rigid structures and a large number of negative charges which makes membrane transfer difficult. sd-rxRNAs however, 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 oligonucleotides of the invention in some aspects have a combination of asymmetric structures including a double stranded region and a single stranded region of nucleotides or longer, specific chemical modification patterns and are conjugated to lipophilic or hydrophobic molecules. This class of RNAi like compounds have superior efficacy in vitro and in vivo. 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 contribute to the observed superior efficacy.
• The invention is based at least in part on the surprising discovery that sd-rxRNA molecules are delivered efficiently in vivo to the skin through a variety of methods including intradermal injection and subcutaneous administration. Furthermore, sd-rxRNA molecules are efficient in mediating gene silencing in the region of the skin where they are targeted.
• Aspects of the invention relate to the use of cell-based screening to identify potent sd-rxRNA molecules. Described herein is the identification of potent sd-rxRNA molecules that target a subset of genes including SPP1, CTFG, PTGS2, TGFB1 and TGFB2. In some embodiments, a target gene is selected and an algorithm is applied to identify optimal target sequences within that gene (Example 2). For example, many sequences can be selected for one gene. In some instances, the sequences that are identified are generated as RNAi compounds for a first round of testing. For example, the RNAi compounds based on the optimal predicted sequences can initially be generated as rxRNAori (“ori”) sequences for the first round of screening. After identifying potent RNAi compounds, these can be generated as sd-rxRNA molecules.
• dsRNA formulated according to the invention also includes rxRNAori. rxRNAori refers to a class of RNA molecules described in and incorporated by reference from PCT Publication No. WO2009/102427 (Application No. PCT/US2009/000852), filed on Feb. 11, 2009, and entitled, “MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF.”
• In some embodiments, an rxRNAori molecule comprises a double-stranded RNA (dsRNA) construct of 12-35 nucleotides in length, for inhibiting expression of a target gene, comprising: a sense strand having a 5′-end and a 3′-end, wherein the sense strand is highly modified with 2′-modified ribose sugars, and wherein 3-6 nucleotides in the central portion of the sense strand are not modified with 2′-modified ribose sugars and, an antisense strand having a 5′-end and a 3′-end, which hybridizes to the sense strand and to mRNA of the target gene, wherein the dsRNA inhibits expression of the target gene in a sequence-dependent manner.
• rxRNAori can contain any of the modifications described herein. In some embodiments, at least 30% of the nucleotides in the rxRNAori are modified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the rxRNAori are modified. In some embodiments, 100% of the nucleotides in the sd-rxRNA are modified. In some embodiments, only the passenger strand of the rxRNAori contains modifications.
• In some embodiments, the RNAi compounds of the invention comprise an asymmetric compound comprising a duplex region (required for efficient RISC entry of 8-15 bases long) and single stranded region of 4-12 nucleotides long; with a 13 or 14 nucleotide duplex. A 6 or 7 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 chemical 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 to chemical modification pattern in the guide strand includes 2′Omethyl modification ofposition 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 ofposition 1 and C/U in positions 11-18 and 5′ end chemical phosphorylation and and 2′F modification of C/U in positions 2-10. In some embodiments the passenger strand and/or the guide strand contains at least one 5-methyl C or U modifications.
• In some embodiments, at least 30% of the nucleotides in the sd-rxRNA are modified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the sd-rxRNA are modified. In some embodiments, 100% of the nucleotides in the sd-rxRNA are modified.
• The above-described chemical modification patterns of the oligonucleotides of the invention are well tolerated and actually improved efficacy of asymmetric RNAi compounds.
• 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 in some instances results in sub-optimal efficacy and in some instances complete lost of efficacy. The combination of elements results in development of a compound, which is fully active following passive delivery to cells such as HeLa cells.
• The data in the Examples presented below demonstrates high efficacy of the oligonucleotides of the invention both in vitro in variety of cell types and in vivo upon local and systemic administration.
• 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 arepositions 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. 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.
• Aspects of the invention relate to double-stranded ribonucleic acid molecules (dsRNA) such as sd-rxRNA and rxRNAori. dsRNA associated with the invention can comprise a sense strand and an antisense strand wherein the antisense strand is complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23. For example, the antisense strand can be complementary to at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides, or can be complementary to 25 nucleotides of a sequence selected from the sequences within Tables 2, 5, 6, 9, 11, 12, 13, 14, 15, 16, 17 and 23.
• dsRNA associated with the invention can comprise a sense strand and an antisense strand wherein the sense strand and/or the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 1-27. For example, the sense strand and/or the antisense strand can comprise at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 contiguous nucleotides, or can comprise 25 nucleotides of a sequence selected from the sequences within Tables 1-27.
• Aspects of the invention relate to dsRNA directed against CTGF. For example, the antisense strand of a dsRNA directed against CTGF can be complementary to at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 11, 12 and 15. The sense strand and/or the antisense strand of a dsRNA directed against CTGF can comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 10, 11, 12, 15, 20 and 24.
• In some embodiments, the sense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs: 2463, 3429, 2443, 3445, 2459, 3493, 2465 and 3469. In certain embodiments, the sense strand comprises or consists of a sequence selected from the group consisting of: SEQ ID NOs: 2463, 3429, 2443, 3445, 2459, 3493, 2465 and 3469.
• In some embodiments, the antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the group consisting of: 2464, 3430, 4203, 3446, 2460, 3494, 2466 and 3470. In certain embodiments, the antisense strand comprises or consists of a sequence selected from the group consisting of: 2464, 3430, 4203, 3446, 2460, 3494, 2466 and 3470.
• In a preferred embodiment, the sense strand comprises SEQ ID NO:2463 (GCACCUUUCUAGA) and the antisense strand comprises SEQ ID NO:2464 (UCUAGAAAGGUGCAAACAU). The sequences of SEQ ID NO:2463 and SEQ ID NO:2464 can be modified in a variety of ways according to modifications described herein. A preferred modification pattern for SEQ ID NO:2463 is depicted by SEQ ID NO:3429 (G.mC. A.mC.mC.mU.mU.mU.mC.mU. A*mG*mA.TEG-Chl). A preferred modification pattern for SEQ ID NO:2464 is depicted by SEQ ID NO:3430 (P.mU.fC.fU. A. G.mA. A.mA. G. G.fU. G.mC* A* A* A*mC* A* U). An sd-rxRNA consisting of SEQ ID NO:3429 and SEQ ID NO:3430 is also referred to as RXi-109.
• In another preferred embodiment, the sense strand comprises SEQ ID NO:2443 (UUGCACCUUUCUAA) and the antisense strand comprises SEQ ID NO:4203 (UUAGAAAGGUGCAAACAAGG). The sequences of SEQ ID NO:2443 and SEQ ID NO:4203 can be modified in a variety of ways according to modifications described herein. A preferred modification pattern for SEQ ID NO:2443 is depicted by SEQ ID NO:3445 (mU.mU. G.mC. A.mC.mC.mU.mU.mU.mC.mU*mA*mA.TEG-Chl). A preferred modification pattern for SEQ ID NO:4203 is depicted by SEQ ID NO:3446 (P.mU.tU. A. G. A.mA. A. G. G.tU. G.tC.mA.mA*mA*tC*mA*mA*mG* G.).
• In another preferred embodiment, the sense strand comprises SEQ ID NO:2459 (GUGACCAAAAGUA) and the antisense strand comprises SEQ ID NO:2460 (UACUUUUGGUCACACUCUC). The sequences of SEQ ID NO:2459 and SEQ ID NO:2460 can be modified in a variety of ways according to modifications described herein. A preferred modification pattern for SEQ ID NO:2459 is depicted by SEQ ID NO:3493 (G.mU. G. A.mC.mC. A. A. A. A. G*mU*mA.TEG-Chl). A preferred modification pattern for SEQ ID NO:2460 is depicted by SEQ ID NO:3494 (P.mU. A.fC.fU.fU.fU.fU. G. G.fU.mC. A.mC* A*mC*mU*mC*mU* C.).
• In another preferred embodiment, the sense strand comprises SEQ ID NO:2465 (CCUUUCUAGUUGA) and the antisense strand comprises SEQ ID NO:2466 (UCAACUAGAAAGGUGCAAA). The sequences of SEQ ID NO:2465 and SEQ ID NO:2466 can be modified in a variety of ways according to modifications described herein. A preferred modification pattern for SEQ ID NO:2465 is depicted by SEQ ID NO:3469 (mC.mC.mU.mU.mU.mC.mU. A. G.mU.mU*mG*mA.TEG-Chl). A preferred modification pattern for SEQ ID NO:2466 is depicted by SEQ ID NO:3470 (P.mU.fC. A. A.fC.fU. A. G. A.mA. A. G. G*fU*mG*fC*mA*mA* A.).
• A preferred embodiment of an rxRNAori directed against CTGF can comprise at least 12 contiguous nucleotides of a sequence selected from the group consisting of: SEQ ID NOs:1835, 1847, 1848 and 1849. In some embodiments, the sense strand of the rxRNAori comprises or consists of SEQ ID NOs:1835, 1847, 1848 or 1849.
• Aspects of the invention relate to compositions comprising dsRNA such as sd-rxRNA and rxRNAori. In some embodiments compositions comprise two or more dsRNA that are directed against different genes.
• 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 to 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 8-15 nucleotides long. In certain embodiments, the passenger strand is 8, 9, 10, 11, 12, 13, 14 or 15 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 8-15 nucleotides long. In certain embodiments, the region of the molecule that is double stranded is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long. In certain embodiments the double stranded region is 13 or 14 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. Molecules that have a double stranded region of 8-15 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.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. Nat. 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.
• 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 sd-rxRNA 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, wherein the RNAi construct involves a hairpin, the 5′-stem sequence may comprise a 2′-modified ribose sugar, such as 2-O-methyl modified nucleotide, at the 2^ndnucleotide 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 2^ndnucleotide on the 5′-end of the guide strand and no other modified nucleotides.
• In other aspects, the sd-rxRNA 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 111-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 subject hairpin constructs, and cells comprising such vectors or the subject hairpin constructs. 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 6 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 betweennucleotide 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.
• In some embodiments, the base moiety of a nucleoside may be modified. For example, a pyrimidine base may be modified at the 2, 3, 4, 5, and/or 6 position of the pyrimidine ring. In some embodiments, the exocyclic amine of cytosine may be modified. A purine base may also be modified. For example, a purine base may be modified at the 1, 2, 3, 6, 7, or 8 position. In some embodiments, the exocyclic amine of adenine may be modified. In some cases, a nitrogen atom in a ring of a base moiety may be substituted with another atom, such as carbon. A modification to a base moiety may be any suitable modification. Examples of modifications are known to those of ordinary skill in the art. In some embodiments, the base modifications include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles.
• In some embodiments, a pyrimidine may be modified at the 5 position. For example, the 5 position of a pyrimidine may be modified with an alkyl group, an alkynyl group, an alkenyl group, an acyl group, or substituted derivatives thereof. In other examples, the 5 position of a pyrimidine may be modified with a hydroxyl group or an alkoxyl group or substituted derivative thereof. Also, the N⁴position of a pyrimidine may be alkylated. In still further examples, the pyrimidine 5-6 bond may be saturated, a nitrogen atom within the pyrimidine ring may be substituted with a carbon atom, and/or the O²and O⁴atoms may be substituted with sulfur atoms. It should be understood that other modifications are possible as well.
• In other examples, the N⁷position and/or N²and/or N³position of a purine may be modified with an alkyl group or substituted derivative thereof. In further examples, a third ring may be fused to the purine bicyclic ring system and/or a nitrogen atom within the purine ring system may be substituted with a carbon atom. It should be understood that other modifications are possible as well.
• Non-limiting examples of pyrimidines modified at the 5 position are disclosed in U.S. Pat. Nos. 5,591,843, 7,205,297, 6,432,963, and 6,020,483; non-limiting examples of pyrimidines modified at the N⁴position are disclosed in U.S. Pat. No. 5,580,731; non-limiting examples of purines modified at the 8 position are disclosed in U.S. Pat. Nos. 6,355,787 and 5,580,972; non-limiting examples of purines modified at the N⁶position are disclosed in U.S. Pat. Nos. 4,853,386, 5,789,416, and 7,041,824; and non-limiting examples of purines modified at the 2 position are disclosed in U.S. Pat. Nos. 4,201,860 and 5,587,469, all of which are incorporated herein by reference.
• Non-limiting examples of modified bases include N⁴,N⁴-ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N-methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, N⁶-isopentenyl-adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxy aminomethyl-2-thiouracil, 5-methoxyuracil, 2-methylthio-N⁶-isopentenyladenine, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, 2-thiocytosine, and 2,6-diaminopurine. In some embodiments, the base moiety may be a heterocyclic base other than a purine or pyrimidine. The heterocyclic base may be optionally modified and/or substituted.
• 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, alkoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as NH₂, NHR, NR_2.), 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′-O-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.
• Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics,75^thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described inOrganic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.
• Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
• Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.
• If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
• 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—CH—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., 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.
• One of ordinary skill in the art will appreciate that the synthetic methods, as described herein, utilize a variety of protecting groups. By the term “protecting group,” as used herein, it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound. In certain embodiments, a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction. As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized. Hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4.4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (TPDMS), diethylisopropylsilyl (DEPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts). For protecting 1,2- or 1,3-diols, the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene ortho ester, 1-(N,N-dimethylamino)ethylidene derivative, α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic carbonates, cyclic boronates, ethyl boronate, and phenyl boronate. Amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (lpaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivative, N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(0-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-12-(trimethylsilyl)ethoxylmethylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenyifluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenyithiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitm-4-methoxybenzenesulfenamide, uiphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), 0-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide. Exemplary protecting groups are detailed herein. However, it will be appreciated that the present invention is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the method of the present invention. Additionally, a variety of protecting groups are described inProtective Groups in Organic Synthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference.
• It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term “substituted” whether preceeded by the term “optionally” or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment, for example, of infectious diseases or proliferative disorders. The term “stable”, as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein.
• The term “aliphatic,” as used herein, includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “aliphatic” is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term “alkyl” includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as “alkenyl,” “alkynyl,” and the like. Furthermore, as used herein, the terms “alkyl,” “alkenyl,” “alkynyl,” and the like encompass both substituted and unsubstituted groups. In certain embodiments, as used herein, “lower alkyl” is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1-6 carbon atoms.
• In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the invention contain 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, —CH₂— cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, —CH₂— cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.
• Some examples of substituents of the above-described aliphatic (and other) moieties of compounds of the invention include, but are not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_x; —CO₂(R_x); —CON(R_x)₂; —OC(O)R_x; —OCO₂R_x; —OCON(R_x)₂; —N(R_x)₂; —S(O)₂R_x; —NR_x(CO)R_xwherein each occurrence of R_xindependently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments described herein.
• The term “heteroaliphatic,” as used herein, refers to aliphatic moieties that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched, cyclic or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc. In certain embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_x; —CO₂(R_x); —CON(R_x)₂; —OC(O)R_x; —OCO₂R_x; —OCON(R_x)₂; —N(R_x)₂; —S(O)₂R_x; —NR_x(CO)R_x, wherein each occurrence of R_xindependently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and wherein any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substitutents are illustrated by the specific embodiments described herein.
• The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine.
• 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-C₆for straight chain, C₃-C₆for 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-C₆includes 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-C₆for straight chain, C₃-C₆for 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-C₆includes 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-C₆for straight chain, C₃-C₆for branched chain). The term C₂-C₆includes 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, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, allylsulfamyl, 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 “hydrophobic modifications’ include bases modified in a fashion, where (1) overall hydrophobicity of the base is significantly increases, (2) the base is still capable of forming close to regular Watson-Crick interaction. Some, of the examples of base modifications include but are not limited to 5-position uridine and cytidine modifications like phenyl,
• 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), Isobutyl, butyl, aminobenzyl; phenyl; naphthyl, For purposes of the present invention, the term “overhang” refers to terminal non-base pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of the complementary strand to which the first strand or region forms a duplex. One or more polynucleotides that are capable of forming a duplex through hydrogen bonding can have overhangs. The overhand length generally doesn't exceed 5 bases in length.
• 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)_1-2N′R″, (CR′R″)_0-3CHO, (CR′R″)_0-3(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″)_0-3OR′ groups; wherein each R′ and R″ are each independently hydrogen, a C₁-C₅alkyl, C₂-C₅alkenyl, C₂-C₅alkynyl, or aryl group, or R′ and R″ taken together are a benzylidene group or a —(CH₂)₂O(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 terms “polynucleotide,” “nucleotide sequence,” “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” and “oligonucleotide” refer to a polymer of two or more nucleotides. The polynucleotides can be DNA, RNA, or derivatives or modified versions thereof. The polynucleotide may be single-stranded or double-stranded. The polynucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. The polynucleotide may comprise a modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. The olynucleotide may comprise a modified sugar moiety (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), and/or a modified phosphate moiety (e.g., phosphorothioates and 5′-N-phosphoramidite linkages). A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA. DNA-RNA, and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone.
• 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”, 2^ndEd., Wiley-Interscience, New York, 1999).
• The term “nucleotide” includes nucleosides which further comprise a phosphate group or a phosphate analog.
• The nucleic acid molecules may be associated with a hydrophobic moiety for targeting and/or delivery of the molecule to a cell. In certain embodiments, the hydrophobic moiety is associated with the nucleic acid molecule through a linker. In certain embodiments, the association is through non-covalent interactions. In other embodiments, the association is through a covalent bond. Any linker known in the art may be used to associate the nucleic acid with the hydrophobic moiety. Linkers known in the art are described in published international PCT applications, WO 92/03464, WO 95/23162, WO 2008/021157, WO 2009/021157, WO 2009/134487, WO 2009/126933, U.S. Patent Application Publication 2005/0107325, U.S. Pat. Nos. 5,414,077, 5,419,966, 5,512,667, 5,646,126, and 5,652,359, which are incorporated herein by reference. The linker may be as simple as a covalent bond to a multi-atom linker. The linker may be cyclic or acyclic. The linker may be optionally substituted. In certain embodiments, the linker is capable of being cleaved from the nucleic acid. In certain embodiments, the linker is capable of being hydrolyzed under physiological conditions. In certain embodiments, the linker is capable of being cleaved by an enzyme (e.g., an esterase or phosphodiesterase). In certain embodiments, the linker comprises a spacer element to separate the nucleic acid from the hydrophobic moiety. The spacer element may include one to thirty carbon or heteroatoms. In certain embodiments, the linker and/or spacer element comprises protonatable functional groups. Such protonatable functional groups may promote the endosomal escape of the nucleic acid molecule. The protonatable functional groups may also aid in the delivery of the nucleic acid to a cell, for example, neutralizing the overall charge of the molecule. In other embodiments, the linker and/or spacer element is biologically inert (that is, it does not impart biological activity or function to the resulting nucleic acid molecule).
• In certain embodiments, the nucleic acid molecule with a linker and hydrophobic moiety is of the formulae described herein. In certain embodiments, the nucleic acid molecule is of the formula:
• 
• wherein
X is N or CH;
• A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic;
  R¹is a hydrophobic moiety;
  R²is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and
  R³is a nucleic acid.
  In certain embodiments, the molecule is of the formula:
• 
• In certain embodiments, the molecule is of the formula:
• 
• In certain embodiments, the molecule is of the formula:
• 
• In certain embodiments, the molecule is of the formula:
• 
• In certain embodiments, X is N. In certain embodiments, X is CH.
  In certain embodiments, A is a bond. In certain embodiments, A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic. In certain embodiments. A is acyclic, substituted or unsubstituted, branched or unbranched aliphatic. In certain embodiments, A is acyclic, substituted, branched or unbranched aliphatic. In certain embodiments, A is acyclic, substituted, unbranched aliphatic. In certain embodiments, A is acyclic, substituted, unbranched alkyl. In certain embodiments, A is acyclic, substituted, unbranched C_1-20alkyl. In certain embodiments, A is acyclic, substituted, unbranched C_1-12alkyl. In certain embodiments, A is acyclic, substituted, unbranched C_1-10alkyl. In certain embodiments, A is acyclic, substituted, unbranched C_1-8alkyl. In certain embodiments, A is acyclic, substituted, unbranched C_1-6alkyl. In certain embodiments, A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic. In certain embodiments, A is acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic. In certain embodiments, A is acyclic, substituted, branched or unbranched heteroaliphatic. In certain embodiments. A is acyclic, substituted, unbranched heteroaliphatic.
  In certain embodiments, A is of the formula:
• 
• In certain embodiments, A is of one of the formulae:
• 
• In certain embodiments, A is of one of the formulae:
• 
• In certain embodiments, A is of one of the formulae:
• 
• In certain embodiments, A is of the formula:
• 
• In certain embodiment, A is of the formula:
• 
• In certain embodiment, A is of the formula:
• 
• wherein
  each occurrence of R is independently the side chain of a natural or unnatural amino acid; and
  n is an integer between 1 and 20, inclusive. In certain embodiments, A is of the formula:
• 
• In certain embodiments, each occurrence of R is independently the side chain of a natural amino acid. In certain embodiments, n is an integer between 1 and 15, inclusive. In certain embodiments, n is an integer between 1 and 10, inclusive. In certain embodiments, n is an integer between 1 and 5, inclusive. In certain embodiments, A is of the formula:
• 
• wherein n is an integer between 1 and 20, inclusive. In certain embodiments, A is of the formula:
• 
• In certain embodiments, n is an integer between 1 and 15, inclusive. In certain embodiments, n is an integer between 1 and 10, inclusive. In certain embodiments, n is an integer between 1 and 5, inclusive.
  In certain embodiments, A is of the formula:
• 
• wherein n is an integer between 1 and 20, inclusive. In certain embodiments, A is of the formula:
• 
• In certain embodiments, n is an integer between 1 and 15, inclusive. In certain embodiments, n is an integer between 1 and 10, inclusive. In certain embodiments, n is an integer between 1 and 5, inclusive.
  In certain embodiments the molecule is of the formula:
• 
• wherein X, R¹, R², and R³are as defined herein; and
  A′ is substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic.
  In certain embodiments, A′ is of one of the formulae:
• 
• In certain embodiments, A is of one of the formulae:
• 
• In certain embodiments, A is of one of the formulae:
• 
• In certain embodiments, A is of the formula:
• 
• In certain embodiments, A is of the formula:
• 
• In certain embodiments, R¹is a steroid. In certain embodiments, R¹is a cholesterol. In certain embodiments, R¹is a lipophilic vitamin. In certain embodiments, R¹is a vitamin A. In certain embodiments, R¹is a vitamin E.
  In certain embodiments, R¹is of the formula:
• 
• wherein R^Ais substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic.
  In certain embodiments, R¹is of the formula:
• 
• In certain embodiments, R¹is of the formula:
• 
• In certain embodiments R¹is of the formula:
• 
• In certain embodiments, R¹is of the formula:
• 
• In certain embodiments, R¹is of the formula:
• 
• In certain embodiments, the nucleic acid molecule is of the formula:
• 
• wherein
X is N or CH;
• A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic;
  R¹is a hydrophobic moiety;
  R²is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and
  R³is a nucleic acid.
  In certain embodiments, the nucleic acid molecule is of the formula:
• 
• wherein
X is N or CH;
• A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic;
  R¹is a hydrophobic moiety;
  R²is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and
  R³is a nucleic acid.
  In certain embodiments, the nucleic acid molecule is of the formula:
• 
• wherein
X is N or CH;
• A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic;
  R¹is a hydrophobic moiety;
  R²is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and
  R¹is a nucleic acid. In certain embodiments, the nucleic acid molecule is of the formula:
• 
• In certain embodiments, the nucleic acid molecule is of the formula:
• 
• In certain embodiments, the nucleic acid molecule is of the formula:
• 
• wherein R³is a nucleic acid.
  In certain embodiments, the nucleic acid molecule is of the formula:
• 
• wherein R³is a nucleic acid; and
  n is an integer between 1 and 20, inclusive.
  In certain embodiments, the nucleic acid molecule is of the formula:
• 
• In certain embodiments, the nucleic acid molecule is of the formula:
• 
• In certain embodiments, the nucleic acid molecule is of the formula:
• 
• In certain embodiments, the nucleic acid molecule is of the formula:
• 
• In certain embodiments, the nucleic acid molecule is of the formula:
• 
• 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 phosphorothioate linkages.
• In certain embodiments, oligonucleotides of the invention comprise hydrophobically 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.
• Another type of conjugates that can be attached to the end (3′ or 5′ end), the loop region, or any other parts of the sd-rxRNA might include a sterol, sterol type molecule, peptide, small molecule, protein, etc. In some embodiments, a sd-rxRNA 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 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. Nos. 5,013,830; 5,214,135; 5,525,719; Kawasaki et al. 1993. J. Med. Chem.36:831; WO 92/03568; U.S. Pat. Nos. 5,276,019; and 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 Reviews90:543-584) 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 Biology26:1. 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 (D N 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/CarrierUptake 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 sd-rxRNA of the invention may be delivered by using various beta-glucan containing particles, referred to as GeRPs (glucan encapsulated RNA loaded particle), described in, and incorporated by reference from, U.S. Provisional Application No. 61/310,611. filed on Mar. 4, 2010 and entitled “Formulations and Methods for Targeted Delivery to Phagocyte Cells.” Such particles are also described in, and incorporated by reference from US Patent Publications US 2005/0281781 A1, and US 2010/0040656, and in PCT publications WO 2006/007372, and WO 2007/050643. The sd-rxRNA molecule may be hydrophobically modified and optionally may be associated with a lipid and/or amphiphilic peptide. In certain embodiments, the beta-glucan particle is derived from yeast. In 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.
• Glucan particles can be derived from insoluble components of fungal cell walls such as yeast cell walls. In some embodiments, the yeast is Baker's yeast. Yeast-derived glucan molecules can include one or more of β-(1,3)-Glucan, β-(1,6)-Glucan, mannan and chitin. In some embodiments, a glucan particle comprises a hollow yeast cell wall whereby the particle maintains a three dimensional structure resembling a cell, within which it can complex with or encapsulate a molecule such as an RNA molecule. Some of the advantages associated with the use of yeast cell wall particles are availability of the components, their biodegradable nature, and their ability to be targeted to phagocytic cells.
• In some embodiments, glucan particles can be prepared by extraction of insoluble components from cell walls, for example by extracting Baker's yeast (Fleischmann's) with 1M NaOH/pH 4.0 H2O, followed by washing and drying. Methods of preparing yeast cell wall particles are discussed in, and incorporated by reference from U.S. Pat. Nos. 4,810,646, 4,992,540, 5,082,936, 5,028,703, 5,032,401, 5,322,841, 5,401,727, 5,504,079, 5,607,677, 5,968,811, 6,242,594, 6,444,448, 6,476,003, US Patent Publications 2003/0216346, 2004/0014715 and 2010/0040656, and PCT published application WO02/12348.
• Protocols for preparing glucan particles are also described in, and incorporated by reference from, the following references: Soto and Ostroff (2008), “Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery.”Bioconjug Chem19(4):840-8; Soto and Ostroff (2007), “Oral Macrophage Mediated Gene Delivery System,”Nanotech,Volume 2, Chapter 5 (“Drug Delivery”), pages 378-381; and Li et al. (2007), “Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88- and Syk kinase-dependent pathways.”Clinical Immunology124(2):170-181.
• Glucan containing particles such as yeast cell wall particles can also be obtained commercially. Several non-limiting examples include: Nutricell MOS 55 from Biorigin (Sao Paolo, Brazil), SAF-Mannan (SAF Agri, Minneapolis, Minn.), Nutrex (Sensient Technologies, Milwaukee, Wis.), alkali-extracted particles such as those produced by Nutricepts (Nutricepts Inc., Burnsville, Minn.) and ASA Biotech, acid-extracted WGP particles from Biopolymer Engineering, and organic solvent-extracted particles such as Adjuvax™ from Alpha-beta Technology, Inc. (Worcester, Mass.) and microparticulate glucan from Novogen (Stamford, Conn.).
• Glucan particles such as yeast cell wall particles can have varying levels of purity depending on the method of production and/or extraction. In some instances, particles are alkali-extracted, acid-extracted or organic solvent-extracted to remove intracellular components and/or the outer mannoprotein layer of the cell wall. Such protocols can produce particles that have a glucan (w/w) content in the range of 50%-90%. In some instances, a particle of lower purity, meaning lower glucan w/w content may be preferred, while in other embodiments, a particle of higher purity, meaning higher glucan w/w content may be preferred.
• Glucan particles, such as yeast cell wall particles, can have a natural lipid content. For example, the particles can contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more than 20% w/w lipid. In the Examples section, the effectiveness of two glucan particle batches are tested: YGP SAF and YGP SAF+L (containing natural lipids). In some instances, the presence of natural lipids may assist in complexation or capture of RNA molecules.
• Glucan containing particles typically have a diameter of approximately 2-4 microns, although particles with a diameter of less than 2 microns or greater than 4 microns are also compatible with aspects of the invention.
• The RNA molecule(s) to be delivered are complexed or “trapped” within the shell of the glucan particle. The shell or RNA component of the particle can be labeled for visualization, as described in, and incorporated by reference from, Soto and Ostroff (2008)Bioconjug Chem19:840. Methods of loading GeRPs are discussed further below.
• 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 oligonucleotides pharmacokinetic or toxicological 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 LIPOFECTAMINE™ 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.
• Nucleic acids associated with the invention can be hydrophobically modified and can be encompassed within neutral nanotransporters. Further description of neutral nanotransporters is incorporated by reference from PCT Application PCT/US2009/005251, filed on Sep. 22, 2009, and entitled “Neutral Nanotransporters.” Such particles enable quantitative oligonucleotide incorporation into non-charged lipid mixtures. The lack of toxic levels of cationic lipids in such neutral nanotransporter compositions is an important feature.
• As demonstrated in PCT/US2009/005251, oligonucleotides can effectively be incorporated into a lipid mixture that is free of cationic lipids and such a composition can effectively deliver a therapeutic oligonucleotide to a cell in a manner that it is functional. For example, a high level of activity was observed when the fatty mixture was 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 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.
• In some aspects, stable particles ranging in size from 50 to 140 nm can be 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, oleyl-lithocholic, cholenic, oleoyl-cholenic, palmityl, heptadecyl, myristyl, bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl lithocholic 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 aposition 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 and 9 carbons. Such conjugates may have significantly better in vivo efficacy, in particular delivery to liver. These types of molecules are expected to work atconcentrations 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. 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.
• 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 hydrophobically 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, LIPOFECTAMNE™ (e.g., LIPOFECTAMINE™ 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. USA93:3176; Hope et al. 1998. Molecular Membrane Biology15:1). 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. Nos. 4,235,871; 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. U.S.A.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. Cell88:223).
• 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 0 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-C₂₀alkyl chains, C₂-C₂₀alkenyl chains. C₂-C₂₀alkynyl 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 J1991, 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 Acta1197:95-108).
• 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. Pharmazie54:559-566.
• 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. Pharmazie54:559-566.
• 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 Acta1197:95-108).
• 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 decreased, 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.
• 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. In preferred embodiments, the sd-rxRNA molecules are administered by intradermal injection or subcutaneously.
• 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. In some embodiments, 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 (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750). decaglycerol sesquioleate (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 asCaptex 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-C₁₀glycerides, 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 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 regimens 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.
• Administration of sd-rxRNAs, such as through intradermal injection or subcutaneous delivery, can be optimized through testing of dosing regimens. In some embodiments, a single administration is sufficient. To further prolong the effect of the administered sd-rxRNA, the sd-rxRNA can be administered in a slow-release formulation or device, as would be familiar to one of ordinary skill in the art. The hydrophobic nature of sd-rxRNA compounds can enable use of a wide variety of polymers, some of which are not compatible with conventional oligonucleotide delivery.
• In other embodiments, the sd-rxRNA is administered multiple times. In some instances it is administered daily, bi-weekly, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every four months, every five months, every six months or less frequently than every six months. In some instances, it is administered multiple times per day, week, month and/or year. For example, it can be administered approximately every hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9hours 10 hours, 12 hours or more than twelve hours. It can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 times per day.
• Aspects of the invention relate to administering sd-rxRNA molecules to a subject. In some instances the subject is a patient and administering the sd-rxRNA molecule involves administering the sd-rxRNA molecule in a doctor's office.
• In some embodiments, more than one sd-rxRNA molecule is administered simultaneously. For example a composition may be administered that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different sd-rxRNA molecules. In certain embodiments, a composition comprises 2 or 3 different sd-rxRNA molecules. When a composition comprises more than one sd-rxRNA, the sd-rxRNA molecules within the composition can be directed to the same gene or to different genes.
• FIG. 1 reveals the expression profile for several genes associated with the invention. As expected, target gene expression is elevated early and returns to normal byday 10.FIG. 2 provides a summary of experimental design.FIGS. 3-6 show in vivo silencing of MAP4K4 and PPIB expression following intradermal injection of sd-rxRNA molecules targeting these genes.FIGS. 7-8 show that the silencing effect of sd-rxRNAs can persist for at least 8 days. Thus, in some embodiments, sd-rxRNA is administered within 8 days prior to an event that compromises or damages the skin such as a surgery. For examples, an sd-rxRNA could be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or more than 10 days prior to an event that compromises or damages the skin.FIG. 9 demonstrates examples of dosing regimens.
• In some instances, the effective amount of sd-rxRNA that is delivered by subcutaneous administration is at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more than 100 mg/kg including any intermediate values.
• In some instances, the effective amount of sd-rxRNA that is delivered through intradermal injection is at least approximately 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or more than 950 μg including any intermediate values.
• sd-rxRNA molecules administered through methods described herein are effectively targeted to all the cell types in the skin.
• 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 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.
• A further preferred use for the agents 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 priming agent/RNAi agent 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. 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 News16:1. 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 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 therapeutic agent (e.g., a RNAi agent or vector or transgene encoding same). 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 as described herein. 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 modulatory method of the invention involves contacting a cell capable of expressing target gene with a therapeutic agent 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 modulatory 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. Typically, subjects are first treated with a priming agent so as to be more responsive to the subsequent RNAi therapy. As such, the present invention provides methods of treating an individual 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.
• The therapeutic agents of the invention can be administered to individuals 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. See, for example. Eichelbaum. M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43(2):254-266
RNAi in Skin Indications
• Nucleic acid molecules, or compositions comprising nucleic acid molecules, described herein may in some embodiments be administered to pre-treat, treat or prevent compromised skin. As used herein “compromised skin” refers to skin which exhibits characteristics distinct from normal skin. Compromised skin may occur in association with a dermatological condition. Several non-limiting examples of dermatological conditions include rosacea, common acne, seborrheic dermatitis, perioral dermatitis, acneform rashes, transient acantholytic dermatosis, and acne necrotica miliaris. In some instances, compromised skin may comprise a wound and/or scar tissue. In some instances, methods and compositions associated with the invention may be used to promote wound healing, prevention, reduction or inhibition of scarring, and/or promotion of re-epithelialisation of wounds.
• A subject can be pre-treated or treated prophylactically with a molecule associated with the invention, prior to the skin of the subject becoming compromised. As used herein “pre-treatment” or “prophylactic treatment” refers to administering a nucleic acid to the skin prior to the skin becoming compromised. For example, a subject could be pre-treated 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or more than 8 days prior to the skin becoming compromised. In other embodiments, a subject can be treated with a molecule associated with the invention immediately before the skin becomes compromised and/or simultaneous to the skin becoming compromised and/or after the skin has been compromised. In some embodiments, the skin is compromised through a medical procedure such as surgery, including elective surgery. In certain embodiments methods and compositions may be applied to areas of the skin that are believed to be at risk of becoming compromised. It should be appreciated that one of ordinary skill in the art would be able to optimize timing of administration using no more than routine experimentation.
• In some aspects, methods associated with the invention can be applied to promote healing of compromised skin. Administration can occur at any time up until the compromised skin has healed, even if the compromised skin has already partially healed. The timing of administration can depend on several factors including the nature of the compromised skin, the degree of damage within the compromised skin, and the size of the compromised area. In some embodiments administration may occur immediately after the skin is compromised, or 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, or more than 48 hours after the skin has been compromised. Methods and compositions of the invention may be administered one or more times as necessary. For example, in some embodiments, compositions may be administered daily or twice daily. In some instances, compositions may be administered both before and after formation of compromised skin.
• Compositions associated with the invention may be administered by any suitable route. In some embodiments, administration occurs locally at an area of compromised skin. For example, compositions may be administered by intradermal injection. Compositions for intradermal injection may include injectable solutions. Intradermal injection may in some embodiments occur around the are of compromised skin or at a site where the skin is likely to become compromised. In some embodiments, compositions may also be administered in a topical form, such as in a cream or ointment. In some embodiments, administration of compositions described herein comprises part of an initial treatment or pre-treatment of compromised skin, while in other embodiments, administration of such compositions comprises follow-up care for an area of compromised skin.
• The appropriate amount of a composition or medicament to be applied can depend on many different factors and can be determined by one of ordinary skill in the art through routine experimentation. Several non-limiting factors that might be considered include biological activity and bioavailability of the agent, nature of the agent, mode of administration, half-life, and characteristics of the subject to be treated.
• In some aspects, nucleic acid molecules associated with the invention may also be used in treatment and/or prevention of fibrotic disorders, including pulmonary fibrosis, liver cirrhosis, scleroderma and glomerulonephritis, lung fibrosis, liver fibrosis, skin fibrosis, muscle fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy, restenosis, and uterine fibrosis.
• A therapeutically effective amount of a nucleic acid molecule described herein may in some embodiments be an amount sufficient to prevent the formation of compromised skin and/or improve the condition of compromised skin and/or to treat or prevent a fibrotic disorder. In some embodiments, improvement of the condition of compromised skin may correspond to promotion of wound healing and/or inhibition of scarring and/or promotion of epithelial regeneration. The extent of prevention of formation of compromised skin and/or improvement to the condition of compromised to skin may in some instances be determined by, for example, a doctor or clinician.
• The ability of nucleic acid molecules associated with the invention to prevent the formation of compromised skin and/or improve the condition of compromised skin may in some instances be measured with reference to properties exhibited by the skin. In some instances, these properties may include rate of epithelialisation and/or decreased size of an area of compromised skin compared to control skin at comparable time points.
• As used herein, prevention of formation of compromised skin, for example prior to a surgical procedure, and/or improvement of the condition of compromised skin, for example after a surgical procedure, can encompass any increase in the rate of healing in the compromised skin as compared with the rate of healing occurring in a control sample. In some instances, the condition of compromised skin may be assessed with respect to either comparison of the rate of re-epithelialisation achieved in treated and control skin, or comparison of the relative areas of treated and control areas of compromised skin at comparable time points. In some aspects, a molecule that prevents formation of compromised skin or promotes healing of compromised skin may be a molecule that, upon administration, causes the area of compromised skin to exhibit an increased rate of re-epithelialisation and/or a reduction of the size of compromised skin compared to a control at comparable time points. In some embodiments, the healing of compromised skin may give rise to a rate of healing that is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% greater than the rate occurring in controls.
• In some aspects, subjects to be treated by methods and compositions associated with the invention may be subjects who will undergo, are undergoing or have undergone a medical procedure such as a surgery. In some embodiments, the subject may be prone to defective, delayed or otherwise impaired re-epithelialisation, such as dermal wounds in the aged. Other non-limiting examples of conditions or disorders in which wound healing is associated with delayed or otherwise impaired re-epithelialisation include patients suffering from diabetes, patients with polypharmacy, post-menopausal women, patients susceptible to pressure injuries, patients with venous disease, clinically obese patients, patients receiving chemotherapy, patients receiving radiotherapy, patients receiving steroid treatment, and immuno-compromised patients. In some instances, defective re-epithelialisation response can contributes to infections at the wound site, and to the formation of chronic wounds such as ulcers.
• In some embodiments, methods associated with the invention may promote the re-epithelialisation of compromised skin in chronic wounds, such as ulcers, and may also inhibit scarring associated with wound healing. In other embodiments, methods associated with the invention are applied to prevention or treatment of compromised skin in acute wounds in patients predisposed to impaired wound healing developing into chronic wounds. In other aspects, methods associated with the invention are applied to promote accelerated healing of compromised skin while preventing, reducing or inhibiting scarring for use in general clinical contexts. In some aspects, this can involve the treatment of surgical incisions and application of such methods may result in the prevention, reduction or inhibition of scarring that may otherwise occur on such healing. Such treatment may result in the scars being less noticeable and exhibiting regeneration of a more normal skin structure. In other embodiments, the compromised skin that is treated is not compromised skin that is caused by a surgical incision. The compromised skin may be subject to continued care and continued application of medicaments to encourage re-epithelialisation and healing.
• In some aspects, methods associated with the invention may also be used in the treatment of compromised skin associated with grafting procedures. This can involve treatment at a graft donor site and/or at a graft recipient site. Grafts can in some embodiments involve skin, artificial skin, or skin substitutes. Methods associated with the invention can also be used for promoting epithelial regeneration. As used herein, promotion of epithelial regeneration encompasses any increase in the rate of epithelial regeneration as compared to the regeneration occurring in a control-treated or untreated epithelium. The rate of epithelial regeneration attained can in some instances be compared with that taking place in control-treated or untreated epithelia using any suitable model of epithelial regeneration known in the art. Promotion of epithelial regeneration may be of use to induce effective re-epithelialisation in contexts in which the re-epithelialisation response is impaired, inhibited, retarded or otherwise defective. Promotion of epithelial regeneration may be also effected to accelerate the rate of defective or normal epithelial regeneration responses in patients suffering from epithelial damage.
• Some instances where re-epithelialisation response may be defective include conditions such as pemphigus, Hailey-Hailey disease (familial benign pemphigus), toxic epidermal necrolysis (TEN)/Lyell's syndrome, epidermolysis bullosa, cutaneous leishmaniasis and actinic keratosis. Defective re-epithelialisation of the lungs may be associated with idiopathic pulmonary fibrosis (IPF) or interstitial lung disease. Defective re-epithelialisation of the eye may be associated with conditions such as partial limbal stem cell deficiency or corneal erosions. Defective re-epithelialisation of the gastrointestinal tract or colon may be associated with conditions such as chronic anal fissures (fissure in ano), ulcerative colitis or Crohn's disease, and other inflammatory bowel disorders.
• In some aspects, methods associated with the invention are used to prevent, reduce or otherwise inhibit compromised skin associated with scarring. This can be applied to any site within the body and any tissue or organ, including the skin, eye, nerves, tendons, ligaments, muscle, and oral cavity (including the lips and palate), as well as internal organs (such as the liver, heart, brain, abdominal cavity, pelvic cavity, thoracic cavity, guts and reproductive tissue). In the skin, treatment may change the morphology and organization of collagen fibers and may result in making the scars less visible and blend in with the surrounding skin. As used herein, prevention, reduction or inhibition of scarring encompasses any degree of prevention, reduction or inhibition in scarring as compared to the level of scarring occurring in a control-treated or untreated wound.
• Prevention, reduction or inhibition of compromised skin, such as compromised skin associated with dermal scarring, can be assessed and/or measured with reference to microscopic and/or macroscopic characteristics. Macroscopic characteristics may include color, height, surface texture and stiffness of the skin. In some instances, prevention, reduction or inhibition of compromised skin may be demonstrated when the color, height, surface texture and stiffness of the skin resembles that of normal skin more closely after treatment than does a control that is untreated. Microscopic assessment of compromised skin may involve examining characteristics such as thickness and/or orientation and/or composition of the extracellular matrix (ECM) fibers, and cellularity of the compromised skin. In some instances, prevention, reduction or inhibition of compromised skin may be demonstrated when the thickness and/or orientation and/or composition of the extracellular matrix (ECM) fibers, and/or cellularity of the compromised skin resembles that of normal skin more closely after treatment than does a control that is untreated.
• In some aspects, methods associated with the invention are used for cosmetic purposes, at least in part to contribute to improving the cosmetic appearance of compromised skin. In some embodiments, methods associated with the invention may be used to prevent, reduce or inhibit compromised skin such as scarring of wounds covering joints of the body. In other embodiments, methods associated with the invention may be used to promote accelerated wound healing and/or prevent, reduce or inhibit scarring of wounds at increased risk of forming a contractile scar, and/or of wounds located at sites of high skin tension.
• In some embodiments, methods associated with the invention can be applied to promoting healing of compromised skin in instances where there is an increased risk of pathological scar formation, such as hypertrophic scars and keloids, which may have more pronounced deleterious effects than normal scarring. In some embodiments, methods described herein for promoting accelerated healing of compromised skin and/or preventing, reducing or inhibiting scarring are applied to compromised skin produced by surgical revision of pathological scars.
• Aspects of the invention can be applied to compromised skin caused by burn injuries. Healing in response to burn injuries can lead to adverse scarring, including the formation of hypertrophic scars. Methods associated with the invention can be applied to treatment of all injuries involving damage to an epithelial layer, such as injuries to the skin in which the epidermis is damaged. Other non-limiting examples of injuries to epithelial tissue include injuries involving the respiratory epithelia, digestive epithelia or epithelia surrounding internal tissues or organs.
RNAi to Treat Liver Fibrosis
• In some embodiments, methods associated with the invention are used to treat liver fibrosis. Liver fibrosis is the excessive accumulation of extracellular matrix proteins, including collagen, that occurs in most types of chronic liver diseases. It is the scarring process that represents the liver's response to injury. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. In the same way as skin and other organs heal wounds through deposition of collagen and other matrix constituents so the liver repairs injury through the deposition of new collagen. Activated hepatic stellate cells, portal fibroblasts, and myofibroblasts of bone marrow origin have been identified as major collagen-producing cells in the injured liver. These cells are activated by fibrogenic cytokines such as TGF-β1, angiotensin II, and leptin. In some embodiments, methods provided herein are aimed at inhibiting the accumulation of fibrogenic cells and/or preventing the deposition of extracellular matrix proteins. In some embodiments, RNAi molecules (including sd-rxRNA and rxRNAori) may be designed to target CTGF, TGF-β1, angiotensin II, and/or leptin. In some embodiments, RNAi molecules (including sd-rxRNA and rxRNAori) may be designed to target those genes listed in Tables 1-25.
Trabeculectomy Failure
• Trabeculectomy is a surgical procedure designed to create a channel or bleb though the sclera to allow excess fluid to drain from the anterior of the eye, leading to reduced intracocular pressure (IOP), a risk factor for glaucoma-related vision loss. The most common cause of trabeculectomy failure is blockage of the bleb by scar tissue. In certain embodiments, the sd-rxRNA is used to prevent formation of scar tissue resulting from a trabeculectomy. In some embodiments, the sd-rxRNA targets connexin 43. In other embodiments, the sd-rxRNA targets proyly 4-hydroxylase. In yet other embodiments, the sd-rxRNA targets procollagen C-protease.
Target Genes
• It should be appreciated that based on the RNAi molecules designed and disclosed herein, one of ordinary skill in the art would be able to design such RNAi molecules to target a variety of different genes depending on the context and intended use. For purposes of pre-treating, treating, or preventing compromised skin and/or promoting wound healing and/or preventing, reducing or inhibiting scarring, one of ordinary skill in the art would appreciate that a variety of suitable target genes could be identified based at least in part on the known or predicted functions of the genes, and/or the known or predicted expression patterns of the genes. Several non-limiting examples of genes that could be targeted by RNAi molecules for pre-treating, treating, or preventing compromised skin and/or promoting wound healing and/or preventing, reducing or inhibiting scarring include genes that encode for the following proteins: Transforming growth factor β (TGFβ1, TGFβ2, TGFβ3), Osteopontin (SPP1), Connective tissue growth factor (CTGF), Platelet-derived growth factor (PDGF), Hypoxia inducible factor-1α (HIF1α), Collagen I and/or III, Prolyl 4-hydroxylase (P4H), Procollagen C-protease (PCP),Matrix metalloproteinase 2, 9 (MMP2, 9), Integrins, Connexin, Histamine H1 receptor, Tissue transglutaminase, Mammalian target of rapamycin (mTOR), HoxB13, VEGF, IL-6, SMAD proteins, Ribosomal protein S6 kinases (RSP6), Cyclooxygenase-2 (COX-2/PTGS2), Cannabinoid receptors (CB1, CB2), and/or miR29b.
• Transforming growth factor β proteins, for which three isoforms exist in mammals (TGFβ1, TGFβ2, TGFβ3), are secreted proteins belonging to a superfamily of growth factors involved in the regulation of many cellular processes including proliferation, migration, apoptosis, adhesion, differentiation, inflammation, immuno-suppression and expression of extracellular proteins. These proteins are produced by a wide range of cell types including epithelial, endothelial, hematopoietic, neuronal, and connective tissue cells. Representative Genbank accession numbers providing DNA and protein sequence information for human TGFβ1, TGFβ2 and TGFβ3 are BT007245, BC096235, and X14149, respectively. Within the TGFβ family, TGFβ1 and TGFβ2 but not TGFβ3 represent suitable targets. The alteration in the ratio of TGFβ variants will promote better wound healing and will prevent excessive scar formation.
• Osteopontin (OPN), also known as Secreted phosphoprotein 1 (SPP1), Bone Sinaloprotein 1 (BSP-1), and early T-lymphocyte activation (ETA-1) is a secreted glycoprotein protein that binds to hydroxyapatite. OPN has been implicated in a variety of biological processes including bone remodeling, immune functions, chemotaxis, cell activation and apoptosis. Osteopontin is produced by a variety of cell types including fibroblasts, preosteoblasts, osteoblasts, osteocytes, odontoblasts, bone marrow cells, hypertrophic chondrocytes, dendritic cells, macrophages, smooth muscle, skeletal muscle myoblasts, endothelial cells, and extraosseous (non-bone) cells in the inner ear, brain, kidney, deciduum, and placenta. Representative Genbank accession number providing DNA and protein sequence information for human Osteopontin are NM_000582.2 and X13694.
• Connective tissue growth factor (CTGF), also known as Hypertrophic chondrocyte-specific protein 24, is a secreted heparin-binding protein that has been implicated in wound healing and scleroderma. Connective tissue growth factor is active in many cell types including fibroblasts, myofibroblasts, endothelial and epithelial cells. Representative Genbank accession number providing DNA and protein sequence information for human CTGF are NM_001901.2 and M92934.
• The Platelet-derived growth factor (PDGF) family of proteins, including several isoforms, are secreted mitogens. PDGF proteins are implicated in wound healing, at least in part, because they are released from platelets following wounding. Representative Genbank accession numbers providing DNA and protein sequence information for human PDGF genes and proteins include X03795 (PDGFA), X02811 (PDGFB), AF091434 (PDGFC), AB033832 (PDGFD).
• Hypoxia inducible factor-1α (HIF1α), is a transcription factor involved in cellular response to hypoxia. HIF1α is implicated in cellular processes such as embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. A representative Genbank accession number providing DNA and protein sequence information for human HIF1α is U22431.
• Collagen proteins are the most abundant mammalian proteins and are found in tissues such as skin, tendon, vascular, ligature, organs, and bone. Collagen I proteins (such as COL1A1 and COL1A2) are detected in scar tissue during wound healing, and are expressed in the skin. Collagen III proteins (including COL3A1) are detected in connective tissue in wounds (granulation tissue), and are also expressed in skin. Representative Genbank accession numbers providing DNA and protein sequence information for human Collagen proteins include: Z74615 (COL1A1), J03464 (COL1A2) and X14420 (COL3A1).
• Prolyl 4-hydroxylase (P4H), is involved in production of collagen and in oxygen sensing. A representative Genbank accession number providing DNA and protein sequence information for human P4H is AY198406.
• Procollagen C-protease (PCP) is another target.
• Matrix metalloproteinase 2, 9 (MMP2, 9) belong to the metzincin metalloproteinase superfamily and are zinc-dependent endopeptidases. These proteins are implicated in a variety of cellular processes including tissue repair. Representative Genbank accession numbers providing DNA and protein sequence information for human MMP proteins are M55593 (MMP2) and J05070 (MMP9).
• Integrins are a family of proteins involved in interaction and communication between a cell and the extracellular matrix. Vertebrates contain a variety of integrins including α₁β₁, α₂β₁,α₄β₁,α₅β₁,α₆β₁,α_Lβ₂,α_Mβ₂,α_IIbβ₃,α_vβ₃,α_vβ₅,α_vβ₆,α₆β₄.
• Connexins are a family of vertebrate transmembrane proteins that form gap junctions. Several examples of Connexins, with the accompanying gene name shown in brackets, include Cx23 (GJE1), Cx25 (GJB7), Cx26 (GJB2), Cx29 (GJE 1), Cx30 (GJB6), Cx30,2 (GJC3), Cx30,3 (GJB4), Cx31 (GJB3), Cx31,1 (GJB5), Cx31,9 (GJC1/GJD3), Cx32 (GJB1), Cx33 (GJA6), Cx36 (GJD2/GJA9), Cx37 (GJA4), Cx39 (GJD4), Cx40 (GJA5), Cx40,1 (GJD4), Cx43 (GJA1), Cx45 (GJC1/GJA7), Cx46 (GJA3), Cx47 (GJC2/GJA12), Cx50 (GJA8), Cx59 (GJA10), and Cx62 (GJA10).
• Histamine H1 receptor (HRH1) is a metabotropic G-protein-coupled receptor involved in the phospholipase C and phosphatidylinositol (PIP2) signaling pathways. A representative Genbank accession number providing DNA and protein sequence information for human HRH1 is Z34897.
• Tissue transglutaminase, also called Protein-glutamine gamma-glutamyltransferase 2, is involved in protein crosslinking and is implicated is biological processes such as apoptosis, cellular differentiation and matrix stabilization. A representative Genbank accession number providing DNA and protein sequence information for human Tissue transglutaminase is M55153.
• Mammalian target of rapamycin (mTOR), also known as Serine/threonine-protein kinase mTOR and FK506 binding protein 12-rapamycin associated protein 1 (FRAP1), is involved in regulating cell growth and survival, cell motility, transcription and translation. A representative Genbank accession number providing DNA and protein sequence information for human mTOR is L34075.
• HoxB13 belongs to the family of Homeobox proteins and has been linked to functions such as cutaneous regeneration and fetal skin development. A representative Genbank accession number providing DNA and protein sequence information for human HoxB13 is U57052.
• Vascular endothelial growth factor (VEGF) proteins are growth factors that bind to tyrosine kinase receptors and are implicated in multiple disorders such as cancer, age-related macular degeneration, rheumatoid arthritis and diabetic retinopathy. Members of this protein family include VEGF-A, VEGF-B, VEGF-C and VEGF-D. Representative Genbank accession numbers providing DNA and protein sequence information for human VEGF proteins are M32977 (VEGF-A). U43368 (VEGF-B), X94216 (VEGF-C), and D89630 (VEGF-D).
• Interleukin-6 (IL-6) is a cytokine involved in stimulating immune response to tissue damage. A representative Genbank accession number providing DNA and protein sequence information for human IL-6 is X04430.
• SMAD proteins (SMAD1-7, 9) are a family of transcription factors involved in regulation of TGFβ signaling. Representative Genbank accession numbers providing DNA and protein sequence information for human SMAD proteins are U59912 (SMAD1), U59911 (SMAD2), U68019 (SMAD3), U44378 (SMAD4), U59913 (SMAD5), U59914 (SMAD6), AF015261 (SMAD7), and BC011559 (SMAD9).
• Ribosomal protein S6 kinases (RSK6) represent a family of serine/threonine kinases involved in activation of the transcription factor CREB. A representative Genbank accession number providing DNA and protein sequence information for human Ribosomal protein S6 kinase alpha-6 is AF184965.
• Cyclooxygenase-2 (COX-2), also called Prostaglandin G/H synthase 2 (PTGS2), is involved in lipid metabolism and biosynthesis of prostanoids and is implicated in inflammatory disorders such as rheumatoid arthritis. A representative Genbank accession number providing DNA and protein sequence information for human COX-2 is AY462100.
• Cannabinoid receptors, of which there are currently two known subtypes, CB1 and CB2, are a class of cell membrane receptors under the G protein-coupled receptor superfamily. The CB1 receptor is expressed mainly in the brain, but is also expressed in the lungs, liver and kidneys, while the CB2 receptor is mainly expressed in the immune system and in hematopoietic cells. A representative Genbank accession number providing DNA and protein sequence information for human CB1 is NM_001160226. NM_001160258, NM_001160259, NM_001160260, NM_016083, and NM_033181.
• miR29b (or miR-29b) is a microRNA (miRNA), which is a short (20-24 nt) non-coding RNA involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce an approximately 70-nt stem-loop precursor miRNA (pre-miRNA), which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products. The mature miRNA is incorporated into a RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA. A representative miRBase accession number for miR29b is MI0000105 (website: mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000105).
• In some embodiments, the sd-rxRNA targets connexin 43 (CX43). This gene is a member of the connexin gene family. The encoded protein is a component of gap junctions, which are composed of arrays of intercellular channels that provide a route for the diffusion of low molecular weight materials from cell to cell. The encoded protein is the major protein of gap junctions in the heart that are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. A related intronless pseudogene has been mapped tochromosome 5. Mutations in this gene have been associated with oculodentodigital dysplasia and heart malformations. Representative Genbank accession numbers providing DNA and protein sequence information for human CX43 genes and proteins include NM_000165 and NP_000156.
• In other embodiments, the sd-rxRNA targets prolyl 4-hydroxylase (P4HTM). The product of this gene belongs to the family of prolyl 4-hydroxylases. This protein is a prolyl hydroxylase that may be involved in the degradation of hypoxia-inducible transcription factors under normoxia. It plays a role in adaptation to hypoxia and may be related to cellular oxygen sensing. Alternatively spliced variants encoding different isoforms have been identified. Representative Genbank accession numbers providing DNA and protein sequence information for human P4HTM genes and proteins include NM_177938, NP_808807, NM_177939, and NP_808808.
• In certain embodiments, the sd-rxRNA targets procollagen C-protease.
EXAMPLESExample 1: In Vivo Gene Silencing in Skin after Local Delivery of Sd-rxRNA
• Demonstrated herein is gene silencing in skin following administration of sd-rxRNA molecules. Rat incision models were used which included 6 dorsal incisions per animal. Analysis included monitoring of digital images, detection of target gene expression, scar assessment, and histology.FIG. 1 reveals an expression profile of several genes including MAP4K4, SPP1, CTGF, PTGS2 and TGFB1. As expected, when expression of these genes was monitored post-incision, target gene expression was elevated early and then returned to normal byday 10.
• FIG. 2 presents an overview of intradermal injection experiments with sd-rxRNA molecules. 6 intradermal injections were performed at each site. Each injection consisted of approximately 34μ, 300 μg total. Images were taken before injection and 15 minutes after the first injection.
• FIG. 3 demonstrates in vivo silencing following intradermal injection of sd-rxRNA in rats. 6 injections were made per dose. 300 μg in PBS was injected ondays 1 & 2 (2 doses) or on day 2 (1 dose). 5 incisions sites were made per treatment. Incisions were 1 cm. 3 mm skin biopsies were harvested 48 hours after the last dose and target expression was determined by QPCR.
• FIG. 4 demonstrates in vivo silencing of MAP4K4, PPIB and CTGF expression in rats following intradermal injection of sd-rxRNA molecules. A single intradermal injection of PBS (vehicle), or 300 ug of MAP4K4, or 2 different CTGF or PPIB targeting sd-rxRNA were injected at 6 sites. 3 mm skin biopsies harvested 48 hours post injection and processed for RNA. Data was analyzed by QPCR and normalized to B-Actin. PBS was set to 1. Data was graphed as a percent reduction in targeted gene expression relative to non-targeting sd-rxRNA (i.e. targeting other gene). Gene expression from untreated skin samples on treated animals are similar to PBS treated or sham controls.
• FIG. 5 demonstrates in vivo silencing in mice following intradermal injection of sd-rxRNA molecules. C57BL/6 mice were used, with n=7 wheal sites active and PBS. The control group consisted of 12. 300 ug was administered in 50 ul/injections. 3 mm biopsies were processed for RNA, and target expression determined by QPCR. Expression was normalized to housekeeping gene cyclophilin B.
• FIG. 6 reveals the in vitro potency and in vivo effectiveness of 2 different sd-rxRNAs targeting PPIB. Two PPIB sd-rxRNAs with different EC50s were compared in vivo. Similar in vivo results were obtained with 1 injection of 300 μg
• FIGS. 7 and 8 demonstrate the duration of gene silencing achieved through administration of sd-rxRNA. There were 6 injection sites per animal. 3 mm skin biopsies were harvested ondays 3, 5, and 8. RNA was isolated and gene expression was analyzed by qPCR and normalized to B-Actin
• FIG. 9 compares two different dosage regimens,Days 1 and 3vs. Days 0 and 2. There were 6 injection sites per animal. 3 mm skin biopsies were harvested ondays 3, 5, and 8. RNA was isolated and gene expression was analyzed by qPCR and normalized to B-Actin.
Example 2: Identification of Potent sd-rxRNAs
• Up to 300 rxRNA on compounds were screened for 5 anti-scarring targets. Optimal sequences in SPP1, CTGF, PTGS2, TGFB1 and TGFB2 for sd-rxRNA development were identified using a sequence selection algorithm. The algorithm selects sequences based on the following criteria: a GC content greater than 32% but less than 47%, homology to specific animal models (e.g., mouse or rat), avoidance of 5 or more U/U stretches and/or 2 or more G/C stretches, an off-target hit score of less than 500, and avoidance of sequences contained within the 5′ UTR.
• The sequences were developed initially as 25 nucleotide blunt-ended duplexes with O-methyl modification. Such sequences were screened in various cell lines to identify those were most efficient in reducing gene expression. Several concentrations of the RNA molecules, such as 0.025, 0.1 and 0.25 nM, were tested, and suitable concentrations to screen for bDNA were determined. A bDNA was then run of a full screen at a desired concentration. Dose response curves were generated to determine the most potent sequences. Hyperfunctional hits were those with an EC50 of less than 100 pM in lipid transfection. Potent molecules were selected to be developed into sd-rxRNAs based on the parameters described throughout the application and a secondary screen was conducted using the sd-rxRNAs.
• FIGS. 10-12 reveal that CTGF sd-rxRNAs are efficacious in mediating gene silencing. A dose response for CTGF is indicated inFIG. 12.
• FIGS. 13-14 reveal that the original sd-rxrNA screen had a low hit rate.FIG. 15 reveals PTGS2 knockdown using sd-rxRNA against PTGS2.FIGS. 16-24 reveal that hTGFB1, TGFB, TGFB2 sd-rxRNAs are capable of mediating gene silencing.FIGS. 25-28 shows the identification of potent hSPP1 sd-rxRNAs.
Example 3: Linker Chemistry
• FIG. 36 demonstrates that variation of linker chemistry does not influence silencing activity of sd-rxRNAs in vitro. Two different linker chemistries were evaluated, a hydroxyproline linker and ribo linker, on multiple sd-rxRNAs (targeting Map4k4 or PPIB) in passive uptake assays to determine linkers which favor self delivery. HeLa cells were transfected in the absence of a delivery vehicle (passive transfection) with sd-rxRNAs at 1 uM, 0.1 uM or 0.01 uM for 48 hrs. Use of either linker results in an efficacious delivery of sd-rxRNA.
• The ribo linker used in Example 5 had the following structure:
• 
Example 4: Optimization of Target Sequences
• Chemical optimization was performed for several lead sequences, including CTGF, PTGS2, TGF I1, and TGFβ2. Multiples versions of sd-rxRNA leads were synthesized. The sense strand was further O-methyl modified, such as by introduction of O-methyl blocks on the ends, introduction of O-methyl phosphorothioate blocks at the ends or introduction of full O-methyl modification with a phosphorothioate block on the 3′end.
• The guide strand was modified to decrease the number of 2′F, substitute 2′F with O-methyl, vary the number of ribonucleotides, eliminate stretches of ribonucleotides, minimize the presence of ribonucleotides next to the phosphorothioate modifications, and if possible remove ribonucleotides from the single stranded region.
• Various versions of compounds were synthesized and their efficacy was tested in vitro using passive uptake. The efficacy and toxicity of the optimized compounds was evaluated in vivo.
• All compounds show in vivo efficacy. Initially, activity required high concentration and at high concentrations some compound demonstrated injection site reaction. However, data indicated that efficacy and toxicity in vivo could be dramatically improved by enhancement of stability and reduction of 2′ F content. In some instances, toxicity, at least in part, was related to the presence of cholesterol containing short oligomer metabolites. This type of toxicity is expected to be reduced by stabilization. In general, chemical stabilization was well tolerated. Exact chemical optimization patterns differed for various compound. In some cases, complete stabilization resulted in a slightly negative impact on activity. For most target sites, at least two chemically optimized leads were identified: chemically optimized with in vitro efficacy retained or improved compared to an Early Lead and Fully Modified, where in vitro efficacy is slightly reduced.
• In general, a fully O-methyl modified sense strand is acceptable. In some instances, it is preferable if less than all of the nucleotides in the sense strand are O-methyl modified. In some instances, the 3′ end of the passenger strand contained a PS/O-METHYL block (2 O-methyl modifications and two 2 phosphorothioate modifications) to insure maximized stability next to the hydrophobic conjugate.
• For all compounds, it was possible to identify functional heavily stabilized leads. In some instances, the number of ribonucleotides per compounds was reduced to 4-6. Multiples versions of sd-rxRNA leads were synthesized. The number of 2′F modified purines was limited where possible to improve manufacturability but some optimized compounds do contain some 2′F modified purines.
Optimized Compounds
• A summary of CTGF lead compounds is shown in Table 24. PTGS leads are shown in Table 25. hTGFβ1 leads are shown in Table 26 and hTGFβ2 leads are shown in Table 27. Lead compounds were tested for in vitro efficacy with varying levels of methylation of the sense strand.
• For CTGF Lead 1 (L1), the fully O-methyl modified sense strand was efficacious having a slight reduction in in vitro efficacy.
• For CTGF L2, the fully O-methyl modified sense strand was efficacious, having a slight reduction in in vitro efficacy.
• For CTGF L3, the fully O-methyl modified sense strand was partially efficacious, having a reduction in in vitro efficacy.
• For CTGF L4, the fully O-methyl modified sense strand was partially efficacious, having a slight reduction in in vitro efficacy.
• For PTGS2 L1 and L2, the fully O-methyl modified sense strand was not efficacious.
• For TGFβ1 hL3, the fully O-methyl modified sense strand was efficacious.
• For TGFβ2, the fully O-methyl modified sense strand was efficacious.
In Vivo Efficacy of Lead Compounds
• The activity of lead compounds was tested in vivo both in cell culture and in animal models.FIGS. 33 and 34 demonstrate the activity of optimized CTGF L1 compounds.FIG. 35 demonstrates the in vitro stability of the CTGF L1 compounds.FIGS. 36 and 37 demonstrate the activity of optimized CTGF L2 compounds.FIG. 38 demonstrates the in vitro stability of the CTGF L2 compounds.FIG. 39 provides a summary of the in vivo activity of CTGF lead compounds.FIG. 40 demonstrates the efficacy of CTGF L1 compounds in skin biopsies from rats.FIG. 41 shows the efficacy of CTGF L2 compounds in achieving gene silencing.
• FIG. 42 demonstrates CTGF silencing following intradermal injection of RXi-109.FIG. 43 demonstrates the duration of CTGF silencing in skin after intradermal injection of the sd-rxRNA in SD rats. Eight millimeter skin biopsies were harvested, and mRNA levels were quantified by QPCR and normalized to a housekeeping gene. Shown is percent (%) silencing vs. Non Targeting Control (NTC); PBS at each time point is one experimental group; * p≤0.04; ** p≤0.002.
• FIGS. 44-46 show that CTGF L3 and L4 compounds are also active.FIG. 47 demonstrates changes in mRNA expression levels of CTGF, α-SM actin, collagen 1A2, and collagen 3A1 after intradermal injection of CTFG sd-rxRNA in SD rats. mRNA levels were quantified by qPCR. Substantial reduction in CTGF expression is observed.
• FIG. 49 demonstrates that administration of sd-rxRNAs decreases wound width over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats ondays 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.
• FIG. 50 demonstrates that administration of sd-rxRNAs decreases wound area over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats ondays 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.
• FIG. 51 demonstrates that administration of sd-rxRNAs increase the percentage of wound re-epithelialization over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats ondays 3, 6, and 9 post-wounding. Each group represents 5 rats. Two non-serial sections from each wound were measured and the average width of the two was calculated per wound. *p<0.05 vs. PBS an NTC.
• FIG. 52 demonstrates that administration of sd-rxRNAs increases the average granulation tissue maturity scores over the course of at least 9 days. The graph shows microscopic measurements of wound width in rats ondays 3, 6, and 9 post-wounding (5=mature, 1=immature). Each group represents 5 rats.FIG. 53 demonstrates CD68 labeling inday 9 wounds (0=no labeling, 3=substantial labeling). Each group represents 5 rats.
• FIG. 54 demonstrates that CTGF leads have different toxicity levels in vitro.FIG. 55 shows percentage (%) of cell viability after RXi-109 dose escalation (oligos formulated in PBS).
• FIG. 56 is a schematic of a non-limiting example of aPhase 1 and 2 clinical trial design for lead compounds. This schematic represents a divided dose, single day ascending dose clinical trial.
• FIG. 57 is a schematic of a non-limiting example of aPhase 1 and 2 clinical trial design. This schematic represents a divided dose, multi-day ascending dose clinical trial.
• Activity of PTGS2, TGFβ1 and TGFβ2 leads was also tested.FIGS. 59 and 60 demonstrate activity of PTGS2 L1 and L2 compounds.FIGS. 61 and 62 demonstrate the activity of h TGFβ1 compounds andFIGS. 63 and 64 demonstrate the activity of hTGFβ2 compounds.
• Gene knock-down in liver was also tested following tail vein injection mice.FIG. 58 demonstrates a percent (%) decrease in PPIB expression in the liver relative to PBS control. Lipoid formulated rxRNAs (10 mg/kg) were delivery systemically to Balb/c mice (n=5) by single tail vein injections. Liver tissue was harvested at 24 hours after injection and expression was analyzed by qPCR (normalized to β-actin). Map4K4 rxRNAori also showed significant silencing (˜83%, p<0.001) although Map4K4 sd-rxRNA did not significantly reduce target gene expression (˜17%, p=0.019). TD.035.2278, Published lipidoid delivery reagent, 98N12-5(1), from Akinc, 2009.
• Table 1 provides sequences tested in the Original sd-rxRNA screen.
  Table 2 demonstrates inhibition of gene expression with PTGS2 ori sequences.
  Table 3 demonstrates non-limiting examples of PTGS2 sd-rxRNA sequences.
  Table 4 demonstrates non-limiting examples of TGFB1 sd-rxRNA sequences.
  Table 5 demonstrates inhibition of gene expression with hTGFB1 on sequences.
  Table 6 demonstrates inhibition of gene expression with hTGFB2 ori sequences.
  Table 7 demonstrates non-limiting examples of hTGFB2 sd-rxRNA sequences.
  Table 8 demonstrates non-limiting examples of hSPP1 sd-rxRNA sequences.
  Table 9 demonstrates inhibition of gene expression with hSPP1 on sequences.
  Table 10 demonstrates non-limiting examples of hCTGF sd-rxRNA sequences.
  Table 11 demonstrates inhibition of gene expression with hCTGF on sequences.
  Table 12 demonstrates inhibition of gene expression with CTGF on sequences.
  Table 13 demonstrates inhibition of gene expression with SPP1 sd-rxRNA sequences.
  Table 14 demonstrates inhibition of gene expression with PTGS2 sd-rxRNA sequences.
  Table 15 demonstrates inhibition of gene expression with CTGF sd-rxRNA sequences.
  Table 16 demonstrates inhibition of gene expression with TGFB2 sd-rxRNA sequences.
  Table 17 demonstrates inhibition of gene expression with TGFB1 sd-rxRNA sequences.
  Table 18 demonstrates inhibition of gene expression with SPP1 sd-rxRNA sequences.
  Table 19 demonstrates inhibition of gene expression with PTGS2 sd-rxRNA sequences.
  Table 20 demonstrates inhibition of gene expression with CTGF sd-rxRNA sequences.
  Table 21 demonstrates inhibition of gene expression with TGFB2 sd-rxRNA sequences.
  Table 22 demonstrates inhibition of gene expression with TGFB1 sd-rxRNA sequences.
  Table 23 provides non-limiting examples of CB1 sequences.
  Table 24 provides a summary of CTGF Leads.
  Table 25 provides a summary of PTGS2 Leads.
  Table 26 provides a summary of TGFβ1 Leads.
  Table 27 provides a summary of TGFβ1 Leads.

• TABLE 1
  Original sd-rxRNA screen

  Oligo		SEQ ID		SEQ ID
  ID#	G1-	NO	Sense-sd-rxRNA GII	NO	AS-sd-rxRNA-GII

  14394	TGFB1	1	GmCmUAAmUGGmUGGAA-	2	5′-P-mU(2′-F-U)(2′-F-C)(2′-F-C)A(2′-
  			chol		F-C)(2′-F-C)A(2′-F-U)(2′-F-
  					U)AGmC*A*mC*G*mC*G*G
  14395	TGFB1	3	mUGAmUmCGmUGmCGmCmUmC-	4	5′-P-mGAG(2′-F-C)G(2′-F-C)A(2′-F-
  			chol		C)GA(2′-F-
  					U)mCA*mU*G*mU*mU*G*G
  14396	TGFB1	5	mCAAmUmUmCmCmUGGmCGA-	6	5′-P-mU(2′-F-C)G(2′-F-C)(2′-F-
  			chol		C)AGGAA(2′-F-
  					U)mUG*mU*mU*G*mC*mU*G
  14397	TGFB1	7	AGmUGGAmUmCmCAmCGA-	8	5′-P-mU(2′-F-C)G(2′-F-U)GGA(2′-F-
  			chol		U)(2′-F-C)(2′-F-
  					C)AmCmU*mU*mC*mC*A*G*C
  14398	TGFB1	9	mUAmCAGmCAAGGmUmCmC-	10	5′-P-mGGA(2′-F-C)(2′-F-C)(2′-F-
  			chol		U)(2′-F-U)G(2′-F-C)(2′-F-
  					U)GmUA*mC*mU*G*mC*G*U
  14399	TGFB1	11	AAmCAmUGAmUmCGmUGmC-	12	5′-P-mG(2′-F-C)A(2′-F-C)GA(2′-F-
  			chol		U)(2′-F-C)A(2′-F-
  					U)GmUmU*G*G*A*mC*A*G
  14400	TGFB1	13	AmCAmUGAmUmCGmUGmCG-	14	5′-P-mCG(2′-F-C)A(2′-F-C)GA(2′-F-
  			chol		U)(2′-F-C)A(2′-F-
  					U)GmU*mU*G*G*A*mC*A
  14401	TGFB1	15	mCAGmCAAGGmUmCmCmUG-	16	5′-P-mCAGGA(2′-F-C)(2′-F-C)(2′-F-
  			chol		U)(2′-F-U)G(2′-F-
  					C)mUG*mU*A*mC*mU*G*C
  14402	TGFB1	17	mCmCAAmCAmUGAmUmCGmU-	18	5′-P-mA(2′-F-C)GA(2′-F-U)(2′-F-
  			chol		C)A(2′-F-U)G(2′-F-U)(2′-F-
  					U)GG*A*mC*A*G*mC*U
  14403	TGFB1	19	AGmCGGAAGmCGmCAmU-	20	5′-P-mA(2′-F-U)G(2′-F-C)G(2′-F-
  			chol		C)(2′-F-U)(2′-F-U)(2′-F-C)(2′-F-
  					C)GmCmU*mU*mC*A*mC*mC*A
  14404	TGFB1	21	GmCAmUmCGAGGmCmCAmU-	22	5′-P-mA(2′-F-U)GG(2′-F-C)(2′-F-
  			chol		C)(2′-F-U)(2′-F-C)GA(2′-F-
  					U)GmC*G*mC*mU*mU*mC*C
  14405	TGFB1	23	GAmCmUAmUmCGAmCAmUG-	24	5′-P-mCA(2′-F-U)G(2′-F-U)(2′-F-
  			chol		C)GA(2′-F-
  					U)AGmUmC*mU*mU*G*mC*A*G
  14406	TGFB1	25	AmCmCmUGmCAAGAmCmUA-	26	5′-P-mUAG(2′-F-U)(2′-F-C)(2′-F-
  			chol		U)(2′-F-U)G(2′-F-
  					C)AGGmU*G*G*A*mU*A*G
  14407	TGFB1	27	GmCmUmCmCAmCGGAGAA-	28	5′-P-mU(2′-F-U)(2′-F-C)(2′-F-U)(2′-F-
  			chol		C)(2′-F-C)G(2′-F-
  					U)GGAGmC*mU*G*A*A*G*C
  14408	TGFB2	29	GGmCmUmCmUmCmCmUmUmCGA-	30	5′-P-mU(2′-F-
  			chol		C)GAAGGAGAGmCmC*A*mU*mU*
  					mC*G*C
  14409	TGFB2	31	GAmCAGGAAmCmCmUGG-	32	5′-P-mC(2′-F-C)AGG(2′-F-U)(2′-F-
  			chol		U)(2′-F-C)(2′-F-C)(2′-F-
  					U)GmUmC*mU*mU*mU*A*mU*G
  14410	TGFB2	33	mCmCAAGGAGGmUmUmUA-	34	5′-P-mUAAA(2′-F-C)(2′-F-C)(2′-F-
  			chol		U)(2′-F-C)(2′-F-C)(2′-F-U)(2′-F-
  					U)GG*mC*G*mU*A*G*U
  14411	TGFB2	35	AmUmUmUmCmCAmUmCmUAmCA-	36	5′-P-mUG(2′-F-U)AGA(2′-F-
  			chol		U)GGAAAmU*mC*A*mC*mC*mU*C
  14412	TGFB2	37	mUmCmCAmUmCmUAmCAAmCA-	38	5′-P-mUG(2′-F-U)(2′-F-U)G(2′-F-
  			chol		U)AGA(2′-F-
  					U)GGA*A*A*mU*mC*A*C
  14413	TGFB2	39	mUmUmUmCmCAmUmCmUAmCAA-	40	5′-P-mU(2′-F-U)G(2′-F-U)AGA(2′-F-
  			chol		U)GGAAA*mU*mC*A*mC*mC*U
  14414	TGFB2	41	mCGmCmCAAGGAGGmUmU-	42	5′-P-mAA(2′-F-C)(2′-F-C)(2′-F-U)(2′-
  			chol		F-C)(2′-F-C)(2′-F-U)(2′-F-
  					U)GGmCG*mU*A*G*mU*A*C
  14415	TGFB2	43	GmUGGmUGAmUmCAGAA-	44	5′-P-mU(2′-F-U)(2′-F-C)(2′-F-
  			chol		U)GA(2′-F-U)(2′-F-C)A(2′-F-C)(2′-F-
  					C)AmC*mU*G*G*mU*A*U
  14416	TGFB2	45	mCmUmCmCmUGmCmUAAmUGmU-	46	5′-P-mA(2′-F-C)A(2′-F-U)(2′-F-
  			chol		U)AG(2′-F-
  					C)AGGAG*A*mU*G*mU*G*G
  14417	TGFB2	47	AmCmCmUmCmCAmCAmUAmUA-	48	5′-P-mUA(2′-F-U)A(2′-F-U)G(2′-F-
  			chol		U)GGAGGmU*G*mC*mC*A*mU*C
  14418	TGFB2	49	AAGmUmCmCAmCmUAGGA-	50	5′-P-mU(2′-F-C)(2′-F-C)(2′-F-
  			chol		U)AG(2′-F-U)GGA(2′-F-
  					C)mUmU*mU*A*mU*A*G*U
  14419	TGFB2	51	mUGGmUGAmUmCAGAAA-	52	5′-P-mU(2′-F-U)(2′-F-U)(2′-F-C)(2′-F-
  			chol		U)GA(2′-F-U)(2′-F-C)A(2′-F-
  					C)mCA*mC*mU*G*G*mU*A
  14420	TGFB2	53	AGmUmCmCAmCmUAGGAA-	54	5′-P-mU(2′-F-U)(2′-F-C)(2′-F-C)(2′-F-
  			chol		U)AG(2′-F-
  					U)GGAmCmU*mU*mU*A*mU*A*G
  14421	TGFB2	55	AmCGmCmCAAGGAGGmU-	56	5′-P-mA(2′-F-C)(2′-F-C)(2′-F-U)(2′-F-
  			chol		C)(2′-F-C)(2′-F-U)(2′-F-U)GG(2′-F-
  					C)GmU*A*G*mU*A*mC*U
  14422	PTGS2	57	mCAmCAmUmUmUGAmUmUGA-	58	5′-P-mU(2′-F-C)AA(2′-F-U)(2′-F-
  			chol		C)AAA(2′-F-
  					U)GmUG*A*mU*mC*mU*G*G
  14423	PTGS2	59	mCAmCmUGmCmCmUmCAAmUmU-	60	5′-P-mAA(2′-F-U)(2′-F-U)GAGG(2′-F-
  			chol		C)AGmUG*mU*mU*G*A*mU*G
  14424	PTGS2	61	AAAmUAmCmCAGmUmCmUmU-	62	5′-P-mAAGA(2′-F-C)(2′-F-U)GG(2′-F-
  			chol		U)A(2′-F-
  					U)mUmU*mC*A*mU*mC*mU*G
  14425	PTGS2	63	mCAmUmUmUGAmUmUGAmCA-	64	5′-P-mUG(2′-F-U)(2′-F-C)AA(2′-F-
  			chol		U)(2′-F-
  					C)AAAmUG*mU*G*A*mU*mC*U

• TABLE 2
  inhibition of gene expression
  with PTGS2 or sequences NM_000963.2

  Target					NM_000963.2
  Gene			SEQ	PTGS2	% Expres-
  Duplex	Gene	Ref	ID	Sense	sion
  ID	Region	Pos	No	Sequence	(0.1 nM)

  15147	CDS	176	65	CCUGGCGCUCA	59%
  				GCCAUACACCA
  				AAA
  15148	CDS	177	66	CUGGCGCUCAG	72%
  				CCAUACAGCAA
  				AUA
  15149	CDS	178	67	UGGCGCUCAGC	77%
  				CAUACAGCAAA
  				UCA
  15150	CDS	180	68	GCGCUCAGCCA	70%
  				UACAGCAAAUC
  				CUA
  15151	CDS	182	69	GCUCAGCCAUA	76%
  				CAGCAAAUCCU
  				UGA
  15152	CDS	183	70	CUCAGCCAUAC	74%
  				AGCAAAUCCUU
  				GCA
  15153	CDS	184	71	UCAGCCAUACA	47%
  				GCAAAUCCUUG
  				CUA
  15154	CDS	186	72	AGCCAUACAGC	55%
  				AAAUCCUUGCU
  				GUA
  15155	CDS	187	73	GCCAUACAGCA	41%
  				AAUCCUUGCUG
  				UUA
  15156	CDS	188	74	CCAUACAGCAA	46%
  				AUCCUUGCUGU
  				UCA
  15157	CDS	212	75	CCACCCAUGUC	31%
  				AAAACCGAGGU
  				GUA
  15158	CDS	243	76	AGUGUGGGAUU	23%
  				UGACCAGUAUA
  				AGA
  15159	CDS	244	77	GUGUGGGAUUU	24%
  				GACCAGUAUAA
  				GUA
  15160	CDS	245	78	UGUGGGAUUUG	38%
  				ACCAGUAUAAG
  				UGA
  15161	CDS	252	79	UUUGACCAGUA	29%
  				UAAGUGCGAUU
  				GUA
  15162	CDS	285	80	GGAUUCUAUGG	16%
  				AGAAAACUGCU
  				CAA
  15163	CDS	337	81	UAUUUCUGAAA	32%
  				CCCACUCCAAA
  				CAA
  15164	CDS	338	82	AUUUCUGAAAC	21%
  				CCACUCCAAAC
  				ACA
  15165	CDS	339	83	UUUCUGAAACC	21%
  				CACUCCAAACA
  				CAA
  15166	CDS	340	84	UUCUGAAACCC	45%
  				ACUCCAAACAC
  				AGA
  15167	CDS	345	85	AAACCCACUCC	87%
  				AAACACAGUGC
  				ACA
  15168	CDS	347	86	ACCCACUCCAA	83%
  				ACACAGUGCAC
  				UAA
  15169	CDS	349	87	CCACUCCAAAC	51%
  				ACAGUGCACUA
  				CAA
  15170	CDS	350	88	CACUCCAAACA	31%
  				CAGUGCACUAC
  				AUA
  15171	CDS	394	89	UUUGGAACGUU	43%
  				GUGAAUAACAU
  				UCA
  15172	CDS	406	90	UGAAUAACAUU	21%
  				CCCUUCCUUCG
  				AAA
  15173	CDS	407	91	GAAUAACAUUC	32%
  				CCUUCCUUCGA
  				AAA
  15174	CDS	408	92	AAUAACAUUCC	27%
  				CUUCCUUCGAA
  				AUA
  15175	CDS	435	93	AUUAUGAGUUA	27%
  				UGUGUUGACAU
  				CCA
  15176	CDS	437	94	UAUGAGUUAUG	21%
  				UGUUGACAUCC
  				AGA
  15177	CDS	439	95	UGAGUUAUGUG	30%
  				UUGACAUCCAG
  				AUA
  15178	CDS	440	96	GAGUUAUGUGU	68%
  				UGACAUCCAGA
  				UCA
  15179	CDS	441	97	AGUUAUGUGUU	35%
  				GACAUCCAGAU
  				CAA
  15180	CDS	442	98	GUUAUGUGUUG	36%
  				ACAUCCAGAUC
  				ACA
  15181	CDS	443	99	UUAUGUGUUGA	51%
  				CAUCCAGAUCA
  				CAA
  15182	CDS	444	100	UAUGUGUUGAC	2.40%
  				AUCCAGAUCAC
  				AUA
  15183	CDS	445	101	AUGUGUUGACA	37%
  				UCCAGAUCACA
  				UUA
  15184	CDS	446	102	UGUGUUGACAU	42%
  				CCAGAUCACAU
  				UUA
  15185	CDS	448	103	UGUUGACAUCC	18%
  				AGAUCACAUUU
  				GAA
  15186	CDS	449	104	GUUGACAUCCA	24%
  				GAUCACAUUUG
  				AUA
  15187	CDS	450	105	UUGACAUCCAGA	25%
  				UCACAUUUGAUU
  				A
  15188	CDS	452	106	GACAUCCAGAUC	27%
  				ACAUUUGAUUGA
  				A
  15189	CDS	454	107	CAUCCAGAUCAC	27%
  				AUUUGAUUGACA
  				A
  15190	CDS	455	108	AUCCAGAUCACA	32%
  				UUUGAUUGACAG
  				A
  15191	CDS	456	109	UCCAGAUCACAU	40%
  				UUGAUUGACAGU
  				A
  15192	CDS	457	110	CCAGAUCACAUU	52%
  				UGAUUGACAGUC
  				A
  15193	CDS	460	111	GAUCACAUUUGA	40%
  				UUGACAGUCCAC
  				A
  15194	CDS	461	112	AUCACAUUUGAU	46%
  				UGACAGUCCACC
  				A
  15195	CDS	462	113	UCACAUUUGAUU	34%
  				GACAGUCCACCA
  				A
  15196	CDS	463	114	CACAUUUGAUUG	30%
  				ACAGUCCACCAA
  				A
  15197	CDS	464	115	ACAUUUGAUUGA	32%
  				CAGUCCACCAAC
  				A
  15198	CDS	465	116	CAUUUGAUUGAC	44%
  				AGUCCACCAACU
  				A
  15199	CDS	467	117	UUUGAUUGACAG	17%
  				UCCACCAACUUA
  				A
  15200	CDS	468	118	UUGAUUGACAGU	22%
  				CCACCAACUUAC
  				A
  15201	CDS	469	119	UGAUUGACAGUC	27%
  				CACCAACUUACA
  				A
  15202	CDS	470	120	GAUUGACAGUCC	41%
  				ACCAACUUACAA
  				A
  15203	CDS	471	121	AUUGACAGUCCA	39%
  				CCAACUUACAAU
  				A
  15204	CDS	472	122	UUGACAGUCCAC	61%
  				CAACUUACAAUG
  				A
  15205	CDS	473	123	UGACAGUCCACC	48%
  				AACUUACAAUGC
  				A
  15206	CDS	479	124	UCCACCAACUUA	29%
  				CAAUGCUGACUA
  				A
  15207	CDS	486	125	ACUUACAAUGCU	35%
  				GACUAUGGCUAC
  				A
  15208	CDS	488	126	UUACAAUGCUGA	32%
  				CUAUGGCUACAA
  				A
  15209	CDS	517	127	GGGAAGCCUUCU	39%
  				CUAACCUCUCCU
  				A
  15210	CDS	518	128	6GAAGCCUUCUC	48%
  				UAACCUCUCCUA
  				A
  15211	CDS	519	129	GAAGCCUUCUC	19%
  				UAACCUCUCCU
  				AUA
  15212	CDS	520	130	AAGCCUUCUCU	17%
  				AACCUCUCCUA
  				UUA
  15213	CDS	524	131	CUUCUCUAACC	17%
  				UCUCCUAUUAU
  				ACA
  15214	CDS	525	132	UUCUCUAACCU	34%
  				CUCCUAUUAUA
  				CUA
  15215	CDS	526	133	UCUCUAACCUC	49%
  				UCCUAUUAUAC
  				UAA
  15216	CDS	501	134	GUAAAAAGCAG	35%
  				CUUCCUGAUUC
  				AAA
  15217	CDS	602	135	UAAAAAGCAGC	25%
  				UUCCUGAUUCA
  				AAA
  15218	CDS	606	136	AAGCAGCUUCC	27%
  				UGAUUCAAAUG
  				AGA
  15219	CDS	615	137	CCUGAUUCAAA	37%
  				UGAGAUUGUGG
  				AAA
  15220	CDS	616	138	CUGAUUCAAAU	27%
  				GAGAUUGUGGA
  				AAA
  15221	CDS	636	139	GAAAAAUUGCU	37%
  				UCUAAGAAGAA
  				AGA
  15222	CDS	637	140	AAAAAUUGCUU	56%
  				CUAAGAAGAAA
  				GUA
  15223	CDS	638	141	AAAAUUGCUUC	25%
  				UAAGAAGAAAG
  				UUA
  15224	CDS	639	142	AAAUUGCUUCU	34%
  				AAGAAGAAAGU
  				UCA
  15225	CDS	678	143	GGCUCAAACAU	68%
  				GAUGUUUGCAU
  				UCA
  15226	CDS	679	144	GCUCAAACAUG	51%
  				AUGUUUGCAUU
  				CUA
  15227	CDS	680	145	CUCAAACAUGA	50%
  				UGUUUGCAUUC
  				UUA
  15228	CDS	682	146	CAAACAUGAUG	51%
  				UUUGCAUUCUU
  				UGA
  15229	CDS	683	147	AAACAUGAUGU	63%
  				UUGCAUUCUUU
  				GCA
  15230	CDS	722	148	UCAGUUUUUCA	45%
  				AGACAGAUCAU
  				AAA
  15231	CDS	723	149	CAGUUUUUCAA	59%
  				GACAGAUCAUA
  				AGA
  15232	CDS	724	150	AGUUUUUCAAG	80%
  				ACAGAUCAUAA
  				GCA
  15233	CDS	725	151	GUUUUUCAAGA	55%
  				CAGAUCAUAAG
  				CGA
  15234	CDS	726	152	UUUUUCAAGAC	53%
  				AGAUCAUAAGC
  				GAA
  15235	CDS	776	153	CCAUGGGGUGG	56%
  				ACUUAAAUCAU
  				AUA
  15236	CDS	787	154	ACUUAAAUCAU	63%
  				AUUUACGGUGA
  				AAA
  15237	CDS	788	155	CUUAAAUCAUA	43%
  				UUUACGGUGAA
  				ACA
  15233	CDS	789	156	UUAAAUCAUAU	48%
  				UUACGGUGAAA
  				CUA
  15239	CDS	790	157	UAAAUCAUAUU	56%
  				UACGGUGAAAC
  				UCA
  15240	CDS	792	158	AAUCAUAUUUA	46%
  				CGGUGAAACUC
  				UGA
  15241	CDS	793	159	AUCAUAUUUAC	64%
  				GGUGAAACUCU
  				GGA
  15242	CDS	799	160	UUUACGGUGAA	35%
  				ACUCUGGCUAG
  				ACA
  15243	CDS	819	161	AGACAGCGUAA	65%
  				ACUGCGCCUUU
  				UCA
  15244	CDS	820	162	GACAGCGUAAA	51%
  				CUGCGCCUUUU
  				CAA
  15245	CDS	821	163	ACAGCGUAAAC	48%
  				UGCGCCUUUUC
  				AAA
  15246	CDS	822	164	CAGCGUAAACU	61%
  				GCGCCUUUUCA
  				AGA
  15247	CDS	823	165	AGCGUAAACUG	48%
  				CGCCUUUUCAA
  				GGA
  15248	CDS	828	166	AAACUGCGCCU	42%
  				UUUCAAGGAUG
  				GAA
  15249	CDS	830	167	ACUGCGCCUUU	29%
  				UCAAGGAUGGA
  				AAA
  15250	CDS	861	168	UAUCAGAUAAU	32%
  				UGAUGGAGAGA
  				UGA
  15251	CDS	862	169	AUCAGAUAAUU	55%
  				GAUGGAGAGAU
  				GUA
  15252	CDS	863	170	UCAGAUAAUUG	50%
  				AUGGAGAGAUG
  				UAA
  15253	CDS	864	171	CAGAUAAUUGA	50%
  				UGGAGAGAUGU
  				AUA
  15254	CDS	865	172	AGAUAAUUGAU	55%
  				GGAGAGAUGUA
  				UCA
  15255	CDS	866	173	GAUAAUUGAUG	65%
  				GAGAGAUGUAU
  				CCA
  15256	CDS	867	174	AUAAUUGAUGG	54%
  				AGAGAUGUAUC
  				CUA
  15257	CDS	880	175	AGAUGUAUCCU	78%
  				CCCACAGUCAA
  				AGA
  15258	CDS	881	176	GAUGUAUCCUC	79%
  				CCACAGUCAAA
  				GAA
  15259	CDS	882	177	AUGUAUCCUCC	49%
  				CACAGUCAAAG
  				AUA
  15260	CDS	883	178	UGUAUCCUCCC	28%
  				ACAGUCAAAGA
  				UAA
  15261	CDS	884	179	GUAUCCUCCCA	56%
  				CAGUCAAAGAU
  				ACA
  15262	CDS	885	180	UAUCCUCCCAC	42%
  				AGUCAAAGAUA
  				CUA
  15263	CDS	887	181	UCCUCCCACA	45%
  				GUCAAAGAUA
  				CUCAA
  15264	CDS	888	182	CCUCCCACAG	73%
  				UCAAAGAUAC
  				UCAGA
  15265	CDS	889	183	CUCCCACAGU	56%
  				CAAAGAUACU
  				CAGGA
  15266	CDS	891	184	CCCACAGUCA	80%
  				AAGAUACUCA
  				GGCAA
  15267	CDS	901	185	AAGAUACUCA	59%
  				GGCAGAGAUG
  				AUCUA
  15268	CDS	935	186	AGUCCCUGAG	83%
  				CAUCUACGGU
  				UUGCA
  15265	CDS	980	187	UCUGGUGCCU	55%
  				GGUCUGAUGA
  				UGUAA
  15270	CDS	981	188	CUGCUGCCUG	56%
  				GUCUGAUGAU
  				GUAUA
  15271	CDS	982	189	UGGUGCCUGG	43%
  				UCUGAUGAUG
  				UAUGA
  15272	CDS	983	190	GGUGCCUGGU	41%
  				CUGAUGAUGU
  				AUGCA
  15273	CDS	984	191	GUGCCUGGUC	42%
  				UGAUGAUGUA
  				UGCCA
  15274	CDS	985	192	UGCCUGGUCU	37%
  				GAUGAUGUAU
  				GCCAA
  15275	CDS	986	193	GCCUGGUCUG	61%
  				AUGAUGUAUG
  				CCACA
  15276	CDS	1016	194	GCUGCGGGAA	44%
  				CACAACAGAG
  				UAUGA
  15277	CDS	1019	195	GCGGGAACAC	33%
  				AACAGAGUAU
  				GCGAA
  15278	CDS	1038	196	UGCGAUGUGC	53%
  				UUAAACAGGA
  				GCAUA
  15279	CDS	1039	197	GCGAUGUGCU	109%
  				UAAACAGGAG
  				CAUCA
  15280	CDS	1040	198	CGAUGUGCUU	77%
  				AAACAGGAGC
  				AUCCA
  15281	CDS	1042	199	AUGUGCUUAA	69%
  				ACAGGAGCAU
  				CCUGA
  15282	CDS	1043	200	UGUGCUUAAA	76%
  				CAGGAGCAUC
  				CUGAA
  15283	CDS	1044	201	GUGCUUAAAC	65%
  				AGGAGCAUCC
  				UGAAA
  15284	CDS	1045	202	UGCUUAAACA	64%
  				GGAGCAUCCU
  				GAAUA
  15285	CDS	1084	203	UGUUCCAGAC	41%
  				AAGCAGGCUA
  				AUACA
  15286	CDS	1093	204	CAAGCAGGCU	24%
  				AAUACUGAUA
  				GGAGA
  15287	CDS	1095	205	AGCAGGCUAA	50%
  				UACUGAUAGG
  				AGAGA
  15288	CDS	1096	206	GCAGGCUAAU	51%
  				ACUGAUAGGA
  				GAGAA
  15289	CDS	1124	207	UAAGAUUGUG	35%
  				AUUGAAGAUU
  				AUGUA
  15290	CDS	1125	208	AAGAUUGUGAU	34%
  				UGAAGAUUAUG
  				UGA
  15291	CDS	1126	209	AGAUUGUGAUU	59%
  				GAAGAUUAUGU
  				GCA
  15292	CDS	1127	210	GAUUGUGAUUG	41%
  				AAGAUUAUGUG
  				CAA
  15293	CDS	1128	211	AUUGUGAUUGA	51%
  				AGAUUAUGUGC
  				AAA
  15294	CDS	1129	212	UUGUGAUUGAA	45%
  				GAUUAUGUGCA
  				ACA
  15295	CDS	1131	213	GUGAUUGAAGA	37%
  				UUAUGUGCAAC
  				ACA
  15296	CDS	1132	214	UGAUUGAAGAU	34%
  				UAUGUGCAACA
  				CUA
  15297	CDS	1134	215	AUUGAAGAUUA	24%
  				UGUGCAACACU
  				UGA
  15298	CDS	1136	216	UGAAGAUUAUG	37%
  				UGCAACACUUG
  				AGA
  15299	CDS	1138	217	AAGAUUAUGUG	44%
  				CAACACUUGAG
  				UGA
  15300	CDS	1145	218	UGUGCAACACU	29%
  				UGAGUGGCUAU
  				CAA
  15301	CDS	1149	219	CAACACUUGAG	33%
  				UGGCUAUCACU
  				UCA
  15302	CDS	1179	220	AAAUUUGACCC	35%
  				AGAACUACUUU
  				UCA
  15303	CDS	1180	221	AAUUUGACCCA	41%
  				GAACUACUUUU
  				CAA
  15304	CDS	1181	222	AUUUGACCCAG	40%
  				AACUACUUUUC
  				AAA
  15305	CDS	1200	223	UUCAACAAACA	49%
  				AUUCCAGUACC
  				AAA
  15306	CDS	1211	224	AUUCCAGUACC	27%
  				AAAAUCGUAUU
  				GCA
  15307	CDS	1217	225	GUACCAAAAUC	31%
  				GUAUUGCUGCU
  				GAA
  15308	CDS	1270	226	UUCUGCCUGAC	35%
  				ACCUUUCAAAU
  				UCA
  15309	CDS	1280	227	CACCUUUCAAA	57%
  				UUCAUGACCAG
  				AAA
  15310	CDS	1284	228	UUUCAAAUUCA	42%
  				UGACCAGAAAU
  				ACA
  15311	CDS	1289	229	AAUUCAUGACC	52%
  				AGAAAUACAAC
  				UAA
  15312	CDS	1327	230	ACAACAACUCU	58%
  				AUAUUGCUGGA
  				ACA
  15313	CDS	1352	231	UGGAAUUACCC	35%
  				AGUUUGUUGAA
  				UCA
  15314	CDS	1356	232	AUUACCCAGUU	41%
  				UGUUGAAUCAU
  				UCA
  15315	CDS	1357	233	UUACCCAGUUU	58%
  				GUUGAAUCAUU
  				CAA
  15316	CDS	1353	234	ACCCAGUUUG	52%
  				UUGAAUCAUU
  				CACCA
  15317	CDS	1360	235	CCCAGUUUGU	66%
  				UGAAUCAUUC
  				ACCAA
  15318	CDS	1361	236	CCAGUUUGUU	54%
  				GAAUCAUUCA
  				CCAGA
  15319	CDS	1365	237	UUUGUUGAAU	47%
  				CAUUCACCAG
  				GCAAA
  15320	CDS	1462	238	AGAGCAGGCA	65%
  				GAUGAAAUAC
  				CAGUA
  15321	CDS	1463	239	GAGCAGGCAG	66%
  				AUGAAAUACC
  				AGUCA
  15322	CDS	1465	240	GCAGGCAGAU	22%
  				GAAAUACCAG
  				UCUUA
  15323	CDS	1466	241	CAGGCAGAUG	43%
  				AAAUACCAGU
  				CUUUA
  15324	CDS	1472	242	GAUGAAAUAC	23%
  				CAGUCUUUUA
  				AUGAA
  15325	CDS	1473	243	AUGAAAUACC	61%
  				AGUCUUUUAA
  				UGAGA
  15326	CDS	1474	244	UGAAAUACCA	49%
  				GUCUUUUAAU
  				GAGUA
  15327	CDS	1475	245	GAAAUACCAG	76%
  				UCUUUUAAUG
  				AGUAA
  15328	CDS	1476	246	AAAUACCAGU	51%
  				CUUUUAAUGA
  				GUACA
  15329	CDS	1477	247	AAUACCAGUC	72%
  				UUUUAAUGAG
  				UACCA
  15330	CDS	1478	248	AUACCAGUCU	40%
  				UUUAAUGAGU
  				ACCGA
  15331	CDS	1479	249	UACCAGUCUU	53%
  				UUAAUGAGUA
  				CCGCA
  15332	CDS	1480	250	ACCAGUCUUU	39%
  				UAAUGAGUAC
  				CGCAA
  15333	CDS	1481	251	CCAGUCUUUU	41%
  				AAUGAGUACC
  				GCAAA
  15334	CDS	1483	252	AGUCUUUUAA	38%
  				UGAGUACCGC
  				AAACA
  15335	CDS	1485	253	UCUUUUAAUG	55%
  				AGUACCGCAA
  				ACGCA
  15336	CDS	1486	254	CUUUUAAUGA	63%
  				GUACCGCAAA
  				CGCUA
  15337	CDS	1487	255	UUUUAAUGAG	52%
  				UACCGCAAAC
  				GCUUA
  15338	CDS	1495	256	AGUACCGCAA	49%
  				ACGCUUUAUG
  				CUGAA
  15339	CDS	1524	257	UAUGAAUCAU	65%
  				UUGAAGAACU
  				UACAA
  15340	CDS	1525	258	AUGAAUCAUU	63%
  				UGAAGAACUU
  				ACAGA
  15341	CDS	1527	259	GAAUCAUUUG	65%
  				AAGAACUUAC
  				AGGAA
  15342	CDS	1529	260	AUCAUUUGAA	43%
  				GAACUUACAG
  				GAGAA
  15343	CDS	1531	261	CAUUUGAAGA	63%
  				ACUUACAGGA
  				GAAAA
  15344	CDS	1532	262	AUUUGAAGAA	33%
  				CUUACAGGAG
  				AAAAA
  15345	CDS	1574	263	GGAAGCACUC	62%
  				UAUGGUGACA
  				UCGAA
  15345	CDS	1609	264	UGUAUCCUGC	36%
  				CCUUCUGGUA
  				GAAAA
  15347	CDS	1614	265	CCUGCCCUUC	58%
  				UGGUAGAAAA
  				GCCUA
  15348	CDS	1650	266	AUCUUUGGUG	60%
  				AAACCAUGGU
  				AGAAA
  15349	CDS	1666	267	UGGUAGAAGU	88%
  				UGGAGCACCA
  				UUCUA
  15350	CDS	1669	268	UAGAAGUUGG	85%
  				AGCACCAUUC
  				UCCUA
  15351	CDS	1672	269	AAGUUGGAGC	83%
  				ACCAUUCUCC
  				UUGAA
  15352	CDS	1675	270	UUGGAGCACC	85%
  				AUUCUCCUUG
  				AAAGA
  15353	CDS	1676	271	UGGAGCACCA	83%
  				UUCUCCUUGA
  				AAGGA
  15354	CDS	1677	272	GGAGCACCAU	74%
  				UCUCCUUGAA
  				AGGAA
  15355	CDS	1678	273	GAGCACCAUU	81%
  				CUCCUUGAAA
  				GGACA
  15356	CDS	1679	274	AGCACCAUUC	86%
  				UCCUUGAAAG
  				GACUA
  15357	CDS	1680	275	GCACCAUUCU	98%
  				CCUUGAAAGG
  				ACUUA
  15358	CDS	1681	276	CACCAUUCUC	78%
  				CUUGAAAGGA
  				CUUAA
  15359	CDS	1682	277	ACCAUUCUCC	88%
  				UUGAAAGGAC
  				UUAUA
  15360	CDS	1683	278	CCAUUCUCCU	88%
  				UGAAAGGACU
  				UAUGA
  15361	CDS	1762	279	UGGGUUUUCA	78%
  				AAUCAUCAAC
  				ACUGA
  15362	CDS	1763	280	GGGUUUUCAA	92%
  				AUCAUCAACA
  				CUGCA
  15363	CDS	1767	281	UUUCAAAUCA	85%
  				UCAACACUGC
  				CUCAA
  15364	CDS	1770	282	CAAAUCAUCA	84%
  				ACACUGCCUC
  				AAUUA
  15365	CDS	1773	283	AUCAUCAACA	86%
  				CUGCCUCAAU
  				UCAGA
  15366	CDS	1774	284	UCAUCAACAC	94%
  				UGCCUCAAUU
  				CAGUA
  15367	CDS	1775	285	CAUCAACACU	84%
  				GCCUCAAUUC
  				AGUCA
  15368	CDS	1776	286	AUCAACACUGC	84%
  				CUCAAUUCAGU
  				CUA
  15369	CDS	1777	287	UCAACACUGCC	68%
  				UCAAUUCAGUC
  				UCA
  15370	CDS	1778	288	CAACACUGCCU	73%
  				CAAUUCAGUCU
  				CUA
  15371	CDS	1779	289	AACACUGCCUC	79%
  				AAUUCAGUCUC
  				UCA
  15372	CDS	1780	290	ACACUGCCUCA	78%
  				AUUCAGUCUCU
  				CAA
  15373	CDS	1781	291	CACUGCCUCAA	92%
  				UUCAGUCUCUC
  				AUA
  15374	CDS	1782	292	ACUGCCUCAAU	89%
  				UCAGUCUCUCA
  				UCA
  15375	CDS	1783	293	CUGCCUCAAUU	95%
  				CAGUCUCUCAU
  				CUA
  15376	CDS	1784	294	UGCCUCAAUUC	83%
  				AGUCUCUCAUC
  				UGA
  15377	CDS	1785	295	GCCUCAAUUCA	46%
  				GUCUCUCAUCU
  				GCA
  15378	CDS	1786	296	CCUCAAUUCAG	51%
  				UCUCUCAUCUG
  				CAA
  15379	CDS	1787	297	CUCAAUUCAGU	61%
  				CUCUCAUCUGC
  				AAA
  15380	CDS	1790	298	AAUUCAGUCUC	30%
  				UCAUCUGCAAU
  				AAA
  15381	CDS	1791	299	AUUCAGUCUCU	32%
  				CAUCUGCAAUA
  				ACA
  15382	CDS	1792	300	UUCAGUCUCUC	30%
  				AUCUGCAAUAA
  				CGA
  15383	CDS	1793	301	UCAGUCUCUCA	38%
  				UCUGCAAUAAC
  				GUA
  15384	CDS	1794	302	CAGUCUCUCAU	67%
  				CUGCAAUAACG
  				UGA
  15385	CDS	1795	303	AGUCUCUCAUC	71%
  				UGCAAUAACGU
  				GAA
  15386	CDS	1796	304	GUCUCUCAUCU	81%
  				GCAAUAACGUG
  				AAA
  15387	CDS	1856	305	AGAGCUCAUUA	33%
  				AAACAGUCACC
  				AUA
  15388	CDS	1857	306	GAGCUCAUUAA	55%
  				AACAGUCACCA
  				UCA
  15389	CDS	1858	307	AGCUCAUUAAA	31%
  				ACAGUCACCAU
  				CAA
  15390	CDS	1859	308	GCUCAUUAAAA	46%
  				CAGUCACCAUC
  				AAA
  15391	CDS	1860	309	CUCAUUAAAAC	43%
  				AGUCACCAUCA
  				AUA
  15392	CDS	1861	310	UCAUUAAAACA	58%
  				GUCACCAUCAA
  				UGA
  15393	CDS	1862	311	CAUUAAAACAG	78%
  				UCACCAUCAAU
  				GCA
  15394	CDS	1864	312	UUAAAACAGUC	41%
  				ACCAUCAAUGC
  				AAA
  15395	CDS	1865	313	UAAAACAGUCA	80%
  				CCAUCAAUGCA
  				AGA
  15396	CDS	1866	314	AAAACAGUCAC	79%
  				CAUCAAUGCAA
  				GUA
  15397	CDS	1868	315	AACAGUCACCA	34%
  				UCAAUGCAAGU
  				UCA
  15398	CDS	1912	316	AUAUCAAUCCC	39%
  				ACAGUACUACU
  				AAA
  15399	CDS	1928	317	ACUACUAAAAG	39%
  				AACGUUCGACU
  				GAA
  15400	CDS/	1941	318	CGUUCGACUGA	30%
  	3UTR			ACUGUAGAAGU
  				CUA
  15401	CDS/	1946	319	GACUGAACUGU	25%
  	3UTR			AGAAGUCUAAU
  				GAA
  15402	CDS/	1949	320	UGAACUGUAGA	29%
  	3UTR			AGUCUAAUGAU
  				CAA
  15403	3UTR	2077	321	UCCUGUUGCGG	45%
  				AGAAAGGAGUC
  				AUA
  15404	3UTR	2082	322	UUGCGGAGAAA	43%
  				GGAGUCAUACU
  				UGA
  15405	3UTR	2098	323	CAUACUUGUGA	30%
  				AGACUUUUAUG
  				UCA
  15406	3UTR	2128	324	CUAAAGAUUUU	41%
  				GCUGUUGCUG
  				UUAA
  15407	3UTR	2141	325	UGUUGCUGUUA	29%
  				AGUUUGGAAAA
  				CAA
  1540S	3UTR	2188	326	AGAGAGAAAUG	26%
  				AGUUUUGACGU
  				CUA
  15409	3UTR	2235	327	UUAUAAGAACG	33%
  				AAAGUAAAGAU
  				GUA
  15410	3UTR	2281	328	AAGAUGGCAAA	28%
  				AUGCUGAAAGU
  				UUA
  15411	3UTR	2305	329	UUACACUGUCG	46%
  				AUGUUUCCAAU
  				GCA
  15412	3UTR	2446	330	GACAUUACCAG	24%
  				UAAUUUCAUGU
  				CUA
  15413	3UTR	2581	331	CAAAAAGAAGC	36%
  				UGUCUUGGAUU
  				UAA
  15414	3UTR	2669	332	CUUUUUCACCA	41%
  				AGAGUAUAAAC
  				CUA
  15415	3UTR	2730	333	AUGCCAAAUUU	61%
  				AUUAAGGUGGU
  				GGA
  15416	3UTR	2750	334	GUGGAGCCACU	39%
  				GCAGUGUUAUC
  				UUA
  15417	3UTR	2752	335	GGAGCCACUGC	45%
  				AGUGUUAUCUU
  				AAA
  15418	3UTR	2802	336	CAGAAUUUGUU	49%
  				UAUAUGGCUGG
  				UAA
  15419	3UTR	2810	337	GUUUAUAUGGC	34%
  				UGGUAACAUGU
  				AAA
  15420	3UTR	2963	338	UACUCAGAUUU	42%
  				UGCUAUGAGGU
  				UAA
  15421	3U7R	2967	339	CAGAUUUUGCU	39%
  				AUGAGGUUAAU
  				GAA
  15422	3UTR	2970	340	AUUUUGCUAU	43%
  				GAGGUUAAUG
  				AAGUA
  15423	3UTR	2986	341	AAUGAAGUAC	40%
  				CAAGCUGUGC
  				UUGAA
  15424	3UTR	3064	342	AUCACAUUGC	59%
  				AAAAGUAGCA
  				AUGAA
  15425	3UTR	3072	343	GCAAAAGUAG	35%
  				CAAUGACCUC
  				AUAAA
  15426	3UTR	3083	344	AAUGACCUCA	40%
  				UAAAAUACCU
  				CUUCA
  15427	3UTR	3134	345	AAUUUUAUCU	55%
  				CAGUCUUGAA
  				GCCAA
  15428	3UTR	3147	346	UCUUGAAGCC	52%
  				AAUUCAGUAG
  				GUGCA
  15429	3UTR	3157	347	AAUUCAGUAG	71%
  				GUGCAUUGGA
  				AUCAA
  15430	3UTR	3212	348	UUUCUUCUUU	38%
  				UAGCCAUUUU
  				GCUAA
  15431	3UTR	3216	349	UUCUUUUAGC	40%
  				CAUUUUGCUA
  				AGAGA
  15432	3UTR	3225	350	CCAUUUUGCU	36%
  				AAGAGACACA
  				GUCUA
  15433	3U7R	3278	351	UUACUAGUUU	70%
  				UAAGAUCAGA
  				GUUCA
  15434	3UTR	3313	352	ACUCUGCCUA	56%
  				UAUUUUCUUA
  				CCUGA
  15435	3UTR	3335	353	UGAACUUUUG	64%
  				CAAGUUUUCA
  				GGUAA
  15436	3UTR	3336	354	GAACUUUUGC	62%
  				AAGUUUUCAG
  				GUAAA
  15437	3UTR	3351	355	UUCAGGUAAA	62%
  				CCUCAGCUCA
  				GGACA
  15438	3UTR	3360	356	ACCUCAGCUC	53%
  				AGGACUGCUA
  				UUUAA
  15439	3UTR	3441	357	CUUAUUUUAA	83%
  				GUGAAAAGCA
  				GAGAA
  15440	3UTR	3489	358	UAUCUGUAAC	93%
  				CAAGAUGGAU
  				GCAAA
  15441	3UTR	3662	359	UUUUCCACAU	36%
  				CUCAUUGUCA
  				CUGAA
  15442	3UTR	3668	360	ACAUCUCAUU	40%
  				GUCACUGACA
  				UUUAA
  15443	3UTR	3735	361	GUCUUAUUAG	40%
  				GACACUAUGG
  				UUAUA
  15444	3UTR	3737	362	CUUAUUAGGA	37%
  				CACUAUGGUU
  				AUAAA
  15445	3U7R	3738	363	UUAUUAGGAC	38%
  				ACUAUGGUUA
  				UAAAA
  15446	3UTR	3752	364	UGGUUAUAAA	28%
  				CUGUGUUUAA
  				GCCUA
  15447	3U7R	3913	365	AUAUUUAAGG	40%
  				UUGAAUGUUU
  				GUCCA
  15448	3UTR	3961	366	CUAGCCCACA	47%
  				AAGAAUAUUG
  				UCUCA
  15449	3UTR	3981	367	UCUCAUUAGC	56%
  				CUGAAUGUGC
  				CAUAA
  15450	3UTR	3994	368	AAUGUGCCAU	52%
  				AAGACUGACC
  				UUUUA

• TABLE 3
  PTGS2 sd-rxRNA

  Duplex ID	ID	Sequence	SEQ ID NO

  17388	17062	G.A.A.A.A.mC.mU.G.mC.mU.mC.A.A.Chl	369
  	17063	P.mU.fU.G.A.G.fC.A.G.fU.fU.fU.fU.fC*fU*fC*fC*A*fU*A	370
  17389	17064	A.mC.mC.mU.mC.mU.mC.mC.mU.A.mU.mU.A.Chl	371
  	17065	P.mU.A.A.fU.A.G.G.A.G.A.G.G.fU*fU*A*G*A*G*A	372
  17390	17066	mU.mC.mC.A.mC.mC.A.A.mC.mU.mU.A.A.Chl	373
  	17068	P.mU.fU.A.A.G.fU.fU.G.G.fU.G.G.A*fC*fU*G*fU*fC*A	374
  17391	17067	G.mU.mC.mC.A.mC.mC.A.A.mC.mU.mU.A.A.Chl	375
  	17068	P.mU.fU.A.A.G.fU.fU.G.G.fU.G.G.A*fC*fU*G*fU*fC*A	376
  17392	17069	mC.mU.mC.mC.mU.A.mU.mU.A.mU.A.mC.A.Chl	377
  	17070	P.mU.G.fU.A.fU.A.A.fU.A.G.G.A.G*A*G*G*fU*fU*A	378
  17393	17071	G.A.mU.mC.A.mC.A.mU.mU.mU.G.A.A.Chl	379
  	17073	P.mU.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*G*G*A*fU*G	380
  17394	17072	A.G.A.mU.mC.A.mC.A.mU.mU.mU.G.A.A.Chl	381
  	17073	P.mU.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*G*G*A*fU*G	382
  17395	17074	A.A.mC.mC.mU.mC.mU.mC.mC.mU.A.mU.A.Chl	383
  	17075	P.mU.A.fU.A.G.G.A.G.A.G.G.fU.fU*A*G*A*G*A*A	384
  17396	17076	G.mU.mU.G.A.mC.A.mU.mC.mC.A.G.A.Chl	385
  	17077	P.mU.fC.fU.G.G.A.fU.G.fU.fC.A.A.fC*A*fC*A*fU*A*A	386
  17397	17078	mC.mC.mU.mU.mC.mC.mU.mU.mC.G.A.A.A.Chl	387
  	17079	P.mU.fU.fU.fC.G.A.A.G.G.A.A.G.G*G*A*A*fU*G*U	388
  17398	17080	A.mC.mU.mC.mC.A.A.A.mC.A.mC.A.A.Chl	389
  	17082	P.mU.fU.G.fU.G.fU.fU.fU.G.G.A.G.fU*G*G*G*fU*fU*U	390
  17399	17081	mC.A.mC.mU.mC.mC.A.A.A.mC.A.mC.A.A.Chl	391
  	17082	P.mU.fU.G.fU.G.fU.fU.fU.G.G.A.G.fU*G*G*G*fU*fU*U	392
  17400	17083	mC.A.mC.mU.mC.mC.A.A.A.mC.A.mC.A.Chl	393
  	17084	P.mUGfUGfUfUfUGGAGfUG*G*G*fU*fU*fU*C	394
  17401	17085	mC.mC.A.mC.mC.A.A.mC.mU.mU.A.mCA.Chl	395
  	17087	P.mUGfUAAGfUfUGGfUGG*A*fC*fU*G*fU*C	396
  17402	17086	mU.mC.mC.A.mC.mC.A.A.mC.mU.mU.A.mCA.Chl	397
  	17087	P.mUGfUAAGfUfUGGfUGG*A*fC*fU*G*fU*C	398
  17403	17088	A.A.mU.A.mC.mC.A.G.mU.mC.mU.mU.A.Chl	399
  	17089	P.mU.A.A.G.A.fC.fU.G.G.fU.A.fU.fU*fU*fC*A*fU*fC*U	400
  17404	17090	G.A.mC.mC.A.G.mU.A.mU.A.A.G.A.Chl	401
  	17091	P.mU.fC.fU.fU.A.fU.A.fC.fU.G.G.fU.fC*A*A*A*fU*fC*C	402
  17405	17092	G.mU.mC.mU.mU.mU.mU.A.A.mU.G.A.A.Chl	403
  	17093	P.mU.fU.fC.A.fU.fU.A.A.A.A.G.A.fC*fU*G*G*fU*A*U	404
  17406	17094	A.A.mU.mU.mU.mC.A.mU.G.mU.mC.mU.A.Chl	405
  	17095	P.mU.A.G.A.fC.A.fU.G.A.A.A.fU.fU*A*fC*fU*G*G*U	406
  17407	17096	A.mU.mC.A.mC.A.mU.mU.mU.G.A.mU.A.Chl	407
  	17098	P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU*fC*fU*G*G*A*U	408
  17408	17097	G.A.mU.mC.A.mC.A.mU.mU.mU.G.A.mU.A.Chl	409
  	17098	P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU*fC*fU*G*G*A*U	410
  17409	17099	mU.mC.mC.A.G.A.mU.mC.A.mC.A.mU.A.Chl	411
  	17100	P.mU.A.fU.G.fU.G.A.fU.fC.fU.G.G.A*fU*G*fU*fC*A*A	412
  17410	17101	mU.A.mC.mU.G.A.mU.A.G.G.A.G.A.Chl	413
  	17102	P.mU.fC.fU.fC.fC.fU.A.fU.fC.A.G.fU.A*fU*fU*A*G*fC*C	414
  17411	17103	G.mU.G.mC.A.A.mC.A.mC.mU.fU.G.A.Chl	415
  	17104	P.mU.fC.A.A.G.fU.G.fU.fU.G.mC.A.fC*A*fU*A*A*fU*C	416
  17412	17105	A.mC.mC.A.G.mU.A.mU.A.A.G.mU.A.Chl	417
  	17106	P.mU.A.fC.fU.fU.A.fU.A.fC.fU.G.G.fU*fC*A*A*A*fU*C	418
  17413	17107	G.A.A.G.mU.mC.mU.A.A.mU.G.A.A.Chl	419
  	17108	P.mU.fU.fC.A.fU.fU.A.G.A.fC.mU.fU.fC*fU*A*fC*A*G*U	420
  17414	17109	A.A.G.A.A.G.A.A.A.G.mU.mU.A.Chl	421
  	17110	P.mU.A.A.fC.fU.fU.fU.fC.fU.fU.fC.fU.fU*A*G*A*A*G*C	422
  17415	17111	mU.mC.A.mC.A.mU.mU.mU.G.AmU.mU.A.Chl	423
  	17113	P.mU.A.A.fU.fC.A.A.A.fU.G.fUG.A*fU*fC*fU*G*G*A	424
  17416	17112	A.mU.mC.A.mC.A.mU.mU.mU.G.AmU.mU.A.Chl	425
  	17113	P.mU.A.A.fU.fC.A.A.A.fU.G.fUG.A*fU*fC*fU*G*G*A	426
  17417	17114	A.mC.A.mU.mU.mU.G.A.mU.mUG.A.A.Chl	427
  	17116	P.mU.fU.fC.A.A.fU.fC.A.A.A.fUG.fU*G*A*fU*fC*fU*G	428
  17418	17115	mC.A.mC.A.mU.mU.mU.G.A.mU.mUG.A.A.Chl	429
  	17116	P.mU.fU.fC.A.A.fU.fC.A.A.A.fUG.fU*G*A*fU*fC*fU*G	430
  17419	17117	A.mU.mU.mU.G.A.mU.mU.G.AmC.A.A.Chl	431
  	17119	P.mU.fU.G.fU.fC.A.A.fU.fC.A.AA.fU*G*fU*G*A*fU*C	432
  17420	17118	mC.A.mU.mU.mU.G.A.mU.mU.G.AmC.A.A.Chl	433
  	17119	P.mU.fU.G.fU.fC.A.A.fU.fC.A.AA.fU*G*fU*G*A*fU*C	434
  17421	17120	mC.A.mU.mC.mU.G.mC.A.A.mU.A.A.A.Chl	435
  	17122	P.mU.fU.fU.A.fU.fU.G.fC.A.G.A.fU.G*A*G*A*G*A*C	436
  17422	17121	mU.mC.A.mU.mC.mU.G.mC.A.A.mU.A.A.A.Chl	437
  	17122	P.mU.fU.fU.A.fU.fU.G.fC.A.G.A.fU.G*A*G*A*G*A*C	438

• TABLE 4
  TGFB1 sd-rxRNA

  	hTGFB1 Single		SEQ ID
  	Strand ID	sd-rxRNA sequence	NO

  Target Gene
  Duplex ID
  18454	17491	mC.A.mC.A.G.mC.A.mU.A.mU.A.mU.A.Chl	439
  	17492	P.mU.A.fU.A.fU.A.fU.G.fC.fU.G.fU.G*fU*G*fU*A*fC*U	440
  18455	17493	mC.A.G.mC.A.mU.A.mU.A.mU.A.mU.A.Chl	441
  	17494	P.mU.A.fU.A.fU.A.fU.A.fU.G.fC.fU.G*fU*G*fU*G*fU*A	442
  18456	17495	G.mU.A.mC.A.mU.mU.G.A.mC.mU.mU.A.Chl	443
  	17497	P.mU.A.A.G.fU.fC.A.A.fU.G.fU.A.fC*A*G*fC*fU*G*C	444
  18457	17496	mU.G.mU.A.mC.A.mU.mU.G.A.mC.mU.mU.A.Chl	445
  	17497	P.mU.A.A.G.fU.fC.A.A.fU.G.fU.A.fC*A*G*fC*fU*G*C	446
  18458	17498	A.A.mC.mU.A.mU.mU.G.mC.mU.mU.mC.A.Chl	447
  	17500	P.mU.G.A.A.G.fC.A.A.fU.A.G.fU.fU*G*G*fU*G*fU*C	448
  18459	17499	mC.A.A.mC.mU.A.mU.mU.G.mC.mU.mU.mC.A.Chl	449
  	17500	P.mU.G.A.A.G.fC.A.A.fU.A.G.fU.fU*G*G*fU*G*fU*C	450
  18460	17501	G.mC.A.mU.A.mU.A.mU.A.mU.G.mU.A.Chl	451
  	17502	P.mU.A.fC.A.fU.A.fU.A.fU.A.fU.G.fC*fU*G*fU*G*fU*G	452
  18461	17503	mU.G.mU.A.mC.A.mU.mU.G.A.mC.mU.A.Chl	453
  	17505	P.mU.A.G.fU.fC.A.A.fU.G.fU.A.fC.A*G*fC*fU*G*fC*C	454
  18462	17504	mC.mU.G.mU.A.mC.A.mU.mU.G.A.mC.mU.A.Chl	455
  	17505	P.mU.A.G.fU.fC.A.A.fU.G.fU.A.fC.A*G*fC*fU*G*fC*C	456
  18463	17506	A.G.mC.A.mU.A.mU.A.mU.A.mU.G.A.Chl	457
  	17507	P.mU.fC.A.fU.A.fU.A.fU.A.fU.G.fC.fU*G*fU*G*fU*G*U	458
  18464	17508	mC.A.G.mC.A.A.mC.A.A.mU.mU.mC.A.Chl	459
  	17509	P.mU.G.A.A.fU.fU.G.fU.fU.G.fC.fU.G*fU*A*fU*fU*fU*C	460
  18465	17510	mC.A.mU.A.mU.A.mU.A.mU.G.mU.mU.A.Chl	461
  	17511	P.mU.A.A.fC.A.fU.A.fU.A.fU.A.fU.G*fC*fU*G*fU*G*U	462
  18466	17512	mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.mC.A.Chl	463
  	17514	P.mU.G.A.G.fC.fU.G.A.A.G.fC.A.A*fU*A*G*fU*fU*G	464
  18467	17513	A.mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.mC.A.Chl	465
  	17514	P.mU.G.A.G.fC.fU.G.A.A.G.fC.A.A*fU*A*G*fU*fU*G	466
  18468	17515	A.mC.A.G.mC.A.mU.A.mU.A.mU.A.A.Chl	467
  	17516	P.mU.fU.A.fU.A.fU.A.fU.G.fC.fU.G.fU*G*fU*G*fU*A*C	468
  18469	17517	A.mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.A.Chl	469
  	17519	P.mU.A.G.fC.fU.G.A.A.G.fC.A.A.fU*A*G*fU*fU*G*G	470
  18470	17518	mU.A.mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.A.Chl	471
  	17519	P.mU.A.G.fC.fU.G.A.A.G.fC.A.A.fU*A*G*fU*fU*G*G	472
  18471	17520	mC.A.G.A.G.mU.A.mC.A.mC.A.mC.A.Chl	473
  	17521	P.mU.G.fU.G.fU.G.fU.A.fC.fU.fC.fU.G*C*fU*fU*G*A*A	474
  18472	17522	mU.mC.A.A.G.mC.A.G.A.G.mU.A.A.Chl	475
  	17523	P.mU.fU.A.fC.fU.fC.fU.G.fC.fU.fU.G.A*A*fC*fU*fU*G*U	476
  18473	17524	A.G.mC.A.G.A.G.mU.A.mC.A.mC.A.Chl	477
  	17525	P.mU.G.fU.G.fU.A.fC.fU.fC.fU.G.fC.fU*fU*G*A*A*fC*U	478
  18474	17526	G.A.mC.A.A.G.mU.mU.mC.A.A.G.A.Chl	479
  	17527	P.mU.fC.fU.fU.G.A.A.fC.fU.fU.G.fU.fC*A*fU*A*G*A*U	480
  18475	17528	mC.mU.A.mU.G.A.mC.A.A.G.mU.mU.A.Chl	481
  	17529	P.mU.A.A.fC.fU.fU.G.fU.fC.A.fU.A.G*A*fU*fU*fU*fC*G	482
  18476	17530	G.mC.A.G.A.G.mU.A.mC.A.mC.A.A.Chl	483
  	17531	P.mU.fU.G.fU.G.fU.A.fC.fU.fC.fU.G.fC*fU*fU*G*A*A*C	484
  18477	17532	mU.G.A.mC.A.A.G.mU.mU.mCA.A.A.Chl	485
  	17533	P.mU.fU.fU.G.A.A.fC.fU.fU.GfU.fC.A*fU*A*G*A*fU*U	486
  18478	17534	mU.A.mC.A.mC.A.mC.A.G.mCA.mU.A.Chl	487
  	17535	P.mU.A.fU.G.fC.fU.G.fU.G.fUG.fU.A*fC*fU*fC*fU*G*C	488
  18479	17536	A.A.mC.G.A.A.A.mU.mC.mUA.mU.A.Chl	489
  	17537	P.mU.A.fU.A.G.A.fU.fU.fU.fCG.fU.fU*G*fU*G*G*G*U	490
  18480	17538	mU.mU.G.A.mC.mU.mU.mC.mC.GmC.A.A.Chl	491
  	17539	P.mU.fU.G.fC.G.G.A.A.G.fUfC.A.A*fU*G*fU*A*fC*A	492
  18481	17540	A.mC.A.A.mC.G.A.A.A.mUmC.mU.A.Chl	493
  	17541	P.mU.A.G.A.fU.fU.fU.fC.G.fUfU.G.fU*G*G*G*fU*fU*U	494
  18482	17542	mU.mC.A.A.mC.A.mC.A.mU.mCA.G.A.Chl	495
  	17543	P.mU.fC.fU.G.A.fU.G.fU.G.fUfU.G.A*A*G*A*A*fC*A	496
  18483	17544	A.mC.A.A.G.mU.mU.mC.A.AG.mC.A.Chl	497
  	17545	P.mU.G.fC.fU.fU.G.A.A.fC.fUfU.G.fU*fC*A*fU*A*G*A	498
  18484	17546	A.mU.mC.mU.A.mU.G.A.mC.AA.G.A.Chl	499
  	17547	P.mU.fC.fU.fU.G.fU.fC.A.fU.AG.A.fU*fU*fU*fC*G*fU*U	500
  Rat Targeting
  TGFB1
  18715	18691	G.A.A.A.mU.A.mU.A.G.mC.A.A.A-chol	503
  	18692	P.mU.fU.fU.G.fC.fU.A.fU.A.fU.fU.fU.fC*fU*G*G*fU*A*G	504
  18716	18693	G.A.A.mC.mU.mC.mU.A.mC.mC.A.G.A-chol	505
  	18694	P.mU.fC.fU.G.G.fU.A.G.A.G.fU.fU.fC*fU*A*fC*G*fU*G	506
  18717	18695	G.mC.A.A.A.G.A.mU.A.A.mU.G.A-chol	507
  	18696	P.mU.fC.A.fU.fU.A.fU.fC.fU.fU.fU.G.fC*fU*G*fU*fC*A*C	508
  18718	18697	A.A.mC.mU.mC.mU.A.mC.mC.A.G.A.A-chol	509
  	18698	P.mU.fU.fC.fU.G.G.fU.A.G.A.G.fU.fU*fC*fU*A*fC*G*U	510
  18719	18699	A.mC.mU.mC.mU.A.mC.mC.A.G.A.A.A-chol	511
  	18700	P.mU.fU.fU.fC.fU.G.G.fU.A.G.A.G.fU*fU*fC*fU*A*fC*G	512
  18720	18701	A.mC.A.G.mC.A.A.A.G.A.mU.A.A-chol	513
  	18702	P.mU.fU.A.fU.fC.fU.fU.fU.G.fC.fU.G.fU*fC*A*fC*A*A*G	514
  18721	18703	mC.A.A.mU.mC.mU.A.mU.G.A.mC.A.A-chol	515
  	18704	P.mU.fU.G.fU.fC.A.fU.A.G.A.fU.fU.G*fC*G*fU*fU*G*U	516
  18722	18705	A.G.A.mU.mU.mC.A.A.G.mU.mC.A.A-chol	517
  	18706	P.mU.fU.G.A.fC.fU.fU.G.A.A.fU.fC.fU*fC*fU*G*fC*A*G	518
  18723	18707	mC.mU.G.mU.G.G.A.G.mC.A.A.mC.A-chol	519
  	18708	P.mU.G.fU.fU.G.fC.fU.fC.fC.A.fC.A.G*fU*fU*G*A*fC*U	520
  18724	18709	mU.G.A.mC.A.G.mC.A.A.A.G.A.A-chol	521
  	18710	P.mU.fU.fC.fU.fU.fU.G.fC.fU.G.fU.fC.A*fC*A*A*G*A*G	522
  18725	18711	A.mU.G.A.mC.A.A.A.A.mC.mC.A.A-chol	523
  	18712	P.mU.fU.G.G.fU.fU.fU.fU.G.fU.fC.A.fU*A*G*A*fU*fU*G	524
  18726	18713	G.A.G.A.mU.mU.mC.A.A.G.mU.mC.A-chol	525
  	18714	P.mU.G.A.fC.fU.fU.G.A.A.fU.fC.fU.fC*fU*G*fC*A*G*G	526

• TABLE 5
  Inhibition of gene expression with hTGFB1 ori sequences

  Target			SEQ		% Expression
  Gene	Gene	Ref	ID	hTGFB1	0.025 nM HeLa
  Duplex ID	Region	Pos	NO	Sense Sequence	cells
  15732	CDS	954	527	CGCGGGACUAUCCACCUGCAAGACA	57.3%
  15733	CDS	956	528	CGGGACUAUCCACCUGCAAGACUAA	38.2%
  15734	CDS	957	529	GGGACUAUCCACCUGCAAGACUAUA	49.1%
  15735	CDS	961	530	CUAUCCACCUGCAAGACUAUCGACA	34.9%
  15736	CDS	962	531	UAUCCACCUGCAAGACUAUCGACAA	39.4%
  15737	CDS	964	532	UCCACCUGCAAGACUAUCGACAUGA	44.4%
  15738	CDS	965	533	CCACCUGCAAGACUAUCGACAUGGA	53.3%
  15739	CDS	966	534	CACCUGCAAGACUAUCGACAUGGAA	52.8%
  15740	CDS	967	535	ACCUGCAAGACUAUCGACAUGGAGA	46.2%
  15741	CDS	968	536	CCUGCAAGACUAUCGACAUGGAGCA	48.1%
  15742	CDS	1209	537	AAUGGUGGAAACCCACAACGAAAUA	36.7%
  15743	CDS	1210	538	AUGGUGGAAACCCACAACGAAAUCA	28.8%
  15744	CDS	1211	539	UGGUGGAAACCCACAACGAAAUCUA	23.1%
  15745	CDS	1212	540	GGUGGAAACCCACAACGAAAUCUAA	13.2%
  15746	CDS	1213	541	GUGGAAACCCACAACGAAAUCUAUA	21.1%
  15747	CDS	1214	542	UGGAAACCCACAACGAAAUCUAUGA	28.7%
  15748	CDS	1215	543	GGAAACCCACAACGAAAUCUAUGAA	32.9%
  15749	CDS	1216	544	GAAACCCACAACGAAAUCUAUGACA	41.5%
  15750	CDS	1217	545	AAACCCACAACGAAAUCUAUGACAA	29.9%
  15751	CDS	1218	546	AACCCACAACGAAAUCUAUGACAAA	16.4%
  15752	CDS	1219	547	ACCCACAACGAAAUCUAUGACAAGA	23.3%
  15753	CDS	1220	548	CCCACAACGAAAUCUAUGACAAGUA	37.5%
  15754	CDS	1221	549	CCACAACGAAAUCUAUGACAAGUUA	19.1%
  15755	CDS	1222	550	CACAACGAAAUCUAUGACAAGUUCA	14.4%
  15756	CDS	1224	551	CAACGAAAUCUAUGACAAGUUCAAA	20.1%
  15757	CDS	1225	552	AACGAAAUCUAUGACAAGUUCAAGA	18.3%
  15758	CDS	1226	S53	ACGAAAUCUAUGACAAGUUCAAGCA	23.2%
  15759	CDS	1227	554	CGAAAUCUAUGACAAGUUCAAGCAA	29.0%
  15760	CDS	1228	555	GAAAUCUAUGACAAGUUCAAGCAGA	15.6%
  15761	CDS	1229	556	AAAUCUAUGACAAGUUCAAGCAGAA	32.3%
  15762	CDS	1230	557	AAUCUAUGACAAGUUCAAGCAGAGA	36.1%
  15763	CDS	1231	558	AUCUAUGACAAGUUCAAGCAGAGUA	30.6%
  15764	CDS	1232	559	UCUAUGACAAGUUCAAGCAGAGUAA	24.9%
  15765	CDS	1233	560	CUAUGACAAGUUCAAGCAGAGUACA	15.9%
  15766	CDS	1234	561	UAUGACAAGUUCAAGCAGAGUACAA	31.2%
  15767	CDS	1235	562	AUGACAAGUUCAAGCAGAGUACACA	17.2%
  15768	CDS	1236	563	UGACAAGUUCAAGCAGAGUACACAA	23.5%
  15769	CDS	1237	564	GACAAGUUCAAGCAGAGUACACACA	24.5%
  15770	CDS	1238	565	ACAAGUUCAAGCAGAGUACACACAA	38.5%
  15771	CDS	1240	566	AAGUUCAAGCAGAGUACACACAGCA	38.7%
  15772	CDS	1241	567	AGUUCAAGCAGAGUACACACAGCAA	34.3%
  15773	CDS	1242	568	GUUCAAGCAGAGUACACACAGCAUA	20.8%
  15774	CDS	1243	569	UUCAAGCAGAGUACACACAGCAUAA	33.4%
  15775	CDS	1244	570	UCAAGCAGAGUACACACAGCAUAUA	19.6%
  15776	CDS	1245	571	CAAGCAGAGUACACACAGCAUAUAA	25.5%
  15777	CDS	1246	572	AAGCAGAGUACACACAGCAUAUAUA	12.8%
  15778	CDS	1247	573	AGCAGAGUACACACAGCAUAUAUAA	27.6%
  15779	CDS	1248	574	GCAGAGUACACACAGCAUAUAUAUA	15.9%
  15780	CDS	1249	575	CAGAGUACACACAGCAUAUAUAUGA	24.1%
  15781	CDS	1250	576	AGAGUACACACAGCAUAUAUAUGUA	22.6%
  15782	CDS	1251	577	GAGUACACACAGCAUAUAUAUGUUA	26.7%
  15783	CDS	1252	578	AGUACACACAGCAUAUAUAUGUUCA	66.6%
  15784	CDS	1254	579	UACACACAGCAUAUAUAUGUUCUUA	33.6%
  15785	CDS	1262	580	GCAUAUAUAUGUUCUUCAACACAUA	40.4%
  15786	CDS	1263	581	CAUAUAUAUGUUCUUCAACACAUCA	42.5%
  15787	CDS	1264	582	AUAUAUAUGUUCUUCAACACAUCAA	27.2%
  15788	CDS	1265	583	UAUAUAUGUUCUUCAACACAUCAGA	23.2%
  15789	CDS	1266	584	AUAUAUGUUCUUCAACACAUCAGAA	35.5%
  15790	CDS	1267	585	UAUAUGUUCUUCAACACAUCAGAGA	34.6%
  15791	CDS	1268	586	AUAUGUUCUUCAACACAUCAGAGCA	29.7%
  15792	CDS	1269	587	UAUGUUCUUCAACACAUCAGAGCUA	35.4%
  15793	CDS	1270	588	AUGUUCUUCAACACAUCAGAGCUCA	35.2%
  15794	CDS	1335	589	GCUGCGUCUGCUGAGGCUCAAGUUA	28.0%
  15795	CDS	1336	590	CUGCGUCUGCUGAGGCUCAAGUUAA	37.1%
  15796	CDS	1337	591	UGCGUCUGCUGAGGCUCAAGUUAAA	25.5%
  15797	CDS	1338	592	GCGUCUGCUGAGGCUCAAGUUAAAA	59.7%
  15798	CDS	1339	593	CGUCUGCUGAGGCUCAAGUUAAAAA	52.8%
  15799	CDS	1340	594	GUCUGCUGAGGCUCAAGUUAAAAGA	47.9%
  15800	CDS	1341	595	UCUGCUGAGGCUCAAGUUAAAAGUA	49.8%
  15801	CDS	1342	596	CUGCUGAGGCUCAAGUUAAAAGUGA	50.7%
  15802	CDS	1343	597	UGCUGAGGCUCAAGUUAAAAGUGG	43.4%
  				A
  15803	CDS	1344	598	GCUGAGGCUCAAGUUAAAAGUGGAA	52.6%
  15804	CDS	1345	599	CUGAGGCUCAAGUUAAAAGUGGAGA	73.3%
  15805	CDS	1346	600	UGAGGCUCAAGUUAAAAGUGGAGCA	58.0%
  15806	CDS	1347	601	GAGGCUCAAGUUAAAAGUGGAGCAA	64.9%
  15807	CDS	1348	602	AGGCUCAAGUUAAAAGUGGAGCAGA	68.1%
  15808	CDS	1349	603	GGCUCAAGUUAAAAGUGGAGCAGCA	73.8%
  15809	CDS	1350	604	GCUCAAGUUAAAAGUGGAGCAGCAA	78.8%
  15810	CDS	1351	605	CUCAAGUUAAAAGUGGAGCAGCACA	76.6%
  15811	CDS	1352	606	UCAAGUUAAAAGUGGAGCAGCACGA	72.9%
  15813	CDS	1370	608	AGCACGUGGAGCUGUACCAGAAAUA	69.7%
  15814	CDS	1371	609	GCACGUGGAGCUGUACCAGAAAUAA	73.3%
  15815	CDS	1372	610	CACGUGGAGCUGUACCAGAAAUACA	55.0%
  15816	CDS	1373	611	ACGUGGAGCUGUACCAGAAAUACAA	63.8%
  15817	CDS	1374	612	CGUGGAGCUGUACCAGAAAUACAGA	85.7%
  15818	CDS	1375	613	GUGGAGCUGUACCAGAAAUACAGCA	85.0%
  15819	CDS	1376	614	UGGAGCUGUACCAGAAAUACAGCAA	82.5%
  15820	CDS	1377	615	GGAGCUGUACCAGAAAUACAGCAAA	43.1%
  15821	CDS	1378	616	GAGCUGUACCAGAAAUACAGCAACA	58.5%
  15822	CDS	1379	617	AGCUGUACCAGAAAUACAGCAACAA	48.1%
  15823	CDS	1380	618	GCUGUACCAGAAAUACAGCAACAAA	48.1%
  15824	CDS	1381	619	CUGUACCAGAAAUACAGCAACAAUA	35.0%
  15825	CDS	1382	620	UGUACCAGAAAUACAGCAACAAUUA	36.4%
  15826	CDS	1383	621	GUACCAGAAAUACAGCAACAAUUCA	24.6%
  15827	CDS	1384	622	UACCAGAAAUACAGCAACAAUUCCA	33.4%
  15828	CDS	1385	623	ACCAGAAAUACAGCAACAAUUCCUA	121.5%
  15829	CDS	1386	624	CCAGAAAUACAGCAACAAUUCCUGA	62.1%
  15830	CDS	1387	625	CAGAAAUACAGCAACAAUUCCUGGA	98.3%
  15831	CDS	1390	626	AAAUACAGCAACAAUUCCUGGCGAA	36.6%
  15832	CDS	1391	627	AAUACAGCAACAAUUCCUGGCGAUA	39.5%
  15833	CDS	1392	628	AUACAGCAACAAUUCCUGGCGAUAA	40.0%
  15834	CDS	1393	629	UACAGCAACAAUUCCUGGCGAUACA	89.4%
  15835	CDS	1394	630	ACAGCAACAAUUCCUGGCGAUACCA	62.3%
  15836	CDS	1396	631	AGCAACAAUUCCUGGCGAUACCUCA	41.0%
  15837	CDS	1441	632	AGCGACUCGCCAGAGUGGUUAUCUA	31.2%
  15838	CDS	1442	633	GCGACUCGCCAGAGUGGUUAUCUUA	46.2%
  15839	CDS	1443	634	CGACUCGCCAGAGUGGUUAUCUUUA	46.8%
  15840	CDS	1444	635	GACUCGCCAGAGUGGUUAUCUUUUA	50.6%
  15841	CDS	1445	636	ACUCGCCAGAGUGGUUAUCUUUUGA	50.8%
  15842	CDS	1446	637	CUCGCCAGAGUGGUUAUCUUUUGAA	71.8%
  15843	CDS	1447	638	UCGCCAGAGUGGUUAUCUUUUGAU	43.7%
  				A
  15844	CDS	1448	639	CGCCAGAGUGGUUAUCUUUUGAUG	42.1%
  				A
  15845	CDS	1449	640	GCCAGAGUGGUUAUCUUUUGAUGU	31.0%
  				A
  15846	CDS	1450	641	CCAGAGUGGUUAUCUUUUGAUGUC	46.0%
  				A
  15847	CDS	1451	642	CAGAGUGGUUAUCUUUUGAUGUCA	40.2%
  				A
  15848	CDS	1452	643	AGAGUGGUUAUCUUUUGAUGUCAC	38.5%
  				A
  15849	CDS	1453	644	GAGUGGUUAUCUUUUGAUGUCACC	67.4%
  				A
  15850	CDS	1454	645	AGUGGUUAUCUUUUGAUGUCACCG	57.4%
  				A
  15851	CDS	1455	646	GUGGUUAUCUUUUGAUGUCACCGG	40.6%
  				A
  15852	CDS	1456	647	UGGUUAUCUUUUGAUGUCACCGGA	70.5%
  				A
  15853	CDS	1457	648	GGUUAUCUUUUGAUGUCACCGGAG	82.8%
  				A
  15854	CDS	1458	649	GUUAUCUUUUGAUGUCACCGGAGU	74.8%
  				A
  15855	CDS	1459	650	UUAUCUUUUGAUGUCACCGGAGUU	86.8%
  				A
  15856	CDS	1460	651	UAUCUUUUGAUGUCACCGGAGUUG	76.5%
  				A
  15857	CDS	1551	652	CAGCAGGGAUAACACACUGCAAGUA	70.5%
  15858	CDS	1552	653	AGCAGGGAUAACACACUGCAAGUGA	60.5%
  15859	CDS	1553	654	GCAGGGAUAACACACUGCAAGUGGA	43.5%
  15860	CDS	1554	655	CAGGGAUAACACACUGCAAGUGGAA	56.3%
  15861	CDS	1555	656	AGGGAUAACACACUGCAAGUGGACA	63.9%
  15862	CDS	1556	657	GGGAUAACACACUGCAAGUGGACAA	66.9%
  15863	CDS	1558	658	GAUAACACACUGCAAGUGGACAUCA	62.2%
  15864	CDS	1559	659	AUAACACACUGCAAGUGGACAUCAA	40.5%
  15865	CDS	1560	660	UAACACACUGCAAGUGGACAUCAAA	57.9%
  15866	CDS	1610	661	ACCUGGCCACCAUUCAUGGCAUGAA	69.4%
  15867	CDS	1611	662	CCUGGCCACCAUUCAUGGCAUGAAA	49.1%
  15868	CDS	1612	663	CUGGCCACCAUUCAUGGCAUGAACA	31.9%
  15869	CDS	1705	664	CGAGCCCUGGACACCAACUAUUGCA	56.4%
  15870	CDS	1706	665	GAGCCCUGGACACCAACUAUUGCUA	42.6%
  15871	CDS	1707	666	AGCCCUGGACACCAACUAUUGCUUA	29.8%
  15872	CDS	1708	667	GCCCUGGACACCAACUAUUGCUUCA	19.8%
  15873	CDS	1709	668	CCCUGGACACCAACUAUUGCUUCAA	37.7%
  15874	CDS	1710	669	CCUGGACACCAACUAUUGCUUCAGA	44.0%
  15875	CDS	1711	670	CUGGACACCAACUAUUGCUUCAGCA	35.8%
  15876	CDS	1712	671	UGGACACCAACUAUUGCUUCAGCUA	31.5%
  15877	CDS	1713	672	GGACACCAACUAUUGCUUCAGCUCA	27.3%
  15878	CDS	1714	673	GACACCAACUAUUGCUUCAGCUCCA	44.7%
  15879	CDS	1715	674	ACACCAACUAUUGCUUCAGCUCCAA	44.9%
  15880	CDS	1754	675	GCGUGCGGCAGCUGUACAUUGACUA	23.9%
  15881	CDS	1755	676	CGUGCGGCAGCUGUACAUUGACUUA	18.3%
  15882	CDS	1756	677	GUGCGGCAGCUGUACAUUGACUUCA	41.2%
  15883	CDS	1757	678	UGCGGCAGCUGUACAUUGACUUCCA	26.4%
  15884	CDS	1759	679	CGGCAGCUGUACAUUGACUUCCGCA	28.0%
  15885	CDS	1760	680	GGCAGCUGUACAUUGACUUCCGCAA	22.8%
  15886	CDS	1761	681	GCAGCUGUACAUUGACUUCCGCAAA	34.1%
  15887	CDS	1762	682	CAGCUGUACAUUGACUUCCGCAAGA	36.3%
  15888	CDS	1763	683	AGCUGUACAUUGACUUCCGCAAGGA	84.1%
  15889	CDS	1849	684	UGCCCCUACAUUUGGAGCCUGGACA	93.0%
  15890	CDS	1889	685	UCCUGGCCCUGUACAACCAGCAUAA	51.7%
  15891	CDS	1890	686	CCUGGCCCUGUACAACCAGCAUAAA	71.9%
  15892	CDS	1891	687	CUGGCCCUGUACAACCAGCAUAACA	36.1%
  15893	CDS	1997	688	AGGUGGAGCAGCUGUCCAACAUGAA	60.9%
  15894	3UTR	2115	689	CAUGGGGGCUGUAUUUAAGGACACA	57.2%
  15895	3UTR	2155	690	CCUGGGGCCCCAUUAAAGAUGGAGA	86.0%
  15896	3UTR	2156	691	CUGGGGCCCCAUUAAAGAUGGAGAA	73.3%
  15897	3UTR	2157	692	UGGGGCCCCAUUAAAGAUGGAGAGA	68.8%
  15898	3UTR	2158	693	GGGGCCCCAUUAAAGAUGGAGAGAA	65.8%
  15899	3UTR	2159	694	GGGCCCCAUUAAAGAUGGAGAGAGA	42.7%
  15900	3UTR	2160	695	GGCCCCAUUAAAGAUGGAGAGAGGA	34.4%
  15901	3UTR	2161	696	GCCCCAUUAAAGAUGGAGAGAGGAA	56.0%
  15902	3UTR	2162	697	CCCCAUUAAAGAUGGAGAGAGGACA	74.9%
  15903	3UTR	2163	698	CCCAUUAAAGAUGGAGAGAGGACUA	79.6%
  15904	3UTR	2180	699	GAGGACUGCGGAUCUCUGUGUCAUA	98.3%
  15905	3UTR	2275	700	CUCCUGCCUGUCUGCACUAUUCCUA	100.2%
  15906	3UTR	2276	701	UCCUGCCUGUCUGCACUAUUCCUUA	103.8%
  15907	3UTR	2277	702	CCUGCCUGUCUGCACUAUUCCUUUA	110.4%
  15908	3UTR	2278	703	CUGCCUGUCUGCACUAUUCCUUUGA	105.2%
  15909	3UTR	2279	704	UGCCUGUCUGCACUAUUCCUUUGCA	118.8%
  15910	3UTR	2325	705	CAGUGGGGAACACUACUGUAGUUAA	112.2%
  15911	3UTR	2326	706	AGUGGGGAACACUACUGUAGUUAG	107.7%
  				A
  15912	3UTR	2327	707	GUGGGGAACACUACUGUAGUUAGA	108.6%
  				A
  15913	3UTR	2328	708	UGGGGAACACUACUGUAGUUAGAU	N/A
  				A

• TABLE 6
  Inhibition of gene expression with hTGFB2 ori sequences

  			%
  			Expression
  Oligo	Gene	Ref	A549 0.1	25-mer Sense Strand (position	SEQ ID
  id	Region	Pos	NM	of SS, replaced with AA)	NO
  15451	5UTR/CDS	651	98%	UUUUAAAAAAUGCACUACUGUGUGC	709
  15452	CDS	654	102.2%	UAAAAAAUGCACUACUGUGUGCUGA	710
  15453	CDS	730	83.7%	GCAGCACACUCGAUAUGGACCAGUU	711
  15454	CDS	732	80.3%	AGCACACUCGAUAUGGACCAGUUCA	712
  15455	CDS	733	79.6%	GCACACUCGAUAUGGACCAGUUCAU	713
  15456	CDS	734	89.1%	CACACUCGAUAUGGACCAGUUCAUG	714
  15457	CDS	735	87.8%	ACACUCGAUAUGGACCAGUUCAUGC	715
  15458	CDS	736	95.3%	CACUCGAUAUGGACCAGUUCAUGCG	716
  15459	CDS	847	103.8%	UCCCCCCGGAGGUGAUUUCCAUCUA	717
  15460	CDS	848	83.6%	CCCCCCGGAGGUGAUUUCCAUCUAC	718
  15461	CDS	851	72.2%	CCCGGAGGUGAUUUCCAUCUACAAC	719
  15462	CDS	853	85.8%	CGGAGGUGAUUUCCAUCUACAACAG	720
  15463	CDS	855	67.1%	GAGGUGAUUUCCAUCUACAACAGCA	721
  15464	CDS	952	68.9%	ACUACGCCAAGGAGGUUUACAAAAU	722
  15465	CDS	963	81.1%	GAGGUUUACAAAAUAGACAUGCCGC	723
  15466	CDS	1107	82.1%	UUCUACAGACCCUACUUCAGAAUUG	724
  15467	CDS	1108	99.1%	UCUACAGACCCUACUUCAGAAUUGU	725
  15468	CDS	1109	95.1%	CUACAGACCCUACUUCAGAAUUGUU	726
  15469	CDS	1129	90.4%	UUGUUCGAUUUGACGUCUCAGCAAU	727
  15470	CDS	1130	76.7%	UGUUCGAUUUGACGUCUCAGCAAUG	728
  15471	CDS	1131	79.7%	GUUCGAUUUGACGUCUCAGCAAUGG	729
  15472	CDS	1132	87.5%	UUCGAUUUGACGUCUCAGCAAUGGA	730
  15473	CDS	1144	66.9%	UCUCAGCAAUGGAGAAGAAUGCUUC	731
  15474	CDS	1145	76.6%	CUCAGCAAUGGAGAAGAAUGCUUCC	732
  15475	CDS	1147	88.9%	CAGCAAUGGAGAAGAAUGCUUCCAA	733
  15476	CDS	1162	84.5%	AUGCUUCCAAUUUGGUGAAAGCAGA	734
  15477	CDS	1163	89.2%	UGCUUCCAAUUUGGUGAAAGCAGAG	735
  15478	CDS	1155	86.6%	CUUCCAAUUUGGUGAAAGCAGAGUU	735
  15479	CDS	1177	61.2%	UGAAAGCAGAGUUCAGAGUCUUUCG	737
  15480	CDS	1185	92.6%	GAGUUCAGAGUCUUUCGUUUGCAGA	738
  15481	CDS	1219	99.6%	CCAGAGUGCCUGAACAACGGAUUGA	739
  15482	CDS	1224	94.0%	GUGCCUGAACAACGGAUUGAGCUAU	740
  15483	CDS	1225	88.1%	UGCCUGAACAACGGAUUGAGCUAUA	741
  15484	CDS	1228	59.3%	CUGAACAACGGAUUGAGCUAUAUCA	742
  15485	CDS	1229	77.5%	UGAACAACGGAUUGAGCUAUAUCAG	743
  15486	CDS	1230	61.5%	GAACAACGGAUUGAGCUAUAUCAGA	744
  15487	CDS	1233	84.5%	CAACGGAUUGAGCUAUAUCAGAUUC	745
  15488	CDS	1238	87.7%	GAUUGAGCUAUAUCAGAUUCUCAAG	746
  15489	CDS	1239	78.7%	AUUGAGCUAUAUCAGAUUCUCAAGU	747
  15490	CDS	1240	94.1%	UUGAGCUAUAUCAGAUUCUCAAGUC	748
  15491	CDS	1247	92.6%	AUAUCAGAUUCUCAAGUCCAAAGAU	749
  15492	CDS	1256	94.3%	UCUCAAGUCCAAAGAUUUAACAUCU	750
  15493	CDS	1259	99.1%	CAAGUCCAAAGAUUUAACAUCUCCA	751
  15494	CDS	1286	87.4%	CCAGCGCUACAUCGACAGCAAAGUU	752
  15495	CDS	1288	84.5%	AGCGCUACAUCGACAGCAAAGUUGU	753
  15496	CDS	1289	60.1%	GCGCUACAUCGACAGCAAAGUUGUG	754
  15497	CDS	1292	78.8%	CUACAUCGACAGCAAAGUUGUGAAA	755
  15498	CDS	1331	80.1%	CGAAUGGCUCUCCUUCGAUGUAACU	756
  15499	CDS	1353	62.4%	ACUGAUGCUGUUCAUGAAUGGCUUC	757
  15500	CDS	1361	74.3%	UGUUCAUGAAUGGCUUCACCAUAAA	758
  15501	CDS	1362	75.1%	GUUCAUGAAUGGCUUCACCAUAAAG	759
  15502	CDS	1363	87.2%	UUCAUGAAUGGCUUCACCAUAAAGA	760
  15503	CDS	1364	70.4%	UCAUGAAUGGCUUCACCAUAAAGAC	761
  15504	CDS	1365	100.7%	CAUGAAUGGCUUCACCAUAAAGACA	762
  15505	CDS	1368	100.1%	GAAUGGCUUCACCAUAAAGACAGGA	763
  15506	CDS	1398	92.0%	GGAUUUAAAAUAAGCUUACACUGUC	764
  15507	CDS	1399	83.2%	GAUUUAAAAUAAGCUUACACUGUCC	765
  15508	CDS	1415	85.6%	ACACUGUCCCUGCUGCACUUUUGUA	766
  15509	CDS	1418	97.4%	CUGUCCCUGCUGCACUUUUGUACCA	767
  15510	CDS	1420	59.1%	GUCCCUGCUGCACUUUUGUACCAUC	768
  15511	CDS	1421	73.7%	UCCCUGCUGCACUUUUGUACCAUCU	769
  15512	CDS	1422	79.5%	CCCUGCUGCACUUUUGUACCAUCUA	770
  15513	CDS	1451	62.7%	UUACAUCAUCCCAAAUAAAAGUGAA	771
  15514	CDS	1452	76.0%	UACAUCAUCCCAAAUAAAAGUGAAG	772
  15515	CDS	1470	44.7%	AGUGAAGAACUAGAAGCAAGAUUUG	773
  15516	CDS	1472	75.6%	UGAAGAACUAGAAGCAAGAUUUGCA	774
  15517	CDS	1474	96.8%	AAGAACUAGAAGCAAGAUUUGCAGG	775
  15518	CDS	1475	94.3%	AGAACUAGAAGCAAGAUUUGCAGGU	776
  15519	CDS	1476	63.3%	GAACUAGAAGCAAGAUUUGCAGGUA	777
  15520	CDS	1480	65.9%	UAGAAGCAAGAUUUGCAGGUAUUGA	778
  15521	CDS	1481	59.6%	AGAAGCAAGAUUUGCAGGUAUUGAU	779
  15522	CDS	1482	56.0%	GAAGCAAGAUUUGCAGGUAUUGAUG	780
  15523	CDS	1483	69.2%	AAGCAAGAUUUGCAGGUAUUGAUGG	781
  15524	CDS	1484	64.5%	AGCAAGAUUUGCAGGUAUUGAUGGC	782
  15525	CDS	1485	92.0%	GCAAGAUUUGCAGGUAUUGAUGGCA	783
  15526	CDS	1486	101.7%	CAAGAUUUGCAGGUAUUGAUGGCAC	784
  15527	CDS	1496	103.3%	AGGUAUUGAUGGCACCUCCACAUAU	785
  15528	CDS	1503	102.3%	GAUGGCACCUCCACAUAUACCAGUG	786
  15529	CDS	1506	86.6%	GGCACCUCCACAUAUACCAGUGGUG	787
  15530	CDS	1510	79.9%	CCUCCACAUAUACCAGUGGUGAUCA	788
  15531	CDS	1511	44.9%	CUCCACAUAUACCAGUGGUGAUCAG	789
  15532	CDS	1512	57.3%	UCCACAUAUACCAGUGGUGAUCAGA	790
  15533	CDS	1517	64.9%	AUAUACCAGUGGUGAUCAGAAAACU	791
  15534	CDS	1518	90.8%	UAUACCAGUGGUGAUCAGAAAACUA	792
  15535	CDS	1520	47.1%	UACCAGUGGUGAUCAGAAAACUAUA	793
  15536	CDS	1526	55.7%	UGGUGAUCAGAAAACUAUAAAGUCC	794
  15537	CDS	1527	89.6%	GGUGAUCAGAAAACUAUAAAGUCCA	795
  15538	CDS	1529	92.4%	UGAUCAGAAAACUAUAAAGUCCACU	796
  15539	CDS	1531	87.2%	AUCAGAAAACUAUAAAGUCCACUAG	797
  15540	CDS	1532	93.4%	UCAGAAAACUAUAAAGUCCACUAGG	798
  15541	CDS	1575	78.4%	ACCCCACAUCUCCUGCUAAUGUUAU	799
  15542	CDS	1576	84.6%	CCCCACAUCUCCUGCUAAUGUUAUU	800
  15543	CDS	1579	95.9%	CACAUCUCCUGCUAAUGUUAUUGCC	801
  15544	CDS	1591	89.6%	UAAUGUUAUUGCCCUCCUACAGACU	802
  15545	CDS	1592	85.0%	AAUGUUAUUGCCCUCCUACAGACUU	803
  15546	CDS	1598	51.2%	AUUGCCCUCCUACAGACUUGAGUCA	804
  15547	CDS	1650	39.4%	GCUUUGGAUGCGGCCUAUUGCUUUA	805
  15548	CDS	1652	82.3%	UUUGGAUGCGGCCUAUUGCUUUAGA	806
  15549	CDS	1653	86.1%	UUGGAUGCGGCCUAUUGCUUUAGAA	807
  15550	CDS	1655	80.0%	GGAUGCGGCCUAUUGCUUUAGAAAU	808
  15551	CDS	1657	72.3%	AUGCGGCCUAUUGCUUUAGAAAUGU	809
  15552	CDS	1658	72.2%	UGCGGCCUAUUGCUUUAGAAAUGUG	810
  15553	CDS	1659	57.8%	GCGGCCUAUUGCUUUAGAAAUGUGC	811
  15554	CDS	1660	83.4%	CGGCCUAUUGCUUUAGAAAUGUGCA	812
  15555	CDS	1662	79.3%	GCCUAUUGCUUUAGAAAUGUGCAGG	813
  15556	CDS	1663	86.3%	CCUAUUGCUUUAGAAAUGUGCAGGA	814
  15557	CDS	1664	84.8%	CUAUUGCUUUAGAAAUGUGCAGGAU	815
  15558	CDS	1665	71.1%	UAUUGCUUUAGAAAUGUGCAGGAUA	816
  15559	CDS	1666	61.8%	AUUGCUUUAGAAAUGUGCAGGAUAA	817
  15560	CDS	1667	84.9%	UUGCUUUAGAAAUGUGCAGGAUAAU	818
  15561	CDS	1668	82.8%	UGCUUUAGAAAUGUGCAGGAUAAUU	819
  15562	CDS	1670	69.8%	CUUUAGAAAUGUGCAGGAUAAUUGC	820
  15563	CDS	1671	90.2%	UUUAGAAAUGUGCAGGAUAAUUGCU	821
  15564	CDS	1672	68.6%	UUAGAAAUGUGCAGGAUAAUUGCUG	822
  15565	CDS	1678	74.2%	AUGUGCAGGAUAAUUGCUGCCUACG	823
  15566	CDS	1761	58.6%	GGGUACAAUGCCAACUUCUGUGCUG	824
  15567	CDS	1767	86.3%	AAUGCCAACUUCUGUGCUGGAGCAU	825
  15568	CDS	1782	83.7%	GCUGGAGCAUGCCCGUAUUUAUGGA	826
  15569	CDS	1783	86.9%	CUGGAGCAUGCCCGUAUUUAUGGAG	827
  15570	CDS	1786	90.5%	GAGCAUGCCCGUAUUUAUGGAGUUC	828
  15571	CDS	1787	91.1%	AGCAUGCCCGUAUUUAUGGAGUUCA	829
  15572	CDS	1788	68.0%	GCAUGCCCGUAUUUAUGGAGUUCAG	830
  15573	CDS	1789	75.7%	CAUGCCCGUAUUUAUGGAGUUCAGA	831
  15574	CDS	1796	88.9%	GUAUUUAUGGAGUUCAGACACUCAG	832
  15575	CDS	1800	52.5%	UUAUGGAGUUCAGACACUCAGCACA	833
  15576	CDS	1907	90.8%	AACCAUUCUCUACUACAUUGGCAAA	834
  15577	CDS	1924	70.2%	UUGGCAAAACACCCAAGAUUGAACA	835
  15578	CDS	1925	77.5%	UGGCAAAACACCCAAGAUUGAACAG	836
  15579	CDS/3UTR	1973	91.1%	UUGCAAAUGCAGCUAAAAUUCUUGG	837
  15580	3UTR	2020	70.1%	CAAUGAUGAUGAUAAUGAUGAUGAC	838
  15581	3UTR	2022	43.3%	AUGAUGAUGAUAAUGAUGAUGACGA	839
  15582	3UTR	2023	60.3%	UGAUGAUGAUAAUGAUGAUGACGAC	840
  15583	3UTR	2025	75.4%	AUGAUGAUAAUGAUGAUGACGACGA	841
  15584	3UTR	2026	40.8%	UGAUGAUAAUGAUGAUGACGACGAC	842
  15585	3UTR	2028	51.8%	AUGAUAAUGAUGAUGACGACGACAA	843
  15586	3UTR	2029	59.1%	UGAUAAUGAUGAUGACGACGACAAC	844
  15587	3UTR	2031	51.3%	AUAAUGAUGAUGACGACGACAACGA	845
  15588	3UTR	2032	32.7%	UAAUGAUGAUGACGACGACAACGAU	846
  15589	3UTR	2034	33.8%	AUGAUGAUGACGACGACAACGAUGA	847
  15590	3UTR	2035	57.0%	UGAUGAUGACGACGACAACGAUGAU	848
  15591	3UTR	2039	40.5%	GAUGACGACGACAACGAUGAUGCUU	849
  15592	3UTR	2045	56.8%	GACGACAACGAUGAUGCUUGUAACA	850
  15593	3UTR	2046	28.5%	ACGACAACGAUGAUGCUUGUAACAA	851
  15594	3UTR	2065	44.7%	UAACAAGAAAACAUAAGAGAGCCUU	852
  15595	3UTR	2066	58.3%	AACAAGAAAACAUAAGAGAGCCUUG	853
  15596	3UTR	2067	62.9%	ACAAGAAAACAUAAGAGAGCCUUGG	854
  15597	3UTR	2072	38.1%	AAAACAUAAGAGAGCCUUGGUUCAU	855
  15598	3UTR	2073	44.6%	AAACAUAAGAGAGCCUUGGUUCAUC	856
  15599	3UTR	2079	53.6%	AAGAGAGCCUUGGUUCAUCAGUGUU	857
  15600	3UTR	2081	33.2%	GAGAGCCUUGGUUCAUCAGUGUUAA	858
  15601	3UTR	2083	28.2%	GAGCCUUGGUUCAUCAGUGUUAAAA	859
  15602	3UTR	2110	46.5%	UUUUUGAAAAGGCGGUACUAGUUCA	860
  15603	3UTR	2116	56.1%	AAAAGGCGGUACUAGUUCAGACACU	861
  15604	3UTR	2117	60.9%	AAAGGCGGUACUAGUUCAGACACUU	862
  15605	3UTR	2136	76.8%	ACACUUUGGAAGUUUGUGUUCUGUU	863
  15606	3UTR	2137	29.5%	CACUUUGGAAGUUUGUGUUCUGUUU	864
  15607	3UTR	2140	62.6%	UUUGGAAGUUUGUGUUCUGUUUGUU	865
  15608	3UTR	2145	50.7%	AAGUUUGUGUUCUGUUUGUUAAAAC	866
  15609	3UTR	2147	62.9%	GUUUGUGUUCUGUUUGUUAAAACUG	867
  15610	3UTR	2148	59.7%	UUUGUGUUCUGUUUGUUAAAACUGG	868
  15611	3UTR	2149	50.3%	UUGUGUUCUGUUUGUUAAAACUGGC	869
  15612	3UTR	2150	49.8%	UGUGUUCUGUUUGUUAAAACUGGCA	870
  15613	3UTR	2152	55.2%	UGUUCUGUUUGUUAAAACUGGCAUC	871
  15614	3UTR	2153	82.2%	GUUCUGUUUGUUAAAACUGGCAUCU	872
  15615	3UTR	2154	70.0%	UUCUGUUUGUUAAAACUGGCAUCUG	873
  15616	3UTR	2155	45.5%	UCUGUUUGUUAAAACUGGCAUCUGA	874
  15617	3UTR	2156	54.9%	CUGUUUGUUAAAACUGGCAUCUGAC	875
  15618	3UTR	2189	40.4%	AGUUGAAGGCCUUAUUCUACAUUUC	876
  15619	3UTR	2190	34.1%	GUUGAAGGCCUUAUUCUACAUUUCA	877
  15620	3UTR	2207	91.3%	ACAUUUCACCUACUUUGUAAGUGAG	878
  15621	3UTR	2265	60.9%	AAUAAACACUGGAAGAAUUUAUUAG	879
  15622	3UTR	2267	36.4%	UAAACACUGGAAGAAUUUAUUAGUG	880
  15623	3UTR	2295	40.6%	AUUAUGUGAACAACGACAACAACAA	881
  15624	3UTR	2296	33.6%	UUAUGUGAACAACGACAACAACAAC	882
  15625	3UTR	2297	32.7%	UAUGUGAACAACGACAACAACAACA	883
  15626	3UTR	2298	40.8%	AUGUGAACAACGACAACAACAACAA	884
  15627	3UTR	2299	38.5%	UGUGAACAACGACAACAACAACAAC	885
  15628	3UTR	2301	84.2%	UGAACAACGACAACAACAACAACAA	886
  15629	3UTR	2302	43.2%	GAACAACGACAACAACAACAACAAC	887
  15630	3UTR	2304	57.8%	ACAACGACAACAACAACAACAACAA	888
  15631	3UTR	2305	44.3%	CAACGACAACAACAACAACAACAAC	889
  15632	3UTR	2308	38.7%	CGACAACAACAACAACAACAACAAA	890
  15633	3UTR	2309	37.4%	GACAACAACAACAACAACAACAAAC	891
  15634	3UTR	2314	73.5%	CAACAACAACAACAACAAACAGGAA	892
  15635	3UTR	2315	54.2%	AACAACAACAACAACAAACAGGAAA	893
  15636	3UTR	2389	30.7%	CUUGAUUUUUCUGUAUUGCUAUGCA	894
  15637	3UTR	2435	16.0%	ACUCUUAGAGUUAACAGUGAGUUAU	895
  15638	3UTR	2445	18.4%	UUAACAGUGAGUUAUUUAUUGUGUG	896
  15639	3UTR	2471	36.3%	UACUAUAUAAUGAACGUUUCAUUGC	897
  15640	3UTR	2472	73.3%	ACUAUAUAAUGAACGUUUCAUUGCC	898
  15641	3UTR	2484	63.4%	ACGUUUCAUUGCCCUUGGAAAAUAA	899
  15642	3UTR	2488	65.4%	UUCAUUGCCCUUGGAAAAUAAAACA	900
  15643	3UTR	2493	39.3%	UGCCCUUGGAAAAUAAAACAGGUGU	901
  15644	3UTR	2519	66.7%	UAAAGUGGAGACCAAAUACUUUGCC	902
  15645	3UTR	2520	40.1%	AAAGUGGAGACCAAAUACUUUGCCA	903
  15646	3UTR	2526	40.9%	GAGACCAAAUACUUUGCCAGAAACU	904
  15647	3UTR	2527	41.5%	AGACCAAAUACUUUGCCAGAAACUC	905
  15648	3UTR	2528	47.6%	GACCAAAUACUUUGCCAGAAACUCA	906
  15649	3UTR	2529	47.6%	ACCAAAUACUUUGCCAGAAACUCAU	907
  15650	3UTR	2530	31.9%	CCAAAUACUUUGCCAGAAACUCAUG	908
  15651	3UTR	2531	29.0%	CAAAUACUUUGCCAGAAACUCAUGG	909
  15652	3UTR	2537	78.0%	CUUUGCCAGAAACUCAUGGAUGGCU	910
  15653	3UTR	2538	52.4%	UUUGCCAGAAACUCAUGGAUGGCUU	911
  15654	3UTR	2540	59.7%	UGCCAGAAACUCAUGGAUGGCUUAA	912
  15655	3UTR	2541	45.1%	GCCAGAAACUCAUGGAUGGCUUAAG	913
  15656	3UTR	2542	42.1%	CCAGAAACUCAUGGAUGGCUUAAGG	914
  15657	3UTR	2543	76.9%	CAGAAACUCAUGGAUGGCUUAAGGA	915
  15658	3UTR	2544	29.0%	AGAAACUCAUGGAUGGCUUAAGGAA	916
  15659	3UTR	2547	45.2%	AACUCAUGGAUGGCUUAAGGAACUU	917
  15660	3UTR	2560	38.4%	CUUAAGGAACUUGAACUCAAACGAG	918
  15661	3UTR	2561	33.3%	UUAAGGAACUUGAACUCAAACGAGC	919
  15662	3UTR	2562	31.9%	UAAGGAACUUGAACUCAAACGAGCC	920
  15663	3UTR	2563	44.5%	AAGGAACUUGAACUCAAACGAGCCA	921
  15664	3UTR	2564	90.1%	AGGAACUUGAACUCAAACGAGCCAG	922
  15665	3UTR	2566	64.4%	GAACUUGAACUCAAACGAGCCAGAA	923
  15666	3UTR	2623	32.5%	AAGUGAGUUAUUAUAUGACCGAGAA	924
  15667	3UTR	2681	34.0%	UGUUAUGUAUCAGCUGCCUAAGGAA	925
  15668	3UTR	2791	59.0%	UUUAAUUGUAAAUGGUUCUUUGUCA	926
  15669	3UTR	2792	56.3%	UUAAUUGUAAAUGGUUCUUUGUCAG	927
  15670	3UTR	2793	46.8%	UAAUUGUAAAUGGUUCUUUGUCAGU	928
  15671	3UTR	2795	53.2%	AUUGUAAAUGGUUCUUUGUCAGUUU	929
  15672	3UTR	2798	33.1%	GUAAAUGGUUCUUUGUCAGUUUAGU	930
  15673	3UTR	2809	32.8%	UUUGUCAGUUUAGUAAACCAGUGAA	931
  15674	3UTR	2813	40.9%	UCAGUUUAGUAAACCAGUGAAAUGU	932
  15675	3UTR	2816	38.1%	GUUUAGUAAACCAGUGAAAUGUUGA	933
  15676	3UTR	2840	59.4%	AAAUGUUUUGACAUGUACUGGUCAA	934
  15677	3UTR	2957	77.9%	UGGAUAUAGAAGCCAGCAUAAUUGA	935
  15678	3UTR	2958	74.1%	GGAUAUAGAAGCCAGCAUAAUUGAA	936
  15679	3UTR	2959	52.4%	GAUAUAGAAGCCAGCAUAAUUGAAA	937
  15680	3UTR	2963	49.9%	UAGAAGCCAGCAUAAUUGAAAACAC	938
  15681	3UTR	2964	45.3%	AGAAGCCAGCAUAAUUGAAAACACA	939
  15682	3UTR	2966	45.5%	AAGCCAGCAUAAUUGAAAACACAUC	940
  15683	3UTR	3137	60.5%	ACAAAUGUAUGUUUCUUUUAGCUGG	941
  15684	3UTR	3138	63.6%	CAAAUGUAUGUUUCUUUUAGCUGGC	942
  15685	3UTR	3142	58.4%	UGUAUGUUUCUUUUAGCUGGCCAGU	943
  15686	3UTR	3144	56.3%	UAUGUUUCUUUUAGCUGGCCAGUAC	944
  15687	3UTR	3145	52.1%	AUGUUUCUUUUAGCUGGCCAGUACU	945
  15688	3UTR	3147	74.6%	GUUUCUUUUAGCUGGCCAGUACUUU	946
  15689	3UTR	3150	70.4%	UCUUUUAGCUGGCCAGUACUUUUGA	947
  15690	3UTR	3154	61.7%	UUAGCUGGCCAGUACUUUUGAGUAA	948
  15691	3UTR	3156	52.3%	AGCUGGCCAGUACUUUUGAGUAAAG	949
  15692	3UTR	3157	72.2%	GCUGGCCAGUACUUUUGAGUAAAGC	950
  15693	3UTR	3158	62.4%	CUGGCCAGUACUUUUGAGUAAAGCC	951
  15694	3UTR	3180	49.0%	GCCCCUAUAGUUUGACUUGCACUAC	952
  15695	3UTR	3182	43.9%	CCCUAUAGUUUGACUUGCACUACAA	953
  15696	3UTR	3183	35.2%	CCUAUAGUUUGACUUGCACUACAAA	954
  15697	3UTR	3184	38.1%	CUAUAGUUUGACUUGCACUACAAAU	955
  15698	3UTR	3185	73.3%	UAUAGUUUGACUUGCACUACAAAUG	956
  15699	3UTR	3256	86.3%	UUCAUUAUUAUGACAUAAGCUACCU	957
  15700	3UTR	3258	61.6%	CAUUAUUAUGACAUAAGCUACCUGG	958
  15701	3UTR	3342	66.0%	UUCAUCUUCCAAGCAUCAUUACUAA	959
  15702	3UTR	3346	67.3%	UCUUCCAAGCAUCAUUACUAACCAA	960
  15703	3UTR	3358	63.6%	CAUUACUAACCAAGUCAGACGUUAA	961
  15704	3UTR	3396	71.8%	UAGGAAAAGGAGGAAUGUUAUAGAU	962
  15705	3UTR	3550	69.1%	UUGUUAUUACAAAGAGGACACUUCA	963
  15706	3UTR	3657	72.3%	GGGGAAAAAAGUCCAGGUCAGCAUA	964
  15707	3UTR	3671	79.7%	AGGUCAGCAUAAGUCAUUUUGUGUA	965
  15708	3UTR	3779	57.5%	UUUCUUUCCUCUGAGUGAGAGUUAU	966
  15709	3UTR	3783	62.6%	UUUCCUCUGAGUGAGAGUUAUCUAU	967
  15710	3UTR	3932	61.3%	UAAAAAUUAAUAGGCAAAGCAAUGG	968
  15711	3UTR	3934	44.3%	AAAAUUAAUAGGCAAAGCAAUGGAA	969
  15712	3UTR	4034	68.7%	UUUUUUGGAAUUUCCUGACCAUUAA	970
  15713	3UTR	4058	50.6%	AUUAAAGAAUUGGAUUUGCAAGUUU	971
  15714	3UTR	4120	69.8%	UAAACAGCCCUUGUGUUGGAUGUAA	972
  15715	3UTR	4147	39.5%	CAAUCCCAGAUUUGAGUGUGUGUUG	973
  15716	3UTR	4148	62.2%	AAUCCCAGAUUUGAGUGUGUGUUGA	974
  15717	3UTR	4152	34.2%	CCAGAUUUGAGUGUGUGUUGAUUAU	975
  15718	3UTR	4273	38.0%	GUCUUUCCUCAUGAAUGCACUGAUA	976
  15719	3UTR	4460	48.5%	UAUUUUUGUGUUAAUCAGCAGUACA	977
  15720	3UTR	4482	37.1%	ACAAUUUGAUCGUUGGCAUGGUUAA	978
  15721	3UTR	4580	60.1%	GUUUUUGUGGUGCUCUAGUGGUAAA	979
  15722	3UTR	4583	50.6%	UUUGUGGUGCUCUAGUGGUAAAUAA	980
  15723	3UTR	4584	42.1%	UUGUGGUGCUCUAGUGGUAAAUAAA	981
  15724	3UTR	4642	91.3%	UCAGUACCAUCAUCGAGUCUAGAAA	982
  15725	3UTR	4737	90.4%	UUCUCCCUUAAGGACAGUCACUUCA	983
  15726	3UTR	4751	94.6%	CAGUCACUUCAGAAGUCAUGCUUUA	984
  15727	3UTR	4753	87.2%	GUCACUUCAGAAGUCAUGCUUUAAA	985
  15728	3UTR	4858	70.2%	GUAAUUGUUUGAGAUUUAGUUUCCA	986
  15729	3UTR	4963	81.2%	CGCCAGGGCCAAAAGAACUGGUCUA	987
  15730	3UTR	5177	81.4%	CCAGACUCCUCAAACGAGUUGCCAA	988

• TABLE 7
  hTGFB2 sd-rxRNA

  	hTGFB2 Single		SEQ ID
  	Strand ID	sd-rxRNA sequences	NO

  Target Gene
  Duplex ID
  18570	17560	mU.A.mU.mU.mU.A.mU.mU.G.mU.G.mU.A.Chl	989
  	17562	P.mU.A.fC.A.fC.A.A.fU.A.A.A.fU.A*A*fC*fU*fC*A*C	990
  18571	17561	mU.mU.A.mU.mU.mU.A.mU.mU.G.mU.G.mU.A.Chl	991
  	17562	P.mU.A.fC.A.fC.A.A.fU.A.A.A.fU.A*A*fC*fU*fC*A*C	992
  18572	17563	A.mU.mC.A.G.mU.G.mU.mU.A.A.A.A.Chl	993
  	17565	P.mU.fU.fU.fU.A.A.fC.A.fC.fU.G.A.fU*G*A*A*fC*fC*A	994
  18573	17564	mC.A.mU.mC.A.G.mU.G.mU.mU.A.A.A.A.Chl	995
  	17565	P.mU.fU.fU.fU.A.A.fC.A.fC.fU.G.A.fU*G*A*A*fC*fC*A	996
  18574	17566	A.mU.G.G.mC.mU.mU.A.A.G.G.A.A.Chl	997
  	17568	P.mU.fU.fC.fC.fU.fU.A.A.G.fC.fC.A.fU*fC*fC*A*fU*G*A	998
  18575	17567	G.A.mU.G.G.mC.mU.mU.A.A.G.G.A.A.Chl	999
  	17568	P.mU.fU.fC.fC.fU.fU.A.A.G.fC.fC.A.fU*fC*fC*A*fU*G*A	1000
  18576	17569	mU.mU.G.mU.G.mU.mU.mC.mU.G.mU.mU.A.Chl	1001
  	17571	P.mU.A.A.fC.A.G.A.A.fC.A.fC.A.A*A*fC*fU*fU*fC*C	1002
  18577	17570	mU.mU.mU.G.mU.G.mU.mU.mC.mU.G.mU.mU.A.Chl	1003
  	17571	P.mU.A.A.fC.A.G.A.A.fC.A.fC.A.A*A*fC*fU*fU*fC*C	1004
  18578	17572	A.A.A.mU.A.mC.mU.mU.mU.G.mC.mC.A.Chl	1005
  	17574	P.mU.G.G.fC.A.A.A.G.fU.A.fU.fU.fU*G*G*fU*fC*fU*C	1006
  18579	17573	mC.A.A.A.mU.A.mC.mU.mU.mU.G.mC.mC.A.Chl	1007
  	17574	P.mU.G.G.fC.A.A.A.G.fU.A.fU.fU.fU*G*G*fU*fC*fU*C	1008
  18580	17575	mC.mU.mU.G.mC.A.mC.mU.A.mC.A.A.A.Chl	1009
  	17577	P.mU.fU.fU.G.fU.A.G.fU.G.fC.A.A.G*fU*fC*A*A*A*C	1010
  18581	17576	A.mC.mU.mU.G.mC.A.mC.mU.A.mC.A.A.A.Chl	1011
  	17577	P.mU.fU.fU.G.fU.A.G.fU.G.fC.A.A.G*fU*fC*A*A*A*C	1012
  18582	17578	G.A.A.mU.mU.mU.A.mU.mU.A.G.mU.A.Chl	1013
  	17580	P.mU.A.fC.fU.A.A.fU.A.A.A.fU.fU.fC*fU*fU*fC*fC*A*G	1014
  18583	17579	A.G.A.A.mU.mU.mU.A.mU.mU.A.G.mU.A.Chl	1015
  	17580	P.mU.A.fC.fU.A.A.fU.A.A.A.fU.fU.fC*fU*fU*fC*fC*A*G	1016
  18584	17581	mU.mU.G.mC.A.mC.mU.A.mC.A.A.A.A.Chl	1017
  	17583	P.mU.fU.fU.fU.G.fU.A.G.fU.G.fC.A.A*G*fU*fC*A*A*A	1018
  18585	17582	mC.mU.mU.G.mC.A.mC.mU.A.mC.A.A.A.A.Chl	1019
  	17583	P.mU.fU.fU.fU.G.fU.A.G.fU.G.fC.A.A*G*fU*fC*A*A*A	1020
  18586	17584	A.mU.A.A.A.A.mC.A.G.G.mU.G.A.Chl	1021
  	17586	P.mU.fC.A.fC.fC.fU.G.fU.fU.fU.fU.A.fU*fU*fU*fU*fC*fC*A	1022
  18587	17585	A.A.mU.A.A.A.A.mC.A.G.G.mU.G.A.Chl	1023
  	17586	P.mU.fC.A.fC.fC.fU.G.fU.fU.fU.fU.A.fU*fU*fU*fU*fC*fC*A	1024
  18588	17587	G.A.mC.A.A.mC.A.A.mC.A.A.mC.A.Chl	1025
  	17588	P.mU.G.fU.fU.G.fU.fU.G.fU.fU.G.fU.fC*G*fU*fU*G*fU*U	1026
  18589	17589	A.mU.G.mC.mU.mU.G.mU.A.A.mC.A.A.Chl	1027
  	17590	P.mU.fU.G.fU.fU.A.fC.A.A.G.fC.A.fU*fC*A*fU*fC*G*U	1028
  18590	17591	mC.A.G.A.A.A.mC.mU.mC.A.mU.G.A.Chl	1029
  	17592	P.mU.fC.A.fU.G.A.G.fU.fU.fU.fC.fU.G*G*fC*A*A*A*G	1030
  18591	17593	G.mU.A.mU.mU.G.mC.mU.A.mU.G.mC.A.Chl	1031
  	17594	P.mU.G.fC.A.fU.A.G.fC.A.A.fU.A.fC*A*G*A*A*A*A	1032
  18592	17595	mC.mC.A.G.A.A.A.mC.mU.mC.A.mU.A.Chl	1033
  	17596	P.mU.A.fU.G.A.G.fU.fU.fU.fC.fU.G.G*fC*A*A*A*G*U	1034
  18593	17597	A.mC.mU.mC.A.A.A.mC.G.A.G.mC.A.Chl	1035
  	17598	P.mU.G.fC.fU.fC.G.fU.fU.fU.G.A.G.fU*fU*fC*A*A*G*U	1036
  18594	17599	A.mU.A.mU.G.A.mC.mC.G.A.G.A.A.Chl	1037
  	17600	P.mU.fU.fC.fU.fC.G.G.fU.fC.A.fU.A.fU*A*A*fU*A*A*C	1038
  18595	17601	mC.G.A.mC.G.A.mC.A.A.mC.G.A.A.Chl	1039
  	17602	P.mU.fU.fC.G.fU.fU.G.fU.fC.G.fU.fC.G*fU*fC*A*fU*fC*A	1040
  18596	17603	G.mU.A.A.A.mC.mC.A.G.mU.G.A.A.Chl	1041
  	17604	P.mU.fU.fC.A.fC.fU.G.G.fU.fU.fU.A.fC*fU*A*A*A*fC*U	1042
  18597	17605	mU.mU.G.mU.mC.A.G.mU.mU.mU.A.G.A.Chl	1043
  	17606	P.mU.fC.fU.A.A.A.fC.fU.G.A.fC.A.A*A*G*A*A*fC*C	1044
  18598	17607	mU.mC.A.mU.mC.A.G.mU.G.mU.mU.A.A.Chl	1045
  	17608	P.mU.fU.A.A.fC.A.fC.fU.G.A.fU.G.A*A*fC*fC*A*A*G	1046
  18599	17609	A.A.mC.mU.mC.A.A.A.mC.G.A.G.A.Chl	1047
  	17610	P.mU.fC.fU.fC.G.fU.fU.fU.G.A.G.fU.fU*fC*A*A*G*fU*U	1048
  18600	17611	mC.G.A.mC.A.A.mC.A.A.mC.A.A.A.Chl	1049
  	17612	P.mU.fU.fU.G.fU.fU.G.fU.fU.G.fU.fC.G*fU*fU*G*fU*fU*C	1050
  18601	17613	A.mC.G.A.mC.A.A.mC.G.A.mU.G.A.Chl	1051
  	17614	P.mU.fC.A.fU.fC.G.fU.fU.G.fU.fC.G.fU*fC*G*fU*fC*A*U	1052
  18602	17615	G.mC.mU.G.mC.mC.mU.A.A.G.G.A.A.Chl	1053
  	17616	P.mU.fU.fC.fC.fU.fU.A.G.G.fC.A.G.fC*fU*G*A*fU*A*C	1054
  18603	17617	A.mU.mU.mC.mU.A.mC.A.mU.mU.mU.mC.A.Chl	1055
  	17618	P.mU.G.A.A.A.fU.G.fU.A.G.A.A.fU*A*A*G*G*fC*C	1056
  18604	17619	G.mU.G.mU.G.mU.mU.G.A.mU.mU.A.A.Chl	1057
  	17620	P.mU.fU.A.A.fU.fC.A.A.fC.A.fC.A.fC*A*fC*fU*fC*A*A	1058
  Rat TGFB2
  sd-rxRNA
  18678	18604	mC.G.G.mU.G.A.mC.A.A.mU.G.A.A-chol	1061
  	18605	mU.fU.fC.A.fU.fU.G.fU.fC.A.fC.fC.G*fU*G*A*fU*fU*U	1062
  18679	18606	mU.mU.G.mU.mC.mU.mC.G.G.mU.A.mU.A-chol	1063
  	18607	mU.A.fU.A.fC.fC.G.A.G.A.fC.A.A*A*G*G*G*A*A	1064
  18680	18608	G.A.G.mU.mU.G.mU.A.mU.G.mU.A.A-chol	1065
  	18609	mU.fU.A.fC.A.fU.A.fC.A.A.fC.fU.fC*fC*A*fC*fU*G*A	1066
  18681	18610	A.mU.mU.mU.G.mU.mU.A.G.mU.G.mU.A-chol	1067
  	18611	mU.A.fC.A.fC.fU.A.A.fC.A.A.A.fU*fU*fC*fU*fU*fC*C	1068
  18682	18612	G.mC.A.A.G.mU.mC.mU.G.A.G.A.A-chol	1069
  	18613	mU.fU.fC.fU.fC.A.G.A.fC.fU.fU.G.fC*fU*fC*A*G*fU*U	1070
  18683	18614	A.A.A.mU.mC.A.mC.G.G.mU.G.A.A-chol	1071
  	18615	mU.fU.fC.A.fC.fC.G.fU.G.A.fU.fU.fU*fU*fC*A*fU*fC*C	1072
  18684	18616	A.A.A.mU.G.mC.A.G.mC.mU.A.A.A-chol	1073
  	18617	mU.fU.fU.A.G.fC.fU.G.fC.A.fU.fU.fU*A*fC*A*A*G*A	1074
  18685	18618	mC.mU.mU.G.G.A.A.A.A.mU.A.A.A-chol	1075
  	18619	mU.fU.fU.A.fU.fU.fU.fU.fC.fC.A.A.G*G*G*fC*A*A*U	1076
  18686	18620	mC.mC.mU.mU.mU.G.A.A.mU.A.A.A.A-chol	1077
  	18621	mU.fU.fU.fU.A.fU.fU.fC.A.A.A.G.G*fU*A*fC*fU*G*G	1078
  18687	18622	A.A.mC.A.mC.A.mC.mU.G.mC.A.A.A-chol	1079
  	18623	mU.fU.fU.G.fC.A.G.fU.G.fU.G.fU.fU*fU*fU*fC*A*fU*C	1080
  18688	18624	A.A.A.A.mC.A.mC.A.mC.mU.G.mC.A-chol	1081
  	18625	mU.G.fC.A.G.fU.G.fU.G.fU.fU.fU.fU*fC*A*fU*fC*A*U	1082
  18689	18626	G.A.A.G.G.mC.mC.mU.G.mU.mU.A.A-chol	1083
  	18627	mU.fU.A.A.fC.A.G.G.fC.fC.fU.fU.fC*fU*G*G*A*fC*A	1084
  18690	18628	mU.A.mU.mU.G.mC.mU.mC.mU.G.mC.A.A-chol	1085
  	18629	mU.fU.G.fC.A.G.A.G.fC.A.A.fU.A*fC*A*G*A*G*G	1086

• TABLE 8
  hSPP1 sd-rxRNA

  	hSPP1 Single		SEQ ID
  	Strand ID	sd-rxRNA sequence	NO

  Target Gene
  Duplex ID
  18538	17430	G.A.mU.G.A.A.mU.mC.mU.G.A.mU.A.Chl	1087
  	17432	P.mU.A.fU.fC.A.G.A.fU.fU.fC.A.fU.fC*A*G*A*A*fU*G	1088
  18539	17431	mU.G.A.mU.G.A.A.mU.mC.mU.G.A.mU.A.Chl	1089
  	17432	P.mU.A.fU.fC.A.G.A.fU.fU.fC.A.fU.fC*A*G*A*A*fU*G	1090
  18540	17433	A.mU.mU.mU.G.mC.mU.mU.mU.mU.G.mC.A.Chl	1091
  	17435	P.mU.G.fC.A.A.A.A.G.fC.A.A.A.fU*fC*A*fC*fU*G*fC	1092
  18541	17434	G.A.mU.mU.mU.G.mC.mU.mU.mU.mU.G.mC.A.Chl	1093
  	17435	P.mU.G.fC.A.A.A.A.G.fC.A.A.A.fU*fC*A*fC*fU*G*fC	1094
  18542	17436	G.mU.G.A.mU.mU.mU.G.mC.mU.mU.mU.AChl	1095
  	17438	P.mU.A.A.A.G.fC.A.A.A.fU.fC.A.fC*fU*G*fC*A*A*fU	1096
  18543	17437	A.G.mU.G.A.mU.mU.mU.G.mC.mU.mU.mU.A.Chl	1097
  	17438	P.mU.A.A.A.G.fC.A.A.A.fU.fC.A.fC*fU*G*fC*A*A*fU	1098
  18544	17439	A.A.mU.mU.mU.mC.G.mU.A.mU.mU.mU.A.Chl	1099
  	17441	P.mU.A.A.A.fU.A.fC.G.A.A.A.fU.fU*fU*fC*A*G*G*fU	1100
  18545	17440	A.A.A.mU.mU.mU.mC.G.mU.A.mU.mU.mU.A.Chl	1101
  	17441	P.mU.A.A.A.fU.A.fC.G.A.A.A.fU.fU*fU*fC*A*G*G*fU	1102
  18546	17442	mC.A.mC.A.G.mC.mC.A.mU.G.A.A.A.Chl	1103
  	17444	P.mU.fU.fU.C.A.fU.G.G.fC.fU.G.fU.G*A*A*A*fU*fU*fC	1104
  18547	17443	mU.mC.A.mC.A.G.mC.mC.A.mU.G.A.A.A.Chl	1105
  	17444	P.mU.fU.fU.C.A.fU.G.G.fC.fU.G.fU.G*A*A*A*fU*fU*fC	1106
  18548	17445	G.A.mU.mU.mU.G.mC.mU.mU.mU.mU.G.A.Chl	1107
  	17447	P.mU.fC.A.A.A.A.G.fC.A.A.A.fU.fC*A*fC*fU*G*fC*A	1108
  18549	17446	mU.G.A.mU.mU.mU.G.mC.mU.mU.mU.mU.G.A.Chl	1109
  	17447	P.mU.fC.A.A.A.A.G.fC.A.A.A.fU.fC*A*fC*fU*G*fC*A	1110
  18550	17448	mU.mU.G.mC.mU.mU.mU.mU.G.mC.mC.mU.A.Chl	1111
  	17450	P.mU.A.G.G.fC.A.A.A.A.G.fC.A.A*A*fU*fC*A*fC*U	1112
  18551	17449	mU.mU.mU.G.mC.mU.mU.mU.mU.G.mC.mC.mU.A.Chl	1113
  	17450	P.mU.A.G.G.fC.A.A.A.A.G.fC.A.A*A*fU*fC*A*fC*U	1114
  18552	17451	mU.mU.mU.mC.mU.mC.A.G.mU.mU.mU.A.A.Chl	1115
  	17452	P.mU.fU.A.A.A.fC.fU.G.A.G.A.A.A*G*A*A*G*fC*A	1116
  18553	17453	mU.mU.G.mC.A.mU.mU.mU.A.G.mU.mC.A.Chl	1117
  	17454	P.mU.G.A.fC.fU.A.A.A.fU.G.fC.A.A*A*G*fU*G*A*G	1118
  18554	17455	A.mC.mU.mU.mU.G.mC.A.mU.mU.mU.A.A.Chl	1119
  	17456	P.mU.fU.A.A.A.fU.G.fC.A.A.A.G.fU*G*A*G*A*A*A	1120
  18555	17457	A.mU.mU.mU.A.G.mU.mC.A.A.A.A.A.Chl	1121
  	17458	P.mU.fU.fU.fU.fU.G.A.fC.fU.A.A.A.fU*G*fC*A*A*A*G	1122
  18556	17459	mU.mU.mC.mU.mU.mU.mC.mU.mC.A.G.mU.A.Chl	1123
  	17460	P.mU.A.fC.fU.G.A.G.A.A.A.G.A.A*G*fC*A*fU*fU*fU	1124
  18557	17461	mU.mC.mU.mU.mU.mC.mU.mC.A.G.mU.mU.A.Chl	1125
  	17462	P.mU.A.A.fC.fU.G.A.G.A.A.A.G.A*A*G*fC*A*fU*fU	1126
  18558	17463	G.A.A.A.G.A.G.A.A.mC.A.mU.A.Chl	1127
  	17464	P.mU.A.fU.G.fU.fU.fC.fU.fC.fU.fU.fU.fC*A*fU*fU*fU*fU*G	1128
  18559	17465	mC.mU.mU.mU.G.mC.A.mU.mU.mU.A.G.A.Chl	1129
  	17466	P.mU.fC.fU.A.A.A.fU.G.fC.A.A.A.G*fU*G*A*G*A*A	1130
  18560	17467	mU.mU.mU.G.mC.A.mU.mU.mU.A.G.mU.A.Chl	1131
  	17468	P.mU.A.fC.fU.A.A.A.fU.G.fC.A.A.A*G*fU*G*A*G*A	1132
  18561	17469	mC.mU.mC.A.mC.mU.mU.mU.G.mC.A.mU.A.Chl	1133
  	17470	P.mU.A.fU.G.fC.A.A.A.G.fU.G.A.G*A*A*A*fU*fU*G	1134
  18562	17471	mU.mU.mC.mU.mC.A.mC.mU.mU.mU.G.mC.A.Chl	1135
  	17472	P.mU.G.fC.A.A.A.G.fU.G.A.G.A.A*A*fU*fU*G*fU*A	1136
  18563	17473	mC.A.mC.mU.mC.mC.A.G.mU.mU.G.mU.A.Chl	1137
  	17474	P.mU.A.fC.A.A.fC.fU.G.G.A.G.fU.G*A*A*A*A*fC*U	1138
  18564	17475	A.A.mU.G.A.A.A.G.A.G.A.A.A.Chl	1139
  	17476	P.mU.fU.fU.fC.fU.fC.fU.fU.fU.fC.A.fU.fU*fU*fU*G*fC*fU*A	1140
  18565	17477	mU.G.mC.A.G.mU.G.A.mU.mU.mU.mG.A.Chl	1141
  	17478	P.mU.fC.A.A.A.fU.fC.A.fC.fU.G.fC.A*A*fU*fU*fC*fU*C	1142
  18566	17479	mU.G.A.A.A.G.A.G.A.A.mC.A.A.Chl	1143
  	17480	P.mU.fU.G.fU.fU.fC.fU.fC.fU.fU.fU.fC.A*fU*fU*fU*fU*G*C	1144
  18567	17481	A.mC.mC.mU.G.A.A.A.mU.mU.mU.mC.A.Chl	1145
  	17482	P.mU.G.A.A.A.fU.fU.fU.fC.A.G.G.fU*G*fU*fU*fU*A*U	1146
  18568	17483	G.A.A.mU.mU.G.mC.A.G.mU.G.A.A.Chl	1147
  	17484	P.mU.fU.fC.A.fC.fU.G.fC.A.A.fU.fU.fC*fU*fC*A*fU*G*G	1148
  18569	17485	G.G.mC.mU.G.A.mU.mU.mC.mU.G.G.A.Chl	1149
  	17486	P.mU.fC.fC.A.G.A.A.fU.fC.A.G.fC.fC*fU*G*fU*fU*fU*A	1150
  Rat Targeting
  SPP1
  18662	18630	G.mU.mU.mC.G.mU.mU.G.mU.mU.mU.mC.A-chol	1151
  	18631	P.mU.G.A.A.A.fC.A.A.fC.G.A.A.fC*fU*A*A*G*fC*U	1152
  18663	18632	G.A.A.A.G.A.A.A.mU.A.G.A.A-chol	1153
  	18633	P.mU.fU.fC.fU.A.fU.fU.fU.fC.fU.fU.fU.fC*fU*fC*fC*A*fC*A	1154
  18664	18634	G.mU.G.G.A.G.A.A.A.G.A.A.A-chol	1155
  	18635	P.mU.fU.fU.fC.fU.fU.fU.fC.fU.fC.fC.A.fC*A*fU*A*fC*A*U	1156
  18665	18636	mC.mU.G.mU.G.mU.mC.A.mC.mU.A.mU.A-chol	1157
  	18637	P.mU.A.fU.A.G.fU.G.A.fC.A.fC.A.G*A*fC*fU*A*fU*U	1158
  18666	18638	G.mU.mU.mU.mC.mU.mC.A.G.mU.mU.mC.A-chol	1159
  	18639	P.mU.G.A.A.fC.fU.G.A.G.A.A.A.fC*A*A*G*fC*A*G	1160
  18667	18640	mU.A.mC.A.G.G.A.A.mC.A.G.mC.A-chol	1161
  	18641	P.mU.G.fC.fU.G.fU.fU.fC.fC.fU.G.fU.A*A*G*fU*fU*fU*G	1162
  18668	18642	G.mC.A.G.G.mC.A.A.A.mC.mU.mU.A-chol	1163
  	18643	P.mU.A.A.G.fU.fU.fU.G.fC.fC.fU.G.fC*fC*fU*fC*fU*A*C	1164
  18669	18644	A.A.mC.mU.mU.A.mC.A.G.G.A.A.A-chol	1165
  	18645	P.mU.fU.fU.fC.fC.fU.G.fU.A.A.G.fU.fU*fU*G*fC*fC*fU*G	1166
  18670	18646	mC.A.mC.mU.G.mC.A.mU.mU.mU.mU.A.A-chol	1167
  	18647	P.mU.fU.A.A.A.A.fU.G.fC.A.G.fU.G*G*fC*fC*A*fU*U	1168
  18671	18648	G.A.mC.A.mC.mC.A.mC.mU.G.mU.A.A-chol	1169
  	18649	P.mU.fU.A.fC.A.G.fU.G.G.fU.G.fU.fC*fU*G*fC*A*fU*G	1170
  18672	18650	A.G.A.G.G.mC.A.G.G.mC.A.A.A-chol	1171
  	18651	P.mU.fU.fU.G.fC.fC.fU.G.fC.fC.fU.fC.fU*A*fC*A*fU*A*C	1172
  18673	18652	mU.A.G.A.G.G.mC.A.G.G.mC.A.A-chol	1173
  	18653	P.mU.fU.G.fC.fC.fU.G.fC.fC.fU.fC.fU.A*fC*A*fU*A*fC*A	1174
  18674	18654	G.A.G.A.G.mU.mU.mC.A.mU.mC.mU.A-chol	1175
  	18655	P.mU.A.G.A.fU.G.A.A.fC.fU.fC.fU.fC*fU*A*A*fU*fU*C	1176
  18675	18656	mU.G.mU.G.A.A.mU.A.A.A.mU.mC.A-chol	1177
  	18657	P.mU.G.A.fU.fU.fU.A.fU.U.fC.A.fC.A*fC*fC*A*fC*A*A	1178
  18676	18658	G.mU.G.A.A.mU.A.A.A.mU.mC.mU.A-chol	1179
  	18659	P.mU.A.G.A.fU.fU.fU.A.fU.fU.fC.A.fC*A*fC*fC*A*fC*A	1180
  18677	18660	mU.G.A.AmU.A.A.A.mU.mC.mU.mU.A-chol	1181
  	18661	P.mU.A.A.G.A.fU.fU.fU.A.fU.U.fC.A*fC*A*fC*fC*A*C	1182

• TABLE 9
  Inhibition of gene expression with hSPP1 ori sequences

  Target			SEQ		A549
  Gene	Gene	Ref	ID	hSPP1	0.1 nM
  Duplex ID	Region	Pos	NO	Sense Strand Sequence	Activity

  14840	5UTR/CDS	155	1183	AAGGAAAACUCACUACCAUGAGAAA	4.4%
  14841	5UTR/CDS	161	1184	AACUCACUACCAUGAGAAUUGCAGA	2.46%
  14842	5UTR/CDS	163	1185	CUCACUACCAUGAGAAUUGCAGUGA	20.54%
  14843	5UTR/CDS	164	1186	UCACUACCAUGAGAAUUGCAGUGAA	2.8%
  14844	CDS	168	1187	UACCAUGAGAAUUGCAGUGAUUUGA	3.6%
  14845	CDS	169	1188	ACCAUGAGAAUUGCAGUGAUUUGCA	5.2%
  14846	CDS	171	1189	CAUGAGAAUUGCAGUGAUUUGCUUA	0.8%
  14847	CDS	172	1190	AUGAGAAUUGCAGUGAUUUGCUUUA	0.95%
  14848	CDS	173	1191	UGAGAAUUGCAGUGAUUUGCUUUUA	3.2%
  14849	CDS	174	1192	GAGAAUUGCAGUGAUUUGCUUUUGA	4.14%
  14850	CDS	175	1193	AGAAUUGCAGUGAUUUGCUUUUGCA	2.9%
  14851	CDS	176	1194	GAAuuGCAGUGAUUUGCUUUUGCCA	8.38%
  14852	CDS	177	1195	AAUUGCAGUGAUUUGCUUUUGCCUA	4.6%
  14853	CDS	180	1196	UGCAGUGAUUUGCUUUUGCCUCCUA	11.1%
  14854	CDS	181	1197	GCAGUGAUUUGCUUUUGCCUCCUAA	10.87%
  14855	CDS	182	1198	CAGUGAUUUGCUUUUGCCUCCUAGA	5.3%
  14856	CDS	206	1199	GCAUCACCUGUGCCAUACCAGUUAA	15.29%
  14857	CDS	208	1200	AUCACCUGUGCCAUACCAGUUAAAA	22.6%
  14858	CDS	212	1201	CCUGUGCCAUACCAGUUAAACAGGA	13.3%
  14859	CDS	215	1202	GUGCCAUACCAGUUAAACAGGCUGA	21.2%
  14860	CDS	216	1203	UGCCAUACCAGUUAAACAGGCUGAA	20.24%
  14861	CDS	220	1204	AUACCAGUUAAACAGGCUGAUUCUA	12.5%
  14862	CDS	221	1205	UACCAGUUAAACAGGCUGAUUCUGA	9.9%
  14863	CDS	222	1206	ACCAGUUAAACAGGCUGAUUCUGGA	3.9%
  14864	CDS	225	1207	AGUUAAACAGGCUGAUUCUGGAAGA	20.48%
  14865	CDS	226	1208	GUUAAACAGGCUGAUUCUGGAAGUA	10.7%
  14866	CDS	227	1209	UUAAACAGGCUGAUUCUGGAAGUUA	22.75%
  14867	CDS	228	1210	UAAACAGGCUGAUUCUGGAAGUUCA	0.26%
  14868	CDS	234	1211	GGCUGAUUCUGGAAGUUCUGAGGAA	0.34%
  14869	CDS	236	1212	CUGAUUCUGGAAGUUCUGAGGAAAA	4.4%
  14870	CDS	238	1213	GAUUCUGGAAGUUCuGAGGAAAAGA	4.5%
  14871	CDS	239	1214	AUUCUGGAAGUUCUGAGGAAAAGCA	7.5%
  14872	CDS	240	1215	UUCUGGAAGUUCUGAGGAAAAGCAA	101.3%
  14873	CDS	338	1216	CCCCACAGACCCUUCCAAGUAAGUA	48.3%
  14874	CDS	340	1217	CCACAGACCCUUCCAAGUAAGUCCA	33.9%
  14875	CDS	342	1218	ACAGACCCUUCCAAGuAAGUCCAAA	16.1%
  14876	CDS	343	1219	CAGACCCUUCCAAGUAAGUCCAACA	38.7%
  14877	CDS	345	1220	GACCCUUCCAAGUAAGUCCAACGAA	54.2%
  14878	CDS	348	1221	CCUUCCAAGUAAGUCCAACGAAAGA	12.54%
  14879	CDS	349	1222	CUUCCAAGUAAGUCCAACGAAAGCA	32.44%
  14880	CDS	351	1223	UCCAAGUAAGUCCAACGAAAGCCAA	17.1%
  14881	CDS	353	1224	CAAGUAAGUCCAACGAAAGCCAUGA	32.94%
  14882	CDS	358	1225	AAGUCCAACGAAAGCCAUGACCACA	65.1%
  14883	CDS	362	1226	CCAACGAAAGCCAUGACCACAUGGA	76.9%
  14884	CDS	363	1227	CAACGAAAGCCAUGACCACAUGGAA	69.8%
  14885	CDS	366	1228	CGAAAGCCAUGACCACAUGGAUGAA	78.02%
  14886	CDS	372	1229	CCAUGACCACAUGGAUGAUAUGGAA	19.49%
  14887	CDS	377	1230	ACCACAUGGAUGAUAUGGAUGAUGA	20.43%
  14888	CDS	393	1231	GGAUGAUGAAGAUGAUGAUGACCAA	29.1%
  14889	CDS	394	1232	GAUGAUGAAGAUGAUGAUGACCAUA	24.5%
  14890	CDS	396	1233	UGAUGAAGAUGAUGAUGACCAUGUA	25.90%
  14891	CDS	398	1234	AUGAAGAUGAUGAUGACCAUGUGGA	20.5%
  14892	CDS	399	1235	UGAAGAUGAUGAUGACCAUGUGGAA	7.9%
  14893	CDS	430	1236	GACUCCAUUGACUCGAACGACUCUA	21.6%
  14894	CDS	431	1237	ACUCCAUUGACUCGAACGACUCUGA	13.5%
  14895	CDS	432	1238	CUCCAUUGACUCGAACGACUCUGAA	12.33%
  14896	CDS	435	1239	CAUUGACUCGAACGACUCUGAUGAA	42.5%
  14897	CDS	440	1240	ACUCGAACGACUCUGAUGAUGUAGA	22.54%
  14898	CDS	441	1241	CUCGAACGACUCUGAUGAUGUAGAA	17.4%
  14899	CDS	442	1242	UCGAACGACUCUGAUGAUGUAGAUA	11.2%
  14900	CDS	443	1243	CGAACGACUCUGAUGAUGUAGAUGA	20.7%
  14901	CDS	445	1244	AACGACUCUGAUGAUGUAGAUGACA	27.1%
  14902	CDS	449	1245	ACUCUGAUGAUGUAGAUGACACUGA	39.8%
  14903	CDS	450	1246	CUCUGAUGAUGUAGAUGACACUGAA	9.6%
  14904	CDS	451	1247	UCUGAUGAUGUAGAUGACACUGAUA	4.44%
  14905	CDS	452	1248	CUGAUGAUGUAGAUGACACUGAUGA	8.7%
  14906	CDS	453	1249	UGAUGAUGUAGAUGACACUGAUGAA	16.72%
  14907	CDS	461	1250	UAGAUGACACUGAUGAUUCUCACCA	42.9%
  14908	CDS	462	1251	AGAUGACACUGAUGAUUCUCACCAA	30.1%
  14909	CDS	469	1252	ACUGAUGAUUCUCACCAGUCUGAUA	9.1%
  14910	CDS	470	1253	CUGAUGAUUCUCACCAGUCUGAUGA	19.0%
  14911	CDS	471	1254	UGAUGAUUCUCACCAGUCUGAUGAA	42.1%
  14912	CDS	472	1255	GAUGAUUCUCACCAGUCUGAUGAGA	59.1%
  14913	CDS	476	1256	AUUCUCACCAGUCUGAUGAGUCUCA	38.2%
  14914	CDS	479	1257	CUCACCAGUCUGAUGAGUCUCACCA	34.1%
  14915	CDS	480	1258	UCACCAGUCUGAUGAGUCUCACCAA	48.45%
  14916	CDS	483	1259	CCAGUCUGAUGAGUCUCACCAUUCA	9.5%
  14917	CDS	484	1260	CAGUCUGAUGAGUCUCACCAUUCUA	21.5%
  14918	CDS	485	1261	AGUCUGAUGAGUCUCACCAUUCUGA	18.6%
  14919	CDS	486	1262	GUCUGAUGAGUCUCACCAUUCUGAA	20.2%
  14920	CDS	487	1263	UCUGAUGAGUCUCACCAUUCUGAUA	10.9%
  14921	CDS	488	1264	CUGAUGAGUCUCACCAUUCUGAUGA	18.9%
  14922	CDS	489	1265	UGAUGAGUCUCACCAUUCUGAUGAA	10.7%
  14923	CDS	490	1266	GAUGAGUCUCACCAUUCUGAUGAAA	28.15%
  14924	CDS	493	1267	GAGUCUCACCAUUCUGAUGAAUCUA	18.33%
  14925	CDS	495	1268	GUCUCACCAUUCUGAUGAAUCUGAA	7.61%
  14926	CDS	496	1269	UCUCACCAUUCUGAUGAAUCUGAUA	2.99%
  14927	CDS	497	1270	CUCACCAUUCUGAUGAAUCUGAUGA	7.44%
  14928	CDS	498	1271	UCACCAUUCUGAUGAAUCUGAUGAA	9.7%
  14929	CDS	499	1272	CACCAUUCUGAUGAAUCUGAUGAAA	16.96%
  14930	CDS	501	1273	CCAUUCUGAUGAAUCUGAUGAACUA	3.08%
  14931	CDS	505	1274	UCUGAUGAAUCUGAUGAACUGGUCA	13.24%
  14932	CDS	510	1275	UGAAUCUGAUGAACUGGUCACUGAA	3.16%
  14933	CDS	550	1276	CCAGCAACCGAAGUUUUCACUCCAA	14.02%
  14934	CDS	554	1277	CAACCGAAGUUUUCACUCCAGUUGA	3.10%
  14935	CDS	555	1278	AACCGAAGUUUUCACUCCAGUUGUA	5.27%
  14936	CDS	572	1279	CAGUUGUCCCCACAGUAGACACAUA	13.2%
  14937	CDS	573	1280	AGUUGUCCCCACAGUAGACACAUAA	27.01%
  14938	CDS	574	1281	GUUGUCCCCACAGUAGACACAUAUA	8.76%
  14939	CDS	588	1282	AGACACAUAUGAUGGCCGAGGUGAA	14.04%
  14940	CDS	589	1283	GACACAUAUGAUGGCCGAGGUGAUA	18.40%
  14941	CDS	598	1284	GAUGGCCGAGGUGAUAGUGUGGUUA	12.50%
  14942	CDS	601	1285	GGCCGAGGUGAUAGUGUGGUUUAUA	13.76%
  14943	CDS	602	1286	GCCGAGGUGAUAGUGUGGUUUAUGA	5.34%
  14944	CDS	603	1287	CCGAGGUGAUAGUGUGGUUUAUGGA	29.69%
  14945	CDS	604	1288	CGAGGUGAUAGUGUGGUUUAUGGAA	33.34%
  14946	CDS	606	1289	AGGUGAUAGUGUGGUUUAUGGACUA	17.50%
  14947	CDS	608	1290	GUGAUAGUGUGGUUUAUGGACUGAA	45.90%
  14948	CDS	609	1291	UGAUAGUGUGGUUUAUGGACUGAGA	22.0%
  14949	CDS	610	1292	GAUAGUGUGGUUUAUGGACUGAGGA	19.93%
  14950	CDS	611	1293	AUAGUGUGGUUUAUGGACUGAGGUA	17.34%
  14951	CDS	615	1294	UGUGGUUUAUGGACUGAGGUCAAAA	5.60%
  14952	CDS	617	1295	UGGUUUAUGGACUGAGGUCAAAAUA	25.74%
  14953	CDS	618	1296	GGUUUAUGGACUGAGGUCAAAAUCA	17.63%
  14954	CDS	619	1297	GUUUAUGGACUGAGGUCAAAAUCUA	3.45%
  14955	CDS	621	1298	UUAUGGACUGAGGUCAAAAUCUAAA	18.03%
  14956	CDS	622	1299	UAUGGACUGAGGUCAAAAUCUAAGA	20.98%
  14957	CDS	623	1300	AUGGACUGAGGUCAAAAUCUAAGAA	20.60%
  14958	CDS	624	1301	UGGACUGAGGUCAAAAUCUAAGAAA	26.73%
  14959	CDS	625	1302	GGACUGAGGUCAAAAUCUAAGAAGA	7.45%
  14960	CDS	626	1303	GACUGAGGUCAAAAUCUAAGAAGUA	14.1%
  14961	CDS	629	1304	UGAGGUCAAAAUCUAAGAAGUUUCA	8.61%
  14962	CDS	630	1305	GAGGUCAAAAUCUAAGAAGUUUCGA	19.07%
  14963	CDS	631	1306	AGGUCAAAAUCUAAGAAGUUUCGCA	6.08%
  14964	CDS	632	1307	GGUCAAAAUCUAAGAAGUUUCGCAA	19.82%
  14965	CDS	636	1308	AAAAUCUAAGAAGUUUCGCAGACCA	21.55%
  14966	CDS	637	1309	AAAUCUAAGAAGUUUCGCAGACCUA	30.20%
  14967	CDS	638	1310	AAUCUAAGAAGUUUCGCAGACCUGA	18.23%
  14968	CDS	686	1311	ACGAGGACAUCACCUCACACAUGGA	14.85%
  14969	CDS	687	1312	CGAGGACAUCACCUCACACAUGGAA	28.04%
  14970	CDS	689	1313	AGGACAUCACCUCACACAUGGAAAA	3.80%
  14971	CDS	698	1314	CCUCACACAUGGAAAGCGAGGAGUA	7.67%
  14972	CDS	703	1315	CACAUGGAAAGCGAGGAGUUGAAUA	5.8%
  14973	CDS	704	1316	ACAUGGAAAGCGAGGAGUUGAAUGA	5.3%
  14974	CDS	705	1317	CAUGGAAAGCGAGGAGUUGAAUGGA	24.47%
  14975	CDS	718	1318	GAGUUGAAUGGUGCAUACAAGGCCA	26.39%
  14976	CDS	785	1319	GCCGUGGGAAGGACAGUUAUGAAAA	7.60%
  14977	CDS	786	1320	CCGUGGGAAGGACAGUUAUGAAACA	8.75%
  14978	CDS	788	1321	GUGGGAAGGACAGUUAUGAAACGAA	8.34%
  14979	CDS	790	1322	GGGAAGGACAGUUAUGAAACGAGUA	5.38%
  14980	CDS	792	1323	GAAGGACAGUUAUGAAACGAGUCAA	11.45%
  14981	CDS	794	1324	AGGACAGUUAUGAAACGAGUCAGCA	11.78%
  14982	CDS	795	1325	GGACAGUUAUGAAACGAGUCAGCUA	10.69%
  14983	CDS	797	1326	ACAGUUAUGAAACGAGUCAGCUGGA	54.58%
  14984	CDS	798	1327	CAGUUAUGAAACGAGUCAGCUGGAA	33.9%
  14985	CDS	846	1328	CCACAAGCAGUCCAGAUUAUAUAAA	24.1%
  14986	CDS	850	1329	AAGCAGUCCAGAUUAUAUAAGCGGA	27.86%
  14987	CDS	854	1330	AGUCCAGAUUAUAUAAGCGGAAAGA	24.29%
  14988	CDS	855	1331	GUCCAGAUUAUAUAAGCGGAAAGCA	54.43%
  14989	CDS	859	1332	AGAUUAUAUAAGCGGAAAGCCAAUA	71.49%
  14990	CDS	860	1333	GAUUAUAUAAGCGGAAAGCCAAUGA	69.64%
  14991	CDS	861	1334	AUUAUAUAAGCGGAAAGCCAAUGAA	38.82%
  14992	CDS	862	1335	UUAUAUAAGCGGAAAGCCAAUGAUA	20.77%
  14993	CDS	865	1336	UAUAAGCGGAAAGCCAAUGAUGAGA	21.79%
  14994	CDS	866	1337	AUAAGCGGAAAGCCAAUGAUGAGAA	50.00%
  14995	CDS	867	1338	UAAGCGGAAAGCCAAUGAUGAGAGA	11.67%
  14996	CDS	870	1339	GCGGAAAGCCAAUGAUGAGAGCAAA	13.5%
  14997	CDS	871	1340	CGGAAAGCCAAUGAUGAGAGCAAUA	15.49%
  14998	CDS	872	1341	GGAAAGCCAAUGAUGAGAGCAAUGA	8.55%
  14999	CDS	873	1342	GAAAGCCAAUGAUGAGAGCAAUGAA	12.12%
  15000	CDS	875	1343	AAGCCAAUGAUGAGAGCAAUGAGCA	16.14%
  15001	CDS	878	1344	CCAAUGAUGAGAGCAAUGAGCAUUA	31.71%
  15002	CDS	879	1345	CAAUGAUGAGAGCAAUGAGCAUUCA	32.25%
  15003	CDS	881	1346	AUGAUGAGAGCAAUGAGCAUUCCGA	6.97%
  15004	CDS	883	1347	GAUGAGAGCAAUGAGCAUUCCGAUA	23.11%
  15005	CDS	885	1348	UGAGAGCAAUGAGCAUUCCGAUGUA	5.53%
  15006	CDS	890	1349	GCAAUGAGCAUUCCGAUGUGAUUGA	10.69%
  15007	CDS	893	1350	AUGAGCAUUCCGAUGUGAUUGAUAA	4.12%
  15008	CDS	894	1351	UGAGCAUUCCGAUGUGAUUGAUAGA	6.49%
  15009	CDS	89S	1352	GAGCAUUCCGAUGUGAUUGGAUAUA	29.12%
  15010	CDS	897	1353	GCAUUCCGAUGUGAUUGAUAGUCAA	3.54%
  15011	CDS	899	1354	AUUCCGAUGUGAUUGAUAGUCAGGA	6.05%
  15012	CDS	901	1355	UCCGAUGUGAUUGAUAGUCAGGAAA	3.31%
  15013	CDS	906	1356	UGUGAUUGAUAGUCAGGAACUUUCA	12.71%
  15014	CDS	907	1357	GUGAUUGAUAGUCAGGAACUUUCCA	13.95%
  15015	CDS	909	1358	GAUUGAUAGUCAGGAACUUUCCAAA	4.03%
  15016	CDS	912	1359	UGAUAGUCAGGAACUUUCCAAAGUC	11.96%
  15017	CDS	913	1360	GAUAGUCAGGAACUUUCCAAAGUCA	14.01%
  15018	CDS	914	1361	AUAGUCAGGAACUUUCCAAAGUCAA	5.56%
  15019	CDS	916	1362	AGUCAGGAACUUUCCAAAGUCAGCA	13.92%
  15020	CDS	917	1363	GUCAGGAACUUUCCAAAGUCAGCCA	19.00%
  15021	CDS	923	1364	AACUUUCCAAAGUCAGCCGUGAAUA	17.56%
  15022	CDS	925	1365	CUUUCCAAAGUCAGCCGUGAAUUCA	19.58%
  15023	CDS	926	1366	UUUCCAAAGUCAGCCGUGAAUUCCA	6.54%
  15024	CDS	935	1367	UCAGCCGUGAAUUCCACAGCCAUGA	16.15%
  15025	CDS	936	1368	CAGCCGUGAAUUCCACAGCCAUGAA	20.62%
  15026	CDS	937	1369	AGCCGUGAAUUCCACAGCCAUGAAA	5.21%
  15027	CDS	943	1370	GAAUUCCACAGCCAUGAAUUUCACA	31.14%
  15028	CDS	944	1371	AAUUCCACAGCCAUGAAUUUCACAA	35.63%
  15029	CDS	945	1372	AUUCCACAGCCAUGAAUUUCACAGA	23.96%
  15030	CDS	946	1373	UUCCACAGCCAUGAAUUUCACAGCA	15.20%
  15031	CDS	947	1374	UCCACAGCCAUGAAUUUCACAGCCA	19.45%
  15032	CDS	950	1375	ACAGCCAUGAAUUUCACAGCCAUGA	25.74%
  15033	CDS	952	1376	AGCCAUGAAUUUCACAGCCAUGAAA	2.59%
  15034	CDS	953	1377	GCCAUGAAUUUCACAGCCAUGAAGA	6.00%
  15035	CDS	954	1378	CCAUGAAUUUCACAGCCAUGAAGAA	4.60%
  15036	CDS	956	1379	AUGAAUUUCACAGCCAUGAAGAUAA	9.20%
  15037	CDS	957	1380	UGAAUUUCACAGCCAUGAAGAUAUA	10.84%
  15038	CDS	958	1381	GAAUUUCACAGCCAUGAAGAUAUGA	40.20%
  15039	CDS	959	1382	AAUUUCACAGCCAUGAAGAUAUGCA	37.25%
  15040	CDS	960	1383	AUUUCACAGCCAUGAAGAUAUGCUA	8.21%
  15041	CDS	961	1384	UUUCACAGCCAUGAAGAUAUGCUGA	12.01%
  15042	CDS	964	1385	CACAGCCAUGAAGAUAUGCUGGUUA	12.25%
  15043	CDS	983	1386	UGGUUGUAGACCCCAAAAGUAAGGA	19.65%
  15044	CDS	984	1387	GGUUGUAGACCCCAAAAGUAAGGAA	28.19%
  15045	CDS	985	1388	GUUGUAGACCCCAAAAGUAAGGAAA	17.92%
  15046	CDS	986	1389	UUGUAGACCCCAAAAGUAAGGAAGA	7.94%
  15047	CDS	987	1390	UGUAGACCCCAAAAGUAAGGAAGAA	15.09%
  15048	CDS	988	1391	GUAGACCCCAAAAGUAAGGAAGAAA	20.01%
  15049	CDS	989	1392	UAGACCCCAAAAGUAAGGAAGAAGA	7.25%
  15050	CDS	990	1393	AGACCCCAAAAGUAAGGAAGAAGAA	12.42%
  15051	CDS	995	1394	CCAAAAGUAAGGAAGAAGAUAAACA	8.96%
  15052	CDS	996	1395	CAAAAGUAAGGAAGAAGAUAAACAA	6.85%
  15053	CDS	997	1396	AAAAGUAAGGAAGAAGAUAAACACA	14.15%
  15054	CDS	998	1397	AAAGUAAGGAAGAAGAUAAACACCA	12.32%
  15055	CDS	999	1398	AAGUAAGGAAGAAGAUAAACACCUA	8.83%
  15056	CDS	1001	1399	GUAAGGAAGAAGAUAAACACCUGAA	15.09%
  15057	CDS	1002	1400	UAAGGAAGAAGAUAAACACCUGAAA	4.91%
  15058	CDS	1007	1401	AAGAAGAUAAACACCUGAAAUUUCA	1.43%
  15059	CDS	1008	1402	AGAAGAUAAACACCUGAAAUUUCGA	3.51%
  15060	CDS	1009	1403	GAAGAUAAACACCUGAAAUUUCGUA	15.12%
  15061	CDS	1010	1404	AAGAUAAACACCUGAAAUUUCGUAA	28.56%
  15062	CDS	1013	1405	AUAAACACCUGAAAUUUCGUAUUUA	5.74%
  15063	CDS	1015	1406	AAACACCUGAAAUUUCGUAUUUCUA	13.01%
  15064	CDS	1024	1407	AAAUUUCGUAUUUCUCAUGAAUUAA	15.54%
  15065	CDS	1030	1408	CGUAUUUCUCAUGAAUUAGAUAGUA	9.47%
  15066	CDS	1031	1409	GUAUUUCUCAUGAAUUAGAUAGUGA	30.03%
  15067	CDS	1032	1410	UAUUUCUCAUGAAUUAGAUAGUGCA	5.31%
  15068	CDS	1036	1411	UCUCAUGAAUUAGAUAGUGCAUCUA	9.74%
  15069	CDS	1037	1412	CUCAUGAAUUAGAUAGUGCAUCUUA	10.78%
  15070	CDS	1038	1413	UCAUGAAUUAGAUAGUGCAUCUUCA	91.87%
  15071	CDS	1039	1414	CAUGAAUUAGAUAGUGCAUCUUCUA	93.82%
  15072	CDS	1040	1415	AUGAAUUAGAUAGUGCAUCUUCUGA	96.06%
  15073	CDS	1041	1416	UGAAUUAGAUAGUGCAUCUUCUGAA	94.91%
  15074	CDS	1042	1417	GAAUUAGAUAGUGCAUCUUCUGAGA	97.91%
  15075	CDS	1043	1418	AAUUAGAUAGUGCAUCUUCUGAGGA	93.76%
  15076	CDS	1044	1419	AUUAGAUAGUGCAUCUUCUGAGGUA	103.92%
  15077	CDS	1045	1420	UUAGAUAGUGCAUCUUCUGAGGUCA	95.85%
  15078	CDS/3UTR	1052	1421	GUGCAUCUUCUGAGGUCAAUUAAAA	93.83%
  15079	CDS/3UTR	1053	1422	UGCAUCUUCUGAGGUCAAUUAAAAA	90.69%
  15080	CDS/3UTR	1054	1423	GCAUCUUCUGAGGUCAAUUAAAAGA	101.49%
  15081	CDS/3UTR	1055	1424	CAUCUUCUGAGGUCAAUUAAAAGGA	110.27%
  15082	CDS/3UTR	1056	1425	AUCUUCUGAGGUCAAUUAAAAGGAA	99.36%
  15083	CDS/3UTR	1057	1426	UCUUCUGAGGUCAAUUAAAAGGAGA	95.31%
  15084	CDS/3UTR	1058	1427	CUUCUGAGGUCAAUUAAAAGGAGAA	15.55%
  15085	3UTR	1081	1428	AAAAAAUACAAUUUCUCACUUUGCA	3.59%
  15086	3UTR	1083	1429	AAAAUACAAUUUCUCACUUUGCAUU	3.46%
  15087	3UTR	1086	1430	AUACAAUUUCUCACUUUGCAUUUAG	2.37%
  15088	3UTR	1087	1431	UACAAUUUCUCACUUUGCAUUUAGU	3.54%
  15089	3UTR	1088	1432	ACAAUUUCUCACUUUGCAUUUAGUC	2.85%
  15090	3UTR	1089	1433	CAAUUUCUCACUUUGCAUUUAGUCA	2.35%
  15091	3UTR	1093	1434	UUCUCACUUUGCAUUUAGUCAAAAG	1.38%
  15092	3UTR	1125	1435	GCUUUAUAGCAAAAUGAAAGAGAAC	4.11%
  15093	3UTR	1127	1436	UUUAUAGCAAAAUGAAAGAGAACAU	3.91%
  15094	3UTR	1128	1437	UUAUAGCAAAAUGAAAGAGAACAUG	3.59%
  15095	3UTR	1147	1438	AACAUGAAAUGCUUCUUUCUCAGUU	1.80%
  15096	3UTR	1148	1439	ACAUGAAAUGCUUCUUUCUCAGUUU	2.17%
  15097	3UTR	1150	1440	AUGAAAUGCUUCUUUCUCAGUUUAU	2.93%
  15098	3UTR	1153	1441	AAAUGCUUCUUUCUCAGUUUAUUGG	2.18%
  15099	3UTR	1154	1442	AAUGCUUCUUUCUCAGUUUAUUGGU	3.92%
  15100	3UTR	1156	1443	UGCUUCUUUCUCAGUUUAUUGGUUG	4.08%
  15101	3UTR	1157	1444	GCUUCUUUCUCAGUUUAUUGGUUGA	1.74%
  15102	3UTR	1158	1445	CUUCUUUCUCAGUUUAUUGGUUGAA	4.74%
  15103	3UTR	1159	1446	UUCUUUCUCAGUUUAUUGGUUGAAU	2.65%
  15104	3UTR	1168	1447	AGUUUAUUGGUUGAAUGUGUAUCUA	2.57%
  15105	3UTR	1178	1448	UUGAAUGUGUAUCUAUUUGAGUCUG	3.76%
  15106	3UTR	1179	1449	UGAAUGUGUAUCUAUUUGAGUCUGG	2.91%
  15107	3UTR	1183	1450	UGUGUAUCUAUUUGAGUCUGGAAAU	0.62%
  15108	3UTR	1184	1451	GUGUAUCUAUUUGAGUCUGGAAAUA	2.45%
  15109	3UTR	1186	1452	GUAUCUAUUUGAGUCUGGAAAUAAC	2.18%
  15110	3UTR	1191	1453	UAUUUGAGUCUGGAAAUAACUAAUG	2.44%
  15111	3UTR	1218	1454	UUUGAUAAUUAGUUUAGUUUGUGGC	19.35%
  15112	3UTR	1219	1455	UUGAUAAUUAGUUUAGUUUGUGGCU	6.19%
  15113	3UTR	1222	1456	AUAAUUAGUUUAGUUUGUGGCUUCA	3.25%
  15114	3UTR	1224	1457	AAUUAGUUUAGUUUGUGGCUUCAUG	2.47%
  15115	3UTR	1225	1458	AUUAGUUUAGUUUGUGGCUUCAUGG	2.28%
  15116	3UTR	1226	1459	UUAGUUUAGUUUGUGGCUUCAUGGA	3.40%
  15117	3UTR	1227	1460	UAGUUUAGUUUGUGGCUUCAUGGAA	4.12%
  15118	3UTR	1244	1461	UCAUGGAAACUCCCUGUAAACUAAA	2.63%
  15119	3UTR	1245	1462	CAUGGAAACUCCCUGUAAACUAAAA	2.20%
  15120	3UTR	1246	1453	AUGGAAACUCCCUGUAAACUAAAAG	3.56%
  15121	3UTR	1247	1454	UGGAAACUCCCUGUAAACUAAAAGC	3.73%
  15122	3UTR	1248	1465	GGAAACUCCCUGUAAACUAAAAGCU	2.43%
  15123	3UTR	1249	1466	GAAACUCCCUGUAAACUAAAAGCUU	2.28%
  15124	3UTR	1251	1467	AACUCCCUGUAAACUAAAAGCUUCA	5.40%
  15125	3UTR	1253	1468	CUCCCUGUAAACUAAAAGCUUCAGG	8.21%
  15126	3UTR	1286	1469	UAUGUUCAUUCUAUAGAAGAAAUGC	3.17%
  15127	3UTR	1294	1470	UUCUAUAGAAGAAAUGCAAACUAUC	2.45%
  15128	3UTR	1295	1471	UCUAUAGAAGAAAUGCAAACUAUCA	3.97%
  15129	3UTR	1296	1472	CUAUAGAAGAAAUGCAAACUAUCAC	3.86%
  15130	3UTR	1297	1473	UAUAGAAGAAAUGCAAACUAUCACU	1.84%
  15131	3UTR	1299	1474	UAGAAGAAAUGCAAACUAUCACUGU	2.53%
  15132	3UTR	1302	1475	AAGAAAUGCAAACUAUCACUGUAUU	2.25%
  15133	3UTR	1303	1476	AGAAAUGCAAACUAUCACUGUAUUU	3.32%
  15134	3UTR	1357	1477	AUUUAUGUAGAAGCAAACAAAAUAC	1.86%
  15135	3UTR	1465	1478	UAUCUUUUUGUGGUGUGAAUAAAUC	3.40%
  15136	3UTR	1466	1479	AUCUUUUUGUGGUGUGAAUAAAUCU	3.49%
  15137	3UTR	1467	1480	UCUUUUUGUGGUGUGAAUAAAUCUU	3.03%
  15138	3UTR	1468	1481	CUUUUUGUGGUGUGAAUAAAUCUUU	3.62%
  15139	3UTR	1496	1482	CUUGAAUGUAAUAAGAAUUUGGUGG	61.48%
  15140	3UTR	1497	1483	UUGAAUGUAAUAAGAAUUUGGUGGU	71.54%
  15141	3UTR	1504	1434	UAAUAAGAAUUUGGUGGUGUCAAUU	58.54%
  15142	3UTR	1511	1485	AAUUUGGUGGUGUCAAUUGCUUAUU	56.93%
  15143	3UTR	1512	1486	AUUUGGUGGUGUCAAUUGCUUAUUU	81.22%
  15144	3UTR	1513	1487	UUUGGUGGUGUCAAUUGCUUAUUUG	59.16%
  15145	3UTR	1514	1488	UUGGUGGUGUCAAUUGCUUAUUUGU	59.46%
  15146	3UTR	1540	1489	UUCCCACGGUUGUCCAGCAAUUAAU	67.40%

• TABLE 10
  hCTGF sd-rxRNA

  Target Gene	CTGF Single		SEQ ID
  Duplex ID	Strand ID	sd-rxRNA sequence	NO

  17356	17007	A.mC.A.mU.mU.A.A.mC.mU.mC.A.mU.A.Chl	1490
  	17009	P.mU.A.fU.G.A.G.fU.fU.A.A.fU.G.fU*fC*fU*fC*fU*fC*A	1491
  17357	17008	G.A.mC.A.mU.mU.A.A.mC.mU.mC.A.mU.A.Chl	1492
  	17009	P.mU.A.fU.G.A.G.fU.fU.A.A.fU.G.fU*fC*fU*fC*fU*fC*A	1493
  17358	17010	mU.G.A.A.G.A.A.mU.G.mU.mU.A.A.Chl	1494
  	17012	P.mU.fU.A.A.fC.A.fU.fU.fC.fU.fU.fC.A*A*A*fC*fC*A*G	1495
  17359	17011	mU.mU.G.A.A.G.A.A.mU.G.mU.mU.A.A.Chl	1496
  	17012	P.mU.fU.A.A.fC.A.fU.fU.fC.fU.fU.fC.A*A*A*fC*fC*A*G	1497
  17360	17013	G.A.mU.A.G.mC.A.mU.mC.mU.mU.A.A.Chl	1498
  	17015	P.mU.fU.A.A.G.A.fU.G.fC.fU.A.fU.fC*fU*G*A*fU*G*A	1499
  17361	17014	A.G.A.mU.A.G.mC.A.mU.mC.mU.mU.A.A.Chl	1500
  	17015	P.mU.fU.A.A.G.A.fU.G.fC.fU.A.fU.fC*fU*G*A*fU*G*A	1501
  17362	17016	mU.G.A.A.G.mU.G.mU.A.A.mU.mU.A.Chl	1502
  	17017	P.mU.A.A.fU.fU.A.fC.A.fC.fU.fU.fC.A*A*A*fU*A*G*C	1503
  17363	17018	A.A.mU.mU.G.A.G.A.A.G.G.A.A.Chl	1504
  	17019	P.mU.fU.fC.fC.fU.fU.fC.fU.fC.A.A.fU.fU*A*fC*A*fC*fU*U	1505
  17364	17020	mU.mU.G.A.G.A.A.G.G.A.A.A.A.Chl	1506
  	17021	P.mU.fU.fU.fU.fC.fC.fU.fU.fC.fU.fC.A.A*fU*fU*A*fC*A*C	1507
  17365	17022	mC.A.mU.mU.mC.mU.G.A.mU.mU.mC.G.A.Chl	1508
  	17023	P.mU.fC.G.A.A.fU.fC.A.G.A.A.fU.G*fU*fC*A*G*A*G	1509
  17366	17024	mU.mU.mC.mU.G.A.mU.mU.mC.G.A.A.A.Chl	1510
  	17025	P.mU.fU.fU.fC.G.A.A.fU.fC.A.G.A.A*fU*G*fU*fC*A*G	1511
  17367	17026	mC.mU.G.mU.mC.G.A.mU.mU.A.G.A.A.Chl	1512
  	17027	P.mU.fU.fC.fU.A.A.fU.fC.G.A.fC.A.G*G*A*fU*fU*fC*C	1513
  17368	17028	mU.mU.mU.G.mC.mC.mU.G.mU.A.A.mC.A.Chl	1514
  	17030	P.mU.G.fU.fU.A.fC.A.G.G.fC.A.A.A*fU*fU*fC*A*fC*U	1515
  17369	17029	A.mU.mU.mU.G.mC.mC.mU.G.mU.A.A.mC.A.Chl	1516
  	17030	P.mU.G.fU.fU.A.fC.A.G.G.fC.A.A.A*fU*fU*fC*A*fC*U	1517
  17370	17031	A.mC.A.A.G.mC.mC.A.G.A.mU.mU.A.Chl	1518
  	17033	P.mU.A.A.fU.fC.fU.G.G.fC.fU.fU.G.fU*fU*A*fC*A*G*G	1519
  17371	17032	“A.A.mC.A.A.G.mC.mC.A.G.A.mU.mU.A.Chl	1520
  	17033	P.mU.A.A.fU.fC.fU.G.G.fC.fU.fU.G.fU*fU*A*fC*A*G*G	1521
  17372	17034	mC.A.G.mU.mU.mU.A.mU.mU.mU.G.mU.A.Chl	1522
  	17035	P.mU.A.fC.A.A.A.fU.A.A.A.fC.fU.G*fU*fC*fC*G*A*A	1523
  17373	17036	mU.G.mU.mU.G.A.G.A.G.mU.G.mU.A.Chl	1524
  	17038	P.mU.A.fC.A.fC.fU.fC.fU.fC.A.A.fC.A*A*A*fU*A*A*A	1525
  17374	17037	mU.mU.G.mU.mU.G.A.G.A.G.mU.G.mU.A.Chl	1526
  	17038	P.mU.A.fC.A.fC.fU.fC.fU.fC.A.A.fC.A*A*A*fU*A*A*A	1527
  17375	17039	mU.G.mC.A.mC.mC.mU.mU.mU.mC.mU.A.A.Chl	1528
  	17041	P.mU.fU.A.G.A.A.A.G.G.fU.G.fC.A*A*A*fC*A*fU*G	1529
  17376	17040	mU.mU.G.mC.A.mC.mC.mU.mU.mU.mC.mU.A.A.Chl	1530
  	17041	P.mU.fU.A.G.A.A.A.G.G.fU.G.fC.A*A*A*fC*A*fU*G	1531
  17377	17042	mU.mU.G.A.G.mC.mU.mU.mU.mC.mU.G.A.Chl	1532
  	17043	P.mU.fC.A.G.A.A.A.G.fC.fU.fC.A.A*A*fC*fU*fU*G*A	1533
  17378	17044	mU.G.A.G.A.G.mU.G.mU.G.A.mC.A.Chl	1534
  	17045	P.mU.G.fU.fC.A.fC.A.fC.fU.fC.fU.fC.A*A*fC*A*A*A*U	1535
  17379	17046	A.G.mU.G.mU.G.A.mC.mC.A.A.A.A.Chl	1536
  	17048	P.mU.fU.fU.fU.G.G.fU.fC.A.fC.A.fC.fU*fC*fU*fC*A*A*C	1537
  17380	17047	G.A.G.mU.G.mU.G.A.mC.mC.A.A.A.A.Chl	1538
  	17048	P.mU.fU.fU.fU.G.G.fU.fC.A.fC.A.fC.fU*fC*fU*fC*A*A*C	1539
  17381	17049	G.mU.G.mU.G.A.mC.mC.A.A.A.A.A.Chl	1540
  	17050	P.mU.fU.fU.fU.fU.G.G.fU.fC.A.fC.A.fC*fU*fC*fU*fC*A*A	1541
  17382	17051	mU.G.mU.G.A.mC.mC.A.A.A.A.G.A.Chl	1542
  	17053	P.mU.fC.fU.fU.fU.fU.G.G.fU.fC.A.fC.A*fC*fU*fC*fU*fC*A	1543
  17383	17052	G.mU.G.mU.G.A.mC.mC.A.A.A.A.G.A.Chl	1544
  	17053	P.mU.fC.fU.fU.fU.fU.G.G.fU.fC.A.fC.A*fC*fU*fC*fU*fC*A	1545
  17384	17054	G.mU.G.A.mC.mC.A.A.A.A.G.mU.A.Chl	1546
  	17055	P.mU.A.fC.fU.fU.fU.fU.G.G.fU.fC.A.fC*A*fC*fU*fC*fU*C	1547
  17385	17056	G.A.mC.mC.A.A.A.A.G.mU.mU.A.A.Chl	1548
  	17057	P.mU.fU.A.A.fC.fU.fU.fU.fU.G.G.fU.fC*A*fC*A*fC*fU*C	1549
  17386	17058	G.mC.A.mC.mC.mU.mU.mU.mC.mU.A.G.A.Chl	1550
  	17059	P.mU.fC.fU.A.G.A.A.A.G.G.fU.G.fC*A*A*A*fC*A*U	1551
  17387	17060	mC.mC.mU.mU.mU.mC.mU.A.G.mU.mU.G.A.Chl	1552
  	17061	P.mU.fC.A.A.fC.fU.A.G.A.A.A.G.G*fU*G*fC*A*A*A	1553

• TABLE 11
  Inhibition of gene expression with hCTGF on sequences

  Target
  Gene			SEQ
  Duplex	Gene	Ref	ID	CTGF	% Expression
  Name	Region	Pos	NO	Sense sequence	(0.1 nM)

  14542	CDS	774	1554	UUUGGCCCAGACCCAACUAUGAUUA	96%
  14543	CDS	776	1555	UGGCCCAGACCCAACUAUGAUUAGA	94%
  14544	CDS	785	1556	CCCAACUAUGAUUAGAGCCAACUGA	55%
  14545	CDS	786	1557	CCAACUAUGAUUAGAGCCAACUGCA	89%
  14546	CDS	934	1558	CUUGCGAAGCUGACCUGGAAGAGAA	63%
  14547	CDS	938	1559	CGAAGCUGACCUGGAAGAGAACAUA	70%
  14548	CDS	940	1560	AAGCUGACCUGGAAGAGAACAUUAA	65%
  14549	CDs	941	1561	AGCUGACCUGGAAGAGAACAUUAAA	81%
  14550	CDS	943	1562	CUGACCUGGAAGAGAACAUUAAGAA	85%
  14551	CDS	944	1563	UGACCUGGAAGAGAACAUUAAGAAA	61%
  14552	CDS	945	1564	GACCUGGAAGAGAACAUUAAGAAGA	73%
  14553	CDS	983	1565	CCGUACUCCCAAAAUCUCCAAGCCA	86%
  14554	CDS	984	1566	CGUACUCCCAAAAUCUCCAAGCCUA	64%
  14555	CDS	985	1567	GUACUCCCAAAAUCUCCAAGCCUAA	71%
  14556	CDS	986	1568	UACUCCCAAAAUCUCCAAGCCUAUA	71%
  14557	CDS	987	1569	ACUCCCAAAAUCUCCAAGCCUAUCA	84%
  14558	CDS	988	1570	CUCCCAAAAUCUCCAAGCCUAUCAA	64%
  14559	CDS	989	1571	UCCCAAAAUCUCCAAGCCUAUCAAA	64%
  14560	CDS	990	1572	CCCAAAAUCUCCAAGCCUAUCAAGA	87%
  14561	CDS	1002	1573	AAGCCUAUCAAGUUUGAGCUUUCUA	46%
  14562	CDS	1003	1574	AGCCUAUCAAGUUUGAGCUUUCUGA	30%
  14563	CDS	1004	1575	GCCUAUCAAGUUUGAGCUUUCUGGA	63%
  14564	CDS	1008	1576	AUCAAGUUUGAGCUUUCUGGCUGCA	77%
  14565	CDS	1025	1577	UGGCUGCACCAGCAUGAAGACAUAA	96%
  14566	CDS	1028	1578	CUGCACCAGCAUGAAGACAUACCGA	79%
  14567	CDS	1029	1579	UGCACCAGCAUGAAGACAUACCGAA	58%
  14568	CDS	1033	1580	CCAGCAUGAAGACAUACCGAGCUAA	59%
  14569	CDS	1035	1581	AGCAUGAAGACAUACCGAGCUAAAA	76%
  14570	CDS	1036	1582	GCAUGAAGACAUACCGAGCUAAAUA	71%
  14571	CDS	1050	1583	CGAGCUAAAUUCUGUGGAGUAUGUA	73%
  14572	CDS	1051	1584	GAGCUAAAUUCUGUGGAGUAUGUAA	72%
  14573	CDS	1053	1585	GCUAAAUUCUGUGGAGUAUGUACCA	87%
  14574	CDS	1054	1586	CUAAAUUCUGUGGAGUAUGUACCGA	83%
  14575	CDS	1135	1587	CUGACGGCGAGGUCAUGAAGAAGAA	77%
  14576	CDS	1138	1588	ACGGCGAGGUCAUGAAGAAGAACAA	72%
  14577	CDS	1139	1589	CGGCGAGGUCAUGAAGAAGAACAUA	85%
  14578	CDS	1143	1590	GAGGUCAUGAAGAAGAACAUGAUGA	83%
  14579	CDS	1145	1591	GGUCAUGAAGAAGAACAUGAUGUUA	91%
  14580	CDS	1148	1592	CAUGAAGAAGAACAUGAUGUUCAUA	92%
  14581	CDS	1157	1593	GAACAUGAUGUUCAUCAAGACCUGA	84%
  14582	CDS	1161	1594	AUGAUGUUCAUCAAGACCUGUGCCA	92%
  14583	CDS	1203	1595	GGAGACAAUGACAUCUUUGAAUCGA	62%
  14584	CDS	1204	1596	GAGACAAUGACAUCUUUGAAUCGCA	56%
  14585	CDS	1205	1597	AGACAAUGACAUCUUUGAAUCGCUA	30%
  14586	CDS	1206	1598	GACAAUGACAUCUUUGAAUCGCUGA	47%
  14587	CDS	1207	1599	ACAAUGACAUCUUUGAAUCGCUGUA	29%
  14588	CDS	1208	1600	CAAUGACAUCUUUGAAUCGCUGUAA	50%
  14589	CDS	1209	1601	AAUGACAUCUUUGAAUCGCUGUACA	39%
  14590	CDS	1210	1602	AUGACAUCUUUGAAUCGCUGUACUA	44%
  14591	CDS	1211	1603	UGACAUCUUUGAAUCGCUGUACUAA	39%
  14592	CDS	1212	1604	GACAUCUUUGAAUCGCUGUACUACA	55%
  14593	CDS	1213	1605	ACAUCUUUGAAUCGCUGUACUACAA	59%
  14594	CDS	1216	1606	UCUUUGAAUCGCUGUACUACAGGAA	80%
  14595	CDS	1217	1607	CUUUGAAUCGCUGUACUACAGGAAA	80%
  14596	CDS	1223	1608	AUCGCUGUACUACAGGAAGAUGUAA	59%
  14597	CDS	1224	1609	UCGCUGUACUACAGGAAGAUGUACA	62%
  14598	CDS	1239	1610	AAGAUGUACGGAGACAUGGCAUGAA	59%
  14599	CDS	1253	1611	CAUGGCAUGAAGCCAGAGAGUGAGA	65%
  14600	3UTR	1266	1612	CAGAGAGUGAGAGACAUUAACUCAA	43%
  14601	3UTR	1267	1613	AGAGAGUGAGAGACAUUAACUCAUA	25%
  14602	3UTR	1268	1614	GAGAGUGAGAGACAUUAACUCAUUA	33%
  14603	3UTR	1269	1615	AGAGUGAGAGACAUUAACUCAUUAA	42%
  14604	3UTR	1270	1616	GAGUGAGAGACAUUAACUCAUUAGA	28%
  14605	3UTR	1271	1617	AGUGAGAGACAUUAACUCAUUAGAA	34%
  14606	3UTR	1272	1618	GUGAGAGACAUUAACUCAUUAGACA	30%
  14607	3UTR	1273	1619	UGAGAGACAUUAACUCAUUAGACUA	33%
  14608	3UTR	1275	1620	AGAGACAUUAACUCAUUAGACUGGA	42%
  14609	3UTR	1277	1621	AGACAUUAACUCAUUAGACUGGAAA	25%
  14610	3UTR	1278	1622	GACAUUAACUCAUUAGACUGGAACA	31%
  14611	3UTR	1279	1623	ACAUUAACUCAUUAGACUGGAACUA	32%
  14612	3UTR	1281	1624	AUUAACUCAUUAGACUGGAACUUGA	23%
  14613	3UTR	1284	1625	AACUCAUUAGACUGGAACUUGAACA	39%
  14614	3UTR	1285	1626	ACUCAUUAGACUGGAACUUGAACUA	30%
  14615	3UTR	1286	1627	CUCAUUAGACUGGAACUUGAACUGA	43%
  14616	3UTR	1287	1628	UCAUUAGACUGGAACUUGAACUGAA	26%
  14617	3UTR	1291	1629	UAGACUGGAACUUGAACUGAUUCAA	33%
  14618	3UTR	1293	1630	GACUGGAACUUGAACUGAUUCACAA	43%
  14619	3UTR	1294	1631	ACUGGAACUUGAACUGAUUCACAUA	28%
  14620	3UTR	1295	1632	CUGGAACUUGAACUGAUUCACAUCA	41%
  14621	3UTR	1296	1633	UGGAACUUGAACUGAUUCACAUCUA	34%
  14622	3UTR	1298	1634	GAACUUGAACUGAUUCACAUCUCAA	31%
  14623	3UTR	1299	1635	AACUUGAACUGAUUCACAUCUCAUA	31%
  14624	3UTR	1300	1636	ACUUGAACUGAUUCACAUCUCAUUA	33%
  14625	3UTR	1301	1637	CUUGAACUGAUUCACAUCUCAUUUA	28%
  14626	3UTR	1326	1638	UCCGUAAAAAUGAUUUCAGUAGCAA	30%
  14627	3UTR	1332	1639	AAAAUGAUUUCAGUAGCACAAGUUA	28%
  14628	3UTR	1395	1640	CCCAAUUCAAAACAUUGUGCCAUGA	63%
  14629	3UTR	1397	1641	CAAUUCAAAACAUUGUGCCAUGUCA	39%
  14630	3UTR	1402	1642	CAAAACAUUGUGCCAUGUCAAACAA	34%
  14631	3UTR	1408	1643	AUUGUGCCAUGUCAAACAAAUAGUA	33%
  14632	3UTR	1409	1644	UUGUGCCAUGUCAAACAAAUAGUCA	33%
  14633	3UTR	1412	1645	UGCCAUGUCAAACAAAUAGUCUAUA	36%
  14634	3UTR	1416	1646	AUGUCAAACAAAUAGUCUAUCAACA	30%
  14635	3UTR	1435	1647	UCAACCCCAGACACUGGUUUGAAGA	39%
  14636	3UTR	1436	1648	CAACCCCAGACACUGGUUUGAAGAA	47%
  14637	3UTR	1438	1649	ACCCCAGACACUGGUUUGAAGAAUA	45%
  14638	3UTR	1439	1650	CCCCAGACACUGGUUUGAAGAAUGA	40%
  14639	3UTR	1442	1651	CAGACACUGGUUUGAAGAAUGUUAA	21%
  14640	3UTR	1449	1652	UGGUUUGAAGAAUGUUAAGACUUGA	22%
  14641	3UTR	1453	1653	UUGAAGAAUGUUAAGACUUGACAGA	24%
  14642	3UTR	1454	1654	UGAAGAAUGUUAAGACUUGACAGUA	37%
  14643	3UTR	1462	1655	GUUAAGACUUGACAGUGGAACUACA	20%
  14644	3UTR	1470	1656	UUGACAGUGGAACUACAUUAGUACA	30%
  14645	3UTR	1471	1657	UGACAGUGGAACUACAUUAGUACAA	43%
  14646	3UTR	1474	1658	CAGUGGAACUACAUUAGUACACAGA	36%
  14647	3UTR	1475	1659	AGUGGAACUACAUUAGUACACAGCA	38%
  14648	3UTR	1476	1660	GUGGAACUACAUUAGUACACAGCAA	35%
  14649	3UTR	1477	1661	UGGAACUACAUUAGUACACAGCACA	34%
  14650	3UTR	1478	1662	GGAACUACAUUAGUACACAGCACCA	33%
  14651	3UTR	1479	1663	GAACUACAUUAGUACACAGCACCAA	39%
  14652	3UTR	1480	1664	AACUACAUUAGUACACAGCACCAGA	27%
  14653	3UTR	1481	1665	ACUACAUUAGUACACAGCACCAGAA	29%
  14654	3UTR	1482	1666	CUACAUUAGUACACAGCACCAGAAA	38%
  14655	3UTR	1483	1667	UACAUUAGUACACAGCACCAGAAUA	28%
  14656	3UTR	1484	1668	ACAUUAGUACACAGCACCAGAAUGA	31%
  14657	3UTR	1486	1669	AUUAGUACACAGCACCAGAAUGUAA	26%
  14658	3UTR	1487	1670	UUAGUACACAGCACCAGAAUGUAUA	31%
  14659	3UTR	1489	1671	AGUACACAGCACCAGAAUGUAUAUA	35%
  14660	3UTR	1490	1672	GUACACAGCACCAGAAUGUAUAUUA	34%
  14661	3UTR	1497	1673	GCACCAGAAUGUAUAUUAAGGUGUA	32%
  14662	3UTR	1503	1674	GAAUGUAUAUUAAGGUGUGGCUUUA	42%
  14663	3UTR	1539	1675	AGGGUACCAGCAGAAAGGUUAGUAA	28%
  14664	3UTR	1543	1676	UACCAGCAGAAAGGUUAGUAUCAUA	29%
  14665	3UTR	1544	1677	ACCAGCAGAAAGGUUAGUAUCAUCA	33%
  14666	3UTR	1548	1678	GCAGAAAGGUUAGUAUCAUCAGAUA	34%
  14667	3UTR	1557	1679	UUAGUAUCAUCAGAUAGCAUCUUAA	22%
  14668	3UTR	1576	1680	UCUUAUACGAGUAAUAUGCCUGCUA	48%
  14669	3UTR	1577	1681	CUUAUACGAGUAAUAUGCCUGCUAA	31%
  14670	3UTR	1579	1682	UAUACGAGUAAUAUGCCUGCUAUUA	43%
  14671	3UTR	1580	1683	AUACGAGUAAUAUGCCUGCUAUUUA	39%
  14672	3UTR	1581	1684	UACGAGUAAUAUGCCUGCUAUUUGA	33%
  14673	3UTR	1582	1685	ACGAGUAAUAUGCCUGCUAUUUGAA	40%
  14674	3UTR	1584	1686	GAGUAAUAUGCCUGCUAUUUGAAGA	38%
  14675	3UTR	1585	1687	AGUAAUAUGCCUGCUAUUUGAAGUA	24%
  14676	3UTR	1586	1688	GUAAUAUGCCUGCUAUUUGAAGUGA	34%
  14677	3UTR	1587	1689	UAAUAUGCCUGCUAUUUGAAGUGUA	26%
  14678	3UTR	1589	1690	AUAUGCCUGCUAUUUGAAGUGUAAA	26%
  14679	3UTR	1591	1691	AUGCCUGCUAUUUGAAGUGUAAUUA	25%
  14680	3UTR	1596	1692	UGCUAUUUGAAGUGUAAUUGAGAAA	36%
  14681	3UTR	1599	1693	UAUUUGAAGUGUAAUUGAGAAGGAA	22%
  14682	3UTR	1600	1694	AUUUGAAGUGUAAUUGAGAAGGAAA	22%
  14683	3UTR	1601	1695	UUUGAAGUGUAAUUGAGAAGGAAAA	19%
  14684	3UTR	1609	1696	GUAAUUGAGAAGGAAAAUUUUAGCA	53%
  14685	3UTR	1610	1697	UAAUUGAGAAGGAAAAUUUUAGCGA	55%
  14686	3UTR	1611	1698	AAUUGAGAAGGAAAAUUUUAGCGUA	20%
  14687	3UTR	1612	1699	AUUGAGAAGGAAAAUUUUAGCGUGA	23%
  14688	3UTR	1613	1700	UUGAGAAGGAAAAUUUUAGCGUGCA	37%
  14689	3UTR	1614	1701	UGAGAAGGAAAAUUUUAGCGUGCUA	31%
  14690	3UTR	1619	1702	AGGAAAAUUUUAGCGUGCUCACUGA	46%
  14691	3UTR	1657	1703	CCAGUGACAGCUAGGAUGUGCAUUA	42%
  14692	3UTR	1661	1704	UGACAGCUAGGAUGUGCAUUCUCCA	39%
  14693	3UTR	1682	1705	UCCAGCCAUCAAGAGACUGAGUCAA	53%
  14694	3UTR	1685	1706	AGCCAUCAAGAGACUGAGUCAAGUA	71%
  14695	3UTR	1686	1707	GCCAUCAAGAGACUGAGUCAAGUUA	54%
  14696	3UTR	1687	1708	CCAUCAAGAGACUGAGUCAAGUUGA	71%
  14697	3UTR	1688	1709	CAUCAAGAGACUGAGUCAAGUUGUA	74%
  14698	3UTR	1689	1710	AUCAAGAGACUGAGUCAAGUUGUUA	61%
  14699	3UTR	1690	1711	UCAAGAGACUGAGUCAAGUUGUUCA	59%
  14700	3UTR	1691	1712	CAAGAGACUGAGUCAAGUUGUUCCA	73%
  14701	3UTR	1692	1713	AAGAGACUGAGUCAAGUUGUUCCUA	78%
  14702	3UTR	1693	1714	AGAGACUGAGUCAAGUUGUUCCUUA	60%
  14703	3UTR	1695	1715	AGACUGAGUCAAGUUGUUCCUUAAA	63%
  14704	3UTR	1696	1716	GACUGAGUCAAGUUGUUCCUUAAGA	92%
  14705	3UTR	1697	1717	ACUGAGUCAAGUUGUUCCUUAAGUA	74%
  14706	3UTR	1707	1718	GUUGUUCCUUAAGUCAGAACAGCAA	70%
  14707	3UTR	1724	1719	AACAGCAGACUCAGCUCUGACAUUA	69%
  14708	3UTR	1725	1720	ACAGCAGACUCAGCUCUGACAUUCA	67%
  14709	3UTR	1726	1721	CAGCAGACUCAGCUCUGACAUUCUA	71%
  14710	3UTR	1727	1722	AGCAGACUCAGCUCUGACAUUCUGA	73%
  14711	3UTR	1728	1723	GCAGACUCAGCUCUGACAUUCUGAA	60%
  14712	3UTR	1729	1724	CAGACUCAGCUCUGACAUUCUGAUA	72%
  14713	3UTR	1732	1725	ACUCAGCUCUGACAUUCUGAUUCGA	24%
  14714	3UTR	1733	1726	CUCAGCUCUGACAUUCUGAUUCGAA	32%
  14715	3UTR	1734	1727	UCAGCUCUGACAUUCUGAUUCGAAA	23%
  14716	3UTR	1735	1728	CAGCUCUGACAUUCUGAUUCGAAUA	27%
  14717	3UTR	1736	1729	AGCUCUGACAUUCUGAUUCGAAUGA	38%
  14718	3UTR	1739	1730	UCUGACAUUCUGAUUCGAAUGACAA	28%
  14719	3UTR	1741	1731	UGACAUUCUGAUUCGAAUGACACUA	29%
  14720	3UTR	1742	1732	GACAUUCUGAUUCGAAUGACACUGA	33%
  14721	3UTR	1743	1733	ACAUUCUGAUUCGAAUGACACUGUA	28%
  14722	3UTR	1747	1734	UCUGAUUCGAAUGACACUGUUCAGA	39%
  14723	3UTR	1748	1735	CUGAUUCGAAUGACACUGUUCAGGA	36%
  14724	3UTR	1750	1736	GAUUCGAAUGACACUGUUCAGGAAA	33%
  14725	3UTR	1751	1737	AUUCGAAUGACACUGUUCAGGAAUA	30%
  14726	3UTR	1759	1738	GACACUGUUCAGGAAUCGGAAUCCA	34%
  14727	3UTR	1760	1739	ACACUGUUCAGGAAUCGGAAUCCUA	35%
  14728	3UTR	1761	1740	CACUGUUCAGGAAUCGGAAUCCUGA	40%
  14729	3UTR	1768	1741	CAGGAAUCGGAAUCCUGUCGAUUAA	34%
  14730	3UTR	1769	1742	AGGAAUCGGAAUCCUGUCGAUUAGA	31%
  14731	3UTR	1770	1743	GGAAUCGGAAUCCUGUCGAUUAGAA	24%
  14732	3UTR	1771	1744	GAAUCGGAAUCCUGUCGAUUAGACA	32%
  14733	3UTR	1772	1745	AAUCGGAAUCCUGUCGAUUAGACUA	29%
  14734	3UTR	1774	1746	UCGGAAUCCUGUCGAUUAGACUGGA	34%
  14735	3UTR	1777	1747	GAAUCCUGUCGAUUAGACUGGACAA	51%
  14736	3UTR	1782	1748	CUGUCGAUUAGACUGGACAGCUUGA	88%
  14737	3UTR	1783	1749	UGUCGAUUAGACUGGACAGCUUGUA	38%
  14738	3UTR	1797	1750	GACAGCUUGUGGCAAGUGAAUUUGA	46%
  14739	3UTR	1798	1751	ACAGCUUGUGGCAAGUGAAUUUGCA	52%
  14740	3UTR	1800	1752	AGCUUGUGGCAAGUGAAUUUGCCUA	43%
  14741	3UTR	1801	1753	GCUUGUGGCAAGUGAAUUUGCCUGA	51%
  14742	3UTR	1802	1754	CUUGUGGCAAGUGAAUUUGCCUGUA	32%
  14743	3UTR	1803	1755	UUGUGGCAAGUGAAUUUGCCUGUAA	31%
  14744	3UTR	1804	1756	UGUGGCAAGUGAAUUUGCCUGUAAA	29%
  14745	3UTR	1805	1757	GUGGCAAGUGAAUUUGCCUGUAACA	20%
  14746	3UTR	1806	1758	UGGCAAGUGAAUUUGCCUGUAACAA	34%
  14747	3UTR	1807	1759	GGCAAGUGAAUUUGCCUGUAACAAA	31%
  14748	3UTR	1808	1760	GCAAGUGAAUUUGCCUGUAACAAGA	27%
  14749	3UTR	1809	1761	CAAGUGAAUUUGCCUGUAACAAGCA	34%
  14750	3UTR	1810	1762	AAGUGAAUUUGCCUGUAACAAGCCA	36%
  14751	3UTR	1811	1763	AGUGAAUUUGCCUGUAACAAGCCAA	31%
  14752	3UTR	1814	1764	GAAUUUGCCUGUAACAAGCCAGAUA	24%
  14753	3UTR	1815	1765	AAUUUGCCUGUAACAAGCCAGAUUA	21%
  14754	3UTR	1816	1766	AUUUGCCUGUAACAAGCCAGAUUUA	22%
  14755	3UTR	1910	1767	AAGUUAAUUUAAAGUUGUUUGUGCA	58%
  14756	3UTR	1911	1768	AGUUAAUUUAAAGUUGUUUGUGCCA	73%
  14757	3UTR	1912	1769	GUUAAUUUAAAGUUGUUUGUGCCUA	64%
  14758	3UTR	1957	1770	UUUGAUAUUUCAAUGUUAGCCUCAA	42%
  14759	3UTR	1961	1771	AUAUUUCAAUGUUAGCCUCAAUUUA	30%
  14760	3UTR	1971	1772	GUUAGCCUCAAUUUCUGAACACCAA	34%
  14761	3UTR	1974	1773	AGCCUCAAUUUCUGAACACCAUAGA	35%
  14762	3UTR	1975	1774	GCCUCAAUUUCUGAACACCAUAGGA	33%
  14763	3UTR	1976	1775	CCUCAAUUUCUGAACACCAUAGGUA	39%
  14764	3UTR	1977	1776	CUCAAUUUCUGAACACCAUAGGUAA	27%
  14765	3UTR	1978	1777	UCAAUUUCUGAACACCAUAGGUAGA	31%
  14766	3UTR	1979	1778	CAAUUUCUGAACACCAUAGGUAGAA	49%
  14767	3UTR	1980	1779	AAUUUCUGAACACCAUAGGUAGAAA	46%
  14768	3UTR	1981	1780	AUUUCUGAACACCAUAGGUAGAAUA	40%
  14769	3UTR	1982	1781	UUUCUGAACACCAUAGGUAGAAUGA	47%
  14770	3UTR	1985	1782	CUGAACACCAUAGGUAGAAUGUAAA	33%
  14771	3UTR	1986	1783	UGAACACCAUAGGUAGAAUGUAAAA	35%
  14772	3UTR	1987	1784	GAACACCAUAGGUAGAAUGUAAAGA	31%
  14773	3UTR	1988	1785	AACACCAUAGGUAGAAUGUAAAGCA	30%
  14774	3UTR	1989	1786	ACACCAUAGGUAGAAUGUAAAGCUA	32%
  14775	3UTR	1991	1787	ACCAUAGGUAGAAUGUAAAGCUUGA	31%
  14776	3UTR	1992	1788	CCAUAGGUAGAAUGUAAAGCUUGUA	34%
  14777	3UTR	1993	1789	CAUAGGUAGAAUGUAAAGCUUGUCA	31%
  14778	3UTR	1994	1790	AUAGGUAGAAUGUAAAGCUUGUCUA	28%
  14779	3UTR	1996	1791	AGGUAGAAUGUAAAGCUUGUCUGAA	32%
  14780	3UTR	2002	1792	AAUGUAAAGCUUGUCUGAUCGUUCA	34%
  14781	3UTR	2017	1793	UGAUCGUUCAAAGCAUGAAAUGGAA	31%
  14782	3UTR	2021	1794	CGUUCAAAGCAUGAAAUGGAUACUA	39%
  14783	3UTR	2022	1795	GUUCAAAGCAUGAAAUGGAUACUUA	25%
  14784	3UTR	2023	1796	UUCAAAGCAUGAAAUGGAUACUUAA	22%
  14785	3UTR	2047	1797	UAUGGAAAUUCUGCUCAGAUAGAAA	39%
  14786	3UTR	2048	1798	AUGGAAAUUCUGCUCAGAUAGAAUA	35%
  14787	3UTR	2059	1799	GCUCAGAUAGAAUGACAGUCCGUCA	44%
  14788	3UTR	2060	1800	CUCAGAUAGAAUGACAGUCCGUCAA	41%
  14789	3UTR	2062	1801	CAGAUAGAAUGACAGUCCGUCAAAA	46%
  14790	3UTR	2063	1802	AGAUAGAAUGACAGUCCGUCAAAAA	45%
  14791	3UTR	2065	1803	AUAGAAUGACAGUCCGUCAAAACAA	41%
  14792	3UTR	2067	1804	AGAAUGACAGUCCGUCAAAACAGAA	36%
  14793	3UTR	2068	1805	GAAUGACAGUCCGUCAAAACAGAUA	40%
  14794	3UTR	2113	1806	AGUGUCCUUGGCAGGCUGAUUUCUA	42%
  14795	3UTR	2114	1807	GUGUCCUUGGCAGGCUGAUUUCUAA	42%
  14796	3UTR	2118	1808	CCUUGGCAGGCUGAUUUCUAGGUAA	111%
  14797	3UTR	2127	1809	GCUGAUUUCUAGGUAGGAAAUGUGA	44%
  14798	3UTR	2128	1810	CUGAUUUCUAGGUAGGAAAUGUGGA	44%
  14799	3UTR	2130	1811	GAUUUCUAGGUAGGAAAUGUGGUAA	46%
  14800	3UTR	2131	1812	AUUUCUAGGUAGGAAAUGUGGUAGA	45%
  14801	3UTR	2142	1813	GGAAAUGUGGUAGCCUCACUUUUAA	37%
  14802	3UTR	2146	1814	AUGUGGUAGCCUCACUUUUAAUGAA	39%
  14803	3UTR	2149	1815	UGGUAGCCUCACUUUUAAUGAACAA	40%
  14804	3UTR	2154	1816	GCCUCACUUUUAAUGAACAAAUGGA	35%
  14805	3UTR	2155	1817	CCUCACUUUUAAUGAACAAAUGGCA	41%
  14806	3UTR	2181	1818	UUAUUAAAAACUGAGUGACUCUAUA	26%
  14807	3UTR	2182	1819	UAUUAAAAACUGAGUGACUCUAUAA	29%
  14808	3UTR	2183	1820	AUUAAAAACUGAGUGACUCUAUAUA	28%
  14809	3UTR	2186	1821	AAAAACUGAGUGACUCUAUAUAGCA	31%
  14810	3UTR	2187	1822	AAAACUGAGUGACUCUAUAUAGCUA	28%
  14811	3UTR	2188	1823	AAACUGAGUGACUCUAUAUAGCUGA	38%
  14812	3UTR	2189	1824	AACUGAGUGACUCUAUAUAGCUGAA	44%
  14813	3UTR	2190	1825	ACUGAGUGACUCUAUAUAGCUGAUA	38%
  14814	3UTR	2255	1826	ACUGUUUUUCGGACAGUUUAUUUGA	29%
  14815	3UTR	2256	1827	CUGUUUUUCGGACAGUUUAUUUGUA	25%
  14816	3UTR	2263	1828	UCGGACAGUUUAUUUGUUGAGAGUA	29%
  14817	3UTR	2265	1829	GGACAGUUUAUUUGUUGAGAGUGUA	24%
  14818	3UTR	2268	1830	CAGUUUAUUUGUUGAGAGUGUGACA	26%
  14819	3UTR	2269	1831	AGUUUAUUUGUUGAGAGUGUGACCA	37%
  14820	3UTR	2272	1832	UUAUUUGUUGAGAGUGUGACCAAAA	27%
  14821	3UTR	2273	1833	UAUUUGUUGAGAGUGUGACCAAAAA	30%
  14822	3UTR	2274	1834	AUUUGUUGAGAGUGUGACCAAAAGA	26%
  14823	3UTR	2275	1835	UUUGUUGAGAGUGUGACCAAAAGUA	27%
  14824	3UTR	2276	1836	UUGUUGAGAGUGUGACCAAAAGUUA	30%
  14825	3UTR	2277	1837	UGUUGAGAGUGUGACCAAAAGUUAA	29%
  14826	3UTR	2278	1838	GUUGAGAGUGUGACCAAAAGUUACA	33%
  14827	3UTR	2279	1839	UUGAGAGUGUGACCAAAAGUUACAA	35%
  14828	3UTR	2281	1840	GAGAGUGUGACCAAAAGUUACAUGA	36%
  14829	3UTR	2282	1841	AGAGUGUGACCAAAAGUUACAUGUA	36%
  14830	3UTR	2283	1842	GAGUGUGACCAAAAGUUACAUGUUA	33%
  14831	3UTR	2284	1843	AGUGUGACCAAAAGUUACAUGUUUA	31%
  14832	3UTR	2285	1844	GUGUGACCAAAAGUUACAUGUUUGA	22%
  14833	3UTR	2286	1845	UGUGACCAAAAGUUACAUGUUUGCA	40%
  14834	3UTR	2293	1846	AAAAGUUACAUGUUUGCACCUUUCA	24%
  14835	3UTR	2295	1847	AAGUUACAUGUUUGCACCUUUCUAA	23%
  14836	3UTR	2296	1848	AGUUACAUGUUUGCACCUUUCUAGA	29%
  14837	3UTR	2299	1849	UACAUGUUUGCACCUUUCUAGUUGA	27%
  14838	3UTR	2300	1850	ACAUGUUUGCACCUUUCUAGUUGAA	29%
  14839	3UTR	2301	1851	CAUGUUUGCACCUUUCUAGUUGAAA	35%

  Key:

  PS	*	= Phosphothioate Backbone Linkage
  RNA	G	= Guanine
  RNA	U	= Uracil
  RNA	C	= Cytosine
  RNA	A	= Adenine
  	m
  	2′ Omethyl
  	f
  	2′-Fluoro
  Phosphate	P
  	5′ Phosphate

• TABLE 12
  Inhibition of gene expression with CTGF ori sequences (Accession
  Number: NM_001901.2)

  Oligo	Gene	Ref	SEQ ID	25-mer Sense Strand (position 25 of	A549 0.1
  ID	Region	Pos	NO	SS, replaced with A)	nM Activity
  				25-mer Sense Strand (position 25 of
  				SS, original base, not replaced by A)
  13843	CDS	1047	1852	UACCGAGCUAAAUUCUGUGGAGUAU	113%
  13844	3UTR	2164	1853	UAAUGAACAAAUGGCCUUUAUUAAA	61%
  13845	3UTR	1795	1854	UGGACAGCUUGUGGCAAGUGAAUUU	99%
  13846	CDS	1228	1855	UGUACUACAGGAAGAUGUACGGAGA	87%
  13847	CDS	1146	1856	GUCAUGAAGAAGAACAUGAUGUUCA	98%
  13848	CDS	1150	1857	UGAAGAAGAACAUGAUGUUCAUCAA	105%
  13849	CDS	1218	1858	UUUGAAUCGCUGUACUACAGGAAGA	91%
  13850	3UTR	2262	1859	UUCGGACAGUUUAUUUGUUGAGAGU	50%
  13851	CDS	1147	1860	UCAUGAAGAAGAACAUGAUGUUCAU	104%
  13852	3UTR	2163	1861	UUAAUGAACAAAUGGCCUUUAUUAA	54%
  13853	3UTR	1414	1862	CCAUGUCAAACAAAUAGUCUAUCAA	35%
  13854	CDS	1195	1863	ACUGUCCCGGAGACAAUGACAUCUU	103%
  13855	3UTR	1788	1864	AUUAGACUGGACAGCUUGUGGCAAG	103%
  13856	3UTR	1793	1865	ACUGGACAGCUUGUGGCAAGUGAAU	81%
  13857	3UTR	1891	1866	UAUAUAUGUACAGUUAUCUAAGUUA	73%
  13858	3UTR	2270	1867	GUUUAUUUGUUGAGAGUGUGACCAA	76%
  13859		482	1868	CAAGAUCGGCGUGUGCACCGCCAAA	95%
  13860	CDS	942	1869	GCUGACCUGGAAGAGAACAUUAAGA	93%
  13861	CDS	1199	1870	UCCCGGAGACAAUGACAUCUUUGAA	83%
  13862	3UTR	2258	1871	GUUUUUCGGACAGUUUAUUUGUUGA	40%
  13863	CDS	1201	1872	CCGGAGACAAUGACAUCUUUGAAUC	123%
  13864	CDS	543	1873	CGCAGCGGAGAGUCCUUCCAGAGCA	124%
  13865	3UTR	1496	1874	AGCACCAGAAUGUAUAUUAAGGUGU	109%
  13866	CDS	793	1875	UGAUUAGAGCCAACUGCCUGGUCCA	125%
  13867	CDS	1198	1876	GUCCCGGAGACAAUGACAUCUUUGA	64%
  13868	3UTR	2160	1877	CUUUUAAUGAACAAAUGGCCUUUAU	68%
  13869	CDS	1149	1878	AUGAAGAAGAACAUGAUGUUCAUCA	107%
  13870	CDS	1244	1879	GUACGGAGACAUGGCAUGAAGCCAG	107%
  13871	3UTR	1495	1880	CAGCACCAGAAUGUAUAUUAAGGUG	77%
  13872		475	1881	CCAACCGCAAGAUCGGCGUGUGCAC	113%
  13873	CDS	806	1882	CUGCCUGGUCCAGACCACAGAGUGG	113%
  13874	CDS	819	1883	ACCACAGAGUGGAGCGCCUGUUCCA	99%
  13875	CDS	1221	1884	GAAUCGCUGUACUACAGGAAGAUGU	97%
  13876	CDS	1152	1885	AAGAAGAACAUGAUGUUCAUCAAGA	121%
  13877	CDS	1163	1886	GAUGUUCAUCAAGACCUGUGCCUGC	125%
  13878	3UTR	1494	1887	ACAGCACCAGAAUGUAUAUUAAGGU	94%
  13879	3UTR	1890	1888	AUAUAUAUGUACAGUUAUCUAAGUU	94%
  13880		473	1889	GGCCAACCGCAAGAUCGGCGUGUGC	122%
  13881		544	1890	GCAGCGGAGAGUCCUUCCAGAGCAG	111%
  13882	CDS	883	1891	ACAACGCCUCCUGCAGGCUAGAGAA	105%
  13883	CDS	1240	1892	AGAUGUACGGAGACAUGGCAUGAAG	99%
  13884	CDS	1243	1893	UGUACGGAGACAUGGCAUGAAGCCA	116%
  13885	3UTR	2266	1894	GACAGUUUAUUUGUUGAGAGUGUGA	53%
  13886	CDS	1011	1895	AAGUUUGAGCUUUCUGGCUGCACCA	118%
  13887	CDS	1020	1896	CUUUCUGGCUGCACCAGCAUGAAGA	110%
  13888	CDS	1168	1897	UCAUCAAGACCUGUGCCUGCCAUUA	119%
  13889		1415	1898	CAUGUCAAACAAAUAGUCUAUCAAC	64%
  13890	3UTR	1792	1899	GACUGGACAGCUUGUGGCAAGUGAA	53%
  13891	3UTR	2156	1900	CUCACUUUUAAUGAACAAAUGGCCU	119%
  13892		379	1901	GCUGCCGCGUCUGCGCCAAGCAGCU	112%
  13893	CDS	1229	1902	GUACUACAGGAAGAUGUACGGAGAC	112%
  13894	3UTR	1791	1903	AGACUGGACAGCUUGUGGCAAGUGA	65%
  13895	3UTR	2158	1904	CACUUUUAAUGAACAAAUGGCCUUU	76%
  13896		488	1905	CGGCGUGUGCACCGCCAAAGAUGGU	89%
  13897	CDS	1151	1906	GAAGAAGAACAUGAUGUUCAUCAAG	119%
  13898	CDS	1156	1907	AGAACAUGAUGUUCAUCAAGACCUG	125%
  13899	CDS	1237	1908	GGAAGAUGUACGGAGACAUGGCAUG	114%
  13900	CDS	1202	1909	CGGAGACAAUGACAUCUUUGAAUCG	130%
  13901	CDS	1236	1910	AGGAAGAUGUACGGAGACAUGGCAU	135%
  13902	3UTR	1786	1911	CGAUUAGACUGGACAGCUUGUGGCA	119%
  13903	3UTR	1789	1912	UUAGACUGGACAGCUUGUGGCAAGU	108%
  13904	3UTR	2290	1913	ACCAAAAGUUACAUGUUUGCACCUU	90%
  13905	CDS	1017	1914	GAGCUUUCUGGCUGCACCAGCAUGA	121%
  13906	CDS	1197	1915	UGUCCCGGAGACAAUGACAUCUUUG	125%
  13907	CDS	1219	1916	UUGAAUCGCUGUACUACAGGAAGAU	98%
  13908	3UTR	2159	1917	ACUUUUAAUGAACAAAUGGCCUUUA	52%
  13909		486	1918	AUCGGCGUGUGCACCGCCAAAGAUG	119%
  13910	CDS	826	1919	AGUGGAGCGCCUGUUCCAAGACCUG	139%
  13911	CDS	1022	1920	UUCUGGCUGCACCAGCAUGAAGACA	144%
  13912	3UTR	1492	1921	ACACAGCACCAGAAUGUAUAUUAAG	99%
  13913	3UTR	1781	1922	CCUGUCGAUUAGACUGGACAGCUUG	89%
  13914		485	1923	GAUCGGCGUGUGCACCGCCAAAGAU	131%
  13915	CDS	1007	1924	UAUCAAGUUUGAGCUUUCUGGCUGC	92%
  13916	CDS	1242	1925	AUGUACGGAGACAUGGCAUGAAGCC	106%
  13917	3UTR	1787	1926	GAUUAGACUGGACAGCUUGUGGCAA	104%
  13918	3UTR	1889	1927	UAUAUAUAUGUACAGUUAUCUAAGU	78%
  13919	3UTR	2294	1928	AAAGUUACAUGUUUGCACCUUUCUA	28%
  13920	CDS	821	1929	CACAGAGUGGAGCGCCUGUUCCAAG	108%
  13921	CDS	884	1930	CAACGCCUCCUGCAGGCUAGAGAAG	125%
  13922	3UTR	2260	1931	UUUUCGGACAGUUUAUUUGUUGAGA	43%
  13923	CDS	889	1932	CCUCCUGCAGGCUAGAGAAGCAGAG	95%
  13924	CDS	1226	1933	GCUGUACUACAGGAAGAUGUACGGA	122%
  13925	3UTR	1493	1934	CACAGCACCAGAAUGUAUAUUAAGG	88%
  13926	3UTR	1799	1935	CAGCUUGUGGCAAGUGAAUUUGCCU	89%
  13927	CDS	807	1936	UGCCUGGUCCAGACCACAGAGUGGA	101%
  13928	CDS	1107	1937	ACCACCCUGCCGGUGGAGUUCAAGU	113%
  13929	CDS	1155	1938	AAGAACAUGAUGUUCAUCAAGACCU	109%
  13930	CDS	1169	1939	CAUCAAGACCUGUGCCUGCCAUUAC	89%
  13931	CDS	1241	1940	GAUGUACGGAGACAUGGCAUGAAGC	96%
  13932	3UTR	1794	1941	CUGGACAGCUUGUGGCAAGUGAAUU	73%
  13933	3UTR	1888	1942	AUAUAUAUAUGUACAGUUAUCUAAG	98%
  13934	3UTR	2289	1943	GACCAAAAGUUACAUGUUUGCACCU	77%
  13935		373	1944	GCGGCUGCUGCCGCGUCUGCGCCAA	85%
  13936	CDS	799	1945	GAGCCAACUGCCUGGUCCAGACCAC	126%
  13937	CDS	802	1946	CCAACUGCCUGGUCCAGACCACAGA	122%
  13938	CDS	1166	1947	GUUCAUCAAGACCUGUGCCUGCCAU	106%

• TABLE 13
  Inhibition of gene expression with SPP1 sd-rxRNA sequences (Accession
  Number: NM_000582.2)

  		SEQ		SEQ		% remaining
  Oligo	Start	ID	Sense	ID	Antisense	expression (1
  Number	Site	NO	sequence	NO	sequence	uM A549)
  14084	1025	1948	CUCAUGAAUUAGA	1949	UCUAAUUCAUGAGAA	61%
  					AUAC
  14085	1049	1950	CUGAGGUCAAUUA	1951	UAAUUGACCUCAGAA	50%
  					GAUG
  14086	1051	1952	GAGGUCAAUUAAA	1953	UUUAAUUGACCUCAG	n/a
  					AAGA
  14087	1048	1954	UCUGAGGUCAAUU	1955	AAUUGACCUCAGAAG	69%
  					AUGC
  14088	1050	1956	UGAGGUCAAUUAA	1957	UUAAUUGACCUCAGA	76%
  					AGAU
  14089	1047	1958	UUCUGAGGUCAAU	1959	AUUGACCUCAGAAGA	60%
  					UGCA
  14090	800	1960	GUCAGCUGGAUGA	1961	UCAUCCAGCUGACUC	71%
  					GUUU
  14091	492	1962	UUCUGAUGAAUCU	1963	AGAUUCAUCAGAAUG	n/a
  					GUGA
  14092	612	1964	UGGACUGAGGUCA	1965	UGACCUCAGUCCAUA	n/a
  					AACC
  14093	481	1966	GAGUCUCACCAUU	1967	AAUGGUGAGACUCAU	n/a
  					CAGA
  14094	614	1968	GACUGAGGUCAAA	1969	UUUGACCUCAGUCCA	n/a
  					UAAA
  14095	951	1970	UCACAGCCAUGAA	1971	UUCAUGGCUGUGAAA	89%
  					UUCA
  14096	482	1972	AGUCUCACCAUUC	1973	GAAUGGUGAGACUCA	87%
  					UCAG
  14097	856	1974	AAGCGGAAAGCCA	1975	UGGCUUUCCGCUUAU	88%
  					AUAA
  14098	857	1976	AGCGGAAAGCCAA	1977	UUGGCUUUCCGCUUA	113%
  					UAUA
  14099	365	1978	ACCACAUGGAUGA	1979	UCAUCCAUGUGGUCA	98%
  					UGGC
  14100	359	1980	GCCAUGACCACAU	1981	AUGUGGUCAUGGCU	84%
  					UUCGU
  14101	357	1982	AAGCCAUGACCAC	1983	GUGGUCAUGGCUUU	88%
  					CGUUG
  14102	858	1984	GCGGAAAGCCAAU	1985	AUUGGCUUUCCGCUU	n/a
  					AUAU
  14103	1012	1986	AAAUUUCGUAUU	1987	AAAUACGAAAUUUCA	93%
  			U		GGUG
  14104	1014	1988	AUUUCGUAUUUC	1989	AGAAAUACGAAAUUU	89%
  			U		CAGG
  14105	356	1990	AAAGCCAUGACCA	1991	UGGUCAUGGCUUUC	85%
  					GUUGG
  14106	368	1992	ACAUGGAUGAUAU	1993	AUAUCAUCCAUGUGG	67%
  					UCAU
  14107	1011	1994	GAAAUUUCGUAU	1995	AAUACGAAAUUUCAG	87%
  			U		GUGU
  14108	754	1996	GCGCCUUCUGAUU	1997	AAUCAGAAGGCGCGU	73%
  					UCAG
  14109	1021	1998	AUUUCUCAUGAAU	1999	AUUCAUGAGAAAUAC	128%
  					GAAA
  14110	1330	2000	CUCUCAUGAAUAG	2001	CUAUUCAUGAGAGAA	101%
  					UAAC
  14111	346	2002	AAGUCCAACGAAA	2003	UUUCGUUGGACUUA	59%
  					CUUGG
  14112	869	2004	AUGAUGAGAGCAA	2005	UUGCUCUCAUCAUUG	89%
  					GCUU
  14113	701	2006	GCGAGGAGUUGAA	2007	UUCAACUCCUCGCUU	95%
  					UCCA
  14114	896	2008	UGAUUGAUAGUC	2009	UGACUAUCAAUCACA	87%
  			A		UCGG
  14115	1035	2010	AGAUAGUGCAUCU	2011	AGAUGCACUAUCUAA	82%
  					UUCA
  14116	1170	2012	AUGUGUAUCUAU	2013	AAUAGAUACACAUUC	36%
  			U		AACC
  14117	1282	2014	UUCUAUAGAAGAA	2015	UUCUUCUAUAGAAU	91%
  					GAACA
  14118	1537	2016	UUGUCCAGCAAUU	2017	AAUUGCUGGACAACC	152%
  					GUGG
  14119	692	2018	ACAUGGAAAGCGA	2019	UCGCUUUCCAUGUGU	n/a
  					GAGG
  14120	840	2020	GCAGUCCAGAUUA	2021	UAAUCUGGUACUGCUU	87%
  					GUGG
  14121	1163	2022	UGGUUGAAUGUG	2023	ACACAUUCAACCAAU	31%
  			U		AAAC
  14122	789	2024	UUAUGAAACGAGU	2025	ACUCGUUUCAUAACU	96%
  					GUCC
  14123	841	2026	CAGUCCAGAUUAU	2027	AUAAUCUGGACUGCU	110%
  					UGUG
  14124	852	2028	AUAUAAGCGGAAA	2029	UUUCCGCUUAUAUAA	91%
  					UCUG
  14125	209	2030	UACCAGUUAAACA	2031	UGUUUAACUGGUAU	110%
  					GGCAC
  14126	1276	2032	UGUUCAUUCUAU	2033	UAUAGAAUGAACAUA	n/a
  			A		GACA
  14127	137	2034	CCGACCAAGGAAA	2035	UUUCCUUGGUCGGC	71%
  					GUUUG
  14128	711	2036	GAAUGGUGCAUAC	2037	GUAUGCACCAUUCAA	115%
  					CUCC
  14129	582	2038	AUAUGAUGGCCGA	2039	UCGGCCAUCAUAUGU	97%
  					GUCU
  14130	839	2040	AGCAGUCCAGAUU	2041	AAUCUGGACUGCUUG	102%
  					UGGC
  14131	1091	2042	GCAUUUAGUCAAA	2043	UUUGACUAAAUGCAA	10%
  					AGUG
  14132	884	2044	AGCAUUCCGAUGU	2045	ACAUCGGAAUGCUCA	93%
  					UUGC
  14133	903	2046	UAGUCAGGAACUU	2047	AAGUUCCUGACUAUC	97%
  					AAUC
  14134	1090	2048	UGCAUUUAGUCAA	2049	UUGACUAAAUGCAAA	39%
  					GUGA
  14135	474	2050	GUCUGAUGAGUC	2051	AGACUCAUCAGACUG	99%
  			U		GUGA
  14136	575	2052	UAGACACAUAUGA	2053	UCAUAUGUGUCUACU	108%
  					GUGG
  14137	671	2054	CAGACGAGGACAU	2055	AUGUCCUCGUCUGUA	98%
  					GCAU
  14138	924	2056	CAGCCGUGAAUUC	2057	GAAUUCACGGCUGAC	100%
  					UUUG
  14139	1185	2058	AGUCUGGAAAUAA	2059	UUAUUUCCAGACUCA	47%
  					AAUA
  14140	1221	2060	AGUUUGUGGCUU	2061	GAAGCCACAAACUAA	100%
  			C		ACUA
  14141	347	2062	AGUCCAACGAAAG	2063	CUUUCGUUGGACUU	103%
  					ACUUG
  14142	634	2064	AAGUUUCGCAGAC	2065	GUCUGCGAAACUUCU	100%
  					UAGA
  14143	877	2066	AGCAAUGAGCAUU	2067	AAUGCUCAUUGCUCU	104%
  					CAUC
  14144	1033	2068	UUAGAUAGUGCA	2069	AUGCACUAUCUAAUU	95%
  			U		CAUG
  14145	714	2070	UGGUGCAUACAAG	2071	CUUGUAUGCACCAUU	101%
  					CAAC
  14146	791	2072	AUGAAACGAGUCA	2073	UGACUCGUUUCAUAA	100%
  					CUGU
  14147	813	2074	CCAGAGUGCUGAA	2075	UUCAGCACUCUGGUC	97%
  					AUCC
  14148	939	2076	CAGCCAUGAAUUU	2077	AAAUUCAUGGCUGUG	109%
  					GAAU
  14149	1161	2078	AUUGGUUGAAUG	2079	ACAUUCAACCAAUAA	34%
  			U		ACUG
  14150	1164	2080	GGUUGAAUGUGU	2081	UACACAUUCAACCAA	n/a
  			A		UAAA
  14151	1190	2082	GGAAAUAACUAAU	2083	AUUAGUUAUUUCCA	n/a
  					GACUC
  14152	1333	2084	UCAUGAAUAGAAA	2085	UUUCUAUUCAUGAG	31%
  					AGAAU
  14153	537	2086	GCCAGCAACCGAA	2087	UUCGGUUGCUGGCA	n/a
  					GGUCC
  14154	684	2088	CACCUCACACAUG	2089	CAUGUGUGAGGUGA	100%
  					UGUCC
  14155	707	2090	AGUUGAAUGGUG	2091	GCACCAUUCAACUCC	99%
  			C		UCGC
  14156	799	2092	AGUCAGCUGGAUG	2093	CAUCCAGCUGACUCG	95%
  					UUUC
  14157	853	2094	UAUAAGCGGAAAG	2095	CUUUCCGCUUAUAUA	106%
  					AUCU
  14158	888	2096	UUCCGAUGUGAU	2097	AAUCACAUCGGAAUG	88%
  			U		CUCA
  14159	1194	2098	AUAACUAAUGUGU	2099	ACACAUUAGUUAUUU	95%
  					CCAG
  14160	1279	2100	UCAUUCUAUAGAA	2101	UUCUAUAGAAUGAAC	15%
  					AUAG
  14161	1300	2102	AACUAUCACUGUA	2103	UACAGUGAUAGUUU	86%
  					GCAUU
  14162	1510	2104	GUCAAUUGCUUAU	2105	AUAAGCAAUUGACAC	86%
  					CACC
  14163	1543	2106	AGCAAUUAAUAAA	2107	UUUAUUAAUUGCUG	110%
  					GACAA
  14164	434	2108	ACGACUCUGAUGA	2109	UCAUCAGAGUCGUUC	134%
  					GAGU
  14165	600	2110	UAGUGUGGUUUA	2111	AUAAACCACACUAUC	102%
  			U		ACCU
  14166	863	2112	AAGCCAAUGAUGA	2113	UCAUCAUUGGCUUUC	93%
  					CGCU
  14167	902	2114	AUAGUCAGGAACU	2115	AGUUCCUGACUAUCA	101%
  					AUCA
  14168	921	2116	AGUCAGCCGUGAA	2117	UUCACGGCUGACUUU	98%
  					GGAA
  14169	154	2118	ACUACCAUGAGAA	2119	UUCUCAUGGUAGUG	n/a
  					AGUUU
  14170	217	2120	AAACAGGCUGAUU	2121	AAUCAGCCUGUUUAA	66%
  					CUGG
  14171	816	2122	GAGUGCUGAAACC	2123	GGUUUCAGCACUCUG	102%
  					GUCA
  14172	882	2124	UGAGCAUUCCGAU	2125	AUCGGAAUGCUCAUU	103%
  					GCUC
  14173	932	2126	AAUUCCACAGCCA	2127	UGGCUGUGGAAUUC	n/a
  					ACGGC
  14174	1509	2128	UGUCAAUUGCUUA	2129	UAAGCAAUUGACACC	n/a
  					ACCA
  14175	157	2130	ACCAUGAGAAUUG	2131	CAAUUCUCAUGGUAG	109%
  					UGAG
  14176	350	2132	CCAACGAAAGCCA	2133	UGGCUUUCGUUGGA	95%
  					CUUAC
  14177	511	2134	CUGGUCACUGAUU	2135	AAUCAGUGACCAGUU	100%
  					CAUC
  14178	605	2136	UGGUUUAUGGAC	2137	AGUCCAUAAACCACA	99%
  			U		CUAU
  14179	811	2138	GACCAGAGUGCUG	2139	CAGCACUCUGGUCAU	88%
  					CCAG
  14180	892	2140	GAUGUGAUUGAU	2141	UAUCAAUCACAUCGG	76%
  			A		AAUG
  14181	922	2142	GUCAGCCGUGAAU	2143	AUUCACGGCUGACUU	59%
  					UGGA
  14182	1169	2144	AAUGUGUAUCUA	2145	AUAGAUACACAUUCA	69%
  			U		ACCA
  14183	1182	2146	UUGAGUCUGGAA	2147	UUUCCAGACUCAAAU	n/a
  			A		AGAU
  14184	1539	2148	GUCCAGCAAUUAA	2149	UUAAUUGCUGGACAA	77%
  					CCGU
  14185	1541	2150	CCAGCAAUUAAUA	2151	UAUUAAUUGCUGGA	n/a
  					CAACC
  14186	427	2152	GACUCGAACGACU	2153	AGUCGUUCGAGUCAA	69%
  					UGGA
  14187	533	2154	ACCUGCCAGCAAC	2155	GUUGCUGGCAGGUCC	78%
  					GUGG
  18538	496	2156	GAUGAAUCUGAUA	2157	UAUCAGAUUCAUCAG	74%
  					AAUG
  18539	496	2158	UGAUGAAUCUGA	2159	UAUCAGAUUCAUCAG	72%
  			UA		AAUG
  18540	175	2160	AUUUGCUUUUGC	2161	UGCAAAAGCAAAUCA	98%
  			A		CUGC
  18541	175	2162	GAUUUGCUUUUG	2163	UGCAAAAGCAAAUCA	28%
  			CA		CUGC
  18542	172	2164	GUGAUUUGCUUU	2165	UAAAGCAAAUCACUG	24%
  			A		CAAU
  18543	172	2166	AGUGAUUUGCUU	2167	UAAAGCAAAUCACUG	14%
  			UA		CAAU
  18544	1013	2168	AAUUUCGUAUUU	2169	UAAAUACGAAAUUUC	100%
  			A		AGGU
  18545	1013	2170	AAAUUUCGUAUU	2171	UAAAUACGAAAUUUC	109%
  			UA		AGGU
  18546	952	2172	CACAGCCAUGAAA	2173	UUUCAUGGCUGUGA	32%
  					AAUUC
  18547	952	2174	UCACAGCCAUGAA	2175	UUUCAUGGCUGUGA	33%
  			A		AAUUC
  18548	174	2176	GAUUUGCUUUUG	2177	UCAAAAGCAAAUCAC	57%
  			A		UGCA
  18549	174	2178	UGAUUUGCUUUU	2179	UCAAAAGCAAAUCAC	53%
  			GA		UGCA
  18550	177	2180	UUGCUUUUGCCU	2181	UAGGCAAAAGCAAAU	97%
  			A		CACU
  18551	177	2182	UUUGCUUUUGCC	2183	UAGGCAAAAGCAAAU	103%
  			UA		CACU
  18552	1150	2184	UUUCUCAGUUUA	2185	UUAAACUGAGAAAGA	96%
  			A		AGCA
  18553	1089	2186	UUGCAUUUAGUCA	2187	UGACUAAAUGCAAAG	94%
  					UGAG
  18554	1086	2188	ACUUUGCAUUUAA	2189	UUAAAUGCAAAGUGA	n/a
  					GAAA
  18555	1093	2190	AUUUAGUCAAAAA	2191	UUUUUGACUAAAUG	n/a
  					CAAAG
  18556	1147	2192	UUCUUUCUCAGUA	2193	UACUGAGAAAGAAGC	n/a
  					AUUU
  18557	1148	2194	UCUUUCUCAGUUA	2195	UAACUGAGAAAGAAG	66%
  					CAUU
  18558	1128	2196	GAAAGAGAACAUA	2197	UAUGUUCUCUUUCA	16%
  					UUUUG
  18559	1087	2198	CUUUGCAUUUAGA	2199	UCUAAAUGCAAAGUG	28%
  					AGAA
  18560	1088	2200	UUUGCAUUUAGU	2201	UACUAAAUGCAAAGU	n/a
  			A		GAGA
  18561	1083	2202	CUCACUUUGCAUA	2203	UAUGCAAAGUGAGAA	53%
  					AUUG
  18562	1081	2204	UUCUCACUUUGCA	2205	UGCAAAGUGAGAAAU	89%
  					UGUA
  18563	555	2206	CACUCCAGUUGUA	2207	UACAACUGGAGUGAA	33%
  					AACU
  18564	1125	2208	AAUGAAAGAGAAA	2209	UUUCUCUUUCAUUU	n/a
  					UGCUA
  18565	168	2210	UGCAGUGAUUUG	2211	UCAAAUCACUGCAAU	14%
  			A		UCUC
  18566	1127	2212	UGAAAGAGAACAA	2213	UUGUUCUCUUUCAU	27%
  					UUUGC
  18567	1007	2214	ACCUGAAAUUUCA	2215	UGAAAUUUCAGGUG	129%
  					UUUAU
  18568	164	2216	GAAUUGCAGUGAA	2217	UUCACUGCAAUUCUC	47%
  					AUGG
  18569	222	2218	GGCUGAUUCUGG	2219	UCCAGAAUCAGCCUG	n/a
  			A		UUUA

• TABLE 14
  Inhibition of gene expression with PTGS2 sd-rxRNA sequences
  (Accession Number: NM_000963.2)

  		SEQ		SEQ		% remaining
  Oligo	Start	ID	Sense	ID	Antisense	expression (1
  Number	Site	NO	sequence	NO	sequence	uM A549)
  14422	451	2220	CACAUUUGAUUGA	2221	UCAAUCAAAUGUGAUC	72%
  					UGG
  14423	1769	2222	CACUGCCUCAAUU	2223	AAUUGAGGCAGUGUU	71%
  					GAUG
  14424	1464	2224	AAAUACCAGUCUU	2225	AAGACUGGUAUUUCAU	74%
  					CUG
  14425	453	2226	CAUUUGAUUGACA	2227	UGUCAAUCAAAUGUGA	83%
  					UCU
  						% remaining
  						expression (1
  						uM PC-3)
  17388	285	2228	GAAAACUGCUCAA	2229	UUGAGCAGUUUUCUCC	88%
  					AUA
  17389	520	2230	ACCUCUCCUAUUA	2231	UAAUAGGAGAGGUUA	25%
  					GAGA
  17390	467	2232	UCCACCAACUUAA	2233	UUAAGUUGGUGGACU	68%
  					GUCA
  17391	467	2234	GUCCACCAACUUAA	2235	UUAAGUUGGUGGACU	101%
  					GUCA
  17392	524	2236	CUCCUAUUAUACA	2237	UGUAUAAUAGGAGAG	49%
  					GUUA
  17393	448	2238	GAUCACAUUUGAA	2239	UUCAAAUGUGAUCUG	29%
  					GAUG
  17394	448	2240	AGAUCACAUUUGA	2241	UUCAAAUGUGAUCUG	31%
  			A		GAUG
  17395	519	2242	AACCUCUCCUAUA	2243	UAUAGGAGAGGUUAG	12%
  					AGAA
  17396	437	2244	GUUGACAUCCAGA	2245	UCUGGAUGUCAACACA	86%
  					UAA
  17397	406	2246	CCUUCCUUCGAAA	2247	UUUCGAAGGAAGGGAA	23%
  					UGU
  17398	339	2248	ACUCCAAACACAA	2249	UUGUGUUUGGAGUGG	102%
  					GUUU
  17399	339	2250	CACUCCAAACACAA	2251	UUGUGUUUGGAGUGG	55%
  					GUUU
  17400	338	2252	CACUCCAAACACA	2253	UGUGUUUGGAGUGGG	62%
  					UUUC
  17401	468	2254	CCACCAACUUACA	2255	UGUAAGUUGGUGGAC	61%
  					UGUC
  17402	468	2256	UCCACCAACUUACA	2257	UGUAAGUUGGUGGAC	179%
  					UGUC
  17403	1465	2258	AAUACCAGUCUUA	2259	UAAGACUGGUAUUUCA	30%
  					UCU
  17404	243	2260	GACCAGUAUAAGA	2261	UCUUAUACUGGUCAAA	32%
  					UCC
  17405	1472	2262	GUCUUUUAAUGAA	2263	UUCAUUAAAAGACUGG	15%
  					UAU
  17406	2446	2264	AAUUUCAUGUCUA	2265	UAGACAUGAAAUUACU	142%
  					GGU
  17407	449	2266	AUCACAUUUGAUA	2267	UAUCAAAUGUGAUCUG	54%
  					GAU
  17408	449	2268	GAUCACAUUUGAU	2269	UAUCAAAUGUGAUCUG	27%
  			A		GAU
  17409	444	2270	UCCAGAUCACAUA	2271	UAUGUGAUCUGGAUG	49%
  					UCAA
  17410	1093	2272	UACUGAUAGGAGA	2273	UCUCCUAUCAGUAUUA	32%
  					GCC
  17411	1134	2274	GUGCAACACUUGA	2275	UCAAGUGUUGCACAUA	70%
  					AUC
  17412	244	2276	ACCAGUAUAAGUA	2277	UACUUAUACUGGUCAA	63%
  					AUC
  17413	1946	2278	GAAGUCUAAUGAA	2279	UUCAUUAGACUUCUAC	19%
  					AGU
  17414	638	2280	AAGAAGAAAGUUA	2281	UAACUUUCUUCUUAGA	27%
  					AGC
  17415	450	2282	UCACAUUUGAUUA	2283	UAAUCAAAUGUGAUCU	216%
  					GGA
  17416	450	2284	AUCACAUUUGAUU	2285	UAAUCAAAUGUGAUCU	32%
  			A		GGA
  17417	452	2286	ACAUUUGAUUGAA	2287	UUCAAUCAAAUGUGAU	99%
  					CUG
  17418	452	2288	CACAUUUGAUUGA	2289	UUCAAUCAAAUGUGAU	54%
  			A		CUG
  17419	454	2290	AUUUGAUUGACAA	2291	UUGUCAAUCAAAUGUG	86%
  					AUC
  17420	454	2292	CAUUUGAUUGACA	2293	UUGUCAAUCAAAUGUG	89%
  			A		AUC
  17421	1790	2294	CAUCUGCAAUAAA	2295	UUUAUUGCAGAUGAG	55%
  					AGAC
  17422	1790	2296	UCAUCUGCAAUAA	2297	UUUAUUGCAGAUGAG	62%
  			A		AGAC

• TABLE 15
  Inhibition of gene expression with CTGF sd-rxRNA sequences
  (Accession number: NM_001901.2)

  						% remaining
  		SEQ		SEQ		mRNA expression
  Oligo	Start	ID	Sense	ID	Antisense	(1 uM sd-rxRNA,
  Number	Site	NO	sequence	NO	sequence	A549)
  13980	1222	2298	ACAGGAAGAUG	2299	UACAUCUUCCUGUAG	98%
  			UA		UACA
  13981	813	2300	GAGUGGAGCGC	2301	AGGCGCUCCACUCUG	82%
  			CU		UGGU
  13982	747	2302	CGACUGGAAGA	4206	UGUCUUCCAGUCGGU	116%
  			CA		AAGC
  13983	817	2303	GGAGCGCCUGU	4207	GAACAGGCGCUCCAC	97%
  			UC		UCUG
  13984	1174	2304	GCCAUUACAAC	4208	CAGUUGUAAUGGCAG	102%
  			UG		GCAC
  13985	1005	2305	GAGCUUUCUG	4209	AGCCAGAAAGCUCAA	114%
  			GCU		ACUU
  13986	814	2306	AGUGGAGCGCC	4210	CAGGCGCUCCACUCU	111%
  			UG		GUGG
  13987	816	2307	UGGAGCGCCUG	4211	AACAGGCGCUCCACU	102%
  			UU		CUGU
  13988	1001	2308	GUUUGAGCUU	4212	AGAAAGCUCAAACUU	99%
  			UCU		GAUA
  13989	1173	2309	UGCCAUUACAA	4213	AGUUGUAAUGGCAG	107%
  			CU		GCACA
  13990	749	2310	ACUGGAAGACA	4214	CGUGUCUUCCAGUCG	91%
  			CG		GUAA
  13991	792	2311	AACUGCCUGGU	4215	GGACCAGGCAGUUGG	97%
  			CC		CUCU
  13992	1162	2312	AGACCUGUGCC	4216	CAGGCACAGGUCUUG	107%
  			UG		AUGA
  13993	811	2313	CAGAGUGGAGC	4217	GCGCUCCACUCUGUG	113%
  			GC		GUCU
  13994	797	2314	CCUGGUCCAGA	4218	GGUCUGGACCAGGCA	n/a
  			CC		GUUG
  13995	1175	2315	CCAUUACAACU	4219	ACAGUUGUAAUGGCA	113%
  			GU		GGCA
  13996	1172	2316	CUGCCAUUACA	4220	GUUGUAAUGGCAGG	110%
  			AC		CACAG
  13997	1177	2317	AUUACAACUGU	4221	GGACAGUUGUAAUG	105%
  			CC		GCAGG
  13998	1176	2318	CAUUACAACUG	4222	GACAGUUGUAAUGGC	89%
  			UC		AGGC
  13999	812	2319	AGAGUGGAGCG	4223	GGCGCUCCACUCUGU	99%
  			CC		GGUC
  14000	745	2320	ACCGACUGGAA	4224	UCUUCCAGUCGGUAA	n/a
  			GA		GCCG
  14001	1230	2321	AUGUACGGAGA	4225	UGUCUCCGUACAUCU	106%
  			CA		UCCU
  14002	920	2322	GCCUUGCGAAG	4226	AGCUUCGCAAGGCCU	93%
  			CU		GACC
  14003	679	2323	GCUGCGAGGAG	4227	CACUCCUCGCAGCAU	102%
  			UG		UUCC
  14004	992	2324	GCCUAUCAAGU	4228	AAACUUGAUAGGCUU	100%
  			UU		GGAG
  14005	1045	2325	AAUUCUGUGG	4229	ACUCCACAGAAUUUA	104%
  			AGU		GCUC
  14006	1231	2326	UGUACGGAGAC	4230	AUGUCUCCGUACAUC	87%
  			AU		UUCC
  14007	991	2327	AGCCUAUCAAG	4231	AACUUGAUAGGCUUG	101%
  			UU		GAGA
  14008	998	2328	CAAGUUUGAGC	4232	AAGCUCAAACUUGAU	98%
  			UU		AGGC
  14009	1049	2329	CUGUGGAGUA	4233	ACAUACUCCACAGAA	98%
  			UGU		UUUA
  14010	1044	2330	AAAUUCUGUG	4234	CUCCACAGAAUUUAG	93%
  			GAG		CUCG
  14011	1327	2331	UUUCAGUAGCA	4235	UGUGCUACUGAAAUC	95%
  			CA		AUUU
  14012	1196	2332	CAAUGACAUCU	4236	AAAGAUGUCAUUGUC	101%
  			UU		UCCG
  14013	562	2333	AGUACCAGUGC	4237	GUGCACUGGUACUUG	66%
  			AC		CAGC
  14014	752	2334	GGAAGACACGU	4238	AAACGUGUCUUCCAG	95%
  			UU		UCGG
  14015	994	2335	CUAUCAAGUUU	4239	UCAAACUUGAUAGGC	85%
  			GA		UUGG
  14016	1040	2336	AGCUAAAUUCU	4240	ACAGAAUUUAGCUCG	61%
  			GU		GUAU
  14017	1984	2337	AGGUAGAAUG	4241	UUACAUUCUACCUAU	32%
  			UAA		GGUG
  14018	2195	2338	AGCUGAUCAGU	4242	AAACUGAUCAGCUAU	86%
  			UU		AUAG
  14019	2043	2339	UUCUGCUCAGA	4243	UAUCUGAGCAGAAUU	81%
  			UA		UCCA
  14020	1892	2340	UUAUCUAAGU	4244	UUAACUUAGAUAACU	84%
  			UAA		GUAC
  14021	1567	2341	UAUACGAGUAA	4245	UAUUACUCGUAUAAG	72%
  			UA		AUGC
  14022	1780	2342	GACUGGACAGC	4246	AAGCUGUCCAGUCUA	65%
  			UU		AUCG
  14023	2162	2343	AUGGCCUUUAU	4247	UAAUAAAGGCCAUUU	80%
  			UA		GUUC
  14024	1034	2344	AUACCGAGCUA	4248	UUUAGCUCGGUAUG	91%
  			AA		UCUUC
  14025	2264	2345	UUGUUGAGAG	4249	ACACUCUCAACAAAU	58%
  			UGU		AAAC
  14026	1032	2346	ACAUACCGAGC	4250	UAGCUCGGUAUGUC	106%
  			UA		UUCAU
  14027	1535	2347	AGCAGAAAGGU	4251	UAACCUUUCUGCUGG	67%
  			UA		UACC
  14028	1694	2348	AGUUGUUCCU	4252	UUAAGGAACAACUUG	94%
  			UAA		ACUC
  14029	1588	2349	AUUUGAAGUG	4253	UUACACUUCAAAUAG	97%
  			UAA		CAGG
  14030	928	2350	AAGCUGACCUG	4254	UCCAGGUCAGCUUCG	100%
  			GA		CAAG
  14031	1133	2351	GGUCAUGAAGA	4255	CUUCUUCAUGACCUC	82%
  			AG		GCCG
  14032	912	2352	AUGGUCAGGCC	4256	AAGGCCUGACCAUGC	84%
  			UU		ACAG
  14033	753	2353	GAAGACACGUU	4257	CAAACGUGUCUUCCA	86%
  			UG		GUCG
  14034	918	2354	AGGCCUUGCGA	4258	CUUCGCAAGGCCUGA	88%
  			AG		CCAU
  14035	744	2355	UACCGACUGGA	4259	CUUCCAGUCGGUAAG	95%
  			AG		CCGC
  14036	466	2356	ACCGCAAGAUC	4260	CCGAUCUUGCGGUUG	73%
  			GG		GCCG
  14037	917	2357	CAGGCCUUGCG	4261	UUCGCAAGGCCUGAC	86%
  			AA		CAUG
  14038	1038	2358	CGAGCUAAAUU	4262	AGAAUUUAGCUCGGU	84%
  			CU		AUGU
  14039	1048	2359	UCUGUGGAGU	4263	CAUACUCCACAGAAU	87%
  			AUG		UUAG
  14040	1235	2360	CGGAGACAUGG	4264	UGCCAUGUCUCCGUA	100%
  			CA		CAUC
  14041	868	2361	AUGACAACGCC	4265	GAGGCGUUGUCAUU	104%
  			UC		GGUAA
  14042	1131	2362	GAGGUCAUGAA	4266	UCUUCAUGACCUCGC	85%
  			GA		CGUC
  14043	1043	2363	UAAAUUCUGU	4267	UCCACAGAAUUUAGC	74%
  			GGA		UCGG
  14044	751	2364	UGGAAGACACG	4268	AACGUGUCUUCCAGU	84%
  			UU		CGGU
  14045	1227	2365	AAGAUGUACGG	4269	CUCCGUACAUCUUCC	99%
  			AG		UGUA
  14046	867	2366	AAUGACAACGC	4270	AGGCGUUGUCAUUG	94%
  			CU		GUAAC
  14047	1128	2367	GGCGAGGUCAU	4271	UCAUGACCUCGCCGU	89%
  			GA		CAGG
  14048	756	2368	GACACGUUUGG	4272	GGCCAAACGUGUCUU	93%
  			CC		CCAG
  14049	1234	2369	ACGGAGACAUG	4273	GCCAUGUCUCCGUAC	100%
  			GC		AUCU
  14050	916	2370	UCAGGCCUUGC	4274	UCGCAAGGCCUGACC	96%
  			GA		AUGC
  14051	925	2371	GCGAAGCUGAC	4275	AGGUCAGCUUCGCAA	80%
  			CU		GGCC
  14052	1225	2372	GGAAGAUGUAC	4276	CCGUACAUCUUCCUG	96%
  			GG		UAGU
  14053	445	2373	GUGACUUCGGC	4277	GAGCCGAAGUCACAG	101%
  			UC		AAGA
  14054	446	2374	UGACUUCGGCU	4278	GGAGCCGAAGUCACA	93%
  			CC		GAAG
  14055	913	2375	UGGUCAGGCCU	4279	CAAGGCCUGACCAUG	67%
  			UG		CACA
  14056	997	2376	UCAAGUUUGA	4280	AGCUCAAACUUGAUA	92%
  			GCU		GGCU
  14057	277	2377	GCCAGAACUGC	4281	CUGCAGUUCUGGCCG	84%
  			AG		ACGG
  14058	1052	2378	UGGAGUAUGU	4282	GGUACAUACUCCACA	n/a
  			ACC		GAAU
  14059	887	2379	GCUAGAGAAGC	4283	CUGCUUCUCUAGCCU	80%
  			AG		GCAG
  14060	914	2380	GGUCAGGCCUU	4284	GCAAGGCCUGACCAU	112%
  			GC		GCAC
  14061	1039	2381	GAGCUAAAUUC	4285	CAGAAUUUAGCUCGG	104%
  			UG		UAUG
  14062	754	2382	AAGACACGUUU	4286	CCAAACGUGUCUUCC	109%
  			GG		AGUC
  14063	1130	2383	CGAGGUCAUGA	4287	CUUCAUGACCUCGCC	103%
  			AG		GUCA
  14064	919	2384	GGCCUUGCGAA	4288	GCUUCGCAAGGCCUG	109%
  			GC		ACCA
  14065	922	2385	CUUGCGAAGCU	4289	UCAGCUUCGCAAGGC	106%
  			GA		CUGA
  14066	746	2386	CCGACUGGAAG	4290	GUCUUCCAGUCGGUA	106%
  			AC		AGCC
  14067	993	2387	CCUAUCAAGUU	4291	CAAACUUGAUAGGCU	67%
  			UG		UGGA
  14068	825	2388	UGUUCCAAGAC	4292	AGGUCUUGGAACAGG	93%
  			CU		CGCU
  14069	926	2389	CGAAGCUGACC	4293	CAGGUCAGCUUCGCA	95%
  			UG		AGGC
  14070	923	2390	UUGCGAAGCUG	4294	GUCAGCUUCGCAAGG	95%
  			AC		CCUG
  14071	866	2391	CAAUGACAACG	4295	GGCGUUGUCAUUGG	132%
  			CC		UAACC
  14072	563	2392	GUACCAGUGCA	4296	CGUGCACUGGUACUU	n/a
  			CG		GCAG
  14073	823	2393	CCUGUUCCAAG	4297	GUCUUGGAACAGGCG	98%
  			AC		CUCC
  14074	1233	2394	UACGGAGACAU	4298	CCAUGUCUCCGUACA	109%
  			GG		UCUU
  14075	924	2395	UGCGAAGCUGA	4299	GGUCAGCUUCGCAAG	95%
  			CC		GCCU
  14076	921	2396	CCUUGCGAAGC	4300	CAGCUUCGCAAGGCC	116%
  			UG		UGAC
  14077	443	2397	CUGUGACUUCG	4301	GCCGAAGUCACAGAA	110%
  			GC		GAGG
  14078	1041	2398	GCUAAAUUCUG	4302	CACAGAAUUUAGCUC	99%
  			UG		GGUA
  14079	1042	2399	CUAAAUUCUGU	4303	CCACAGAAUUUAGCU	109%
  			GG		CGGU
  14080	755	2400	AGACACGUUUG	4304	GCCAAACGUGUCUUC	121%
  			GC		CAGU
  14081	467	2401	CCGCAAGAUCG	4305	GCCGAUCUUGCGGUU	132%
  			GC		GGCC
  14082	995	2402	UAUCAAGUUU	4306	CUCAAACUUGAUAGG	105%
  			GAG		CUUG
  14083	927	2403	GAAGCUGACCU	4307	CCAGGUCAGCUUCGC	114%
  			GG		AAGG
  17356	1267	2404	ACAUUAACUCA	4308	UAUGAGUUAAUGUC	120%
  			UA		UCUCA
  17357	1267	2405	GACAUUAACUC	2406	UAUGAGUUAAUGUC	56%
  			AUA		UCUCA
  17358	1442	2407	UGAAGAAUGU	2408	UUAACAUUCUUCAAA	34%
  			UAA		CCAG
  17359	1442	2409	UUGAAGAAUG	2410	UUAACAUUCUUCAAA	31%
  			UUAA		CCAG
  17360	1557	2411	GAUAGCAUCUU	2412	UUAAGAUGCUAUCU	59%
  			AA		GAUGA
  17361	1557	2413	AGAUAGCAUCU	2414	UUAAGAUGCUAUCU	47%
  			UAA		GAUGA
  17362	1591	2415	UGAAGUGUAA	2416	UAAUUACACUUCAAA	120%
  			UUA		UAGC
  17363	1599	2417	AAUUGAGAAGG	2418	UUCCUUCUCAAUUAC	71%
  			AA		ACUU
  17364	1601	2419	UUGAGAAGGAA	2420	UUUUCCUUCUCAAUU	62%
  			AA		ACAC
  17365	1732	2421	CAUUCUGAUUC	2422	UCGAAUCAGAAUGUC	99%
  			GA		AGAG
  17366	1734	2423	UUCUGAUUCGA	2424	UUUCGAAUCAGAAUG	97%
  			AA		UCAG
  17367	1770	2425	CUGUCGAUUAG	2426	UUCUAAUCGACAGGA	45%
  			AA		UUCC
  17368	1805	2427	UUUGCCUGUAA	2428	UGUUACAGGCAAAUU	71%
  			CA		CACU
  17369	1805	2429	AUUUGCCUGUA	2430	UGUUACAGGCAAAUU	67%
  			ACA		CACU
  17370	1815	2431	ACAAGCCAGAU	2432	UAAUCUGGCUUGUU	65%
  			UA		ACAGG
  17371	1815	2433	AACAAGCCAGA	2434	UAAUCUGGCUUGUU	35%
  			UUA		ACAGG
  17372	2256	2435	CAGUUUAUUU	2436	UACAAAUAAACUGUC	113%
  			GUA		CGAA
  17373	2265	2437	UGUUGAGAGU	2438	UACACUCUCAACAAA	35%
  			GUA		UAAA
  17374	2265	2439	UUGUUGAGAG	2440	UACACUCUCAACAAA	31%
  			UGUA		UAAA
  17375	2295	2441	UGCACCUUUCU	2442	UUAGAAAGGUGCAAA	34%
  			AA		CAUG
  17376	2295	2143	UUGCACCUUUC	2444	UUAGAAAGGUGCAAA	28%
  			UAA		CAUG
  17377	1003	2445	UUGAGCUUUC	2446	UCAGAAAGCUCAAAC	67%
  			UGA		UUGA
  17378	2268	2417	UGAGAGUGUG	2448	UGUCACACUCUCAAC	42%
  			ACA		AAAU
  17379	2272	2449	AGUGUGACCAA	2450	UUUUGGUCACACUCU	35%
  			AA		CAAC
  17380	2272	2451	GAGUGUGACCA	2452	UUUUGGUCACACUCU	29%
  			AAA		CAAC
  17381	2273	2453	GUGUGACCAAA	2454	UUUUUGGUCACACUC	42%
  			AA		UCAA
  17382	2274	2455	UGUGACCAAAA	2456	UCUUUUGGUCACACU	42%
  			GA		CUCA
  17383	2274	2457	GUGUGACCAAA	2458	UCUUUUGGUCACACU	37%
  			AGA		CUCA
  17384	2275	2459	GUGACCAAAAG	2460	UACUUUUGGUCACAC	24%
  			UA		UCUC
  17385	2277	2461	GACCAAAAGUU	2462	UUAACUUUUGGUCAC	27%
  			AA		ACUC
  17386	2296	2463	GCACCUUUCUA	2464	UCUAGAAAGGUGCAA	23%
  			GA		ACAU
  17387	2299	2465	CCUUUCUAGUU	2466	UCAACUAGAAAGGUG	46%
  			GA		CAAA

• TABLE 16
  Inhibition of gene expression with TGFB2 sd-rxRNA sequences
  (Accession Number: NM_001135599.1)

  		SEQ		SEQ		% remaining
  Oligo	Start	ID	Sense	ID	Antisense	expression (1
  Number	Site	NO	sequence	NO	sequence	uM A549)
  14408	1324	2467	GGCUCUCCUUCGA	2468	UCGAAGGAGAGCCAU	94%
  					UCGC
  14409	1374	2469	GACAGGAACCUGG	2470	CCAGGUUCCUGUCUU	n/a
  					UAUG
  14410	946	2471	CCAAGGAGGUUUA	2472	UAAACCUCCUUGGCG	90%
  					UAGU
  14411	849	2473	AUUUCCAUCUACA	2474	UGUAGAUGGAAAUCA	72%
  					CCUC
  14412	852	2475	UCCAUCUACAACA	2476	UGUUGUAGAUGGAA	76%
  					AUCAC
  14413	850	2177	UUUCCAUCUACAA	2478	UUGUAGAUGGAAAU	98%
  					CACCU
  14414	944	2479	CGCCAAGGAGGUU	2480	AACCUCCUUGGCGUA	100%
  					GUAC
  14415	1513	2481	GUGGUGAUCAGA	2482	UUCUGAUCACCACUG	n/a
  			A		GUAU
  14416	1572	2483	CUCCUGCUAAUGU	2484	ACAUUAGCAGGAGAU	100%
  					GUGG
  14417	1497	2485	ACCUCCACAUAUA	2486	UAUAUGUGGAGGUG	73%
  					CCAUC
  14418	1533	2487	AAGUCCACUAGGA	2488	UCCUAGUGGACUUUA	98%
  					UAGU
  14419	1514	2489	UGGUGAUCAGAAA	2490	UUUCUGAUCACCACU	86%
  					GGUA
  14420	1534	2491	AGUCCACUAGGAA	2492	UUCCUAGUGGACUU	99%
  					UAUAG
  14421	943	2493	ACGCCAAGGAGGU	2494	ACCUCCUUGGCGUAG	41%
  					UACU
  18570	2445	2495	UAUUUAUUGUGU	2496	UACACAAUAAAUAAC	79%
  			A		UCAC
  18571	2445	2497	UUAUUUAUUGUG	2498	UACACAAUAAAUAAC	75%
  			UA		UCAC
  18572	2083	2499	AUCAGUGUUAAAA	2500	UUUUAACACUGAUGA	47%
  					ACCA
  18573	2083	2501	CAUCAGUGUUAAA	2502	UUUUAACACUGAUGA	17%
  			A		ACCA
  18574	2544	2503	AUGGCUUAAGGAA	2504	UUCCUUAAGCCAUCC	59%
  					AUGA
  18575	2544	2505	GAUGGCUUAAGG	2506	UUCCUUAAGCCAUCC	141%
  			AA		AUGA
  18576	2137	2507	UUGUGUUCUGUU	2508	UAACAGAACACAAAC	77%
  			A		UUCC
  18577	2137	2509	UUUGUGUUCUGU	2510	UAACAGAACACAAAC	59%
  			UA		UUCC
  18578	2520	2511	AAAUACUUUGCCA	2512	UGGCAAAGUAUUUG	75%
  					GUCUC
  18579	2520	2513	CAAAUACUUUGCC	2514	UGGCAAAGUAUUUG	55%
  			A		GUCUC
  18580	3183	2515	CUUGCACUACAAA	2516	UUUGUAGUGCAAGU	84%
  					CAAAC
  18581	3183	2517	ACUUGCACUACAA	2518	UUUGUAGUGCAAGU	80%
  			A		CAAAC
  18582	2267	2519	GAAUUUAUUAGU	2520	UACUAAUAAAUUCUU	82%
  			A		CCAG
  18583	2267	2521	AGAAUUUAUUAG	2522	UACUAAUAAAUUCUU	67%
  			UA		CCAG
  18584	3184	2523	UUGCACUACAAAA	2524	UUUUGUAGUGCAAG	77%
  					UCAAA
  18585	3184	2525	CUUGCACUACAAA	2526	UUUUGUAGUGCAAG	59%
  			A		UCAAA
  18586	2493	2527	AUAAAACAGGUGA	2528	UCACCUGUUUUAUU	84%
  					UUCCA
  18587	2493	2529	AAUAAAACAGGUG	2530	UCACCUGUUUAUU	70%
  			A		UUCCA
  18588	2297	2531	GACAACAACAACA	2532	UGUUGUUGUUGUCG	40%
  					UUGUU
  18589	2046	2533	AUGCUUGUAACAA	2534	UUGUUACAAGCAUCA	39%
  					UCGU
  18590	2531	2535	CAGAAACUCAUGA	2536	UCAUGAGUUUCUGG	56%
  					CAAAG
  18591	2389	2537	GUAUUGCUAUGCA	2538	UGCAUAGCAAUACAG	64%
  					AAAA
  18592	2530	2539	CCAGAAACUCAUA	2540	UAUGAGUUUCUGGC	44%
  					AAAGU
  18593	2562	2541	ACUCAAACGAGCA	2542	UGCUCGUUUGAGUU	87%
  					CAAGU
  18594	2623	2543	AUAUGACCGAGAA	2544	UUCUCGGUCAUAUAA	69%
  					UAAC
  18595	2032	2545	CGACGACAACGAA	2546	UUCGUUGUCGUCGU	55%
  					CAUCA
  18596	2809	2547	GUAAACCAGUGAA	2548	UUCACUGGUUUACUA	58%
  					AACU
  18597	2798	2549	UUGUCAGUUUAG	2550	UCUAAACUGACAAAG	38%
  			A		AACC
  18598	2081	2551	UCAUCAGUGUUAA	2552	UUAACACUGAUGAAC	25%
  					CAAG
  18599	2561	2553	AACUCAAACGAGA	2554	UCUCGUUUGAGUUC	57%
  					AAGUU
  18600	2296	2555	CGACAACAACAAA	2556	UUUGUUGUUGUCGU	69%
  					UGUUC
  18601	2034	2557	ACGACAACGAUGA	2558	UCAUCGUUGUCGUCG	22%
  					UCAU
  18602	2681	2559	GCUGCCUAAGGAA	2560	UUCCUUAGGCAGCUG	43%
  					AUAC
  18603	2190	2561	AUUCUACAUUUCA	2562	UGAAAUGUAGAAUAA	128%
  					GGCC

• TABLE 17
  Inhibition of gene expression with TGFB1 sd-rxRNA sequences
  (Accession Number NM_000660.3)

  		SEQ		SEQ		% remaining
  Oligo	Start	ID	Sense	ID	Antisense	expression (1
  Number	Site	NO	sequence	NO	sequence	uM A549)
  14394	1194	2563	GCUAAUGGUGGA	2564	UUCCACCAUUAGCA	24%
  			A		CGCGG
  14395	2006	2565	UGAUCGUGCGCUC	2566	GAGCGCACGAUCAU	79%
  					GUUGG
  14396	1389	2567	CAAUUCCUGGCGA	2568	UCGCCAGGAAUUGU	77%
  					UGCUG
  14397	1787	2569	AGUGGAUCCACGA	2570	UCGUGGAUCCACUU	n/a
  					CCAGC
  14398	1867	2571	UACAGCAAGGUCC	2572	GGACCUUGCUGUAC	82%
  					UGCGU
  14399	2002	2573	AACAUGAUCGUGC	2574	GCACGAUCAUGUUG	n/a
  					GACAG
  14400	2003	2575	ACAUGAUCGUGCG	2576	CGCACGAUCAUGUU	n/a
  					GGACA
  14401	1869	2577	CAGCAAGGUCCUG	2578	CAGGACCUUGCUGU	82%
  					ACUGC
  14402	2000	2579	CCAACAUGAUCGU	2580	ACGAUCAUGUUGGA	66%
  					CAGCU
  14403	986	2581	AGCGGAAGCGCAU	2582	AUGCGCUUCCGCUU	78%
  					CACCA
  14404	995	2583	GCAUCGAGGCCAU	2584	AUGGCCUCGAUGCG	79%
  					CUUCC
  14405	963	2585	GACUAUCGACAUG	2586	CAUGUCGAUAGUCU	80%
  					UGCAG
  14406	955	2587	ACCUGCAAGACUA	2588	UAGUCUUGCAGGUG	88%
  					GAUAG
  14407	1721	2589	GCUCCACGGAGAA	2590	UUCUCCGUGGAGCU	n/a
  					GAAGC
  18454	1246	2591	CACAGCAUAUAUA	2592	UAUAUAUGCUGUG	58%
  					UGUACU
  18455	1248	2593	CAGCAUAUAUAUA	2594	UAUAUAUAUGCUGU	87%
  					GUGUA
  18456	1755	2595	GUACAUUGACUUA	2596	UAAGUCAAUGUACA	107%
  					GCUGC
  18457	1755	2597	UGUACAUUGACUU	2598	UAAGUCAAUGUACA	77%
  			A		GCUGC
  18458	1708	2599	AACUAUUGCUUCA	2600	UGAAGCAAUAGUUG	75%
  					GUGUC
  18459	1708	2601	CAACUAUUGCUUC	2602	UGAAGCAAUAGUUG	73%
  			A		GUGUC
  18460	1250	2603	GCAUAUAUAUGUA	2604	UACAUAUAUAUGCU	n/a
  					GUGUG
  18461	1754	2605	UGUACAUUGACUA	2606	UAGUCAAUGUACAG	91%
  					CUGCC
  18462	1754	2607	CUGUACAUUGACU	2608	UAGUCAAUGUACAG	92%
  			A		CUGCC
  18463	1249	2609	AGCAUAUAUAUGA	2610	UCAUAUAUAUGCUG	n/a
  					UGUGU
  18464	1383	2611	CAGCAACAAUUCA	2612	UGAAUUGUUGCUG	77%
  					UAUUUC
  18465	1251	2613	CAUAUAUAUGUUA	2614	UAACAUAUAUAUGC	84%
  					UGUGU
  18466	1713	2615	UUGCUUCAGCUCA	2616	UGAGCUGAAGCAAU	n/a
  					AGUUG
  18467	1713	2617	AUUGCUUCAGCUC	2518	UGAGCUGAAGCAAU	83%
  			A		AGUUG
  18468	1247	2619	ACAGCAUAUAUAA	2620	UUAUAUAUGCUGU	96%
  					GUGUAC
  18469	1712	2621	AUUGCUUCAGCUA	2622	UAGCUGAAGCAAUA	90%
  					GUUGG
  18470	1712	2623	UAUUGCUUCAGCU	2624	UAGCUGAAGCAAUA	98%
  			A		GUUGG
  18471	1212	2625	CAAGUUCAAGCAA	2626	UUGCUUGAACUUGU	n/a
  					CAUAG
  18472	1222	2627	CAGAGUACACACA	2628	UGUGUGUACUCUGC	45%
  					UUGAA
  18473	1228	2629	ACACACAGCAUAA	2630	UUAUGCUGUGUGU	36%
  					ACUCUG
  18474	1233	2631	CAGCAUAUAUAUA	2632	UAUAUAUAUGCUGU	68%
  					GUGUA
  18475	1218	2633	UCAAGCAGAGUAA	2634	UUACUCUGCUUGAA	64%
  					CUUGU
  18476	1235	2635	AGCAUAUAUAUGA	2636	UCAUAUAUAUGCUG	78%
  					UGUGU
  18477	1225	2637	AGAGUACACACAA	2638	UUGUGUGUACUCU	92%
  					GCUUGA
  18478	1221	2639	AAGCAGAGUACAA	2640	UUGUACUCUGCUUG	103%
  					AACUU
  18479	1244	2641	UUCAACACAUCAA	2642	UUGAUGUGUUGAA	84%
  					GAACAU
  18480	1224	2643	AGCAGAGUACACA	2644	UGUGUACUCUGCUU	37%
  					GAACU
  18481	1242	2645	AUAUAUGUUCUU	2646	UAAGAACAUAUAUA	62%
  			A		UGCUG
  18482	1213	2647	GACAAGUUCAAGA	2648	UCUUGAACUUGUCA	47%
  					UAGAU
  18483	1760	2649	UUAAAGAUGGAGA	2650	UCUCCAUCUUUAAU	69%
  					GGGGC
  18484	1211	2651	CUAUGACAAGUUA	2652	UAACUUGUCAUAGA	n/a
  					UUUCG
  19411	1212	2653	CAACGAAAUCUAA	2654	UUAGAUUUCGUUG	52%
  					UGGGUU
  19412	1222	2655	UAUGACAAGUUCA	2656	UGAACUUGUCAUAG	51%
  					AUUUC
  19413	1228	2657	AAGUUCAAGCAGA	2658	UCUGCUUGAACUUG	n/a
  					UCAUA
  19414	1233	2659	CAAGCAGAGUACA	2660	UGUACUCUGCUUGA	41%
  					ACUUG
  19415	1218	2661	AAUCUAUGACAAA	2662	UUUGUCAUAGAUU	104%
  					UCGUUG
  19416	1244	2663	CACACAGCAUAUA	2664	UAUAUGCUGUGUG	31%
  					UACUCU

• TABLE 18
  Inhibition of gene expression with SPP1 sd-rxRNA sequences (Accession Number NM_000582.2)

  						% remaining
  Oligo	Start	SEQ ID		SEQ ID		expression
  Number	Site	NO	Sense sequence	NO	Antisense sequence	(1 uM A549)

  14084	1025	2665	mC.mU.mC. A.mU.	2666	P.mU.fC.fU. A.	61%
  			G. A. A.mU.mU. A.		A.fU.fU.fC. A.fU. G. A.
  			G. A.Chl		G* A* A* A*mU* A* C.
  14085	1049	2667	mC.mU. G. A. G.	2668	P.mU. A. A.fU.fU. G.	50%
  			G.mU.mC. A.		A.fC.fC.fU.mC. A. G* A*
  			A.mU.mU. A.Chl		A* G* A*mU* G.
  14086	1051	2669	G. A. G. G.mU.mC.	2670	P.mU.fU.fU. A. A.fU.fU.	n/a
  			A. A.mU.mU. A. A.		G. A.fC.mC.mU.mC* A*
  			A.Chl		G* A* A* G* A.
  14087	1048	2671	mU.mC.mU. G. A. G.	2672	P.mA. A.fU.fU. G.	69%
  			G.mU.mC. A.		A.fC.fC.fU.fC. A. G. A*
  			A.mU.mU.Chl		A* G* A*mU* G* C.
  14088	1050	2673	mU. G. A. G.	2674	P.mU.fU. A. A.fU.fU. G.	76%
  			G.mU.mC. A.		A.fC.fC.mU.mC. A* G*
  			A.mU.mU. A. A.Chl		A* A* G* A* U.
  14089	1047	2675	mU.mU.mC.mU. G.	2676	P.mA.fU.fU. G.	60%
  			A. G. G.mU.mC. A.		A.fC.fC.fU.fC. A. G. A.
  			A.mU.Chl		A* G* A*mU* G*mC*
  					A.
  14090	800	2677	G.mU.mC. A.	2678	P.mU.fC. A.fU.fC.fC. A.	71%
  			G.mC.mU. G. G.		G.fC.fU. G.
  			A.mU. G. A.Chl		A.mC*mU*mC*
  					G*mU*mU* U.
  14091	492	2679	mU.mU.mC.mU. G.	2680	P.mA. G. A.fU.fU.fC.	n/a
  			A.mU. G. A.		A.fU.fC. A. G. A. A*mU*
  			A.mU.mC.mU.Chl		G* G*mU* G* A.
  14092	612	2681	mU. G. G. A.mC.mU.	2682	P.mU. G. A.fC.fC.fU.fC.	n/a
  			G. A. G. G.mU.mC.		A. G.fU.mC.mC. A*mU*
  			A.Chl		A* A* A*mC* C.
  14093	481	2683	G. A.	2684	P.mA. A.fU. G. G.fU. G.	n/a
  			G.mU.mC.mU.mC.		A. G. A.mC.mU.mC*
  			A.mC.mC.		A*mU*mC* A* G* A.
  			A.mU.mU.Chl
  14094	614	2685	G. A.mC.mU. G. A.	2686	P.mU.fU.fU. G.	n/a
  			G. G.mU.mC. A. A.		A.fC.fC.fU.fC. A.
  			A.Chl		G.mU.mC*mC* A*mU*
  					A* A* A.
  14095	951	2687	mU.mC. A.mC. A.	2688	P.mU.fU.fC. A.fU. G.	89%
  			G.mC.mC. A.mU. G.		G.fC.fU. G.mU. G. A* A*
  			A. A.Chl		A*mU*mU*mC* A.
  14096	482	2689	A.	2690	P.mG. A. A.fU. G. G.fU.	87%
  			G.mU.mC.mU.mC.		G. A. G. A.mC.mU*mC*
  			A.mC.mC.		A*mU*mC* A* G.
  			A.mU.mU.mC.Chl
  14097	856	2691	A. A. G.mC. G. G. A.	2692	P.mU. G.	88%
  			A. A. G.mC.mC. A.Chl		G.fC.fU.fU.fU.fC.fC.
  					G.mC.mU.mU* A*mU*
  					A*mU* A* A.
  14098	857	2693	A. G.mC. G. G. A. A.	2694	P.mU.fU. G.	113%
  			A. G.mC.mC. A. A.Chl		G.fC.fU.fU.fU.fC.fC.
  					G.mC.mU*mU* A*mU*
  					A*mU* A.
  14099	365	2695	A.mC.mC. A.mC.	2696	P.mU.fC. A.fU.fC.fC.	98%
  			A.mU. G. G. A.mU.		A.fU. G.fU. G.
  			G. A.Chl		G.mU*mC* A*mU* G*
  					G* C.
  14100	359	2697	G.mC.mC. A.mU. G.	2698	P.mA.fU. G.fU. G.	84%
  			A.mC.mC. A.mC.		G.fU.fC. A.fU. G.
  			A.mU.Chl		G.mC*mU*mU*mU*mC*
  					G* U.
  14101	357	2699	A. A. G.mC.mC.	2700	P.mG.fU. G. G.fU.fC.	88%
  			A.mU. G. A.mC.mC.		A.fU. G.
  			A.mC.Chl		G.mC.mU.mU*mU*mC*
  					G*mU*mU* G.
  14102	858	2701	G.mC. G. G. A. A. A.	2702	P.mA.fU.fU. G.	n/a
  			G.mC.mC. A.		G.fC.fU.fU.fU.fC.mC.
  			A.mU.Chl		G.mC*mU*mU*
  					A*mU* A* U.
  14103	1012	2703	A. A.	2704	P.mA. A. A.fU. A.fC. G.	93%
  			A.mU.mU.mU.mC.		A. A.
  			G.mU.		A.mU.mU.mU*mC* A*
  			A.mU.mU.mU.Chl		G* G*mU* G.
  14104	1014	2705	A.mU.mU.mU.mC.	2706	P.mA. G. A. A. A.fU.	89%
  			G.mU.		A.fC. G. A. A.
  			A.mU.mU.mU.mC.mU.		A.mU*mU*mU*mC*
  			Chl		A* G* G.
  14105	356	2707	A. A. A. G.mC.mC.	2708	P.mU. G. G.fU.fC. A.fU.	85%
  			A.mU. G. A.mC.mC.		G.
  			A.Chl		G.fC.mU.mU.mU*mC*
  					G*mU*mU* G* G.
  14106	368	2709	A.mC. A.mU. G. G.	2710	P.mA.fU. A.fU.fC.	67%
  			A.mU. G. A.mU.		A.fU.fC.fC. A.mU.
  			A.mU.Chl		G.mU* G* G*mU*mC*
  					A* U.
  14107	1011	2711	G. A. A.	2712	P.mA. A.fU. A.fC. G. A.	87%
  			A.mU.mU.mU.mC.		A. A.fU.mU.mU.mC* A*
  			G.mU. A.mU.mU.Chl		G* G*mU* G* U.
  14108	754	2713	G.mC.	2714	P.mA. A.fU.fC. A. G. A.	73%
  			G.mC.mC.mU.mU.mC.		A. G. G.mC. G.mC*
  			mU. G.		G*mU*mU*mC* A* G.
  			A.mU.mU.Chl
  14109	1021	2715	A.mU.mU.mU.mC.mU.	2716	P.mA.fU.fU.fC. A.fU. G.	128%
  			mC. A.mU. G. A.		A. G. A. A. A.mU*
  			A.mU.Chl		A*mC* G* A* A* A.
  14110	1330	2717	mC.mU.mC.mU.mC.	2718	P.mC.fU. A.fU.fU.fC.	101%
  			A.mU. G. A. A.mU. A.		A.fU. G. A. G. A. G* A*
  			G.Chl		A*mU* A* A* C.
  14111	346	2719	A. A. G.mU.mC.mC.	2720	P.mU.fU.fU.fC. G.fU.fU.	59%
  			A. A.mC. G. A. A.		G. G. A.mC.mU.mU*
  			A.Chl		A*mC*mU*mU* G* G.
  14112	869	2721	A.mU. G. A.mU. G.	2722	P.mU.fU.	89%
  			A. G. A. G.mC. A.		G.fC.fU.fC.fU.fC.
  			A.Chl		A.fU.mC. A.mU*mU*
  					G* G*mC*mU* U.
  14113	701	2723	G.mC. G. A. G. G. A.	2724	P.mU.fU.fC. A.	95%
  			G.mU.mU. G. A.		A.fC.fU.fC.fC.fU.mC.
  			A.Chl		G.mC*mU*mU*mU*mC*
  					mC* A.
  14114	896	2725	mU. G. A.mU.mU. G.	2726	P.mU. G. A.fC.fU.	87%
  			A.mU. A. G.mU.mC.		A.fU.fC. A. A.mU.mC.
  			A.Chl		A*mC* A*mU*mC* G*
  					G.
  14115	1035	2727	A. G. A.mU. A.	2728	P.mA. G. A.fU. G.fC.	82%
  			G.mU. G.mC.		A.fC.fU. A.mU.mC.mU*
  			A.mU.mC.mU.Chl		A* A*mU*mU*mC* A.
  14116	1170	2729	A.mU. G.mU. G.mU.	2730	P.mA. A.fU. A. G. A.fU.	36%
  			A.mU.mC.mU.		A.fC. A.mC.
  			A.mU.mU.Chl		A.mU*mU*mC* A*
  					A*mC* C.
  14117	1282	2731	mU.mU.mC.mU.	2732	P.mU.fU.fC.fU.fU.fC.fU.	91%
  			A.mU. A. G. A. A. G.		A.fU. A. G. A. A*mU*
  			A. A.Chl		G* A* A*mC* A.
  14118	1537	2733	mU.mU.	2734	P.mA. A.fU.fU. G.fC.fU.	152%
  			G.mU.mC.mC. A.		G. G. A.mC. A.
  			G.mC. A.		A*mC*mC* G*mU* G*
  			A.mU.mU.Chl		G.
  14119	692	2735	A.mC. A.mU. G. G.	2736	P.mU.fC.	n/a
  			A. A. A. G. C.mG.		G.fC.fU.fU.fU.fC.fC.
  			A.Chl		A.mU. G.mU* G*mU*
  					G* A* G* G.
  14120	840	2737	G.mC. A.	2738	P.mU. A. A.fU.fC.fU. G.	87%
  			G.mU.mC.mC. A. G.		G. A.fC.mU.
  			A.mU.mU. A.Chl		G.mC*mU*mU*
  					G*mU* G* G.
  14121	1163	2739	mU. G. G.mU.mU. G.	2740	P.mA.fC. A.fC.	31%
  			A. A.mU. G.mU.		A.fU.fU.fC. A. A.mC.mC.
  			G.mU.Chl		A* A*mU* A* A* A* C.
  14122	789	2741	mU.mU. A.mU. G. A.	2742	P.mA.fC.fU.fC.	96%
  			A. A.mC. G. A.		G.fU.fU.fU.fC. A.mU. A.
  			G.mU.Chl		A*mC*mU*
  					G*mU*mC* C.
  14123	841	2743	mC. A.	2744	P.mA.fU. A. A.fU.fC.fU.	110%
  			G.mU.mC.mC. A. G.		G. G. A.mC.mU.
  			A.mU.mU. A.mU.Chl		G*mC*mU*mU*
  					G*mU* G.
  14124	852	2745	A.mU. A.mU. A. A.	2746	P.mU.fU.fU.fC.fC.	91%
  			G.mC. G. G. A. A.		G.fC.fU.fU. A.mU.
  			A.Chl		A.mU* A*
  					A*mU*mC*mU* G.
  14125	209	2747	mU. A.mC.mC. A.	2748	P.mU. G.fU.fU.fU. A.	110%
  			G.mU.mU. A. A.		A.fC.fU. G. G.mU.
  			A.mC. A.Chl		A*mU* G* G*mC* A*
  					C.
  14126	1276	2749	mU. G.mU.mU.mC.	2750	P.mU. A.fU. A. G. A.	n/a
  			A.mU.mU.mC.mU.		A.fU. G. A. A.mC.
  			A.mU. A.Chl		A*mU* A* G* A*mC*
  					A.
  14127	137	2751	mC.mC. G. A.mC.mC.	2752	P.mU.fU.fU.fC.fC.fU.fU.	71%
  			A. A. G. G. A. A.		G. G.fU.mC. G. G*mC*
  			A.Chl		G*mU*mU*mU* G.
  14128	711	2753	G. A. A.mU. G.	2754	P.mG.fU. A.fU. G.fC.	115%
  			G.mU. G.mC. A.mU.		A.fC.fC. A.mU.mU.mC*
  			A.mC.Chl		A* A*mC*mU*mC* C.
  14129	582	2755	A.mU. A.mU. G.	2756	P.mU.fC. G. G.fC.fC.	97%
  			A.mU. G. G.mC.mC.		A.fU.fC. A.mU. A.mU*
  			G. A.Chl		G*mU* G*mU*mC* U.
  14130	839	2757	A. G.mC. A.	2758	P.mA. A.fU.fC.fU. G. G.	102%
  			G.mU.mC.mC. A. G.		A.fC.fU. G.mC.mU*mU*
  			A.mU.mU.Chl		G*mU* G* G* C.
  14131	1091	2759	G.mC.	2760	P.mU.fU.fU. G. A.fC.fU.	10%
  			A.mU.mU.mU. A.		A. A. A.mU. G.mC* A*
  			G.mU.mC. A. A.		A* A* G*mU* G.
  			A.Chl
  14132	884	2761	A. G.mC.	2762	P.mA.fC. A.fU.fC. G. G.	93%
  			A.mU.mU.mC.mC. G.		A. A.fU. G.mC.mU*mC*
  			A.mU. G.mU.Chl		A*mU*mU* G* C.
  14133	903	2763	mU. A. G.mU.mC. A.	2764	P.mA. A.	97%
  			G. G. A.		G.fU.fU.fC.fC.fU. G.
  			A.mC.mU.mU.Chl		A.mC.mU. A*mU*mC*
  					A* A*mU* C.
  14134	1090	2765	mU. G.mC.	2766	P.mU.fU. G. A.fC.fU. A.	39%
  			A.mU.mU.mU. A.		A. A.fU. G.mC. A* A* A*
  			G.mU.mC. A. A.Chl		G*mU* G* A.
  14135	474	2767	G.mU.mC.mU. G.	2768	P.mA. G. A.fC.fU.fC.	99%
  			A.mU. G. A.		A.fU.fC. A. G.
  			G.mU.mC.mU.Chl		A.mC*mU* G* G*mU*
  					G* A.
  14136	575	2769	mU. A. G. A.mC.	2770	P.mU.fC. A.fU. A.fU.	108%
  			A.mC. A.mU. A.mU.		G.fU. G.fU.mC.mU.
  			G. A.Chl		A*mC*mU* G*mU* G*
  					G.
  14137	671	2771	mC. A. G. A.mC. G.	2772	P.mA.fU.	98%
  			A. G. G. A.mC.		G.fU.fC.fC.fU.fC.
  			A.mU.Chl		G.fU.mC.mU. G*mU*
  					A* G*mC* A* U.
  14138	924	2773	mC. A. G.mC.mC.	2774	P.mG. A. A.fU.fU.fC.	100%
  			G.mU. G. A.		A.fC. G. G.mC.mU. G*
  			A.mU.mU.mC.Chl		A*mC*mU*mU*mU*
  					G.
  14139	1185	2775	A. G.mU.mC.mU. G.	2776	P.mU.fU.	47%
  			G. A. A. A.mU. A.		A.fU.fU.fU.fC.fC. A. G.
  			A.Chl		A.mC.mU*mC* A* A*
  					A*mU* A.
  14140	1221	2777	A. G.mU.mU.mU.	2778	P.mG. A. A. G.fC.fC.	100%
  			G.mU. G.		A.fC. A. A. A.mC.mU*
  			G.mC.mU.mU.mC.Chl		A* A* A*mC*mU* A.
  14141	347	2779	A. G.mU.mC.mC. A.	2780	P.mC.fU.fU.fU.fC.	103%
  			A.mC. G. A. A. A.		G.fU.fU. G. G.
  			G.Chl		A.mC.mU*mU*
  					A*mC*mU*mU* G.
  14142	634	2781	A. A.	2782	P.mG.fU.fC.fU. G.fC. G.	100%
  			G.mU.mU.mU.mC.		A. A.
  			G.mC. A. G.		A.mC.mU.mU*mC*mU*
  			A.mC.Chl		mU* A* G* A.
  14143	877	2783	A. G.mC. A. A.mU.	2784	P.mA. A.fU. G.fC.fU.fC.	104%
  			G. A. G.mC.		A.fU.fU.
  			A.mU.mU.Chl		G.mC.mU*mC*mU*mC*
  					A*mU* C.
  14144	1033	2785	mU.mU. A. G. A.mU.	2786	P.mA.fU. G.fC. A.fC.fU.	95%
  			A. G.mU. G.mC.		A.fU.fC.mU. A.
  			A.mU.Chl		A*mU*mU*mC*
  					A*mU* G.
  14145	714	2787	mU. G. G.mU. G.mC.	2788	P.mC.fU.fU. G.fU. A.fU.	101%
  			A.mU. A.mC. A. A.		G.fC. A.mC.mC.
  			G.Chl		A*mU*mU*mC* A* A*
  					C.
  14146	791	2789	A.mU. G. A. A.	2790	P.mU. G. A.fC.fU.fC.	100%
  			A.mC. G. A.		G.fU.fU.fU.mC. A.mU*
  			G.mU.mC. A.Chl		A* A*mC*mU* G* U.
  14147	813	2791	mC.mC. A. G. A.	2792	P.mU.fU.fC. A. G.fC.	97%
  			G.mU. G.mC.mU. G.		A.fC.fU.fC.mU. G.
  			A. A.Chl		G*mU*mC*
  					A*mU*mC* C.
  14148	939	2793	mC. A. G.mC.mC.	2794	P.mA. A. A.fU.fU.fC.	109%
  			A.mU. G. A.		A.fU. G. G.mC.mU.
  			A.mU.mU.mU.Chl		G*mU* G* G* A* A* U.
  14149	1161	2795	A.mU.mU. G.	2796	P.mA.fC. A.fU.fU.fC. A.	34%
  			G.mU.mU. G. A.		A.fC.fC. A. A.mU* A* A*
  			A.mU. G.mU.Chl		A*mC*mU* G.
  14150	1164	2797	G. G.mU.mU. G. A.	2798	P.m U. A.fC. A.fC.	n/a
  			A.mU. G.mU. G.mU.		A.fU.fU.fC. A.
  			A.Chl		A.mC.mC* A* A*mU*
  					A* A* A.
  14151	1190	2799	G. G. A. A. A.mU. A.	2800	P.mA.fU.fU. A. G.fU.fU.	n/a
  			A.mC.mU. A.		A.fU.fU.mU.mC.mC* A*
  			A.mU.Chl		G* A*mC*mU* C.
  14152	1333	2801	mU.mC. A.mU. G. A.	2802	P.mU.fU.fU.fC.fU.	31%
  			A.mU. A. G. A. A.		A.fU.fU.fC. A.mU. G. A*
  			A.Chl		G* A* G* A* A* U.
  14153	537	2803	G.mC.mC. A. G.mC.	2804	P.mU.fU.fC. G. G.fU.fU.	n/a
  			A. A.mC.mC. G. A.		G.fC.fU. G. G.mC* A*
  			A.Chl		G* G*mU*mC* C.
  14154	684	2805	mC.	2806	P.mC. A.fU. G.fU. G.fU.	100%
  			A.mC.mC.mU.mC.		G. A. G. G.mU. G*
  			A.mC. A.mC. A.mU.		A*mU* G*mU*mC*C.
  			G.Chl
  14155	707	2807	A. G.mU.mU. G. A.	2808	P.mG.fC. A.fC.fC.	99%
  			A.mU. G. G.mU.		A.fU.fU.fC. A.
  			G.mC.Chl		A.mC.mU*mC*mC*mU*
  					mC* G* C.
  14156	799	2809	A. G.mU.mC. A.	2810	P.mC. A.fU.fC.fC. A.	95%
  			G.mC.mU. G. G.		G.fC.fU. G.
  			A.mU. G.Chl		A.mC.mU*mC*
  					G*mU*mU*mU* C.
  14157	853	2811	mU. A.mU. A. A.	2812	P.mC.fU.fU.fU.fC.fC.	106%
  			G.mC. G. G. A. A. A.		G.fC.fU.fU. A.mU.
  			G.Chl		A*mU* A* A*mU*mC*
  					U.
  14158	888	2813	mU.mU.mC.mC. G.	2814	P.mA. A.fU.fC. A.fC.	88%
  			A.mU. G.mU. G.		A.fU.fC. G. G. A. A*mU*
  			A.mU.mU.Chl		G*mC*mU*mC* A.
  14159	1194	2815	A.mU. A. A.mC.mU.	2816	P.mA.fC. A.fC. A.fU.fU.	95%
  			A. A.mU. G.mU.		A. G.fU.mU.
  			G.mU.Chl		A.mU*mU*mU*mC*mC*
  					A* G.
  14160	1279	2817	mU.mC.	2818	P.mU.fU.fC.fU. A.fU. A.	15%
  			A.mU.mU.mC.mU.		G. A. A.mU. G. A*
  			A.mU. A. G. A. A.Chl		A*mC* A*mU* A*G.
  14161	1300	2819	A. A.mC.mU.	2820	P.mU. A.fC. A. G.fU. G.	86%
  			A.mU.mC. A.mC.mU.		A.fU. A.
  			G.mU. A.Chl		G.mU.mU*mU* G*mC*
  					A*mU* U.
  14162	1510	2821	G.mU.mC. A.	2822	P.mA.fU. A. A. G.fC. A.	86%
  			A.mU.mU.		A.fU.fU. G. A.mC*
  			G.mC.mU.mU.		A*mC*mC* A*mC* C.
  			A.mU.Chl
  14163	1543	2823	A. G.mC. A.	2824	P.mU.fU.fU. A.fU.fU. A.	110%
  			A.mU.mU. A. A.mU.		A.fU.fU. G.mC.mU* G*
  			A. A. A.Chl		G* A*mC* A* A.
  14164	434	2825	A.mC. G.	2826	P.mU.fC. A.fU.fC. A. G.	134%
  			A.mC.mU.mC.mU. G.		A. G.fU.mC.
  			A.mU. G. A.Chl		G.mU*mU*mC* G* A*
  					G* U.
  14165	600	2827	mU. A. G.mU. G.mU.	2828	P.mA.fU. A. A. A.fC.fC.	102%
  			G. G.mU.mU.mU.		A.fC. A.mC.mU.
  			A.mU.Chl		A*mU*mC*
  					A*mC*mC* U.
  14166	863	2829	A. A. G.mC.mC. A.	2830	P.mU.fC. A.fU.fC.	93%
  			A.mU. G. A.mU. G.		A.fU.fU. G.
  			A.Chl		G.mC.mU.mU*mU*mC*
  					mC* G*mC* U.
  14167	902	2831	A.mU. A. G.mU.mC.	2832	P.mA. G.fU.fU.fC.fC.fU.	101%
  			A. G. G. A.		G. A.fC.mU. A.mU*mC*
  			A.mC.mU.Chl		A* A*mU*mC* A.
  14168	921	2833	A. G.mU.mC. A.	2834	P.mU.fU.fC. A.fC. G.	98%
  			G.mC.mC. G.mU. G.		G.fC.fU. G.
  			A. A.Chl		A.mC.mU*mU*mU* G*
  					G* A* A.
  14169	154	2835	A.mC.mU.	2836	P.mU.fU.fC.fU.fC. A.fU.	n/a
  			A.mC.mC. A.mU. G.		G. G.fU. A. G.mU* G*
  			A. G. A. A.Chl		A* G*mU*mU* U.
  14170	217	2837	A. A. A.mC. A. G.	2838	P.mA. A.fU.fC. A.	66%
  			G.mC.mU. G.		G.fC.fC.fU.
  			A.mU.mU.Chl		G.mU.mU.mU* A*
  					A*mC*mU* G* G.
  14171	816	2839	G. A. G.mU.	2840	P.mG. G.fU.fU.fU.fC. A.	102%
  			G.mC.mU. G. A. A.		G.fC.
  			A.mC.mC.Chl		A.mC.mU.mC*mU* G*
  					G*mU*mC* A.
  14172	882	2841	mU. G. A. G.mC.	2842	P.mA.fU.fC. G. G. A.	103%
  			A.mU.mU.mC.mC. G.		A.fU. G.fC.mU.mC.
  			A.mU.Chl		A*mU*mU*
  					G*mC*mU* C.
  14173	932	2843	A.	2844	P.mU. G. G.fC.fU. G.fU.	n/a
  			A.mU.mU.mC.mC.		G. G. A. A.mU.mU*mC*
  			A.mC. A. G.mC.mC.		A*mC* G* G* C.
  			A.Chl
  14174	1509	2845	mU. G.mU.mC. A.	2846	P.mU. A. A. G.fC. A.	n/a
  			A.mU.mU.		A.fU.fU. G. A.mC.
  			G.mC.mU.mU. A.Chl		A*mC*mC* A*mC*mC*
  					A.
  14175	157	2847	A.mC.mC. A.mU. G.	2848	P.mC. A.	109%
  			A. G. A. A.mU.mU.		A.fU.fU.fC.fU.fC. A.fU.
  			G.Chl		G. G.mU* A* G*mU*
  					G* A* G.
  14176	350	2849	mC.mC. A. A.mC. G.	2850	P.mU. G.	95%
  			A. A. A. G.mC.mC.		G.fC.fU.fU.fU.fC.
  			A.Chl		G.fU.mU. G. G*
  					A*mC*mU*mU* A* C.
  14177	511	2851	mC.mU. G.	2852	P.mA. A.fU.fC. A. G.fU.	100%
  			G.mU.mC. A.mC.mU.		G. A.fC.mC. A.
  			G. A.mU.mU.Chl		G*mU*mU*mC*
  					A*mU* C.
  14178	605	2853	mU. G.	2854	P.mA. G.fU.fC.fC. A.fU.	99%
  			G.mU.mU.mU.		A. A. A.mC.mC. A*mC*
  			A.mU. G. G.		A*mC*mU* A* U.
  			A.mC.mU.Chl
  14179	811	2855	G. A.mC.mC. A. G.	2856	P.mC. A. G.fC.	88%
  			A. G.mU. G.mC.mU.		A.fC.fU.fC.fU. G.
  			G.Chl		G.mU.mC*
  					A*mU*mC*mC* A* G.
  14180	892	2857	G. A.mU. G.mU. G.	2858	P.mU. A.fU.fC. A.	76%
  			A.mU.mU. G. A.mU.		A.fU.fC. A.fC.
  			A.Chl		A.mU.mC* G* G* A*
  					A*mU* G.
  14181	922	2859	G.mU.mC. A.	2860	P.mA.fU.fU.fC. A.fC. G.	59%
  			G.mC.mC. G.mU. G.		G.fC.fU. G.
  			A. A.mU.Chl		A.mC*mU*mU*mU*
  					G* G* A.
  14182	1169	2861	A. A.mU. G.mU.	2862	P.mA.fU. A. G. A.fU.	69%
  			G.mU.		A.fC. A.fC.
  			A.mU.mC.mU.		A.mU.mU*mC* A*
  			A.mU.Chl		A*mC*mC* A.
  14183	1182	2863	mU.mU. G. A.	2864	P.mU.fU.fU.fC.fC. A. G.	n/a
  			G.mU.mC.mU. G. G.		A.fC.fU.mC. A. A*
  			A. A. A.Chl		A*mU* A* G* A* U.
  14184	1539	2865	G.mU.mC.mC. A.	2866	P.mU.fU. A. A.fU.fU.	77%
  			G.mC. A. A.mU.mU.		G.fC.fU. G. G. A.mC* A*
  			A. A.Chl		A*mC*mC* G* U.
  14185	1541	2867	mC.mC. A. G.mC. A.	2868	P.mU. A.fU.fU. A.	n/a
  			A.mU.mU. A. A.mU.		A.fU.fU. G.fC.mU. G. G*
  			A.Chl		A*mC* A* A*mC*C.
  14186	427	2869	G. A.mC.mU.mC. G.	2870	P.mA. G.fU.fC.	69%
  			A. A.mC. G.		G.fU.fU.fC. G. A.
  			A.mC.mU.Chl		G.mU.mC* A* A*mU*
  					G* G* A.
  14187	533	2871	A.mC.mC.mU.	2872	P.mG.fU.fU. G.fC.fU. G.	78%
  			G.mC.mC. A. G.mC.		G.fC. A. G.
  			A. A.mC.Chl		G.mU*mC*mC*
  					G*mU* G* G.
  18538	496	2873	G. A.mU. G. A.	2874	P.mU. A.fU.fC. A. G.	74%
  			A.mU.mC.mU. G.		A.fU.fU.fC. A.fU.fC* A*
  			A.mU. A.Chl		G* A* A*fU* G.
  18539	496	2875	mU. G. A.mU. G. A.	2876	P.mU. A.fU.fC. A. G.	72%
  			A.mU.mC.mU. G.		A.fU.fU.fC. A.fU.fC* A*
  			A.mU. A.Chl		G* A* A*fU* G.
  18540	175	2877	A.mU.mU.mU.	2878	P.mU. G.fC. A. A. A. A.	98%
  			G.mC.mU.mU.mU.mU.		G.fC. A. A. A.fU*fC*
  			G.mC. A.Chl		A*fC*fU*fG* C.
  18541	175	2879	G. A.mU.mU.mU.	2880	P.mU. G.fC. A. A. A. A.	28%
  			G.mC.mU.mU.mU.mU.		G.fC. A. A. A.fU*fC*
  			G.mC. A.Chl		A*fC*fU*fG* C.
  18542	172	2881	G.mU. G.	2882	P.mU. A. A. A. G.fC. A.	24%
  			A.mU.mU.mU.		A. A.fU.fC. A.fC*fU*
  			G.mC.mU.mU.mU.		G*fC* A* A* U.
  			A.Chl
  18543	172	2883	A. G.mU. G.	2884	P.mU. A. A. A. G.fC. A.	14%
  			A.mU.mU.mU.		A. A.fU.fC. A.fC*fU*
  			G.mC.mU.mU.mU.		G*fC* A* A* U.
  			A.Chl
  18544	1013	2885	A.	2886	P.mU. A. A. A.fU. A.fC.	100%
  			A.mU.mU.mU.mC.		G. A. A. A.fU.fU*fU*fC*
  			G.mU.		A* G* G* U.
  			A.mU.mU.mU.A.Chl
  18545	1013	2887	A. A.	2888	P.mU. A. A. A.fU. A.fC.	109%
  			A.mU.mU.mU.mC.		G. A. A. A.fU.fU*fU*fC*
  			G.mU.		A* G* G* U.
  			A.mU.mU.mU.A.Chl
  18546	952	2889	mC. A.mC. A.	2890	P.mU.fU.fU. C. A.fU. G.	32%
  			G.mC.mC. A.mU. G.		G.fC.fU. G.fU. G* A* A*
  			A. A. A.Chl		A*fU*fU* C.
  18547	952	2891	mU.mC. A.mC. A.	2892	P.mU.fU.fU. C. A.fU. G.	33%
  			G.mC.mC. A.mU. G.		G.fC.fU. G.fU. G* A* A*
  			A. A. A.Chl		A*fU*fU* C.
  18548	174	2893	G. A.mU.mU.mU.	2894	P.mU.fC. A. A. A. A.	57%
  			G.mC.mU.mU.mU.mU.		G.fC. A. A. A.fU.fC*
  			G. A.Chl		A*fC*fU* G*fC* A.
  18549	174	2895	mU. G.	2896	P.mU.fC. A. A. A. A.	53%
  			A.mU.mU.mU.		G.fC. A. A. A.fU.fC*
  			G.mC.mU.mU.mU.mU.		A*fC*fU* G*fC*A.
  			G. A.Chl
  18550	177	2897	mU.mU.	2898	P.mU. A. G. G.fC. A. A.	97%
  			G.mC.mU.mU.mU.mU.		A. A. G.fC. A. A*
  			G.mC.mC.mU.		A*fU*fC* A*fC* U.
  			A.Chl
  18551	177	2899	mU.mU.mU.	2900	P.mU. A. G. G.fC. A. A.	103%
  			G.mC.mU.mU.mU.mU.		A. A. G.fC. A. A*
  			G.mC.mC.mU.		A*fU*fC* A*fC* U.
  			A.Chl
  18552	1150	2901	mU.mU.mU.mC.mU.	2902	P.mU.fU. A. A. A.fC.fU.	96%
  			mC. A.		G. A. G. A. A. A* G* A*
  			G.mU.mU.mU. A.		A* G*fC* A.
  			A.Chl
  18553	1089	2903	mU.mU. G.mC.	2904	P.mU. G. A.fC.fU. A. A.	94%
  			A.mU.mU.mU. A.		A.fU. G.fC. A. A* A*
  			G.mU.mC. A.Chl		G*fU* G* A* G.
  18554	1086	2905	A.mC.mU.mU.mU.	2906	P.mU.fU. A. A. A.fU.	n/a
  			G.mC.		G.fC. A. A. A. G.fU* G*
  			A.mU.mU.mU. A.		A* G* A* A* A.
  			A.Chl
  18555	1093	2907	A.mU.mU.mU. A.	2908	P.mU.fU.fU.fU.fU. G.	n/a
  			G.mU.mC. A. A. A. A.		A.fC.fU. A. A. A.fU*
  			A.Chl		G*fC* A* A* A* G.
  18556	1147	2909	mU.mU.mC.mU.mU.	2910	P.mU. A.fC.fU. G. A. G.	n/a
  			mU.mC.mU.mC. A.		A. A. A. G. A. A* G*fC*
  			G.mU. A.Chl		A*fU*fU* U.
  18557	1148	2911	mU.mC.mU.mU.mU.	2912	P.mU. A. A.fC.fU. G. A.	66%
  			mC.mU.mC. A.		G. A. A. A. G. A* A*
  			G.mU.mU. A.Chl		G*fC* A*fU* U.
  18558	1128	2913	G. A. A. A. G. A. G.	2914	P.mU. A.fU.	16%
  			A. A.mC. A.mU.		G.fU.fU.fC.fU.fC.fU.fU.fU.
  			A.Chl		fC* A*fU*fU*fU*fU*
  					G.
  18559	1087	2915	mC.mU.mU.mU.	2916	P.mU.fC.fU. A. A. A.fU.	28%
  			G.mC.		G.fC. A. A. A. G*fU* G*
  			A.mU.mU.mU. A. G.		A* G* A* A.
  			A.Chl
  18560	1088	2917	mU.mU.mU. G.mC.	2918	P.mU. A.fC.fU. A. A.	n/a
  			A.mU.mU.mU. A.		A.fU. G.fC. A. A. A*
  			G.mU. A.Chl		G*fU* G* A* G* A.
  18561	1083	2919	mC.mU.mC.	2920	P.mU. A.fU. G.fC. A. A.	53%
  			A.mC.mU.mU.mU.		A. G.fU. G. A. G* A* A*
  			G.mC. A.mU. A.Chl		A*fU*fU* G.
  18562	1081	2921	mU.mU.mC.mU.mC.	2922	P.mU. G.fC. A. A. A.	89%
  			A.mC.mU.mU.mU.		G.fU. G. A. G. A. A*
  			G.mC. A.Chl		A*fU*fU* G*fU* A.
  18563	555	2923	mC.	2924	P.mU. A.fC. A. A.fC.fU.	33%
  			A.mC.mU.mC.mC. A.		G. G. A. G.fU. G* A* A*
  			G.mU.mU. G.mU.		A* A*fC*fU.
  			A.Chl
  18564	1125	2925	A. A.mU. G. A. A. A.	2926	P.mU.fU.fU.fC.fU.fC.fU.	n/a
  			G. A. G. A. A. A.Chl		fU.fU.fC.
  					A.fU.fU*fU*fU*
  					G*fC*fU* A.
  18565	168	2927	mU. G.mC. A. G.mU.	2928	P.mU.fC. A. A. A.fU.fC.	14%
  			G.		A.fC.fU. G.fC. A*
  			A.mU.mU.mU.mG.		A*fU*fU*fC*fU*C.
  			A.Chl
  18566	1127	2929	mU. G. A. A. A. G. A.	2930	P.mU.fU.	27%
  			G. A. A.mC. A. A.Chl		G.fU.fU.fC.fU.fC.fU.fU.fU.fC.
  					A*fU*fU*fU*fU*
  					G* C.
  18567	1007	2931	A.mC.mC.mU. G. A.	2932	P.mU. G. A. A.	129%
  			A.		A.fU.fU.fU.fC. A. G.
  			A.mU.mU.mU.mC.		G.fU* G*fU*fU*fU* A*
  			A.Chl		U.
  18568	164	2933	G. A. A.mU.mU.	2934	P.mU.fU.fC. A.fC.fU.	47%
  			G.mC. A. G.mU. G. A.		G.fC. A.
  			A.Chl		A.fU.fU.fC*fU*fC*
  					A*fU* G* G.
  18569	222	2935	G. G.mC.mU. G.	2936	P.mU.fC.fC. A. G. A.	n/a
  			A.mU.mU.mC.mU.		A.fU.fC. A. G.fC.fC*fU*
  			G. G. A.Chl		G*fU*fU*fU* A.
  20612	172	2937	A. G.mU. G.	2938	P.mU. A. A. A. G.fC. A.	n/a
  			A.mU.mU.mU.		A. A.fU.mC. A.mC*mU*
  			G.mC.mU.mU.mU.		G*mC* A* A* U.
  			A.Chl
  20613	172	2939	A. G.mU. G.	2940	P.mU. A. A. A. G.fC. A.	n/a
  			A.mU.mU.mU.		A. A.fU.fC. A.mC*fU*
  			G.mC.mU.mU.mU.		G*mC* A* A* U.
  			A.Chl
  20614	172	2941	A. G.mU. G.	2942	P.mU. A. A. A. G. C. A.	101%
  			A.mU.mU.mU.		A. A. U.mC. A.mC*mU*
  			G.mC.mU.mU.mU.		G*mC* A* A* U.
  			A.Chl
  20615	172	2943	A. G.mU. G.	2944	P.mU. A. A. A. G.fC. A.	104%
  			A.mU.mU.mU.		A. A.fU.mC.
  			G.mC.mU.mU.mU.		A.mC*mU*mG*mC*mA*
  			A.Chl		mA* U.

• TABLE 19
  Inhibition of gene expression with PTGS2 sd-rxRNA sequences (Accession Number: NM_000963.2)

  Oligo	Start	SEQ ID		SEQ ID
  Number	Site	NO	Sense sequence	NO	Antisense sequence

  						% remaining
  						expression
  						(1 uM A549)
  14422	451	2945	mC. A.mC.	2946	P.mU.fC. A. A.fU.fC. A.	72%
  			A.mU.mU.mU. G.		A. A.fU. G.mU. G*
  			A.mU.mU. G. A.Chl		A*mU*mC*mU* G* G.
  14423	1769	2947	mC. A.mC.mU.	2948	P.mA. A.fU.fU. G.A. G.	71%
  			G.mC.mC.mU.mC. A.		G.fC. A. G.mU.
  			A.mU.mU.Chl		G*mU*mU* G* A*mU*
  					G.
  14424	1464	2949	A. A. A.mU.	2950	P.mA. A. G. A.fC.fU. G.	74%
  			A.mC.mC. A.		G.fU.
  			G.mU.mC.mU.mU.Chl		A.mU.mU.mU*mC*
  					A*mU*mC*mU* G.
  14425	453	2951	mC. A.mU.mU.mU.	2952	P.mU. G.fU.fC. A.	83%
  			G. A.mU.mU. G.		A.fU.fC. A. A. A.mU.
  			A.mC. A.Chl		G*mU* G* A*mU*mC*
  					U.
  						% remaining
  						expression
  						(1 uM PC-3)
  17388	285	2953	G. A. A. A.	2954	P.mU.fU. G. A. G.fC. A.	88%
  			A.mC.mU.		G.fU.fU.fU.fU.fC*fU*fC*
  			G.mC.mU.mC. A.		fC* A*fU* A.
  			A.Chl
  17389	520	2955	A.mC.mC.mU.mC.mU.	2956	P.mU. A. A.fU. A. G. G.	25%
  			mC.mC.mU.		A. G. A. G. G.fU*fU* A*
  			A.mU.mU. A.Chl		G* A* G* A.
  17390	467	2957	mU.mC.mC.	2958	P.mU.fU. A. A. G.fU.fU.	68%
  			A.mC.mC. A.		G. G.fU. G. G. A*fC*fU*
  			A.mC.mU.mU. A.		G*fU*fC* A.
  			A.Chl
  17391	467	2959	G.mU.mC.mC.	2960	P.mU.fU. A. A. G.fU.fU.	101%
  			A.mC.mC. A.		G. G.fU. G. G. A*fC*fU*
  			A.mC.mU.mU. A.		G*fU*fC* A.
  			A.Chl
  17392	524	2961	mC.mU.mC.mC.mU.	2962	P.mU. G.fU. A.fU. A.	49%
  			A.mU.mU. A.mU.		A.fU. A. G. G. A. G* A*
  			A.mC. A.Chl		G* G*fU*fU* A.
  17393	448	2963	G. A.mU.mC. A.mC.	2964	P.mU.fU.fC. A. A. A.fU.	29%
  			A.mU.mU.mU. G. A.		G.fU. G. A.fU.fC*fU* G*
  			A.Chl		G* A*fU* G.
  17394	448	2965	A. G. A.mU.mC.	2966	P.mU.fU.fC. A. A. A.fU.	31%
  			A.mC.		G.fU. G. A.fU.fC*fU* G*
  			A.mU.mU.mU. G. A.		G* A*fU*G.
  			A.Chl
  17395	519	2967	A.	2968	P.mU. A.fU. A. G. G. A.	12%
  			A.mC.mC.mU.mC.mU.		G. A. G. G.fU.fU* A* G*
  			mC.mC.mU. A.mU.		A* G* A* A.
  			A.Chl
  17396	437	2969	G.mU.mU. G. A.mC.	2970	P.mU.fC.fU. G. G. A.fU.	86%
  			A.mU.mC.mC. A. G.		G.fU.fC. A. A.fC* A*fC*
  			A.Chl		A*fU* A* A.
  17397	406	2971	mC.mC.mU.mU.mC.	2972	P.mU.fU.fU.fC. G. A. A.	23%
  			mC.mU.mU.mC. G.		G. G. A. A. G. G* G* A*
  			A. A. A.Chl		A*fU* G* U.
  17398	339	2973	A.mC.mU.mC.mC.	2974	P.mU.fU. G.fU.	102%
  			A. A. A.mC. A.mC. A.		G.fU.fU.fU. G. G. A.
  			A.Chl		G.fU* G* G* G*fU*fU*
  					U.
  17399	339	2975	mC.	2976	P.mU.fU. G.fU.	55%
  			A.mC.mU.mC.mC. A.		G.fU.fU.fU. G. G. A.
  			A. A.mC. A.mC. A.		G.fU* G* G* G*fU*fU*
  			A.Chl		U.
  17400	338	2977	mC.	2978	P.mU. G.fU. G.fU.fU.fU.	62%
  			A.mC.mU.mC.mC. A.		G. G. A. G.fU. G* G*
  			A. A.mC. A.mC. A.Chl		G*fU*fU*fU* C.
  17401	468	2979	mC.mC. A.mC.mC. A.	2980	P.mU. G.fU. A. A.	61%
  			A.mC.mU.mU. A.mC.		G.fU.fU. G. G.fU. G. G*
  			A.Chl		A*fC*fU* G*fU* C.
  17402	468	2981	mU.mC.mC.	2982	P.mU. G.fU. A. A.	179%
  			A.mC.mC. A.		G.fU.fU. G. G.fU. G. G*
  			A.mC.mU.mU. A.mC.		A*fC*fU* G*fU*C.
  			A.Chl
  17403	1465	2983	A. A.mU. A.mC.mC.	2984	P.mU. A. A. G. A.fC.fU.	30%
  			A.		G. G.fU. A.fU.fU*fU*fC*
  			G.mU.mC.mU.mU.		A*fU*fC* U.
  			A.Chl
  17404	243	2985	G. A.mC.mC. A.	2986	P.mU.fC.fU.fU. A.fU.	32%
  			G.mU. A.mU. A. A.		A.fC.fU. G. G.fU.fC* A*
  			G. A.Chl		A* A*fU*fC* C.
  17405	1472	2987	G.mU.mC.mU.mU.mU.	2988	P.mU.fU.fC. A.fU.fU. A.	15%
  			mU. A. A.mU. G.		A. A. A. G. A.fC*fU* G*
  			A. A.Chl		G*fU* A* U.
  17406	2446	2989	A.	2990	P.mU. A. G. A.fC. A.fU.	142%
  			A.mU.mU.mU.mC.		G. A. A. A.fU.fU*
  			A.mU.		A*fC*fU* G* G* U.
  			G.mU.mC.mU.A.Chl
  17407	449	2991	A.mU.mC. A.mC.	2992	P.mU. A.fU.fC. A. A.	54%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.Chl		A.fU*fC*fU* G* G* A*
  					U.
  17408	449	2993	G. A.mU.mC. A.mC.	2994	P.mU. A.fU.fC. A. A.	27%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.Chl		A.fU*fC*fU* G* G* A*
  					U.
  17409	444	2995	mU.mC.mC. A. G.	2996	P.mU. A.fU. G.fU. G.	49%
  			A.mU.mC. A.mC.		A.fU.fC.fU. G. G. A*fU*
  			A.mU. A.Chl		G*fU*fC* A* A.
  17410	1093	2997	mU. A.mC.mU. G.	2998	P.mU.fC.fU.fC.fC.fU.	32%
  			A.mU. A. G. G. A. G.		A.fU.fC. A. G.fU.
  			A.Chl		A*fU*fU* A* G*fC* C.
  17411	1134	2999	G.mU. G.mC. A.	3000	P.mU.fC. A. A. G.fU.	70%
  			A.mC. A.mC.mU.fU.		G.fU.fU. G.mC. A.fC*
  			G. A.Chl		A*fU* A* A*fU* C.
  17412	244	3001	A.mC.mC. A. G.mU.	3002	P.mU. A.fC.fU.fU. A.fU.	63%
  			A.mU. A. A. G.mU.		A.fC.fU. G. G.fU*fC* A*
  			A.Chl		A* A*fU* C.
  17413	1946	3003	G. A. A.	3004	P.mU.fU.fC. A.fU.fU. A.	19%
  			G.mU.mC.mU. A.		G. A.fC.mU.fU.fC*fU*
  			A.mU. G. A. A.Chl		A*fC* A* G* U.
  17414	638	3005	A. A. G. A. A. G. A.	3006	P.mU. A.	27%
  			A. A. G.mU.mU.		A.fC.fU.fU.fU.fC.fU.fU.fC.
  			A.Chl		fU.fU* A* G* A* A*
  					G* C.
  17415	450	3007	mU.mC. A.mC.	3008	P.mU. A. A.fU.fC. A. A.	216%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU.mU. A.Chl		A*fU*fC*fU* G* G* A.
  17416	450	3009	A.mU.mC. A.mC.	3010	P.mU. A. A.fU.fC. A. A.	32%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU.mU. A.Chl		A*fU*fC*fU* G* G* A.
  17417	452	3011	A.mC.	3012	P.mU.fU.fC. A. A.fU.fC.	99%
  			A.mU.mU.mU. G.		A. A. A.fU. G.fU* G*
  			A.mU.mU. G. A.		A*fU*fC*fU*G.
  			A.Chl
  17418	452	3013	mC. A.mC.	3014	P.mU.fU.fC. A. A.fU.fC.	54%
  			A.mU.mU.mU. G.		A. A. A.fU. G.fU* G*
  			A.mU.mU. G. A.		A*fU*fC*fU*G.
  			A.Chl
  17419	454	3015	A.mU.mU.mU. G.	3016	P.mU.fU. G.fU.fC. A.	86%
  			A.mU.mU. G. A.mC.		A.fU.fC. A. A. A.fU*
  			A. A.Chl		G*fU* G* A*fU* C.
  17420	454	3017	mC. A.mU.mU.mU.	3018	P.mU.fU. G.fU.fC. A.	89%
  			G. A.mU.mU. G.		A.fU.fC. A. A. A.fU*
  			A.mC. A. A.Chl		G*fU* G* A*fU* C.
  17421	1790	3019	mC. A.mU.mC.mU.	3020	P.mU.fU.fU. A.fU.fU.	55%
  			G.mC. A. A.mU. A. A.		G.fC. A. G. A.fU. G* A*
  			A.Chl		G* A* G* A* C.
  17422	1790	3021	mU.mC.	3022	P.mU.fU.fU. A.fU.fU.	62%
  			A.mU.mC.mU. G.mC.		G.fC. A. G. A.fU. G* A*
  			A. A.mU. A. A. A.Chl		G* A* G* A* C.
  21180	448	3023	G. A.mU.mC. A.mC.	3024	P.mU.fU.fC. A.mA. A.fU.	76%
  			A.mU.mU.mU. G. A.		G.fU. G. A.mU.mC*mU*
  			A.TEG-Chl		G* G* A*mU* G.
  21181	448	3025	G. A.mU.mC. A.mC.	3026	P.mU.fU.fC. A.mA. A.fU.	37%
  			A.mU.mU.mU. G. A.		G.fU. G.
  			A.TEG-Chl		A.fU.fC*fU*mG*mG*mA*
  					fU* G.
  21182	448	3027	G. A.mU.mC. A.mC.	3028	P.mU.fU.fC. A. A. A.fU.	29%
  			A.mU.mU.mU.		G.fU. G. A.fU.fC*fU* G*
  			G*mA*mA.TEG-Chl		G* A*fU* G.
  21183	448	3029	mG*mA*mU.mC.	3030	P.mU.fU.fC. A. A. A.fU.	46%
  			A.mC.		G.fU. G. A.fU.fC*fU* G*
  			A.mU.mU.mU.		G* A*fU* G.
  			G*mA*mA.TEG-Chl
  21184	448	3031	mG*mA*mU.mC.mA.	3032	P.mU.fU.fC. A. A. A.fU.	60%
  			mC.mA.mU.mU.mU.		G.fU. G. A.fU.fC*fU* G*
  			mG*mA*mA.TEG-		G* A*fU*G.
  			Chl
  21185	449	3033	G. A.mU.mC. A.mC.	3034	P.mU. A.fU.fC. A. A.	27%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.TEG-Chl		A.fU.fC*fU* G* G*
  					A*fU* G.
  21186	449	3035	G. A.mU.mC. A.mC.	3036	P.mU. A.fU.fC. A. A.	57%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.TEG-Chl		A.mU.mC*mU* G* G*
  					A*mU* G.
  21187	449	3037	G. A.mU.mC. A.mC.	3038	P.mU. A.fU.fC. A.mA.	54%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.TEG-Chl		A.mU.mC*mU* G* G*
  					A*mU* G.
  21188	449	3039	G. A.mU.mC. A.mC.	3040	P.mU. A.fU.fC. A. A.	66%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.TEG-Chl		A.mU.mC*mU*mG*mG*
  					mA*mU* G.
  21189	449	3041	G. A.mU.mC. A.mC.	3042	P.mU. A.fU.fC. A.mA.	44%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.TEG-Chl		A.mU.mC*mU*mG*mG*
  					mA*mU* G.
  21190	449	3043	G. A.mU.mC. A.mC.	3044	P.mU. A.fU.fC. A. A.	52%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.TEG-Chl		A.fU.fC*fU*mG*mG*mA*
  					fU* G.
  21191	449	3045	G. A.mU.mC. A.mC.	3046	P.mU. A.fU.fC. A.mA.	41%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.TEG-Chl		A.fU.fC*fU*mG*mG*mA*
  					fU* G.
  21192	449	3047	G. A.mU.mC. A.mC.	3048	P.mU. A.fU.fC. A. A.	98%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.TEG-Chl		A.fU.mC*fU*mG*mG*
  					mA*fU* G.
  21193	449	3049	G. A.mU.mC. A.mC.	3050	P.mU. A.fU.fC. A. A.	93%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A*mU*mA.TEG-Chl		A.fU*fC*fU* G* G* A*
  					U.
  21194	449	3051	mG*mA*mU.mC.	3052	P.mU. A.fU.fC. A. A.	119%
  			A.mC.		A.fU. G.fU. G.
  			A.mU.mU.mU. G.		A.fU*fC*fU* G* G* A*
  			A*mU*mA.TEG-Chl		U.
  21195	449	3053	mG*mA*mU.mC.mA.	3054	P.mU. A.fU.fC. A. A.	292%
  			mC.mA.mU.mU.mU.		A.fU. G.fU. G.
  			mG.mA*mU*mA.TEG-		A.fU*fC*fU* G* G* A*
  			Chl		U.
  20620	449	3055	G. A.mU.mC. A.mC.	3056	P.mU. A.fU.fC. A. A.	24%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.Chl-TEG		A.mU*mC*mU* G* G*
  					A* U.
  20621	449	3057	G. A.mU.mC. A.mC.	3058	P.mU. A.fU.fC. A. A.	5%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.Chl-TEG		A.mU*fC*mU* G* G*
  					A* U.
  20622	449	3059	G. A.mU.mC. A.mC.	3060	P.mU. A. U. C. A. A. A.	25%
  			A.mU.mU.mU. G.		U. G. U. G.
  			A.mU. A.Chl-TEG		A.mU*mC*mU* G* G*
  					A* U.
  20623	449	3061	G. A.mU.mC. A.mC.	3062	P.mU. A.fU.fC. A. A.	14%
  			A.mU.mU.mU. G.		A.fU. G.fU. G.
  			A.mU. A.Chl-TEG		A.mU*mC*mU*mG*mG*
  					mA* U.
  20588	448	3063	G. A.mU.mC. A.mC.	3064	P.mU.fU.fC. A. A. A.fU.	17%
  			A.mU.mU.mU. G. A.		G.fU. G. A.mU.mC*mU*
  			A.Chl-TEG		G* G* A*mU* G.
  20589	448	3065	G. A.mU.mC. A.mC.	3066	P.mU.fU.fC. A. A. A.fU.	40%
  			A.mU.mU.mU. G. A.		G.fU. G. A.mU.fC*mU*
  			A.Chl-TEG		G* G* A*fU* G.
  20590	448	3067	G. A.mU.mC. A.mC.	3068	P.mU. U. C. A. A. A. U.	34%
  			A.mU.mU.mU. G. A.		G. U. G. A.mU.mC*mU*
  			A.Chl-TEG		G* G* A*mU* G.
  20591	448	3069	G. A.mU.mC. A.mC.	3070	P.mU.fU.fC. A. A. A.fU.	n/a
  			A.mU.mU.mU. G. A.		G.fU. G.
  			A.Chl-TEG		A.fU.fC*fU*mG*mG*mA*
  					fU* G.

• TABLE 20
  Inhibition of gene expression with CTGF sd-rxRNA sequences (Accession Number: NM_001901.2)

  						% remaining
  						mRNA expression
  Oligo	Start	SEQ ID		SEQ ID		(1 uM sd-rxRNA,
  Number	Site	NO	Sense sequence	NO	Antisense sequence	A549)

  13980	1222	3071	A.mC. A. G. G. A.	3072	P.mU. A.fC.	98%
  			A. G. A.mU. G.mU.		A.fU.fC.fU.fU.fC.fC.mU.
  			A.Chl		G.mU* A* G*mU*
  					A*mC* A.
  13981	813	3073	G. A. G.mU. G. G.	3074	P.mA. G. G.fC.	82%
  			A. G.mC.		G.fC.fU.fC.fC.
  			G.mC.mC.mU.Chl		A.mC.mU.mC*mU*
  					G*mU* G* G* U.
  13982	747	3075	mC. G. A.mC.mU.	3076	P.mU.	116%
  			G. G. A. A. G.		G.fU.fC.fU.fU.fC.fC. A.
  			A.mC. A.Chl		G.mU.mC. G* G*mU*
  					A* A* G* C.
  13983	817	3077	G. G. A. G.mC.	3078	P.mG. A. A.fC. A. G.	97%
  			G.mC.mC.mU.		G.fC.
  			G.mU.mU.mC.Chl		G.fC.mU.mC.mC*
  					A*mC*mU*mC*mU*
  					G.
  13984	1174	3079	G.mC.mC.	3080	P.mC. A. G.fU.fU.	102%
  			A.mU.mU. A.mC.		G.fU. A. A.fU. G.
  			A. A.mC.mU.		G.mC* A* G* G*mC*
  			G.Chl		A* C.
  13985	1005	3081	G. A.	3082	P.mA. G.fC.fC. A. G. A.	114%
  			G.mC.mU.mU.mU.		A. A. G.mC.mU.mC*
  			mC.mU. G.		A* A* A*mC*mU* U.
  			G.mC.mU.Chl
  13986	814	3083	A. G.mU. G. G. A.	3084	P.mC. A. G. G.fC.	111%
  			G.mC.		G.fC.fU.fC.fC.
  			G.mC.mC.mU.		A.mC.mU*mC*mU*
  			G.Chl		G*mU* G* G.
  13987	816	3085	mU. G. G. A.	3086	P.mA. A.fC. A. G. G.fC.	102%
  			G.mC.		G.fC.fU.mC.mC.
  			G.mC.mC.mU.		A*mC*mU*mC*mU*
  			G.mU.mU.Chl		G* U.
  13988	1001	3087	G.mU.mU.mU. G.	3088	P.mA. G. A. A. A.	99%
  			A.		G.fC.fU.fC. A. A.
  			G.mC.mU.mU.mU.		A.mC*mU*mU* G*
  			mC.mU.Chl		A*mU* A.
  13989	1173	3089	mU. G.mC.mC.	3090	P.mA. G.fU.fU. G.fU.	107%
  			A.mU.mU. A.mC.		A. A.fU. G. G.mC. A*
  			A. A.mC.mU.Chl		G* G*mC* A*mC* A.
  13990	749	3091	A.mC.mU. G. G.	3092	P.mC. G.fU.	91%
  			A. A. G. A.mC.		G.fU.fC.fU.fU.fC.fC. A.
  			A.mC. G.Chl		G.mU*mC* G*
  					G*mU* A* A.
  13991	792	3093	A. A.mC.mU.	3094	P.mG. G. A.fC.fC. A. G.	97%
  			G.mC.mC.mU. G.		G.fC. A. G.mU.mU* G*
  			G.mU.mC.mC.Chl		G*mC*mU*mC* U.
  13992	1162	3095	A. G.	3096	P.mC. A. G. G.fC. A.fC.	107%
  			A.mC.mC.mU.		A. G.
  			G.mU.		G.mU.mC.mU*mU*
  			G.mC.mC.mU.		G* A*mU* G*A.
  			G.Chl
  13993	811	3097	mC. A. G. A.	3098	P.mG.fC. G.fC.fU.fC.fC.	113%
  			G.mU. G. G. A.		A.fC.fU.mC.mU.
  			G.mC. G.mC.Chl		G*mU* G*
  					G*mU*mC* U.
  13994	797	3099	mC.mC.mU. G.	3100	P.mG. G.fU.fC.fU. G.	n/a
  			G.mU.mC.mC. A.		G. A.fC.fC. A. G.
  			G. A.mC.mC.Chl		G*mC* A*
  					G*mU*mU* G.
  13995	1175	3101	mC.mC.	3102	P.mA.fC. A. G.fU.fU.	113%
  			A.mU.mU. A.mC.		G.fU. A. A.mU. G.
  			A. A.mC.mU.		G*mC* A* G* G*mC*
  			G.mU.Chl		A.
  13996	1172	3103	mC.mU.	3104	P.mG.fU.fU. G.fU. A.	110%
  			G.mC.mC.		A.fU. G. G.mC. A. G*
  			A.mU.mU. A.mC.		G*mC* A*mC* A* G.
  			A. A.mC.Chl
  13997	1177	3105	A.mU.mU. A.mC.	3106	P.mG. G. A.fC. A.	105%
  			A. A.mC.mU.		G.fU.fU. G.fU. A.
  			G.mU.mC.mC.Chl		A.mU* G* G*mC* A*
  					G* G.
  13998	1176	3107	mC. A.mU.mU.	3108	P.mG. A.fC. A. G.fU.fU.	89%
  			A.mC. A.		G.fU. A. A.mU. G*
  			A.mC.mU.		G*mC* A* G* G* C.
  			G.mU.mC.Chl
  13999	812	3109	A. G. A. G.mU. G.	3110	P.mG. G.fC.	99%
  			G. A. G.mC.		G.fC.fU.fC.fC.
  			G.mC.mC.Chl		A.fC.mU.mC.mU*
  					G*mU* G* G*mU* C.
  14000	745	3111	A.mC.mC. G.	3112	P.mU.fC.fU.fU.fC.fC. A.	n/a
  			A.mC.mU. G. G. A.		G.fU.fC. G. G.mU* A*
  			A. G. A.Chl		A* G*mC*mC* G.
  14001	1230	3113	A.mU. G.mU.	3114	P.mU.	106%
  			A.mC. G. G. A. G.		G.fU.fC.fU.fC.fC. G.fU.
  			A.mC. A.Chl		A.mC.
  					A.mU*mC*mU*mU*
  					mC*mC* U.
  14002	920	3115	G.mC.mC.mU.mU.	3116	P.mA. G.fC.fU.fU.fC.	93%
  			G.mC. G. A. A.		G.fC. A. A. G.
  			G.mC.mU.Chl		G.mC*mC*mU* G*
  					A*mC* C.
  14003	679	3117	G.mC.mU. G.mC.	3118	P.mC.	102%
  			G. A. G. G. A.		A.fC.fU.fC.fC.fU.fC.
  			G.mU. G.Chl		G.fC. A. G.mC*
  					A*mU*mU*mU*mC*
  					C.
  14004	992	3119	G.mC.mC.mU.	3120	P.mA. A. A.fC.fU.fU. G.	100%
  			A.mU.mC. A. A.		A.fU. A. G.
  			G.mU.mU.mU.Chl		G.mC*mU*mU* G*
  					G* A* G.
  14005	1045	3121	A.	3122	P.mA.fC.fU.fC.fC. A.fC.	104%
  			A.mU.mU.mC.mU.		A. G. A.
  			G.mU. G. G. A.		A.mU.mU*mU* A*
  			G.mU.Chl		G*mC*mU* C.
  14006	1231	3123	mU. G.mU. A.mC.	3124	P.mA.fU.	87%
  			G. G. A. G. A.mC.		G.fU.fC.fU.fC.fC. G.fU.
  			A.mU.Chl		A.mC.
  					A*mU*mC*mU*mU*
  					mC* C.
  14007	991	3125	A. G.mC.mC.mU.	3126	P.mA. A.fC.fU.fU. G.	101%
  			A.mU.mC. A. A.		A.fU. A. G.
  			G.mU.mU.Chl		G.mC.mU*mU* G* G*
  					A* G* A.
  14008	998	3127	mC. A. A.	3128	P.mA. A. G.fC.fU.fC. A.	98%
  			G.mU.mU.mU. G.		A. A.fC.mU.mU. G*
  			A.		A*mU* A* G* G* C.
  			G.mC.mU.mU.Chl
  14009	1049	3129	mC.mU. G.mU. G.	3130	P.mA.fC. A.fU.	98%
  			G. A. G.mU. A.mU.		A.fC.fU.fC.fC. A.mC. A.
  			G.mU.Chl		G* A*
  					A*mU*mU*mU* A.
  14010	1044	3131	A. A.	3132	P.mC.fU.fC.fC. A.fC. A.	93%
  			A.mU.mU.mC.mU.		G. A. A.mU.mU.mU*
  			G.mU. G. G. A.		A* G*mC*mU*mC*G.
  			G.Chl
  14011	1327	3133	mU.mU.mU.mC.	3134	P.mU. G.fU. G.fC.fU.	95%
  			A. G.mU. A. G.mC.		A.fC.fU. G. A. A.
  			A.mC. A.Chl		A*mU*mC*
  					A*mU*mU* U.
  14012	1196	3135	mC. A. A.mU. G.	3136	P.mA. A. A. G. A.fU.	101%
  			A.mC.		G.fU.fC. A.mU.mU.
  			A.mU.mC.mU.mU.		G*mU*mC*mU*mC*
  			mU.Chl		mC* G.
  14013	562	3137	A. G.mU.	3138	P.mG.fU. G.fC. A.fC.fU.	66%
  			A.mC.mC. A.		G. G.fU.
  			G.mU. G.mC.		A.mC.mU*mU*
  			A.mC.Chl		G*mC* A* G* C.
  14014	752	3139	G. G. A. A. G.	3140	P.mA. A. A.fC. G.fU.	95%
  			A.mC. A.mC.		G.fU.fC.fU.mU.mC.mC*
  			G.mU.mU.mU.Chl		A* G*mU*mC* G*
  					G.
  14015	994	3141	mC.mU.	3142	P.mU.fC. A. A.	85%
  			A.mU.mC. A. A.		A.fC.fU.fU. G. A.mU.
  			G.mU.mU.mU. G.		A. G*
  			A.Chl		G*mC*mU*mU* G*
  					G.
  14016	1040	3143	A. G.mC.mU. A.	3144	P.mA.fC. A. G. A.	61%
  			A.		A.fU.fU.fU. A.
  			A.mU.mU.mC.mU.		G.mC.mU*mC* G*
  			G.mU.Chl		G*mU* A* U.
  14017	1984	3145	A. G. G.mU. A. G.	3146	P.mU.fU. A.fC.	32%
  			A. A.mU. G.mU. A.		A.fU.fU.fC.fU.
  			A.Chl		A.mC.mC.mU* A*mU*
  					G* G*mU* G.
  14018	2195	3147	A. G.mC.mU. G.	3148	P.mA. A. A.fC.fU. G.	86%
  			A.mU.mC. A.		A.fU.fC. A. G.mC.mU*
  			G.mU.mU.mU.Chl		A*mU* A*mU* A* G.
  14019	2043	3149	mU.mU.mC.mU.	3150	P.mU. A.fU.fC.fU. G. A.	81%
  			G.mC.mU.mC. A.		G.fC. A. G. A.
  			G. A.mU. A.Chl		A*mU*mU*mU*mC*
  					mC* A.
  14020	1892	3151	mU.mU.	3152	P.mU.fU. A.	84%
  			A.mU.mC.mU. A.		A.fC.fU.fU. A. G.
  			A. G.mU.mU. A.		A.mU. A. A*mC*mU*
  			A.Chl		G*mU* A* C.
  14021	1567	3153	mU. A.mU. A.mC.	3154	P.mU. A.fU.fU.	72%
  			G. A. G.mU. A.		A.fC.fU.fC. G.fU.
  			A.mU. A.Chl		A.mU. A* A* G*
  					A*mU* G* C.
  14022	1780	3155	G. A.mC.mU. G.	3156	P.mA. A. G.fC.fU.	65%
  			G. A.mC. A.		G.fU.fC.fC. A.
  			G.mC.mU.mU.Chl		G.mU.mC*mU* A*
  					A*mU*mC* G.
  14023	2162	3157	A.mU. G.	3158	P.mU. A. A.fU. A. A. A.	80%
  			G.mC.mC.mU.mU.		G. G.fC.mC.
  			mU. A.mU.mU.		A.mU*mU*mU*
  			A.Chl		G*mU*mU* C.
  14024	1034	3159	A.mU. A.mC.mC.	3160	P.mU.fU.fU. A.	91%
  			G. A. G.mC.mU. A.		G.fC.fU.fC. G. G.mU.
  			A. A.Chl		A.mU*
  					G*mU*mC*mU*mU*
  					C.
  14025	2264	3161	mU.mU.	3162	P.mA.fC.	58%
  			G.mU.mU. G. A.		A.fC.fU.fC.fU.fC. A.
  			G. A. G.mU.		A.mC. A. A* A*mU*
  			G.mU.Chl		A* A* A* C.
  14026	1032	3163	A.mC. A.mU.	3164	P.mU. A. G.fC.fU.fC. G.	106%
  			A.mC.mC. G. A.		G.fU. A.mU.
  			G.mC.mU. A.Chl		G.mU*mC*mU*mU*
  					mC* A* U.
  14027	1535	3165	A. G.mC. A. G. A.	3166	P.mU. A.	67%
  			A. A. G. G.mU.mU.		A.fC.fC.fU.fU.fU.fC.fU.
  			A.Chl		G.mC.mU* G* G*mU*
  					A*mC* C.
  14028	1694	3167	A. G.mU.mU.	3168	P.mU.fU. A. A. G. G. A.	94%
  			G.mU.mU.mC.mC.		A.fC. A.
  			mU.mU. A. A.Chl		A.mC.mU*mU* G*
  					A*mC*mU* C.
  14029	1588	3169	A.mU.mU.mU. G.	3170	P.mU.fU. A.fC.	97%
  			A. A. G.mU. G.mU.		A.fC.fU.fU.fC. A. A.
  			A. A.Chl		A.mU* A* G*mC* A*
  					G* G.
  14030	928	3171	A. A. G.mC.mU.	3172	P.mU.fC.fC. A. G.	100%
  			G. A.mC.mC.mU.		G.fU.fC. A.
  			G. G. A.Chl		G.mC.mU.mU*mC*
  					G*mC* A* A* G.
  14031	1133	3173	G. G.mU.mC.	3174	P.mC.fU.fU.fC.fU.fU.fC.	82%
  			A.mU. G. A. A. G.		A.fU. G.
  			A. A. G.Chl		A.mC.mC*mU*mC*
  					G*mC*mC* G.
  14032	912	3175	A.mU. G.	3176	P.mA. A. G. G.fC.fC.fU.	84%
  			G.mU.mC. A. G.		G. A.fC.mC. A.mU*
  			G.mC.mC.mU.mU.		G*mC* A*mC* A*G.
  			Chl
  14033	753	3177	G. A. A. G. A.mC.	3178	P.mC. A. A. A.fC. G.fU.	86%
  			A.mC.		G.fU.fC.mU.mU.mC*mC*
  			G.mU.mU.mU.		A* G*mU*mC*G.
  			G.Chl
  14034	918	3179	A. G.	3180	P.mC.fU.fU.fC. G.fC. A.	88%
  			G.mC.mC.mU.mU.		A. G. G.mC.mC.mU*
  			G.mC. G. A. A.		G* A*mC*mC* A*U.
  			G.Chl
  14035	744	3181	mU. A.mC.mC. G.	3182	P.mC.fU.fU.fC.fC. A.	95%
  			A.mC.mU. G. G. A.		G.fU.fC. G. G.mU. A*
  			A. G.Chl		A* G*mC*mC* G* C.
  14036	466	3183	A.mC.mC. G.mC.	3184	P.mC.fC. G.	73%
  			A. A. G. A.mU.mC.		A.fU.fC.fU.fU. G.fC. G.
  			G. G.Chl		G.mU*mU* G*
  					G*mC*mC* G.
  14037	917	3185	mC. A. G.	3186	P.mU.fU.fC. G.fC. A. A.	86%
  			G.mC.mC.mU.mU.		G. G.fC.mC.mU. G*
  			G.mC. G. A. A.Chl		A*mC*mC* A*mU* G.
  14038	1038	3187	mC. G. A.	3188	P.mA. G. A.	84%
  			G.mC.mU. A. A.		A.fU.fU.fU. A.
  			A.mU.mU.mC.mU.		G.fC.mU.mC. G*
  			Chl		G*mU* A*mU* G* U.
  14039	1048	3189	mU.mC.mU.	3190	P.mC. A.fU.	87%
  			G.mU. G. G. A.		A.fC.fU.fC.fC. A.fC. A.
  			G.mU. A.mU.		G. A*
  			G.Chl		A*mU*mU*mU* A*
  					G.
  14040	1235	3191	mC. G. G. A. G.	3192	P.mU. G.fC.fC. A.fU.	100%
  			A.mC. A.mU. G.		G.fU.fC.fU.mC.mC.
  			G.mC. A.Chl		G*mU* A*mC*
  					A*mU* C.
  14041	868	3193	A.mU. G. A.mC.	3194	P.mG. A. G. G.fC.	104%
  			A. A.mC.		G.fU.fU. G.fU.mC.
  			G.mC.mC.mU.mC.		A.mU*mU* G*
  			Chl		G*mU* A* A.
  14042	1131	3195	G. A. G.	3196	P.mU.fC.fU.fU.fC.	85%
  			G.mU.mC. A.mU.		A.fU. G.
  			G. A. A. G. A.Chl		A.fC.mC.mU.mC*
  					G*mC*mC* G*mU* C.
  14043	1043	3197	mU. A. A.	3198	P.mU.fC.fC. A.fC. A. G.	74%
  			A.mU.mU.mC.mU.		A. A.fU.mU.mU. A*
  			G.mU. G. G. A.Chl		G*mC*mU*mC* G* G.
  14044	751	3199	mU. G. G. A. A. G.	3200	P.mA. A.fC. G.fU.	84%
  			A.mC. A.mC.		G.fU.fC.fU.fU.mC.mC.
  			G.mU.mU.Chl		A* G*mU*mC* G* G*
  					U.
  14045	1227	3201	A. A. G. A.mU.	3202	P.mC.fU.fC.fC. G.fU.	99%
  			G.mU. A.mC. G. G.		A.fC.
  			A. G.Chl		A.fU.mC.mU.mU*mC*
  					mC*mU* G*mU* A.
  14046	867	3203	A. A.mU. G.	3204	P.mA. G. G.fC. G.fU.fU.	94%
  			A.mC. A. A.mC.		G.fU.fC. A.mU.mU* G*
  			G.mC.mC.mU.Chl		G*mU* A* A* C.
  14047	1128	3205	G. G.mC. G. A. G.	3206	P.mU.fC. A.fU. G.	89%
  			G.mU.mC. A.mU.		A.fC.fC.fU.fC.
  			G. A.Chl		G.mC.mC*
  					G*mU*mC* A* G* G.
  14048	756	3207	G. A.mC. A.mC.	3208	P.mG. G.fC.fC. A. A.	93%
  			G.mU.mU.mU. G.		A.fC. G.fU.
  			G.mC.mC.Chl		G.mU.mC*mU*mU*mC*
  					mC* A* G.
  14049	1234	3209	A.mC. G. G. A. G.	3210	P.mG.fC.fC. A.fU.	100%
  			A.mC. A.mU. G.		G.fU.fC.fU.fC.mC.
  			G.mC.Chl		G.mU* A*mC*
  					A*mU*mC* U.
  14050	916	3211	mU.mC. A. G.	3212	P.mU.fC. G.fC. A. A. G.	96%
  			G.mC.mC.mU.mU.		G.fC.fC.mU. G.
  			G.mC. G. A.Chl		A*mC*mC* A*mU*
  					G* C.
  14051	925	3213	G.mC. G. A. A.	3214	P.mA. G. G.fU.fC. A.	80%
  			G.mC.mU. G.		G.fC.fU.fU.mC. G.mC*
  			A.mC.mC.mU.Chl		A* A* G* G*mC* C.
  14052	1225	3215	G. G. A. A. G.	3216	P.mC.fC. G.fU. A.fC.	96%
  			A.mU. G.mU.		A.fU.fC.fU.mU.mC.mC*
  			A.mC. G. G.Chl		mU* G*mU* A* G*
  					U.
  14053	445	3217	G.mU. G.	3218	P.mG. A. G.fC.fC. G. A.	101%
  			A.mC.mU.mU.mC.		A. G.fU.mC. A.mC* A*
  			G.		G* A* A* G* A.
  			G.mC.mU.mC.Chl
  14054	446	3219	mU. G.	3220	P.mG. G. A. G.fC.fC. G.	93%
  			A.mC.mU.mU.mC.		A. A. G.mU.mC.
  			G.		A*mC* A* G* A* A*
  			G.mC.mU.mC.mC.		G.
  			Chl
  14055	913	3221	mU. G. G.mU.mC.	3222	P.mC. A. A. G.	67%
  			A. G.		G.fC.fC.fU. G.
  			G.mC.mC.mU.mU.		A.mC.mC. A*mU*
  			G.Chl		G*mC* A*mC* A.
  14056	997	3223	mU.mC. A. A.	3224	P.mA. G.fC.fU.fC. A. A.	92%
  			G.mU.mU.mU. G.		A.fC.fU.mU. G. A*mU*
  			A. G.mC.mU.Chl		A* G* G*mC* U.
  14057	277	3225	G.mC.mC. A. G.	3226	P.mC.fU. G.fC. A.	84%
  			A. A.mC.mU.		G.fU.fU.fC.fU. G.
  			G.mC. A. G.Chl		G.mC*mC* G* A*mC*
  					G* G.
  14058	1052	3227	mU. G. G. A.	3228	P.mG. G.fU. A.fC. A.fU.	n/a
  			G.mU. A.mU.		A.fC.fU.mC.mC.
  			G.mU.		A*mC* A* G* A* A*
  			A.mC.mC.Chl		U.
  14059	887	3229	G.mC.mU. A. G.	3230	P.mC.fU.	80%
  			A. G. A. A. G.mC.		G.fC.fU.fU.fC.fU.fC.fU.
  			A. G.Chl		A. G.mC*mC*mU*
  					G*mC* A* G.
  14060	914	3231	G. G.mU.mC. A.	3232	P.mG.fC. A. A. G.	112%
  			G.		G.fC.fC.fU. G.
  			G.mC.mC.mU.mU.		A.mC.mC* A*mU*
  			G.mC.Chl		G*mC* A* C.
  14061	1039	3233	G. A. G.mC.mU.	3234	P.mC. A. G. A.	104%
  			A. A.		A.fU.fU.fU. A.
  			A.mU.mU.mC.mU.		G.mC.mU.mC* G*
  			G.Chl		G*mU* A*mU* G.
  14062	754	3235	A. A. G. A.mC.	3236	P.mC.fC. A. A. A.fC.	109%
  			A.mC.		G.fU.
  			G.mU.mU.mU. G.		G.fU.mC.mU.mU*mC*
  			G.Chl		mC* A* G*mU* C.
  14063	1130	3237	mC. G. A. G.	3238	P.mC.fU.fU.fC. A.fU. G.	103%
  			G.mU.mC. A.mU.		A.fC.fC.mU.mC.
  			G. A. A. G.Chl		G*mC*mC*
  					G*mU*mC* A.
  14064	919	3239	G.	3240	P.mG.fC.fU.fU.fC.	109%
  			G.mC.mC.mU.mU.		G.fC. A. A. G.
  			G.mC. G. A. A.		G.mC.mC*mU* G*
  			G.mC.Chl		A*mC*mC* A.
  14065	922	3241	mC.mU.mU.	3242	P.mU.fC. A.	106%
  			G.mC. G. A. A.		G.fC.fU.fU.fC. G.fC. A.
  			G.mC.mU. G.		A. G* G*mC*mC*mU*
  			A.Chl		G* A.
  14066	746	3243	mC.mC. G.	3244	P.mG.fU.fC.fU.fU.fC.fC.	106%
  			A.mC.mU. G. G. A.		A. G.fU.mC. G.
  			A. G. A.mC.Chl		G*mU* A* A* G*mC*
  					C.
  14067	993	3245	mC.mC.mU.	3246	P.mC. A. A. A.fC.fU.fU.	67%
  			A.mU.mC. A. A.		G. A.fU. A. G.
  			G.mU.mU.mU.		G*mC*mU*mU* G*
  			G.Chl		G* A.
  14068	825	3247	mU.	3248	P.mA. G.	93%
  			G.mU.mU.mC.mC.		G.fU.fC.fU.fU. G. G. A.
  			A. A. G.		A.mC. A* G* G*mC*
  			A.mC.mC.mU.Chl		G*mC* U.
  14069	926	3249	mC. G. A. A.	3250	P.mC. A. G. G.fU.fC. A.	95%
  			G.mC.mU. G.		G.fC.fU.mU.mC.
  			A.mC.mC.mU.		G*mC* A* A* G* G*
  			G.Chl		C.
  14070	923	3251	mU.mU. G.mC. G.	3252	P.mG.fU.fC. A.	95%
  			A. A. G.mC.mU. G.		G.fC.fU.fU.fC. G.mC. A.
  			A.mC.Chl		A* G*
  					G*mC*mC*mU* G.
  14071	866	3253	mC. A. A.mU. G.	3254	P.mG. G.fC. G.fU.fU.	132%
  			A.mC. A. A.mC.		G.fU.fC. A.mU.mU. G*
  			G.mC.mC.Chl		G*mU* A* A*mC* C.
  14072	563	3255	G.mU. A.mC.mC.	3256	P.mC. G.fU. G.fC.	n/a
  			A. G.mU. G.mC.		A.fC.fU. G. G.mU.
  			A.mC. G.Chl		A.mC*mU*mU*
  					G*mC* A* G.
  14073	823	3257	mC.mC.mU.	3258	P.mG.fU.fC.fU.fU. G.	98%
  			G.mU.mU.mC.mC.		G. A. A.fC. A. G.
  			A. A. G. A.mC.Chl		G*mC*
  					G*mC*mU*mC* C.
  14074	1233	3259	mU. A.mC. G. G.	3260	P.mC.fC. A.fU.	109%
  			A. G. A.mC. A.mU.		G.fU.fC.fU.fC.fC.
  			G. G.Chl		G.mU. A*mC*
  					A*mU*mC*mU* U.
  14075	924	3261	mU. G.mC. G. A.	3262	P.mG. G.fU.fC. A.	95%
  			A. G.mC.mU. G.		G.fC.fU.fU.fC. G.mC.
  			A.mC.mC.Chl		A* A* G* G*mC*mC*
  					U.
  14076	921	3263	mC.mC.mU.mU.	3264	P.mC. A. G.fC.fU.fU.fC.	116%
  			G.mC. G. A. A.		G.fC. A. A. G.
  			G.mC.mU. G.Chl		G*mC*mC*mU* G*
  					A* C.
  14077	443	3265	mC.mU. G.mU. G.	3266	P.mG.fC.fC. G. A. A.	110%
  			A.mC.mU.mU.mC.		G.fU.fC. A.mC. A. G*
  			G. G.mC.Chl		A* A* G* A* G* G.
  14078	1041	3267	G.mC.mU. A. A.	3268	P.mC. A.fC. A. G. A.	99%
  			A.mU.mU.mC.mU.		A.fU.fU.fU. A.
  			G.mU. G.Chl		G.mC*mU*mC* G*
  					G*mU* A.
  14079	1042	3269	mC.mU. A. A.	3270	P.mC.fC. A.fC. A. G. A.	109%
  			A.mU.mU.mC.mU.		A.fU.fU.mU. A.
  			G.mU. G. G.Chl		G*mC*mU*mC* G*
  					G* U.
  14080	755	3271	A. G. A.mC. A.mC.	3272	P.mG.fC.fC. A. A. A.fC.	121%
  			G.mU.mU.mU. G.		G.fU.
  			G.mC.Chl		G.mU.mC.mU*mU*mC*
  					mC* A* G* U.
  14081	467	3273	mC.mC. G.mC. A.	3274	P.mG.fC. C.fG. A.	132%
  			A. G. A.mU.mC. G.		U.fC.fU.fU.fG. C.mG.
  			G.mC.Chl		G*mU*mU* G*
  					G*mC* C.
  14082	995	3275	mU. A.mU.mC. A.	3276	P.mC.fU.fC. A. A.	105%
  			A. G.mU.mU.mU.		A.fC.fU.fU. G. A.mU.
  			G. A. G.Chl		A* G*
  					G*mC*mU*mU* G.
  14083	927	3277	G. A. A.	3278	P.mC.fC. A. G. G.fU.fC.	114%
  			G.mC.mU. G.		A. G.fC.mU.mU.mC*
  			A.mC.mC.mU. G.		G*mC* A* A* G*G.
  			G.Chl
  17356	1267	3279	A.mC. A.mU.mU.	3280	P.mU. A.fU. G. A.	120%
  			A. A.mC.mU.mC.		G.mU.fU. A. A.fU.
  			A.mU. A.Chl		G.fU*fC*fU*fC*fU*fC*
  					A.
  17357	1267	3281	G. A.mC.	3282	P.mU. A.fU. G. A.	56%
  			A.mU.mU. A.		G.mU.fU. A. A.fU.
  			A.mC.mU.mC.		G.fU*fC*fU*fC*fU*fC*
  			A.mU. A.Chl		A.
  17358	1442	3283	mU. G. A. A. G. A.	3284	P.mU.fU. A. A.fC.	34%
  			A.mU. G.mU.mU.		A.fU.fU.fC.fU.fU.fC. A*
  			A. A.Chl		A* A*fC*fC* A* G.
  17359	1442	3285	mU.mU. G. A. A.	3286	P.mU.fU. A. A.fC.	31%
  			G. A. A.mU.		A.fU.fU.fC.fU.fU.fC. A*
  			G.mU.mU. A.		A* A*fC*fC* A*G.
  			A.Chl
  17360	1557	3287	G. A.mU. A.	3288	P.mU.fU. A. A. G. A.fU.	59%
  			G.mC.		G.fC.fU. A.fU.fC*fU*
  			A.mU.mC.mU.mU.		G* A*fU* G*A.
  			A. A.Chl
  17361	1557	3289	A. G. A.mU. A.	3290	P.mU.fU. A. A. G. A.fU.	47%
  			G.mC.		G.fC.fU. A.fU.fC*fU*
  			A.mU.mC.mU.mU.		G* A*fU* G*A.
  			A. A.Chl
  17362	1591	3291	mU. G. A. A.	3292	P.mU. A. A.fU.fU. A.fC.	120%
  			G.mU. G.mU. A.		A.fC.fU.fU.fC. A* A*
  			A.mU.mU. A.Chl		A*fU* A* G* C.
  17363	1599	3293	A. A.mU.mU. G.	3294	P.mU.fU.fC.fC.fU.fU.fC.	71%
  			A. G. A. A. G. G. A.		fU.fC. A. A.fU.fU*
  			A.Chl		A*fC* A*fC*fU* U.
  17364	1601	3295	mU.mU. G. A. G.	3296	P.mU.fU.fU.fU.fC.fC.fU.	62%
  			A. A. G. G. A. A. A.		fU.fC.fU.fC. A.
  			A.Chl		A*fU*fU* A*fC* A* C.
  17365	1732	3297	mC.	3298	P.mU.fC. G. A. A.fU.fC.	99%
  			A.mU.mU.mC.mU.		A. G. A. A.fU.
  			G. A.mU.mU.mC.		G*fU*fC* A* G* A*G.
  			G. A.Chl
  17366	1734	3299	mU.mU.mC.mU.	3300	P.mU.fU.fU.fC. G. A.	97%
  			G. A.mU.mU.mC.		A.fU.fC. A. G. A. A*fU*
  			G. A. A. A.Chl		G*fU*fC* A* G.
  17367	1770	3301	mC.mU.	3302	P.mU.fU.fC.fU. A.	45%
  			G.mU.mC. G.		A.fU.fC. G. A.fC. A. G*
  			A.mU.mU. A. G. A.		G* A*fU*fU*fC*C.
  			A.Chl
  17368	1805	3303	mU.mU.mU.	3304	P.mU. G.fU.fU. A.fC. A.	71%
  			G.mC.mC.mU.		G. G.fC. A. A.
  			G.mU. A. A.mC.		A*fU*fU*fC* A*fC* U.
  			A.Chl
  17369	1805	3305	A.mU.mU.mU.	3306	P.mU. G.fU.fU. A.fC. A.	67%
  			G.mC.mC.mU.		G. G.fC. A. A.
  			G.mU. A. A.mC.		A*fU*fU*fC* A*fC* U.
  			A.Chl
  17370	1815	3307	A.mC. A. A.	3308	P.mU. A. A.fU.fC.fU. G.	65%
  			G.mC.mC. A. G.		G.fC.fU.fU. G.fU*fU*
  			A.mU.mU. A.Chl		A*fC* A* G* G.
  17371	1815	3309	A. A.mC. A. A.	3310	P.mU. A. A.fU.fC.fU. G.	35%
  			G.mC.mC. A. G.		G.fC.fU.fU. G.fU*fU*
  			A.mU.mU. A.Chl		A*fC* A* G* G.
  17372	2256	3311	mC. A.	3312	P.mU. A.fC. A. A. A.fU.	113%
  			G.mU.mU.mU.		A. A. A.fC.fU.
  			A.mU.mU.mU.		G*fU*fC*fC* G* A* A.
  			G.mU.A.Chl
  17373	2265	3313	mU. G.mU.mU. G.	3314	P.mU. A.fC.	35%
  			A. G. A. G.mU.		A.fC.fU.fC.fU.fC. A.
  			G.mU. A.Chl		A.fC. A* A* A*fU* A*
  					A* A.
  17374	2265	3315	mU.mU.	3316	P.mU. A.fC.	31%
  			G.mU.mU. G. A.		A.fC.fU.fC.fU.fC. A.
  			G. A. G.mU.		A.fC. A* A* A*fU* A*
  			G.mU. A.Chl		A* A.
  17375	2295	3317	mU. G.mC.	3318	P.mU.fU. A. G. A. A. A.	34%
  			A.mC.mC.mU.mU.		G. G.fU. G.fC. A* A*
  			mU.mC.mU. A.		A*fC* A*fU*G.
  			A.Chl
  17376	2295	3319	mU.mU. G.mC.	3320	P.mU.fU. A. G. A. A. A.	28%
  			A.mC.mC.mU.mU.		G. G.fU. G.fC. A* A*
  			mU.mC.mU. A.		A*fC* A*fU*G.
  			A.Chl
  17377	1003	3321	mU.mU. G. A.	3322	P.mU.fC. A. G. A. A. A.	67%
  			G.mC.mU.mU.mU.		G.fC.fU.fC. A. A*
  			mC.mU. G. A.Chl		A*fC*fU*fU* G* A.
  17378	2268	3323	mU. G. A. G. A.	3324	P.mU. G.fU.fC. A.fC.	42%
  			G.mU. G.mU. G.		A.fC.fU.fC.fU.fC. A*
  			A.mC. A.Chl		A*fC* A* A* A* U.
  17379	2272	3325	A. G.mU. G.mU.	3326	P.mU.fU.fU.fU. G.	35%
  			G. A.mC.mC. A. A.		G.fU.fC. A.fC.
  			A. A.Chl		A.fC.fU*fC*fU*fC* A*
  					A* C.
  17380	2272	3327	G. A. G.mU.	3328	P.mU.fU.fU.fU. G.	29%
  			G.mU. G.		G.fU.fC. A.fC.
  			A.mC.mC. A. A. A.		A.fC.fU*fC*fU*fC* A*
  			A.Chl		A* C.
  17381	2273	3329	G.mU. G.mU. G.	3330	P.mU.fU.fU.fU.fU. G.	42%
  			A.mC.mC. A. A. A.		G.fU.fC. A.fC.
  			A. A.Chl		A.fC*fU*fC*fU*fC* A*
  					A.
  17382	2274	3331	mU. G.mU. G.	3332	P.mU.fC.fU.fU.fU.fU.	42%
  			A.mC.mC. A. A. A.		G. G.fU.fC. A.fC.
  			A. G. A.Chl		A*fC*fU*fC*fU*fC* A.
  17383	2274	3333	G.mU. G.mU. G.	3334	P.mU.fC.fU.fU.fU.fU.	37%
  			A.mC.mC. A. A. A.		G. G.fU.fC. A.fC.
  			A. G. A.Chl		A*fC*fU*fC*fU*fC* A.
  17384	2275	3335	G.mU. G.	3336	P.mU.	24%
  			A.mC.mC. A. A. A.		A.fC.fU.fU.fU.fU. G.
  			A. G.mU. A.Chl		G.fU.fC. A.fC*
  					A*fC*fU*fC*fU* C.
  17385	2277	3337	G. A.mC.mC. A. A.	3338	P.mU.fU. A.	27%
  			A. A. G.mU.mU. A.		A.fC.fU.fU.fU.fU. G.
  			A.Chl		G.fU.fC* A*fC*
  					A*fC*fU* C.
  17386	2296	3339	G.mC.	3340	P.mU.fC.fU. A. G. A. A.	23%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC* A*
  			mU.mC.mU. A. G.		A* A*fC* A* U.
  			A.Chl
  17387	2299	3341	mC.mC.mU.mU.mU.	3342	P.mU.fC. A. A.fC.fU. A.	46%
  			mC.mU. A.		G. A. A. A. G. G*fU*
  			G.mU.mU. G.		G*fC* A* A* A.
  			A.Chl
  21138	2296	3343	G.mC.	3344	P.mU.fC.fU. A. G.	42%
  			A.mC.mC.mU.mU.		A.mA. A. G. G.fU.
  			mU.mC.mU. A. G.		G.mC* A* A* A*mC*
  			A.TEG-Chl		A* U.
  21139	2296	3345	G.mC.	3346	P.mU.fC.fU. A. G.mA.	32%
  			A.mC.mC.mU.mU.		A.mA. G. G.fU. G.mC*
  			mU.mC.mU. A. G.		A* A* A*mC* A* U.
  			A.TEG-Chl
  21140	2296	3347	G.mC.	3348	P.mU.fC.fU. A. G. A. A.	41%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.mC*
  			mU.mC.mU. A. G.		A*mA* A*mC* A* U.
  			A.TEG-Chl
  21141	2296	3349	G.mC.	3350	P.mU.fC.fU. A. G.	51%
  			A.mC.mC.mU.mU.		A.mA. A. G. G.fU.
  			mU.mC.mU. A. G.		G.mC* A*mA* A*mC*
  			A.TEG-Chl		A* U.
  21142	2296	3351	G.mC.	3352	P.mU.fC.fU. A. G.mA.	25%
  			A.mC.mC.mU.mU.		A.mA. G. G.fU. G.mC*
  			mU.mC.mU. A. G.		A*mA* A*mC* A* U.
  			A.TEG-Chl
  21143	2296	3353	G.mC.	3354	P.mU.fC.fU. A. G. A. A.	61%
  			A.mC.mC.mU.mU.		A. G. G.fU.
  			mU.mC.mU. A. G.		G.fC*mA*mA*mA*fC*
  			A.TEG-Chl		mA* U.
  21144	2296	3355	G.mC.	3356	P.mU.fC.fU. A. G.	49%
  			A.mC.mC.mU.mU.		A.mA. A. G. G.fU.
  			mU.mC.mU. A. G.		G.fC*mA*mA*mA*fC*
  			A.TEG-Chl		mA* U.
  21145	2296	3357	G.mC.	3358	P.mU.fC.fU. A. G.mA.	46%
  			A.mC.mC.mU.mU.		A.mA. G. G.fU.
  			mU.mC.mU. A. G.		G.fC*mA*mA*mA*fC*
  			A.TEG-Chl		mA* U.
  21146	2296	3359	G.mC.	3360	P.mU.fC.fU. A. G. A. A.	37%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC* A*
  			mU.mC.mU.		A* A*fC* A* U.
  			A*mG*mA.TEG-
  			Chl
  21147	2296	3361	mG*mC*	3362	P.mU.fC.fU. A. G. A. A.	43%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC* A*
  			mU.mC.mU.		A* A*fC* A* U.
  			A*mG*mA.TEG-
  			Chl
  21148	2296	3363	mG*mC*mA.mC.	3364	P.mU.fC.fU. A. G. A. A.	29%
  			mC.mU.mU.mU.mC.		A. G. G.fU. G.fC* A*
  			mU.mA*mG*mA.		A* A*fC* A* U.
  			TEG-Chl
  21149	2275	3365	G.mU. G.	3366	P.mU.	138%
  			A.mC.mC. A. A. A.		A.fC.fU.fU.fU.fU. G.
  			A.		G.fU.fC. A.fC*
  			G*mU*mA.TEG-		A*fC*fU*fC*fU* C.
  			Chl
  21150	2275	3367	mG*mU* G.	3368	P.mU.	116%
  			A.mC.mC. A.		A.fC.fU.fU.fU.fU. G.
  			A.mA. A.		G.fU.fC. A.fC*
  			G*mU*mA.TEG-		A*fC*fU*fC*fU*C.
  			Chl
  21151	2275	3369	mG*mU*mG.mA.	3370	P.mU.	105%
  			mC.mC.mA.mA.mA.		A.fC.fU.fU.fU.fU. G.
  			mA.mG*mU*mA.		G.fU.fC. A.fC*
  			TEG-Chl		A*fC*fU*fC*fU* C.
  21152	2295	3371	mU.mU. G.mC.	3372	P.mU.fU. A. G. A.mA.	46%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC. A. A*
  			mU.mC.mU. A.		A*fC* A*fA* G* G.
  			A.TEG-Chl
  21153	2295	3373	mU.mU. G.mC.	3374	P.mU.fU. A. G.mA.	28%
  			A.mC.mC.mU.mU.		A.mA. G. G.fU. G.fC. A.
  			mU.mC.mU. A.		A* A*fC* A*fA* G* G.
  			A.TEG-Chl
  21154	2295	3375	mU.mU. G.mC.	3376	P.mU.fU.mA. G.mA.	28%
  			A.mC.mC.mU.mU.		A.mA. G.mG.fU. G.fC.
  			mU.mC.mU. A.		A. A* A*fC* A*fA* G*
  			A.TEG-Chl		G.
  21155	2295	3377	mU.mU. G.mC.	3378	P.mU.fU. A. G. A.mA.	60%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.mC. A.
  			mU.mC.mU. A.		A* A*mC* A*mA* G*
  			A.TEG-Chl		G.
  21156	2295	3379	mU.mU. G.mC.	3380	P.mU.fU. A. G. A.mA.	54%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC.
  			mU.mC.mU. A.		A.mA*mA*fC*mA*fA*
  			A.TEG-Chl		mG* G.
  21157	2295	3381	mU.mU. G.mC.	3382	P.mU.fU. A. G. A.mA.	40%
  			A.mC.mC.mU.mU.		A. G. G.fU.
  			mU.mC.mU. A.		G.fC.mA.mA*mA*fC*
  			A.TEG-Chl		mA*fA*mG* G.
  21158	2295	3383	mU.mU. G.mC.	3384	P.mU.fU. A. G. A.mA.	n/a
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC.
  			mU.mC.mU. A.		A.mA*mA*fC*mA*mA*
  			A.TEG-Chl		mG* G.
  21159	2295	3385	mU.mU. G.mC.	3386	P.mU.fU. A. G. A.mA.	41%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC.
  			mU.mC.mU. A.		A.mA*mA*mC*mA*mA*
  			A.TEG-Chl		mG* G.
  21160	2295	3387	mU.mU. G.mC.	3388	P.mU.fU. A. G. A.mA.	65%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC.mA.
  			mU.mC.mU. A.		A*mA*mC*mA*mA*
  			A.Chl-TEG		mG*mG.
  21161	2295	3389	mU.mU. G.mC.	3390	P.mU.fU. A. G. A.mA.	43%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC. A. A*
  			mU.mC.mU. A.		A*fC* A*mA*mG* G.
  			A.TEG-Chl
  21162	2295	3391	mU.mU. G.mC.	3392	P.mU.fU. A. G. A.mA.	41%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC.mA.
  			mU.mC.mU. A.		A*mA*fC*
  			A.TEG-Chl		A*mA*mG* G.
  21163	2295	3393	mU.mU. G.mC.	3394	P.mU.fU. A. G. A. A. A.	32%
  			A.mC.mC.mU.mU.		G. G.fU. G.fC. A. A*
  			mU.mC.mU. A*		A*fC* A* A* G* G.
  			A*TEG-Chl
  21164	2295	3395	mU.mU. G.mC.	3396	P.mU.fU. A. G. A. A. A.	39%
  			A.mC.mC.mU.mU.		G. G.fU. G.fC. A. A*
  			mU.mC.mU.mA*		A*fC* A* A* G* G.
  			mA*TEG-Chl
  21165	2295	3397	mU*mU* G.mC.	3398	P.mU.fU. A. G. A. A. A.	28%
  			A.mC.mC.mU.mU.		G. G.fU. G.fC. A. A*
  			mU.mC.mU.mA*		A*fC* A* A* G* G.
  			mA*TEG-Chl
  21166	2295	3399	mU.mU.mG.mC.mA.	3400	P.mU.fU. A. G. A. A. A.	27%
  			mC.mC.mU.mU.		G. G.fU. G.fC. A. A*
  			mU.mC.mU.mA*		A*fC* A* A* G* G.
  			mA*TEG-Chl
  21167	2299	3401	mC.mC.mU.mU.mU.	3402	P.mU.fC. A. A.fC.fU. A.	49%
  			mC.mU. A.		G. A.mA. A. G. G*fU*
  			G.mU.mU. G.		G*fC* A* A* A.
  			A.TEG-Chl
  21168	2299	3403	mC.mC.mU.mU.mU.	3404	P.mU.fC. A. A.fC.fU. A.	53%
  			mC.mU. A.		G. A.mA. A. G. G*mU*
  			G.mU.mU. G.		G*mC* A* A* A.
  			A.TEG-Chl
  21169	2299	3405	mC.mC.mU.mU.mU.	3406	P.mU.fC. A. A.fC.fU. A.	47%
  			mC.mU. A.		G.mA. A. A.mG. G*fU*
  			G.mU.mU. G.		G*fC* A* A* A.
  			A.TEG-Chl
  21170	2299	3407	mC.mC.mU.mU.mU.	3408	P.mU.fC. A. A.fC.fU. A.	70%
  			mC.mU. A.		G.mA. A. A.mG.
  			G.mU.mU. G.		G*mU* G*mC* A* A*
  			A.TEG-Chl		A.
  21171	2299	3409	mC.mC.mU.mU.mU.	3410	P.mU.fC. A. A.fC.fU. A.	65%
  			mC.mU. A.		G. A.mA. A. G. G*mU*
  			G.mU.mU. G.		G*mC* A*mA* A.
  			A.TEG-Chl
  21172	2299	3411	mC.mC.mU.mU.mU.	3412	P.mU.fC. A. A.fC.fU. A.	43%
  			mC.mU. A.		G. A.mA. A. G. G*mU*
  			G.mU.mU. G.		G*mC*mA*mA* A.
  			A.TEG-Chl
  21173	2299	3413	mC.mC.mU.mU.mU.	3414	P.mU.fC. A. A.fC.fU. A.	52%
  			mC.mU. A.		G. A.mA. A.
  			G.mU.mU. G.		G.mG*mU*mG*mC*
  			A.TEG-Chl		mA*mA* A.
  21174	2299	3415	mC.mC.mU.mU.mU.	3416	P.mU.fC. A. A.fC.fU. A.	47%
  			mC.mU. A.		G. A.mA. A. G.
  			G.mU.mU. G.		G*mU*mG*mC*mA*
  			A.TEG-Chl		mA* A.
  21175	2299	3417	mC.mC.mU.mU.mU.	3418	P.mU.fC. A. A.fC.fU. A.	35%
  			mC.mU. A.		G. A.mA. A. G.
  			G.mU.mU. G.		G*fU*mG*fC*mA*mA*
  			A.TEG-Chl		A.
  21176	2299	3419	mC.mC.mU.mU.mU.	3420	P.mU.fC. A. A.fC.fU. A.	50%
  			mC.mU. A.		G.mA. A. A.mG.
  			G.mU.mU. G.		G*fU*mG*fC*mA*mA*
  			A.TEG-Chl		A.
  21177	2299	3421	mC.mC.mU.mU.mU.	3422	P.mU.fC. A. A.fC.fU. A.	37%
  			mC.mU. A.		G. A. A. A. G. G*fU*
  			G.mU.mU*mG*mA.		G*fC* A* A* A.
  			TEG-Chl
  21178	2299	3423	mC*mC*mU.mU.	3424	P.mU.fC. A. A.fC.fU. A.	36%
  			mU.mC.mU. A.		G. A. A. A. G. G*fU*
  			G.mU.mU*mG*mA.		G*fC* A* A* A.
  			TEG-Chl
  21179	2299	3425	mC*mC*mU.mU.	3426	P.mU.fC. A. A.fC.fU. A.	35%
  			mU.mC.mU.mA.mG.		G. A. A. A. G. G*fU*
  			mU.mU*mG*mA.		G*fC* A* A* A.
  			TEG-Chl
  21203	2296	3427	G.mC.	3428	P.mU.fC.fU. A. G.	40%
  			A.mC.mC.mU.mU.		A.mA. A. G. G.fU.
  			mU.mC.mU.		G.mC* A* A* A*mC*
  			A*mG*mA.TEG-		A*U.
  			Chl
  21204	2296	3429	G.mC.	3430	P.mU.fC.fU. A. G.mA.	28%
  			A.mC.mC.mU.mU.		A.mA. G. G.fU. G.mC*
  			mU.mC.mU.		A* A* A*mC* A* U.
  			A*mG*mA.TEG-
  			Chl
  21205	2296	3431	G.mC.	3432	P.mU.fC.fU. A. G.mA.	51%
  			A.mC.mC.mU.mU.		A.mA. G. G.fU. G.mC*
  			mU.mC.mU.		A*mA* A*mC* A* U.
  			A*mG*mA.TEG-
  			Chl
  21206	2296	3433	mG*mC*	3434	P.mU.fC.fU. A. G.	46%
  			A.mC.mC.mU.mU.		A.mA. A. G. G.fU.
  			mU.mC.mU.		G.mC* A* A* A*mC*
  			A*mG*mA.TEG-		A* U.
  			Chl
  21207	2296	3435	mG*mC*	3436	P.mU.fC.fU. A. G.mA.	29%
  			A.mC.mC.mU.mU.		A.mA. G. G.fU. G.mC*
  			mU.mC.mU.		A* A* A*mC* A* U.
  			A*mG*mA.TEG-
  			Chl
  21208	2296	3437	mG*mC*	3438	P.mU.fC.fU. A. G.mA.	72%
  			A.mC.mC.mU.mU.		A.mA. G. G.fU. G.mC*
  			mU.mC.mU.		A*mA* A*mC* A* U.
  			A*mG*mA.TEG-
  			Chl
  21209	2296	3439	mG*mC*mA.mC.	3440	P.mU.fC.fU. A. G.	89%
  			mC.mU.mU.mU.mC.		A.mA. A. G. G.fU.
  			mU.mA*mG*mA.		G.mC* A* A* A*mC*
  			TEG-Chl		A* U.
  21210	2296	3441	mG*mC*mA.mC.	3442	P.mU.fC.fU. A. G.mA.	65%
  			mC.mU.mU.mU.mC.		A.mA. G. G.fU. G.mC*
  			mU.mA*mG*mA.		A* A* A*mC* A* U.
  			TEG-Chl
  21211	2296	3443	mG*mC*mA.mC.	3444	P.mU.fC.fU. A. G.mA.	90%
  			mC.mU.mU.mU.mC.		A.mA. G. G.fU. G.mC*
  			mU.mA*mG*mA.		A*mA* A*mC* A* U.
  			TEG-Chl
  21212	2295	3445	mU.mU. G.mC.	3446	P.mU.fU. A. G. A.mA.	60%
  			A.mC.mC.mU.mU.		A. G. G.fU.
  			mU.mC.mU*mA*		G.fC.mA.mA*mA*fC*
  			mA.TEG-Chl		mA*mA*mG* G.
  21213	2295	3447	mU.mU. G.mC.	3448	P.mU.fU. A. G. A.mA.	63%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC.
  			mU.mC.mU*mA*		A.mA*mA*mC*mA*mA*
  			mA.TEG-Chl		mG* G.
  21214	2295	3449	mU.mU. G.mC.	3450	P.mU.fU. A. G. A.mA.	52%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC. A. A*
  			mU.mC.mU*mA*		A*fC* A*mA*mG* G.
  			mA.TEG-Chl
  21215	2295	3451	mU.mU. G.mC.	3452	P.mU.fU. A. G. A.mA.	45%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC.mA.
  			mU.mC.mU*mA*		A*mA*fC*
  			mA.TEG-Chl		A*mA*mG* G.
  21216	2295	3453	mU*mU* G.mC.	3454	P.mU.fU. A. G. A.mA.	65%
  			A.mC.mC.mU.mU.		A. G. G.fU.
  			mU.mC.mU*mA*		G.fC.mA.mA*mA*fC*
  			mA.TEG-Chl		mA*mA*mG* G.
  21217	2295	3455	mU*mU* G.mC.	3456	P.mU.fU. A. G. A.mA.	69%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC.
  			mU.mC.mU*mA*		A.mA*mA*mC*mA*mA*
  			mA.TEG-Chl		mG* G.
  21218	2295	3457	mU*mU* G.mC.	3458	P.mU.fU. A. G. A.mA.	62%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC. A. A*
  			mU.mC.mU*mA*		A*fC* A*mA*mG* G.
  			mA.TEG-Chl
  21219	2295	3459	mU*mU* G.mC.	3460	P.mU.fU. A. G. A.mA.	54%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC.mA.
  			mU.mC.mU*mA*		A*mA*fC*
  			mA.TEG-Chl		A*mA*mG* G.
  21220	2295	3461	mU.mU.mG.mC.mA.	3462	P.mU.fU. A. G. A.mA.	52%
  			mC.mC.mU.mU.		A. G. G.fU.
  			mU.mC.mU*mA*		G.fC.mA.mA*mA*fC*
  			mA.TEG-Chl		mA*mA*mG* G.
  21221	2295	3463	mU.mU.mG.mC.mA.	3464	P.mU.fU. A. G. A.mA.	53%
  			mC.mC.mU.mU.		A. G. G.fU. G.fC.
  			mU.mC.mU*mA*		A.mA*mA*mC*mA*mA*
  			mA.TEG-Chl		mG* G.
  21222	2295	3465	mU.mU.mG.mC.mA.	3466	P.mU.fU. A. G. A.mA.	43%
  			mC.mC.mU.mU.		A. G. G.fU. G.fC. A. A*
  			mU.mC.mU*mA*		A*fC* A*mA*mG* G.
  			mA.TEG-Chl
  21223	2295	3467	mU.mU.mG.mC.mA.	3468	P.mU.fU. A. G. A.mA.	43%
  			mC.mC.mU.mU.		A. G. G.fU. G.fC.mA.
  			mU.mC.mU*mA*		A*mA*fC*
  			mA.TEG-Chl		A*mA*mG* G.
  21224	2299	3469	mC.mC.mU.mU.mU.	3470	P.mU.fC. A. A.fC.fU. A.	60%
  			mC.mU. A.		G. A.mA. A. G.
  			G.mU.mU*mG*mA.		G*fU*mG*fC*mA*mA*
  			TEG-Chl		A.
  21225	2299	3471	mC*mC*mU.mU.	3472	P.mU.fC. A. A.fC.fU. A.	67%
  			mU.mC.mU. A.		G. A.mA. A. G.
  			G.mU.mU*mG*mA.		G*fU*mG*fC*mA*mA*
  			TEG-Chl		A.
  21226	2299	3473	mC*mC*mU.mU.	3474	P.mU.fC. A. A.fC.fU. A.	66%
  			mU.mC.mU.mA.mG.		G. A.mA. A. G.
  			mU.mU*mG*mA.		G*fU*mG*fC*mA*mA*
  			TEG-Chl		A.
  21227	2296	3475	G.mC.	3476	P.mU.fC.fU. A. G.mA.	49%
  			A.mC.mC.mU.mU.		A.mA. G. G.fU.
  			mU.mC.mU.		G.fC*mA*mA*mA*fC*
  			A*mG*mA.TEG-		mA* U.
  			Chl
  20584	2296	3477	G.mC.	3478	P.mU.fC.fU. A. G. A. A.	70%
  			A.mC.mC.mU.mU.		A. G. G.mU. G.mC* A*
  			mU.mC.mU. A. G.		A* A*mC* A* U.
  			A.Chl-TEG
  20585	2296	3479	G.mC.	3480	P.mU.fC.fU. A. G. A. A.	15%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.mC* A*
  			mU.mC.mU. A. G.		A* A*mC* A* U.
  			A.Chl-TEG
  20586	2296	3481	G.mC.	3482	P.mU. C. U. A. G. A. A.	30%
  			A.mC.mC.mU.mU.		A. G. G.mU. G.mC* A*
  			mU.mC.mU. A. G.		A* A*mC* A* U.
  			A.Chl-TEG
  20587	2296	3483	G.mC.	3484	P.mU.fC.fU. A. G. A. A.	32%
  			A.mC.mC.mU.mU.		A. G. G.fU.
  			mU.mC.mU. A. G.		G.fC*mA*mA*mA*fC*
  			A.Chl-TEG		mA* U.
  20616	2275	3485	G.mU. G.	3486	P.mU.	22%
  			A.mC.mC. A. A. A.		A.fC.fU.fU.fU.fU. G.
  			A. G.mU. A.Chl-		G.fU.mC. A.mC*
  			TEG		A*mC*mU*mC*mU*
  					C.
  20617	2275	3487	G.mU. G.	3488	P.mU.	18%
  			A.mC.mC. A. A. A.		A.fC.fU.fU.fU.fU. G.
  			A. G.mU. A.Chl-		G.fU.fC. A.mC*
  			TEG		A*fC*mU*fC*mU* C.
  20618	2275	3489	G.mU. G.	3490	P.mU. A. C. U. U. U. U.	36%
  			A.mC.mC. A. A. A.		G. G. U.mC. A.mC*
  			A. G.mU. A.Chl-		A*mC*mU*mC*mU*
  			TEG		C.
  20619	2275	3491	G.mU. G.	3492	P.mU.	28%
  			A.mC.mC. A. A. A.		A.fC.fU.fU.fU.fU. G.
  			A. G.mU. A.Chl-		G.fU.fC.
  			TEG		A.mC*mA*mC*mU*mC*
  					mU* C.
  21381	2275	3493	G.mU. G.	3494	P.mU.	28%
  			A.mC.mC. A. A. A.		A.fC.fU.fU.fU.fU. G.
  			A.		G.fU.mC. A.mC*
  			G*mU*mA.TEG-		A*mC*mU*mC*mU*
  			Chl		C.
  21382	2275	3495	G.mU. G.	3496	P.mU.	28%
  			A.mC.mC. A. A. A.		A.fC.fU.fU.fU.fU. G.
  			A.		G.fU.fC. A.mC*
  			G*mU*mA.TEG-		A*fC*mU*fC*mU*C.
  			Chl
  21383	2275	3497	mG*mU*mG.mA.	3498	P.mU.	43%
  			mC.mC.mA.mA.mA.		A.fC.fU.fU.fU.fU. G.
  			mA.mG*mU*mA.		G.fU.mC. A.mC*
  			TEG-Chl		A*mC*mU*mC*mU*
  					C.
  21384	2275	3499	mG*mU*mG.mA.	3500	P.mU.	50%
  			mC.mC.mA.mA.mA.		A.fC.fU.fU.fU.fU. G.
  			mA.mG*mU*mA.		G.fU.fC. A.mC*
  			TEG-Chl		A*fC*mU*fC*mU* C.
  20392	2275	3501	G.mU. G.	3502	P.mU.	28%
  			A.mC.mC. A. A. A.		A.fC.fU.fU.fU.fU. G.
  			A. G.mU. A.TEG-		G.fU.fC. A.fC*
  			Chl		A*fC*fU*fC*fU* C.
  20393	2296	3503	G.mC.	3504	P.mU.fC.fU. A. G. A. A.	35%
  			A.mC.mC.mU.mU.		A. G. G.fU. G.fC* A*
  			mU.mC.mU. A. G.		A* A*fC* A* U.
  			A.TEG-Chl
  21429	2275	3505	G.mU. G.	3506	P.mU.	36%
  			A.mC.mC. A. A. A.		A.fC.fU.fU.fU.fU. G.
  			A.		G.fU.fC. A.mC*
  			G*mU*mA.Teg-		A*fC*mU*fC*mU* C.
  			Chl
  21430	2275	3507	G.mU. G.	3508	P.mU.	31%
  			A.mC.mC. A.		A.fC.fU.fU.fU.fU. G.
  			A.mA. A.		G.fU.mC. A.mC*
  			G*mU*mA.Teg-		A*mC*mU*mC*mU*
  			Chl		C.

• TABLE 21
  Inhibition of gene expression with TGFB2 sd-rxRNA
  sequences (Accession Number: NM_001135599.1)

  						% remaining
  Oligo	Start	SEQ ID		SEQ ID		expression
  Number	Site	NO	Sense sequence	NO	Antisense sequence	(1 uM, A549)

  14408	1324	3509	G.	3510	P.mU.fC. G. A. A. G. G.	94%
  			G.mC.mU.mC.mU.		A. G. A. G.mC.mC*
  			mC.mC.mU.mU.mC.		A*mU*mU*mC* G* C.
  			G. A.Chl
  14409	1374	3511	G. A.mC. A. G. G. A.	3512	P.mC.fC. A. G.	n/a
  			A.mC.mC.mU. G.		G.fU.fU.fC.fC.fU.
  			G.Chl		G.mU.mC*mU*mU*mU*
  					A*mU* G.
  14410	946	3513	mC.mC. A. A. G. G.	3514	P.mU. A. A.	90%
  			A. G.		A.fC.fC.fU.fC.fC.fU.mU.
  			G.mU.mU.mU.		G. G*mC* G*mU* A*
  			A.Chl		G* U.
  14411	849	3515	A.mU.mU.mU.mC.	3516	P.mU. G.fU. A. G. A.fU.	72%
  			mC. A.mU.mC.mU.		G. G. A. A. A.mU*mC*
  			A.mC. A.Chl		A*mC*mC*mU* C.
  14412	852	3517	mU.mC.mC.	3518	P.mU. G.fU.fU. G.fU.	76%
  			A.mU.mC.mU.		A. G. A.fU. G. G. A* A*
  			A.mC. A. A.mC.		A*mU*mC* A* C.
  			A.Chl
  14413	850	3519	mU.mU.mU.mC.mC.	3520	P.mU.fU. G.fU. A. G.	98%
  			A.mU.mC.mU.		A.fU. G. G. A. A.
  			A.mC. A. A.Chl		A*mU*mC*
  					A*mC*mC* U.
  14414	944	3521	mC. G.mC.mC. A. A.	3522	P.mA.	100%
  			G. G. A. G.		A.fC.fC.fU.fC.fC.fU.fU.
  			G.mU.mU.Chl		G. G.mC. G*mU* A*
  					G*mU* A* C.
  14415	1513	3523	G.mU. G. G.mU. G.	3524	P.mU.fU.fC.fU. G.	n/a
  			A.mU.mC. A. G. A.		A.fU.fC. A.fC.mC.
  			A.Chl		A.mC*mU* G*
  					G*mU* A* U.
  14416	1572	3525	mC.mU.mC.mC.mU.	3526	P.mA.fC. A.fU.fU. A.	100%
  			G.mC.mU. A.		G.fC. A. G. G. A. G*
  			A.mU. G.mU.Chl		A*mU* G*mU* G* G.
  14417	1497	3527	A.mC.mC.mU.mC.mC.	3528	P.mU. A.fU. A.fU.	73%
  			A.mC. A.mU.		G.fU. G. G. A. G.
  			A.mU. A.Chl		G.mU* G*mC*mC*
  					A*mU* C.
  14418	1533	3529	A. A. G.mU.mC.mC.	3530	P.mU.fC.fC.fU. A. G.fU.	98%
  			A.mC.mU. A. G. G.		G. G.
  			A.Chl		A.mC.mU.mU*mU*
  					A*mU* A* G* U.
  14419	1514	3531	mU. G. G.mU. G.	3532	P.mU.fU.fU.fC.fU. G.	86%
  			A.mU.mC. A. G. A.		A.fU.fC. A.mC.mC.
  			A. A.Chl		A*mC*mU* G*
  					G*mU* A.
  14420	1534	3533	A. G.mU.mC.mC.	3534	P.mU.fU.fC.fC.fU. A.	99%
  			A.mC.mU. A. G. G.		G.fU. G. G.
  			A. A.Chl		A.mC.mU*mU*mU*
  					A*mU* A* G.
  14421	943	3535	A.mC. G.mC.mC. A.	3536	P.mA.fC.fC.fU.fC.fC.fU.	41%
  			A. G. G. A. G.		fU. G. G.mC. G.mU*
  			G.mU.Chl		A* G*mU* A*mC* U.
  18570	2445	3537	mU. A.mU.mU.mU.	3538	P.mU. A.fC. A.fC. A.	79%
  			A.mU.mU. G.mU.		A.fU. A. A. A.fU. A*
  			G.mU. A.Chl		A*fC*fU*fC* A* C.
  18571	2445	3539	mU.mU.	3540	P.mU. A.fC. A.fC. A.	75%
  			A.mU.mU.mU.		A.fU. A. A. A.fU. A*
  			A.mU.mU. G.mU.		A*fC*fU*fC* A* C.
  			G.mU.A.Chl
  18572	2083	3541	A.mU. C. A. G.mU.	3542	P.mU.fU.fU.fU. A.	47%
  			G.mU.mU. A. A. A.		A.fC. A.fC.fU. G. A.fU*
  			A.Chl		G* A* A*fC*fC* A.
  18573	2083	3543	mC. A.mU.mC. A.	3544	P.mU.fU.fU.fU. A.	17%
  			G.mU. G.mU.mU.		A.fC. A.fC.fU. G. A.fU*
  			A. A. A. A.Chl		G* A* A*fC*fC* A.
  18574	2544	3545	A.mU. G.	3546	P.mU.fU.fC.fC.fU.fU.	59%
  			G.mC.mU.mU. A. A.		A. A. G.fC.fC. A.
  			G. G. A. A.Chl		U*fC*fC* A*fU* G* A.
  18575	2544	3547	G. A.mU. G.	3548	P.mU.fU.fC.fC.fU.fU.	141%
  			G.mC.mU.mU. A. A.		A. A. G.fC.fC. A.
  			G. G. A. A.Chl		U*fC*fC* A*fU* G* A.
  18576	2137	3549	mU.mU. G.mU.	3550	P.mU. A. A.fC. A. G. A.	77%
  			G.mU.mU.mC.mU.		A.fC. A.fC. A. A*
  			G.mU.mU. A.Chl		A*fC*fU*fU*fC* C.
  18577	2137	3551	mU.mU.mU. G.mU.	3552	P.mU. A. A.fC. A. G. A.	59%
  			G.mU.mU.mC.mU.		A.fC. A.fC. A. A*
  			G.mU.mU. A.Chl		A*fC*fU*fU*fC* C.
  18578	2520	3553	A. A. A.mU.	3554	P.mU. G. G.fC. A. A. A.	75%
  			A.mC.mU.mU.mU.		G.fU. A.fU.fU.fU* G*
  			G.mC.mC. A.Chl		G*fU*fC*fU* C.
  18579	2520	3555	mC. A. A. A.mU.	3556	P.mU. G. G.fC. A. A. A.	55%
  			A.mC.mU.mU.mU.		G.fU. A.fU.fU.fU* G*
  			G.mC.mC. A.Chl		G*fU*fC*fU* C.
  18580	3183	3557	mC.mU.mU. G.mC.	3558	P.mU.fU.fU. G.fU. A.	84%
  			A.mC.mU. A.mC. A.		G.fU. G.fC. A. A.
  			A. A.Chl		G*fU*fC* A* A* A* C.
  18581	3183	3559	A.mC.mU.mU.	3560	P.mU.fU.fU. G.fU. A.	80%
  			G.mC. A.mC.mU.		G.fU. G.fC. A. A.
  			A.mC. A. A. A.Chl		G*fU*fC* A* A* A* C.
  18582	2267	3561	G. A.	3562	P.mU. A.fC.fU. A. A.fU.	82%
  			A.mU.mU.mU.		A. A.
  			A.mU.mU. A. G.mU.		A.fU.fU.fC*fU*fU*fC*fC*
  			A.Chl		A* G.
  18583	2267	3563	A. G. A.	3564	P.mU. A.fC.fU. A. A.fU.	67%
  			A.mU.mU.mU.		A. A.
  			A.mU.mU. A. G.mU.		A.fU.fU.fC*fU*fU*fC*fC*
  			A.Chl		A* G.
  18584	3184	3565	mU.mU. G.mC.	3566	P.mU.fU.fU.fU. G.fU.	77%
  			A.mC.mU. A.mC. A.		A. G.fU. G.fC. A. A*
  			A. A. A.Chl		G*fU*fC* A* A* A.
  18585	3184	3567	mC.mU.mU. G.mC.	3568	P.mU.fU.fU.fU. G.fU.	59%
  			A.mC.mU. A.mC. A.		A. G.fU. G.fC. A. A*
  			A. A. A.Chl		G*fU*fC* A* A* A.
  18586	2493	3569	A.mU. A. A. A.	3570	P.mU.fC. A.fC.fC.fU.	84%
  			A.mC. A. G. G.mU.		G.fU.fU.fU.fU.
  			G. A.Chl		A.fU*fU*fU*fU*fC*fC*
  					A.
  18587	2493	3571	A. A.mU. A. A. A.	3572	P.mU.fC. A.fC.fC.fU.	70%
  			A.mC. A. G. G.mU.		G.fU.fU.fU.fU.
  			G. A.Chl		A.fU*fU*fU*fU*fC*fC*
  					A.
  18588	2297	3573	G. A.mC. A. A.mC.	3574	P.mU. G.fU.fU.	40%
  			A. A.mC. A. A.mC.		G.fU.fU. G.fU.fU.
  			A.Chl		G.fU.fC* G*fU*fU*
  					G*fU* U.
  18589	2046	3575	A.mU. G.	3576	P.mU.fU. G.fU.fU.	39%
  			C.mU.mU. G.mU. A.		A.fC. A. A. G.fC.
  			A.mC. A. A.Chl		A.fU*fC* A*fU*fC* G*
  					U.
  18590	2531	3577	mC. A. G. A. A.	3578	P.mU.fC. A.fU. G. A.	56%
  			A.mC.mU.mC.		G.fU.fU.fU.fC.fU. G*
  			A.mU. G. A.Chl		G*fC* A* A* A* G.
  18591	2389	3579	G.mU. A.mU.mU.	3580	P.mU. G.fC. A.fU. A.	64%
  			G.mC.mU. A.mU.		G.fC. A. A.fU. A.fC* A*
  			G.mC. A.Chl		G* A* A* A* A.
  18592	2530	3581	mC.mC. A. G. A. A.	3582	P.mU. A.fU. G. A.	44%
  			A.mC.mU.mC.		G.fU.fU.fU.fC.fU. G.
  			A.mU. A.Chl		G*fC* A* A* A* G* U.
  18593	2562	3583	A.mC.mU.mC. A. A.	3584	P.mU. G.fC.fU.fC.	87%
  			A.mC. G. A. G.mC.		G.fU.fU.fU. G. A.
  			A.Chl		G.fU*fU*fC* A* A* G*
  					U.
  18594	2623	3585	A.mU. A.mU. G.	3586	P.mU.fU.fC.fU.fC. G.	69%
  			A.mC.mC. G. A. G.		G.fU.fC. A.fU. A.fU*
  			A. A.Chl		A* A*fU* A* A* C.
  18595	2032	3587	mC. G. A.mC. G.	3588	P.mU.fU.fC. G.fU.fU.	55%
  			A.mC. A. A.mC. G.		G.fU.fC. G.fU.fC.
  			A. A.Chl		G*fU*fC* A*fU*fC* A.
  18596	2809	3589	G.mU. A. A.	3590	P.mU.fU.fC. A.fC.fU. G.	58%
  			A.mC.mC. A. G.mU.		G.fU.fU.fU. A.fC*fU*
  			G. A. A.Chl		A* A* A*fC* U.
  18597	2798	3591	mU.mU. G.mU.mC.	3592	P.mU.fC.fU. A. A.	38%
  			A. G.mU.mU.mU. A.		A.fC.fU. G. A.fC. A. A*
  			G. A.Chl		A* G* A* A*fC* C.
  18598	2081	3593	mU.mC. A.mU.mC.	3594	P.mU.fU. A. A.fC.	25%
  			A. G.mU.		A.fC.fU. G. A.fU. G. A*
  			G.mU.mU. A. A.Chl		A*fC*fC* A* A* G.
  18599	2561	3595	A. A.mC.mU.mC. A.	3596	P.mU.fC.fU.fC.	57%
  			A. A.mC. G. A. G.		G.fU.fU.fU. G. A.
  			A.Chl		G.fU.fU*fC* A* A*
  					G*fU* U.
  18600	2296	3597	mC. G. A.mC. A.	3598	P.mU.fU.fU. G.fU.fU.	69%
  			A.mC. A. A.mC. A.		G.fU.fU. G.fU.fC.
  			A. A.Chl		G*fU*fU* G*fU*fU*
  					C.
  18601	2034	3599	A.mC. G. A.mC. A.	3600	P.mU.fC. A.fU.fC.	22%
  			A.mC. G. A.mU. G.		G.fU.fU. G.fU.fC.
  			A.Chl		G.fU*fC* G*fU*fC*
  					A*fU.
  18602	2681	3601	G.mC.mU.	3602	P.mU.fU.fC.fC.fU.fU.	43%
  			G.mC.mC.mU. A. A.		A. G. G.fC. A. G.fC*fU*
  			G. G. A. A.Chl		G* A*fU* A* C.
  18603	2190	3603	A.mU.mU.mC.mU.	3604	P.mU. G. A. A. A.fU.	128%
  			A.mC.		G.fU. A. G. A. A.fU* A*
  			A.mU.mU.mU.mC.		A* G* G*fC* C.
  			A.Chl
  20604	2083	3605	mC. A.mU.mC. A.	3606	P.mU.fU.fU.fU. A.	19%
  			G.mU. G.mU.mU.		A.fC. A.fC.fU. G.
  			A. A. A. A.Chl		A.mU* G* A*
  					A*mC*mC* A.
  20605	2083	3607	mC. A.mU.mC. A.	3608	P.mU.fU.fU.fU. A.	20%
  			G.mU. G.mU.mU.		A.fC. A.fC.fU. G.
  			A. A. A. A.Chl		A.mU* G* A*
  					A*fC*mC* A.
  20606	2083	3609	mC. A.mU.mC. A.	3610	P.mU. U. U. U. A. A. C.	82%
  			G.mU. G.mU.mU.		A. C. U. G. A.mU* G*
  			A. A. A. A.Chl		A* A*mC*mC* A.
  20607	2083	3611	mC. A.mU.mC. A.	3612	P.mU.fU.fU.fU. A.	59%
  			G.mU. G.mU.mU.		A.fC. A.fC.fU. G.
  			A. A. A. A.Chl		A.fU*mG*mA*mA*fC*
  					fC* A.
  21722	2081	3613	mU.mC. A.mU.mC.	3614	P.mU.fU. A. A.fC.	34%
  			A. G.mU.		A.fC.fU. G. A.fU. G. A*
  			G.mU.mU. A. A.Chl		A*mC*mC* A* A* G.
  21723	2081	3615	mU.mC. A.mU.mC.	3616	P.mU.fU. A. A.fC.	53%
  			A. G.mU.		A.fC.fU. G. A.fU.
  			G.mU.mU. A. A.Chl		G.mA*mA*mC*mC*mA*
  					mA* G.
  21724	2081	3617	mU.mC. A.mU.mC.	3618	P.mU.fU. A. A.fC.	48%
  			A. G.mU.		A.fC.fU. G. A.mU.
  			G.mU.mU. A. A.Chl		G.mA*mA*mC*mC*mA*
  					mA* G.
  21725	2081	3619	mU.mC. A.mU.mC.	3620	P.mU.fU. A. A.fC.	45%
  			A. G.mU.		A.fC.fU. G. A.fU. G. A*
  			G.mU.mU. A. A.Chl		A*fC*fC*mA*mA* G.
  21726	2081	3621	mU.mC. A.mU.mC.	3622	P.mU.fU. A. A.fC.	54%
  			A. G.mU.		A.fC.fU. G. A.fU.
  			G.mU.mU. A. A.Chl		G.mA*mA*fC*fC*mA*
  					mA* G.
  21727	2081	3623	mU.mC. A.mU.mC.	3624	P.mU.fU.A. A.fC.	29%
  			A. G.mU.		A.fC.fU. G. A.fU. G. A*
  			G.mU.mU*mA*mA.		A*fC*fC* A* A* G.
  			TEG-Chl
  21728	2081	3625	mU*mC* A.mU.mC.	3626	P.mU.fU. A. A.fC.	27%
  			A. G.mU.		A.fC.fU. G. A.fU. G. A*
  			G.mU.mU*mA*mA.		A*fC*fC* A* A* G.
  			TEG-Chl
  21729	2081	3627	mU*mC*mA.mU.mC.	3628	P.mU.fU. A. A.fC.	30%
  			mA.mG.mU.mG.		A.fC.fU. G. A.fU. G. A*
  			mU.mU*mA*mA.TEG-		A*fC*fC* A* A*G.
  			Chl
  21375	2081	3629	mU.mC. A.mU.mC.	3630	P.mU.fU. A. A.fC.	29%
  			A. G.mU.		A.fC.fU. G. A.fU. G. A*
  			G.mU.mU*mA*mA.		A*mC*mC* A* A* G.
  			TEG-Chl
  21376	2081	3631	mU.mC. A.mU.mC.	3632	P.mU.fU. A. A.fC.	30%
  			A. G.mU.		A.fC.fU. G. A.fU. G. A*
  			G.mU.mU*mA*mA.		A*fC*fC*mA*mA* G.
  			TEG-Chl
  21377	2081	3633	mU.mC. A.mU.mC.	3634	P.mU.fU. A. A.fC.	37%
  			A. G.mU.		A.fC.fU. G. A.fU.
  			G.mU.mU*mA*mA.		G.mA*mA*fC*fC*mA*
  			TEG-Chl		mA* G.
  21378	2081	3635	mU*mC*mA.mU.mC.	3636	P.mU.fU. A. A.fC.	32%
  			mA.mG.mU.mG.		A.fC.fU. G. A.fU. G. A*
  			mU.mU*mA*mA.TEG-		A*mC*mC* A* A*G.
  			Chl
  21379	2081	3637	mU*mC*mA.mU.mC.	3638	P.mU.fU. A. A.fC.	31%
  			mA.mG.mU.mG.		A.fC.fU. G. A.fU. G. A*
  			mU.mU*mA*mA.TEG-		A*fC*fC*mA*mA*G.
  			Chl
  21380	2081	3639	mU*mC*mA.mU.mC.	3640	P.mU.fU. A. A.fC.	39%
  			mA.mG.mU.mG.		A.fC.fU. G. A.fU.
  			mU.mU*mA*mA.TEG-		G.mA*mA*fC*fC*mA*
  			Chl		mA* G.

• TABLE 22
  Inhibition of gene expression with TGFB1 sd-rxRNA sequences (Accession Number: NM_000660.3)

  						% remaining
  Oligo	Start	SEQ ID		SEQ ID		expression
  Number	Site	NO	Sense sequence	NO	Antisense sequence	(1 uM A549)

  14394	1194	3641	G.mC.mU. A. A.mU.	3642	P.mU.fU.fC.fC. A.fC.fC.	24%
  			G. G.mU. G. G. A.		A.fU.fU. A. G.mC*
  			A.Chl		A*mC* G*mC* G* G.
  14395	2006	3643	mU. G. A.mU.mC.	3644	P.mG. A. G.fC. G.fC.	79%
  			G.mU. G.mC.		A.fC. G. A.mU.mC.
  			G.mC.mU.mC.Chl		A*mU* G*mU*mU* G*
  					G.
  14396	1389	3645	mC. A.	3646	P.mU.fC. G.fC.fC. A. G.	77%
  			A.mU.mU.mC.mC.mU.		G. A. A.mU.mU.
  			G. G.mC. G. A.Chl		G*mU*mU*
  					G*mC*mU* G.
  14397	1787	3647	A. G.mU. G. G.	3648	P.mU.fC. G.fU. G. G.	n/a
  			A.mU.mC.mC. A.mC.		A.fU.fC.fC.
  			G. A.Chl		A.mC.mU*mU*mC*mC*
  					A* G* C.
  14398	1867	3649	mU. A.mC. A. G.mC.	3650	P.mG. G. A.fC.fC.fU.fU.	82%
  			A. A. G.		G.fC.fU. G.mU.
  			G.mU.mC.mC.Chl		A*mC*mU* G*mC* G*
  					U.
  14399	2002	3651	A. A.mC. A.mU. G.	3652	P.mG.fC. A.fC. G.	n/a
  			A.mU.mC. G.mU.		A.fU.fC. A.fU.
  			G.mC.Chl		G.mU.mU* G* G*
  					A*mC* A* G.
  14400	2003	3653	A.mC. A.mU. G.	3654	P.mC. G.fC. A.fC. G.	n/a
  			A.mU.mC. G.mU.		A.fU.fC. A.mU.
  			G.mC. G.Chl		G.mU*mU* G* G*
  					A*mC* A.
  14401	1869	3655	mC. A. G.mC. A. A. G.	3656	P.mC. A. G. G.	82%
  			G.mU.mC.mC.mU.		A.fC.fC.fU.fU.
  			G.Chl		G.mC.mU. G*mU*
  					A*mC*mU* G* C.
  14402	2000	3657	mC.mC. A. A.mC.	3658	P.mA.fC. G. A.fU.fC.	66%
  			A.mU. G. A.mU.mC.		A.fU. G.fU.mU. G. G*
  			G.mU.Chl		A*mC* A* G*mC* U.
  14403	986	3659	A. G.mC. G. G. A. A.	3660	P.mA.fU. G.fC.	78%
  			G.mC. G.mC.		G.fC.fU.fU.fC.fC.
  			A.mU.Chl		G.mC.mU*mU*mC*
  					A*mC*mC* A.
  14404	995	3661	G.mC. A.mU.mC. G.	3662	P.mA.fU. G.	79%
  			A. G. G.mC.mC.		G.fC.fC.fU.fC. G. A.mU.
  			A.mU.Chl		G.mC*
  					G*mC*mU*mU*mC* C.
  14405	963	3663	G. A.mC.mU.	3664	P.mC. A.fU. G.fU.fC. G.	80%
  			A.mU.mC. G. A.mC.		A.fU. A.
  			A.mU. G.Chl		G.mU.mC*mU*mU*
  					G*mC* A* G.
  14406	955	3665	A.mC.mC.mU. G.mC.	3666	P.mU. A. G.fU.fC.fU.fU.	88%
  			A. A. G. A.mC.mU.		G.fC. A. G. G.mU* G*
  			A.Chl		G* A*mU* A* G.
  14407	1721	3667	G.mC.mU.mC.mC.	3668	P.mU.fU.fC.fU.fC.fC.	n/a
  			A.mC. G. G. A. G. A.		G.fU. G. G. A.
  			A.Chl		G.mC*mU* G* A* A*
  					G* C.
  18454	1246	3669	mC. A.mC. A. G.mC.	3670	P.mU. A.fU. A.fU. A.fU.	58%
  			A.mU. A.mU. A.mU.		G.fC.fU. G.fU. G*fU*
  			A.Chl		G*fU* A*fC* U.
  18455	1248	3671	mC. A. G.mC. A.mU.	3672	P.mU. A.fU. A.fU. A.fU.	87%
  			A.mU. A.mU. A.mU.		A.fU. G.fC.fU. G*fU*
  			A.Chl		G*fU* G*fU* A.
  18456	1755	3673	G.mU. A.mC.	3674	P.mU. A. A. G.fU.fC. A.	107%
  			A.mU.mU. G.		A.fU. G.fU. A.fC* A*
  			A.mC.mU.mU. A.Chl		G*fC*fU* G* C.
  18457	1755	3675	mU. G.mU. A.mC.	3676	P.mU. A. A. G.fU.fC. A.	77%
  			A.mU.mU. G.		A.fU. G.fU. A.fC* A*
  			A.mC.mU.mU. A.Chl		G*fC*fU* G* C.
  18458	1708	3677	A. A.mC.mU.	3678	P.mU. G. A. A. G.fC. A.	75%
  			A.mU.mU.		A.fU. A. G.fU.fU* G*
  			G.mC.mU.mU.mC.		G*fU* G*fU*C.
  			A.Chl
  18459	1708	3679	mC. A. A.mC.mU.	3680	P.mU. G. A. A. G.fC. A.	73%
  			A.mU.mU.		A.fU. A. G.fU.fU* G*
  			G.mC.mU.mU.mC.		G*fU* G*fU* C.
  			A.Chl
  18460	1250	3681	G.mC. A.mU. A.mU.	3682	P.mU. A.fC. A.fU. A.fU.	n/a
  			A.mU. A.mU. G.mU.		A.fU. A.fU. G.fC*fU*
  			A.Chl		G*fU* G*fU* G.
  18461	1754	3683	mU. G.mU. A.mC.	3684	P.mU. A. G.fU.fC. A.	91%
  			A.mU.mU. G.		A.fU. G.fU. A.fC. A*
  			A.mC.mU. A.Chl		G*fC*fU* G*fC* C.
  18462	1754	3685	mC.mU. G.mU.	3686	P.mU. A. G.fU.fC. A.	92%
  			A.mC. A.mU.mU. G.		A.fU. G.fU. A.fC. A*
  			A.mC.mU. A.Chl		G*fC*fU* G*fC* C.
  18463	1249	3687	A. G.mC. A.mU.	3688	P.mU.fC. A.fU. A.fU.	n/a
  			A.mU. A.mU. A.mU.		A.fU. A.fU. G.fC.fU*
  			G. A.Chl		G*fU* G*fU* G* U.
  18464	1383	3689	mC. A. G.mC. A.	3690	P.mU. G. A. A.fU.fU.	77%
  			A.mC. A.		G.fU.fU. G.fC.fU. G*fU*
  			A.mU.mU.mC. A.Chl		A*fU*fU*fU* C.
  18465	1251	3691	mC. A.mU. A.mU.	3692	P.mU. A. A.fC. A.fU.	84%
  			A.mU. A.mU.		A.fU. A.fU. A.fU.
  			G.mU.mU. A.Chl		G*fC*fU* G*fU* G* U.
  18466	1713	3693	mU.mU.	3694	P.mU. G. A. G.fC.fU. G.	n/a
  			G.mC.mU.mU.mC. A.		A. A. G.fC. A. A*fU* A*
  			G.mC.mU.mC. A.Chl		G*fU*fU* G.
  18467	1713	3695	A.mU.mU.	3696	P.mU. G. A. G.fC.fU. G.	83%
  			G.mC.mU.mU.mC. A.		A. A. G.fC. A. A*fU* A*
  			G.mC.mU.mC. A.Chl		G*fU*fU* G.
  18468	1247	3697	A.mC. A. G.mC.	3698	P.mU.fU. A.fU. A.fU.	96%
  			A.mU. A.mU. A.mU.		A.fU. G.fC.fU. G.fU*
  			A. A.Chl		G*fU* G*fU* A* C.
  18469	1712	3699	A.mU.mU.	3700	P.mU. A. G.fC.fU. G. A.	90%
  			G.mC.mU.mU.mC. A.		A. G.fC. A. A.fU* A*
  			G.mC.mU. A.Chl		G*fU*fU* G* G.
  18470	1712	3701	mU. A.mU.mU.	3702	P.mU. A. G.fC.fU. G. A.	98%
  			G.mC.mU.mU.mC. A.		A. G.fC. A. A.fU* A*
  			G.mC.mU. A.Chl		G*fU*fU* G* G.
  18471	1212	3703	mC. A. A.	3704	P.mU.fU. G.fC.fU.fU. G.	n/a
  			G.mU.mU.mC. A. A.		A. A.fC.fU.fU. G*fU*fC*
  			G.mC. A. A.Chl		A*fU* A* G.
  18472	1222	3705	mC. A. G. A. G.mU.	3706	P.mU. G.fU. G.fU. G.fU.	45%
  			A.mC. A.mC. A.mC.		A.fC.fU.fC.fU. G*
  			A.Chl		C*fU*fU* G* A* A.
  18473	1228	3707	A.mC. A.mC. A.mC.	3708	P.mU.fU. A.fU. G.fC.fU.	36%
  			A. G.mC. A.mU. A.		G.fU. G.fU. G.fU*
  			A.Chl		A*fC*fU*fC*fU* G.
  18474	1233	3709	mC. A. G.mC. A.mU.	3710	P.mU. A.fU. A.fU. A.fU.	68%
  			A.mU. A.mU. A.mU.		A.fU. G.fC.fU. G*fU*
  			A.Chl		G*fU* G*fU* A.
  18475	1218	3711	mU.mC. A. A. G.mC.	3712	P.mU.fU. A.fC.fU.fC.fU.	64%
  			A. G. A. G.mU. A.		G.fC.fU.fU. G. A*
  			A.Chl		A*fC*fU*fU* G* U.
  18476	1235	3713	A. G.mC. A.mU.	3714	P.mU.fC. A.fU. A.fU.	78%
  			A.mU. A.mU. A.mU.		A.fU. A.fU. G.fC.fU*
  			G. A.Chl		G*fU* G*fU* G* U.
  18477	1225	3715	A. G. A. G.mU. A.mC.	3716	P.mU.fU. G.fU. G.fU.	92%
  			A.mC. A.mC. A. A.Chl		G.fU. A.fC.fU.fC.fU*
  					G*fC*fU*fU* G* A.
  18478	1221	3717	A. A. G.mC. A. G. A.	3718	P.mU.fU. G.fU.	103%
  			G.mU. A.mC. A. A.Chl		A.fC.fU.fC.fU.
  					G.fC.fU.fU* G* A*
  					A*fC*fU* U.
  18479	1244	3719	mU.mU.mC. A. A.mC.	3720	P.mU.fU. G. A.fU. G.fU.	84%
  			A.mC. A.mU.mC. A.		G.fU.fU. G. A. A* G* A*
  			A.Chl		A*fC* A* U.
  18480	1224	3721	A. G.mC. A. G. A.	3722	P.mU. G.fU. G.fU.	37%
  			G.mU. A.mC. A.mC.		A.fC.fU.fC.fU.
  			A.Chl		G.fC.fU*fU* G* A*
  					A*fC* U.
  18481	1242	3723	A.mU. A.mU. A.mU.	3724	P.mU. A. A. G. A. A.fC.	62%
  			G.mU.mU.mC.mU.mU.		A.fU. A.fU. A.fU* A*fU*
  			A.Chl		G*fC*fU* G.
  18482	1213	3725	G. A.mC. A. A.	3726	P.mU.fC.fU.fU. G. A.	47%
  			G.mU.mU.mC. A. A.		A.fC.fU.fU. G.fU.fC*
  			G. A.Chl		A*fU* A* G* A* U.
  18483	1760	3727	mU.mU. A. A. A. G.	3728	P.mU.fC.fU.fC.fC.	69%
  			A.mU. G. G. A. G.		A.fU.fC.fU.fU.fU. A.
  			A.Chl		A*fU* G* G* G* G* C.
  18484	1211	3729	mC.mU. A.mU. G.	3730	P.mU. A. A.fC.fU.fU.	n/a
  			A.mC. A. A.		G.fU.fC. A.fU. A. G*
  			G.mU.mU. A.Chl		A*fU*fU*fU*fC* G.
  19411	1212	3731	mC. A. A.mC. G. A. A.	3732	P.mU.fU. A. G.	52%
  			A.mU.mC.mU. A.		A.fU.fU.fU.fC. G.fU.fU.
  			A.Chl		G*fU* G* G* G*fU*fU.
  19412	1222	3733	mU. A.mU. G. A.mC.	3734	P.mU. G. A. A.fC.fU.fU.	51%
  			A. A. G.mU.mU.mC.		G.fU.fC. A.fU. A* G*
  			A.Chl		A*fU*fU*fU*fC.
  19413	1228	3735	A. A. G.mU.mU.mC.	3736	P.mU.fC.fU. G.fC.fU.fU.	n/a
  			A. A. G.mC. A. G.		G. A. A.fC.fU.fU*
  			A.Chl		G*fU*fC* A*fU* A.
  19414	1233	3737	mC. A. A. G.mC. A. G.	3738	P.mU. G.fU.	41%
  			A. G.mU. A.mC. A.Chl		A.fC.fU.fC.fU.
  					G.fC.fU.fU. G* A*
  					A*fC*fU*fU* G.
  19415	1218	3739	A. A.mU.mC.mU.	3740	P.mU.fU.fU. G.fU.fC.	104%
  			A.mU. G. A.mC. A. A.		A.fU. A. G.
  			A.Chl		A.fU.fU*fU*fC*
  					G*fU*fU* G.
  19416	1244	3741	mC. A.mC. A.mC. A.	3742	P.mU. A.fU. A.fU.	31%
  			G.mC. A.mU. A.mU.		G.fC.fU. G.fU. G.fU.
  			A.Chl		G*fU* A*fC*fU*fC*fU.
  19417	655	3743	G. A. A. A.mU.	3744	P.mU.fU.fU. G.fC.fU.	n/a
  			A.mU. A. G.mC. A. A.		A.fU. A.fU.fU.fU.fC*fU*
  			A.Chl		G* G*fU* A* G.
  19418	644	3745	G. A.	3746	P.mU.fC.fU. G. G.fU. A.	n/a
  			A.mC.mU.mC.mU.		G. A. G.fU.fU.fC*fU*
  			A.mC.mC. A. G. A.Chl		A*fC* G*fU* G.
  19419	819	3747	G.mC. A. A. A. G.	3748	P.mU.fC. A.fU.fU.	n/a
  			A.mU. A. A.mU. G.		A.fU.fC.fU.fU.fU.
  			A.Chl		G.fC*fU* G*fU*fC* A*
  					C.
  19420	645	3749	A. A.mC.mU.mC.mU.	3750	P.mU.fU.fC.fU. G. G.fU.	n/a
  			A.mC.mC. A. G. A.		A. G. A. G.fU.fU*fC*fU*
  			A.Chl		A*fC* G* U.
  19421	646	3751	A.mC.mU.mC.mU.	3752	P.mU.fU.fU.fC.fU. G.	n/a
  			A.mC.mC. A. G. A. A.		G.fU. A. G. A.
  			A.Chl		G.fU*fU*fC*fU* A*fC*
  					G.
  19422	816	3753	A.mC. A. G.mC. A. A.	3754	P.mU.fU.	n/a
  			A. G. A.mU. A. A.Chl		A.fU.fC.fU.fU.fU.
  					G.fC.fU. G.fU*fC* A*fC*
  					A* A* G.
  19423	495	3755	mC. A. A.mU.mC.mU.	3756	P.mU.fU. G.fU.fC. A.fU.	n/a
  			A.mU. G. A.mC. A.		A. G. A.fU.fU. G*fC*
  			A.Chl		G*fU*fU* G* U.
  19424	614	3757	A. G. A.mU.mU.mC.	3758	P.mU.fU. G. A.fC.fU.fU.	n/a
  			A. A. G.mU.mC. A.		G. A. A.fU.fC.fU*fC*fU*
  			A.Chl		G*fC* A* G.
  19425	627	3759	mC.mU. G.mU. G. G.	3760	P.mU. G.fU.fU.	n/a
  			A. G.mC. A. A.mC.		G.fC.fU.fC.fC. A.fC. A.
  			A.Chl		G*fU*fU* G* A*fC* U.
  19426	814	3761	mU. G. A.mC. A.	3762	P.mU.fU.fC.fU.fU.fU.	n/a
  			G.mC. A. A. A. G. A.		G.fC.fU. G.fU.fC. A*fC*
  			A.Chl		A* A* G* A* G.
  19427	501	3763	A.mU. G. A.mC. A. A.	3764	P.mU.fU. G.	n/a
  			A. A.mC.mC. A. A.Chl		G.fU.fU.fU.fU. G.fU.fC.
  					A.fU* A* G* A*fU*fU*
  					G.
  19428	613	3765	G. A. G.	3766	P.mU. G. A.fC.fU.fU. G.	n/a
  			A.mU.mU.mC. A. A.		A. A.fU.fC.fU.fC*fU*
  			G.mU.mC. A.Chl		G*fC* A* G* G.
  21240	1244	3767	mC. A.mC. A.mC. A.	3768	P.mU. A.fU. A.fU.	0.875
  			G.mC. A.mU. A.mU.		G.fC.fU. G.fU. G.fU.
  			A.Chl		G*mU*
  					A*mC*mU*mC* U.
  21241	1244	3769	mC. A.mC. A.mC. A.	3770	P.mU. A.fU. A.fU.	0.88
  			G.mC. A.mU. A.mU.		G.fC.fU. G.fU. G.fU.
  			A.Chl		G*mU*mA*mC*mU*mC*
  					U.
  21242	1244	3771	mC. A.mC. A.mC. A.	3772	P.mU. A.fU. A.fU.	0.635
  			G.mC. A.mU. A.mU.		G.fC.fU. G.fU.
  			A.Chl		G.fU.mG*mU*mA*mC*
  					mU*mC* U.
  21243	1244	3773	mC. A.mC. A.mC. A.	3774	P.mU. A.fU. A.fU.	0.32
  			G.mC. A.mU. A.mU.		G.fC.fU. G.fU.
  			A.Chl		G.fU.mG*fU*mA*fC*mU*
  					fC* U.
  21244	1244	3775	mC. A.mC. A.mC. A.	3776	P.mU. A.fU. A.fU.	0.36
  			G.mC. A.mU. A.mU.		G.fC.fU. G.fU. G.fU.
  			A.Chl		G*fU* A*fC*mU*mC*
  					U.
  21245	1244	3777	mC. A.mC. A.mC. A.	3778	P.mU. A.fU. A.fU.	0.265
  			G.mC. A.mU.		G.fC.fU. G.fU. G.fU.
  			A*mU*mA.TEG-Chl		G*fU* A*fC*fU*fC*fU.
  21246	1244	3779	mC*mA*mC. A.mC.	3780	P.mU. A.fU. A.fU.	0.334
  			A. G.mC. A.mU.		G.fC.fU. G.fU. G.fU.
  			A*mU*mA.TEG-Chl		G*fU* A*fC*fU*fC*fU.
  21247	1244	3781	mC*mA*mC.mA.mC.	3782	P.mU. A.fU. A.fU.	0.29
  			mA.mG.mC.mA.mU.		G.fC.fU. G.fU. G.fU.
  			mA*mU*mA.TEG-Chl		G*fU* A*fC*fU*fC*fU.
  21248	614	3783	mA. G.	3784	P.mU.fU. G. A.fC.fU.fU.	n/a
  			A.mU.mU.mC. A. A.		G. A. A.fU.fC.fU*fC*fU*
  			G.mU.mC*mA*mA.TEG-		G*fC*fU* U.
  			Chl
  20608	1244	3785	mC. A.mC. A.mC. A.	3786	P.mU. A.fU. A.fU.	79%
  			G.mC. A.mU. A.mU.		G.fC.fU. G.fU. G.mU.
  			A.Chl		G*mU*
  					A*mC*mU*mC* U.
  20609	1244	3787	mC. A.mC. A.mC. A.	3788	P.mU. A.fU. A.fU.	60%
  			G.mC. A.mU. A.mU.		G.fC.fU. G.fU. G.mU.
  			A.Chl		G*fU* A*mC*fU*mC*
  					U.
  20610	1244	3789	mC. A.mC. A.mC. A.	3790	P.mU. A. U. A. U. G. C.	93%
  			G.mC. A.mU. A.mU.		U. G. U. G.mU. G*mU*
  			A.Chl		A*mC*mU*mC* U.
  20611	1244	3791	mC. A.mC. A.mC. A.	3792	P.mU. A.fU. A.fU.	n/a
  			G.mC. A.mU. A.mU.		G.fC.fU. G.fU.
  			A.Chl		G.mU.mG*mU*mA*mC*
  					mU*mC* U.
  21374	614	3793	mC*mA*mC.mA.mC.	3794	P.mU. A.fU. A.fU.	24%
  			mA.mG.mC.mA.mU.		G.fC.fU. G.fU.
  			mA*mU*mA.TEG-Chl		G.fU.mG*fU*mA*fC*mU*
  					fC* U.

• TABLE 23
  CB1 sequences

  Ref

  SEQ ID

  SEQ ID

  Pos	NO	1-mer Sense Seq	NO	25-mer Sense Seq w/A @ 25
  1690	3795	AUGUCUGUGUCCACAGACA	3796	GUAACCAUGUCUGUGUCCACAGACA
  1686	3797	AACCAUGUCUGUGUCCACA	3798	CAAGGUAACCAUGUCUGUGUCCACA
  1685	3799	UAACCAUGUCUGUGUCCAC	3800	CCAAGGUAACCAUGUCUGUGUCCAA
  1684	3801	GUAACCAUGUCUGUGUCCA	3802	GCCAAGGUAACCAUGUCUGUGUCCA
  1649	3803	AAAGCUGCAUCAAGAGCAC	3804	CCGCAGAAAGCUGCAUCAAGAGCAA
  1648	3805	GAAAGCUGCAUCAAGAGCA	3806	GCCGCAGAAAGCUGCAUCAAGAGCA
  1494	3807	CAUCUAUGCUCUGAGGAGU	3808	CCCCAUCAUCUAUGCUCUGAGGAGA
  1493	3809	UCAUCUAUGCUCUGAGGAG	3810	ACCCCAUCAUCUAUGCUCUGAGGAA
  1492	3811	AUCAUCUAUGCUCUGAGGA	3812	AACCCCAUCAUCUAUGCUCUGAGGA
  1491	3813	CAUCAUCUAUGCUCUGAGG	3814	GAACCCCAUCAUCUAUGCUCUGAGA
  1490	3815	CCAUCAUCUAUGCUCUGAG	3816	UGAACCCCAUCAUCUAUGCUCUGAA
  1489	3817	CCCAUCAUCUAUGCUCUGA	3818	GUGAACCCCAUCAUCUAUGCUCUGA
  1487	3819	ACCCCAUCAUCUAUGCUCU	3820	CCGUGAACCCCAUCAUCUAUGCUCA
  1486	3821	AACCCCAUCAUCUAUGCUC	3822	ACCGUGAACCCCAUCAUCUAUGCUA
  1358	3823	UGGUGUUGAUCAUCUGCU	3824	UCCUGGUGGUGUUGAUCAUCUGCU
  		G		A
  1357	3825	GUGGUGUUGAUCAUCUGC	3826	AUCCUGGUGGUGUUGAUCAUCUGC
  		U		A
  1355	3827	UGGUGGUGUUGAUCAUCU	3828	UGAUCCUGGUGGUGUUGAUCAUCU
  		G		A
  1354	3829	CUGGUGGUGUUGAUCAUC	3830	CUGAUCCUGGUGGUGUUGAUCAUC
  		U		A
  1351	3831	AUCCUGGUGGUGUUGAUC	3832	GUCCUGAUCCUGGUGGUGUUGAUC
  		A		A
  1198	3833	AUUCUCUGGAAGGCUCACA	3834	AUGUAUAUUCUCUGGAAGGCUCACA
  1197	3835	UAUUCUCUGGAAGGCUCAC	3836	CAUGUAUAUUCUCUGGAAGGCUCAA
  1196	3837	AUAUUCUCUGGAAGGCUCA	3838	ACAUGUAUAUUCUCUGGAAGGCUCA
  1195	3839	UAUAUUCUCUGGAAGGCUC	3840	UACAUGUAUAUUCUCUGGAAGGCUA
  1131	3841	CUACCUGAUGUUCUGGAUC	3842	UGAAACCUACCUGAUGUUCUGGAUA
  1129	3843	ACCUACCUGAUGUUCUGGA	3844	GAUGAAACCUACCUGAUGUUCUGGA
  1127	3845	AAACCUACCUGAUGUUCUG	3846	UUGAUGAAACCUACCUGAUGUUCUA
  1126	3847	GAAACCUACCUGAUGUUCU	3848	AUUGAUGAAACCUACCUGAUGUUCA
  1086	3849	ACUGCAAUCUGUUUGCUCA	3850	CGAGAAACUGCAAUCUGUUUGCUCA
  1084	3851	AAACUGCAAUCUGUUUGCU	3852	UGCGAGAAACUGCAAUCUGUUUGCA
  972	3853	CCUGGCCUAUAAGAGGAUU	3854	CAGGCCCCUGGCCUAUAAGAGGAUA
  951	3855	GUACAUAUCCAUUCACAGG	3856	CGACAGGUACAUAUCCAUUCACAGA
  950	3857	GGUACAUAUCCAUUCACAG	3858	UCGACAGGUACAUAUCCAUUCACAA
  948	3859	CAGGUACAUAUCCAUUCAC	3860	CAUCGACAGGUACAUAUCCAUUCAA
  947	3861	ACAGGUACAUAUCCAUUCA	3862	CCAUCGACAGGUACAUAUCCAUUCA
  946	3863	GACAGGUACAUAUCCAUUC	3864	GCCAUCGACAGGUACAUAUCCAUUA
  943	3865	AUCGACAGGUACAUAUCCA	3866	ACAGCCAUCGACAGGUACAUAUCCA
  941	3867	CCAUCGACAGGUACAUAUC	3868	UCACAGCCAUCGACAGGUACAUAUA
  940	3869	GCCAUCGACAGGUACAUAU	3870	CUCACAGCCAUCGACAGGUACAUAA
  869	3871	ACGUGUUUCUGUUCAAACU	3872	GCCGCAACGUGUUUCUGUUCAAACA
  868	3873	AACGUGUUUCUGUUCAAAC	3874	AGCCGCAACGUGUUUCUGUUCAAAA
  1647	3875	AGAAAGCUGCAUCAAGAGC	3876	GGCCGCAGAAAGCUGCAUCAAGAGA
  1645	3877	GCAGAAAGCUGCAUCAAGA	3878	AGGGCCGCAGAAAGCUGCAUCAAGA
  1394	3879	UCAUGGUGUAUGAUGUCU	3880	UUGCAAUCAUGGUGUAUGAUGUCU
  		U		A
  1393	3881	AUCAUGGUGUAUGAUGUC	3882	CUUGCAAUCAUGGUGUAUGAUGUC
  		U		A
  1391	3883	CAAUCAUGGUGUAUGAUG	3884	UGCUUGCAAUCAUGGUGUAUGAUG
  		U		A
  1125	3885	UGAAACCUACCUGAUGUUC	3886	CAUUGAUGAAACCUACCUGAUGUUA
  1090	3887	CAAUCUGUUUGCUCAGACA	3888	AAACUGCAAUCUGUUUGCUCAGACA
  1089	3889	GCAAUCUGUUUGCUCAGAC	3890	GAAACUGCAAUCUGUUUGCUCAGAA
  1088	3891	UGCAAUCUGUUUGCUCAGA	3892	AGAAACUGCAAUCUGUUUGCUCAGA
  1087	3893	CUGCAAUCUGUUUGCUCAG	3894	GAGAAACUGCAAUCUGUUUGCUCAA
  1397	3895	UGGUGUAUGAUGUCUUUG	3896	CAAUCAUGGUGUAUGAUGUCUUUG
  		G		A
  1396	3897	AUGGUGUAUGAUGUCUUU	3898	GCAAUCAUGGUGUAUGAUGUCUUU
  		G		A
  1120	3899	AUUGAUGAAACCUACCUGA	3900	CCACACAUUGAUGAAACCUACCUGA
  1118	3901	ACAUUGAUGAAACCUACCU	3902	UCCCACACAUUGAUGAAACCUACCA
  1117	3903	CACAUUGAUGAAACCUACC	3904	UUCCCACACAUUGAUGAAACCUACA
  1116	3905	ACACAUUGAUGAAACCUAC	3906	UUUCCCACACAUUGAUGAAACCUAA
  1132	3907	UACCUGAUGUUCUGGAUC	3908	GAAACCUACCUGAUGUUCUGGAUCA
  		G
  845	3909	UGUUCCACCGCAAAGAUAG	3910	UCCACGUGUUCCACCGCAAAGAUAA
  844	3911	GUGUUCCACCGCAAAGAUA	3912	UUCCACGUGUUCCACCGCAAAGAUA
  573	3913	CUUCAAGGAGAAUGAGGAG	3914	CUCGUCCUUCAAGGAGAAUGAGGAA
  572	3915	CCUUCAAGGAGAAUGAGGA	3916	UCUCGUCCUUCAAGGAGAAUGAGGA
  571	3917	UCCUUCAAGGAGAAUGAGG	3918	CUCUCGUCCUUCAAGGAGAAUGAGA
  1449	3919	AUUCUGCAGUAUGCUCUGC	3920	GUUUGCAUUCUGCAGUAUGCUCUG
  				A
  1448	3921	CAUUCUGCAGUAUGCUCUG	3922	UGUUUGCAUUCUGCAGUAUGCUCU
  				A
  1447	3923	GCAUUCUGCAGUAUGCUCU	3924	GUGUUUGCAUUCUGCAGUAUGCUC
  				A
  1253	3925	AGAGCAUCAUCAUCCACAC	3926	CCCAGAAGAGCAUCAUCAUCCACAA
  1252	3927	AAGAGCAUCAUCAUCCACA	3928	ACCCAGAAGAGCAUCAUCAUCCACA
  1247	3929	CCCAGAAGAGCAUCAUCAU	3930	GUGGCACCCAGAAGAGCAUCAUCAA
  1246	3931	ACCCAGAAGAGCAUCAUCA	3932	CGUGGCACCCAGAAGAGCAUCAUCA
  311	3933	UGAAGUCGAUCCUAGAUGG	3934	AGGUUAUGAAGUCGAUCCUAGAUG
  				A
  310	3935	AUGAAGUCGAUCCUAGAUG	3936	GAGGUUAUGAAGUCGAUCCUAGAU
  				A
  1249	3937	CAGAAGAGCAUCAUCAUCC	3938	GGCACCCAGAAGAGCAUCAUCAUCA
  585	3939	UGAGGAGAACAUCCAGUGU	3940	GGAGAAUGAGGAGAACAUCCAGUGA
  583	3941	AAUGAGGAGAACAUCCAGU	3942	AAGGAGAAUGAGGAGAACAUCCAGA
  581	3943	AGAAUGAGGAGAACAUCCA	3944	UCAAGGAGAAUGAGGAGAACAUCCA
  580	3945	GAGAAUGAGGAGAACAUCC	3946	UUCAAGGAGAAUGAGGAGAACAUCA
  579	3947	GGAGAAUGAGGAGAACAUC	3948	CUUCAAGGAGAAUGAGGAGAACAUA
  578	3949	AGGAGAAUGAGGAGAACAU	3950	CCUUCAAGGAGAAUGAGGAGAACAA
  577	3951	AAGGAGAAUGAGGAGAACA	3952	UCCUUCAAGGAGAAUGAGGAGAACA
  574	3953	UUCAAGGAGAAUGAGGAG	3954	UCGUCCUUCAAGGAGAAUGAGGAGA
  		A
  1257	3955	CAUCAUCAUCCACACGUCU	3956	GAAGAGCAUCAUCAUCCACACGUCA
  1255	3957	AGCAUCAUCAUCCACACGU	3958	CAGAAGAGCAUCAUCAUCCACACGA
  1682	3959	AGGUAACCAUGUCUGUGUC	3960	UUGCCAAGGUAACCAUGUCUGUGUA
  1681	3961	AAGGUAACCAUGUCUGUGU	3952	AUUGCCAAGGUAACCAUGUCUGUGA
  1680	3963	CAAGGUAACCAUGUCUGUG	3964	GAUUGCCAAGGUAACCAUGUCUGUA
  1499	3965	AUGCUCUGAGGAGUAAGG	3966	UCAUCUAUGCUCUGAGGAGUAAGGA
  		A
  1498	3967	UAUGCUCUGAGGAGUAAG	3968	AUCAUCUAUGCUCUGAGGAGUAAGA
  		G
  1497	3969	CUAUGCUCUGAGGAGUAAG	3970	CAUCAUCUAUGCUCUGAGGAGUAAA
  1496	3971	UCUAUGCUCUGAGGAGUAA	3972	CCAUCAUCUAUGCUCUGAGGAGUAA
  1388	3973	UUGCAAUCAUGGUGUAUG	3974	CUCUGCUUGCAAUCAUGGUGUAUG
  		A		A
  1387	3975	CUUGCAAUCAUGGUGUAU	3976	CCUCUGCUUGCAAUCAUGGUGUAUA
  		G
  1386	3977	GCUUGCAAUCAUGGUGUA	3978	CCCUCUGCUUGCAAUCAUGGUGUAA
  		U
  1385	3979	UGCUUGCAAUCAUGGUGU	3980	GCCCUCUGCUUGCAAUCAUGGUGUA
  		A
  1384	3981	CUGCUUGCAAUCAUGGUG	3982	GGCCCUCUGCUUGCAAUCAUGGUGA
  		U
  1383	3983	UCUGCUUGCAAUCAUGGU	3984	GGGCCCUCUGCUUGCAAUCAUGGUA
  		G
  1382	3985	CUCUGCUUGCAAUCAUGGU	3986	GGGGCCCUCUGCUUGCAAUCAUGGA
  1314	3987	CCGCAUGGACAUUAGGUUA	3988	CCAAGCCCGCAUGGACAUUAGGUUA
  1094	3989	CUGUUUGCUCAGACAUUU	3990	UGCAAUCUGUUUGCUCAGACAUUUA
  		U
  1093	3991	UCUGUUUGCUCAGACAUU	3992	CUGCAAUCUGUUUGCUCAGACAUUA
  		U
  1083	3993	GAAACUGCAAUCUGUUUGC	3994	CUGCGAGAAACUGCAAUCUGUUUGA
  1082	3995	AGAAACUGCAAUCUGUUUG	3996	ACUGCGAGAAACUGCAAUCUGUUUA
  1080	3997	CGAGAAACUGCAAUCUGUU	3998	GAACUGCGAGAAACUGCAAUCUGUA
  323	3999	UAGAUGGCCUUGCAGAUAC	4000	CGAUCCUAGAUGGCCUUGCAGAUAA
  322	4001	CUAGAUGGCCUUGCAGAUA	4002	UCGAUCCUAGAUGGCCUUGCAGAUA
  1179	4003	CGUGUAUGCGUACAUGUA	4004	GUUCAUCGUGUAUGCGUACAUGUA
  		U		A
  1178	4005	UCGUGUAUGCGUACAUGU	4006	UGUUCAUCGUGUAUGCGUACAUGU
  		A		A
  1177	4007	AUCGUGUAUGCGUACAUG	4008	CUGUUCAUCGUGUAUGCGUACAUG
  		U		A
  1320	4009	GGACAUUAGGUUAGCCAAG	4010	CCGCAUGGACAUUAGGUUAGCCAAA
  1319	4011	UGGACAUUAGGUUAGCCAA	4012	CCCGCAUGGACAUUAGGUUAGCCAA
  1318	4013	AUGGACAUUAGGUUAGCCA	4014	GCCCGCAUGGACAUUAGGUUAGCCA
  1317	4015	CAUGGACAUUAGGUUAGCC	4016	AGCCCGCAUGGACAUUAGGUUAGCA
  1316	4017	GCAUGGACAUUAGGUUAGC	4018	AAGCCCGCAUGGACAUUAGGUUAGA
  1315	4019	CGCAUGGACAUUAGGUUAG	4020	CAAGCCCGCAUGGACAUUAGGUUAA
  1415	4021	GGAAGAUGAACAAGCUCAU	4022	UCUUUGGGAAGAUGAACAAGCUCAA
  552	4023	UUACAACAAGUCUCUCUCG	4024	AGAAUUUUACAACAAGUCUCUCUCA
  551	4025	UUUACAACAAGUCUCUCUC	4026	CAGAAUUUUACAACAAGUCUCUCUA
  550	4027	UUUUACAACAAGUCUCUCU	4028	ACAGAAUUUUACAACAAGUCUCUCA
  476	4029	GUCCCUUCCAAGAGAAGAU	4030	GGGGAAGUCCCUUCCAAGAGAAGAA
  474	4031	AAGUCCCUUCCAAGAGAAG	4032	UAGGGGAAGUCCCUUCCAAGAGAAA
  473	4033	GAAGUCCCUUCCAAGAGAA	4034	UUAGGGGAAGUCCCUUCCAAGAGAA
  1020	4035	UUGCCUGAUGUGGACCAUA	4036	GGCGUUUUGCCUGAUGUGGACCAU
  				A
  1019	4037	UUUGCCUGAUGUGGACCA	4038	UGGCGUUUUGCCUGAUGUGGACCA
  		U		A
  1018	4039	UUUUGCCUGAUGUGGACC	4040	GUGGCGUUUUGCCUGAUGUGGACC
  		A		A
  606	4041	GGAGAACUUCAUGGACAUA	4042	GUGUGGGGAGAACUUCAUGGACAU
  				A
  1568	4043	AGCCUCUGGAUAACAGCAU	4044	CUGCGCAGCCUCUGGAUAACAGCAA
  1170	4045	UCUGUUCAUCGUGUAUGC	4046	ACUGCUUCUGUUCAUCGUGUAUGCA
  		G
  1169	4047	UUCUGUUCAUCGUGUAUG	4048	UACUGCUUCUGUUCAUCGUGUAUG
  		C	A
  1168	4049	CUUCUGUUCAUCGUGUAU	4050	GUACUGCUUCUGUUCAUCGUGUAU
  		G		A
  1421	4051	UGAACAAGCUCAUUAAGAC	4052	GGAAGAUGAACAAGCUCAUUAAGAA
  1420	4053	AUGAACAAGCUCAUUAAGA	4054	GGGAAGAUGAACAAGCUCAUUAAGA
  1419	4055	GAUGAACAAGCUCAUUAAG	4056	UGGGAAGAUGAACAAGCUCAUUAAA
  1418	4057	AGAUGAACAAGCUCAUUAA	4058	UUGGGAAGAUGAACAAGCUCAUUAA
  1417	4059	AAGAUGAACAAGCUCAUUA	4060	UUUGGGAAGAUGAACAAGCUCAUUA
  1172	4061	UGUUCAUCGUGUAUGCGU	4062	UGCUUCUGUUCAUCGUGUAUGCGU
  		A		A
  1078	4063	UGCGAGAAACUGCAAUCUG	4064	UGGAACUGCGAGAAACUGCAAUCUA
  825	4065	CAGCUUCAUUGACUUCCAC	4066	UGUCUACAGCUUCAUUGACUUCCAA
  824	4067	ACAGCUUCAUUGACUUCCA	4068	UUGUCUACAGCUUCAUUGACUUCCA
  823	4069	UACAGCUUCAUUGACUUCC	4070	UUUGUCUACAGCUUCAUUGACUUCA
  821	4071	UCUACAGCUUCAUUGACUU	4072	UUUUUGUCUACAGCUUCAUUGACU
  		A
  820	4073	GUCUACAGCUUCAUUGACU	4074	AUUUUUGUCUACAGCUUCAUUGACA
  612	4075	CUUCAUGGACAUAGAGUGU	4076	GGAGAACUUCAUGGACAUAGAGUGA
  611	4077	ACUUCAUGGACAUAGAGUG	4078	GGGAGAACUUCAUGGACAUAGAGUA
  610	4079	AACUUCAUGGACAUAGAGU	4080	GGGGAGAACUUCAUGGACAUAGAGA
  549	4081	AUUUUACAACAAGUCUCUC	4082	UACAGAAUUUUACAACAAGUCUCUA
  547	4083	GAAUUUUACAACAAGUCUC	4084	AUUACAGAAUUUUACAACAAGUCUA
  1176	4085	CAUCGUGUAUGCGUACAUG	4086	UCUGUUCAUCGUGUAUGCGUACAU
  				A
  1175	4087	UCAUCGUGUAUGCGUACAU	4088	UUCUGUUCAUCGUGUAUGCGUACA
  				A
  1174	4089	UUCAUCGUGUAUGCGUACA	4090	CUUCUGUUCAUCGUGUAUGCGUACA
  1173	4091	GUUCAUCGUGUAUGCGUA	4092	GCUUCUGUUCAUCGUGUAUGCGUA
  		C		A
  1171	4093	CUGUUCAUCGUGUAUGCG	4094	CUGCUUCUGUUCAUCGUGUAUGCG
  		U		A
  609	4095	GAACUUCAUGGACAUAGAG	4096	UGGGGAGAACUUCAUGGACAUAGAA
  608	4097	AGAACUUCAUGGACAUAGA	4098	GUGGGGAGAACUUCAUGGACAUAGA
  607	4099	GAGAACUUCAUGGACAUAG	4100	UGUGGGGAGAACUUCAUGGACAUA
  				A
  1322	4101	ACAUUAGGUUAGCCAAGAC	4102	GCAUGGACAUUAGGUUAGCCAAGAA
  1321	4103	GACAUUAGGUUAGCCAAGA	4104	CGCAUGGACAUUAGGUUAGCCAAGA
  1027	4105	AUGUGGACCAUAGCCAUUG	4106	UGCCUGAUGUGGACCAUAGCCAUUA
  545	4107	CAGAAUUUUACAACAAGUC	4108	ACAUUACAGAAUUUUACAACAAGUA
  532	4109	CAGGUGAACAUUACAGAAU	4110	GCAGACCAGGUGAACAUUACAGAAA
  813	4111	CAUUUUUGUCUACAGCUUC	4112	GAGUGUCAUUUUUGUCUACAGCUU
  				A
  812	4113	UCAUUUUUGUCUACAGCU	4114	GGAGUGUCAUUUUUGUCUACAGCU
  		U		A
  811	4115	GUCAUUUUUGUCUACAGC	4116	GGGAGUGUCAUUUUUGUCUACAGC
  		U		A
  809	4117	GUGUCAUUUUUGUCUACA	4118	UGGGGAGUGUCAUUUUUGUCUACA
  		G		A
  808	4119	AGUGUCAUUUUUGUCUAC	4120	CUGGGGAGUGUCAUUUUUGUCUAC
  		A		A
  569	4121	CGUCCUUCAAGGAGAAUGA	4122	CUCUCUCGUCCUUCAAGGAGAAUGA
  568	4123	UCGUCCUUCAAGGAGAAUG	4124	UCUCUCUCGUCCUUCAAGGAGAAUA
  1444	4125	UUUGCAUUCUGCAGUAUG	4126	ACGGUGUUUGCAUUCUGCAGUAUG
  		C		A
  1443	4127	GUUUGCAUUCUGCAGUAU	4128	GACGGUGUUUGCAUUCUGCAGUAU
  		G		A
  1446	4129	UGCAUUCUGCAGUAUGCUC	4130	GGUGUUUGCAUUCUGCAGUAUGCU
  				A
  1445	4131	UUGCAUUCUGCAGUAUGC	4132	CGGUGUUUGCAUUCUGCAGUAUGC
  		U		A
  1442	4133	UGUUUGCAUUCUGCAGUA	4134	AGACGGUGUUUGCAUUCUGCAGUA
  		U		A
  1677	4135	UGCCAAGGUAACCAUGUCU	4136	CAAGAUUGCCAAGGUAACCAUGUCA
  1676	4137	UUGCCAAGGUAACCAUGUC	4138	UCAAGAUUGCCAAGGUAACCAUGUA
  1675	4139	AUUGCCAAGGUAACCAUGU	4140	GUCAAGAUUGCCAAGGUAACCAUGA
  1603	4141	CUGCACAAACACGCAAACA	4142	GACUGCCUGCACAAACACGCAAACA
  1110	4143	UUUCCCACACAUUGAUGAA	4144	AGACAUUUUCCCACACAUUGAUGAA
  1109	4145	UUUUCCCACACAUUGAUGA	4146	CAGACAUUUUCCCACACAUUGAUGA
  1108	4147	AUUUUCCCACACAUUGAUG	4148	UCAGACAUUUUCCCACACAUUGAUA
  1605	4149	GCACAAACACGCAAACAAU	4150	CUGCCUGCACAAACACGCAAACAAA
  1604	4151	UGCACAAACACGCAAACAA	4152	ACUGCCUGCACAAACACGCAAACAA
  1671	4153	CAAGAUUGCCAAGGUAACC	4154	CACGGUCAAGAUUGCCAAGGUAACA
  1670	4155	UCAAGAUUGCCAAGGUAAC	4156	GCACGGUCAAGAUUGCCAAGGUAAA
  1669	4157	GUCAAGAUUGCCAAGGUAA	4158	AGCACGGUCAAGAUUGCCAAGGUAA
  628	4159	UGUUUCAUGGUCCUGAACC	4160	AUAGAGUGUUUCAUGGUCCUGAACA
  1115	4161	CACACAUUGAUGAAACCUA	4162	UUUUCCCACACAUUGAUGAAACCUA
  1114	4163	CCACACAUUGAUGAAACCU	4164	AUUUUCCCACACAUUGAUGAAACCA
  1113	4165	CCCACACAUUGAUGAAACC	4166	CAUUUUCCCACACAUUGAUGAAACA
  1112	4167	UCCCACACAUUGAUGAAAC	4168	ACAUUUUCCCACACAUUGAUGAAAA
  1111	4169	UUCCCACACAUUGAUGAAA	4170	GACAUUUUCCCACACAUUGAUGAAA
  814	4171	AUUUUUGUCUACAGCUUCA	4172	AGUGUCAUUUUUGUCUACAGCUUC
  				A
  1659	4173	CAAGAGCACGGUCAAGAUU	4174	CUGCAUCAAGAGCACGGUCAAGAUA
  1657	4175	AUCAAGAGCACGGUCAAGA	4176	AGCUGCAUCAAGAGCACGGUCAAGA
  1167	4177	GCUUCUGUUCAUCGUGUA	4178	CGUACUGCUUCUGUUCAUCGUGUAA
  		U
  1166	4179	UGCUUCUGUUCAUCGUGU	4180	GCGUACUGCUUCUGUUCAUCGUGU
  		A		A
  1668	4181	GGUCAAGAUUGCCAAGGUA	4182	GAGCACGGUCAAGAUUGCCAAGGUA
  819	4183	UGUCUACAGCUUCAUUGAC	4184	CAUUUUUGUCUACAGCUUCAUUGAA
  818	4185	UUGUCUACAGCUUCAUUGA	4186	UCAUUUUUGUCUACAGCUUCAUUG
  				A
  817	4187	UUUGUCUACAGCUUCAUU	4188	GUCAUUUUUGUCUACAGCUUCAUU
  		G		A
  816	4189	UUUUGUCUACAGCUUCAU	4190	UGUCAUUUUUGUCUACAGCUUCAU
  		U		A
  1543	4191	UUUCCCUCUUGUGAAGGCA	4192	AGCAUGUUUCCCUCUUGUGAAGGCA
  1660	4193	AAGAGCACGGUCAAGAUUG	4194	UGCAUCAAGAGCACGGUCAAGAUUA
  1030	4195	UGGACCAUAGCCAUUGUGA	4196	CUGAUGUGGACCAUAGCCAUUGUGA
  531	4197	CCAGGUGAACAUUACAGAA	4198	AGCAGACCAGGUGAACAUUACAGAA
  1259	4199	UCAUCAUCCACACGUCUGA	4200	AGAGCAUCAUCAUCCACACGUCUGA
  1258	4201	AUCAUCAUCCACACGUCUG	4202	AAGAGCAUCAUCAUCCACACGUCUA

  Chemical Modification Key

  Chl	cholesterol with hydroxyprolinal linker
  TEG-	cholesterol with TEG linker
  Chl
  m	2′OMe
  f	2′fluoro
  *	phosphorothioate linkage
  .	phosphodiester linkage

• TABLE 24
  Summary of CTGF Leads

  				Opt.		Opt.	Opt.
  Generic	TEG		Targeting	lead		lead	lead
  Name	ID
  	2′F	site	sequence	Optimizedlead sequence	2′F	2′OH	Priority

  CTGF L1

  	21045	4	2295	21212	mU.mU. G.mC. A.mC.mC.mU.mU.mU.mC.mU*mA*mA-chol	4	7(2)	1
  					P.mU.fU. A. G. A. mA. A. G. G.fU. G.fC. mA. mA* mA*fC*
  					mA*mA* mG*G
  				21214	mU.mU. G.mC. A.mC.mC.mU.mU.mU.mC.mU*mA*mA-chol	4	12(2)	3
  					P.mU.fU. A. G. A. mA. A. G.fU. G.fC. A. A* A*fC*
  					A*mA*mG*G
  				21215	mU.mU. G.mC. A. mC.mC.mU.mU.mU.mC.mU*mA*mA-chol	4	10(2)	2
  					P.mU.fU. A. G. A. mA. A. G. G.fU. G.fC. mA. A* mA*fC*
  					A*mA*mG*G
  CTGFL2
  	20393	5	2296	21204	G.mC. A.mC.mC.mU.mU.mU.mC.mU. A*mG*mA-TEG-Chl	3	11(3)	1
  					P.mU.fC.fU.A.G.mA.A.mA.G.G.fU.G.mC*A*A*A*mC*A*U
  				21205	G.mC. A.mC.mC.mU.mU.mU.mC.mU. A*mG*mA-TEG-Chl	3	10(3)	3
  					P.mU.fC.fC.A.G.mA.A.mA.G.G.fU.G.mC*A*mA*A*mC*A*U
  				21227	G.mC. A.mC.mC.mU.mU.mU.mC.mU. A*mG*mA-TEG-Chl	5	7(3)	2
  					P.mU.fC.fU.A.G.mA.A.mA.G.G.fU.G.fC*mA*mA*mA*fC*mA*U
  CTGF L3
  	20392	13	2275	21381	G.mU. G. A. mC.mC. A. A. A. A. G*mU*mA-TEG-Chl	6	6(9)	1
  					P.mU.A.fC.fU.fU.fU.fU.G.G.fU.mC.A.mC*A*mC*mU*mC*mU*C
  				21383	mG*mU*mG.mA.mC.mC.mA.mA.mA.mA.mG*mU*mA-TEG-Chl	6	6(0)	2
  					P.mU.A.fC.fU.fU.fU.fU.G.G.fU.mC.A.mC*A*mC*mU*mC*mU*C
  CTGF L4
  	17387	5	2299	21224	mC.mC.mU.mU.mU.mC.mU. A. G.mU.mU* mG*mA-TEG-Chl	5	9(2)	1
  					P.mU.fC. A. A.fC.fU. A. G. A. mA. A. G.
  					G*fU*mG*fC*mA*mA* A

  
  Table 24: Lead 21212 corresponds to SEQ ID NOs 3445 and 3446; lead 21214 corresponds to SEQ ID NOs 3449 and 3450 (an unmodified form of SEQ ID NO:3450 corresponds to SEQ ID NO:4205:UCAACUAGAAAGGUGCAAA);lead 21215 corresponds to SEQ ID NOs 3451 and 3452 (an unmodified form of SEQ ID NO:3452 corresponds to SEQ ID NO:4204:UUAGAAAGGUGCAAACAAGG);lead 21204 corresponds to SEQ ID NOs 3429 and 3430; lead 21205 corresponds to SEQ ID NOs 3431 and 3432; lead 21227 corresponds to SEQ ID NOs 3475 and 3476; lead 21381 corresponds to SEQ ID NOs 3493 and 3494; lead 21383 corresponds to SEQ ID NOs 3497 and 3498; and lead 21224 corresponds to SEQ ID NOs 3469 and 3470.

• TABLE 25
  Summary of PTGS2 Leads

  				Optimized		Opt.	Opt.
  Generic	TEG		Targeting	lead		lead	lead
  Name	ID
  	2′F	site	sequence	Optimizedlead sequence	2′F	2′OH	Priority

  PTGS2 L1

  	20394	8	448	21228	G. A.mU.mC. A.mC. A.mU.mU.mU. G*mA*mA-TEG-Chl	8	6(5)	1
  					P.mU.fU.fC.A.mA.A.fU.G.fU.G.A.fU.fC*fU*mG*mG*mA*fU* G
  				21229	G. A.mU.mC. A.mC. A.mU.mU.mU. G*mA*mA-TEG-Chl	4	10(5)	3
  					P.mU.fU.fC.A.A.A.fU.G.fU.G.A.mU.mC*mU*G*G*A*mU*G
  				21230	G. A.mU.mC. A.mC. A.mU.mU.mU. G*mA*mA-TEG-Chl	8	7(5)	2
  					P.mU.fC.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*mG*mG*mA*fU*G
  PTGS2 L2
  	20395	8	449	21293	G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG-Chl	5	8(6)	4
  					P.mU. A.fU.fC. A. A. A.fU. G.fU. G.
  					A.mU.mC*mU*mG*mG*mA*fU* G
  				21394	G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG-Chl	6	11(6)	3
  					P.mU. A.fU.fC. A. A. A.fU. G.fU. G. A.mU.fC*mU*
  					G* G* A *fU* G
  				21233	G. A.mU.mC. A.mC. A. mU.mU.mU. G. A*mU*mA-TEG-Chl	8	11(6)	1
  					P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*G*G*A*fU*G
  				21234	G. A.mU.mC. A.mC. A.mU.mU.mU. G. A*mU*mA-TEG-Chl	7	8(6)	2
  					P.mU.A.fU.fC.A.A.A.fU.G.fU.G.A.fU.fC*fU*mG*mG*mA*fU*G

  
  Table 25: Lead 21228 corresponds to SEQ ID NOs 4309 and 4310; lead 21229 corresponds to SEQ ID NOs 4311 and 4312; lead 21230 corresponds to SEQ ID NOs 4313 and 4314; lead 21293 corresponds to SEQ ID NOs 4315 and 4316; lead 21394 corresponds to SEQ ID NOs 4317 and 4318; lead 21233 corresponds to SEQ ID NOs 4319 and 501. and lead 21234 corresponds to SEQ ID NOs 502 and 1059.

• TABLE 26
  Summary of hTGFB1 Leads

  Generic	Targeting	Optimized		Opt. lead	Opt. lead
  Name	site	lead sequence	Optimizedlead sequence	2′F	2′OH	Priority
  TGFb1 hL3	1244	21374	mC*mA*mC.mA.mC.mA.mG.mC.mA.mU.mA*mU*mA-TEG-Chl	9	6(0)	1
  			P.mU.A.fU.A.fU.G.fC.fU.G.fU.G.fU.mG*fU*mA*fC*mU*fC*U

  
  Table 26:lead 21374 corresponds to SEQ ID NOs 3793 and 3794.

• TABLE 27
  Summary of hTGFB2 Leads

  Generic	Targeting	Optimized		Opt. lead	Opt. lead
  Name	site	lead sequence	Optimizedlead sequence	2′F	2′OH	Priority
  TGFb2 hL3	2081	21379	mU*mC*mA.mU.mC.mA.mG.mU.mG.mU.mU*mA-TEG-Chl	7	9(0)	1
  			P.mU.fU. A. A.fC. A.fC.fU. G. A.fU. G. A*
  			A*fC*fC*mA*mA* G
  			mU*mC*mA.mU.mC.mA.mG.mU.mG.mU.mU*mA*mA-TEG-Chl	7	7(0)	2
  			P.mU.fU. A. A.fC. A.fC.fU. G. A.fU.
  			G.mA*mA*fC*fC*mA*mA* G

  
  Table 27:lead 21379 corresponds to SEQ ID NOs 3637, 3638, 3639 and 3640.
• 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 PCT Publication No. WO2010/033247 (Application No. PCT/US2009/005247), filed on Sep. 22, 2009, and entitled “REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS” and PCT Publication No. WO2009/102427 (Application No. PCT/US2009/000852), filed on Feb. 11, 2009, and entitled, “MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF.”

Claims (20)

What is claimed is:

1-55. (canceled)

56. A double-stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein the sense and/or antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 4, 5, 6, 7, 16, 17, 21, 22, 26, and 27, wherein the dsRNA is an sd-rxRNA, wherein the antisense strand is 16-23 nucleotides long and the sense strand is 12-15 nucleotides long, wherein the sd-rxRNA 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 antisense 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, and wherein at least 40% of the nucleotides of the double-stranded nucleic acid molecule are modified.

57. The dsRNA ofclaim 56, wherein the sense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3793 and/or the antisense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3794.

58. The dsRNA ofclaim 56, wherein:

(i) the sense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3637 and/or the antisense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3738; or

(ii) the sense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3639 and/or the antisense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3640.

59. The dsRNA ofclaim 56, wherein the dsRNA is hydrophobically modified or wherein the dsRNA is linked to a hydrophobic conjugate.

60. A composition comprising the dsRNA ofclaim 56.

61. The composition ofclaim 60 wherein the composition is

(i) formulated for delivery to the skin;

(ii) in a neutral formulation;

(iii) formulated for topical delivery; or

(iv) formulated for intradermal injection.

62. A method comprising administering the dsRNA ofclaim 56 to the skin of a subject in need thereof.

63. A method for treating or preventing a fibrotic disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein the sense and/or antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 4, 5, 6, 7, 16, 17, 21, 22, 26, and 27, wherein the dsRNA is an sd-rxRNA, wherein the antisense strand is 16-23 nucleotides long and the sense strand is 12-15 nucleotides long, wherein the sd-rxRNA 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 antisense 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, and wherein at least 40% of the nucleotides of the double-stranded nucleic acid molecule are modified.

64. The method ofclaim 63, wherein the dsRNA is administered via intradermal injection or locally to the skin.

65. The method ofclaim 63, wherein one or more of the dsRNAs is hydrophobically modified or wherein the dsRNA is linked to a hydrophobic conjugate.

66. The method ofclaim 63, wherein the sense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3793 and/or the antisense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3794.

67. The method ofclaim 63, wherein:

(i) the sense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3637 and/or the antisense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3738; or

(ii) the sense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3639 and/or the antisense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3640.

68. The method ofclaim 63, wherein the fibrotic disorder is selected from the group consisting of pulmonary fibrosis, liver cirrhosis, scleroderma and glomerulonephritis, liver fibrosis, skin fibrosis, muscle fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy, restenosis and uterine fibrosis, and scarring resulting in the failure of a trabeculectomy.

69. A method comprising administering to a subject in need thereof a therapeutically effective amount of a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand, wherein the sense and/or antisense strand comprises at least 12 contiguous nucleotides of a sequence selected from the sequences within Tables 4, 5, 6, 7, 16, 17, 21, 22, 26, and 27, wherein the dsRNA is an sd-rxRNA, wherein the antisense strand is 16-23 nucleotides long and the sense strand is 12-15 nucleotides long, wherein the sd-rxRNA 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 antisense 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, and wherein at least 40% of the nucleotides of the double-stranded nucleic acid molecule are modified.

70. The method ofclaim 69, wherein the dsRNA is administered via intradermal injection or locally to the skin.

71. The method ofclaim 69, wherein one or more of the dsRNAs is hydrophobically modified or wherein the dsRNA is linked to a hydrophobic conjugate.

72. The method ofclaim 69, wherein the sense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3793 and/or the antisense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3794.

73. The method ofclaim 69, wherein:

(i) the sense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3637 and/or the antisense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3738; or

(ii) the sense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3639 and/or the antisense strand comprises at least 12 contiguous nucleotides of SEQ ID NO: 3640.

74. The method ofclaim 69, wherein the method is a method for treating or preventing a fibrotic disorder selected from the group consisting of pulmonary fibrosis, liver cirrhosis, scleroderma and glomerulonephritis, liver fibrosis, skin fibrosis, muscle fibrosis, radiation fibrosis, kidney fibrosis, proliferative vitreoretinopathy, restenosis and uterine fibrosis, and scarring resulting in the failure of a trabeculectomy.

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