WO2011119887A1 - 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

RNA INTERFERENCE IN DERMAL AND FIBROTIC INDICATIONS

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. US 61/317,252, entitled "RNA INTERFERENCE IN SKIN INDICATIONS," filed on March 24, 2010, and U.S. Provisional Application Serial No. US 61/317,633, entitled "RNA INTERFERENCE IN SKIN INDICATIONS," filed on March 25, 2010, the entire disclosures of which are herein incorporated by reference in their entireties.

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 ah, in 1989. Subsequently, ISIS Pharmaceuticals, Inc. (Carlsbad, CA) 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 hydrophobic ally 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 TGFBl, TGFB2, hTGFBl, hTGFB2, PTGS2, SPP1, hSPPl, 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 hydrophobic ally 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, SPPl, CTGF, PTGS2 and TGFBl. As expected, target gene expression is elevated early and returns to normal by day 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. 11 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 hTGFBl following administration of sd-rxRNA targeting hTGFBl.

FIG. 17 demonstrates gene expression of hTGFBl following administration of sd-rxRNA targeting hTGFBl.

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 hSPPl sd-rxRNAs.

FIG. 26 demonstrates identification of potent hSPPl sd-rxRNAs.

FIG. 27 demonstrates identification of potent hSPPl 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 LI sd-rxRNAs are active.

FIG. 34 demonstrates in vitro efficacy of chemically optimized CTGF LI sd- rxRNAs.

FIG. 35 demonstrates in vitro stability of chemically optimized CTGF LI 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 LIB 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, a-SM actin, collagen 1A2, and collagen 3A1 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 on days 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 on days 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 on days 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 on days 3, 6, and 9 post-wounding (5 = mature, 1 = immature). Each group represents 5 rats.

FIG. 53 demonstrates CD68 labeling in day 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 Phases 1 and 2 clinical trial design.

FIG. 57 is a schematic of Phases 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 Akinc, 2009.

FIG. 59 demonstrates that chemically optimized PTGS2 LI sd-rxRNAs are active.

FIG. 60 demonstrates that chemically optimized PTGS2 L2 sd-rxRNAs are active.

FIG. 61 demonstrates that chemically optimized hTGFBl LI sd-rxRNAs are active.

FIG. 62 demonstrates that chemically optimized hTGFBl LI sd-rxRNAs are active.

FIG. 63 demonstrates that chemically optimized hTGFB2 LI 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 September 22, 2009, and entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS," and PCT application PCT/US2009/005246, filed on September 22, 2009, and entitled "RNA INTERFERENCE IN SKIN INDICATIONS." Briefly, an sd-rxRNA, (also referred to as an sd-rxRNA^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 5 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, TGFB 1 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 February 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 chemical modification pattern in the guide strand includes 2' Omethyl modification of position 1 and C/U in positions 11-18 and 5' end chemical phosphorylation. Yet another preferred chemical modification pattern in the guide strand includes 2'Omethyl modification of position 1 and C/U in positions 11-18 and 5' end chemical

phosphorylation and 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 are positions 4 and 5 of the pyrimidines. The major advantage of these positions is (a) ease of synthesis and (b) lack of interference with base-pairing and A form helix formation, which are essential for RISC complex loading and target recognition. 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

(UCU AGA A AGGUGC A A AC AU) . 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

(UU AG A A AGGUGC A A AC A AGG). 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.fU. A. G. A.mA. A. G. G.fU. G.fC.mA.mA*mA*fC*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

(UC A ACU AG A A AGGUGC AA A) . 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 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 (AG) of less than -13 kkal/mol. In some embodiments, the thermodynamic stability (AG) is less than -20 kkal/mol. In some embodiments there is a loss of efficacy when (AG) goes below -21 kkal/mol. In some embodiments a (AG) 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 (AG) value may become active at a relatively higher concentration, while a molecule with a relatively lower (AG) value may become active at a relatively lower concentration. In some embodiments, the (AG) 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'-0- methyl, 2'-0-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'-0-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 Wolfram, 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 l9 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 (AG). In some embodiments, molecules will be selected that have a (AG) of less than -13 kkal/mol. For example, the (AG) value may be -13, -14, -15, -16, -17, -18, -19, -21, -22 or less than -22 kkal/mol. In other embodiments, the (AG) value may be higher than -13 kkal/mol. For example, the (AG) value may be -12, -11, -10, -9, -8, -7 or more than -7 kkal mol. It should be appreciated that AG can be calculated using any method known in the art. In some embodiments AG is calculated using Mfold, available through the Mfold internet site (http://mfoM,bioinfo.rpi.edLi cgi-bin/rna-forml.cgi). Methods for calculating AG are described in, and are incorporated by reference from, the following references: Zuker, M. (2003) Nucleic Acids Res., 31(13):3406-15; Mathews, D. H., Sabina, J., Zuker, M. and Turner, D. H. (1999) J. Mol. Biol. 288:911-940; Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl. Acad. Sci. 101:7287-7292; Duan, S., Mathews, D. H., and Turner, D. H. (2006) Biochemistry 45:9819-9832; Wuchty, S., Fontana, W., Hofacker, I. L., and Schuster, P. (1999) Biopolymers 49: 145-165.

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'-0-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'-0-alkyl nucleotides include 2'-0-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'-0-methyl modified nucleotide, at the 2^nd nucleotide on the 5 '-end of the polynucleotide and, in some embodiments, no other modified nucleotides. The hairpin structure having such modification may have enhanced target specificity or reduced off-target silencing compared to a similar construct without the 2' -O-methyl modification at said position.

Certain combinations of specific 5'-stem sequence and 3'-stem sequence modifications may result in further unexpected advantages, as partly manifested by enhanced ability to inhibit target gene expression, enhanced serum stability, and/or increased target specificity, etc.

In certain embodiments, the guide strand comprises a 2' -O-methyl modified nucleotide at the 2^nd nucleotide 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 Ill-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'-0- 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'-0-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 between nucleotide 9, 11, 12, 13, or 14 on the guide strand (first polynucleotide) and the opposite nucleotide on the sense strand (second polynucleotide) to promote efficient guide strand loading.

More detailed aspects of the invention are described in the sections below.

Duplex Characteristics

Double-stranded oligonucleotides of the invention may be formed by two separate complementary nucleic acid strands. Duplex formation can occur either inside or outside the cell containing the target gene.

As used herein, the term "duplex" includes the region of the double-stranded nucleic acid molecule(s) that is (are) hydrogen bonded to a complementary sequence. Double-stranded oligonucleotides of the invention may comprise a nucleotide sequence that is sense to a target gene and a complementary sequence that is antisense to the target gene. The sense and antisense nucleotide sequences correspond to the target gene sequence, e.g., are identical or are sufficiently identical to effect target gene inhibition (e.g. , are about at least about 98% identical, 96% identical, 94%, 90% identical, 85% identical, or 80% identical) to the target gene sequence.

In certain embodiments, the double-stranded oligonucleotide of the invention is double-stranded over its entire length, i.e. , with no overhanging single-stranded sequence at either end of the molecule, i.e. , is blunt-ended. In other embodiments, the individual nucleic acid molecules can be of different lengths. In other words, a double-stranded oligonucleotide of the invention is not double-stranded over its entire length. For instance, when two separate nucleic acid molecules are used, one of the molecules, e.g. , the first molecule comprising an antisense sequence, can be longer than the second molecule hybridizing thereto (leaving a portion of the molecule single-stranded).

Likewise, when a single nucleic acid molecule is used a portion of the molecule at either end can remain single- stranded.

In one embodiment, a double-stranded oligonucleotide of the invention contains mismatches and/or loops or bulges, but is double- stranded over at least about 70% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double- stranded over at least about 80% of the length of the oligonucleotide. In another embodiment, a double- stranded oligonucleotide of the invention is double- stranded over at least about 90%-95% of the length of the oligonucleotide. In another embodiment, a double- stranded oligonucleotide of the invention is double- stranded over at least about 96%-98% of the length of the oligonucleotide. In certain embodiments, the double-stranded oligonucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

Modifications

The nucleotides of the invention may be modified at various locations, including the sugar moiety, the phosphodiester linkage, and/or the base.

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 N⁴ position 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 O² and O⁴ atoms may be substituted with sulfur atoms. It should be understood that other modifications are possible as well.

In other examples, the N⁷ position and/or N² and/or N³ position 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. Patent 5591843, U.S. Patent 7,205,297, U.S. Patent 6,432,963, and U.S. Patent 6,020,483; non-limiting examples of pyrimidines modified at the N⁴ position are disclosed in U.S Patent 5,580,731 ; non- limiting examples of purines modified at the 8 position are disclosed in U.S. Patent 6,355,787 and U.S. Patent 5,580,972; non-limiting examples of purines modified at the N⁶ position are disclosed in U.S. Patent 4,853,386,

U.S. Patent 5,789,416, and U.S. Patent 7,041,824; and non-limiting examples of purines modified at the 2 position are disclosed in U.S. Patent 4,201,860 and U.S. Patent

5,587,469, all of which are incorporated herein by reference.

Non-limiting examples of modified bases include

7- deazaxanthosine, 7-deazaguanosine, S-oxo-ZV^-methyladenine, 4-acetylcytosine, 5-

(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, A^-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'-0-methyl nucleotides. Such 2'-0-methyl nucleotides may be referred to as "methylated," and the corresponding nucleotides may be made from unmethylated nucleotides followed by alkylation or directly from methylated nucleotide reagents. Modified nucleomonomers may be used in combination with unmodified nucleomonomers. For example, an oligonucleotide of the invention may contain both methylated and unmethylated nucleomonomers.

Some exemplary modified nucleomonomers include sugar- or backbone-modified ribonucleotides. Modified ribonucleotides may contain a non- naturally occurring base (instead of a naturally occurring base), such as uridines or cytidines modified at the 5'- position, e.g. , 5'-(2-amino)propyl uridine and 5'-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. , 8-bromo guanosine; deaza nucleotides, e.g. , 7-deaza-adenosine; and N-alkylated nucleotides, e.g. , N6-methyl adenosine. Also, sugar- modified ribonucleotides may have the 2' -OH group replaced by a H, alxoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as NH₂, NHR, 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'-0-methyl modified RNA may also be beneficial in circumstances in which it is desirable to minimize cellular stress responses. RNA having 2'-0-methyl nucleomonomers may not be recognized by cellular machinery that is thought to recognize unmodified RNA. The use of 2'-0-methylated or partially 2'-0-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'-0-alkyl (including 2'-0- methyl and 2'-0-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^th Ed. , 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 in Organic 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 fraws-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-CH₂- 0-) phosphate (PO₃ ² ), 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'-0 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), /?-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), i-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, l-[(2-chloro-4-methyl)phenyl]-4- methoxypiperidin-4-yl (CTMP), l,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, l-(2-chloroethoxy)ethyl, 1-methyl-l- methoxyethyl, 1 -methyl- 1-benzyloxyethyl, 1 -methyl- l-benzyloxy-2-fluoroethyl, 2,2,2- trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, i-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, /?,/ -dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a- 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-l-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, l,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, i-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-/?-xylylsilyl, triphenylsilyl,

diphenylmethylsilyl (DPMS), i-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxy acetate, phenoxy acetate, 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 5-benzyl thiocarbonate, 4-ethoxy-l-napththyl 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(l , l-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate,

monosuccinoate, (£^')-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, a-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, l-t- butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, /?-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, a-methoxybenzylidene ortho ester, 1-(N,N- dimethylamino)ethylidene derivative, a-(N,N'-dimethylamino)benzylidene derivative, 2- oxacyclopentylidene ortho ester, di-i-butylsilylene group (DTBS), 1 , 3-(l , 1,3,3- tetraisopropyldisiloxanylidene) derivative (TIPDS), tetra-i-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-i-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), l-(l-adamantyl)- l-methylethyl carbamate (Adpoc), 1, 1- dimethyl-2-haloethyl carbamate, l , l-dimethyl-2,2-dibromoethyl carbamate (DB-i-BOC), l, l-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1 -methyl- l-(4-biphenylyl)ethyl carbamate (Bpoc), l-(3,5-di-i-butylphenyl)- l-methylethyl carbamate (i-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, i-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), /?-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-(l ,3- dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4- dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl carbamate (Ppoc), l, l-dimethyl-2-cyanoethyl carbamate, m-chloro-/?-acyloxybenzyl carbamate, /?-(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'-/?-toluenesulfonylaminocarbonyl derivative, N'-phenylaminothiocarbonyl derivative, i-amyl carbamate, 5-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 , l-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1 , 1 -dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'- methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1- methylcyclohexyl carbamate, 1 -methyl- 1 -cyclopropylmethyl carbamate, 1-methyl-l- (3,5-dimethoxyphenyl)ethyl carbamate, 1 -methyl- l-(p-phenylazophenyl)ethyl carbamate, 1 -methyl- 1-phenylethyl carbamate, 1 -methyl- l-(4-pyridyl)ethyl carbamate, phenyl carbamate, /?-(phenylazo)benzyl carbamate, 2,4,6-tri-i-butylphenyl carbamate, 4- (trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifhioroacetamide, 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-(o- 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-l,l,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),

5- substituted l,3-dimethyl-l,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl- l,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N- allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(l-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- phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N- ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1- dimethylthiomethyleneamine, N-benzylideneamine, N-/?-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-l-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), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate,

benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,

triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys), /?-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), β-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 in Protective 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, H-propyl, isopropyl, cyclopropyl, -CH₂- cyclopropyl, vinyl, allyl, H-butyl, sec-butyl, isobutyl, ferf-butyl, cyclobutyl, -CH₂- cyclobutyl, H-pentyl, sec-pentyl, isopentyl, ferf-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, l-methyl-2-buten-l-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; -CI; -Br; -I; -OH; -N0₂; -CN; -CF₃; -CH₂CF₃; -CHC1₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂; - CH2SO2CH3; -C(0)R_x; -C0₂(Rx); -CON(R_x)₂; -OC(0)R_x; -OC0₂R_x; -OCON(R_x)₂; - (R_X)₂; -S(0)₂R_X; -NR_x(CO)R_x wherein each occurrence of R_x independently 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; -CI; -Br; -I; -OH; -N0₂; -CN; -CF₃; - CH₂CF₃; -CHC1₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂S0₂CH₃; -C(0)R_x; -C0₂(R_x); -CON(R_x)₂; -OC(0)R_x; -OC0₂R_x; -OCON(R_x)₂; -N(R_X)₂; -S(0)₂R_x; -NR_x(CO)R_x, wherein each occurrence of R_x independently 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. , Ci-C₆ for straight chain, C3-C6 for branched chain), and more preferably 4 or fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C1-C5 includes alkyl groups containing 1 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkyl includes both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,

aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyls can be further substituted, e.g. , with the substituents described above. An "alkylaryl" or an "arylalkyl" moiety is an alkyl substituted with an aryl (e.g. , phenylmethyl (benzyl)). The term "alkyl" also includes the side chains of natural and unnatural amino acids. The term "n-alkyl" means a straight chain (i.e. , unbranched) unsubstituted alkyl group.

The term "alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term "alkenyl" includes straight-chain alkenyl groups (e.g. , ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. In certain embodiments, a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g. , C₂-C₆ for straight chain, C3-C₆ for branched chain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C₂-C6 includes alkenyl groups containing 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkenyl includes both

"unsubstituted alkenyls" and "substituted alkenyls," the latter of which refers to alkenyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. The term "alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, the term "alkynyl" includes straight-chain alkynyl groups (e.g. , ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g. , C₂-C₆ for straight chain, C3-C6 for branched chain). The term C₂-C₆ includes alkynyl groups containing 2 to 6 carbon atoms.

Moreover, unless otherwise specified, the term alkynyl includes both

"unsubstituted alkynyls" and "substituted alkynyls," the latter of which refers to alkynyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to five carbon atoms in its backbone structure. "Lower alkenyl" and "lower alkynyl" have chain lengths of, for example, 2-5 carbon atoms.

The term "alkoxy" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. The alkoxy groups can be substituted with independently selected groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,

alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulffiydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfmyl, 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 (C6H50H); 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 -0^~ (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")o-₃NR'R",

(CR'R")o-₃CN, N0₂, halogen, (CR'R")o-₃C(halogen)₃, (CR'R")o-₃CH(halogen)₂, (CR'R")o- ₃CH₂(halogen), (CR'R")o_₃CONR'R", (CR'R")₀-₃S(O)₁_₂NR'R", (CR'R")o-₃CHO,

(CR'R")o-₃0(CR'R")o-₃H, (CR'R")o-₃S(0)₀-₂R', (CR'R")o-₃0(CR'R")o-₃H, (CR'R")o-₃COR', (CR'R")o-₃C0₂R', or (CR'R")o-₃0R' groups; wherein each R' and R" are each

independently hydrogen, a C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, or aryl group, or R' and R" taken together are a benzylidene group or a— (CH₂)₂0(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 compirse a modified sugar moiety (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2'-0- 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-(l-propynyl)uracil, 5-(l-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^nd Ed., 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. Patent 5,414,077, U.S. Patent 5,419,966, U.S. Patent 5,512,667, U.S. Patent 5,646, 126, and U.S. Patent 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;

R¹ is a hydrophobic moiety;

R² is 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

R³ is 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:

R³

/

,-0

X<^/v^\,A> v^\, OR¹

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 Ci_-2o alkyl. In certain embodiments, A is acyclic, substituted, unbranched Ci_i₂ alkyl. In certain embodiments, A is acyclic, substituted, unbranched Ci_io alkyl. In certain embodiments, A is acyclic, substituted, unbranched Ci_-8 alkyl. In certain embodiments, A is acyclic, substituted, unbranched Ci_ 6 alkyl. 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:

53 In c

55 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 R³ are 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 c

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, R is a steroid. In certain embodiments, R is a cholesterol. In certain embodiments, R¹ is a lipophilic vitamin. In certain embodiments, R¹ is a vitamin A. In certain embodiments, R¹ is a vitamin E.

In certain embodiments, R¹ is of the formula:

wherein R^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

In certain embodiments, R is 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;

R¹ is a hydrophobic moiety;

R² is 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 substituted or unsubstituted, branched or unbranched heteroaryl; and

R³ is 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;

R¹ is a hydrophobic moiety;

R² is 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

R³ is 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;

R¹ is a hydrophobic moiety;

R² is 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 R is 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 R is a nucleic acid.

In certain embodiments, the nucleic acid molecule is of the formula:

wherein R is 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 (-0-(P0^2~)-0-) 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

hydrophobicly modified nucleotides or "hydrophobic modifications." As used herein "hydrophobic modifications" refers to bases that are modified such that (1) overall hydrophobicity of the base is significantly increased, and/or (2) the base is still capable of forming close to regular Watson -Crick interaction. Several non-limiting examples of base modifications include 5 -position uridine and cytidine modifications such as phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C6H50H); 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'-0 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. Figure 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. Figure 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. /. Am. Chem. Soc. 106:6077; Stec et al. 1985. /. Org. Chem. 50:3908; Stec et al. J. Chromatog. 1985. 326:263; LaPlanche et al. 1986. Nucl. Acid. Res. 1986. 14:9081; Fasman G. D., 1989. Practical Handbook of Biochemistry and Molecular Biology. 1989. CRC Press, Boca Raton, Fla.; Lamone. 1993. Biochem. Soc. Trans. 21 : 1; U.S. Pat. No. 5,013,830; U.S. Pat. No. 5,214,135; U.S. Pat. No. 5,525,719; Kawasaki et al. 1993. /. Med. Chem.

36:831 ; WO 92/03568; U.S. Pat. No. 5,276,019; and U.S. Pat. No. 5,264,423.

The synthesis method selected can depend on the length of the desired oligonucleotide and such choice is within the skill of the ordinary artisan. For example, the phosphoramidite and phosphite triester method can produce oligonucleotides having 175 or more nucleotides, while the H-phosphonate method works well for

oligonucleotides of less than 100 nucleotides. If modified bases are incorporated into the oligonucleotide, and particularly if modified phosphodiester linkages are used, then the synthetic procedures are altered as needed according to known procedures. In this regard, Uhlmann et al. (1990, Chemical Reviews 90:543-584) provide references and outline procedures for making oligonucleotides with modified bases and modified phosphodiester linkages. Other exemplary methods for making oligonucleotides are taught in Sonveaux. 1994. "Protecting Groups in Oligonucleotide Synthesis"; Agrawal. Methods in Molecular Biology 26: 1. Exemplary synthesis methods are also taught in "Oligonucleotide Synthesis - A Practical Approach" (Gait, M. J. IRL Press at Oxford University Press. 1984). Moreover, linear oligonucleotides of defined sequence, including some sequences with modified nucleotides, are readily available from several commercial sources.

The oligonucleotides may be purified by polyacrylamide gel electrophoresis, or by any of a number of chromatographic methods, including gel chromatography and high pressure liquid chromatography. To confirm a nucleotide sequence, especially unmodified nucleotide sequences, oligonucleotides may be subjected to DNA sequencing by any of the known procedures, including Maxam and Gilbert sequencing, Sanger sequencing, capillary electrophoresis sequencing, the wandering spot sequencing procedure or by using selective chemical degradation of oligonucleotides bound to Hybond paper. Sequences of short oligonucleotides can also be analyzed by laser desorption mass spectroscopy or by fast atom bombardment (McNeal, et al., 1982, /. 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. /. Chrom. 599:35.

Other exemplary synthesis techniques are well known in the art (see, e.g. , Sambrook et al., Molecular Cloning: a Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (DN Glover Ed. 1985); Oligonucleotide Synthesis (M J Gait Ed, 1984; Nucleic Acid Hybridisation (B D Hames and S J Higgins eds. 1984); A Practical Guide to Molecular Cloning (1984); or the series, Methods in Enzymology (Academic Press, Inc.)).

In certain embodiments, the subject RNAi constructs or at least portions thereof are transcribed from expression vectors encoding the subject constructs. Any art recognized vectors may be use for this purpose. The transcribed RNAi constructs may be isolated and purified, before desired modifications (such as replacing an unmodified sense strand with a modified one, etc.) are carried out. Delivery/Carrier

Uptake of Oligonucleotides by Cells

Oligonucleotides and oligonucleotide compositions are contacted with (i.e. , brought into contact with, also referred to herein as administered or delivered to) and taken up by one or more cells or a cell lysate. The term "cells" includes prokaryotic and eukaryotic cells, preferably vertebrate cells, and, more preferably, mammalian cells. In a preferred embodiment, the oligonucleotide compositions of the invention are contacted with human cells.

Oligonucleotide compositions of the invention can be contacted with cells in vitro, e.g. , in a test tube or culture dish, (and may or may not be introduced into a subject) or in vivo, e.g. , in a subject such as a mammalian subject. Oligonucleotides are taken up by cells at a slow rate by endocytosis, but endocytosed oligonucleotides are generally sequestered and not available, e.g. , for hybridization to a target nucleic acid molecule. In one embodiment, cellular uptake can be facilitated by electroporation or calcium phosphate precipitation. However, these procedures are only useful for in vitro or ex vivo embodiments, are not convenient and, in some cases, are associated with cell toxicity. In another embodiment, delivery of oligonucleotides into cells can be enhanced by suitable art recognized methods including calcium phosphate, DMSO, glycerol or dextran, electroporation, or by transfection, e.g. , using cationic, anionic, or neutral lipid compositions or liposomes using methods known in the art (see e.g. , WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No. 4,897,355; Bergan et al. 1993. Nucleic Acids Research. 21 :3567). Enhanced delivery of oligonucleotides can also be mediated by the use of vectors (See e.g. , Shi, Y. 2003. Trends Genet 2003 Jan. 19:9; Reichhart J M et al. Genesis. 2002. 34(1-2): 1604, Yu et al. 2002. Proc. Natl. Acad Sci. USA 99:6047; Sui et al. 2002. Proc. Natl. Acad Sci. USA 99:5515) viruses, polyamine or polycation conjugates using compounds such as polylysine, protamine, or Ni, N12-bis (ethyl) spermine (see, e.g. , Bartzatt, R. et a/.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, US Provisional Application No. 61/310,611, filed on March 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 Al, and US 2010/0040656, and in PCT publications WO 2006/007372, and WO 2007/050643. The sd-rxRNA molecule may be hydrophobic ally 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 6-(l,3)-Glucan, 6-(l,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 H20, followed by washing and drying. Methods of preparing yeast cell wall particles are discussed in, and incorporated by reference from U.S. Patents 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 Chem 19(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 Immunology 124(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 Chem 19:840. 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 LIPOFECT AMINE™ 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 nano transporters. Further description of neutral nanotransporters is incorporated by reference from PCT Application

PCT/US2009/005251, filed on September 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, olely-lithocholic, cholenic, oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, such as steroids, vitamins, such as vitamin E, fatty acids either saturated or unsaturated, fatty acid esters, such as triglycerides, pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl, t- butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. The cholesterol moiety may be reduced (e.g. as in cholestan) or may be substituted (e.g. by halogen). A combination of different lipophilic groups in one molecule is also possible.

The hydrophobic molecule may be attached at various positions of the polynucleotide. As described above, the hydrophobic molecule may be linked to the terminal residue of the polynucleotide such as the 3' of 5 '-end of the polynucleotide. Alternatively, it may be linked to an internal nucleotide or a nucleotide on a branch of the polynucleotide. The hydrophobic molecule may be attached, for instance to a 2'- position of the nucleotide. The hydrophobic molecule may also be linked to the heterocyclic base, the sugar or the backbone of a nucleotide of the polynucleotide.

The hydrophobic molecule may be connected to the polynucleotide by a linker moiety. Optionally the linker moiety is a non-nucleotidic linker moiety. Non-nucleotidic linkers are e.g. abasic residues (dSpacer), oligoethyleneglycol, such as triethyleneglycol (spacer 9) or hexaethylenegylcol (spacer 18), or alkane-diol, such as butanediol. The spacer units are preferably linked by phosphodiester or phosphorothioate bonds. The linker units may appear just once in the molecule or may be incorporated several times, e.g. via phosphodiester, phosphorothioate, methylphosphonate, or amide linkages.

Typical conjugation protocols involve the synthesis of polynucleotides bearing an aminolinker at one or more positions of the sequence, however, a linker is not required. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the polynucleotide still bound to a solid support or following cleavage of the

polynucleotide in solution phase. Purification of the modified polynucleotide by HPLC typically results in a pure material.

In some embodiments the hydrophobic molecule is a sterol type conjugate, a PhytoSterol conjugate, cholesterol conjugate, sterol type conjugate with altered side chain length, fatty acid conjugate, any other hydrophobic group conjugate, and/or hydrophobic modifications of the internal nucleoside, which provide sufficient hydrophobicity to be incorporated into micelles.

For purposes of the present invention, the term "sterols", refers or steroid alcohols are a subgroup of steroids with a hydroxyl group at the 3 -position of the A-ring. They are amphipathic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway. The overall molecule is quite flat. The hydroxyl group on the A ring is polar. The rest of the aliphatic chain is non-polar. Usually sterols are considered to have an 8 carbon chain at position 17.

For purposes of the present invention, the term "sterol type molecules", refers to steroid alcohols, which are similar in structure to sterols. The main difference is the structure of the ring and number of carbons in a position 21 attached side chain. For purposes of the present invention, the term "PhytoSterols" (also called plant sterols) are a group of steroid alcohols, phytochemicals naturally occurring in plants. There are more then 200 different known PhytoSterols

For purposes of the present invention, the term "Sterol side chain" refers to a chemical composition of a side chain attached at the position 17 of sterol-type molecule. In a standard definition sterols are limited to a 4 ring structure carrying a 8 carbon chain at position 17. In this invention, the sterol type molecules with side chain longer and shorter than conventional are described. The side chain may branched or contain double back bones.

Thus, sterols useful in the invention, for example, include cholesterols, as well as unique sterols in which position 17 has attached side chain of 2-7 or longer then 9 carbons. In a particular embodiment, the length of the polycarbon tail is varied between 5 and 9 carbons. Such conjugates may have significantly better in vivo efficacy, in particular delivery to liver. These types of molecules are expected to work at concentrations 5 to 9 fold lower then oligonucleotides conjugated to conventional cholesterols.

Alternatively the polynucleotide may be bound to a protein, peptide or positively charged chemical that functions as the hydrophobic molecule. The proteins may be selected from the group consisting of protamine, dsRNA binding domain, and arginine rich peptides. Exemplary positively charged chemicals include spermine, spermidine, cadaverine, and putrescine.

In another embodiment hydrophobic molecule conjugates may demonstrate even higher efficacy when it is combined with optimal chemical modification patterns of the polynucleotide (as described herein in detail), containing but not limited to hydrophobic modifications, phosphorothioate modifications, and 2' ribo modifications.

In another embodiment the sterol type molecule may be a naturally occurring PhytoSterols. 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 hydrophobicly modified polynucleotides. This methodology provides for the specific delivery of the polynucleotides to particular tissues (Figure 12).

In another embodiment the fat emulsions of the cargo molecule contain more then 70% of Linoleic acid (C18H3202) 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, LIPOFECTAMINE™ (e.g. , LIPOFECT AMINE™ 2000), DOPE,

Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[l-(2,3-dioleoloxy)-propyl]- Ν,Ν,Ν-trimethylammonium chloride (DOTMA), N-[l -(2,3-dioleoloxy)-propyl]-N,N,N- trimethylammonium methylsulfate (DOTAP), 3β-[Ν-(Ν',Ν'- dimethylaminoethane)carbamoyl]cholesterol (DC-Choi), 2,3,-dioleyloxy-N- [2(sperminecarboxamido)ethyl] -Ν,Ν-dimethyl- 1 -propanaminium trifluoroacetate (DOSPA), l,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). The cationic lipid N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), for example, was found to increase 1000-fold the antisense effect of a phosphorothioate oligonucleotide. (Vlassov et al., 1994, Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides can also be complexed with, e.g. , poly (L- lysine) or avidin and lipids may, or may not, be included in this mixture, e.g., steryl-poly (L- lysine).

Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g. , U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998. Molecular Membrane Biology 15: 1). Other lipid compositions which can be used to facilitate uptake of the instant oligonucleotides can be used in connection with the claimed methods. In addition to those listed supra, other lipid compositions are also known in the art and include, e.g. , those taught in U.S. Pat. No. 4,235,871; U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.

In one embodiment lipid compositions can further comprise agents, e.g. , viral proteins to enhance lipid-mediated transfections of oligonucleotides (Kamata, et al.,

1994. Nucl. Acids. Res. 22:536). In another embodiment, oligonucleotides are contacted with cells as part of a composition comprising an oligonucleotide, a peptide, and a lipid as taught, e.g. , in U.S. patent 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. /. 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. US.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. Cell 88:223).

Oligonucleotides can be attached to the transporting peptide using known techniques, e.g. , ( Prochiantz, A. 1996. Curr. Opin. Neurobiol. 6:629; Derossi et al.

1998. Trends Cell Biol. 8:84; Troy et al. 1996. /. Neurosci. 16:253), Vives et al. 1997. /. 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 β 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. /. Neurosci. 16:253).

In one embodiment, a linking group can be attached to a nucleomonomer and the transporting peptide can be covalently attached to the linker. In one embodiment, a linker can function as both an attachment site for a transporting peptide and can provide stability against nucleases. Examples of suitable linkers include substituted or unsubstituted C1-C2₀ alkyl chains, C2-C2₀ alkenyl chains, C2-C2₀ alkynyl chains, peptides, and heteroatoms (e.g. , S, O, NH, etc.). Other exemplary linkers include bifunctional crosslinking agents such as sulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g. , Smith et al. Biochem J 1991.276: 417-2).

In one embodiment, oligonucleotides of the invention are synthesized as molecular conjugates which utilize receptor-mediated endocytotic mechanisms for delivering genes into cells (see, e.g. , Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559, and the references cited therein). Targeting Agents

The delivery of oligonucleotides can also be improved by targeting the oligonucleotides to a cellular receptor. The targeting moieties can be conjugated to the oligonucleotides or attached to a carrier group (i.e. , poly(L-lysine) or liposomes) linked to the oligonucleotides. This method is well suited to cells that display specific receptor- mediated endocytosis.

For instance, oligonucleotide conjugates to 6-phosphomannosylated proteins are internalized 20-fold more efficiently by cells expressing mannose 6-phosphate specific receptors than free oligonucleotides. The oligonucleotides may also be coupled to a ligand for a cellular receptor using a biodegradable linker. In another example, the delivery construct is mannosylated streptavidin which forms a tight complex with biotinylated oligonucleotides. Mannosylated streptavidin was found to increase 20-fold the internalization of biotinylated oligonucleotides. (Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

In addition specific ligands can be conjugated to the polylysine component of polylysine-based delivery systems. For example, transferrin-polylysine, adenovirus- polylysine, and influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides- polylysine conjugates greatly enhance receptor-mediated DNA delivery in eucaryotic cells. Mannosylated glycoprotein conjugated to poly(L-lysine) in aveolar macrophages has been employed to enhance the cellular uptake of oligonucleotides. Liang et al. 1999. Pharmazie 54:559-566.

Because malignant cells have an increased need for essential nutrients such as folic acid and transferrin, these nutrients can be used to target oligonucleotides to cancerous cells. For example, when folic acid is linked to poly(L-lysine) enhanced oligonucleotide uptake is seen in promyelocytic leukaemia (HL-60) cells and human melanoma (M-14) cells. Ginobbi et al. 1997. Anticancer Res. 17:29. In another example, liposomes coated with maleylated bovine serum albumin, folic acid, or ferric

protoporphyrin IX, show enhanced cellular uptake of oligonucleotides in murine macrophages, KB cells, and 2.2.15 human hepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.

Liposomes naturally accumulate in the liver, spleen, and reticuloendothelial system (so-called, passive targeting). By coupling liposomes to various ligands such as antibodies are protein A, they can be actively targeted to specific cell populations. For example, protein A-bearing liposomes may be pretreated with H-2K specific antibodies which are targeted to the mouse major histocompatibility complex-encoded H-2K protein expressed on L cells. (Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).

Other in vitro and/or in vivo delivery of RNAi reagents are known in the art, and can be used to deliver the subject RNAi constructs. See, for example, U.S. patent application publications 20080152661, 20080112916, 20080107694, 20080038296, 20070231392, 20060240093, 20060178327, 20060008910, 20050265957, 20050064595, 20050042227, 20050037496, 20050026286, 20040162235, 20040072785, 20040063654, 20030157030, WO 2008/036825, WO04/065601, and AU2004206255B2, just to name a few (all incorporated by reference).

Administration

The optimal course of administration or delivery of the oligonucleotides may vary depending upon the desired result and/or on the subject to be treated. As used herein "administration" refers to contacting cells with oligonucleotides and can be performed in vitro or in vivo. The dosage of oligonucleotides may be adjusted to optimally reduce expression of a protein translated from a target nucleic acid molecule, e.g. , as measured by a readout of RNA stability or by a therapeutic response, without undue experimentation.

For example, expression of the protein encoded by the nucleic acid target can be measured to determine whether or not the dosage regimen needs to be adjusted accordingly. In addition, an increase or decrease in RNA or protein levels in a cell or produced by a cell can be measured using any art recognized technique. By determining whether transcription has been 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 μιη 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 sequioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.

Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, poly glycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C₈-Ci₂) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized Cs-Cio glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both oil/water and water/oil) have been proposed to enhance the oral bioavailability of drugs.

Microemulsions offer improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant- induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11: 1385; Ho et al., J. Pharm. Sci., 1996, 85: 138-143). Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides from the gastrointestinal tract, as well as improve the local cellular uptake of

oligonucleotides within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

In an embodiment, the present invention employs various penetration enhancers to affect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to increasing the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also act to enhance the permeability of lipophilic drugs.

Five categories of penetration enhancers that may be used in the present invention include: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants. Other agents may be utilized to enhance the penetration of the administered oligonucleotides include: glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-15 pyrrol, azones, and terpenes such as limonene, and menthone.

The oligonucleotides, especially in lipid formulations, can also be administered by coating a medical device, for example, a catheter, such as an angioplasty balloon catheter, with a cationic lipid formulation. Coating may be achieved, for example, by dipping the medical device into a lipid formulation or a mixture of a lipid formulation and a suitable solvent, for example, an aqueous-based buffer, an aqueous solvent, ethanol, methylene chloride, chloroform and the like. An amount of the formulation will naturally adhere to the surface of the device which is subsequently administered to a patient, as appropriate. Alternatively, a lyophilized mixture of a lipid formulation may be specifically bound to the surface of the device. Such binding techniques are described, for example, in K. Ishihara et al., Journal of Biomedical Materials Research, Vol. 27, pp. 1309-1314 (1993), the disclosures of which are incorporated herein by reference in their entirety.

The useful dosage to be administered and the particular mode of administration will vary depending upon such factors as the cell type, or for in vivo use, the age, weight and the particular animal and region thereof to be treated, the particular oligonucleotide and delivery method used, the therapeutic or diagnostic use contemplated, and the form of the formulation, for example, suspension, emulsion, micelle or liposome, as will be readily apparent to those skilled in the art. Typically, dosage is administered at lower levels and increased until the desired effect is achieved. When lipids are used to deliver the oligonucleotides, the amount of lipid compound that is administered can vary and generally depends upon the amount of oligonucleotide agent being administered. For example, the weight ratio of lipid compound to oligonucleotide agent is preferably from about 1 : 1 to about 15: 1, with a weight ratio of about 5: 1 to about 10: 1 being more preferred. Generally, the amount of cationic lipid compound which is administered will vary from between about 0.1 milligram (mg) to about 1 gram (g). By way of general guidance, typically between about 0.1 mg and about 10 mg of the particular

oligonucleotide agent, and about 1 mg to about 100 mg of the lipid compositions, each per kilogram of patient body weight, is administered, although higher and lower amounts can be used.

The agents of the invention are administered to subjects or contacted with cells in a biologically compatible form suitable for pharmaceutical administration. By

"biologically compatible form suitable for administration" is meant that the

oligonucleotide is administered in a form in which any toxic effects are outweighed by the therapeutic effects of the oligonucleotide. In one embodiment, oligonucleotides can be administered to subjects. Examples of subjects include mammals, e.g. , humans and other primates; cows, pigs, horses, and farming (agricultural) animals; dogs, cats, and other domesticated pets; mice, rats, and transgenic non-human animals.

Administration of an active amount of an oligonucleotide of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example, an active amount of an oligonucleotide may vary according to factors such as the type of cell, the oligonucleotide used, and for in vivo uses the disease state, age, sex, and weight of the individual, and the ability of the oligonucleotide to elicit a desired response in the individual. Establishment of therapeutic levels of oligonucleotides within the cell is dependent upon the rates of uptake and efflux or degradation. Decreasing the degree of degradation prolongs the 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 trhough 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, 9 hours 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.

Figure 1 reveals the expression profile for several genes associated with the invention. As expected, target gene expression is elevated early and returns to normal by day 10. Figure 2 provides a summary of experimental design. Figures 3-6 show in vivo silencing of MAP4K4 and PPIB expression following intradermal injection of sd-rxRNA molecules targeting these genes. Figures 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 eb adminsitered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 days prior to an event that compromises or damages the skin. Figure 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 SI 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 News 16: 1. Exemplary targets for cleavage by oligonucleotides include, e.g. , protein kinase Ca, ICAM-1, c-raf kinase, p53, c-myb, and the bcr/abl fusion gene found in chronic myelogenous leukemia.

The subject nucleic acids can be used in RNAi-based therapy in any animal having RNAi pathway, such as human, non-human primate, non-human mammal, non- human vertebrates, rodents (mice, rats, hamsters, rabbits, etc.), domestic livestock animals, pets (cats, dogs, etc.), Xenopus, fish, insects {Drosophila, etc.), and worms (C. elegans), etc.

The invention provides methods for 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 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- βΐ, 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-βΙ, 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 (TGF l, TGFp2, TGFp3), Osteopontin (SPP1),

Connective tissue growth factor (CTGF), Platelet-derived growth factor (PDGF), Hypoxia inducible factor- la (HIFla), Collagen I and/or III, Prolyl 4-hydroxylase (P4H), Procollagen C-protease (PCP), Matrix metalloproteinase 2, 9 (MMP2, 9), Integrins, Connexin, Histamine HI 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 (TGF i, TGFP2, TGF 3), 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, immunosuppression 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 TGF i, TGFP2 and TGFP3 are BT007245, BC096235, and X14149, respectively. Within the TGF family, TGF l and TGF 2 but not TGF 3 represent suitable targets. The alteration in the ratio of TGF 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 hydroxy apatite. 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 (PDGF A), X02811 (PDGFB), AF091434 (PDGFC), AB033832 (PDGFD).

Hypoxia inducible factor- la (HIFla), is a transcription factor involved in cellular response to hypoxia. HIFla 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 HIF1 a 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 αιβι, α₂βι, α₄βι, α₅βι, α₆βι, α ₂, 0-Μ 2, ¾¾β3, α_νβ₃, α_νβ₅, α_νβ₆, 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 (GJE1), 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 HI 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 (FRAPl), 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.

HoxB 13 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 HoxB 13 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 TGF 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, CB 1 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 to chromosome 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.

EXAMPLES

Example 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. Figure 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 ws elevated early and then returned to normal by day 10.

Figure 2 presents an overview of intradermal injection experiments wih sd- rxRNA molecules. 6 intradermal injections were performed at each site. Each injection consisted of approximately 34 μ, 300 μg total. Images were taken before injection and 15 minutes after the first injection.

Figure 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 on days 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.

Figure 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.

Figure 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.

Figure 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

Figures 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 on days 3, 5, and 8. RNA was isolated and gene expression was analyzed by qPCR and normalized to B-Actin

Figure 9 compares two different dosage regimens, Days 1 and 3 vs. Days 0 and 2. There were 6 injection sites per animal. 3 mm skin biopsies were harvested on days 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 ori 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.

Figures 10-12 reveal that CTGF sd-rxRNAs are efficacious in mediating gene silencing. A dose response for CTGF is indicated in Figure 12.

Figures 13-14 reveal that the original sd-rxrNA screen had a low hit rate. Figure 15 reveals PTGS2 knockdown using sd-rxRNA against PTGS2. Figures 16-24 reveal that hTGFBl, TGFB, TGFB2 sd-rxRNAs are capable of mediating gene silencing.

Figures 25-28 shows the identificatio of potent hSPPl sd-rxRNAs.

Example 3: Linker chemistry

Figure 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 following structure:

Example 4: Optimization of target sequences

Chemical optimization was performed for several lead sequences, including CTGF, PTGS2, TGFpi, and TGF 2. 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 ful 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 ribonucleitides 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. hTGF i leads are shown in Table 26 and hTGF 2 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 (LI), 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 LI and L2, the fully O-methyl modified sense strand was not efficacious.

For TGF i hL3, the fully O-methyl modified sense strand was efficacious.

For TGF 2, 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. Figures 33 and 34 demonstrate the activity of optimized CTGF LI compounds. Figure 35 demontrates the in vitro stability of the CTGF LI compounds. Figures 36 and 37 demonstrate the activity of optimized CTGF L2 compounds. Figure 38 demontrates the in vitro stability of the CTGF L2 compounds. Figure 39 provides a summary of the in vivo activity of CTGF lead compounds. Figure 40 demonstrates the efficacy of CTGF LI compounds in skin biopsies from rats. Figure 41 shows the efficacy of CTGF L2 compounds in achieving gene silencing.

Figure 42 demonstrates CTGF silencing following intradermal injection of RXi- 109. Figure 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.

Figures 44-46 show that CTGF L3 and L4 compounds are also active. FIG. 47 demonstrates changes in mRNA expression levels of CTGF, a-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 on days 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 on days 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 on days 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 on days 3, 6, and 9 post- wounding (5 = mature, 1 = immature). Each group represents 5 rats. FIG. 53 demonstrates CD68 labeling in day 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 a Phase 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 a Phase 1 and 2 clinical trial design. This schematic represents a divided dose, multi-day ascending dose clinical trial.

Activity of PTGS2, TGF i and TGFP2 leads was also tested. Figures 59 and 60 demonstrate activity of PTGS2 LI and L2 compounds. Figures 61 and 62 demonstrate the activity of h TGFpi compounds and Figures 63 and 64 demonstrate the activity of hTGF 2 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 hTGFB l ori 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 hSPPl sd-rxRNA sequences. Table 9 demonstrates inhibition of gene expression with hSPPl ori sequences.

Table 10 demonstrates non- limiting examples of hCTGF sd-rxRNA sequences.

Table 11 demonstrates inhibition of gene expression with hCTGF ori sequences.

Table 12 demonstrates inhibition of gene expression with CTGF ori 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 TGFBl 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 TGFBl 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 TGF i Leads.

Table 27 provides a summary of TGF i Leads.

Table 1 : Original sd-rxRNA screen

Oligo SEQ ID SEQ ID

ID# Gl- NO Sense-sd-rxRNA GII NO AS-sd-rxRNA-GII

5'-P-mGGA(2'-F-C)(2'-F-C)(2'-F- m U A m C A G m C A A G G m U m C m U)(2'-F-U)G(2'-F-C)(2'-F-

14398 TGFB1 9 C-chol 10 U)GmUA*mC*mU*G*mC*G*U

5'-P-mG(2'-F-C)A(2'-F-C)GA(2'-F-

AAmCAmUGAmUmCGmUGm U)(2'-F-C)A(2'-F-

14399 TGFB1 11 C-chol 12 U)GmUmU*G*G*A*mC*A*G

5'-P-mCG(2'-F-C)A(2'-F-C)GA(2'-F-

Am C Am U G Am U m CG m U G m C U)(2'-F-C)A(2'-F-

14400 TGFB1 13 G-chol 14 U)GmU*mU*G*G*A*mC*A

5'-P-mCAGGA(2'-F-C)(2'-F-C)(2'-F- mCAGmCAAGGmUmCmCmU U)(2'-F-U)G(2'-F-

14401 TGFB1 15 G-chol 16 C)mUG*mU*A*mC*mU*G*C

5'-P-mA(2'-F-C)GA(2'-F-U)(2'-F- mCmCAAmCAmUGAmUmCG C)A(2'-F-U)G(2'-F-U)(2'-F-

14402 TGFB1 17 mU-chol 18 U)GG*A*mC*A*G*mC*U

5'-P-mA(2'-F-U)G(2'-F-C)G(2'-F-

AGmCGGAAGmCGmCAmU- C)(2'-F-U)(2'-F-U)(2'-F-C)(2'-F-

14403 TGFB1 19 chol 20 C)GmCmU*mU*mC*A*mC*mC*A

5'-P-mA(2'-F-U)GG(2'-F-C)(2'-F-

GmCAmUmCGAGGmCmCAm C)(2'-F-U)(2'-F-C)GA(2'-F-

14404 TGFB1 21 U-chol 22 U)GmC*G*mC*mU*mU*mC*C

5'-P-mCA(2'-F-U)G(2'-F-U)(2'-F-

GAmCmUAmUmCGAmCAmU C)GA(2'-F-

14405 TGFB1 23 G-chol 24 U)AGmUmC*mU*mU*G*mC*A*G

5^,-P-mUAG(2'-F-U)(2'-F-C)(2^,-F-

AmCmCmUGmCAAGAmCmU U)(2'-F-U)G(2'-F-

14406 TGFB1 25 A-chol 26 C)AGGmU*G*G*A*mU*A*G

5'-P-mU(2'-F-U)(2^,-F-C)(2^,-F-U)(2^,-F-

GmCmUmCmCAmCGGAGAA- C)(2'-F-C)G(2'-F-

14407 TGFB1 27 chol 28 U)GGAGmC*mU*G*A*A*G*C

5'-P-mU(2'-F-

GGmCmUmCmUmCmCmUm C)GAAGGAGAGmCmC*A*mU*mU*

14408 TGFB2 29 UmCGA-chol 30 mC*G*C

5^,-P-mC(2'-F-C)AGG(2'-F-U)(2^,-F-

GAmCAGGAAmCmCmUGG- U)(2'-F-C)(2'-F-C)(2'-F-

14409 TGFB2 31 chol 32 U)GmUmC*mU*mU*mU*A*mU*G

5^,-P-mUAAA(2'-F-C)(2'-F-C)(2^,-F- mCmCAAGGAGGmUmUmUA U)(2'-F-C)(2'-F-C)(2^,-F-U)(2^,-F-

14410 TGFB2 33 -chol 34 U)GG*mC*G*mU*A*G*U

5^,-P-mUG(2'-F-U)AGA(2'-F-

AmUmUmUmCmCAmUmCm U)GGAAAmU*mC*A*mC*mC*mU*

14411 TGFB2 35 UAmCA-chol 36 C

5^,-P-mUG(2'-F-U)(2'-F-U)G(2^,-F- mUmCmCAmUmCmUAmCAA U)AGA(2'-F-

14412 TGFB2 37 mCA-chol 38 U)GGA*A*A*mU*mC*A*C

mUmUmUmCmCAmUmCmU 5^,-P-mU(2'-F-U)G(2'-F-U)AGA(2^,-F-

14413 TGFB2 39 AmCAA-chol 40 U)GGAAA*mU*mC*A*mC*mC*U

5'-P-mAA(2^,-F-C)(2^,-F-C)(2^,-F-U)(2'- mCGmCmCAAGGAGGmUmU- F-C)(2'-F-C)(2'-F-U)(2'-F-

14414 TGFB2 41 chol 42 U)GGmCG*mU*A*G*mU*A*C

5^,-P-mU(2'-F-U)(2'-F-C)(2^,-F-

GmUGGmUGAmUmCAGAA- U)GA(2'-F-U)(2'-F-C)A(2^,-F-C)(2^,-F-

14415 TGFB2 43 chol 44 C)AmC*mU*G*G*mU*A*U Oligo SEQ ID SEQ ID

ID# Gl- NO Sense-sd-rxRNA GII NO AS-sd-rxRNA-GII

5'-P-mA(2'-F-C)A(2'-F-U)(2'-F- mCmUmCmCmUGmCmUAA U)AG(2'-F-

14416 TGFB2 45 mUGmU-chol 46 C)AGGAG*A*mU*G*mU*G*G

Am C m Cm U m C m CAm C Am U A 5'-P-mllA(2'-F-U)A(2'-F-U)G(2'-F-

14417 TGFB2 47 mUA-chol 48 U)GGAGGmU*G*mC*mC*A*mU*C

5'-P-mll(2'-F-C)(2'-F-C)(2'-F-

AAGmUmCmCAmCmUAGGA- U)AG(2'-F-U)GGA(2'-F-

14418 TGFB2 49 chol 50 C)mUmU*mU*A*mU*A*G*U

5'-P-mll(2'-F-U)(2'-F-U)(2'-F-C)(2'-F- mUGGmUGAmUmCAGAAA- U)GA(2'-F-U)(2'-F-C)A(2'-F-

14419 TGFB2 51 chol 52 C)mCA*mC*mU*G*G*mU*A

5'-P-mll(2'-F-U)(2'-F-C)(2'-F-C)(2'-F-

U)AG(2'-F-

AGmUmCmCAmCmUAGGAA- U)GGAmCmU*mU*mU*A*mU*A*

14420 TGFB2 53 chol 54 G

5'-P-mA(2'-F-C)(2'-F-C)(2'-F-U)(2'-F-

AmCGmCmCAAGGAGGmU- C)(2'-F-C)(2'-F-U)(2'-F-U)GG(2'-F-

14421 TGFB2 55 chol 56 C)GmU*A*G*mU*A*mC*U

5'-P-mll(2'-F-C)AA(2'-F-U)(2'-F- mCAmCAmUmUmUGAmUm C)AAA(2'-F-

14422 PTGS2 57 UGA-chol 58 U)GmUG*A*mU*mC*mU*G*G mCAmCmUGmCmCmUmCAA 5'-P-mAA(2'-F-U)(2'-F-U)GAGG(2'-F-

14423 PTGS2 59 mUmU-chol 60 C)AGmUG*mU*mU*G*A*mU*G

5'-P-mAAGA(2'-F-C)(2'-F-U)GG(2'-F-

AAAmUAmCmCAGmUmCmU U)A(2'-F-

14424 PTGS2 61 mU-chol 62 U)mUmU*mC*A*mU*mC*mU*G

5'-P-mllG(2'-F-U)(2'-F-C)AA(2'-F- mCAmUmUmUGAmUmUGA U)(2'-F-

14425 PTGS2 63 mCA-chol 64 C)AAAmUG*mU*G*A*mU*mC*U

Table 2: Inhibition of gene expression with PTGS2 ori sequences

NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

CCAUACAGCAAAUCCUUGCUGUUC

15156 CDS 188 74 A 46%

CCACCCAUGUCAAAACCGAGGUGU

15157 CDS 212 75 A 31%

AGUGUGGGAUUUGACCAGUAUAA

15158 CDS 243 76 GA 23%

GUGUGGGAUUUGACCAGUAUAAG

15159 CDS 244 77 UA 24%

UGUGGGAUUUGACCAGUAUAAGU

15160 CDS 245 78 GA 38%

UUUGACCAGUAUAAGUGCGAUUG

15161 CDS 252 79 UA 29%

GGAUUCUAUGGAGAAAACUGCUCA

15162 CDS 285 80 A 16%

UAUUUCUGAAACCCACUCCAAACA

15163 CDS 337 81 A 32%

15164 CDS 338 82 AUUUCUGAAACCCACUCCAAACACA 21%

15165 CDS 339 83 UUUCUGAAACCCACUCCAAACACAA 21%

15166 CDS 340 84 UUCUGAAACCCACUCCAAACACAGA 45%

15167 CDS 345 85 AAACCCACU CCAAACAC AG U G CACA 87%

15168 CDS 347 86 ACCCACUCCAAACACAGUGCACUAA 83%

15169 CDS 349 87 CCACUCCAAACACAGUGCACUACAA 51%

15170 CDS 350 88 CACUCCAAACACAGUGCACUACAUA 31%

UUUGGAACGUUGUGAAUAACAUU

15171 CDS 394 89 CA 43%

UGAAUAACAUUCCCUUCCUUCGAA

15172 CDS 406 90 A 21%

GAAUAACAUUCCCUUCCUUCGAAA

15173 CDS 407 91 A 32%

AAUAACAUUCCCUUCCUUCGAAAU

15174 CDS 408 92 A 27%

AUUAUGAGUUAUGUGUUGACAUC

15175 CDS 435 93 CA 27%

UAUGAGUUAUGUGUUGACAUCCA

15176 CDS 437 94 GA 21%

UGAGUUAUGUGUUGACAUCCAGA

15177 CDS 439 95 UA 30%

GAGUUAUGUGUUGACAUCCAGAU

15178 CDS 440 96 CA 68%

AGUUAUGUGUUGACAUCCAGAUCA

15179 CDS 441 97 A 35%

GUUAUGUGUUGACAUCCAGAUCAC

15180 CDS 442 98 A 36%

UUAUGUGUUGACAUCCAGAUCACA

15181 CDS 443 99 A 51%

UAUGUGUUGACAUCCAGAUCACAU

15182 CDS 444 100 A 24%

AUGUGUUGACAUCCAGAUCACAUU

15183 CDS 445 101 A 37%

UGUGUUGACAUCCAGAUCACAUUU

15184 CDS 446 102 A 42% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

UGUUGACAUCCAGAUCACAUUUGA

15185 CDS 448 103 A 18%

GUUGACAUCCAGAUCACAUUUGAU

15186 CDS 449 104 A 24%

UUGACAUCCAGAUCACAUUUGAUU

15187 CDS 450 105 A 25%

GACAUCCAGAUCACAUUUGAUUGA

15188 CDS 452 106 A 27%

CAUCCAGAUCACAUUUGAUUGACA

15189 CDS 454 107 A 27%

AUCCAGAUCACAUUUGAUUGACAG

15190 CDS 455 108 A 32%

UCCAGAUCACAUUUGAUUGACAGU

15191 CDS 456 109 A 40%

CCAGAUCACAUUUGAUUGACAGUC

15192 CDS 457 110 A 52%

GAUCACAUUUGAUUGACAGUCCAC

15193 CDS 460 111 A 40%

AUCACAUUUGAUUGACAGUCCACC

15194 CDS 461 112 A 46%

UCACAUUUGAUU GACAG U CCACCA

15195 CDS 462 113 A 34%

CACAUUUGAUUGACAGUCCACCAA

15196 CDS 463 114 A 30%

ACAUUUGAUUGACAGUCCACCAAC

15197 CDS 464 115 A 32%

CAUUUGAUUGACAGUCCACCAACU

15198 CDS 465 116 A 44%

UUUGAUUGACAGUCCACCAACUUA

15199 CDS 467 117 A 17%

UUGAUUGACAGUCCACCAACUUAC

15200 CDS 468 118 A 22%

UGAU U GACAG UCCACCAACU UACA

15201 CDS 469 119 A 27%

GAUUGACAGUCCACCAACUUACAA

15202 CDS 470 120 A 41%

AU U GACAG UCCACCAACU UACAAU

15203 CDS 471 121 A 39%

UUGACAGUCCACCAACUUACAAUG

15204 CDS 472 122 A 61%

UGACAGUCCACCAACUUACAAUGC

15205 CDS 473 123 A 48%

UCCACCAACUUACAAUGCUGACUA

15206 CDS 479 124 A 29%

ACUUACAAUGCUGACUAUGGCUAC

15207 CDS 486 125 A 35%

UUACAAUGCUGACUAUGGCUACAA

15208 CDS 488 126 A 32%

GGGAAGCCUUCUCUAACCUCUCCU

15209 CDS 517 127 A 39%

GGAAGCCUUCUCUAACCUCUCCUA

15210 CDS 518 128 A 48% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

GAAGCCUUCUCUAACCUCUCCUAU

15211 CDS 519 129 A 19%

AAGCCUUCUCUAACCUCUCCUAUU

15212 CDS 520 130 A 17%

CUUCUCUAACCUCUCCUAUUAUAC

15213 CDS 524 131 A 17%

UUCUCUAACCUCUCCUAUUAUACU

15214 CDS 525 132 A 34%

UCUCUAACCUCUCCUAUUAUACUA

15215 CDS 526 133 A 49%

GUAAAAAGCAGCUUCCUGAUUCAA

15216 CDS 601 134 A 35%

UAAAAAGCAGCUUCCUGAUUCAAA

15217 CDS 602 135 A 25%

AAGCAGCUUCCUGAUUCAAAUGAG

15218 CDS 606 136 A 27%

CCUGAUUCAAAUGAGAUUGUGGAA

15219 CDS 615 137 A 37%

CUGAUUCAAAUGAGAUUGUGGAA

15220 CDS 616 138 AA 27%

GAAAAAUUGCUUCUAAGAAGAAAG

15221 CDS 636 139 A 37%

AAAAAUUGCUUCUAAGAAGAAAGU

15222 CDS 637 140 A 56%

AAAAUUGCUUCUAAGAAGAAAGUU

15223 CDS 638 141 A 25%

AAAUUGCUUCUAAGAAGAAAGUUC

15224 CDS 639 142 A 34%

GGCUCAAACAUGAUGUUUGCAUUC

15225 CDS 678 143 A 68%

GCUCAAACAUGAUGUUUGCAUUCU

15226 CDS 679 144 A 51%

CUCAAACAUGAUGUUUGCAUUCUU

15227 CDS 680 145 A 50%

CAAACAUGAUGUUUGCAUUCUUU

15228 CDS 682 146 GA 51%

AAACAUGAUGUUUGCAUUCUUUG

15229 CDS 683 147 CA 63%

UCAGUUUUUCAAGACAGAUCAUAA

15230 CDS 722 148 A 45%

CAGU U U U UCAAG ACAG AUCAU AAG

15231 CDS 723 149 A 59%

AGUUUUUCAAGACAGAUCAUAAGC

15232 CDS 724 150 A 80%

GUUUUUCAAGACAGAUCAUAAGCG

15233 CDS 725 151 A 55%

UUUUUCAAGACAGAUCAUAAGCGA

15234 CDS 726 152 A 53%

CCAUGGGGUGGACUUAAAUCAUAU

15235 CDS 776 153 A 56%

ACUUAAAUCAUAUUUACGGUGAAA

15236 CDS 787 154 A 63% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

CUUAAAUCAUAUUUACGGUGAAAC

15237 CDS 788 155 A 43%

UUAAAUCAUAUUUACGGUGAAACU

15238 CDS 789 156 A 48%

UAAAUCAUAUUUACGGUGAAACUC

15239 CDS 790 157 A 56%

AAUCAUAUUUACGGUGAAACUCUG

15240 CDS 792 158 A 46%

AUCAUAUUUACGGUGAAACUCUGG

15241 CDS 793 159 A 64%

UUUACGGUGAAACUCUGGCUAGAC

15242 CDS 799 160 A 35%

AGACAGCGUAAACUGCGCCUUUUC

15243 CDS 819 161 A 65%

GACAGCGUAAACUGCGCCUUUUCA

15244 CDS 820 162 A 51%

ACAGCGUAAACUGCGCCUUUUCAA

15245 CDS 821 163 A 48%

CAGCGUAAACUGCGCCUUUUCAAG

15246 CDS 822 164 A 61%

AGCGUAAACUGCGCCUUUUCAAGG

15247 CDS 823 165 A 48%

AAACUGCGCCUUUUCAAGGAUGGA

15248 CDS 828 166 A 42%

ACUGCGCCUUUUCAAGGAUGGAAA

15249 CDS 830 167 A 29%

UAUCAGAUAAUUGAUGGAGAGAU

15250 CDS 861 168 GA 32%

AUCAGAUAAUUGAUGGAGAGAUG

15251 CDS 862 169 UA 55%

UCAGAUAAUUGAUGGAGAGAUGU

15252 CDS 863 170 AA 50%

CAGAUAAUUGAUGGAGAGAUGUA

15253 CDS 864 171 UA 50%

AGAUAAUUGAUGGAGAGAUGUAU

15254 CDS 865 172 CA 55%

GAUAAUUGAUGGAGAGAUGUAUC

15255 CDS 866 173 CA 65%

AUAAUUGAUGGAGAGAUGUAUCC

15256 CDS 867 174 UA 54%

AGAUGUAUCCUCCCACAGUCAAAG

15257 CDS 880 175 A 78%

GAUGUAUCCUCCCACAGUCAAAGA

15258 CDS 881 176 A 79%

AUGUAUCCUCCCACAGUCAAAGAU

15259 CDS 882 177 A 49%

UGUAUCCUCCCACAGUCAAAGAUA

15260 CDS 883 178 A 28%

GUAUCCUCCCACAGUCAAAGAUAC

15261 CDS 884 179 A 56%

UAUCCUCCCACAGUCAAAGAUACU

15262 CDS 885 180 A 42% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

15263 CDS 887 181 UCCUCCCACAGUCAAAGAUACUCAA 45%

15264 CDS 888 182 CCUCCCACAGUCAAAGAUACUCAGA 73%

CUCCCACAGUCAAAGAUACUCAGG

15265 CDS 889 183 A 56%

15266 CDS 891 184 CCCACAGUCAAAGAUACUCAGGCAA 80%

AAGAUACUCAGGCAGAGAUGAUCU

15267 CDS 901 185 A 59%

AGUCCCUGAGCAUCUACGGUUUGC

15268 CDS 935 186 A 83%

UCUGGUGCCUGGUCUGAUGAUGU

15269 CDS 980 187 AA 55%

CUGGUGCCUGGUCUGAUGAUGUA

15270 CDS 981 188 UA 56%

UGGUGCCUGGUCUGAUGAUGUAU

15271 CDS 982 189 GA 43%

GGUGCCUGGUCUGAUGAUGUAUG

15272 CDS 983 190 CA 41%

GUGCCUGGUCUGAUGAUGUAUGC

15273 CDS 984 191 CA 42%

UGCCUGGUCUGAUGAUGUAUGCC

15274 CDS 985 192 AA 37%

GCCUGGUCUGAUGAUGUAUGCCAC

15275 CDS 986 193 A 61%

GCUGCGGGAACACAACAGAGUAUG

15276 CDS 1016 194 A 44%

GCGGGAACACAACAGAGUAUGCGA

15277 CDS 1019 195 A 33%

UGCGAUGUGCUUAAACAGGAGCAU

15278 CDS 1038 196 A 53%

GCGAUGUGCUUAAACAGGAGCAUC

15279 CDS 1039 197 A 109%

CGAUGUGCUUAAACAGGAGCAUCC

15280 CDS 1040 198 A 77%

AUGUGCUUAAACAGGAGCAUCCUG

15281 CDS 1042 199 A 69%

UGUGCUUAAACAGGAGCAUCCUGA

15282 CDS 1043 200 A 76%

GUGCUUAAACAGGAGCAUCCUGAA

15283 CDS 1044 201 A 65%

UGCUUAAACAGGAGCAUCCUGAAU

15284 CDS 1045 202 A 64%

UGUUCCAGACAAGCAGGCUAAUAC

15285 CDS 1084 203 A 41%

CAAGCAGGCUAAUACUGAUAGGAG

15286 CDS 1093 204 A 24%

AGCAGGCUAAUACUGAUAGGAGAG

15287 CDS 1095 205 A 50%

GCAGGCUAAUACUGAUAGGAGAGA

15288 CDS 1096 206 A 51%

UAAGAUUGUGAUUGAAGAUUAUG

15289 CDS 1124 207 UA 35% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

AAGAUUGUGAUUGAAGAUUAUGU

15290 CDS 1125 208 GA 34%

AGAUUGUGAUUGAAGAUUAUGUG

15291 CDS 1126 209 CA 59%

GAUUGUGAUUGAAGAUUAUGUGC

15292 CDS 1127 210 AA 41%

AUUGUGAUUGAAGAUUAUGUGCA

15293 CDS 1128 211 AA 51%

UUGUGAUUGAAGAUUAUGUGCAA

15294 CDS 1129 212 CA 45%

GUGAUUGAAGAUUAUGUGCAACAC

15295 CDS 1131 213 A 37%

UGAUUGAAGAUUAUGUGCAACACU

15296 CDS 1132 214 A 34%

AUUGAAGAUUAUGUGCAACACUUG

15297 CDS 1134 215 A 24%

UGAAGAUUAUGUGCAACACUUGAG

15298 CDS 1136 216 A 37%

AAGAUUAUGUGCAACACUUGAGUG

15299 CDS 1138 217 A 44%

UGUGCAACACUUGAGUGGCUAUCA

15300 CDS 1145 218 A 29%

CAACACUUGAGUGGCUAUCACUUC

15301 CDS 1149 219 A 33%

AAAUUUGACCCAGAACUACUUUUC

15302 CDS 1179 220 A 35%

AAUUUGACCCAGAACUACUUUUCA

15303 CDS 1180 221 A 41%

AUUUGACCCAGAACUACUUUUCAA

15304 CDS 1181 222 A 40%

15305 CDS 1200 223 U U CAACAAACAAU U CCAG U ACC AAA 49%

AUUCCAGUACCAAAAUCGUAUUGC

15306 CDS 1211 224 A 27%

GUACCAAAAUCGUAUUGCUGCUGA

15307 CDS 1217 225 A 31%

UUCUGCCUGACACCUUUCAAAUUC

15308 CDS 1270 226 A 35%

CACCU U U CAAAUU CAU G ACCAG AA

15309 CDS 1280 227 A 57%

UUUCAAAUUCAUGACCAGAAAUAC

15310 CDS 1284 228 A 42%

AAUUCAUGACCAGAAAUACAACUA

15311 CDS 1289 229 A 52%

ACAACAACUCUAUAUUGCUGGAAC

15312 CDS 1327 230 A 58%

UGGAAUUACCCAGUUUGUUGAAU

15313 CDS 1352 231 CA 35%

AUUACCCAGUUUGUUGAAUCAUUC

15314 CDS 1356 232 A 41%

UUACCCAGUUUGUUGAAUCAUUCA

15315 CDS 1357 233 A 58% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

ACCCAGUUUGUUGAAUCAUUCACC

15316 CDS 1359 234 A 52%

CCCAGUUUGUUGAAUCAUUCACCA

15317 CDS 1360 235 A 66%

CCAGUUUGUUGAAUCAUUCACCAG

15318 CDS 1361 236 A 54%

UUUGUUGAAUCAUUCACCAGGCAA

15319 CDS 1365 237 A 47%

AGAGCAGGCAGAUGAAAUACCAGU

15320 CDS 1462 238 A 65%

GAGCAGGCAGAUGAAAUACCAGUC

15321 CDS 1463 239 A 66%

GCAGGCAG AU GAAAU ACCAG UCU U

15322 CDS 1465 240 A 22%

CAGGCAGAUGAAAUACCAGUCUUU

15323 CDS 1466 241 A 43%

GAUGAAAUACCAGUCUUUUAAUGA

15324 CDS 1472 242 A 23%

AUGAAAUACCAGUCUUUUAAUGAG

15325 CDS 1473 243 A 61%

UGAAAUACCAGUCUUUUAAUGAG

15326 CDS 1474 244 UA 49%

GAAAUACCAGUCUUUUAAUGAGUA

15327 CDS 1475 245 A 76%

AAAUACCAGUCUUUUAAUGAGUAC

15328 CDS 1476 246 A 51%

AAUACCAGUCUUUUAAUGAGUACC

15329 CDS 1477 247 A 72%

AUACCAGUCUUUUAAUGAGUACCG

15330 CDS 1478 248 A 40%

UACCAGUCUUUUAAUGAGUACCGC

15331 CDS 1479 249 A 53%

ACCAGUCUUUUAAUGAGUACCGCA

15332 CDS 1480 250 A 39%

CCAGUCUUUUAAUGAGUACCGCAA

15333 CDS 1481 251 A 41%

AGUCUUUUAAUGAGUACCGCAAAC

15334 CDS 1483 252 A 38%

UCUUUUAAUGAGUACCGCAAACGC

15335 CDS 1485 253 A 55%

CUUUUAAUGAGUACCGCAAACGCU

15336 CDS 1486 254 A 63%

UUUUAAUGAGUACCGCAAACGCUU

15337 CDS 1487 255 A 52%

AGUACCGCAAACGCUUUAUGCUGA

15338 CDS 1495 256 A 49%

UAUGAAUCAUUUGAAGAACUUACA

15339 CDS 1524 257 A 65%

AUGAAUCAUUUGAAGAACUUACAG

15340 CDS 1525 258 A 63%

GAAUCAUUUGAAGAACUUACAGGA

15341 CDS 1527 259 A 65% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

AUCAUUUGAAGAACUUACAGGAGA

15342 CDS 1529 260 A 43%

CAUU U GAAGAACU U ACAGGAG AAA

15343 CDS 1531 261 A 63%

AU U UGAAGAACU U ACAGG AG AAAA

15344 CDS 1532 262 A 33%

GGAAGCACUCUAUGGUGACAUCGA

15345 CDS 1574 263 A 62%

UGUAUCCUGCCCUUCUGGUAGAAA

15346 CDS 1609 264 A 36%

CCUGCCCUUCUGGUAGAAAAGCCU

15347 CDS 1614 265 A 58%

AUCUUUGGUGAAACCAUGGUAGAA

15348 CDS 1650 266 A 60%

UGGUAGAAGUUGGAGCACCAUUCU

15349 CDS 1666 267 A 88%

UAGAAGUUGGAGCACCAUUCUCCU

15350 CDS 1669 268 A 85%

AAGUUGGAGCACCAUUCUCCUUGA

15351 CDS 1672 269 A 83%

UUGGAGCACCAUUCUCCUUGAAAG

15352 CDS 1675 270 A 85%

UGGAGCACCAUUCUCCUUGAAAGG

15353 CDS 1676 271 A 83%

GGAGCACCAUUCUCCUUGAAAGGA

15354 CDS 1677 272 A 74%

GAGCACCAUUCUCCUUGAAAGGAC

15355 CDS 1678 273 A 81%

AGCACCAUUCUCCUUGAAAGGACU

15356 CDS 1679 274 A 86%

GCACCAUUCUCCUUGAAAGGACUU

15357 CDS 1680 275 A 98%

CACCAUUCUCCUUGAAAGGACUUA

15358 CDS 1681 276 A 78%

ACCAUUCUCCUUGAAAGGACUUAU

15359 CDS 1682 277 A 88%

CCAUUCUCCUUGAAAGGACUUAUG

15360 CDS 1683 278 A 88%

UGGGUUUUCAAAUCAUCAACACUG

15361 CDS 1762 279 A 78%

GGGUUUUCAAAUCAUCAACACUGC

15362 CDS 1763 280 A 92%

UUUCAAAUCAUCAACACUGCCUCA

15363 CDS 1767 281 A 85%

CAAAUCAUCAACACUGCCUCAAUU

15364 CDS 1770 282 A 84%

AUCAUCAACACUGCCUCAAUUCAG

15365 CDS 1773 283 A 86%

UCAUCAACACUGCCUCAAUUCAGU

15366 CDS 1774 284 A 94%

CAUCAACACUGCCUCAAUUCAGUC

15367 CDS 1775 285 A 84% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

AUCAACACUGCCUCAAUUCAGUCU

15368 CDS 1776 286 A 84%

UCAACACUGCCUCAAUUCAGUCUC

15369 CDS 1777 287 A 68%

CAACACUGCCUCAAUUCAGUCUCU

15370 CDS 1778 288 A 73%

AACACUGCCUCAAUUCAGUCUCUC

15371 CDS 1779 289 A 79%

ACACUGCCUCAAUUCAGUCUCUCA

15372 CDS 1780 290 A 78%

CACUGCCUCAAUUCAGUCUCUCAU

15373 CDS 1781 291 A 92%

ACUGCCUCAAUUCAGUCUCUCAUC

15374 CDS 1782 292 A 89%

CUGCCUCAAUUCAGUCUCUCAUCU

15375 CDS 1783 293 A 95%

UGCCUCAAUUCAGUCUCUCAUCUG

15376 CDS 1784 294 A 83%

GCCUCAAUUCAGUCUCUCAUCUGC

15377 CDS 1785 295 A 46%

CCUCAAUUCAGUCUCUCAUCUGCA

15378 CDS 1786 296 A 51%

CUCAAUUCAGUCUCUCAUCUGCAA

15379 CDS 1787 297 A 61%

AAUUCAGUCUCUCAUCUGCAAUAA

15380 CDS 1790 298 A 30%

AUUCAGUCUCUCAUCUGCAAUAAC

15381 CDS 1791 299 A 32%

UUCAGUCUCUCAUCUGCAAUAACG

15382 CDS 1792 300 A 30%

UCAGUCUCUCAUCUGCAAUAACGU

15383 CDS 1793 301 A 38%

CAGUCUCUCAUCUGCAAUAACGUG

15384 CDS 1794 302 A 67%

AGUCUCUCAUCUGCAAUAACGUGA

15385 CDS 1795 303 A 71%

GUCUCUCAUCUGCAAUAACGUGAA

15386 CDS 1796 304 A 81%

AGAGCUCAUUAAAACAGUCACCAU

15387 CDS 1856 305 A 33%

GAGCUCAUUAAAACAGUCACCAUC

15388 CDS 1857 306 A 55%

AGCUCAUUAAAACAGUCACCAUCA

15389 CDS 1858 307 A 31%

GCUCAUUAAAACAGUCACCAUCAA

15390 CDS 1859 308 A 46%

CUCAUUAAAACAGUCACCAUCAAU

15391 CDS 1860 309 A 43%

UCAUUAAAACAGUCACCAUCAAUG

15392 CDS 1861 310 A 58%

CAUUAAAACAGUCACCAUCAAUGC

15393 CDS 1862 311 A 78% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

U U AAAACAG U CACCAU CAAU G CAA

15394 CDS 1864 312 A 41%

UAAAACAGUCACCAUCAAUGCAAG

15395 CDS 1865 313 A 80%

AAAACAGUCACCAUCAAUGCAAGU

15396 CDS 1866 314 A 79%

AACAGU CACCAU CAAU G CAAGU U C

15397 CDS 1868 315 A 34%

AUAUCAAUCCCACAGUACUACUAA

15398 CDS 1912 316 A 39%

ACUACUAAAAGAACGUUCGACUGA

15399 CDS 1928 317 A 39%

CDS/3UT CGUUCGACUGAACUGUAGAAGUCU

15400 R 1941 318 A 30%

CDS/3UT GACUGAACUGUAGAAGUCUAAUGA

15401 R 1946 319 A 25%

CDS/3UT UGAACUGUAGAAGUCUAAUGAUCA

15402 R 1949 320 A 29%

UCCUGUUGCGGAGAAAGGAGUCAU

15403 3UTR 2077 321 A 45%

UUGCGGAGAAAGGAGUCAUACUU

15404 3UTR 2082 322 GA 43%

CAUACUUGUGAAGACUUUUAUGU

15405 3UTR 2098 323 CA 30%

CUAAAGAUUUUGCUGUUGCUGUU

15406 3UTR 2128 324 AA 41%

UGUUGCUGUUAAGUUUGGAAAAC

15407 3UTR 2141 325 AA 29%

AGAGAGAAAUGAGUUUUGACGUC

15408 3UTR 2188 326 UA 26%

UUAUAAGAACGAAAGUAAAGAUGU

15409 3UTR 2235 327 A 33%

AAGAUGGCAAAAUGCUGAAAGUUU

15410 3UTR 2281 328 A 28%

UUACACUGUCGAUGUUUCCAAUGC

15411 3UTR 2305 329 A 46%

GACAUUACCAGUAAUUUCAUGUCU

15412 3UTR 2446 330 A 24%

CAAAAAGAAGCUGUCUUGGAUUUA

15413 3UTR 2581 331 A 36%

CU U U U UCACCAAGAG U AU AAACCU

15414 3UTR 2669 332 A 41%

AUGCCAAAUUUAUUAAGGUGGUG

15415 3UTR 2730 333 GA 61%

GUGGAGCCACUGCAGUGUUAUCU

15416 3UTR 2750 334 UA 39%

GGAGCCACUGCAGUGUUAUCUUAA

15417 3UTR 2752 335 A 45%

CAGAAUUUGUUUAUAUGGCUGGU

15418 3UTR 2802 336 AA 49%

GUUUAUAUGGCUGGUAACAUGUA

15419 3UTR 2810 337 AA 34% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

UACUCAGAUUUUGCUAUGAGGUU

15420 3UTR 2963 338 AA 42%

CAGAUUUUGCUAUGAGGUUAAUG

15421 3UTR 2967 339 AA 39%

AUUUUGCUAUGAGGUUAAUGAAG

15422 3UTR 2970 340 UA 43%

AAUGAAGUACCAAGCUGUGCUUGA

15423 3UTR 2986 341 A 40%

AUCACAUUGCAAAAGUAGCAAUGA

15424 3UTR 3064 342 A 59%

GCAAAAGUAGCAAUGACCUCAUAA

15425 3UTR 3072 343 A 35%

AAUGACCUCAUAAAAUACCUCUUC

15426 3UTR 3083 344 A 40%

AAUUUUAUCUCAGUCUUGAAGCCA

15427 3UTR 3134 345 A 55%

UCUUGAAGCCAAUUCAGUAGGUGC

15428 3UTR 3147 346 A 52%

AAUUCAGUAGGUGCAUUGGAAUCA

15429 3UTR 3157 347 A 71%

UUUCUUCUUUUAGCCAUUUUGCU

15430 3UTR 3212 348 AA 38%

UUCUUUUAGCCAUUUUGCUAAGA

15431 3UTR 3216 349 GA 40%

CCAUUUUGCUAAGAGACACAGUCU

15432 3UTR 3225 350 A 36%

UUACUAGUUUUAAGAUCAGAGUU

15433 3UTR 3278 351 CA 70%

ACUCUGCCUAUAUUUUCUUACCUG

15434 3UTR 3313 352 A 56%

UGAACUUUUGCAAGUUUUCAGGU

15435 3UTR 3335 353 AA 64%

GAACUUUUGCAAGUUUUCAGGUA

15436 3UTR 3336 354 AA 62%

UUCAGGUAAACCUCAGCUCAGGAC

15437 3UTR 3351 355 A 62%

ACCUCAGCUCAGGACUGCUAUUUA

15438 3UTR 3360 356 A 53%

CU U AU U U U AAGU GAAAAGCAGAG A

15439 3UTR 3441 357 A 83%

UAUCUGUAACCAAGAUGGAUGCAA

15440 3UTR 3489 358 A 93%

UUUUCCACAUCUCAUUGUCACUGA

15441 3UTR 3662 359 A 36%

ACAUCUCAUUGUCACUGACAUUUA

15442 3UTR 3668 360 A 40%

GUCUUAUUAGGACACUAUGGUUA

15443 3UTR 3735 361 UA 40%

CUUAUUAGGACACUAUGGUUAUA

15444 3UTR 3737 362 AA 37%

UUAUUAGGACACUAUGGUUAUAA

15445 3UTR 3738 363 AA 38% NM_000963.2

Target Gene Gene SEQ ID PTGS2 % Expression (0.1 Duplex ID Region Ref Pos No Sense Sequence nM)

UGGUUAUAAACUGUGUUUAAGCC

15446 3UTR 3752 364 UA 28%

AUAUUUAAGGUUGAAUGUUUGUC

15447 3UTR 3919 365 CA 40%

CUAGCCCACAAAGAAUAUUGUCUC

15448 3UTR 3961 366 A 47%

UCUCAUUAGCCUGAAUGUGCCAUA

15449 3UTR 3981 367 A 56%

AAUGUGCCAUAAGACUGACCUUUU

15450 3UTR 3994 368 A 52%

Table 3: PTGS2 sd-rxRNA

Duplex ID ID Sequence SEQ ID NO

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 m U . mC. A. mC. A. m U . m U . m U .G . Am U . m U . A.Ch 1 423

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

Target Gene hTGFBl

Duplex ID Single Strand ID sd-rxRNA sequence SEQ ID NO

17497 P.mU.A.A.G.fU.fC.A.A.fU.G.fU.A.fC*A*G*fC*fU*G*C 446

447

18458 17498 A.A.mC.mU.A.mU.mU.G.mC.mU.mU.mC.A.Chl

448

17500 P.mU.G.A.A.G.fC.A.A.fU.A.G.fU.fU*G*G*fU*G*fU*C

449

18459 17499 mC.A.A.mC.mU.A.mU.mU.G.mC.mU.mU.mC.A.Chl

450

17500 P.mU.G.A.A.G.fC.A.A.fU.A.G.fU.fU*G*G*fU*G*fU*C

451

18460 17501 G.mC.A.mU.A.mU.A.mU.A.mU.G.mU.A.Chl

17502 P.mU.A.fC.A.fU.A.fU.A.fU.A.fU.G.fC*fU*G*fU*G*fU*G 452

453

18461 17503 mU.G.mU.A.mC.A.mU.mU.G.A.mC.mU.A.Chl

17505 P.mU.A.G.fU.fC.A.A.fU.G.fU.A.fC.A*G*fC*fU*G*fC*C 454

455

18462 17504 mC.mU.G.mU.A.mC.A.mU.mU.G.A.mC.mU.A.Chl

17505 P.mU.A.G.fU.fC.A.A.fU.G.fU.A.fC.A*G*fC*fU*G*fC*C 456

457

18463 17506 A.G.mC.A.mU.A.mU.A.mU.A.mU.G.A.Chl

17507 P.mU.fC.A.fU.A.fU.A.fU.A.fU.G.fC.fU*G*fU*G*fU*G*U 458

459

18464 17508 mC.A.G.mC.A.A.mC.A.A.mU.mU.mC.A.Chl

P.mU.G.A.A.fU.fU.G.fU.fU.G.fC.fU.G*fU*A*fU*fU*fU* 460

17509 C

461

18465 17510 mC.A.mU.A.mU.A.mU.A.mU.G.mU.mU.A.Chl

462

17511 P.mU.A.A.fC.A.fU.A.fU.A.fU.A.fU.G*fC*fU*G*fU*G*U

463

18466 17512 mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.mC.A.Chl

464

17514 P.mU.G.A.G.fC.fU.G.A.A.G.fC.A.A*fU*A*G*fU*fU*G

465

18467 17513 A.mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.mC.A.Chl

466

17514 P.mU.G.A.G.fC.fU.G.A.A.G.fC.A.A*fU*A*G*fU*fU*G

467

18468 17515 A.mC.A.G.mC.A.mU.A.mU.A.mU.A.A.Chl

17516 P.mU.fU.A.fU.A.fU.A.fU.G.fC.fU.G.fU*G*fU*G*fU*A*C 468

469

18469 17517 A.mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.A.Chl

470

17519 P.mU.A.G.fC.fU.G.A.A.G.fC.A.A.fU*A*G*fU*fU*G*G

471

18470 17518 mU.A.mU.mU.G.mC.mU.mU.mC.A.G.mC.mU.A.Chl

472

17519 P.mU.A.G.fC.fU.G.A.A.G.fC.A.A.fU*A*G*fU*fU*G*G

473

18471 17520 mC.A.G.A.G.mU.A.mC.A.mC.A.mC.A.Chl

17521 P.mU.G.fU.G.fU.G.fU.A.fC.fU.fC.fU.G*C*fU*fU*G*A*A 474

475

18472 17522 mU.mC.A.A.G.mC.A.G.A.G.mU.A.A.Chl Target Gene hTGFBl

Duplex ID Single Strand ID sd-rxRNA sequence SEQ ID NO

P.mU.fU.A.fC.fU.fC.fU.G.fC.fU.fU.G.A*A*fC*fU*fU*G* 476

17523 U

18473 17524 A.G.mC.A.G.A.G.mU.A.mC.A.mC.A.Chl 477

P.mU.G.fU.G.fU.A.fC.fU.fC.fU.G.fC.fU*fU*G*A*A*fC* 478

17525 U

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

P.mU.A.A.fC.fU.fU.G.fU.fC.A.fU.A.G*A*fU*fU*fU*fC* 482

17529 G

18476 17530 G.mC.A.G.A.G.mU.A.mC.A.mC.A.A.Chl 483

P.mU.fU.G.fU.G.fU.A.fC.fU.fC.fU.G.fC*fU*fU*G*A*A* 484

17531 C

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

P.mU.fC.fU.fU.G.fU.fC.A.fU.AG.A.fU*fU*fU*fC*G*fU* 500

17547 U

501

Rat 502

Targeting

TGFB1

18715 18691 G.A.A.A.mU.A.mU.A.G.mC.A.A.A-chol 503

P.mU.fU.fU.G.fC.fU.A.fU.A.fU.fU.fU.fC*fU*G*G*fU*A 504

18692 *G

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

P.mU.fC.A.fU.fU.A.fU.fC.fU.fU.fU.G.fC*fU*G*fU*fC*A 508

18696 *C

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

P.mU.fU.fU.fC.fU.G.G.fU.A.G.A.G.fU*fU*fC*fU*A*fC* 512

18700 G

18720 18701 A.mC.A.G.mC.A.A.A.G.A.mU.A.A-chol 513

P.mU.fU.A.fU.fC.fU.fU.fU.G.fC.fU.G.fU*fC*A*fC*A*A* 514

18702 G

18721 18703 mC.A.A.mU.mC.mU.A.mU.G.A.mC.A.A-chol 515

P.mU.fU.G.fU.fC.A.fU.A.G.A.fU.fU.G*fC*G*fU*fU*G* 516

18704 U Target Gene hTGFBI

Duplex ID Single Strand ID sd-rxRNA sequence SEQ ID NO

18722 18705 A.G.A.mU.mU.mC.A.A.G.mU.mC.A.A-chol 517

P.mU.fU.G.A.fC.fU.fU.G.A.A.fU.fC.fU*fC*fU*G*fC*A* 518

18706 G

18723 18707 mC.mU.G.mU.G.G.A.G.mC.A.A.mC.A-chol 519

P.mU.G.fU.fU.G.fC.fU.fC.fC.A.fC.A.G*fU*fU*G*A*fC* 520

18708 U

18724 18709 mU.G.A.mC.A.G.mC.A.A.A.G.A.A-chol 521

P.mU.fU.fC.fU.fU.fU.G.fC.fU.G.fU.fC.A*fC*A*A*G*A* 522

18710 G

18725 18711 A.mU.G.A.mC.A.A.A.A.mC.mC.A.A-chol 523

P.mU.fU.G.G.fU.fU.fU.fU.G.fU.fC.A.fU*A*G*A*fU*fU* 524

18712 G

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 hTGFBI ori sequences

% Expression

Target Gene Gene SEQ ID hTGFBl 0.025 nM HeLa Duplex ID Region Ref Pos NO Sense Sequence cells

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 G AC AAG U U CAAG CAG AG U AC ACAC A 24.5%

15770 CDS 1238 565 ACAAG U UCAAGCAG AG U ACACACAA 38.5%

15771 CDS 1240 566 AAG UUCAAGCAGAG U ACACACAG CA 38.7%

15772 CDS 1241 567 AGUUCAAGCAGAGUACACACAGCAA 34.3%

15773 CDS 1242 568 G U UCAAGCAGAG U ACACACAGCAUA 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 CAG AG U ACACACAGCAU AU AU AU GA 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 32.1%

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%

597 UGCUGAGGCUCAAGUUAAAAGUGG

15802 CDS 1343 A 43.4%

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%

15812 CDS 1369 607 CAGCACGUGGAGCUGUACCAGAAAA 69.8% % Expression

Target Gene Gene SEQ ID hTGFBl 0.025 nM HeLa Duplex ID Region Ref Pos NO Sense Sequence cells

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 G AG CU G U ACCAG AAAU ACAG CAAC A 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 UG U ACCAGAAAU ACAGCAACAAU UA 36.4%

15826 CDS 1383 621 G U ACCAG AAAU ACAGCAACAAU UCA 24.6%

15827 CDS 1384 622 UACCAGAAAU ACAGCAACAAU UCCA 33.4%

15828 CDS 1385 623 ACCAGAAAU ACAGCAACAAU U CCU A 121.5%

15829 CDS 1386 624 CCAGAAAUACAGCAACAAUUCCUGA 62.1%

15830 CDS 1387 625 CAG AAAU ACAG CAACAAU U CCU G G A 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%

638 UCGCCAGAGUGGUUAUCUUUUGAU

15843 CDS 1447 A 43.7%

639 CGCCAGAGUGGUUAUCUUUUGAUG

15844 CDS 1448 A 42.1%

640 GCCAGAGUGGUUAUCUUUUGAUGU

15845 CDS 1449 A 31.0%

641 CCAGAGUGGUUAUCUUUUGAUGUC

15846 CDS 1450 A 46.0%

642 CAGAGUGGUUAUCUUUUGAUGUCA

15847 CDS 1451 A 40.2%

643 AGAGUGGUUAUCUUUUGAUGUCAC

15848 CDS 1452 A 38.5%

644 GAGUGGUUAUCUUUUGAUGUCACC

15849 CDS 1453 A 67.4%

645 AGUGGUUAUCUUUUGAUGUCACCG

15850 CDS 1454 A 57.4%

646 GUGGUUAUCUUUUGAUGUCACCGG

15851 CDS 1455 A 40.6%

647 UGGUUAUCUUUUGAUGUCACCGGA

15852 CDS 1456 A 70.5%

648 GGUUAUCUUUUGAUGUCACCGGAG

15853 CDS 1457 A 82.8% % Expression

Target Gene Gene SEQ ID hTGFBl 0.025 nM HeLa Duplex ID Region Ref Pos NO Sense Sequence cells

649 GUUAUCUUUUGAUGUCACCGGAGU

15854 CDS 1458 A 74.8%

650 UUAUCUUUUGAUGUCACCGGAGUU

15855 CDS 1459 A 86.8%

651 UAUCUUUUGAUGUCACCGGAGUUG

15856 CDS 1460 A 76.5%

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 GCAGCUGUACAU UGACU UCCGCAAA 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% % Expression

Target Gene Gene SEQ ID hTGFBl 0.025 nM HeLa Duplex ID Region Ref Pos NO Sense Sequence cells

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%

706 AGUGGGGAACACUACUGUAGUUAG

15911 3UTR 2326 A 107.7%

707 GUGGGGAACACUACUGUAGUUAGA

15912 3UTR 2327 A 108.6%

708 UGGGGAACACUACUGUAGUUAGAU

N/A

15913 3UTR 2328 A

Table 6: Inhibition of gene expression with hTGFB2 ori sequences

%

Expression

Oligo Gene A549 0.1 25-mer Sense Strand (position 25 of SS, id Region Ref Pos nM replaced with A) SEQ ID NO

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 111

15520 CDS 1480 65.9% UAGAAGCAAGAUUUGCAGGUAUUGA 118

15521 CDS 1481 59.6% AGAAGCAAGAUUUGCAGGUAUUGAU 119

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 %

Expression

Oligo Gene A549 0.1 25-mer Sense Strand (position 25 of SS, id Region Ref Pos nM replaced with A) SEQ ID NO

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 %

Expression

Oligo Gene A549 0.1 25-mer Sense Strand (position 25 of SS, id Region Ref Pos nM replaced with A) SEQ ID NO

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% U AU G U G AAC AACG ACAAC AACAACA 883

15626 3UTR 2298 40.8% AUGUGAACAACGACAACAACAACAA 884

15627 3UTR 2299 38.5% U G U G AACAACG ACAACAACAACAAC 885

15628 3UTR 2301 84.2% UGAACAACGACAACAACAACAACAA 886 %

Expression

Oligo Gene A549 0.1 25-mer Sense Strand (position 25 of SS, id Region Ref Pos nM replaced with A) SEQ ID NO

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% CAACAACAACAAC AACAAACAG G AA 892

15635 3UTR 2315 54.2% AACAAC AACAACAACAAACAG G AAA 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% U AAAG UGG AGACCAAAU ACU U U GCC 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% ACCAAAU ACU U U GCCAG AAACUCAU 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 %

Expression

Oligo Gene A549 0.1 25-mer Sense Strand (position 25 of SS, id Region Ref Pos nM replaced with A) SEQ ID NO

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 %

Expression

Oligo Gene A549 0.1 25-mer Sense Strand (position 25 of SS, id Region Ref Pos nM replaced with A) SEQ ID NO

15729 3UTR 4963 81.2% CGCCAGGGCCAAAAGAACUGGUCUA 987

15730 3UTR 5177 81.4% CCAGACUCCUCAAACGAGUUGCCAA 988

Table 7: hTGFB2 sd-rxRNA

Target Gene hTGFB2 SEQ ID

Duplex ID Single Strand ID sd-rxRNA sequences NO

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 1059

18678 18604 mC.G.G.mU.G.A.mC.A.A.mU.G.A.A-chol 1060

18605 mU.fU.fC.A.fU.fU.G.fU.fC.A.fC.fC.G*fU*G*A*fU*fU*U 1061

18679 18606 mU.mU.G.mU.mC.mU.mC.G.G.mU.A.mU.A-chol 1062

18607 mU.A.fU.A.fC.fC.G.A.G.A.fC.A.A*A*G*G*G*A*A 1063

18680 18608 G.A.G.mU.mU.G.mU.A.mU.G.mU.A.A-chol 1064

18609 mU.fU.A.fC.A.fU.A.fC.A.A.fC.fU.fC*fC*A*fC*fU*G*A 1065

18681 18610 A.mU.mU.mU.G.mU.mU.A.G.mU.G.mU.A-chol 1066

18611 mU.A.fC.A.fC.fU.A.A.fC.A.A.A.fU*fU*fC*fU*fU*fC*C 1067

18682 18612 G.mC.A.A.G.mU.mC.mU.G.A.G.A.A-chol 1068

18613 mU.fU.fC.fU.fC.A.G.A.fC.fU.fU.G.fC*fU*fC*A*G*fU*U 1069

18683 18614 A.A.A.mU.mC.A.mC.G.G.mU.G.A.A-chol 1070

18615 mU.fU.fC.A.fC.fC.G.fU.G.A.fU.fU.fU*fU*fC*A*fU*fC*C 1071

18684 18616 A.A.A.mU.G.mC.A.G.mC.mU.A.A.A-chol 1072

18617 mU.fU.fU.A.G.fC.fU.G.fC.A.fU.fU.fU*A*fC*A*A*G*A 1073

18685 18618 mC.mU.mU.G.G.A.A.A.A.mU.A.A.A-chol 1074

18619 mU.fU.fU.A.fU.fU.fU.fU.fC.fC.A.A.G*G*G*fC*A*A*U 1075

18686 18620 mC.mC.mU.mU.mU.G.A.A.mU.A.A.A.A-chol 1076

18621 mU.fU.fU.fU.A.fU.fU.fC.A.A.A.G.G*fU*A*fC*fU*G*G 1077

18687 18622 A.A.mC.A.mC.A.mC.mU.G.mC.A.A.A-chol 1078

18623 mU.fU.fU.G.fC.A.G.fU.G.fU.G.fU.fU*fU*fU*fC*A*fU*C 1079

18688 18624 A.A.A.A.mC.A.mC.A.mC.mU.G.mC.A-chol 1080 Target Gene hTGFB2 SEQ ID Duplex ID Single Strand ID sd-rxRNA sequences NO

18625 mU.G.fC.A.G.fU.G.fU.G.fU.fU.fU.fU*fC*A*fU*fC*A*U 1081

18689 18626 G.A.A.G.G.mC.mC.mU.G.mU.mU.A.A-chol 1082

18627 mU.fU.A.A.fC.A.G.G.fC.fC.fU.fU.fC*fU*G*G*A*fC*A 1083

18690 18628 mU.A.mU.mU.G.mC.mU.mC.mU.G.mC.A.A-chol 1084

18629 mU.fU.G.fC.A.G.A.G.fC.A.A.fU.A*fC*A*G*A*G*G 1085

Table 8: hSPP1 sd-rxRNA

18558 17463 G.A.A.A.G.A.G.A.A.mC.A.mU.A.Chl 1126

P.mU.A.fU.G.fU.fU.fC.fU.fC.fU.fU.fU.fC*A*fU*fU*fU*fU*

17464 G 1127

18559 17465 mC.mU.mU.mU.G.mC.A.mU.mU.mU.A.G.A.Chl 1128

17466 P.mU.fC.fU.A.A.A.fU.G.fC.A.A.A.G*fU*G*A*G*A*A 1129

18560 17467 mU.mU.mU.G.mC.A.mU.mU.mU.A.G.mU.A.Chl 1130

17468 P.mU.A.fC.fU.A.A.A.fU.G.fC.A.A.A*G*fU*G*A*G*A 1131

18561 17469 mC.mU.mC.A.mC.mU.mU.mU.G.mC.A.mU.A.Chl 1132

17470 P.mU.A.fU.G.fC.A.A.A.G.fU.G.A.G*A*A*A*fU*fU*G 1133

18562 17471 mU.mU.mC.mU.mC.A.mC.mU.mU.mU.G.mC.A.Chl 1134

17472 P.mU.G.fC.A.A.A.G.fU.G.A.G.A.A*A*fU*fU*G*fU*A 1135

18563 17473 mC.A.mC.mU.mC.mC.A.G.mU.mU.G.mU.A.Chl 1136

17474 P.mU.A.fC.A.A.fC.fU.G.G.A.G.fU.G*A*A*A*A*fC*U 1137

18564 17475 A.A.mU.G.A.A.A.G.A.G.A.A.A.Chl 1138

P.mU.fU.fU.fC.fU.fC.fU.fU.fU.fC.A.fU.fU*fU*fU*G*fC*fU*

17476 A 1139

18565 17477 mU.G.mC.A.G.mU.G.A.mU.mU.mU.mG.A.Chl 1140

17478 P.mU.fC.A.A.A.fU.fC.A.fC.fU.G.fC.A*A*fU*fU*fC*fU*C 1141

18566 17479 mU.G.A.A.A.G.A.G.A.A.mC.A.A.Chl 1142

P.mU.fU.G.fU.fU.fC.fU.fC.fU.fU.fU.fC.A*fU*fU*fU*fU*G*

17480 C 1143

18567 17481 A.mC.mC.mU.G.A.A.A.mU.mU.mU.mC.A.Chl 1144

17482 P.mU.G.A.A.A.fU.fU.fU.fC.A.G.G.fU*G*fU*fU*fU*A*U 1145

18568 17483 G.A.A.mU.mU.G.mC.A.G.mU.G.A.A.Chl 1146

17484 P.mU.fU.fC.A.fC.fU.G.fC.A.A.fU.fU.fC*fU*fC*A*fU*G*G 1147

18569 17485 G.G.mC.mU.G.A.mU.mU.mC.mU.G.G.A.Chl 1148

17486 P.mU.fC.fC.A.G.A.A.fU.fC.A.G.fC.fC*fU*G*fU*fU*fU*A 1149

Rat Targeting

SPP1

18662 18630 G.mU.mU.mC.G.mU.mU.G.mU.mU.mU.mC.A-chol 1150

18631 P.mU.G.A.A.A.fC.A.A.fC.G.A.A.fC*fU*A*A*G*fC*U 1151

18663 18632 G.A.A.A.G.A.A.A.mU.A.G.A.A-chol 1152

P.mU.fU.fC.fU.A.fU.fU.fU.fC.fU.fU.fU.fC*fU*fC*fC*A*fC*

18633 A 1153

18664 18634 G.mU.G.G.A.G.A.A.A.G.A.A.A-chol 1154

18635 P.mU.fU.fU.fC.fU.fU.fU.fC.fU.fC.fC.A.fC*A*fU*A*fC*A*U 1155

18665 18636 mC.mU.G.mU.G.mU.mC.A.mC.mU.A.mU.A-chol 1156

18637 P.mU.A.fU.A.G.fU.G.A.fC.A.fC.A.G*A*fC*fU*A*fU*U 1157

18666 18638 G.mU.mU.mU.mC.mU.mC.A.G.mU.mU.mC.A-chol 1158

18639 P.mU.G.A.A.fC.fU.G.A.G.A.A.A.fC*A*A*G*fC*A*G 1159

18667 18640 mU.A.mC.A.G.G.A.A.mC.A.G.mC.A-chol 1160

18641 P.mU.G.fC.fU.G.fU.fU.fC.fC.fU.G.fU.A*A*G*fU*fU*fU*G 1161

18668 18642 G.mC.A.G.G.mC.A.A.A.mC.mU.mU.A-chol 1162

18643 P.mU.A.A.G.fU.fU.fU.G.fC.fC.fU.G.fC*fC*fU*fC*fU*A*C 1163

18669 18644 A.A.mC.mU.mU.A.mC.A.G.G.A.A.A-chol 1164

18645 P.mU.fU.fU.fC.fC.fU.G.fU.A.A.G.fU.fU*fU*G*fC*fC*fU*G 1165

18670 18646 mC.A.mC.mU.G.mC.A.mU.mU.mU.mU.A.A-chol 1166

18647 P.mU.fU.A.A.A.A.fU.G.fC.A.G.fU.G*G*fC*fC*A*fU*U 1167

18671 18648 G.A.mC.A.mC.mC.A.mC.mU.G.mU.A.A-chol 1168

18649 P.mU.fU.A.fC.A.G.fU.G.G.fU.G.fU.fC*fU*G*fC*A*fU*G 1169

18672 18650 A.G.A.G.G.mC.A.G.G.mC.A.A.A-chol 1170

18651 P.mU.fU.fU.G.fC.fC.fU.G.fC.fC.fU.fC.fU*A*fC*A*fU*A*C 1171

18673 18652 mU.A.G.A.G.G.mC.A.G.G.mC.A.A-chol 1172

18653 P.mU.fU.G.fC.fC.fU.G.fC.fC.fU.fC.fU.A*fC*A*fU*A*fC*A 1173 18674 18654 G.A.G.A.G.mU.mU.mC.A.mU.mC.mU.A-chol 1174

18655 P.mU.A.G.A.fU.G.A.A.fC.fU.fC.fU.fC*fU*A*A*fU*fU*C 1175

18675 18656 mU.G.mU.G.A.A.mU.A.A.A.mU.mC.A-chol 1176

18657 P.mU.G.A.fU.fU.fU.A.fU.U.fC.A.fC.A*fC*fC*A*fC*A*A 1177

18676 18658 G.mU.G.A.A.mU.A.A.A.mU.mC.mU.A-chol 1178

18659 P.mU.A.G.A.fU.fU.fU.A.fU.fU.fC.A.fC*A*fC*fC*A*fC*A 1179

18677 18660 mU.G.A.AmU.A.A.A.mU.mC.mU.mU.A-chol 1180

18661 P.mU.A.A.G.A.fU.fU.fU.A.fU.U.fC.A*fC*A*fC*fC*A*C 1181

Table 9: Inhibition of gene expression with hSPPl ori sequences

A549 0.1

Target Gene Gene SEQ ID hSPPl nM Duplex ID Region Ref Pos NO Sense Strand Sequence Activity

14878 CDS 348 1220 CCU UCCAAG U AAG UCCAACGAAAGA 12.54%

14879 CDS 349 1221 C U U CCAAG U AAG U CCAACG AAAG CA 32.44%

14880 CDS 351 1222 UCCAAGUAAGUCCAACGAAAGCCAA 17.1%

14881 CDS 353 1223 CAAGUAAGUCCAACGAAAGCCAUGA 32.94%

14882 CDS 358 1224 AAGUCCAACGAAAGCCAUGACCACA 65.1%

14883 CDS 362 1225 CCAACGAAAGCCAUGACCACAUGGA 76.9%

14884 CDS 363 1226 CAACGAAAGCCAUGACCACAUGGAA 69.8%

14885 CDS 366 1227 CGAAAGCCAUGACCACAUGGAUGAA 78.02%

14886 CDS 372 1228 CCAUGACCACAUGGAUGAUAUGGAA 19.49%

14887 CDS 377 1229 ACCACAUGGAUGAUAUGGAUGAUGA 20.43%

14888 CDS 393 1230 GGAUGAUGAAGAUGAUGAUGACCAA 29.1%

14889 CDS 394 1231 GAUGAUGAAGAUGAUGAUGACCAUA 24.5%

14890 CDS 396 1232 UGAUGAAGAUGAUGAUGACCAUGUA 25.90%

14891 CDS 398 1233 AUGAAGAUGAUGAUGACCAUGUGGA 20.5%

14892 CDS 399 1234 UGAAGAUGAUGAUGACCAUGUGGAA 7.9%

14893 CDS 430 1235 GACUCCAUUGACUCGAACGACUCUA 21.6%

14894 CDS 431 1236 ACUCCAUUGACUCGAACGACUCUGA 13.5%

14895 CDS 432 1237 CUCCAUUGACUCGAACGACUCUGAA 12.33%

14896 CDS 435 1238 CAUUGACUCGAACGACUCUGAUGAA 42.5%

14897 CDS 440 1239 ACUCGAACGACUCUGAUGAUGUAGA 22.54%

14898 CDS 441 1240 CUCGAACGACUCUGAUGAUGUAGAA 17.4%

14899 CDS 442 1241 UCGAACGACUCUGAUGAUGUAGAUA 11.2%

14900 CDS 443 1242 CGAACGACUCUGAUGAUGUAGAUGA 20.7%

14901 CDS 445 1243 AACGACUCUGAUGAUGUAGAUGACA 27.1%

14902 CDS 449 1244 ACUCUGAUGAUGUAGAUGACACUGA 39.8%

14903 CDS 450 1245 CUCUGAUGAUGUAGAUGACACUGAA 9.6%

14904 CDS 451 1246 UCUGAUGAUGUAGAUGACACUGAUA 4.44%

14905 CDS 452 1247 CUGAUGAUGUAGAUGACACUGAUGA 8.7%

14906 CDS 453 1248 UGAUGAUGUAGAUGACACUGAUGAA 16.72%

14907 CDS 461 1249 UAGAUGACACUGAUGAUUCUCACCA 42.9%

14908 CDS 462 1250 AGAUGACACUGAUGAUUCUCACCAA 30.1%

14909 CDS 469 1251 ACUGAUGAUUCUCACCAGUCUGAUA 9.1%

14910 CDS 470 1252 CUGAUGAUUCUCACCAGUCUGAUGA 19.0%

14911 CDS 471 1253 UGAUGAUUCUCACCAGUCUGAUGAA 42.1%

14912 CDS 472 1254 GAUGAUUCUCACCAGUCUGAUGAGA 59.1%

14913 CDS 476 1255 AUUCUCACCAGUCUGAUGAGUCUCA 38.2%

14914 CDS 479 1256 CUCACCAGUCUGAUGAGUCUCACCA 34.1%

14915 CDS 480 1257 UCACCAGUCUGAUGAGUCUCACCAA 48.45%

14916 CDS 483 1258 CCAGUCUGAUGAGUCUCACCAUUCA 9.5%

14917 CDS 484 1259 CAGUCUGAUGAGUCUCACCAUUCUA 21.5%

14918 CDS 485 1260 AGUCUGAUGAGUCUCACCAUUCUGA 18.6%

14919 CDS 486 1261 GUCUGAUGAGUCUCACCAUUCUGAA 20.2%

14920 CDS 487 1262 UCUGAUGAGUCUCACCAUUCUGAUA 10.9%

14921 CDS 488 1263 CUGAUGAGUCUCACCAUUCUGAUGA 18.9%

14922 CDS 489 1264 UGAUGAGUCUCACCAUUCUGAUGAA 10.7%

14923 CDS 490 1265 GAUGAGUCUCACCAUUCUGAUGAAA 28.15%

14924 CDS 493 1266 GAGUCUCACCAUUCUGAUGAAUCUA 18.33%

14925 CDS 495 1267 GUCUCACCAUUCUGAUGAAUCUGAA 7.61%

14926 CDS 496 1268 UCUCACCAUUCUGAUGAAUCUGAUA 2.99%

14927 CDS 497 1269 CUCACCAUUCUGAUGAAUCUGAUGA 7.44%

14928 CDS 498 1270 UCACCAUUCUGAUGAAUCUGAUGAA 9.7%

14929 CDS 499 1271 CACCAUUCUGAUGAAUCUGAUGAAA 16.96% A549 0.1

Target Gene Gene SEQ ID hSPPl nM Duplex ID Region Ref Pos NO Sense Strand Sequence Activity

14930 CDS 501 1272 CCAUUCUGAUGAAUCUGAUGAACUA 3.08%

14931 CDS 505 1273 UCUGAUGAAUCUGAUGAACUGGUCA 13.24%

14932 CDS 510 1274 UGAAUCUGAUGAACUGGUCACUGAA 3.16%

14933 CDS 550 1275 CCAGCAACCGAAGUUUUCACUCCAA 14.02%

14934 CDS 554 1276 CAACCGAAGUUUUCACUCCAGUUGA 3.10%

14935 CDS 555 1277 AACCGAAGUUUUCACUCCAGUUGUA 5.27%

14936 CDS 572 1278 CAGUUGUCCCCACAGUAGACACAUA 13.2%

14937 CDS 573 1279 AG U UG UCCCCACAG U AGACACAU AA 27.01%

14938 CDS 574 1280 GUUGUCCCCACAGUAGACACAUAUA 8.76%

14939 CDS 588 1281 AGACACAUAUGAUGGCCGAGGUGAA 14.04%

14940 CDS 589 1282 GACACAUAUGAUGGCCGAGGUGAUA 18.40%

14941 CDS 598 1283 GAUGGCCGAGGUGAUAGUGUGGUUA 12.50%

14942 CDS 601 1284 GGCCGAGGUGAUAGUGUGGUUUAUA 13.76%

14943 CDS 602 1285 GCCGAGGUGAUAGUGUGGUUUAUGA 5.34%

14944 CDS 603 1286 CCGAGGUGAUAGUGUGGUUUAUGGA 29.69%

14945 CDS 604 1287 CGAGGUGAUAGUGUGGUUUAUGGAA 33.34%

14946 CDS 606 1288 AGGUGAUAGUGUGGUUUAUGGACUA 17.50%

14947 CDS 608 1289 GUGAUAGUGUGGUUUAUGGACUGAA 45.90%

14948 CDS 609 1290 UGAUAGUGUGGUUUAUGGACUGAGA 22.0%

14949 CDS 610 1291 GAUAGUGUGGUUUAUGGACUGAGGA 19.93%

14950 CDS 611 1292 AUAGUGUGGUUUAUGGACUGAGGUA 17.34%

14951 CDS 615 1293 UGUGGUUUAUGGACUGAGGUCAAAA 5.60%

14952 CDS 617 1294 UGGUUUAUGGACUGAGGUCAAAAUA 25.74%

14953 CDS 618 1295 GGUUUAUGGACUGAGGUCAAAAUCA 17.63%

14954 CDS 619 1296 GUUUAUGGACUGAGGUCAAAAUCUA 3.45%

14955 CDS 621 1297 UUAUGGACUGAGGUCAAAAUCUAAA 18.03%

14956 CDS 622 1298 UAUGGACUGAGGUCAAAAUCUAAGA 20.98%

14957 CDS 623 1299 AUGGACUGAGGUCAAAAUCUAAGAA 20.60%

14958 CDS 624 1300 UGGACUGAGGUCAAAAUCUAAGAAA 26.73%

14959 CDS 625 1301 GGACUGAGGUCAAAAUCUAAGAAGA 7.45%

14960 CDS 626 1302 GACUGAGGUCAAAAUCUAAGAAGUA 14.1%

14961 CDS 629 1303 UGAGGUCAAAAUCUAAGAAGUUUCA 8.61%

14962 CDS 630 1304 GAGGUCAAAAUCUAAGAAGUUUCGA 19.07%

14963 CDS 631 1305 AGGUCAAAAUCUAAGAAGUUUCGCA 6.08%

14964 CDS 632 1306 GGUCAAAAUCUAAGAAGUUUCGCAA 19.82%

14965 CDS 636 1307 AAAAUCUAAGAAGUUUCGCAGACCA 21.55%

14966 CDS 637 1308 AAAUCUAAGAAGUUUCGCAGACCUA 30.20%

14967 CDS 638 1309 AAUCUAAGAAGUUUCGCAGACCUGA 18.23%

14968 CDS 686 1310 ACGAGGACAUCACCUCACACAUGGA 14.85%

14969 CDS 687 1311 CGAGGACAUCACCUCACACAUGGAA 28.04%

14970 CDS 689 1312 AGGACAUCACCUCACACAUGGAAAA 3.80%

14971 CDS 698 1313 CCUCACACAUGGAAAGCGAGGAGUA 7.67%

14972 CDS 703 1314 CACAUGGAAAGCGAGGAGUUGAAUA 5.8%

14973 CDS 704 1315 ACAUGGAAAGCGAGGAGUUGAAUGA 5.3%

14974 CDS 705 1316 CAUGGAAAGCGAGGAGUUGAAUGGA 24.47%

14975 CDS 718 1317 GAGUUGAAUGGUGCAUACAAGGCCA 26.39%

14976 CDS 785 1318 GCCGUGGGAAGGACAGUUAUGAAAA 7.60%

14977 CDS 786 1319 CCGUGGGAAGGACAGUUAUGAAACA 8.75%

14978 CDS 788 1320 GUGGGAAGGACAGUUAUGAAACGAA 8.34%

14979 CDS 790 1321 GGGAAGGACAGUUAUGAAACGAGUA 5.38%

14980 CDS 792 1322 GAAGGACAGUUAUGAAACGAGUCAA 11.45%

14981 CDS 794 1323 AGGACAGUUAUGAAACGAGUCAGCA 11.78% A549 0.1

Target Gene Gene SEQ ID hSPPl nM Duplex ID Region Ref Pos NO Sense Strand Sequence Activity

14982 CDS 795 1324 GGACAGUUAUGAAACGAGUCAGCUA 10.69%

14983 CDS 797 1325 ACAGUUAUGAAACGAGUCAGCUGGA 54.58%

14984 CDS 798 1326 CAGUUAUGAAACGAGUCAGCUGGAA 33.9%

14985 CDS 846 1327 CCACAAGCAGUCCAGAUUAUAUAAA 24.1%

14986 CDS 850 1328 AAGCAGUCCAGAUUAUAUAAGCGGA 27.86%

14987 CDS 854 1329 AGUCCAGAUUAUAUAAGCGGAAAGA 24.29%

14988 CDS 855 1330 GUCCAGAUUAUAUAAGCGGAAAGCA 54.43%

14989 CDS 859 1331 AGAUUAUAUAAGCGGAAAGCCAAUA 71.49%

14990 CDS 860 1332 GAUUAUAUAAGCGGAAAGCCAAUGA 69.64%

14991 CDS 861 1333 AUUAUAUAAGCGGAAAGCCAAUGAA 38.82%

14992 CDS 862 1334 UUAUAUAAGCGGAAAGCCAAUGAUA 20.77%

14993 CDS 865 1335 UAUAAGCGGAAAGCCAAUGAUGAGA 21.79%

14994 CDS 866 1336 AUAAGCGGAAAGCCAAUGAUGAGAA 50.00%

14995 CDS 867 1337 UAAGCGGAAAGCCAAUGAUGAGAGA 11.67%

14996 CDS 870 1338 GCGGAAAGCCAAUGAUGAGAGCAAA 13.5%

14997 CDS 871 1339 CGGAAAGCCAAUGAUGAGAGCAAUA 15.49%

14998 CDS 872 1340 GGAAAGCCAAUGAUGAGAGCAAUGA 8.55%

14999 CDS 873 1341 GAAAGCCAAUGAUGAGAGCAAUGAA 12.12%

15000 CDS 875 1342 AAGCCAAUGAUGAGAGCAAUGAGCA 16.14%

15001 CDS 878 1343 CCAAUGAUGAGAGCAAUGAGCAUUA 31.71%

15002 CDS 879 1344 CAAUGAUGAGAGCAAUGAGCAUUCA 32.25%

15003 CDS 881 1345 AUGAUGAGAGCAAUGAGCAUUCCGA 6.97%

15004 CDS 883 1346 GAUGAGAGCAAUGAGCAUUCCGAUA 23.11%

15005 CDS 885 1347 UGAGAGCAAUGAGCAUUCCGAUGUA 5.53%

15006 CDS 890 1348 GCAAUGAGCAUUCCGAUGUGAUUGA 10.69%

15007 CDS 893 1349 AUGAGCAUUCCGAUGUGAUUGAUAA 4.12%

15008 CDS 894 1350 UGAGCAUUCCGAUGUGAUUGAUAGA 6.49%

15009 CDS 895 1351 GAGCAUUCCGAUGUGAUUGAUAGUA 29.12%

15010 CDS 897 1352 GCAUUCCGAUGUGAUUGAUAGUCAA 3.54%

15011 CDS 899 1353 AUUCCGAUGUGAUUGAUAGUCAGGA 6.05%

15012 CDS 901 1354 UCCGAUGUGAUUGAUAGUCAGGAAA 3.31%

15013 CDS 906 1355 UGUGAUUGAUAGUCAGGAACUUUCA 12.71%

15014 CDS 907 1356 GUGAUUGAUAGUCAGGAACUUUCCA 13.95%

15015 CDS 909 1357 GAUUGAUAGUCAGGAACUUUCCAAA 4.03%

15016 CDS 912 1358 UGAUAGUCAGGAACUUUCCAAAGUC 11.96%

15017 CDS 913 1359 GAUAGUCAGGAACUUUCCAAAGUCA 14.01%

15018 CDS 914 1360 AUAGUCAGGAACUUUCCAAAGUCAA 5.56%

15019 CDS 916 1361 AGUCAGGAACUUUCCAAAGUCAGCA 13.92%

15020 CDS 917 1362 GUCAGGAACUUUCCAAAGUCAGCCA 19.00%

15021 CDS 923 1363 AACUUUCCAAAGUCAGCCGUGAAUA 17.56%

15022 CDS 925 1364 CUUUCCAAAGUCAGCCGUGAAUUCA 19.58%

15023 CDS 926 1365 UUUCCAAAGUCAGCCGUGAAUUCCA 6.54%

15024 CDS 935 1366 UCAGCCGUGAAUUCCACAGCCAUGA 16.15%

15025 CDS 936 1367 CAGCCGUGAAUUCCACAGCCAUGAA 20.62%

15026 CDS 937 1368 AGCCGUGAAUUCCACAGCCAUGAAA 5.21%

15027 CDS 943 1369 G AAU U CCAC AG CCAU G AAU U U CACA 31.14%

15028 CDS 944 1370 AAUUCCACAGCCAUGAAUUUCACAA 35.63%

15029 CDS 945 1371 AUUCCACAGCCAUGAAUUUCACAGA 23.96%

15030 CDS 946 1372 UUCCACAGCCAUGAAUUUCACAGCA 15.20%

15031 CDS 947 1373 U CCAC AG CCAU G AAU U U CAC AG CCA 19.45%

15032 CDS 950 1374 ACAGCCAUGAAUUUCACAGCCAUGA 25.74%

15033 CDS 952 1375 AGCCAUGAAUUUCACAGCCAUGAAA 2.59% A549 0.1

Target Gene Gene SEQ ID hSPPl nM Duplex ID Region Ref Pos NO Sense Strand Sequence Activity

15034 CDS 953 1376 GCCAUGAAUUUCACAGCCAUGAAGA 6.00%

15035 CDS 954 1377 CCAUGAAUUUCACAGCCAUGAAGAA 4.60%

15036 CDS 956 1378 AUGAAUUUCACAGCCAUGAAGAUAA 9.20%

15037 CDS 957 1379 UGAAUUUCACAGCCAUGAAGAUAUA 10.84%

15038 CDS 958 1380 GAAUUUCACAGCCAUGAAGAUAUGA 40.20%

15039 CDS 959 1381 AAUUUCACAGCCAUGAAGAUAUGCA 37.25%

15040 CDS 960 1382 AUUUCACAGCCAUGAAGAUAUGCUA 8.21%

15041 CDS 961 1383 UUUCACAGCCAUGAAGAUAUGCUGA 12.01%

15042 CDS 964 1384 CACAGCCAUGAAGAUAUGCUGGUUA 12.25%

15043 CDS 983 1385 UGGUUGUAGACCCCAAAAGUAAGGA 19.65%

15044 CDS 984 1386 GGUUGUAGACCCCAAAAGUAAGGAA 28.19%

15045 CDS 985 1387 G U UG U AG ACCCCAAAAG UAAGG AAA 17.92%

15046 CDS 986 1388 UUGUAGACCCCAAAAGUAAGGAAGA 7.94%

15047 CDS 987 1389 UGUAGACCCCAAAAGUAAGGAAGAA 15.09%

15048 CDS 988 1390 G UAGACCCCAAAAG UAAGG AAG AAA 20.01%

15049 CDS 989 1391 UAGACCCCAAAAGUAAGGAAGAAGA 7.25%

15050 CDS 990 1392 AGACCCCAAAAGUAAGGAAGAAGAA 12.42%

15051 CDS 995 1393 CCAAAAGUAAGGAAGAAGAUAAACA 8.96%

15052 CDS 996 1394 CAAAAGUAAGGAAGAAGAUAAACAA 6.85%

15053 CDS 997 1395 AAAAGUAAGGAAGAAGAUAAACACA 14.15%

15054 CDS 998 1396 AAAG UAAGG AAG AAG AUAAACACCA 12.32%

15055 CDS 999 1397 AAGUAAGGAAGAAGAUAAACACCUA 8.83%

15056 CDS 1001 1398 GUAAGGAAGAAGAUAAACACCUGAA 15.09%

15057 CDS 1002 1399 UAAGGAAGAAGAUAAACACCUGAAA 4.91%

15058 CDS 1007 1400 AAGAAGAUAAACACCUGAAAUUUCA 1.43%

15059 CDS 1008 1401 AGAAGAUAAACACCUGAAAUUUCGA 3.51%

15060 CDS 1009 1402 GAAGAUAAACACCUGAAAUUUCGUA 15.12%

15061 CDS 1010 1403 AAGAUAAACACCUGAAAUUUCGUAA 28.56%

15062 CDS 1013 1404 AUAAACACCUGAAAUUUCGUAUUUA 5.74%

15063 CDS 1015 1405 AAACACCUGAAAUUUCGUAUUUCUA 13.01%

15064 CDS 1024 1406 AAAUUUCGUAUUUCUCAUGAAUUAA 15.54%

15065 CDS 1030 1407 CGUAUUUCUCAUGAAUUAGAUAGUA 9.47%

15066 CDS 1031 1408 GUAUUUCUCAUGAAUUAGAUAGUGA 30.03%

15067 CDS 1032 1409 UAUUUCUCAUGAAUUAGAUAGUGCA 5.31%

15068 CDS 1036 1410 UCUCAUGAAUUAGAUAGUGCAUCUA 9.74%

15069 CDS 1037 1411 CUCAUGAAUUAGAUAGUGCAUCUUA 10.78%

15070 CDS 1038 1412 UCAUGAAUUAGAUAGUGCAUCUUCA 91.87%

15071 CDS 1039 1413 CAUGAAUUAGAUAGUGCAUCUUCUA 93.82%

15072 CDS 1040 1414 AUGAAUUAGAUAGUGCAUCUUCUGA 96.06%

15073 CDS 1041 1415 UGAAUUAGAUAGUGCAUCUUCUGAA 94.91%

15074 CDS 1042 1416 GAAUUAGAUAGUGCAUCUUCUGAGA 97.91%

15075 CDS 1043 1417 AAUUAGAUAGUGCAUCUUCUGAGGA 93.76%

15076 CDS 1044 1418 AUUAGAUAGUGCAUCUUCUGAGGUA 103.92%

15077 CDS 1045 1419 UUAGAUAGUGCAUCUUCUGAGGUCA 95.85%

15078 CDS/3UTR 1052 1420 GUGCAUCUUCUGAGGUCAAUUAAAA 93.83%

15079 CDS/3UTR 1053 1421 UGCAUCUUCUGAGGUCAAUUAAAAA 90.69%

15080 CDS/3UTR 1054 1422 GCAUCUUCUGAGGUCAAUUAAAAGA 101.49%

15081 CDS/3UTR 1055 1423 CAUCUUCUGAGGUCAAUUAAAAGGA 110.27%

15082 CDS/3UTR 1056 1424 AUCUUCUGAGGUCAAUUAAAAGGAA 99.36%

15083 CDS/3UTR 1057 1425 UCUUCUGAGGUCAAUUAAAAGGAGA 95.31%

15084 CDS/3UTR 1058 1426 CUUCUGAGGUCAAUUAAAAGGAGAA 15.55%

15085 3UTR 1081 1427 AAAAAAUACAAUUUCUCACUUUGCA 3.59% A549 0.1

Target Gene Gene SEQ ID hSPPl nM Duplex ID Region Ref Pos NO Sense Strand Sequence Activity

15086 3UTR 1083 1428 AAAAUACAAUUUCUCACUUUGCAUU 3.46%

15087 3UTR 1086 1429 AUACAAUUUCUCACUUUGCAUUUAG 2.37%

15088 3UTR 1087 1430 UACAAUUUCUCACUUUGCAUUUAGU 3.54%

15089 3UTR 1088 1431 ACAAUUUCUCACUUUGCAUUUAGUC 2.85%

15090 3UTR 1089 1432 CAAUUUCUCACUUUGCAUUUAGUCA 2.35%

15091 3UTR 1093 1433 UUCUCACUUUGCAUUUAGUCAAAAG 1.38%

15092 3UTR 1125 1434 GCUUUAUAGCAAAAUGAAAGAGAAC 4.11%

15093 3UTR 1127 1435 UUUAUAGCAAAAUGAAAGAGAACAU 3.91%

15094 3UTR 1128 1436 UUAUAGCAAAAUGAAAGAGAACAUG 3.59%

15095 3UTR 1147 1437 AACAUGAAAUGCUUCUUUCUCAGUU 1.80%

15096 3UTR 1148 1438 ACAUGAAAUGCUUCUUUCUCAGUUU 2.17%

15097 3UTR 1150 1439 AUGAAAUGCUUCUUUCUCAGUUUAU 2.93%

15098 3UTR 1153 1440 AAAUGCUUCUUUCUCAGUUUAUUGG 2.18%

15099 3UTR 1154 1441 AAUGCUUCUUUCUCAGUUUAUUGGU 3.92%

15100 3UTR 1156 1442 UGCUUCUUUCUCAGUUUAUUGGUUG 4.08%

15101 3UTR 1157 1443 GCUUCUUUCUCAGUUUAUUGGUUGA 1.74%

15102 3UTR 1158 1444 CUUCUUUCUCAGUUUAUUGGUUGAA 4.74%

15103 3UTR 1159 1445 UUCUUUCUCAGUUUAUUGGUUGAAU 2.65%

15104 3UTR 1168 1446 AGUUUAUUGGUUGAAUGUGUAUCUA 2.57%

15105 3UTR 1178 1447 UUGAAUGUGUAUCUAUUUGAGUCUG 3.76%

15106 3UTR 1179 1448 UGAAUGUGUAUCUAUUUGAGUCUGG 2.91%

15107 3UTR 1183 1449 UGUGUAUCUAUUUGAGUCUGGAAAU 0.62%

15108 3UTR 1184 1450 GUGUAUCUAUUUGAGUCUGGAAAUA 2.45%

15109 3UTR 1186 1451 GUAUCUAUUUGAGUCUGGAAAUAAC 2.18%

15110 3UTR 1191 1452 UAUUUGAGUCUGGAAAUAACUAAUG 2.44%

15111 3UTR 1218 1453 UUUGAUAAUUAGUUUAGUUUGUGGC 19.35%

15112 3UTR 1219 1454 UUGAUAAUUAGUUUAGUUUGUGGCU 6.19%

15113 3UTR 1222 1455 AUAAUUAGUUUAGUUUGUGGCUUCA 3.25%

15114 3UTR 1224 1456 AAUUAGUUUAGUUUGUGGCUUCAUG 2.47%

15115 3UTR 1225 1457 AUUAGUUUAGUUUGUGGCUUCAUGG 2.28%

15116 3UTR 1226 1458 UUAGUUUAGUUUGUGGCUUCAUGGA 3.40%

15117 3UTR 1227 1459 UAGUUUAGUUUGUGGCUUCAUGGAA 4.12%

15118 3UTR 1244 1460 UCAUGGAAACUCCCUGUAAACUAAA 2.63%

15119 3UTR 1245 1461 CAUGGAAACUCCCUGUAAACUAAAA 2.20%

15120 3UTR 1246 1462 AUGGAAACUCCCUGUAAACUAAAAG 3.56%

15121 3UTR 1247 1463 UGGAAACUCCCUGUAAACUAAAAGC 3.73%

15122 3UTR 1248 1464 GGAAACUCCCUGUAAACUAAAAGCU 2.43%

15123 3UTR 1249 1465 GAAACUCCCUGUAAACUAAAAGCUU 2.28%

15124 3UTR 1251 1466 AACUCCCUGUAAACUAAAAGCUUCA 5.40%

15125 3UTR 1253 1467 CUCCCUGUAAACUAAAAGCUUCAGG 8.21%

15126 3UTR 1286 1468 UAUGUUCAUUCUAUAGAAGAAAUGC 3.17%

15127 3UTR 1294 1469 UUCUAUAGAAGAAAUGCAAACUAUC 2.45%

15128 3UTR 1295 1470 UCUAUAGAAGAAAUGCAAACUAUCA 3.97%

15129 3UTR 1296 1471 CUAUAGAAGAAAUGCAAACUAUCAC 3.86%

15130 3UTR 1297 1472 UAUAGAAGAAAUGCAAACUAUCACU 1.84%

15131 3UTR 1299 1473 UAGAAGAAAUGCAAACUAUCACUGU 2.53%

15132 3UTR 1302 1474 AAGAAAUGCAAACUAUCACUGUAUU 2.25%

15133 3UTR 1303 1475 AGAAAUGCAAACUAUCACUGUAUUU 3.32%

15134 3UTR 1357 1476 AU U U AUG U AGAAGCAAACAAAAU AC 1.86%

15135 3UTR 1465 1477 UAUCUUUUUGUGGUGUGAAUAAAUC 3.40%

15136 3UTR 1466 1478 AUCUUUUUGUGGUGUGAAUAAAUCU 3.49%

15137 3UTR 1467 1479 UCUUUUUGUGGUGUGAAUAAAUCUU 3.03% A549 0.1

Target Gene Gene SEQ ID hSPPl nM Duplex ID Region Ref Pos NO Sense Strand Sequence Activity

15138 3UTR 1468 1480 CUUUUUGUGGUGUGAAUAAAUCUUU 3.62%

15139 3UTR 1496 1481 CUUGAAUGUAAUAAGAAUUUGGUGG 61.48%

15140 3UTR 1497 1482 UUGAAUGUAAUAAGAAUUUGGUGGU 71.54%

15141 3UTR 1504 1483 UAAUAAGAAUUUGGUGGUGUCAAUU 58.54%

15142 3UTR 1511 1484 AAUUUGGUGGUGUCAAUUGCUUAUU 56.93%

15143 3UTR 1512 1485 AUUUGGUGGUGUCAAUUGCUUAUUU 81.22%

15144 3UTR 1513 1486 UUUGGUGGUGUCAAUUGCUUAUUUG 59.16%

15145 3UTR 1514 1487 UUGGUGGUGUCAAUUGCUUAUUUGU 59.46%

15146 3UTR 1540 1488 UUCCCACGGUUGUCCAGCAAUUAAU 67.40%

Table 10: hCTGF sd-rxRNA

17373 17036 mU.G.mU.mU.G.A.G.A.G.mU.G.mU.A.Chl 1523

17038 P.mU.A.fC.A.fC.fU.fC.fU.fC.A.A.fC.A*A*A*fU*A*A*A 1524

17374 17037 mU.mU.G.mU.mU.G.A.G.A.G.mU.G.mU.A.Chl 1525

17038 P.mU.A.fC.A.fC.fU.fC.fU.fC.A.A.fC.A*A*A*fU*A*A*A 1526

17375 17039 mU.G.mC.A.mC.mC.mU.mU.mU.mC.mU.A.A.Chl 1527

17041 P.mU.fU.A.G.A.A.A.G.G.fU.G.fC.A*A*A*fC*A*fU*G 1528

17376 17040 mU.mU.G.mC.A.mC.mC.mU.mU.mU.mC.mU.A.A.Chl 1529

17041 P.mU.fU.A.G.A.A.A.G.G.fU.G.fC.A*A*A*fC*A*fU*G 1530

17377 17042 mU.mU.G.A.G.mC.mU.mU.mU.mC.mU.G.A.Chl 1531

17043 P.mU.fC.A.G.A.A.A.G.fC.fU.fC.A.A*A*fC*fU*fU*G*A 1532

17378 17044 mU.G.A.G.A.G.mU.G.mU.G.A.mC.A.Chl 1533

17045 P.mU.G.fU.fC.A.fC.A.fC.fU.fC.fU.fC.A*A*fC*A*A*A*U 1534

17379 17046 A.G.mU.G.mU.G.A.mC.mC.A.A.A.A.Chl 1535

17048 P.mU.fU.fU.fU.G.G.fU.fC.A.fC.A.fC.fU*fC*fU*fC*A*A*C 1536

17380 17047 G.A.G.mU.G.mU.G.A.mC.mC.A.A.A.A.Chl 1537

17048 P.mU.fU.fU.fU.G.G.fU.fC.A.fC.A.fC.fU*fC*fU*fC*A*A*C 1538

17381 17049 G.mU.G.mU.G.A.mC.mC.A.A.A.A.A.Chl 1539

17050 P.mU.fU.fU.fU.fU.G.G.fU.fC.A.fC.A.fC*fU*fC*fU*fC*A*A 1540

17382 17051 mU.G.mU.G.A.mC.mC.A.A.A.A.G.A.Chl 1541

17053 P.mU.fC.fU.fU.fU.fU.G.G.fU.fC.A.fC.A*fC*fU*fC*fU*fC*A 1542

17383 17052 G.mU.G.mU.G.A.mC.mC.A.A.A.A.G.A.Chl 1543

17053 P.mU.fC.fU.fU.fU.fU.G.G.fU.fC.A.fC.A*fC*fU*fC*fU*fC*A 1544

17384 17054 G.mU.G.A.mC.mC.A.A.A.A.G.mU.A.Chl 1545

17055 P.mU.A.fC.fU.fU.fU.fU.G.G.fU.fC.A.fC*A*fC*fU*fC*fU*C 1546

17385 17056 G.A.mC.mC.A.A.A.A.G.mU.mU.A.A.Chl 1547

17057 P.mU.fU.A.A.fC.fU.fU.fU.fU.G.G.fU.fC*A*fC*A*fC*fU*C 1548

17386 17058 G.mC.A.mC.mC.mU.mU.mU.mC.mU.A.G.A.Chl 1549

17059 P.mU.fC.fU.A.G.A.A.A.G.G.fU.G.fC*A*A*A*fC*A*U 1550

17387 17060 mC.mC.mU.mU.mU.mC.mU.A.G.mU.mU.G.A.Chl 1551

17061 P.mU.fC.A.A.fC.fU.A.G.A.A.A.G.G*fU*G*fC*A*A*A 1552

Table 11 : Inhibition of gene expression with hCTGF ori sequences

Target Gene Gene Ref SEQ ID CTGF % Expression

Duplex Name Region Pos NO Sense sequence (0.1 nM)

14559 CDS 989 1570 UCCCAAAAUCUCCAAGCCUAUCAAA 64%

14560 CDS 990 1571 CCCAAAAUCUCCAAGCCUAUCAAGA 87%

14561 CDS 1002 1572 AAGCCUAUCAAGUUUGAGCUUUCUA 46%

14562 CDS 1003 1573 AGCCUAUCAAGUUUGAGCUUUCUGA 30%

14563 CDS 1004 1574 GCCUAUCAAGUUUGAGCUUUCUGGA 63%

14564 CDS 1008 1575 AUCAAGUUUGAGCUUUCUGGCUGCA 77%

14565 CDS 1025 1576 UGGCUGCACCAGCAUGAAGACAUAA 96%

14566 CDS 1028 1577 CUGCACCAGCAUGAAGACAUACCGA 79%

14567 CDS 1029 1578 UGCACCAGCAUGAAGACAUACCGAA 58%

14568 CDS 1033 1579 CCAGCAUGAAGACAUACCGAGCUAA 59%

14569 CDS 1035 1580 AGCAUGAAGACAUACCGAGCUAAAA 76%

14570 CDS 1036 1581 GCAUGAAGACAUACCGAGCUAAAUA 71%

14571 CDS 1050 1582 CGAGCUAAAUUCUGUGGAGUAUGUA 73%

14572 CDS 1051 1583 GAGCUAAAUUCUGUGGAGUAUGUAA 72%

14573 CDS 1053 1584 GCUAAAUUCUGUGGAGUAUGUACCA 87%

14574 CDS 1054 1585 CUAAAUUCUGUGGAGUAUGUACCGA 83%

14575 CDS 1135 1586 CUGACGGCGAGGUCAUGAAGAAGAA 77%

14576 CDS 1138 1587 ACGGCGAGGUCAUGAAGAAGAACAA 72%

14577 CDS 1139 1588 CGGCGAGGUCAUGAAGAAGAACAUA 85%

14578 CDS 1143 1589 GAGGUCAUGAAGAAGAACAUGAUGA 83%

14579 CDS 1145 1590 GGUCAUGAAGAAGAACAUGAUGUUA 91%

14580 CDS 1148 1591 CAUGAAGAAGAACAUGAUGUUCAUA 92%

14581 CDS 1157 1592 GAACAUGAUGUUCAUCAAGACCUGA 84%

14582 CDS 1161 1593 AUGAUGUUCAUCAAGACCUGUGCCA 92%

14583 CDS 1203 1594 GGAGACAAUGACAUCUUUGAAUCGA 62%

14584 CDS 1204 1595 GAGACAAUGACAUCUUUGAAUCGCA 56%

14585 CDS 1205 1596 AGACAAUGACAUCUUUGAAUCGCUA 30%

14586 CDS 1206 1597 GACAAUGACAUCUUUGAAUCGCUGA 47%

14587 CDS 1207 1598 ACAAUGACAUCUUUGAAUCGCUGUA 29%

14588 CDS 1208 1599 CAAUGACAUCUUUGAAUCGCUGUAA 50%

14589 CDS 1209 1600 AAUGACAUCUUUGAAUCGCUGUACA 39%

14590 CDS 1210 1601 AUGACAUCUUUGAAUCGCUGUACUA 44%

14591 CDS 1211 1602 UGACAUCUUUGAAUCGCUGUACUAA 39%

14592 CDS 1212 1603 GACAUCUUUGAAUCGCUGUACUACA 55%

14593 CDS 1213 1604 ACAUCUUUGAAUCGCUGUACUACAA 59%

14594 CDS 1216 1605 UCUUUGAAUCGCUGUACUACAGGAA 80%

14595 CDS 1217 1606 CUUUGAAUCGCUGUACUACAGGAAA 80%

14596 CDS 1223 1607 AUCGCUGUACUACAGGAAGAUGUAA 59%

14597 CDS 1224 1608 UCGCUGUACUACAGGAAGAUGUACA 62%

14598 CDS 1239 1609 AAGAUGUACGGAGACAUGGCAUGAA 59%

14599 CDS 1253 1610 CAUGGCAUGAAGCCAGAGAGUGAGA 65%

14600 3UTR 1266 1611 CAGAGAGUGAGAGACAUUAACUCAA 43%

14601 3UTR 1267 1612 AGAGAGUGAGAGACAUUAACUCAUA 25%

14602 3UTR 1268 1613 GAGAGUGAGAGACAUUAACUCAUUA 33%

14603 3UTR 1269 1614 AGAGUGAGAGACAUUAACUCAUUAA 42%

14604 3UTR 1270 1615 GAGUGAGAGACAUUAACUCAUUAGA 28%

14605 3UTR 1271 1616 AGUGAGAGACAUUAACUCAUUAGAA 34%

14606 3UTR 1272 1617 GUGAGAGACAUUAACUCAUUAGACA 30%

14607 3UTR 1273 1618 UGAGAGACAUUAACUCAUUAGACUA 33%

14608 3UTR 1275 1619 AGAGACAUUAACUCAUUAGACUGGA 42%

14609 3UTR 1277 1620 AGACAUUAACUCAUUAGACUGGAAA 25%

14610 3UTR 1278 1621 GACAUUAACUCAUUAGACUGGAACA 31%

14611 3UTR 1279 1622 ACAUUAACUCAUUAGACUGGAACUA 32% Target Gene Gene Ref SEQ ID CTGF % Expression

Duplex Name Region Pos NO Sense sequence (0.1 nM)

14612 3UTR 1281 1623 AUUAACUCAUUAGACUGGAACUUGA 23%

14613 3UTR 1284 1624 AACUCAUUAGACUGGAACUUGAACA 39%

14614 3UTR 1285 1625 ACUCAUUAGACUGGAACUUGAACUA 30%

14615 3UTR 1286 1626 CUCAUUAGACUGGAACUUGAACUGA 43%

14616 3UTR 1287 1627 UCAUUAGACUGGAACUUGAACUGAA 26%

14617 3UTR 1291 1628 UAGACUGGAACUUGAACUGAUUCAA 33%

14618 3UTR 1293 1629 GACUGGAACUUGAACUGAUUCACAA 43%

14619 3UTR 1294 1630 ACUGGAACUUGAACUGAUUCACAUA 28%

14620 3UTR 1295 1631 CUGGAACUUGAACUGAUUCACAUCA 41%

14621 3UTR 1296 1632 UGGAACUUGAACUGAUUCACAUCUA 34%

14622 3UTR 1298 1633 GAACUUGAACUGAUUCACAUCUCAA 31%

14623 3UTR 1299 1634 AACUUGAACUGAUUCACAUCUCAUA 31%

14624 3UTR 1300 1635 ACUUGAACUGAUUCACAUCUCAUUA 33%

14625 3UTR 1301 1636 CUUGAACUGAUUCACAUCUCAUUUA 28%

14626 3UTR 1326 1637 UCCGUAAAAAUGAUUUCAGUAGCAA 30%

14627 3UTR 1332 1638 AAAAUGAUUUCAGUAGCACAAGUUA 28%

14628 3UTR 1395 1639 CCCAAUUCAAAACAUUGUGCCAUGA 63%

14629 3UTR 1397 1640 CAAUUCAAAACAUUGUGCCAUGUCA 39%

14630 3UTR 1402 1641 CAAAACAUUGUGCCAUGUCAAACAA 34%

14631 3UTR 1408 1642 AUUGUGCCAUGUCAAACAAAUAGUA 33%

14632 3UTR 1409 1643 UUGUGCCAUGUCAAACAAAUAGUCA 33%

14633 3UTR 1412 1644 UGCCAUGUCAAACAAAUAGUCUAUA 36%

14634 3UTR 1416 1645 AUGUCAAACAAAUAGUCUAUCAACA 30%

14635 3UTR 1435 1646 UCAACCCCAGACACUGGUUUGAAGA 39%

14636 3UTR 1436 1647 CAACCCCAGACACUGGUUUGAAGAA 47%

14637 3UTR 1438 1648 ACCCCAGACACUGGUUUGAAGAAUA 45%

14638 3UTR 1439 1649 CCCCAGACACUGGUUUGAAGAAUGA 40%

14639 3UTR 1442 1650 CAGACACUGGUUUGAAGAAUGUUAA 21%

14640 3UTR 1449 1651 UGGUUUGAAGAAUGUUAAGACUUGA 22%

14641 3UTR 1453 1652 UUGAAGAAUGUUAAGACUUGACAGA 24%

14642 3UTR 1454 1653 UGAAGAAUGUUAAGACUUGACAGUA 37%

14643 3UTR 1462 1654 GUUAAGACUUGACAGUGGAACUACA 20%

14644 3UTR 1470 1655 UUGACAGUGGAACUACAUUAGUACA 30%

14645 3UTR 1471 1656 UGACAGUGGAACUACAUUAGUACAA 43%

14646 3UTR 1474 1657 CAGUGGAACUACAUUAGUACACAGA 36%

14647 3UTR 1475 1658 AGU GG AACU ACAU U AG U ACACAGCA 38%

14648 3UTR 1476 1659 GUGGAACUACAUUAGUACACAGCAA 35%

14649 3UTR 1477 1660 UGGAACUACAUUAGUACACAGCACA 34%

14650 3UTR 1478 1661 GGAACUACAUUAGUACACAGCACCA 33%

14651 3UTR 1479 1662 GAACUACAUUAGUACACAGCACCAA 39%

14652 3UTR 1480 1663 AACUACAUUAGUACACAGCACCAGA 27%

14653 3UTR 1481 1664 ACU ACAU U AG U ACACAGCACCAG AA 29%

14654 3UTR 1482 1665 CUACAUUAGUACACAGCACCAGAAA 38%

14655 3UTR 1483 1666 U ACAU U AG U ACACAGCACCAG AAU A 28%

14656 3UTR 1484 1667 ACAUUAGUACACAGCACCAGAAUGA 31%

14657 3UTR 1486 1668 AUUAGUACACAGCACCAGAAUGUAA 26%

14658 3UTR 1487 1669 UUAGUACACAGCACCAGAAUGUAUA 31%

14659 3UTR 1489 1670 AGUACACAGCACCAGAAUGUAUAUA 35%

14660 3UTR 1490 1671 GUACACAGCACCAGAAUGUAUAUUA 34%

14661 3UTR 1497 1672 GCACCAGAAUGUAUAUUAAGGUGUA 32%

14662 3UTR 1503 1673 GAAUGUAUAUUAAGGUGUGGCUUUA 42%

14663 3UTR 1539 1674 AGGGUACCAGCAGAAAGGUUAGUAA 28%

14664 3UTR 1543 1675 UACCAGCAGAAAGGUUAGUAUCAUA 29% Target Gene Gene Ref SEQ ID CTGF % Expression

Duplex Name Region Pos NO Sense sequence (0.1 nM)

14665 3UTR 1544 1676 ACCAGCAGAAAGGUUAGUAUCAUCA 33%

14666 3UTR 1548 1677 GCAGAAAGGUUAGUAUCAUCAGAUA 34%

14667 3UTR 1557 1678 UUAGUAUCAUCAGAUAGCAUCUUAA 22%

14668 3UTR 1576 1679 UCUUAUACGAGUAAUAUGCCUGCUA 48%

14669 3UTR 1577 1680 CUUAUACGAGUAAUAUGCCUGCUAA 31%

14670 3UTR 1579 1681 UAUACGAGUAAUAUGCCUGCUAUUA 43%

14671 3UTR 1580 1682 AUACGAGUAAUAUGCCUGCUAUUUA 39%

14672 3UTR 1581 1683 UACGAGUAAUAUGCCUGCUAUUUGA 33%

14673 3UTR 1582 1684 ACGAGUAAUAUGCCUGCUAUUUGAA 40%

14674 3UTR 1584 1685 GAGUAAUAUGCCUGCUAUUUGAAGA 38%

14675 3UTR 1585 1686 AGUAAUAUGCCUGCUAUUUGAAGUA 24%

14676 3UTR 1586 1687 GUAAUAUGCCUGCUAUUUGAAGUGA 34%

14677 3UTR 1587 1688 UAAUAUGCCUGCUAUUUGAAGUGUA 26%

14678 3UTR 1589 1689 AUAUGCCUGCUAUUUGAAGUGUAAA 26%

14679 3UTR 1591 1690 AUGCCUGCUAUUUGAAGUGUAAUUA 25%

14680 3UTR 1596 1691 UGCUAUUUGAAGUGUAAUUGAGAAA 36%

14681 3UTR 1599 1692 UAUUUGAAGUGUAAUUGAGAAGGAA 22%

14682 3UTR 1600 1693 AUUUGAAGUGUAAUUGAGAAGGAAA 22%

14683 3UTR 1601 1694 UUUGAAGUGUAAUUGAGAAGGAAAA 19%

14684 3UTR 1609 1695 GUAAUUGAGAAGGAAAAUUUUAGCA 53%

14685 3UTR 1610 1696 UAAUUGAGAAGGAAAAUUUUAGCGA 55%

14686 3UTR 1611 1697 AAUUGAGAAGGAAAAUUUUAGCGUA 20%

14687 3UTR 1612 1698 AUUGAGAAGGAAAAUUUUAGCGUGA 23%

14688 3UTR 1613 1699 UUGAGAAGGAAAAUUUUAGCGUGCA 37%

14689 3UTR 1614 1700 UGAGAAGGAAAAUUUUAGCGUGCUA 31%

14690 3UTR 1619 1701 AGGAAAAUUUUAGCGUGCUCACUGA 46%

14691 3UTR 1657 1702 CCAGUGACAGCUAGGAUGUGCAUUA 42%

14692 3UTR 1661 1703 UGACAGCUAGGAUGUGCAUUCUCCA 39%

14693 3UTR 1682 1704 UCCAGCCAUCAAGAGACUGAGUCAA 53%

14694 3UTR 1685 1705 AGCCAUCAAGAGACUGAGUCAAGUA 71%

14695 3UTR 1686 1706 GCCAUCAAGAGACUGAGUCAAGUUA 54%

14696 3UTR 1687 1707 CCAUCAAGAGACUGAGUCAAGUUGA 71%

14697 3UTR 1688 1708 CAUCAAGAGACUGAGUCAAGUUGUA 74%

14698 3UTR 1689 1709 AUCAAGAGACUGAGUCAAGUUGUUA 61%

14699 3UTR 1690 1710 UCAAGAGACUGAGUCAAGUUGUUCA 59%

14700 3UTR 1691 1711 CAAGAGACUGAGUCAAGUUGUUCCA 73%

14701 3UTR 1692 1712 AAGAGACUGAGUCAAGUUGUUCCUA 78%

14702 3UTR 1693 1713 AGAGACUGAGUCAAGUUGUUCCUUA 60%

14703 3UTR 1695 1714 AGACUGAGUCAAGUUGUUCCUUAAA 63%

14704 3UTR 1696 1715 GACUGAGUCAAGUUGUUCCUUAAGA 92%

14705 3UTR 1697 1716 ACUGAGUCAAGUUGUUCCUUAAGUA 74%

14706 3UTR 1707 1717 GUUGUUCCUUAAGUCAGAACAGCAA 70%

14707 3UTR 1724 1718 AACAGCAGACUCAGCUCUGACAUUA 69%

14708 3UTR 1725 1719 ACAGCAGACUCAGCUCUGACAUUCA 67%

14709 3UTR 1726 1720 CAGCAGACUCAGCUCUGACAUUCUA 71%

14710 3UTR 1727 1721 AGCAGACUCAGCUCUGACAUUCUGA 73%

14711 3UTR 1728 1722 GCAGACUCAGCUCUGACAUUCUGAA 60%

14712 3UTR 1729 1723 CAGACUCAGCUCUGACAUUCUGAUA 72%

14713 3UTR 1732 1724 ACUCAGCUCUGACAUUCUGAUUCGA 24%

14714 3UTR 1733 1725 CUCAGCUCUGACAUUCUGAUUCGAA 32%

14715 3UTR 1734 1726 UCAGCUCUGACAUUCUGAUUCGAAA 23%

14716 3UTR 1735 1727 CAGCUCUGACAUUCUGAUUCGAAUA 27%

14717 3UTR 1736 1728 AGCUCUGACAUUCUGAUUCGAAUGA 38% Target Gene Gene Ref SEQ ID CTGF % Expression

Duplex Name Region Pos NO Sense sequence (0.1 nM)

14718 3UTR 1739 1729 UCUGACAUUCUGAUUCGAAUGACAA 28%

14719 3UTR 1741 1730 UGACAU UCUGAU UCGAAUGACACUA 29%

14720 3UTR 1742 1731 GACAUUCUGAUUCGAAUGACACUGA 33%

14721 3UTR 1743 1732 ACAUUCUGAUUCGAAUGACACUGUA 28%

14722 3UTR 1747 1733 UCUGAUUCGAAUGACACUGUUCAGA 39%

14723 3UTR 1748 1734 CUGAUUCGAAUGACACUGUUCAGGA 36%

14724 3UTR 1750 1735 GAUUCGAAUGACACUGUUCAGGAAA 33%

14725 3UTR 1751 1736 AUUCGAAUGACACUGUUCAGGAAUA 30%

14726 3UTR 1759 1737 GACACUGUUCAGGAAUCGGAAUCCA 34%

14727 3UTR 1760 1738 ACACUGUUCAGGAAUCGGAAUCCUA 35%

14728 3UTR 1761 1739 CACUGUUCAGGAAUCGGAAUCCUGA 40%

14729 3UTR 1768 1740 CAGGAAUCGGAAUCCUGUCGAUUAA 34%

14730 3UTR 1769 1741 AGGAAUCGGAAUCCUGUCGAUUAGA 31%

14731 3UTR 1770 1742 GGAAUCGGAAUCCUGUCGAUUAGAA 24%

14732 3UTR 1771 1743 GAAUCGGAAUCCUGUCGAUUAGACA 32%

14733 3UTR 1772 1744 AAUCGGAAUCCUGUCGAUUAGACUA 29%

14734 3UTR 1774 1745 UCGGAAUCCUGUCGAUUAGACUGGA 34%

14735 3UTR 1777 1746 GAAUCCUGUCGAUUAGACUGGACAA 51%

14736 3UTR 1782 1747 CUGUCGAUUAGACUGGACAGCUUGA 88%

14737 3UTR 1783 1748 UGUCGAUUAGACUGGACAGCUUGUA 38%

14738 3UTR 1797 1749 GACAGCUUGUGGCAAGUGAAUUUGA 46%

14739 3UTR 1798 1750 ACAGCUUGUGGCAAGUGAAUUUGCA 52%

14740 3UTR 1800 1751 AGCUUGUGGCAAGUGAAUUUGCCUA 43%

14741 3UTR 1801 1752 GCUUGUGGCAAGUGAAUUUGCCUGA 51%

14742 3UTR 1802 1753 CUUGUGGCAAGUGAAUUUGCCUGUA 32%

14743 3UTR 1803 1754 UUGUGGCAAGUGAAUUUGCCUGUAA 31%

14744 3UTR 1804 1755 UGUGGCAAGUGAAUUUGCCUGUAAA 29%

14745 3UTR 1805 1756 GUGGCAAGUGAAUUUGCCUGUAACA 20%

14746 3UTR 1806 1757 UGGCAAGUGAAUUUGCCUGUAACAA 34%

14747 3UTR 1807 1758 GGCAAGUGAAUUUGCCUGUAACAAA 31%

14748 3UTR 1808 1759 GCAAGUGAAUUUGCCUGUAACAAGA 27%

14749 3UTR 1809 1760 CAAG UGAAUUUGCCUGU AACAAG CA 34%

14750 3UTR 1810 1761 AAGUGAAUUUGCCUGUAACAAGCCA 36%

14751 3UTR 1811 1762 AGUGAAUUUGCCUGUAACAAGCCAA 31%

14752 3UTR 1814 1763 GAAUUUGCCUGUAACAAGCCAGAUA 24%

14753 3UTR 1815 1764 AAUUUGCCUGUAACAAGCCAGAUUA 21%

14754 3UTR 1816 1765 AUUUGCCUGUAACAAGCCAGAUUUA 22%

14755 3UTR 1910 1766 AAGUUAAUUUAAAGUUGUUUGUGCA 58%

14756 3UTR 1911 1767 AGUUAAUUUAAAGUUGUUUGUGCCA 73%

14757 3UTR 1912 1768 GUUAAUUUAAAGUUGUUUGUGCCUA 64%

14758 3UTR 1957 1769 UUUGAUAUUUCAAUGUUAGCCUCAA 42%

14759 3UTR 1961 1770 AUAUUUCAAUGUUAGCCUCAAUUUA 30%

14760 3UTR 1971 1771 GUUAGCCUCAAUUUCUGAACACCAA 34%

14761 3UTR 1974 1772 AGCCUCAAUUUCUGAACACCAUAGA 35%

14762 3UTR 1975 1773 GCCUCAAUUUCUGAACACCAUAGGA 33%

14763 3UTR 1976 1774 CCUCAAUUUCUGAACACCAUAGGUA 39%

14764 3UTR 1977 1775 CUCAAUUUCUGAACACCAUAGGUAA 27%

14765 3UTR 1978 1776 UCAAUUUCUGAACACCAUAGGUAGA 31%

14766 3UTR 1979 1777 CAAUUUCUGAACACCAUAGGUAGAA 49%

14767 3UTR 1980 1778 AAUUUCUGAACACCAUAGGUAGAAA 46%

14768 3UTR 1981 1779 AUUUCUGAACACCAUAGGUAGAAUA 40%

14769 3UTR 1982 1780 UUUCUGAACACCAUAGGUAGAAUGA 47%

14770 3UTR 1985 1781 CUGAACACCAUAGGUAGAAUGUAAA 33% Target Gene Gene Ref SEQ ID CTGF % Expression

Duplex Name Region Pos NO Sense sequence (0.1 nM)

14771 3UTR 1986 1782 UGAACACCAUAGGUAGAAUGUAAAA 35%

14772 3UTR 1987 1783 GAACACCAUAGGUAGAAUGUAAAGA 31%

14773 3UTR 1988 1784 AACACCAUAGGUAGAAUGUAAAGCA 30%

14774 3UTR 1989 1785 ACACCAUAGGUAGAAUGUAAAGCUA 32%

14775 3UTR 1991 1786 ACCAUAGGUAGAAUGUAAAGCUUGA 31%

14776 3UTR 1992 1787 CCAUAGGUAGAAUGUAAAGCUUGUA 34%

14777 3UTR 1993 1788 CAUAGGUAGAAUGUAAAGCUUGUCA 31%

14778 3UTR 1994 1789 AUAGGUAGAAUGUAAAGCUUGUCUA 28%

14779 3UTR 1996 1790 AGGUAGAAUGUAAAGCUUGUCUGAA 32%

14780 3UTR 2002 1791 AAUGUAAAGCUUGUCUGAUCGUUCA 34%

14781 3UTR 2017 1792 UGAUCGUUCAAAGCAUGAAAUGGAA 31%

14782 3UTR 2021 1793 CGUUCAAAGCAUGAAAUGGAUACUA 39%

14783 3UTR 2022 1794 GUUCAAAGCAUGAAAUGGAUACUUA 25%

14784 3UTR 2023 1795 UUCAAAGCAUGAAAUGGAUACUUAA 22%

14785 3UTR 2047 1796 UAUGGAAAUUCUGCUCAGAUAGAAA 39%

14786 3UTR 2048 1797 AUGGAAAUUCUGCUCAGAUAGAAUA 35%

14787 3UTR 2059 1798 GCUCAGAUAGAAUGACAGUCCGUCA 44%

14788 3UTR 2060 1799 CUCAGAUAGAAUGACAGUCCGUCAA 41%

14789 3UTR 2062 1800 CAGAUAGAAUGACAGUCCGUCAAAA 46%

14790 3UTR 2063 1801 AGAUAGAAUGACAGUCCGUCAAAAA 45%

14791 3UTR 2065 1802 AUAGAAUGACAGUCCGUCAAAACAA 41%

14792 3UTR 2067 1803 AGAAUGACAGUCCGUCAAAACAGAA 36%

14793 3UTR 2068 1804 GAAUGACAGUCCGUCAAAACAGAUA 40%

14794 3UTR 2113 1805 AGUGUCCUUGGCAGGCUGAUUUCUA 42%

14795 3UTR 2114 1806 GUGUCCUUGGCAGGCUGAUUUCUAA 42%

14796 3UTR 2118 1807 CCUUGGCAGGCUGAUUUCUAGGUAA 111%

14797 3UTR 2127 1808 GCUGAUUUCUAGGUAGGAAAUGUGA 44%

14798 3UTR 2128 1809 CUGAUUUCUAGGUAGGAAAUGUGGA 44%

14799 3UTR 2130 1810 GAUUUCUAGGUAGGAAAUGUGGUAA 46%

14800 3UTR 2131 1811 AUUUCUAGGUAGGAAAUGUGGUAGA 45%

14801 3UTR 2142 1812 GGAAAUGUGGUAGCCUCACUUUUAA 37%

14802 3UTR 2146 1813 AUGUGGUAGCCUCACUUUUAAUGAA 39%

14803 3UTR 2149 1814 UGGUAGCCUCACUUUUAAUGAACAA 40%

14804 3UTR 2154 1815 GCCUCACUUUUAAUGAACAAAUGGA 35%

14805 3UTR 2155 1816 CCUCACUUUUAAUGAACAAAUGGCA 41%

14806 3UTR 2181 1817 UUAUUAAAAACUGAGUGACUCUAUA 26%

14807 3UTR 2182 1818 UAUUAAAAACUGAGUGACUCUAUAA 29%

14808 3UTR 2183 1819 AUUAAAAACUGAGUGACUCUAUAUA 28%

14809 3UTR 2186 1820 AAAAACUGAGUGACUCUAUAUAGCA 31%

14810 3UTR 2187 1821 AAAACUGAGUGACUCUAUAUAGCUA 28%

14811 3UTR 2188 1822 AAACUGAGUGACUCUAUAUAGCUGA 38%

14812 3UTR 2189 1823 AACUGAGUGACUCUAUAUAGCUGAA 44%

14813 3UTR 2190 1824 ACUGAGUGACUCUAUAUAGCUGAUA 38%

14814 3UTR 2255 1825 ACUGUUUUUCGGACAGUUUAUUUGA 29%

14815 3UTR 2256 1826 CUGUUUUUCGGACAGUUUAUUUGUA 25%

14816 3UTR 2263 1827 UCGGACAGUUUAUUUGUUGAGAGUA 29%

14817 3UTR 2265 1828 GGACAGUUUAUUUGUUGAGAGUGUA 24%

14818 3UTR 2268 1829 CAGUUUAUUUGUUGAGAGUGUGACA 26%

14819 3UTR 2269 1830 AGUUUAUUUGUUGAGAGUGUGACCA 37%

14820 3UTR 2272 1831 UUAUUUGUUGAGAGUGUGACCAAAA 27%

14821 3UTR 2273 1832 UAUUUGUUGAGAGUGUGACCAAAAA 30%

14822 3UTR 2274 1833 AUUUGUUGAGAGUGUGACCAAAAGA 26%

14823 3UTR 2275 1834 UUUGUUGAGAGUGUGACCAAAAGUA 27% Target Gene Gene Ref SEQ ID CTGF % Expression Duplex Name Region Pos NO Sense sequence (0.1 nM)

14824 3UTR 2276 1835 UUGUUGAGAGUGUGACCAAAAGUUA 30%

14825 3UTR 2277 1836 UGUUGAGAGUGUGACCAAAAGUUAA 29%

14826 3UTR 2278 1837 GUUGAGAGUGUGACCAAAAGUUACA 33%

14827 3UTR 2279 1838 U U GAG AG UG UG ACCAAAAG UUACAA 35%

14828 3UTR 2281 1839 GAGAGUGUGACCAAAAGUUACAUGA 36%

14829 3UTR 2282 1840 AGAGUGUGACCAAAAGUUACAUGUA 36%

14830 3UTR 2283 1841 GAGUGUGACCAAAAGUUACAUGUUA 33%

14831 3UTR 2284 1842 AGUGUGACCAAAAGUUACAUGUUUA 31%

14832 3UTR 2285 1843 GUGUGACCAAAAGUUACAUGUUUGA 22%

14833 3UTR 2286 1844 UGUGACCAAAAGUUACAUGUUUGCA 40%

14834 3UTR 2293 1845 AAAAGUUACAUGUUUGCACCUUUCA 24%

14835 3UTR 2295 1846 AAGUUACAUGUUUGCACCUUUCUAA 23%

14836 3UTR 2296 1847 AGUUACAUGUUUGCACCUUUCUAGA 29%

14837 3UTR 2299 1848 UACAUGUUUGCACCUUUCUAGUUGA 27%

14838 3UTR 2300 1849 ACAUGUUUGCACCUUUCUAGUUGAA 29%

14839 3UTR 2301 1850 CAUGUUUGCACCUUUCUAGUUGAAA 35%

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 SS, A549 0.1

ID Region Pos NO replaced with A) nM Activity

13850 3UTR 2262 1858 UUCGGACAGUUUAUUUGUUGAGAGU 50%

13851 CDS 1147 1859 UCAUGAAGAAGAACAUGAUGUUCAU 104%

13852 3UTR 2163 1860 UUAAUGAACAAAUGGCCUUUAUUAA 54%

13853 3UTR 1414 1861 CCAUGUCAAACAAAUAGUCUAUCAA 35%

13854 CDS 1195 1862 ACUGUCCCGGAGACAAUGACAUCUU 103%

13855 3UTR 1788 1863 AUUAGACUGGACAGCUUGUGGCAAG 103%

13856 3UTR 1793 1864 ACUGGACAGCUUGUGGCAAGUGAAU 81%

13857 3UTR 1891 1865 UAUAUAUGUACAGUUAUCUAAGUUA 73%

13858 3UTR 2270 1866 GUUUAUUUGUUGAGAGUGUGACCAA 76%

13859 482 1867 CAAGAUCGGCGUGUGCACCGCCAAA 95%

13860 CDS 942 1868 GCUGACCUGGAAGAGAACAUUAAGA 93%

13861 CDS 1199 1869 UCCCGGAGACAAUGACAUCUUUGAA 83%

13862 3UTR 2258 1870 GUUUUUCGGACAGUUUAUUUGUUGA 40%

13863 CDS 1201 1871 CCGGAGACAAUGACAUCUUUGAAUC 123%

13864 CDS 543 1872 CGCAGCGGAGAGUCCUUCCAGAGCA 124%

13865 3UTR 1496 1873 AGCACCAGAAUGUAUAUUAAGGUGU 109%

13866 CDS 793 1874 UGAUUAGAGCCAACUGCCUGGUCCA 125%

13867 CDS 1198 1875 GUCCCGGAGACAAUGACAUCUUUGA 64%

13868 3UTR 2160 1876 CUUUUAAUGAACAAAUGGCCUUUAU 68%

13869 CDS 1149 1877 AUGAAGAAGAACAUGAUGUUCAUCA 107%

13870 CDS 1244 1878 GUACGGAGACAUGGCAUGAAGCCAG 107%

13871 3UTR 1495 1879 CAGCACCAGAAUGUAUAUUAAGGUG 77%

13872 475 1880 CCAACCGCAAGAUCGGCGUGUGCAC 113%

13873 CDS 806 1881 CUGCCUGGUCCAGACCACAGAGUGG 113%

13874 CDS 819 1882 ACCACAGAGUGGAGCGCCUGUUCCA 99%

13875 CDS 1221 1883 GAAUCGCUGUACUACAGGAAGAUGU 97%

13876 CDS 1152 1884 AAGAAGAACAUGAUGUUCAUCAAGA 121%

13877 CDS 1163 1885 GAUGUUCAUCAAGACCUGUGCCUGC 125%

13878 3UTR 1494 1886 ACAGCACCAGAAUGUAUAUUAAGGU 94%

13879 3UTR 1890 1887 AUAUAUAUGUACAGUUAUCUAAGUU 94%

13880 473 1888 GGCCAACCGCAAGAUCGGCGUGUGC 122%

13881 544 1889 GCAGCGGAGAGUCCUUCCAGAGCAG 111%

13882 CDS 883 1890 ACAACGCCUCCUGCAGGCUAGAGAA 105%

13883 CDS 1240 1891 AGAUGUACGGAGACAUGGCAUGAAG 99%

13884 CDS 1243 1892 UGUACGGAGACAUGGCAUGAAGCCA 116%

13885 3UTR 2266 1893 GACAGUUUAUUUGUUGAGAGUGUGA 53%

13886 CDS 1011 1894 AAGUUUGAGCUUUCUGGCUGCACCA 118%

13887 CDS 1020 1895 CUUUCUGGCUGCACCAGCAUGAAGA 110%

13888 CDS 1168 1896 UCAUCAAGACCUGUGCCUGCCAUUA 119%

13889 1415 1897 CAUGUCAAACAAAUAGUCUAUCAAC 64%

13890 3UTR 1792 1898 GACUGGACAGCUUGUGGCAAGUGAA 53%

13891 3UTR 2156 1899 CUCACUUUUAAUGAACAAAUGGCCU 119%

13892 379 1900 GCUGCCGCGUCUGCGCCAAGCAGCU 112%

13893 CDS 1229 1901 GUACUACAGGAAGAUGUACGGAGAC 112%

13894 3UTR 1791 1902 AGACUGGACAGCUUGUGGCAAGUGA 65%

13895 3UTR 2158 1903 CACUUUUAAUGAACAAAUGGCCUUU 76%

13896 488 1904 CGGCGUGUGCACCGCCAAAGAUGGU 89%

13897 CDS 1151 1905 GAAGAAGAACAUGAUGUUCAUCAAG 119%

13898 CDS 1156 1906 AGAACAUGAUGUUCAUCAAGACCUG 125%

13899 CDS 1237 1907 GGAAGAUGUACGGAGACAUGGCAUG 114%

13900 CDS 1202 1908 CGGAGACAAUGACAUCUUUGAAUCG 130%

13901 CDS 1236 1909 AGGAAGAUGUACGGAGACAUGGCAU 135% Oligo Gene Ref SEQ ID 25-mer Sense Strand (position 25 of SS, A549 0.1 ID Region Pos NO replaced with A) nM Activity

13902 3UTR 1786 1910 CGAUUAGACUGGACAGCUUGUGGCA 119%

13903 3UTR 1789 1911 UUAGACUGGACAGCUUGUGGCAAGU 108%

13904 3UTR 2290 1912 AC C AAA AG U U ACAUG U U UGCACCU U 90%

13905 CDS 1017 1913 GAGCUUUCUGGCUGCACCAGCAUGA 121%

13906 CDS 1197 1914 UGUCCCGGAGACAAUGACAUCUUUG 125%

13907 CDS 1219 1915 UUGAAUCGCUGUACUACAGGAAGAU 98%

13908 3UTR 2159 1916 ACUUUUAAUGAACAAAUGGCCUUUA 52%

13909 486 1917 AUCGGCGUGUGCACCGCCAAAGAUG 119%

13910 CDS 826 1918 AGUGGAGCGCCUGUUCCAAGACCUG 139%

13911 CDS 1022 1919 UUCUGGCUGCACCAGCAUGAAGACA 144%

13912 3UTR 1492 1920 ACACAGCACCAGAAUGUAUAUUAAG 99%

13913 3UTR 1781 1921 CCUGUCGAUUAGACUGGACAGCUUG 89%

13914 485 1922 GAUCGGCGUGUGCACCGCCAAAGAU 131%

13915 CDS 1007 1923 UAUCAAGUUUGAGCUUUCUGGCUGC 92%

13916 CDS 1242 1924 AUGUACGGAGACAUGGCAUGAAGCC 106%

13917 3UTR 1787 1925 GAUUAGACUGGACAGCUUGUGGCAA 104%

13918 3UTR 1889 1926 UAUAUAUAUGUACAGUUAUCUAAGU 78%

13919 3UTR 2294 1927 AAAGUUACAUGUUUGCACCUUUCUA 28%

13920 CDS 821 1928 CACAGAGUGGAGCGCCUGUUCCAAG 108%

13921 CDS 884 1929 CAACGCCUCCUGCAGGCUAGAGAAG 125%

13922 3UTR 2260 1930 UUUUCGGACAGUUUAUUUGUUGAGA 43%

13923 CDS 889 1931 CCUCCUGCAGGCUAGAGAAGCAGAG 95%

13924 CDS 1226 1932 GCUGUACUACAGGAAGAUGUACGGA 122%

13925 3UTR 1493 1933 CACAGCACCAGAAUGUAUAUUAAGG 88%

13926 3UTR 1799 1934 CAGCUUGUGGCAAGUGAAUUUGCCU 89%

13927 CDS 807 1935 UGCCUGGUCCAGACCACAGAGUGGA 101%

13928 CDS 1107 1936 ACCACCCUGCCGGUGGAGUUCAAGU 113%

13929 CDS 1155 1937 AAGAACAUGAUGUUCAUCAAGACCU 109%

13930 CDS 1169 1938 CAUCAAGACCUGUGCCUGCCAUUAC 89%

13931 CDS 1241 1939 GAUGUACGGAGACAUGGCAUGAAGC 96%

13932 3UTR 1794 1940 CUGGACAGCUUGUGGCAAGUGAAUU 73%

13933 3UTR 1888 1941 AUAUAUAUAUGUACAGUUAUCUAAG 98%

13934 3UTR 2289 1942 GACCAAAAGUUACAUGUUUGCACCU 77%

13935 373 1943 GCGGCUGCUGCCGCGUCUGCGCCAA 85%

13936 CDS 799 1944 GAGCCAACUGCCUGGUCCAGACCAC 126%

13937 CDS 802 1945 CCAACUGCCUGGUCCAGACCACAGA 122%

13938 CDS 1166 1946 GUUCAUCAAGACCUGUGCCUGCCAU 106%

Table 13: Inhibition of gene expression with SPPl sd-rxRNA sequences (Accession Number: NM_000582.2)

% remaining

Oligo Start SEQ ID SEQ ID expression (1

Number Site NO Sense sequence NO Antisense sequence uM A549)

UUUAAUUGACCUCAG

14086 1051 1951 GAGGUCAAUUAAA 1952 AAGA n/a

AAUUGACCUCAGAAG

14087 1048 1953 UCUGAGGUCAAUU 1954 AUGC 69%

UUAAUUGACCUCAGA

14088 1050 1955 UGAGGUCAAUUAA 1956 AGAU 76%

AUUGACCUCAGAAGA

14089 1047 1957 UUCUGAGGUCAAU 1958 UGCA 60%

UCAUCCAGCUGACUC

14090 800 1959 GUCAGCUGGAUGA 1960 GUUU 71%

AGAUUCAUCAGAAUG

14091 492 1961 UUCUGAUGAAUCU 1962 GUGA n/a

UGACCUCAGUCCAUA

14092 612 1963 UGGACUGAGGUCA 1964 AACC n/a

AAUGGUGAGACUCAU

14093 481 1965 GAGUCUCACCAUU 1966 CAGA n/a

UUUGACCUCAGUCCA

14094 614 1967 GACUGAGGUCAAA 1968 UAAA n/a

UUCAUGGCUGUGAAA

14095 951 1969 UCACAGCCAUGAA 1970 UUCA 89%

GAAUGGUGAGACUCA

14096 482 1971 AGUCUCACCAUUC 1972 UCAG 87%

UGGCUUUCCGCUUAU

14097 856 1973 AAGCGGAAAGCCA 1974 AUAA 88%

UUGGCUUUCCGCUUA

14098 857 1975 AGCGGAAAGCCAA 1976 UAUA 113%

UCAUCCAUGUGGUCA

14099 365 1977 ACCACAUGGAUGA 1978 UGGC 98%

AUGUGGUCAUGGCU

14100 359 1979 GCCAUGACCACAU 1980 UUCGU 84%

GUGGUCAUGGCUUU

14101 357 1981 AAGCCAUGACCAC 1982 CGUUG 88%

AUUGGCUUUCCGCUU

14102 858 1983 GCGGAAAGCCAAU 1984 AUAU n/a

AAAUUUCGUAUU AAAUACGAAAUUUCA

14103 1012 1985 U 1986 GGUG 93%

AUUUCGUAUUUC AG AAAU ACGAAAU U U

14104 1014 1987 U 1988 CAGG 89%

UGGUCAUGGCUUUC

14105 356 1989 AAAGCCAUGACCA 1990 GUUGG 85%

AUAUCAUCCAUGUGG

14106 368 1991 ACAUGGAUGAUAU 1992 UCAU 67%

GAAAUUUCGUAU AAUACGAAAUUUCAG

14107 1011 1993 U 1994 GUGU 87%

AAUCAGAAGGCGCGU

14108 754 1995 GCGCCUUCUGAUU 1996 UCAG 73%

AUUCAUGAGAAAUAC

14109 1021 1997 AUUUCUCAUGAAU 1998 GAAA 128%

CUAUUCAUGAGAGAA

14110 1330 1999 CUCUCAUGAAUAG 2000 UAAC 101%

UUUCGUUGGACUUA

14111 346 2001 AAGUCCAACGAAA 2002 CUUGG 59% % remaining

Oligo Start SEQ ID SEQ ID expression (1

Number Site NO Sense sequence NO Antisense sequence uM A549)

UUGCUCUCAUCAUUG

14112 869 2003 AUGAUGAGAGCAA 2004 GCUU 89%

UUCAACUCCUCGCUU

14113 701 2005 GCGAGGAGUUGAA 2006 UCCA 95%

UGAUUGAUAGUC UGACUAUCAAUCACA

14114 896 2007 A 2008 UCGG 87%

AGAUGCACUAUCUAA

14115 1035 2009 AGAUAGUGCAUCU 2010 UUCA 82%

AUGUGUAUCUAU AAUAGAUACACAUUC

14116 1170 2011 U 2012 AACC 36%

UUCUUCUAUAGAAU

14117 1282 2013 UUCUAUAGAAGAA 2014 GAACA 91%

AAUUGCUGGACAACC

14118 1537 2015 UUGUCCAGCAAUU 2016 GUGG 152%

UCGCUUUCCAUGUGU

14119 692 2017 ACAUGGAAAGCGA 2018 GAGG n/a

UAAUCUGGACUGCUU

14120 840 2019 GCAGUCCAGAUUA 2020 GUGG 87%

UGGUUGAAUGUG ACACAUUCAACCAAU

14121 1163 2021 U 2022 AAAC 31%

ACUCGUUUCAUAACU

14122 789 2023 UUAUGAAACGAGU 2024 GUCC 96%

AUAAUCUGGACUGCU

14123 841 2025 CAGUCCAGAUUAU 2026 UGUG 110%

UUUCCGCUUAUAUAA

14124 852 2027 AUAUAAGCGGAAA 2028 UCUG 91%

UGUUUAACUGGUAU

14125 209 2029 UACCAGUUAAACA 2030 GGCAC 110%

UGUUCAUUCUAU UAUAGAAUGAACAUA

14126 1276 2031 A 2032 GACA n/a

UUUCCUUGGUCGGC

14127 137 2033 CCGACCAAGGAAA 2034 GUUUG 71%

GUAUGCACCAUUCAA

14128 711 2035 GAAUGGUGCAUAC 2036 CUCC 115%

UCGGCCAUCAUAUGU

14129 582 2037 AUAUGAUGGCCGA 2038 GUCU 97%

AAUCUGGACUGCUUG

14130 839 2039 AGCAGUCCAGAUU 2040 UGGC 102%

UUUGACUAAAUGCAA

14131 1091 2041 GCAUUUAGUCAAA 2042 AGUG 10%

ACAUCGGAAUGCUCA

14132 884 2043 AGCAUUCCGAUGU 2044 UUGC 93%

AAGUUCCUGACUAUC

14133 903 2045 UAGUCAGGAACUU 2046 AAUC 97%

UUGACUAAAUGCAAA

14134 1090 2047 UGCAUUUAGUCAA 2048 GUGA 39%

GUCUGAUGAGUC AGACUCAUCAGACUG

14135 474 2049 U 2050 GUGA 99%

UCAUAUGUGUCUACU

14136 575 2051 UAGACACAUAUGA 2052 GUGG 108%

AUGUCCUCGUCUGUA

14137 671 2053 CAGACGAGGACAU 2054 GCAU 98% % remaining

Oligo Start SEQ ID SEQ ID expression (1

Number Site NO Sense sequence NO Antisense sequence uM A549)

GAAUUCACGGCUGAC

14138 924 2055 CAGCCGUGAAUUC 2056 UUUG 100%

UUAUUUCCAGACUCA

14139 1185 2057 AGUCUGGAAAUAA 2058 AAUA 47%

AGUUUGUGGCUU GAAGCCACAAACUAA

14140 1221 2059 C 2060 ACUA 100%

CUUUCGUUGGACUU

14141 347 2061 AGUCCAACGAAAG 2062 ACUUG 103%

GUCUGCGAAACUUCU

14142 634 2063 AAGUUUCGCAGAC 2064 UAGA 100%

AAUGCUCAUUGCUCU

14143 877 2065 AGCAAUGAGCAUU 2066 CAUC 104%

UUAGAUAGUGCA AUGCACUAUCUAAUU

14144 1033 2067 U 2068 CAUG 95%

CUUGUAUGCACCAUU

14145 714 2069 UGGUGCAUACAAG 2070 CAAC 101%

UGACUCGUUUCAUAA

14146 791 2071 AUGAAACGAGUCA 2072 CUGU 100%

UUCAGCACUCUGGUC

14147 813 2073 CCAGAGUGCUGAA 2074 AUCC 97%

AAAUUCAUGGCUGUG

14148 939 2075 CAGCCAUGAAUUU 2076 GAAU 109%

AUUGGUUGAAUG ACAUUCAACCAAUAA

14149 1161 2077 U 2078 ACUG 34%

GGUUGAAUGUGU UACACAUUCAACCAA

14150 1164 2079 A 2080 UAAA n/a

AUUAGUUAUUUCCA

14151 1190 2081 GGAAAUAACUAAU 2082 GACUC n/a

UUUCUAUUCAUGAG

14152 1333 2083 UCAUGAAUAGAAA 2084 AGAAU 31%

UUCGGUUGCUGGCA

14153 537 2085 GCCAGCAACCGAA 2086 GGUCC n/a

CAUGUGUGAGGUGA

14154 684 2087 CACCUCACACAUG 2088 UGUCC 100%

AGUUGAAUGGUG GCACCAUUCAACUCC

14155 707 2089 C 2090 UCGC 99%

CAUCCAGCUGACUCG

14156 799 2091 AGUCAGCUGGAUG 2092 UUUC 95%

CUUUCCGCUUAUAUA

14157 853 2093 UAUAAGCGGAAAG 2094 AUCU 106%

UUCCGAUGUGAU AAUCACAUCGGAAUG

14158 888 2095 U 2096 CUCA 88%

ACACAUUAGUUAUUU

14159 1194 2097 AUAACUAAUGUGU 2098 CCAG 95%

UUCUAUAGAAUGAAC

14160 1279 2099 UCAUUCUAUAGAA 2100 AUAG 15%

UACAGUGAUAGUUU

14161 1300 2101 AACUAUCACUGUA 2102 GCAUU 86%

AUAAGCAAUUGACAC

14162 1510 2103 GUCAAUUGCUUAU 2104 CACC 86%

UUUAUUAAUUGCUG

14163 1543 2105 AGCAAUUAAUAAA 2106 GACAA 110% % remaining

Oligo Start SEQ ID SEQ ID expression (1

Number Site NO Sense sequence NO Antisense sequence uM A549)

UCAUCAGAGUCGUUC

14164 434 2107 ACGACUCUGAUGA 2108 GAGU 134%

UAGUGUGGUUUA AUAAACCACACUAUC

14165 600 2109 U 2110 ACCU 102%

UCAUCAUUGGCUUUC

14166 863 2111 AAGCCAAUGAUGA 2112 CGCU 93%

AGUUCCUGACUAUCA

14167 902 2113 AUAGUCAGGAACU 2114 AUCA 101%

UUCACGGCUGACUUU

14168 921 2115 AGUCAGCCGUGAA 2116 GGAA 98%

UUCUCAUGGUAGUG

14169 154 2117 ACUACCAUGAGAA 2118 AGUUU n/a

AAUCAGCCUGUUUAA

14170 217 2119 AAACAGGCUGAUU 2120 CUGG 66%

GGUUUCAGCACUCUG

14171 816 2121 GAGUGCUGAAACC 2122 GUCA 102%

AUCGGAAUGCUCAUU

14172 882 2123 UGAGCAUUCCGAU 2124 GCUC 103%

UGGCUGUGGAAUUC

14173 932 2125 AAUUCCACAGCCA 2126 ACGGC n/a

UAAGCAAUUGACACC

14174 1509 2127 UGUCAAUUGCUUA 2128 ACCA n/a

CAAUUCUCAUGGUAG

14175 157 2129 ACCAUGAGAAUUG 2130 UGAG 109%

UGGCUUUCGUUGGA

14176 350 2131 CCAACGAAAGCCA 2132 CUUAC 95%

AAUCAGUGACCAGUU

14177 511 2133 CUGGUCACUGAUU 2134 CAUC 100%

UGGUUUAUGGAC AGUCCAUAAACCACA

14178 605 2135 U 2136 CUAU 99%

CAGCACUCUGGUCAU

14179 811 2137 GACCAGAGUGCUG 2138 CCAG 88%

GAUGUGAUUGAU UAUCAAUCACAUCGG

14180 892 2139 A 2140 AAUG 76%

AUUCACGGCUGACUU

14181 922 2141 GUCAGCCGUGAAU 2142 UGGA 59%

AAUGUGUAUCUA AUAGAUACACAUUCA

14182 1169 2143 U 2144 ACCA 69%

UUGAGUCUGGAA UUUCCAGACUCAAAU

14183 1182 2145 A 2146 AGAU n/a

UUAAUUGCUGGACAA

14184 1539 2147 GUCCAGCAAUUAA 2148 CCGU 77%

UAUUAAUUGCUGGA

14185 1541 2149 CCAGCAAUUAAUA 2150 CAACC n/a

AGUCGUUCGAGUCAA

14186 427 2151 GACUCGAACGACU 2152 UGGA 69%

GUUGCUGGCAGGUCC

14187 533 2153 ACCUGCCAGCAAC 2154 GUGG 78%

UAUCAGAUUCAUCAG

18538 496 2155 GAUGAAUCUGAUA 2156 AAUG 74% % remaining

Oligo Start SEQ ID SEQ ID expression (1

Number Site NO Sense sequence NO Antisense sequence uM A549)

UGAUGAAUCUGA UAUCAGAUUCAUCAG

18539 496 2157 UA 2158 AAUG 72%

AUUUGCUUUUGC UGCAAAAGCAAAUCA

18540 175 2159 A 2160 CUGC 98%

GAUUUGCUUUUG UGCAAAAGCAAAUCA

18541 175 2161 CA 2162 CUGC 28%

GUGAUUUGCUUU UAAAGCAAAUCACUG

18542 172 2163 A 2164 CAAU 24%

AGUGAUUUGCUU UAAAGCAAAUCACUG

18543 172 2165 UA 2166 CAAU 14%

AAUUUCGUAUUU UAAAUACGAAAUUUC

18544 1013 2167 A 2168 AGGU 100%

AAAUUUCGUAUU UAAAUACGAAAUUUC

18545 1013 2169 UA 2170 AGGU 109%

UUUCAUGGCUGUGA

18546 952 2171 CACAGCCAUGAAA 2172 AAUUC 32%

UCACAGCCAUGAA UUUCAUGGCUGUGA

18547 952 2173 A 2174 AAUUC 33%

GAUUUGCUUUUG UCAAAAGCAAAUCAC

18548 174 2175 A 2176 UGCA 57%

UGAUUUGCUUUU UCAAAAGCAAAUCAC

18549 174 2177 GA 2178 UGCA 53%

UUGCUUUUGCCU UAGGCAAAAGCAAAU

18550 177 2179 A 2180 CACU 97%

UUUGCUUUUGCC UAGGCAAAAGCAAAU

18551 177 2181 UA 2182 CACU 103%

UUUCUCAGUUUA UUAAACUGAGAAAGA

18552 1150 2183 A 2184 AGCA 96%

UGACUAAAUGCAAAG

18553 1089 2185 UUGCAUUUAGUCA 2186 UGAG 94%

UUAAAUGCAAAGUGA

18554 1086 2187 ACUUUGCAUUUAA 2188 GAAA n/a

UUUUUGACUAAAUG

18555 1093 2189 AUUUAGUCAAAAA 2190 CAAAG n/a

UACUGAGAAAGAAGC

18556 1147 2191 UUCUUUCUCAGUA 2192 AUUU n/a

UAACUGAGAAAGAAG

18557 1148 2193 UCUUUCUCAGUUA 2194 CAUU 66%

UAUGUUCUCUUUCA

18558 1128 2195 GAAAGAGAACAUA 2196 UUUUG 16%

UCUAAAUGCAAAGUG

18559 1087 2197 CUUUGCAUUUAGA 2198 AGAA 28%

UUUGCAUUUAGU UACUAAAUGCAAAGU

18560 1088 2199 A 2200 GAGA n/a

UAUGCAAAGUGAGAA

18561 1083 2201 CUCACUUUGCAUA 2202 AUUG 53%

UGCAAAGUGAGAAAU

18562 1081 2203 UUCUCACUUUGCA 2204 UGUA 89%

UACAACUGGAGUGAA

18563 555 2205 CACUCCAGUUGUA 2206 AACU 33%

UUUCUCUUUCAUUU

18564 1125 2207 AAUGAAAGAGAAA 2208 UGCUA n/a % remaining

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

UGCAGUGAUUUG UCAAAUCACUGCAAU

18565 168 2209 A 2210 UCUC 14%

UUGUUCUCUUUCAU

18566 1127 2211 UGAAAGAGAACAA 2212 UUUGC 27%

UGAAAUUUCAGGUG

18567 1007 2213 ACCUGAAAUUUCA 2214 UUUAU 129%

UUCACUGCAAUUCUC

18568 164 2215 GAAUUGCAGUGAA 2216 AUGG 47%

GGCUGAUUCUGG UCCAGAAUCAGCCUG

18569 222 2217 A 2218 UUUA n/a

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

% remaining

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

UUGUGUUUGGAGUGG

102%

17398 339 2247 AC U CCAAACACAA 2248 GUUU

UUGUGUUUGGAGUGG

55%

17399 339 2249 CACUCCAAACACAA 2250 GUUU

UGUGUUUGGAGUGGG

62%

17400 338 2251 CACUCCAAACACA 2252 UUUC

UGUAAGUUGGUGGAC

61%

17401 468 2253 CCACCAACUUACA 2254 UGUC

UGUAAGUUGGUGGAC

179%

17402 468 2255 UCCACCAACUUACA 2256 UGUC

UAAGACUGGUAUUUCA

30%

17403 1465 2257 AAUACCAGUCUUA 2258 UCU

UCUUAUACUGGUCAAA

32%

17404 243 2259 GACCAGUAUAAGA 2260 UCC

UUCAUUAAAAGACUGG

15%

17405 1472 2261 GUCUUUUAAUGAA 2262 UAU

UAGACAUGAAAU UACU

142%

17406 2446 2263 AAUUUCAUGUCUA 2264 GGU

UAUCAAAUGUGAUCUG

54%

17407 449 2265 AUCACAUUUGAUA 2266 GAU

GAUCACAUUUGAU UAUCAAAUGUGAUCUG

27%

17408 449 2267 A 2268 GAU

UAUGUGAUCUGGAUG

49%

17409 444 2269 UCCAGAUCACAUA 2270 UCAA

UCUCCUAUCAGUAUUA

32%

17410 1093 2271 UACUGAUAGGAGA 2272 GCC

UCAAGUGUUGCACAUA

70%

17411 1134 2273 GUGCAACACUUGA 2274 AUC

UACUUAUACUGGUCAA

63%

17412 244 2275 ACCAGUAUAAGUA 2276 AUC

UUCAUUAGACUUCUAC

19%

17413 1946 2277 GAAGUCUAAUGAA 2278 AGU

UAACUUUCUUCUUAGA

27%

17414 638 2279 AAGAAGAAAGUUA 2280 AGC

UAAUCAAAUGUGAUCU

216%

17415 450 2281 UCACAUUUGAUUA 2282 GGA

AUCACAUUUGAUU UAAUCAAAUGUGAUCU

32%

17416 450 2283 A 2284 GGA

UUCAAUCAAAUGUGAU

99%

17417 452 2285 ACAUUUGAUUGAA 2286 CUG

CACAUUUGAUUGA UUCAAUCAAAUGUGAU

54%

17418 452 2287 A 2288 CUG

UUGUCAAUCAAAUGUG

86%

17419 454 2289 AUUUGAUUGACAA 2290 AUC

CAUUUGAUUGACA UUGUCAAUCAAAUGUG

89%

17420 454 2291 A 2292 AUC

UUUAUUGCAGAUGAG

55%

17421 1790 2293 CAUCUGCAAUAAA 2294 AG AC

UCAUCUGCAAUAA UUUAUUGCAGAUGAG

62%

17422 1790 2295 A 2296 AG AC Table 15: Inhibition of gene expression with CTGF sd-rxRNA sequences (Accession number: NM_001901.2)

% remaining mRNA

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

GCUGCGAGGAG CACUCCUCGCAGCAU

14003 679 2322 UG 4227 UUCC 102%

GCCUAUCAAGU AAACUUGAUAGGCUU

14004 992 2323 UU 4228 GGAG 100%

AAUUCUGUGG ACUCCACAGAAUUUA

14005 1045 2324 AGU 4229 GCUC 104%

UGUACGGAGAC AUGUCUCCGUACAUC

14006 1231 2325 AU 4230 UUCC 87%

AGCCUAUCAAG AACUUGAUAGGCUUG

14007 991 2326 UU 4231 GAGA 101%

CAAGUUUGAGC AAGCUCAAACUUGAU

14008 998 2327 UU 4232 AGGC 98%

CUGUGGAGUA ACAUACUCCACAGAA

14009 1049 2328 UGU 4233 UUUA 98%

AAAUUCUGUG CUCCACAGAAUUUAG

14010 1044 2329 GAG 4234 CUCG 93%

UUUCAGUAGCA UGUGCUACUGAAAUC

14011 1327 2330 CA 4235 AUUU 95%

CAAUGACAUCU AAAGAUGUCAUUGUC

14012 1196 2331 UU 4236 UCCG 101%

AGUACCAGUGC GUGCACUGGUACUUG

14013 562 2332 AC 4237 CAGC 66%

GGAAGACACGU AAACGUGUCUUCCAG

14014 752 2333 UU 4238 UCGG 95%

CUAUCAAGUUU UCAAACUUGAUAGGC

14015 994 2334 GA 4239 UUGG 85%

AGCUAAAUUCU ACAGAAUUUAGCUCG

14016 1040 2335 GU 4240 GUAU 61%

AGGUAGAAUG UUACAUUCUACCUAU

14017 1984 2336 UAA 4241 GGUG 32%

AGCUGAUCAGU AAACUGAUCAGCUAU

14018 2195 2337 UU 4242 AUAG 86%

UUCUGCUCAGA UAUCUGAGCAGAAUU

14019 2043 2338 UA 4243 UCCA 81%

UUAUCUAAGU UUAACUUAGAUAACU

14020 1892 2339 UAA 4244 GUAC 84%

UAUACGAGUAA UAUUACUCGUAUAAG

14021 1567 2340 UA 4245 AUGC 72%

GACUGGACAGC AAGCUGUCCAGUCUA

14022 1780 2341 UU 4246 AUCG 65%

AUGGCCUUUAU UAAUAAAGGCCAUUU

14023 2162 2342 UA 4247 GUUC 80%

AUACCGAGCUA UUUAGCUCGGUAUG

14024 1034 2343 AA 4248 UCUUC 91%

UUGUUGAGAG ACACUCUCAACAAAU

14025 2264 2344 UGU 4249 AAAC 58%

ACAUACCGAGC UAGCUCGGUAUGUC

14026 1032 2345 UA 4250 UUCAU 106%

AGCAGAAAGGU UAACCUUUCUGCUGG

14027 1535 2346 UA 4251 UACC 67% % remaining mRNA

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

AGUUGUUCCU UUAAGGAACAACUUG

14028 1694 2347 UAA 4252 ACUC 94%

AUUUGAAGUG UUACACUUCAAAUAG

14029 1588 2348 UAA 4253 CAGG 97%

AAGCUGACCUG UCCAGGUCAGCUUCG

14030 928 2349 GA 4254 CAAG 100%

GGUCAUGAAGA CUUCUUCAUGACCUC

14031 1133 2350 AG 4255 GCCG 82%

AUGGUCAGGCC AAGGCCUGACCAUGC

14032 912 2351 UU 4256 ACAG 84%

GAAGACACGUU CAAACGUGUCUUCCA

14033 753 2352 UG 4257 GUCG 86%

AGGCCUUGCGA CUUCGCAAGGCCUGA

14034 918 2353 AG 4258 CCAU 88%

UACCGACUGGA CUUCCAGUCGGUAAG

14035 744 2354 AG 4259 CCGC 95%

ACCGCAAGAUC CCGAUCUUGCGGUUG

14036 466 2355 GG 4260 GCCG 73%

CAGGCCUUGCG UUCGCAAGGCCUGAC

14037 917 2356 AA 4261 CAUG 86%

CGAGCUAAAUU AGAAUUUAGCUCGGU

14038 1038 2357 CU 4262 AUGU 84%

UCUGUGGAGU CAUACUCCACAGAAU

14039 1048 2358 AUG 4263 UUAG 87%

CGGAGACAUGG UGCCAUGUCUCCGUA

14040 1235 2359 CA 4264 CAUC 100%

AUGACAACGCC GAGGCGUUGUCAUU

14041 868 2360 UC 4265 GGUAA 104%

GAGGUCAUGAA UCUUCAUGACCUCGC

14042 1131 2361 GA 4266 CGUC 85%

UAAAUUCUGU UCCACAGAAUUUAGC

14043 1043 2362 GGA 4267 UCGG 74%

UGGAAGACACG AACGUGUCUUCCAGU

14044 751 2363 UU 4268 CGGU 84%

AAGAUGUACGG CUCCGUACAUCUUCC

14045 1227 2364 AG 4269 UGUA 99%

AAUGACAACGC AGGCGUUGUCAUUG

14046 867 2365 CU 4270 GUAAC 94%

GGCGAGGUCAU UCAUGACCUCGCCGU

14047 1128 2366 GA 4271 CAGG 89%

GACACGUUUGG GGCCAAACGUGUCUU

14048 756 2367 CC 4272 CCAG 93%

ACGGAGACAUG GCCAUGUCUCCGUAC

14049 1234 2368 GC 4273 AUCU 100%

UCAGGCCUUGC UCGCAAGGCCUGACC

14050 916 2369 GA 4274 AUGC 96%

GCGAAGCUGAC AGGUCAGCUUCGCAA

14051 925 2370 CU 4275 GGCC 80%

GGAAGAUGUAC CCGUACAUCUUCCUG

14052 1225 2371 GG 4276 UAGU 96% % remaining mRNA

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

GUGACUUCGGC GAGCCGAAGUCACAG

14053 445 2372 UC 4277 A AG A 101%

UGACUUCGGCU GGAGCCGAAGUCACA

14054 446 2373 CC 4278 GAAG 93%

UGGUCAGGCCU CAAGGCCUGACCAUG

14055 913 2374 UG 4279 CACA 67%

UCAAGUUUGA AGCUCAAACUUGAUA

14056 997 2375 GCU 4280 GGCU 92%

GCCAGAACUGC CUGCAGUUCUGGCCG

14057 277 2376 AG 4281 ACGG 84%

UGGAGUAUGU GGUACAUACUCCACA

14058 1052 2377 ACC 4282 GAAU n/a

GCUAGAGAAGC CUGCUUCUCUAGCCU

14059 887 2378 AG 4283 GCAG 80%

GGUCAGGCCUU GCAAGGCCUGACCAU

14060 914 2379 GC 4284 GCAC 112%

GAGCUAAAUUC CAGAAUUUAGCUCGG

14061 1039 2380 UG 4285 UAUG 104%

AAGACACGUUU CCAAACGUGUCUUCC

14062 754 2381 GG 4286 AGUC 109%

CGAGGUCAUGA CUUCAUGACCUCGCC

14063 1130 2382 AG 4287 GUCA 103%

GGCCUUGCGAA GCUUCGCAAGGCCUG

14064 919 2383 GC 4288 ACCA 109%

CUUGCGAAGCU UCAGCUUCGCAAGGC

14065 922 2384 GA 4289 CUGA 106%

CCGACUGGAAG GUCUUCCAGUCGGUA

14066 746 2385 AC 4290 AGCC 106%

CCUAUCAAGUU CAAACUUGAUAGGCU

14067 993 2386 UG 4291 UGGA 67%

UGUUCCAAGAC AGGUCUUGGAACAGG

14068 825 2387 CU 4292 CGCU 93%

CGAAGCUGACC CAGGUCAGCUUCGCA

14069 926 2388 UG 4293 AGGC 95%

UUGCGAAGCUG GUCAGCUUCGCAAGG

14070 923 2389 AC 4294 CCUG 95%

CAAUGACAACG GGCGUUGUCAUUGG

14071 866 2390 CC 4295 UAACC 132%

GUACCAGUGCA CGUGCACUGGUACUU

14072 563 2391 CG 4296 GCAG n/a

CCUGUUCCAAG GUCUUGGAACAGGCG

14073 823 2392 AC 4297 CUCC 98%

UACGGAGACAU CCAUGUCUCCGUACA

14074 1233 2393 GG 4298 UCUU 109%

UGCGAAGCUGA GGUCAGCUUCGCAAG

14075 924 2394 CC 4299 GCCU 95%

CCUUGCGAAGC CAGCUUCGCAAGGCC

14076 921 2395 UG 4300 UGAC 116% % remaining mRNA

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

CUGUGACUUCG GCCGAAGUCACAGAA

14077 443 2396 GC 4301 GAGG 110%

GCUAAAUUCUG CACAGAAUUUAGCUC

14078 1041 2397 UG 4302 GGUA 99%

CUAAAUUCUGU CCACAGAAUUUAGCU

14079 1042 2398 GG 4303 CGGU 109%

AGACACGUUUG GCCAAACGUGUCUUC

14080 755 2399 GC 4304 CAGU 121%

CCGCAAGAUCG GCCGAUCUUGCGGUU

14081 467 2400 GC 4305 GGCC 132%

UAUCAAGUUU CUCAAACUUGAUAGG

14082 995 2401 GAG 4306 CUUG 105%

GAAGCUGACCU CCAGGUCAGCUUCGC

14083 927 2402 GG 4307 AAGG 114%

ACAUUAACUCA UAUGAGUUAAUGUC

17356 1267 2403 UA 4308 UCUCA 120%

GACAUUAACUC UAUGAGUUAAUGUC

17357 1267 2404 AUA 2405 UCUCA 56%

UGAAGAAUGU UUAACAUUCUUCAAA

17358 1442 2406 UAA 2407 CCAG 34%

UUGAAGAAUG UUAACAUUCUUCAAA

17359 1442 2408 UUAA 2409 CCAG 31%

GAUAGCAUCUU UUAAGAUGCUAUCU

17360 1557 2410 AA 2411 GAUGA 59%

AGAUAGCAUCU UUAAGAUGCUAUCU

17361 1557 2412 UAA 2413 GAUGA 47%

UGAAGUGUAA UAAUUACACUUCAAA

17362 1591 2414 UUA 2415 UAGC 120%

AAUUGAGAAGG UUCCUUCUCAAUUAC

17363 1599 2416 AA 2417 ACUU 71%

UUGAGAAGGAA UUUUCCUUCUCAAUU

17364 1601 2418 AA 2419 ACAC 62%

CAUUCUGAUUC UCGAAUCAGAAUGUC

17365 1732 2420 GA 2421 AGAG 99%

UUCUGAUUCGA UUUCGAAUCAGAAUG

17366 1734 2422 AA 2423 UCAG 97%

CUGUCGAUUAG UUCUAAUCGACAGGA

17367 1770 2424 AA 2425 UUCC 45%

UUUGCCUGUAA UGUUACAGGCAAAUU

17368 1805 2426 CA 2427 CACU 71%

AUUUGCCUGUA UGUUACAGGCAAAUU

17369 1805 2428 ACA 2429 CACU 67%

ACAAGCCAGAU UAAUCUGGCUUGUU

17370 1815 2430 UA 2431 ACAGG 65%

AACAAGCCAGA UAAUCUGGCUUGUU

17371 1815 2432 UUA 2433 ACAGG 35%

CAGUUUAUUU UACAAAUAAACUGUC

17372 2256 2434 GUA 2435 CGAA 113%

UGUUGAGAGU UACACUCUCAACAAA

17373 2265 2436 GUA 2437 UAAA 35% % remaining mRNA

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

UUGUUGAGAG UACACUCUCAACAAA

17374 2265 2438 UGUA 2439 UAAA 31%

UGCACCUUUCU UUAGAAAGGUGCAAA

17375 2295 2440 AA 2441 CAUG 34%

UUGCACCUUUC UUAGAAAGGUGCAAA

17376 2295 2442 UAA 2443 CAUG 28%

UUGAGCUUUC UCAGAAAGCUCAAAC

17377 1003 2444 UGA 2445 UUGA 67%

UGAGAGUGUG UGUCACACUCUCAAC

17378 2268 2446 ACA 2447 AAAU 42%

AGUGUGACCAA UUUUGGUCACACUCU

17379 2272 2448 AA 2449 CAAC 35%

GAGUGUGACCA UUUUGGUCACACUCU

17380 2272 2450 AAA 2451 CAAC 29%

GUGUGACCAAA UUUUUGGUCACACUC

17381 2273 2452 AA 2453 UCAA 42%

UGUGACCAAAA UCUUUUGGUCACACU

17382 2274 2454 GA 2455 CUCA 42%

GUGUGACCAAA UCUUUUGGUCACACU

17383 2274 2456 AGA 2457 CUCA 37%

GUGACCAAAAG UACUUUUGGUCACAC

17384 2275 2458 UA 2459 UCUC 24%

GACCAAAAGUU UUAACUUUUGGUCAC

17385 2277 2460 AA 2461 ACUC 27%

GCACCUUUCUA UCUAGAAAGGUGCAA

17386 2296 2462 GA 2463 ACAU 23%

CCUUUCUAGUU UCAACUAGAAAGGUG

17387 2299 2464 GA 2465 CAAA 46%

Table 16: Inhibition of gene expression with TGFB2 sd-rxRNA sequences

(Accession Number: NM_001135599.1)

% remaining

Oligo Start SEQ ID SEQ ID expression (1

Number Site NO Sense sequence NO Antisense sequence uM, A549)

AACCUCCUUGGCGUA

14414 944 2478 CGCCAAGGAGGUU 2479 GUAC 100%

GUGGUGAUCAGA UUCUGAUCACCACUG

14415 1513 2480 A 2481 GUAU n/a

ACAUUAGCAGGAGAU

14416 1572 2482 CUCCUGCUAAUGU 2483 GUGG 100%

UAUAUGUGGAGGUG

14417 1497 2484 ACCUCCACAUAUA 2485 CCAUC 73%

UCCUAGUGGACUUUA

14418 1533 2486 AAGUCCACUAGGA 2487 UAGU 98%

UUUCUGAUCACCACU

14419 1514 2488 UGGUGAUCAGAAA 2489 GGUA 86%

UUCCUAGUGGACUU

14420 1534 2490 AGUCCACUAGGAA 2491 UAUAG 99%

ACCUCCUUGGCGUAG

14421 943 2492 ACGCCAAGGAGGU 2493 UACU 41%

UAUUUAUUGUGU UACACAAUAAAUAAC

18570 2445 2494 A 2495 UCAC 79%

UUAUUUAUUGUG UACACAAUAAAUAAC

18571 2445 2496 UA 2497 UCAC 75%

UUUUAACACUGAUGA

18572 2083 2498 AUCAGUGUUAAAA 2499 ACCA 47%

CAUCAGUGUUAAA UUUUAACACUGAUGA

18573 2083 2500 A 2501 ACCA 17%

UUCCUUAAGCCAUCC

18574 2544 2502 AUGGCUUAAGGAA 2503 AUGA 59%

GAUGGCUUAAGG UUCCUUAAGCCAUCC

18575 2544 2504 AA 2505 AUGA 141%

UUGUGUUCUGUU UAACAGAACACAAAC

18576 2137 2506 A 2507 UUCC 77%

UUUGUGUUCUGU UAACAGAACACAAAC

18577 2137 2508 UA 2509 UUCC 59%

UGGCAAAGUAUUUG

18578 2520 2510 AAAUACUUUGCCA 2511 GUCUC 75%

CAAAUACUUUGCC UGGCAAAGUAUUUG

18579 2520 2512 A 2513 GUCUC 55%

UUUGUAGUGCAAGU

18580 3183 2514 CUUGCACUACAAA 2515 CAAAC 84%

ACUUGCACUACAA UUUGUAGUGCAAGU

18581 3183 2516 A 2517 CAAAC 80%

GAAUUUAUUAGU UACUAAUAAAUUCUU

18582 2267 2518 A 2519 CCAG 82%

AGAAUUUAUUAG UACUAAUAAAUUCUU

18583 2267 2520 UA 2521 CCAG 67%

UUUUGUAGUGCAAG

18584 3184 2522 UUGCACUACAAAA 2523 UCAAA 77%

CUUGCACUACAAA UUUUGUAGUGCAAG

18585 3184 2524 A 2525 UCAAA 59%

UCACCUGUUUUAUU

18586 2493 2526 AUAAAACAGGUGA 2527 UUCCA 84% % remaining

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

AAUAAAACAGGUG UCACCUGUUUUAUU

18587 2493 2528 A 2529 UUCCA 70%

UGUUGUUGUUGUCG

18588 2297 2530 GACAACAACAACA 2531 UUGUU 40%

UUGUUACAAGCAUCA

18589 2046 2532 AUGCUUGUAACAA 2533 UCGU 39%

UCAUGAGUUUCUGG

18590 2531 2534 CAGAAACUCAUGA 2535 CAAAG 56%

UGCAUAGCAAUACAG

18591 2389 2536 GUAUUGCUAUGCA 2537 AAAA 64%

UAUGAGUUUCUGGC

18592 2530 2538 CCAGAAACUCAUA 2539 AAAGU 44%

UGCUCGUUUGAGUU

18593 2562 2540 ACUCAAACGAGCA 2541 CAAGU 87%

UUCUCGGUCAUAUAA

18594 2623 2542 AUAUGACCGAGAA 2543 UAAC 69%

UUCGUUGUCGUCGU

18595 2032 2544 CGACGACAACGAA 2545 CAUCA 55%

UUCACUGGUUUACUA

18596 2809 2546 GUAAACCAGUGAA 2547 AACU 58%

UUGUCAGUUUAG UCUAAACUGACAAAG

18597 2798 2548 A 2549 AACC 38%

UUAACACUGAUGAAC

18598 2081 2550 UCAUCAGUGUUAA 2551 CAAG 25%

UCUCGUUUGAGUUC

18599 2561 2552 AACUCAAACGAGA 2553 AAGUU 57%

UUUGUUGUUGUCGU

18600 2296 2554 CGACAACAACAAA 2555 UGUUC 69%

UCAUCGUUGUCGUCG

18601 2034 2556 ACGACAACGAUGA 2557 UCAU 22%

UUCCUUAGGCAGCUG

18602 2681 2558 GCUGCCUAAGGAA 2559 AUAC 43%

UGAAAUGUAGAAUAA

18603 2190 2560 AUUCUACAUUUCA 2561 GGCC 128%

Table 17: Inhibition of gene expression with TGFBl sd-rxRNA sequences (Accession Number NM_000660.3)

% remaining

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

UCGUGGAUCCACUU

n/a

14397 1787 2568 AGUGGAUCCACGA 2569 CCAGC

GGACCUUGCUGUAC

82%

14398 1867 2570 UACAGCAAGGUCC 2571 UGCGU

GCACGAUCAUGUUG

n/a

14399 2002 2572 AACAUGAUCGUGC 2573 GACAG

CGCACGAUCAUGUU

n/a

14400 2003 2574 ACAUGAUCGUGCG 2575 GGACA

CAGGACCUUGCUGU

82%

14401 1869 2576 CAGCAAGGUCCUG 2577 ACUGC

ACGAUCAUGUUGGA

66%

14402 2000 2578 CCAACAUGAUCGU 2579 CAGCU

AUGCGCUUCCGCUU

78%

14403 986 2580 AGCGGAAGCGCAU 2581 CACCA

AUGGCCUCGAUGCG

79%

14404 995 2582 GCAUCGAGGCCAU 2583 CUUCC

CAUGUCGAUAGUCU

80%

14405 963 2584 GACUAUCGACAUG 2585 UGCAG

UAGUCUUGCAGGUG

88%

14406 955 2586 ACCUGCAAGACUA 2587 GAUAG

UUCUCCGUGGAGCU

n/a

14407 1721 2588 GCUCCACGGAGAA 2589 GAAGC

UAUAUAUGCUGUG

58%

18454 1246 2590 CACAGCAUAUAUA 2591 UGUACU

UAUAUAUAUGCUGU

87%

18455 1248 2592 CAGCAUAUAUAUA 2593 GUGUA

UAAGUCAAUGUACA

107%

18456 1755 2594 GUACAUUGACUUA 2595 GCUGC

UGUACAUUGACUU UAAGUCAAUGUACA

77%

18457 1755 2596 A 2597 GCUGC

UGAAGCAAUAGUUG

75%

18458 1708 2598 AACUAUUGCUUCA 2599 GUGUC

CAACUAUUGCUUC UGAAGCAAUAGUUG

73%

18459 1708 2600 A 2601 GUGUC

UACAUAUAUAUGCU

n/a

18460 1250 2602 GCAUAUAUAUGUA 2603 GUGUG

UAGUCAAUGUACAG

91%

18461 1754 2604 UGUACAUUGACUA 2605 CUGCC

CUGUACAUUGACU UAGUCAAUGUACAG

92%

18462 1754 2606 A 2607 CUGCC

UCAUAUAUAUGCUG

n/a

18463 1249 2608 AGCAUAUAUAUGA 2609 UGUGU

UGAAUUGUUGCUG

77%

18464 1383 2610 CAGCAACAAUUCA 2611 UAUUUC

UAACAUAUAUAUGC

84%

18465 1251 2612 CAUAUAUAUGUUA 2613 UGUGU

UGAGCUGAAGCAAU

n/a

18466 1713 2614 UUGCUUCAGCUCA 2615 AGUUG

AUUGCUUCAGCUC UGAGCUGAAGCAAU

83%

18467 1713 2616 A 2617 AGUUG % remaining

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

UUAUAUAUGCUGU

96%

18468 1247 2618 ACAGCAUAUAUAA 2619 GUGUAC

UAGCUGAAGCAAUA

90%

18469 1712 2620 AUUGCUUCAGCUA 2621 GUUGG

UAUUGCUUCAGCU UAGCUGAAGCAAUA

98%

18470 1712 2622 A 2623 GUUGG

UUGCUUGAACUUGU

n/a

18471 1212 2624 CAAGUUCAAGCAA 2625 CAUAG

UGUGUGUACUCUGC

45%

18472 1222 2626 CAGAGUACACACA 2627 UUGAA

UUAUGCUGUGUGU

36%

18473 1228 2628 ACACACAGCAUAA 2629 ACUCUG

UAUAUAUAUGCUGU

68%

18474 1233 2630 CAGCAUAUAUAUA 2631 GUGUA

UUACUCUGCUUGAA

64%

18475 1218 2632 UCAAGCAGAGUAA 2633 CUUGU

UCAUAUAUAUGCUG

78%

18476 1235 2634 AGCAUAUAUAUGA 2635 UGUGU

UUGUGUGUACUCU

92%

18477 1225 2636 AGAGUACACACAA 2637 GCUUGA

UUGUACUCUGCUUG

103%

18478 1221 2638 AAGCAGAGUACAA 2639 AACUU

UUGAUGUGUUGAA

84%

18479 1244 2640 UUCAACACAUCAA 2641 GAACAU

UGUGUACUCUGCUU

37%

18480 1224 2642 AGCAGAGUACACA 2643 GAACU

AUAUAUGUUCUU UAAGAACAUAUAUA

62%

18481 1242 2644 A 2645 UGCUG

UCUUGAACUUGUCA

47%

18482 1213 2646 G ACAAG U U CAAG A 2647 UAGAU

UCUCCAUCUUUAAU

69%

18483 1760 2648 UUAAAGAUGGAGA 2649 GGGGC

UAACUUGUCAUAGA

n/a

18484 1211 2650 CUAUGACAAGUUA 2651 UUUCG

UUAGAUUUCGUUG

52%

19411 1212 2652 CAACGAAAUCUAA 2653 UGGGUU

UGAACUUGUCAUAG

51%

19412 1222 2654 UAUGACAAGUUCA 2655 AUUUC

UCUGCUUGAACUUG

n/a

19413 1228 2656 AAGUUCAAGCAGA 2657 UCAUA

UGUACUCUGCUUGA

41%

19414 1233 2658 CAAGCAGAGUACA 2659 ACUUG

UUUGUCAUAGAUU

104%

19415 1218 2660 AAUCUAUGACAAA 2661 UCGUUG

UAUAUGCUGUGUG

31%

19416 1244 2662 CACACAGCAUAUA 2663 UACUCU Table 18: Inhibition of gene expression with SPPl sd-rxRNA sequences (Accession Number NM_000582.2)

% remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

P.mU. G.

G.fC.fU.fU.fU.fC.fC.

A. A. G.mC. G. G. A. G.mC.mU.mU* A*mU*

14097 856 2690 A. A. G.mC.mC. A.Chi 2691 A*mU* A* A. 88%

P.mU.fU. G.

G.fC.fU.fU.fU.fC.fC.

A. G.mC. G. G. A. A. G.mC.mU*mU* A*mU*

14098 857 2692 A. G.mC.mC. A. A.Chi 2693 A*mU* A. 113%

P.mU.fC. A.fU.fC.fC.

A.mC.mC. A.mC. A.fU. G.fU. G.

A.mU. G. G. A.mU. G.mU*mC* A*mU* G*

14099 365 2694 G. A.Chi 2695 G* C. 98%

P.mA.fU. G.fU. G.

G.mC.mC. A.mU. G. G.fU.fC. A.fU. G.

A.mC.mC. A.mC. G.mC*mU*mU*mU*m

14100 359 2696 A.mU. Chi 2697 C* G* U. 84%

P.mG.fU. G. G.fU.fC.

A. A. G.mC.mC. A.fU. G.

A.mU. G. A.mC.mC. G.mC.mU.mU*mU*mC

14101 357 2698 A.mC.Chl 2699 * G*mU*mU* G. 88%

P.mA.fU.fU. G.

G.mC. G. G. A. A. A. G.fC.fU.fU.fU.fC.mC.

G.mC.mC. A. G.mC*mU*mU*

14102 858 2700 A.mU. Chi 2701 A*mU* A* U. n/a

A. A. P.mA. A. A.fU. A.fC. G.

A.mU.mU.mU.mC. A. A.

G.mU. A.mU.mU.mU*mC* A*

14103 1012 2702 A.mU. mU.mU. Chi 2703 G* G*mU* G. 93%

A.mU.mU.mU.mC. P.mA. G. A. A. A.fU.

G.mU. A.fC. G. A. A.

A.mU.mU.mU.mC.m A.mU*mU*mU*mC*

14104 1014 2704 U.Chi 2705 A* G* G. 89%

P.mU. G. G.fU.fC. A.fU.

A. A. A. G.mC.mC. G.

A.mU. G. A.mC.mC. G.fC.mU.mU.mU*mC*

14105 356 2706 A.Chi 2707 G*mU*mU* G* G. 85%

P.mA.fU. A.fU.fC.

A.mC. A.mU. G. G. A.fU.fC.fC. A.mU.

A.mU. G. A.mU. G.mU* G* G*mU*mC*

14106 368 2708 A.mU. Chi 2709 A* U. 67%

G. A. A. P.mA. A.fU. A.fC. G. A.

A.mU.mU.mU.mC. A. A.fU.mU.mU.mC* A*

14107 1011 2710 G.mU. A.mU. mU. Chi 2711 G* G*mU* G* U. 87%

G.mC.

G.mC.mC.mU.mU.m P.mA. A.fU.fC. A. G. A.

C.mU. G. A. G. G.mC. G.mC*

14108 754 2712 A.mU. mU. Chi 2713 G*mU*mU*mC* A* G. 73%

A.mU.mU.mU.mC.m P.mA.fU.fU.fC. A.fU. G.

U.mC. A.mU. G. A. A. G. A. A. A.mU*

14109 1021 2714 A.mU. Chi 2715 A*mC* G* A* A* A. 128% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

mC.mU.mC.mU.mC. P.mC.fU. A.fU.fU.fC.

A.mU. G. A. A.mU. A. A.fU. G. A. G. A. G* A*

14110 1330 2716 G.Chi 2717 A*mU* A* A* C. 101%

A. A. G.mU.mC.mC. P.mU.fU.fU.fC. G.fU.fU.

A. A.mC. G. A. A. G. G. A.mC.mU.mU*

14111 346 2718 A.Chi 2719 A*mC*mU*mU* G* G. 59%

P.mU.fU.

A.mU. G. A.mU. G. G.fC.fU.fC.fU.fC.

A. G. A. G.mC. A. A.fU.mC. A.mU*mU*

14112 869 2720 A.Chi 2721 G* G*mC*mU* U. 89%

P.mU.fU.fC. A.

G.mC. G. A. G. G. A. A.fC.fU.fC.fC.fU.mC.

G.mU.mU. G. A. G.mC*mU*mU*mU*m

14113 701 2722 A.Chi 2723 C*mC* A. 95%

P.mU. G. A.fC.fU.

mU. G. A.mU.mU. G. A.fU.fC. A. A.mU.mC.

A.mU. A. G.mU.mC. A*mC* A*mU*mC* G*

14114 896 2724 A.Chi 2725 G. 87%

A. G. A.mU. A. P.mA. G. A.fU. G.fC.

G.mU. G.mC. A.fC.fU. A.mU.mC.mU*

14115 1035 2726 A.mU. mC.mU. Chi 2727 A* A*mU*mU*mC* A. 82%

P.mA. A.fU. A. G. A.fU.

A.mU. G.mU. G.mU. A.fC. A.mC.

A.mU.mC.mU. A.mU*mU*mC* A*

14116 1170 2728 A.mU.mU. Chi 2729 A*mC* C. 36% mU.mU.mC.mU. P.mU.fU.fC.fU.fU.fC.fU.

A.mU. A. G. A. A. G. A.fU. A. G. A. A*mU*

14117 1282 2730 A. A.Chi 2731 G* A* A*mC* A. 91% mU.mU. P.mA. A.fU.fU. G.fC.fU.

G.mU.mC.mC. A. G. G. A.mC. A.

G.mC. A. A*mC*mC* G*mU* G*

14118 1537 2732 A.mU.mU. Chi 2733 G. 152%

P.mU.fC.

A.mC. A.mU. G. G. G.fC.fU.fU.fU.fC.fC.

A. A. A. G. C.mG. A.mU. G.mU* G*mU*

14119 692 2734 A.Chi 2735 G* A* G* G. n/a

P.mU. A. A.fU.fC.fU. G.

G.mC. A. G. A.fC.mU.

G.mU.mC.mC. A. G. G.mC*mU*mU*

14120 840 2736 A.mU.mU. A.Chi 2737 G*mU* G* G. 87% mU. G. G.mU.mU. G. P.mA.fC. A.fC.

A. A.mU. G.mU. A.fU.fU.fC. A. A.mC.mC.

14121 1163 2738 G.mU. Chi 2739 A* A*mU* A* A* A* C. 31%

P.mA.fC.fU.fC.

mU.mU. A.mU. G. A. G.fU.fU.fU.fC. A.mU. A.

A. A.mC. G. A. A*mC*mU*

14122 789 2740 G.mU. Chi 2741 G*mU*mC* C. 96%

P.mA.fU. A. A.fU.fC.fU.

mC. A. G. G. A.mC.mU.

G.mU.mC.mC. A. G. G*mC*mU*mU*

14123 841 2742 A.mU.mU. A.mU.Chl 2743 G*mU* G. 110% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

P.mU.fU.fU.fC.fC.

A.mU. A.mU. A. A. G.fC.fU.fU. A.mU.

G.mC. G. G. A. A. A.mU* A*

14124 852 2744 A.Chi 2745 A*mU*mC*mU* G. 91%

P.mU. G.fU.fU.fU. A.

mU. A.mC.mC. A. A.fC.fU. G. G.mU.

G.mU.mU. A. A. A*mU* G* G*mC* A*

14125 209 2746 A.mC. A.Chi 2747 C. 110%

P.mU. A.fU. A. G. A.

mU. G.mU.mU.mC. A.fU. G. A. A.mC.

A.mU.mU.mC.mU. A*mU* A* G* A*mC*

14126 1276 2748 A.mU. A.Chi 2749 A. n/a mC.mC. G. A.mC.mC. P.mU.fU.fU.fC.fC.fU.fU.

A. A. G. G. A. A. G. G.fU.mC. G. G*mC*

14127 137 2750 A.Chi 2751 G*mU*mU*mU* G. 71%

G. A. A.mU. G. P.mG.fU. A.fU. G.fC.

G.mU. G.mC. A.mU. A.fC.fC. A.mU.mU.mC*

14128 711 2752 A.mC.Chl 2753 A* A*mC*mU*mC* C. 115%

A.mU. A.mU. G. P.mU.fC. G. G.fC.fC.

A.mU. G. G.mC.mC. A.fU.fC. A.mU. A.mU*

14129 582 2754 G. A.Chi 2755 G*mU* G*mU*mC* U. 97%

A. G.mC. A. P.mA. A.fU.fC.fU. G. G.

G.mU.mC.mC. A. G. A.fC.fU. G.mC.mU*mU*

14130 839 2756 A.mU. mU. Chi 2757 G*mU* G* G* C. 102%

G.mC.

A.mU.mU.mU. A. P.mU.fU.fU. G. A.fC.fU.

G.mU.mC. A. A. A. A. A.mU. G.mC* A*

14131 1091 2758 A.Chi 2759 A* A* G*mU* G. 10%

A. G.mC. P.mA.fC. A.fU.fC. G. G.

A.mU.mU.mC.mC. G. A. A.fU. G.mC.mU*mC*

14132 884 2760 A.mU. G.mU. Chi 2761 A*mU*mU* G* C. 93%

P.mA. A.

mU. A. G.mU.mC. A. G.fU.fU.fC.fC.fU. G.

G. G. A. A.mC.mU. A*mU*mC*

14133 903 2762 A.mC.mU.mU.Chl 2763 A* A*mU* C. 97% mU. G.mC. P.mU.fU. G. A.fC.fU. A.

A.mU.mU.mU. A. A. A.fU. G.mC. A* A* A*

14134 1090 2764 G.mU.mC. A. A.Chi 2765 G*mU* G* A. 39%

P.mA. G. A.fC.fU. fC.

G.mU.mC.mU. G. A.fU.fC. A. G.

A.mU. G. A. A.mC*mU* G* G*mU*

14135 474 2766 G.mU.mC.mU. Chi 2767 G* A. 99%

P.mU.fC. A.fU. A.fU.

mU. A. G. A.mC. G.fU. G.fU.mC.mU.

A.mC. A.mU. A.mU. A*mC*mU* G*mU* G*

14136 575 2768 G. A.Chi 2769 G. 108%

P.mA.fU.

mC. A. G. A.mC. G. G.fU.fC.fC.fU.fC.

A. G. G. A.mC. G.fU.mC.mU. G*mU*

14137 671 2770 A.mU. Chi 2771 A* G*mC* A* U. 98% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

P.mG. A. A.fU.fU.fC.

mC. A. G.mC.mC. A.fC. G. G.mC.mU. G*

G.mU. G. A. A*mC*mU*mU*mU*

14138 924 2772 A.mU.mU.mC.Chl 2773 G. 100%

P.mU.fU.

A. G.mU.mC.mU. G. A.fU.fU.fU.fC.fC. A. G.

G. A. A. A.mU. A. A.mC.mU*mC* A* A*

14139 1185 2774 A.Chi 2775 A*mU* A. 47%

A. G.mU.mU.mU.

G.mU. G. P.mG. A. A. G.fC.fC.

G.mC.mU.mU.mC.Ch A.fC. A. A. A.mC.mU*

14140 1221 2776 1 2777 A* A* A*mC*mU* A. 100%

P.mC.fU.fU.fU.fC.

A. G.mU.mC.mC. A. G.fU.fU. G. G.

A.mC. G. A. A. A. A.mC.mU*mU*

14141 347 2778 G.Chi 2779 A*mC*mU*mU* G. 103%

A. A. P.mG.fU.fC.fU. G.fC. G.

G.mU.mU.mU.mC. A. A.

G.mC. A. G. A.mC.mU. mU*mC*mU

14142 634 2780 A.mC.Chl 2781 *mU* A* G* A. 100%

P.mA. A.fU. G.fC.fU.fC.

A. G.mC. A. A.mU. A.fU.fU.

G. A. G.mC. G.mC.mU*mC*mU*mC

14143 877 2782 A.mU. mU. Chi 2783 * A*mU* C. 104%

P.mA.fU. G.fC. A.fC.fU.

mU.mU. A. G. A.mU. A.fU.fC.mU. A.

A. G.mU. G.mC. A*mU*mU*mC*

14144 1033 2784 A.mU. Chi 2785 A*mU* G. 95%

P.mC.fU.fU. G.fU. A.fU.

mU. G. G.mU. G.mC. G.fC. A.mC.mC.

A.mU. A.mC. A. A. A*mU*mU*mC* A* A*

14145 714 2786 G.Chi 2787 C. 101%

A.mU. G. A. A. P.mU. G. A.fC.fU.fC.

A.mC. G. A. G.fU.fU.fU.mC. A.mU*

14146 791 2788 G.mU.mC. A.Chi 2789 A* A*mC*mU* G* U. 100%

P.mU.fU.fC. A. G.fC.

mC.mC. A. G. A. A.fC.fU.fC.mU. G.

G.mU. G.mC.mU. G. G*mU*mC*

14147 813 2790 A. A.Chi 2791 A*mU*mC* C. 97% mC. A. G.mC.mC. P.mA. A. A.fU.fU.fC.

A.mU. G. A. A.fU. G. G.mC.mU.

14148 939 2792 A.mU. mU.mU. Chi 2793 G*mU* G* G* A* A* U. 109%

A.mU.mU. G. P.mA.fC. A.fU.fU.fC. A.

G.mU.mU. G. A. A.fC.fC. A. A.mU* A* A*

14149 1161 2794 A.mU. G.mU. Chi 2795 A*mC*mU* G. 34%

P.mU. A.fC. A.fC.

G. G.mU.mU. G. A. A.fU.fU.fC. A.

A.mU. G.mU. G.mU. A.mC.mC* A* A*mU*

14150 1164 2796 A.Chi 2797 A* A* A. n/a

G. G. A. A. A.mU. A. P.mA.fU.fU. A. G.fU.fU.

A.mC.mU. A. A.fU.fU. mU.mC.mC* A*

14151 1190 2798 A.mU. Chi 2799 G* A*mC*mU* C. n/a % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

mU.mC. A.mU. G. A. P.mU.fU.fU.fC.fU.

A.mU. A. G. A. A. A.fU.fU.fC. A.mU. G. A*

14152 1333 2800 A.Chi 2801 G* A* G* A* A* U. 31%

G.mC.mC. A. G.mC. P.mU.fU.fC. G. G.fU.fU.

A. A.mC.mC. G. A. G.fC.fU. G. G.mC* A*

14153 537 2802 A.Chi 2803 G* G*mU*mC* C. n/a mC.

A.mC.mC.mU.mC. P.mC. A.fU. G.fU. G.fU.

A.mC. A.mC. A.mU. G. A. G. G.mU. G*

14154 684 2804 G.Chi 2805 A*mU* G*mU*mC* C. 100%

P.mG.fC. A.fC.fC.

A. G.mU.mU. G. A. A.fU.fU.fC. A.

A.mU. G. G.mU. A.mC.mU*mC*mC*mU

14155 707 2806 G.mC.Chl 2807 *mC* G* C. 99%

P.mC. A.fU.fC.fC. A.

A. G.mU.mC. A. G.fC.fU. G.

G.mC.mU. G. G. A.mC.mU*mC*

14156 799 2808 A.mU. G.Chi 2809 G*mU*mU*mU* C. 95%

P.mC.fU.fU.fU.fC.fC.

mU. A.mU. A. A. G.fC.fU.fU. A.mU.

G.mC. G. G. A. A. A. A*mU* A* A*mU*mC*

14157 853 2810 G.Chi 2811 U. 106% mU.mU.mC.mC. G. P.mA. A.fU.fC. A.fC.

A.mU. G.mU. G. A.fU.fC. G. G. A. A*mU*

14158 888 2812 A.mU. mU. Chi 2813 G*mC*mU*mC* A. 88%

P.mA.fC. A.fC. A.fU.fU.

A.mU. A. A.mC.mU. A. G.fU.mU.

A. A.mU. G.mU. A.mU*mU*mU*mC*m

14159 1194 2814 G.mU. Chi 2815 C* A* G. 95% mU.mC. P.mU.fU.fC.fU. A.fU. A.

A.mU.mU.mC.mU. G. A. A.mU. G. A*

14160 1279 2816 A.mU. A. G. A. A.Chi 2817 A*mC* A*mU* A* G. 15%

P.mU. A.fC. A. G.fU. G.

A. A.mC.mU. A.fU. A.

A.mU.mC. A.mC.mU. G.mU.mU*mU* G*mC*

14161 1300 2818 G.mU. A.Chi 2819 A*mU* U. 86%

G.mU.mC. A.

A.mU.mU. P.mA.fU. A. A. G.fC. A.

G.mC.mU.mU. A.fU.fU. G. A.mC*

14162 1510 2820 A.mU. Chi 2821 A*mC*mC* A*mC* C. 86%

A. G.mC. A. P.mU.fU.fU. A.fU.fU. A.

A.mU.mU. A. A.mU. A.fU.fU. G.mC.mU* G*

14163 1543 2822 A. A. A.Chi 2823 G* A*mC* A* A. 110%

P.mU.fC. A.fU.fC. A. G.

A.mC. G. A. G.fU.mC.

A.mC.mU.mC.mU. G. G.mU*mU*mC* G* A*

14164 434 2824 A.mU. G. A. Chi 2825 G* U. 134%

P.mA.fU. A. A. A.fC.fC.

mU. A. G.mU. G.mU. A.fC. A.mC.mU.

G. G.mU.mU.mU. A*mU*mC*

14165 600 2826 A.mU. Chi 2827 A*mC*mC* U. 102% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

P.mU.fC. A.fU.fC.

A. A. G.mC.mC. A. A.fU.fU. G.

A.mU. G. A.mU. G. G.mC.mU.mU*mU*mC

14166 863 2828 A.Chi 2829 *mC* G*mC* U. 93%

A.mU. A. G.mU.mC. P.mA. G.fU.fU.fC.fC.fU.

A. G. G. A. G. A.fC.mU. A.mU*mC*

14167 902 2830 A.mC.mU.Chl 2831 A* A*mU*mC* A. 101%

P.mU.fU.fC. A.fC. G.

A. G.mU.mC. A. G.fC.fU. G.

G.mC.mC. G.mU. G. A.mC.mU*mU*mU* G*

14168 921 2832 A. A.Chi 2833 G* A* A. 98%

A.mC.mU. P.mU.fU.fC.fU.fC. A.fU.

A.mC.mC. A.mU. G. G. G.fU. A. G.mU* G*

14169 154 2834 A. G. A. A.Chi 2835 A* G*mU*mU* U. n/a

P.mA. A.fU.fC. A.

A. A. A.mC. A. G. G.fC.fC.fU.

G.mC.mU. G. G.mU.mU.mU* A*

14170 217 2836 A.mU. mU. Chi 2837 A*mC*mU* G* G. 66%

P.mG. G.fU.fU.fU.fC. A.

G. A. G.mU. G.fC.

G.mC.mU. G. A. A. A.mC.mU.mC*mU* G*

14171 816 2838 A.mC.mC.Chl 2839 G*mU*mC* A. 102%

P.mA.fU.fC. G. G. A.

mU. G. A. G.mC. A.fU. G.fC.mU.mC.

A.mU.mU.mC.mC. G. A*mU*mU*

14172 882 2840 A.mU. Chi 2841 G*mC*mU* C. 103%

A.

A.mU.mU.mC.mC. P.mU. G. G.fC.fU. G.fU.

A.mC. A. G.mC.mC. G. G. A. A.mU.mU*mC*

14173 932 2842 A.Chi 2843 A*mC* G* G* C. n/a

P.mU. A. A. G.fC. A.

mU. G.mU.mC. A. A.fU.fU. G. A.mC.

A.mU.mU. A*mC*mC* A*mC*mC*

14174 1509 2844 G.mC.mU. mU. A.Chi 2845 A. n/a

P.mC. A.

A.mC.mC. A.mU. G. A.fU.fU.fC.fU.fC. A.fU.

A. G. A. A.mU.mU. G. G.mU* A* G*mU*

14175 157 2846 G.Chi 2847 G* A* G. 109%

P.mU. G.

mC.mC. A. A.mC. G. G.fC.fU.fU.fU.fC.

A. A. A. G.mC.mC. G.fU.mU. G. G*

14176 350 2848 A.Chi 2849 A*mC*mU*mU* A* C. 95%

P.mA. A.fU.fC. A. G.fU.

mC.mU. G. G. A.fC.mC. A.

G.mU.mC. A.mC.mU. G*mU*mU*mC*

14177 511 2850 G. A.mU.mU. Chi 2851 A*mU* C. 100% mU. G.

G.mU.mU.mU. P.mA. G.fU.fC.fC. A.fU.

A.mU. G. G. A. A. A.mC.mC. A*mC*

14178 605 2852 A.mC.mU.Chl 2853 A*mC*mU* A* U. 99% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

P.mC. A. G.fC.

G. A.mC.mC. A. G. A.fC.fU.fC.fU. G.

A. G.mU. G.mC.mU. G.mU.mC*

14179 811 2854 G.Chi 2855 A*mU*mC*mC* A* G. 88%

P.mU. A.fU.fC. A.

G. A.mU. G.mU. G. A.fU.fC. A.fC.

A.mU.mU. G. A.mU. A.mU.mC* G* G* A*

14180 892 2856 A.Chi 2857 A*mU* G. 76%

P.mA.fU.fU.fC. A.fC. G.

G.mU.mC. A. G.fC.fU. G.

G.mC.mC. G.mU. G. A.mC*mU*mU*mU*

14181 922 2858 A. A.mU. Chi 2859 G* G* A. 59%

A. A.mU. G.mU. P.mA.fU. A. G. A.fU.

G.mU. A.fC. A.fC.

A.mU.mC.mU. A.mU.mU*mC* A*

14182 1169 2860 A.mU. Chi 2861 A*mC*mC* A. 69% mU.mU. G. A. P.mU.fU.fU.fC.fC. A. G.

G.mU.mC.mU. G. G. A.fC.fU.mC. A. A*

14183 1182 2862 A. A. A.Chi 2863 A*mU* A* G* A* U. n/a

G.mU.mC.mC. A. P.mU.fU. A. A.fU.fU.

G.mC. A. A.mU.mU. G.fC.fU. G. G. A.mC* A*

14184 1539 2864 A. A.Chi 2865 A*mC*mC* G* U. 77% mC.mC. A. G.mC. A. P.mU. A.fU.fU. A.

A.mU.mU. A. A.mU. A.fU.fU. G.fC.mU. G. G*

14185 1541 2866 A.Chi 2867 A*mC* A* A*mC* C. n/a

P.mA. G.fU.fC.

G. A.mC.mU.mC. G. G.fU.fU.fC. G. A.

A. A.mC. G. G.mU.mC* A* A*mU*

14186 427 2868 A.mC.mU.Chl 2869 G* G* A. 69%

P.mG.fU.fU. G.fC.fU. G.

A.mC.mC.mU. G.fC. A. G.

G.mC.mC. A. G.mC. G.mU*mC*mC*

14187 533 2870 A. A.mC.Chl 2871 G*mU* G* G. 78%

G. A.mU. G. A. P.mU. A.fU.fC. A. G.

A.mU.mC.mU. G. A.fU.fU.fC. A.fU.fC* A*

18538 496 2872 A.mU. A.Chi 2873 G* A* A*fU* G. 74% mU. G. A.mU. G. A. P.mU. A.fU.fC. A. G.

A.mU.mC.mU. G. A.fU.fU.fC. A.fU.fC* A*

18539 496 2874 A.mU. A.Chi 2875 G* A* A*fU* G. 72%

A.mU.mU.mU. P.mU. G.fC. A. A. A. A.

G.mC.mU.mU.mU.m G.fC. A. A. A.fU*fC*

18540 175 2876 U. G.mC. A.Chi 2877 A*fC*fU*fG* C. 98%

G. A.mU.mU.mU. P.mU. G.fC. A. A. A. A.

G.mC.mU.mU.mU.m G.fC. A. A. A.fU*fC*

18541 175 2878 U. G.mC. A.Chi 2879 A*fC*fU*fG* C. 28%

G.mU. G.

A.mU.mU.mU. P.mU. A. A. A. G.fC. A.

G.mC.mU.mU.mU. A. A.fU.fC. A.fC*fU*

18542 172 2880 A.Chi 2881 G*fC* A* A* U. 24% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

A. G.mU. G.

A.mU.mU.mU. P.mU. A. A. A. G.fC. A.

G.mC.mU.mU.mU. A. A.fU.fC. A.fC*fU*

18543 172 2882 A.Chi 2883 G*fC* A* A* U. 14%

A.

A.mU.mU.mU.mC. P.mU. A. A. A.fU. A.fC.

G.mU. G. A. A. A.fU.fU*fU*fC*

18544 1013 2884 A.mU.mU.mU. A.Chi 2885 A* G* G* U. 100%

A. A.

A.mU.mU.mU.mC. P.mU. A. A. A.fU. A.fC.

G.mU. G. A. A. A.fU.fU*fU*fC*

18545 1013 2886 A.mU.mU.mU. A.Chi 2887 A* G* G* U. 109% mC. A.mC. A. P.mU.fU.fU. C. A.fU. G.

G.mC.mC. A.mU. G. G.fC.fU. G.fU. G* A* A*

18546 952 2888 A. A. A.Chi 2889 A*fU*fU* C. 32% mU.mC. A.mC. A. P.mU.fU.fU. C. A.fU. G.

G.mC.mC. A.mU. G. G.fC.fU. G.fU. G* A* A*

18547 952 2890 A. A. A.Chi 2891 A*fU*fU* C. 33%

G. A.mU.mU.mU. P.mU.fC. A. A. A. A.

G.mC.mU.mU.mU.m G.fC. A. A. A.fU.fC*

18548 174 2892 U. G. A. Chi 2893 A*fC*fU* G*fC* A. 57% mU. G.

A.mU.mU.mU. P.mU.fC. A. A. A. A.

G.mC.mU.mU.mU.m G.fC. A. A. A.fU.fC*

18549 174 2894 U. G. A. Chi 2895 A*fC*fU* G*fC* A. 53% mU.mU.

G.mC.mU.mU.mU.m P.mU. A. G. G.fC. A. A.

U. G.mC.mC.mU. A. A. G.fC. A. A*

18550 177 2896 A.Chi 2897 A*fU*fC* A*fC* U. 97% mU.mU.mU.

G.mC.mU.mU.mU.m P.mU. A. G. G.fC. A. A.

U. G.mC.mC.mU. A. A. G.fC. A. A*

18551 177 2898 A.Chi 2899 A*fU*fC* A*fC* U. 103% mU.mU.mU.mC.mU.

mC. A. P.mU.fU. A. A. A.fC.fU.

G.mU.mU.mU. A. G. A. G. A. A. A* G* A*

18552 1150 2900 A.Chi 2901 A* G*fC* A. 96% mU.mU. G.mC. P.mU. G. A.fC.fU. A. A.

A.mU.mU.mU. A. A.fU. G.fC. A. A* A*

18553 1089 2902 G.mU.mC. A.Chi 2903 G*fU* G* A* G. 94%

A.mC.mU.mU.mU.

G.mC. P.mU.fU. A. A. A.fU.

A.mU.mU.mU. A. G.fC. A. A. A. G.fU* G*

18554 1086 2904 A.Chi 2905 A* G* A* A* A. n/a

A.mU.mU.mU. A. P.mU.fU.fU.fU.fU. G.

G.mU.mC. A. A. A. A. A.fC.fU. A. A. A.fU*

18555 1093 2906 A.Chi 2907 G*fC* A* A* A* G. n/a mU.mU.mC.mU.mU. P.mU. A.fC.fU. G. A. G.

mU.mC.mU.mC. A. A. A. A. G. A. A* G*fC*

18556 1147 2908 G.mU. A.Chi 2909 A*fU*fU* U. n/a % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

mU.mC.mU.mU.mU. P.mU. A. A.fC.fU. G. A.

mC.mU.mC. A. G. A. A. A. G. A* A*

18557 1148 2910 G.mU.mU. A.Chi 2911 G*fC* A*fU* U. 66%

P.mU. A.fU.

G. A. A. A. G. A. G. G.fU.fU.fC.fU.fC.fU.fU.f

A. A.mC. A.mU. U.fC* A*fU*fU*fU*fU*

18558 1128 2912 A.Chi 2913 G. 16% mC.mU.mU.mU.

G.mC. P.mU.fC.fU. A. A. A.fU.

A.mU.mU.mU. A. G. G.fC. A. A. A. G*fU* G*

18559 1087 2914 A.Chi 2915 A* G* A* A. 28% mU.mU.mU. G.mC. P.mU. A.fC.fU. A. A.

A.mU.mU.mU. A. A.fU. G.fC. A. A. A*

18560 1088 2916 G.mU. A.Chi 2917 G*fU* G* A* G* A. n/a mC.mU.mC. P.mU. A.fU. G.fC. A. A.

A.mC.mU.mU.mU. A. G.fU. G. A. G* A* A*

18561 1083 2918 G.mC. A.mU. A.Chi 2919 A*fU*fU* G. 53% mU.mU.mC.mU.mC. P.mU. G.fC. A. A. A.

A.mC.mU.mU.mU. G.fU. G. A. G. A. A*

18562 1081 2920 G.mC. A.Chi 2921 A*fU*fU* G*fU* A. 89% mC.

A.mC.mU.mC.mC. A. P.mU. A.fC. A. A.fC.fU.

G.mU.mU. G.mU. G. G. A. G.fU. G* A* A*

18563 555 2922 A.Chi 2923 A* A*fC*fU. 33%

P.mU.fU.fU.fC.fU.fC.fU.

fU.fU.fC.

A. A.mU. G. A. A. A. A.fU.fU*fU*fU*

18564 1125 2924 G. A. G. A. A. A.Chi 2925 G*fC*fU* A. n/a mU. G.mC. A. G.mU.

G. P.mU.fC. A. A. A.fU.fC.

A.mU.mU.mU.mG. A.fC.fU. G.fC. A*

18565 168 2926 A.Chi 2927 A*fU*fU*fC*fU* C. 14%

P.mU.fU.

G.fU.fU.fC.fU.fC.fU.fU.f

mU. G. A. A. A. G. A. U.fC. A*fU*fU*fU*fU*

18566 1127 2928 G. A. A.mC. A. A.Chi 2929 G* C. 27%

A.mC.mC.mU. G. A. P.mU. G. A. A.

A. A.fU.fU.fU.fC. A. G.

A.mU.mU.mU.mC. G.fU* G*fU*fU*fU* A*

18567 1007 2930 A.Chi 2931 U. 129%

P.mU.fU.fC. A.fC.fU.

G. A. A.mU.mU. G.fC. A.

G.mC. A. G.mU. G. A. A.fU.fU.fC*fU*fC*

18568 164 2932 A.Chi 2933 A*fU* G* G. 47%

G. G.mC.mU. G. P.mU.fC.fC. A. G. A.

A.mU.mU.mC.mU. A.fU.fC. A. G.fC.fC*fU*

18569 222 2934 G. G. A.Chi 2935 G*fU*fU*fU* A. n/a

A. G.mU. G.

A.mU.mU.mU. P.mU. A. A. A. G.fC. A.

G.mC.mU.mU.mU. A. A.fU.mC. A.mC*mU*

20612 172 2936 A.Chi 2937 G*mC* A* A* U. n/a % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

A. G.mU. G.

A.mU.mU.mU. P.mU. A. A. A. G.fC. A.

G.mC.mU.mU.mU. A. A.fU.fC. A.mC*fU*

20613 172 2938 A.Chi 2939 G*mC* A* A* U. n/a

A. G.mU. G.

A.mU.mU.mU. P.mU. A. A. A. G. C. A.

G.mC.mU.mU.mU. A. A. U.mC. A.mC*mU*

20614 172 2940 A.Chi 2941 G*mC* A* A* U. 101%

A. G.mU. G. P.mU. A. A. A. G.fC. A.

A.mU.mU.mU. A. A.fU.mC.

G.mC.mU.mU.mU. A.mC*mU*mG*mC*m

20615 172 2942 A.Chi 2943 A*mA* U. 104%

Table 19: Inhibition of gene expression with PTGS2 sd-rxRNA sequences

(Accession Number: NM_000963.2)

% remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

G.mU.mC.mC.

A.mC.mC. A. P.mU.fU. A. A. G.fU.fU.

A.mC.mU.mU. A. G. G.fU. G. G. A*fC*fU*

17391 467 2958 A.Chi 2959 G*fU*fC* A. 101% mC.mU.mC.mC.mU. P.mU. G.fU. A.fU. A.

A.mU.mU. A.mU. A.fU. A. G. G. A. G* A*

17392 524 2960 A.mC. A.Chi 2961 G* G*fU*fU* A. 49%

G. A.mU.mC. A.mC. P.mU.fU.fC. A. A. A.fU.

A.mU.mU.mU. G. A. G.fU. G. A.fU.fC*fU* G*

17393 448 2962 A.Chi 2963 G* A*fU* G. 29%

A. G. A.mU.mC.

A.mC. P.mU.fU.fC. A. A. A.fU.

A.mU.mU.mU. G. A. G.fU. G. A.fU.fC*fU* G*

17394 448 2964 A.Chi 2965 G* A*fU* G. 31%

A.

A.mC.mC.mU.mC.m P.mU. A.fU. A. G. G. A.

U.mC.mC.mU. A.mU. G. A. G. G.fU.fU* A* G*

17395 519 2966 A.Chi 2967 A* G* A* A. 12%

G.mU.mU. G. A.mC. P.mU.fC.fU. G. G. A.fU.

A.mU.mC.mC. A. G. G.fU.fC. A. A.fC* A*fC*

17396 437 2968 A.Chi 2969 A*fU* A* A. 86% mC.mC.mU.mU.mC. P.mU.fU.fU.fC. G. A. A.

mC.mU.mU.mC. G. G. G. A. A. G. G* G* A*

17397 406 2970 A. A. A.Chi 2971 A*fU* G* U. 23%

P.mU.fU. G.fU.

A.mC.mU.mC.mC. G.fU.fU.fU. G. G. A.

A. A. A.mC. A.mC. A. G.fU* G* G* G*fU*fU*

17398 339 2972 A.Chi 2973 U. 102% mC. P.mU.fU. G.fU.

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*

17399 339 2974 A.Chi 2975 U. 55% mC. P.mU. G.fU. G.fU.fU.fU.

A.mC.mU.mC.mC. A. G. G. A. G.fU. G* G*

17400 338 2976 A. A.mC. A.mC. A.Chi 2977 G*fU*fU*fU* C. 62% mC.mC. A.mC.mC. A. P.mU. G.fU. A. A.

A.mC.mU.mU. A.mC. G.fU.fU. G. G.fU. G. G*

17401 468 2978 A.Chi 2979 A*fC*fU* G*fU* C. 61% mU.mC.mC.

A.mC.mC. A. P.mU. G.fU. A. A.

A.mC.mU.mU. A.mC. G.fU.fU. G. G.fU. G. G*

17402 468 2980 A.Chi 2981 A*fC*fU* G*fU* C. 179%

A. A.mU. A.mC.mC.

A. P.mU. A. A. G. A.fC.fU.

G.mU.mC.mU.mU. G. G.fU. A.fU.fU*fU*fC*

17403 1465 2982 A.Chi 2983 A*fU*fC* U. 30%

G. A.mC.mC. A. P.mU.fC.fU.fU. A.fU.

G.mU. A.mU. A. A. A.fC.fU. G. G.fU.fC* A*

17404 243 2984 G. A.Chi 2985 A* A*fU*fC* C. 32% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

G.mU.mC.mU.mU.m P.mU.fU.fC. A.fU.fU. A.

U.mU. A. A.mU. G. A. A. A. G. A.fC*fU* G*

17405 1472 2986 A. A.Chi 2987 G*fU* A* U. 15%

A.

A.mU.mU.mU.mC. P.mU. A. G. A.fC. A.fU.

A.mU. G. A. A. A.fU.fU*

17406 2446 2988 G.mU.mC.mU. A.Chi 2989 A*fC*fU* G* G* U. 142%

P.mU. A.fU.fC. A. A.

A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.fU*fC*fU* G* G* A*

17407 449 2990 A.mU. A.Chi 2991 U. 54%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.fU*fC*fU* G* G* A*

17408 449 2992 A.mU. A.Chi 2993 U. 27% mU.mC.mC. A. G. P.mU. A.fU. G.fU. G.

A.mU.mC. A.mC. A.fU.fC.fU. G. G. A*fU*

17409 444 2994 A.mU. A.Chi 2995 G*fU*fC* A* A. 49% mU. A.mC.mU. G. P.mU.fC.fU.fC.fC.fU.

A.mU. A. G. G. A. G. A.fU.fC. A. G.fU.

17410 1093 2996 A.Chi 2997 A*fU*fU* A* G*fC* C. 32%

G.mU. G.mC. A. P.mU.fC. A. A. G.fU.

A.mC. A.mC.mU.fU. G.fU.fU. G.mC. A.fC*

17411 1134 2998 G. A.Chi 2999 A*fU* A* A*fU* C. 70%

A.mC.mC. A. G.mU. P.mU. A.fC.fU.fU. A.fU.

A.mU. A. A. G.mU. A.fC.fU. G. G.fU*fC* A*

17412 244 3000 A.Chi 3001 A* A*fU* C. 63%

G. A. A. P.mU.fU.fC. A.fU.fU. A.

G.mU.mC.mU. A. G. A.fC.mU.fU.fC*fU*

17413 1946 3002 A.mU. G. A. A.Chi 3003 A*fC* A* G* U. 19%

P.mU. A.

A. A. G. A. A. G. A. A.fC.fU.fU.fU.fC.fU.fU.f

A. A. G.mU.mU. C.fU.fU* A* G* A* A*

17414 638 3004 A.Chi 3005 G* C. 27% mU.mC. A.mC. P.mU. A. A.fU.fC. A. A.

A.mU.mU.mU. G. A.fU. G.fU. G.

17415 450 3006 A.mU.mU. A.Chi 3007 A*fU*fC*fU* G* G* A. 216%

A.mU.mC. A.mC. P.mU. A. A.fU.fC. A. A.

A.mU.mU.mU. G. A.fU. G.fU. G.

17416 450 3008 A.mU.mU. A.Chi 3009 A*fU*fC*fU* G* G* A. 32%

A.mC.

A.mU.mU.mU. G. P.mU.fU.fC. A. A.fU.fC.

A.mU.mU. G. A. A. A. A.fU. G.fU* G*

17417 452 3010 A.Chi 3011 A*fU*fC*fU* G. 99% mC. A.mC.

A.mU.mU.mU. G. P.mU.fU.fC. A. A.fU.fC.

A.mU.mU. G. A. A. A. A.fU. G.fU* G*

17418 452 3012 A.Chi 3013 A*fU*fC*fU* G. 54%

A.mU.mU.mU. G. P.mU.fU. G.fU.fC. A.

A.mU.mU. G. A.mC. A.fU.fC. A. A. A.fU*

17419 454 3014 A. A.Chi 3015 G*fU* G* A*fU* C. 86% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

mC. A.mU.mU.mU. P.mU.fU. G.fU.fC. A.

G. A.mU.mU. G. A.fU.fC. A. A. A.fU*

17420 454 3016 A.mC. A. A.Chi 3017 G*fU* G* A*fU* C. 89% mC. A.mU.mC.mU. P.mU.fU.fU. A.fU.fU.

G.mC. A. A.mU. A. A. G.fC. A. G. A.fU. G* A*

17421 1790 3018 A.Chi 3019 G* A* G* A* C. 55% mU.mC. P.mU.fU.fU. A.fU.fU.

A.mU.mC.mU. G.mC. G.fC. A. G. A.fU. G* A*

17422 1790 3020 A. A.mU. A. A. A.Chi 3021 G* A* G* A* C. 62%

G. A.mU.mC. A.mC. P.mU.fU.fC. A.mA. A.fU.

A.mU.mU.mU. G. A. G.fU. G. A.mU.mC*mU*

21180 448 3022 A.TEG-Chl 3023 G* G* A*mU* G. 76%

P.mU.fU.fC. A.mA. A.fU.

G. A.mU.mC. A.mC. G.fU. G.

A.mU.mU.mU. G. A. A.fU.fC*fU*mG*mG*m

21181 448 3024 A.TEG-Chl 3025 A*fU* G. 37%

G. A.mU.mC. A.mC. P.mU.fU.fC. A. A. A.fU.

A.mU.mU.mU. G.fU. G. A.fU.fC*fU* G*

21182 448 3026 G*mA*mA.TEG-Chl 3027 G* A*fU* G. 29% mG*mA*mU.mC.

A.mC. P.mU.fU.fC. A. A. A.fU.

A.mU.mU.mU. G.fU. G. A.fU.fC*fU* G*

21183 448 3028 G*mA*mA.TEG-Chl 3029 G* A*fU* G. 46% mG*mA*mU.mC.mA

.mC.mA.mU.mU.mU P.mU.fU.fC. A. A. A.fU.

.mG*mA*m A.TEG- G.fU. G. A.fU.fC*fU* G*

21184 448 3030 Chl 3031 G* A*fU* G. 60%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.fU.fC*fU* G* G*

21185 449 3032 A.mU. A.TEG-Chl 3033 A*fU* G. 27%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.mU.mC*mU* G* G*

21186 449 3034 A.mU. A.TEG-Chl 3035 A*mU* G. 57%

P.mU. A.fU.fC. A.mA.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.mU.mC*mU* G* G*

21187 449 3036 A.mU. A.TEG-Chl 3037 A*mU* G. 54%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.mU.mC*mU*mG*mG

21188 449 3038 A.mU. A.TEG-Chl 3039 *mA*mU* G. 66%

P.mU. A.fU.fC. A.mA.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.mU.mC*mU*mG*mG

21189 449 3040 A.mU. A.TEG-Chl 3041 *mA*mU* G. 44%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.fU.fC*fU*mG*mG*m

21190 449 3042 A.mU. A.TEG-Chl 3043 A*fU* G. 52% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

P.mU. A.fU.fC. A.mA.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.fU.fC*fU*mG*mG*m

21191 449 3044 A.mU. A.TEG-Chl 3045 A*fU* G. 41%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.fU.mC*fU*mG*mG*

21192 449 3046 A.mU. A.TEG-Chl 3047 mA*fU* G. 98%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.fU*fC*fU* G* G* A*

21193 449 3048 A*mU*mA.TEG-Chl 3049 U. 93% mG*mA*mU.mC. P.mU. A.fU.fC. A. A.

A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.fU*fC*fU* G* G* A*

21194 449 3050 A*mU*mA.TEG-Chl 3051 U. 119% mG*mA*mU.mC.mA P.mU. A.fU.fC. A. A.

.mC.mA.mU.mU.mU A.fU. G.fU. G.

.mG.mA*mU*mA.TE A.fU*fC*fU* G* G* A*

21195 449 3052 G-Chl 3053 U. 292%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.mU*mC*mU* G* G*

20620 449 3054 A.mU. A.Chl-TEG 3055 A* U. 24%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.mU*fC*mU* G* G*

20621 449 3056 A.mU. A.Chl-TEG 3057 A* U. 5%

P.mU. A. U. C. A. A. A.

G. A.mU.mC. A.mC. U. G. U. G.

A.mU.mU.mU. G. A.mU*mC*mU* G* G*

20622 449 3058 A.mU. A.Chl-TEG 3059 A* U. 25%

P.mU. A.fU.fC. A. A.

G. A.mU.mC. A.mC. A.fU. G.fU. G.

A.mU.mU.mU. G. A.mU*mC*mU*mG*m

20623 449 3060 A.mU. A.Chl-TEG 3061 G*mA* U. 14%

G. A.mU.mC. A.mC. P.mU.fU.fC. A. A. A.fU.

A.mU.mU.mU. G. A. G.fU. G. A.mU.mC*mU*

20588 448 3062 A.Chl-TEG 3063 G* G* A*mU* G. 17%

G. A.mU.mC. A.mC. P.mU.fU.fC. A. A. A.fU.

A.mU.mU.mU. G. A. G.fU. G. A.mU.fC*mU*

20589 448 3064 A.Chl-TEG 3065 G* G* A*fU* G. 40%

G. A.mU.mC. A.mC. P.mU. U. C. A. A. A. U.

A.mU.mU.mU. G. A. G. U. G. A.mU.mC*mU*

20590 448 3066 A.Chl-TEG 3067 G* G* A*mU* G. 34%

P.mU.fU.fC. A. A. A.fU.

G. A.mU.mC. A.mC. G.fU. G.

A.mU.mU.mU. G. A. A.fU.fC*fU*mG*mG*m

20591 448 3068 A.Chl-TEG 3069 A*fU* G. n/a Table 20: Inhibition of gene expression with CTGF sd-rxRNA sequences (Accession Number: NM_001901.2)

% remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

A. A.mC.mU. P.mG. G. A.fC.fC. A. G.

G.mC.mC.mU. G. G.fC. A. G.mU.mU* G*

13991 792 3092 G.mU.mC.mC.Chl 3093 G*mC*mU*mC* U. 97%

A. G.

A.mC.mC.mU. P.mC. A. G. G.fC. A.fC.

G.mU. A. G.

G.mC.mC.mU. G.mU.mC.mU*mU*

13992 1162 3094 G.Chi 3095 G* A*mU* G* A. 107%

P.mG.fC. G.fC.fU.fC.fC.

mC. A. G. A. A.fC.fU.mC.mU.

G.mU. G. G. A. G*mU* G*

13993 811 3096 G.mC. G.mC.Chl 3097 G*mU*mC* U. 113%

P.mG. G.fU.fC.fU. G.

mC.mC.mU. G. G. A.fC.fC. A. G.

G.mU.mC.mC. A. G*mC* A*

13994 797 3098 G. A.mC.mC.Chl 3099 G*mU*mU* G. n/a mC.mC. P.mA.fC. A. G.fU.fU.

A.mU.mU. A.mC. G.fU. A. A.mU. G.

A. A.mC.mU. G*mC* A* G* G*mC*

13995 1175 3100 G.mU. Chi 3101 A. 113% mC.mU.

G.mC.mC. P.mG.fU.fU. G.fU. A.

A.mU.mU. A.mC. A.fU. G. G.mC. A. G*

13996 1172 3102 A. A.mC.Chl 3103 G*mC* A*mC* A* G. 110%

P.mG. G. A.fC. A.

A.mU.mU. A.mC. G.fU.fU. G.fU. A.

A. A.mC.mU. A.mU* G* G*mC* A*

13997 1177 3104 G.mU.mC.mC.Chl 3105 G* G. 105% mC. A.mU.mU.

A.mC. A. P.mG. A.fC. A. G.fU.fU.

A.mC.mU. G.fU. A. A.mU. G*

13998 1176 3106 G.mU.mC.Chl 3107 G*mC* A* G* G* C. 89%

P.mG. G.fC.

A. G. A. G.mU. G. G.fC.fU.fC.fC.

G. A. G.mC. A.fC.mU.mC.mU*

13999 812 3108 G.mC.mC. Chi 3109 G*mU* G* G*mU* C. 99%

A.mC.mC. G. P.mU.fC.fU.fU.fC.fC. A.

A.mC.mU. G. G. A. G.fU.fC. G. G.mU* A*

14000 745 3110 A. G. A.Chi 3111 A* G*mC*mC* G. n/a

P.mU.

G.fU.fC.fU. fC.fC. G.fU.

A.mU. G.mU. A.mC.

A.mC. G. G. A. G. A.mU*mC*mU*mU*

14001 1230 3112 A.mC. A.Chi 3113 mC*mC* U. 106%

P.mA. G.fC.fU.fU.fC.

G.mC.mC.mU.mU. G.fC. A. A. G.

G.mC. G. A. A. G.mC*mC*mU* G*

14002 920 3114 G.mC.mU.Chl 3115 A*mC* C. 93% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

P.mC.

A.fC.fU. fC.fC.fU.fC.

G.mC.mU. G.mC. G.fC. A. G.mC*

G. A. G. G. A. A*mU*mU*mU*mC*

14003 679 3116 G.mU. G.Chi 3117 C. 102%

P.mA. A. A.fC.fU.fU. G.

G.mC.mC.mU. A.fU. A. G.

A.mU.mC. A. A. G.mC*mU*mU* G*

14004 992 3118 G.mU.mU.mU.Chl 3119 G* A* G. 100%

A. P.mA.fC.fU.fC.fC. A.fC.

A.mU.mU.mC.mU. A. G. A.

G.mU. G. G. A. A.mU.mU*mU* A*

14005 1045 3120 G.mU. Chi 3121 G*mC*mU* C. 104%

P.mA.fU.

G.fU.fC.fU.fC.fC. G.fU.

mU. G.mU. A.mC. A.mC.

G. G. A. G. A.mC. A*mU*mC*mU*mU*

14006 1231 3122 A.mU.Chl 3123 mC* C. 87%

P.mA. A.fC.fU.fU. G.

A. G.mC.mC.mU. A.fU. A. G.

A.mU.mC. A. A. G.mC.mU*mU* G* G*

14007 991 3124 G.mU.mU.Chl 3125 A* G* A. 101% mC. A. A.

G.mU.mU.mU. G. P.mA. A. G.fC.fU.fC. A.

A. A. A.fC.mU.mU. G*

14008 998 3126 G.mC.mU.mU.Chl 3127 A*mU* A* G* G* C. 98%

P.mA.fC. A.fU.

mC.mU. G.mU. G. A.fC.fU. fC.fC. A.mC. A.

G. A. G.mU. A.mU. G* A*

14009 1049 3128 G.mU. Chi 3129 A*mU*mU*mU* A. 98%

A. A.

A.mU.mU.mC.mU. P.mC.fU.fC.fC. A.fC. A.

G.mU. G. G. A. G. A. A.mU.mU.mU*

14010 1044 3130 G.Chi 3131 A* G*mC*mU*mC* G. 93%

P.mU. G.fU. G.fC.fU.

mU.mU.mU.mC. A.fC.fU. G. A. A.

A. G.mU. A. G.mC. A*mU*mC*

14011 1327 3132 A.mC. A.Chi 3133 A*mU*mU* U. 95% mC. A. A.mU. G. P.mA. A. A. G. A.fU.

A.mC. G.fU.fC. A.mU.mU.

A.mU.mC.mU.mU. G*mU*mC*mU*mC*

14012 1196 3134 mU.Chl 3135 mC* G. 101%

A. G.mU. P.mG.fU. G.fC. A.fC.fU.

A.mC.mC. A. G. G.fU.

G.mU. G.mC. A.mC.mU*mU*

14013 562 3136 A.mC.Chl 3137 G*mC* A* G* C. 66%

P.mA. A. A.fC. G.fU.

G. G. A. A. G. G.fU.fC.fU.mU.mC.mC

A.mC. A.mC. * A* G*mU*mC* G*

14014 752 3138 G.mU.mU.mU.Chl 3139 G. 95% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

P.mU.fC. A. A.

mC.mU. A.fC.fU.fU. G. A.mU.

A.mU.mC. A. A. A. G*

G.mU.mU.mU. G. G*mC*mU*mU* G*

14015 994 3140 A.Chi 3141 G. 85%

A. G.mC.mU. A. P.mA.fC. A. G. A.

A. A.fU.fU.fU. A.

A.mU.mU.mC.mU. G.mC.mU*mC* G*

14016 1040 3142 G.mU.Chl 3143 G*mU* A* U. 61%

P.mU.fU. A.fC.

A. G. G.mU. A. G. A.fU.fU.fC.fU.

A. A.mU. G.mU. A. A.mC.mC.mU* A*mU*

14017 1984 3144 A.Chi 3145 G* G*mU* G. 32%

A. G.mC.mU. G. P.mA. A. A.fC.fU. G.

A.mU.mC. A. A.fU.fC. A. G.mC.mU*

14018 2195 3146 G.mU.mU.mU.Chl 3147 A*mU* A*mU* A* G. 86%

P.mU. A.fU.fC.fU. G. A.

mU.mU.mC.mU. G.fC. A. G. A.

G.mC.mU.mC. A. A*mU*mU*mU*mC*

14019 2043 3148 G. A.mU. A.Chi 3149 mC* A. 81% mU.mU. P.mU.fU. A.

A.mU.mC.mU. A. A.fC.fU.fU. A. G.

A. G.mU. mU. A. A.mU. A. A*mC*mU*

14020 1892 3150 A.Chi 3151 G*mU* A* C. 84%

P.mU. A.fU.fU.

mU. A.mU. A.mC. A.fC.fU. fC. G.fU.

G. A. G.mU. A. A.mU. A* A* G*

14021 1567 3152 A.mU. A.Chi 3153 A*mU* G* C. 72%

P.mA. A. G.fC.fU.

G. A.mC.mU. G. G.fU.fC.fC. A.

G. A.mC. A. G.mU.mC*mU* A*

14022 1780 3154 G.mC.mU.mU.Chl 3155 A*mU*mC* G. 65%

A.mU. G. P.mU. A. A.fU. A. A. A.

G.mC.mC.mU.mU. G. G.fC.mC.

mU. A.mU.mU. A.mU*mU*mU*

14023 2162 3156 A.Chi 3157 G*mU*mU* C. 80%

P.mU.fU.fU. A.

G.fC.fU.fC. G. G.mU.

A.mU. A.mC.mC. A.mU*

G. A. G.mC.mU. A. G*mU*mC*mU*mU*

14024 1034 3158 A. A.Chi 3159 C. 91% mU.mU. P.mA.fC.

G.mU.mU. G. A. A.fC.fU. fC.fU.fC. A.

G. A. G.mU. A.mC. A. A* A*mU*

14025 2264 3160 G.mU.Chl 3161 A* A* A* C. 58%

P.mU. A. G.fC.fU.fC. G.

A.mC. A.mU. G.fU. A.mU.

A.mC.mC. G. A. G.mU*mC*mU*mU*

14026 1032 3162 G.mC.mU. A.Chi 3163 mC* A* U. 106% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

P.mU. A.

A. G.mC. A. G. A. A.fC.fC.fU.fU.fU.fC.fU.

A. A. G. G.mU.mU. G.mC.mU* G* G*mU*

14027 1535 3164 A.Chi 3165 A*mC* C. 67%

P.mU.fU. A. A. G. G. A.

A. G.mU.mU. A.fC. A.

G.mU.mU.mC.mC. A.mC.mU*mU* G*

14028 1694 3166 mU.mU. A. A.Chi 3167 A*mC*mU* C. 94%

P.mU.fU. A.fC.

A.mU.mU.mU. G. A.fC.fU.fU.fC. A. A.

A. A. G.mU. G.mU. A.mU* A* G*mC* A*

14029 1588 3168 A. A.Chi 3169 G* G. 97%

P.mU.fC.fC. A. G.

A. A. G.mC.mU. G.fU.fC. A.

G. A.mC.mC.mU. G.mC.mU.mU*mC*

14030 928 3170 G. G. A.Chi 3171 G*mC* A* A* G. 100%

P.mC.fU.fU.fC.fU.fU.fC

G. G.mU.mC. . A.fU. G.

A.mU. G. A. A. G. A.mC.mC*mU*mC*

14031 1133 3172 A. A. G.Chi 3173 G*mC*mC* G. 82%

A.mU. G.

G.mU.mC. A. G. P.mA. A. G. G.fC.fC.fU.

G.mC.mC.mU.mU. G. A.fC.mC. A.mU*

14032 912 3174 Chi 3175 G*mC* A*mC* A* G. 84%

G. A. A. G. A.mC.

A.mC. P.mC. A. A. A.fC. G.fU.

G.mU.mU.mU. G.fU.fC. mU.mU.mC*m

14033 753 3176 G.Chi 3177 C* A* G*mU*mC* G. 86%

A. G.

G.mC.mC.mU.mU. P.mC.fU.fU.fC. G.fC. A.

G.mC. G. A. A. A. G. G.mC.mC.mU*

14034 918 3178 G.Chi 3179 G* A*mC*mC* A* U. 88% mU. A.mC.mC. G. P.mC.fU.fU.fC.fC. A.

A.mC.mU. G. G. A. G.fU.fC. G. G.mU. A*

14035 744 3180 A. G.Chi 3181 A* G*mC*mC* G* C. 95%

P.mC.fC. G.

A.mC.mC. G.mC. A.fU.fC.fU.fU. G.fC. G.

A. A. G. A.mU.mC. G.mU*mU* G*

14036 466 3182 G. G.Chi 3183 G*mC*mC* G. 73% mC. A. G. P.mU.fU.fC. G.fC. A. A.

G.mC.mC.mU.mU. G. G.fC.mC.mU. G*

14037 917 3184 G.mC. G. A. A.Chi 3185 A*mC*mC* A*mU* G. 86% mC. G. A. P.mA. G. A.

G.mC.mU. A. A. A.fU.fU.fU. A.

A.mU.mU.mC.mU. G.fC.mU.mC. G*

14038 1038 3186 Chi 3187 G*mU* A*mU* G* U. 84% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

P.mC. A.fU.

mU.mC.mU. A.fC.fU.fC.fC. A.fC. A.

G.mU. G. G. A. G. A*

G.mU. A.mU. A*mU*mU*mU* A*

14039 1048 3188 G.Chi 3189 G. 87%

P.mU. G.fC.fC. A.fU.

mC. G. G. A. G. G.fU.fC.fU.mC.mC.

A.mC. A.mU. G. G*mU* A*mC*

14040 1235 3190 G.mC. A.Chi 3191 A*mU* C. 100%

A.mU. G. A.mC. P.mG. A. G. G.fC.

A. A.mC. G.fU.fU. G.fU.mC.

G.mC.mC.mU.mC. A.mU*mU* G*

14041 868 3192 Chi 3193 G*mU* A* A. 104%

P.mU.fC.fU.fU.fC.

G. A. G. A.fU. G.

G.mU.mC. A.mU. A.fC.mC.mU.mC*

14042 1131 3194 G. A. A. G. A. Chi 3195 G*mC*mC* G*mU* C. 85% mU. A. A. P.mU.fC.fC. A.fC. A. G.

A.mU.mU.mC.mU. A. A.fU.mU.mU. A*

14043 1043 3196 G.mU. G. G. A.Chi 3197 G*mC*mU*mC* G* G. 74%

P.mA. A.fC. G.fU.

mU. G. G. A. A. G. G.fU.fC.fU.fU.mC.mC.

A.mC. A.mC. A* G*mU*mC* G* G*

14044 751 3198 G.mU.mU.Chl 3199 U. 84%

P.mC.fU.fC.fC. G.fU.

A. A. G. A.mU. A.fC.

G.mU. A.mC. G. G. A.fU.mC.mU.mU*mC*

14045 1227 3200 A. G.Chi 3201 mC*mU* G*mU* A. 99%

A. A.mU. G. P.mA. G. G.fC. G.fU.fU.

A.mC. A. A.mC. G.fU.fC. A.mU.mU* G*

14046 867 3202 G.mC.mC.mU.Chl 3203 G*mU* A* A* C. 94%

P.mU.fC. A.fU. G.

G. G.mC. G. A. G. A.fC.fC.fU.fC.

G.mU.mC. A.mU. G.mC.mC*

14047 1128 3204 G. A.Chi 3205 G*mU*mC* A* G* G. 89%

P.mG. G.fC.fC. A. A.

G. A.mC. A.mC. A.fC. G.fU.

G.mU.mU.mU. G. G.mU.mC*mU*mU*m

14048 756 3206 G.mC.mC.Chl 3207 C*mC* A* G. 93%

P.mG.fC.fC. A.fU.

A.mC. G. G. A. G. G.fU.fC.fU.fC.mC.

A.mC. A.mU. G. G.mU* A*mC*

14049 1234 3208 G.mC.Chl 3209 A*mU*mC* U. 100%

P.mU.fC. G.fC. A. A. G.

mU.mC. A. G. G.fC.fC.mU. G.

G.mC.mC.mU.mU. A*mC*mC* A*mU*

14050 916 3210 G.mC. G. A.Chi 3211 G* C. 96%

G.mC. G. A. A. P.mA. G. G.fU.fC. A.

G.mC.mU. G. G.fC.fU.fU.mC. G.mC*

14051 925 3212 A.mC.mC.mU.Chl 3213 A* A* G* G*mC* C. 80% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

P.mC.fC. G.fU. A.fC.

G. G. A. A. G. A.fU.fC.fU.mU.mC.mC

A.mU. G.mU. *mU* G*mU* A* G*

14052 1225 3214 A.mC. G. G.Chi 3215 U. 96%

G.mU. G.

A.mC.mU.mU.mC. P.mG. A. G.fC.fC. G. A.

G. A. G.fU.mC. A.mC* A*

14053 445 3216 G.mC.mU.mC.Chl 3217 G* A* A* G* A. 101% mU. G.

A.mC.mU.mU.mC. P.mG. G. A. G.fC.fC. G.

G. A. A. G.mU.mC.

G.mC.mU.mC.mC. A*mC* A* G* A* A*

14054 446 3218 Chi 3219 G. 93% mU. G. G.mU.mC. P.mC. A. A. G.

A. G. G.fC.fC.fU. G.

G.mC.mC.mU.mU. A.mC.mC. A*mU*

14055 913 3220 G.Chi 3221 G*mC* A*mC* A. 67% mU.mC. A. A. P.mA. G.fC.fU.fC. A. A.

G.mU.mU.mU. G. A.fC.fU.mU. G. A*mU*

14056 997 3222 A. G.mC.mU.Chl 3223 A* G* G*mC* U. 92%

P.mC.fU. G.fC. A.

G.mC.mC. A. G. G.fU.fU.fC.fU. G.

A. A.mC.mU. G.mC*mC* G* A*mC*

14057 277 3224 G.mC. A. G.Chi 3225 G* G. 84% mU. G. G. A. P.mG. G.fU. A.fC. A.fU.

G.mU. A.mU. A.fC.fU.mC.mC.

G.mU. A*mC* A* G* A* A*

14058 1052 3226 A.mC.mC.Chl 3227 U. n/a

P.mC.fU.

G.mC.mU. A. G. G.fC.fU.fU.fC.fU.fC.fU.

A. G. A. A. G.mC. A. G.mC*mC*mU*

14059 887 3228 A. G.Chi 3229 G*mC* A* G. 80%

G. G.mU.mC. A. P.mG.fC. A. A. G.

G. G.fC.fC.fU. G.

G.mC.mC.mU.mU. A.mC.mC* A*mU*

14060 914 3230 G.mC.Chl 3231 G*mC* A* C. 112%

G. A. G.mC.mU. P.mC. A. G. A.

A. A. A.fU.fU.fU. A.

A.mU.mU.mC.mU. G.mC.mU.mC* G*

14061 1039 3232 G.Chi 3233 G*mU* A*mU* G. 104%

A. A. G. A.mC. P.mC.fC. A. A. A.fC.

A.mC. G.fU.

G.mU.mU.mU. G. G.fU.mC.mU.mU*mC*

14062 754 3234 G.Chi 3235 mC* A* G*mU* C. 109%

P.mC.fU.fU.fC. A.fU. G.

mC. G. A. G. A.fC.fC.mU.mC.

G.mU.mC. A.mU. G*mC*mC*

14063 1130 3236 G. A. A. G.Chi 3237 G*mU*mC* A. 103% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

G. P.mG.fC.fU.fU.fC.

G.mC.mC.mU.mU. G.fC. A. A. G.

G.mC. G. A. A. G.mC.mC*mU* G*

14064 919 3238 G.mC.Chl 3239 A*mC*mC* A. 109% mC.mU.mU. P.mU.fC. A.

G.mC. G. A. A. G.fC.fU.fU.fC. G.fC. A.

G.mC.mU. G. A. G* G*mC*mC*mU*

14065 922 3240 A.Chi 3241 G* A. 106%

P.mG.fU.fC.fU.fU.fC.fC

mC.mC. G. . A. G.fU.mC. G.

A.mC.mU. G. G. A. G*mU* A* A* G*mC*

14066 746 3242 A. G. A.mC.Chl 3243 C. 106% mC.mC.mU. P.mC. A. A. A.fC.fU.fU.

A.mU.mC. A. A. G. A.fU. A. G.

G.mU.mU.mU. G*mC*mU*mU* G*

14067 993 3244 G.Chi 3245 G* A. 67% mU. P.mA. G.

G.mU.mU.mC.mC. G.fU.fC.fU.fU. G. G. A.

A. A. G. A.mC. A* G* G*mC*

14068 825 3246 A.mC.mC.mU.Chl 3247 G*mC* U. 93% mC. G. A. A. P.mC. A. G. G.fU.fC. A.

G.mC.mU. G. G.fC.fU.mU.mC.

A.mC.mC.mU. G*mC* A* A* G* G*

14069 926 3248 G.Chi 3249 C. 95%

P.mG.fU.fC. A.

mU.mU. G.mC. G. G.fC.fU.fU.fC. G.mC. A.

A. A. G.mC.mU. G. A* G*

14070 923 3250 A.mC.Chl 3251 G*mC*mC*mU* G. 95% mC. A. A.mU. G. P.mG. G.fC. G.fU.fU.

A.mC. A. A.mC. G.fU.fC. A.mU. mU. G*

14071 866 3252 G.mC.mC.Chl 3253 G*mU* A* A*mC* C. 132%

P.mC. G.fU. G.fC.

G.mU. A.mC.mC. A.fC.fU. G. G.mU.

A. G.mU. G.mC. A.mC*mU*mU*

14072 563 3254 A.mC. G.Chi 3255 G*mC* A* G. n/a

P.mG.fU.fC.fU.fU. G.

mC.mC.mU. G. A. A.fC. A. G.

G.mU.mU.mC.mC. G*mC*

14073 823 3256 A. A. G. A.mC.Chl 3257 G*mC*mU*mC* C. 98%

P.mC.fC. A.fU.

mU. A.mC. G. G. G.fU.fC.fU.fC.fC.

A. G. A.mC. A.mU. G.mU. A*mC*

14074 1233 3258 G. G.Chi 3259 A*mU*mC*mU* U. 109%

P.mG. G.fU.fC. A.

mU. G.mC. G. A. G.fC.fU.fU.fC. G.mC.

A. G.mC.mU. G. A* A* G* G*mC*mC*

14075 924 3260 A.mC.mC.Chl 3261 U. 95% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

P.mC. A. G.fC.fU.fU.fC.

mC.mC.mU.mU. G.fC. A. A. G.

G.mC. G. A. A. G*mC*mC*mU* G*

14076 921 3262 G.mC.mU. G.Chi 3263 A* C. 116% mC.mU. G.mU. G. P.mG.fC.fC. G. A. A.

A.mC.mU.mU.mC. G.fU.fC. A.mC. A. G*

14077 443 3264 G. G.mC.Chl 3265 A* A* G* A* G* G. 110%

P.mC. A.fC. A. G. A.

G.mC.mU. A. A. A.fU.fU.fU. A.

A.mU.mU.mC.mU. G.mC*mU*mC* G*

14078 1041 3266 G.mU. G.Chi 3267 G*mU* A. 99%

P.mC.fC. A.fC. A. G. A.

mC.mU. A. A. A.fU.fU.mU. A.

A.mU.mU.mC.mU. G*mC*mU*mC* G*

14079 1042 3268 G.mU. G. G.Chi 3269 G* U. 109%

P.mG.fC.fC. A. A. A.fC.

A. G. A.mC. A.mC. G.fU.

G.mU.mU.mU. G. G.mU.mC.mU*mU*m

14080 755 3270 G.mC.Chl 3271 C*mC* A* G* U. 121%

P.mG.fC. C.fG. A.

mC.mC. G.mC. A. U.fC.fU.fU.fG. C.mG.

A. G. A.mU.mC. G. G*mU*mU* G*

14081 467 3272 G.mC.Chl 3273 G*mC* C. 132%

P.mC.fU.fC. A. A.

mU. A.mU.mC. A. A.fC.fU.fU. G. A.mU.

A. G.mU.mU.mU. A* G*

14082 995 3274 G. A. G.Chi 3275 G*mC*mU*mU* G. 105%

G. A. A.

G.mC.mU. G. P.mC.fC. A. G. G.fU.fC.

A.mC.mC.mU. G. A. G.fC.mU.mU.mC*

14083 927 3276 G.Chi 3277 G*mC* A* A* G* G. 114%

P.mU. A.fU. G. A.

A.mC. A.mU.mU. G.mU.fU. A. A.fU.

A. A.mC.mU.mC. G.fU*fC*fU*fC*fU*fC

17356 1267 3278 A.mU. A.Chi 3279 * A. 120%

G. A.mC. P.mU. A.fU. G. A.

A.mU.mU. A. G.mU.fU. A. A.fU.

A.mC.mU.mC. G.fU*fC*fU*fC*fU*fC

17357 1267 3280 A.mU. A.Chi 3281 * A. 56% mU. G. A. A. G. A. P.mU.fU. A. A.fC.

A.mU. G.mU.mU. A.fU.fU.fC.fU.fU.fC. A*

17358 1442 3282 A. A.Chi 3283 A* A*fC*fC* A* G. 34% mU.mU. G. A. A.

G. A. A.mU. P.mU.fU. A. A.fC.

G.mU.mU. A. A.fU.fU.fC.fU.fU.fC. A*

17359 1442 3284 A.Chi 3285 A* A*fC*fC* A* G. 31% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

G. A.mU. A.

G.mC. P.mU.fU. A. A. G. A.fU.

A.mU.mC.mU.mU. G.fC.fU. A.fU.fC*fU*

17360 1557 3286 A. A.Chi 3287 G* A*fU* G* A. 59%

A. G. A.mU. A.

G.mC. P.mU.fU. A. A. G. A.fU.

A.mU.mC.mU.mU. G.fC.fU. A.fU.fC*fU*

17361 1557 3288 A. A.Chi 3289 G* A*fU* G* A. 47% mU. G. A. A. P.mU. A. A.fU.fU. A.fC.

G.mU. G.mU. A. A.fC.fU.fU.fC. A* A*

17362 1591 3290 A.mU.mU. A.Chi 3291 A*fU* A* G* C. 120%

A. A.mU.mU. G. P.mU.fU.fC.fC.fU.fU.fC

A. G. A. A. G. G. A. .fU.fC. A. A.fU.fU*

17363 1599 3292 A.Chi 3293 A*fC* A*fC*fU* U. 71% mU.mU. G. A. G. P.mU.fU.fU.fU.fC.fC.fU

A. A. G. G. A. A. A. .fU.fC.fU.fC. A.

17364 1601 3294 A.Chi 3295 A*fU*fU* A*fC* A* C. 62% mC.

A.mU.mU.mC.mU. P.mU.fC. G. A. A.fU.fC.

G. A.mU.mU.mC. A. G. A. A.fU.

17365 1732 3296 G. A.Chi 3297 G*fU*fC* A* G* A* G. 99% mU.mU.mC.mU. P.mU.fU.fU.fC. G. A.

G. A.mU.mU.mC. A.fU.fC. A. G. A. A*fU*

17366 1734 3298 G. A. A. A.Chi 3299 G*fU*fC* A* G. 97% mC.mU.

G.mU.mC. G. P.mU.fU.fC.fU. A.

A.mU.mU. A. G. A. A.fU.fC. G. A.fC. A. G*

17367 1770 3300 A.Chi 3301 G* A*fU*fU*fC* C. 45% mU.mU.mU.

G.mC.mC.mU. P.mU. G.fU.fU. A.fC. A.

G.mU. A. A.mC. G. G.fC. A. A.

17368 1805 3302 A.Chi 3303 A*fU*fU*fC* A*fC* U. 71%

A.mU.mU.mU.

G.mC.mC.mU. P.mU. G.fU.fU. A.fC. A.

G.mU. A. A.mC. G. G.fC. A. A.

17369 1805 3304 A.Chi 3305 A*fU*fU*fC* A*fC* U. 67%

A.mC. A. A. P.mU. A. A.fU.fC.fU. G.

G.mC.mC. A. G. G.fC.fU. fU. G.fU*fU*

17370 1815 3306 A.mU.mU. A.Chi 3307 A*fC* A* G* G. 65%

A. A.mC. A. A. P.mU. A. A.fU.fC.fU. G.

G.mC.mC. A. G. G.fC.fU. fU. G.fU*fU*

17371 1815 3308 A.mU.mU. A.Chi 3309 A*fC* A* G* G. 35% mC. A.

G.mU.mU.mU. P.mU. A.fC. A. A. A.fU.

A.mU.mU.mU. A. A. A.fC.fU.

17372 2256 3310 G.mU. A.Chi 3311 G*fU*fC*fC* G* A* A. 113%

P.mU. A.fC.

mU. G.mU.mU. G. A.fC.fU. fC.fU.fC. A.

A. G. A. G.mU. A.fC. A* A* A*fU* A*

17373 2265 3312 G.mU. A.Chi 3313 A* A. 35% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

mU.mU. P.mU. A.fC.

G.mU.mU. G. A. A.fC.fU.fC.fU.fC. A.

G. A. G.mU. A.fC. A* A* A*fU* A*

17374 2265 3314 G.mU. A.Chi 3315 A* A. 31% mU. G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G. A. A. A.

mU.mC.mU. A. G. G.fU. G.fC. A* A*

17375 2295 3316 A.Chi 3317 A*fC* A*fU* G. 34% mU.mU. G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G. A. A. A.

mU.mC.mU. A. G. G.fU. G.fC. A* A*

17376 2295 3318 A.Chi 3319 A*fC* A*fU* G. 28% mU.mU. G. A. P.mU.fC. A. G. A. A. A.

G.mC.mU.mU.mU G.fC.fU.fC. A. A*

17377 1003 3320 .mC.mU. G. A.Chi 3321 A*fC*fU*fU* G* A. 67% mU. G. A. G. A. P.mU. G.fU.fC. A.fC.

G.mU. G.mU. G. A.fC.fU.fC.fU.fC. A*

17378 2268 3322 A.mC. A.Chi 3323 A*fC* A* A* A* U. 42%

P.mU.fU.fU.fU. G.

A. G.mU. G.mU. G.fU.fC. A.fC.

G. A.mC.mC. A. A. A.fC.fU*fC*fU*fC* A*

17379 2272 3324 A. A.Chi 3325 A* C. 35%

G. A. G.mU. P.mU.fU.fU.fU. G.

G.mU. G. G.fU.fC. A.fC.

A.mC.mC. A. A. A. A.fC.fU*fC*fU*fC* A*

17380 2272 3326 A.Chi 3327 A* C. 29%

P.mU.fU.fU.fU.fU. G.

G.mU. G.mU. G. G.fU.fC. A.fC.

A.mC.mC. A. A. A. A.fC*fU*fC*fU*fC* A*

17381 2273 3328 A. A.Chi 3329 A. 42% mU. G.mU. G. P.mU.fC.fU.fU.fU.fU.

A.mC.mC. A. A. A. G. G.fU.fC. A.fC.

17382 2274 3330 A. G. A.Chi 3331 A*fC*fU*fC*fU*fC* A. 42%

G.mU. G.mU. G. P.mU.fC.fU.fU.fU.fU.

A.mC.mC. A. A. A. G. G.fU.fC. A.fC.

17383 2274 3332 A. G. A.Chi 3333 A*fC*fU*fC*fU*fC* A. 37%

P.mU.

G.mU. G. A.fC.fU.fU.fU.fU. G.

A.mC.mC. A. A. A. G.fU.fC. A.fC*

17384 2275 3334 A. G.mU. A.Chi 3335 A*fC*fU*fC*fU* C. 24%

P.mU.fU. A.

G. A.mC.mC. A. A. A.fC.fU.fU.fU.fU. G.

A. A. G.mU.mU. A. G.fU.fC* A*fC*

17385 2277 3336 A.Chi 3337 A*fC*fU* C. 27%

G.mC.

A.mC.mC.mU.mU. P.mU.fC.fU. A. G. A. A.

mU.mC.mU. A. G. A. G. G.fU. G.fC* A*

17386 2296 3338 A.Chi 3339 A* A*fC* A* U. 23% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

mC.mC.mU.mU.m

U.mC.mU. A. P.mU.fC. A. A.fC.fU. A.

G.mU.mU. G. G. A. A. A. G. G*fU*

17387 2299 3340 A.Chi 3341 G*fC* A* A* A. 46%

G.mC. P.mU.fC.fU. A. G.

A.mC.mC.mU.mU. A.mA. A. G. G.fU.

mU.mC.mU. A. G. G.mC* A* A* A*mC*

21138 2296 3342 A.TEG-Chl 3343 A* U. 42%

G.mC.

A.mC.mC.mU.mU. P.mU.fC.fU. A. G.mA.

mU.mC.mU. A. G. A.mA. G. G.fU. G.mC*

21139 2296 3344 A.TEG-Chl 3345 A* A* A*mC* A* U. 32%

G.mC.

A.mC.mC.mU.mU. P.mU.fC.fU. A. G. A. A.

mU.mC.mU. A. G. A. G. G.fU. G.mC*

21140 2296 3346 A.TEG-Chl 3347 A*mA* A*mC* A* U. 41%

G.mC. P.mU.fC.fU. A. G.

A.mC.mC.mU.mU. A.mA. A. G. G.fU.

mU.mC.mU. A. G. G.mC* A*mA* A*mC*

21141 2296 3348 A.TEG-Chl 3349 A* U. 51%

G.mC.

A.mC.mC.mU.mU. P.mU.fC.fU. A. G.mA.

mU.mC.mU. A. G. A.mA. G. G.fU. G.mC*

21142 2296 3350 A.TEG-Chl 3351 A*mA* A*mC* A* U. 25%

G.mC. P.mU.fC.fU. A. G. A. A.

A.mC.mC.mU.mU. A. G. G.fU.

mU.mC.mU. A. G. G.fC*mA*mA*mA*fC*

21143 2296 3352 A.TEG-Chl 3353 mA* U. 61%

G.mC. P.mU.fC.fU. A. G.

A.mC.mC.mU.mU. A.mA. A. G. G.fU.

mU.mC.mU. A. G. G.fC*mA*mA*mA*fC*

21144 2296 3354 A.TEG-Chl 3355 mA* U. 49%

G.mC. P.mU.fC.fU. A. G.mA.

A.mC.mC.mU.mU. A.mA. G. G.fU.

mU.mC.mU. A. G. G.fC*mA*mA*mA*fC*

21145 2296 3356 A.TEG-Chl 3357 mA* U. 46%

G.mC.

A.mC.mC.mU.mU.

mU.mC.mU. P.mU.fC.fU. A. G. A. A.

A*mG*mA.TEG- A. G. G.fU. G.fC* A*

21146 2296 3358 Chl 3359 A* A*fC* A* U. 37% mG*mC*

A.mC.mC.mU.mU.

mU.mC.mU. P.mU.fC.fU. A. G. A. A.

A*mG*mA.TEG- A. G. G.fU. G.fC* A*

21147 2296 3360 Chl 3361 A* A*fC* A* U. 43% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

mG*mC*mA.mC.

mC.mU.mU.mU.m P.mU.fC.fU. A. G. A. A.

C.mU.mA*mG*m A. G. G.fU. G.fC* A*

21148 2296 3362 A.TEG-Chl 3363 A* A*fC* A* U. 29%

G.mU. G.

A.mC.mC. A. A. A. P.mU.

A. A.fC.fU.fU.fU.fU. G.

G*mU*mA.TEG- G.fU.fC. A.fC*

21149 2275 3364 Chl 3365 A*fC*fU*fC*fU* C. 138% mG*mU* G.

A.mC.mC. A. P.mU.

A.mA. A. A.fC.fU.fU.fU.fU. G.

G*mU*mA.TEG- G.fU.fC. A.fC*

21150 2275 3366 Chl 3367 A*fC*fU*fC*fU* C. 116% mG*mU*mG.mA. P.mU.

mC.mC.mA.mA.m A.fC.fU.fU.fU.fU. G.

A.mA.mG*mU*m G.fU.fC. A.fC*

21151 2275 3368 A.TEG-Chl 3369 A*fC*fU*fC*fU* C. 105% mU.mU. G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G. A.mA.

mU.mC.mU. A. A. G. G.fU. G.fC. A. A*

21152 2295 3370 A.TEG-Chl 3371 A*fC* A*fA* G* G. 46% mU.mU. G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G.mA.

mU.mC.mU. A. A.mA. G. G.fU. G.fC. A.

21153 2295 3372 A.TEG-Chl 3373 A* A*fC* A*fA* G* G. 28% mU.mU. G.mC. P.mU.fU.mA. G.mA.

A.mC.mC.mU.mU. A.mA. G.mG.fU. G.fC.

mU.mC.mU. A. A. A* A*fC* A*fA* G*

21154 2295 3374 A.TEG-Chl 3375 G. 28% mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.mC. A.

mU.mC.mU. A. A* A*mC* A*mA* G*

21155 2295 3376 A.TEG-Chl 3377 G. 60% mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.

mU.mC.mU. A. A.mA*mA*fC*mA*fA*

21156 2295 3378 A.TEG-Chl 3379 mG* G. 54% mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU.

mU.mC.mU. A. G.fC.mA.mA*mA*fC*

21157 2295 3380 A.TEG-Chl 3381 mA*fA*mG* G. 40% mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.

mU.mC.mU. A. A.mA*mA*fC*mA*mA

21158 2295 3382 A.TEG-Chl 3383 *mG* G. n/a mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.

mU.mC.mU. A. A.mA*mA*mC*mA*m

21159 2295 3384 A.TEG-Chl 3385 A*mG* G. 41% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.mA.

mU.mC.mU. A. A*mA*mC*mA*mA*

21160 2295 3386 A.Chl-TEG 3387 mG*mG. 65% mU.mU. G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G. A.mA.

mU.mC.mU. A. A. G. G.fU. G.fC. A. A*

21161 2295 3388 A.TEG-Chl 3389 A*fC* A*mA*mG* G. 43% mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.mA.

mU.mC.mU. A. A*mA*fC*

21162 2295 3390 A.TEG-Chl 3391 A*mA*mG* G. 41% mU.mU. G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G. A. A. A.

mU.mC.mU. A* G. G.fU. G.fC. A. A*

21163 2295 3392 A*TEG-Chl 3393 A*fC* A* A* G* G. 32% mU.mU. G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G. A. A. A.

mU.mC.mU.mA* G. G.fU. G.fC. A. A*

21164 2295 3394 mA*TEG-Chl 3395 A*fC* A* A* G* G. 39% mU*mU* G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G. A. A. A.

mU.mC.mU.mA* G. G.fU. G.fC. A. A*

21165 2295 3396 mA*TEG-Chl 3397 A*fC* A* A* G* G. 28% mU.mU.mG.mC.m

A.mC.mC.mU.mU. P.mU.fU. A. G. A. A. A.

mU.mC.mU.mA* G. G.fU. G.fC. A. A*

21166 2295 3398 mA*TEG-Chl 3399 A*fC* A* A* G* G. 27% mC.mC.mU.mU.m

U.mC.mU. A. P.mU.fC. A. A.fC.fU. A.

G.mU.mU. G. G. A.mA. A. G. G*fU*

21167 2299 3400 A.TEG-Chl 3401 G*fC* A* A* A. 49% mC.mC.mU.mU.m

U.mC.mU. A. P.mU.fC. A. A.fC.fU. A.

G.mU.mU. G. G. A.mA. A. G. G*mU*

21168 2299 3402 A.TEG-Chl 3403 G*mC* A* A* A. 53% mC.mC.mU.mU.m

U.mC.mU. A. P.mU.fC. A. A.fC.fU. A.

G.mU.mU. G. G.mA. A. A.mG. G*fU*

21169 2299 3404 A.TEG-Chl 3405 G*fC* A* A* A. 47% mC.mC.mU.mU.m P.mU.fC. A. A.fC.fU. A.

U.mC.mU. A. G.mA. A. A.mG.

G.mU.mU. G. G*mU* G*mC* A* A*

21170 2299 3406 A.TEG-Chl 3407 A. 70% mC.mC.mU.mU.m

U.mC.mU. A. P.mU.fC. A. A.fC.fU. A.

G.mU.mU. G. G. A.mA. A. G. G*mU*

21171 2299 3408 A.TEG-Chl 3409 G*mC* A*mA* A. 65% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

mC.mC.mU.mU.m

U.mC.mU. A. P.mU.fC. A. A.fC.fU. A.

G.mU.mU. G. G. A.mA. A. G. G*mU*

21172 2299 3410 A.TEG-Chl 3411 G*mC*mA*mA* A. 43% mC.mC.mU.mU.m P.mU.fC. A. A.fC.fU. A.

U.mC.mU. A. G. A.mA. A.

G.mU.mU. G. G.mG*mU*mG*mC*

21173 2299 3412 A.TEG-Chl 3413 mA*mA* A. 52% mC.mC.mU.mU.m P.mU.fC. A. A.fC.fU. A.

U.mC.mU. A. G. A.mA. A. G.

G.mU.mU. G. G*mU*mG*mC*mA*

21174 2299 3414 A.TEG-Chl 3415 mA* A. 47% mC.mC.mU.mU.m P.mU.fC. A. A.fC.fU. A.

U.mC.mU. A. G. A.mA. A. G.

G.mU.mU. G. G*fU*mG*fC*mA*mA

21175 2299 3416 A.TEG-Chl 3417 * A. 35% mC.mC.mU.mU.m P.mU.fC. A. A.fC.fU. A.

U.mC.mU. A. G.mA. A. A.mG.

G.mU.mU. G. G*fU*mG*fC*mA*mA

21176 2299 3418 A.TEG-Chl 3419 * A. 50% mC.mC.mU.mU.m

U.mC.mU. A. P.mU.fC. A. A.fC.fU. A.

G.mU.mU*mG*m G. A. A. A. G. G*fU*

21177 2299 3420 A.TEG-Chl 3421 G*fC* A* A* A. 37% mC*mC*mU.mU.

mU.mC.mU. A. P.mU.fC. A. A.fC.fU. A.

G.mU.mU*mG*m G. A. A. A. G. G*fU*

21178 2299 3422 A.TEG-Chl 3423 G*fC* A* A* A. 36% mC*mC*mU.mU.

mU.mC.mU.mA.m P.mU.fC. A. A.fC.fU. A.

G.mU.mU*mG*m G. A. A. A. G. G*fU*

21179 2299 3424 A.TEG-Chl 3425 G*fC* A* A* A. 35%

G.mC.

A.mC.mC.mU.mU. P.mU.fC.fU. A. G.

mU.mC.mU. A.mA. A. G. G.fU.

A*mG*mA.TEG- G.mC* A* A* A*mC*

21203 2296 3426 Chl 3427 A* U. 40%

G.mC.

A.mC.mC.mU.mU.

mU.mC.mU. P.mU.fC.fU. A. G.mA.

A*mG*mA.TEG- A.mA. G. G.fU. G.mC*

21204 2296 3428 Chl 3429 A* A* A*mC* A* U. 28%

G.mC.

A.mC.mC.mU.mU.

mU.mC.mU. P.mU.fC.fU. A. G.mA.

A*mG*mA.TEG- A.mA. G. G.fU. G.mC*

21205 2296 3430 Chl 3431 A*mA* A*mC* A* U. 51% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

mG*mC*

A.mC.mC.mU.mU. P.mU.fC.fU. A. G.

mU.mC.mU. A.mA. A. G. G.fU.

A*mG*mA.TEG- G.mC* A* A* A*mC*

21206 2296 3432 Chl 3433 A* U. 46% mG*mC*

A.mC.mC.mU.mU.

mU.mC.mU. P.mU.fC.fU. A. G.mA.

A*mG*mA.TEG- A.mA. G. G.fU. G.mC*

21207 2296 3434 Chl 3435 A* A* A*mC* A* U. 29% mG*mC*

A.mC.mC.mU.mU.

mU.mC.mU. P.mU.fC.fU. A. G.mA.

A*mG*mA.TEG- A.mA. G. G.fU. G.mC*

21208 2296 3436 Chl 3437 A*mA* A*mC* A* U. 72% mG*mC*mA.mC. P.mU.fC.fU. A. G.

mC.mU.mU.mU.m A.mA. A. G. G.fU.

C.mU.mA*mG*m G.mC* A* A* A*mC*

21209 2296 3438 A.TEG-Chl 3439 A* U. 89% mG*mC*mA.mC.

mC.mU.mU.mU.m P.mU.fC.fU. A. G.mA.

C.mU.mA*mG*m A.mA. G. G.fU. G.mC*

21210 2296 3440 A.TEG-Chl 3441 A* A* A*mC* A* U. 65% mG*mC*mA.mC.

mC.mU.mU.mU.m P.mU.fC.fU. A. G.mA.

C.mU.mA*mG*m A.mA. G. G.fU. G.mC*

21211 2296 3442 A.TEG-Chl 3443 A*mA* A*mC* A* U. 90% mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU.

mU.mC.mU*mA* G.fC.mA.mA*mA*fC*

21212 2295 3444 mA.TEG-Chl 3445 mA*mA*mG* G. 60% mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.

mU.mC.mU*mA* A.mA*mA*mC*mA*m

21213 2295 3446 mA.TEG-Chl 3447 A*mG* G. 63% mU.mU. G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G. A.mA.

mU.mC.mU*mA* A. G. G.fU. G.fC. A. A*

21214 2295 3448 mA.TEG-Chl 3449 A*fC* A*mA*mG* G. 52% mU.mU. G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.mA.

mU.mC.mU*mA* A*mA*fC*

21215 2295 3450 mA.TEG-Chl 3451 A*mA*mG* G. 45% mU*mU* G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU.

mU.mC.mU*mA* G.fC.mA.mA*mA*fC*

21216 2295 3452 mA.TEG-Chl 3453 mA*mA*mG* G. 65% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

mU*mU* G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.

mU.mC.mU*mA* A.mA*mA*mC*mA*m

21217 2295 3454 mA.TEG-Chl 3455 A*mG* G. 69% mU*mU* G.mC.

A.mC.mC.mU.mU. P.mU.fU. A. G. A.mA.

mU.mC.mU*mA* A. G. G.fU. G.fC. A. A*

21218 2295 3456 mA.TEG-Chl 3457 A*fC* A*mA*mG* G. 62% mU*mU* G.mC. P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.mA.

mU.mC.mU*mA* A*mA*fC*

21219 2295 3458 mA.TEG-Chl 3459 A*mA*mG* G. 54% mU.mU.mG.mC.m P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU.

mU.mC.mU*mA* G.fC.mA.mA*mA*fC*

21220 2295 3460 mA.TEG-Chl 3461 mA*mA*mG* G. 52% mU.mU.mG.mC.m P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.

mU.mC.mU*mA* A.mA*mA*mC*mA*m

21221 2295 3462 mA.TEG-Chl 3463 A*mG* G. 53% mU.mU.mG.mC.m

A.mC.mC.mU.mU. P.mU.fU. A. G. A.mA.

mU.mC.mU*mA* A. G. G.fU. G.fC. A. A*

21222 2295 3464 mA.TEG-Chl 3465 A*fC* A*mA*mG* G. 43% mU.mU.mG.mC.m P.mU.fU. A. G. A.mA.

A.mC.mC.mU.mU. A. G. G.fU. G.fC.mA.

mU.mC.mU*mA* A*mA*fC*

21223 2295 3466 mA.TEG-Chl 3467 A*mA*mG* G. 43% mC.mC.mU.mU.m P.mU.fC. A. A.fC.fU. A.

U.mC.mU. A. G. A.mA. A. G.

G.mU.mU*mG*m G*fU*mG*fC*mA*mA

21224 2299 3468 A.TEG-Chl 3469 * A. 60% mC*mC*mU.mU. P.mU.fC. A. A.fC.fU. A.

mU.mC.mU. A. G. A.mA. A. G.

G.mU.mU*mG*m G*fU*mG*fC*mA*mA

21225 2299 3470 A.TEG-Chl 3471 * A. 67% mC*mC*mU.mU. P.mU.fC. A. A.fC.fU. A.

mU.mC.mU.mA.m G. A.mA. A. G.

G.mU.mU*mG*m G*fU*mG*fC*mA*mA

21226 2299 3472 A.TEG-Chl 3473 * A. 66%

G.mC.

A.mC.mC.mU.mU. P.mU.fC.fU. A. G.mA.

mU.mC.mU. A.mA. G. G.fU.

A*mG*mA.TEG- G.fC*mA*mA*mA*fC*

21227 2296 3474 Chl 3475 mA* U. 49%

G.mC.

A.mC.mC.mU.mU. P.mU.fC.fU. A. G. A. A.

mU.mC.mU. A. G. A. G. G.mU. G.mC* A*

20584 2296 3476 A.Chl-TEG 3477 A* A*mC* A* U. 70% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

G.mC.

A.mC.mC.mU.mU. P.mU.fC.fU. A. G. A. A.

mU.mC.mU. A. G. A. G. G.fU. G.mC* A*

20585 2296 3478 A.Chl-TEG 3479 A* A*mC* A* U. 15%

G.mC.

A.mC.mC.mU.mU. P.mU. C. U. A. G. A. A.

mU.mC.mU. A. G. A. G. G.mU. G.mC* A*

20586 2296 3480 A.Chl-TEG 3481 A* A*mC* A* U. 30%

G.mC. P.mU.fC.fU. A. G. A. A.

A.mC.mC.mU.mU. A. G. G.fU.

mU.mC.mU. A. G. G.fC*mA*mA*mA*fC*

20587 2296 3482 A.Chl-TEG 3483 mA* U. 32%

P.mU.

G.mU. G. A.fC.fU.fU.fU.fU. G.

A.mC.mC. A. A. A. G.fU.mC. A.mC*

A. G.mU. A.Chl- A*mC*mU*mC*mU*

20616 2275 3484 TEG 3485 C. 22%

G.mU. G. P.mU.

A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G.

A. G.mU. A.Chl- G.fU.fC. A.mC*

20617 2275 3486 TEG 3487 A*fC*mU*fC*mU* C. 18%

G.mU. G. P.mU. A. C. U. U. U. U.

A.mC.mC. A. A. A. G. G. U.mC. A.mC*

A. G.mU. A.Chl- A*mC*mU*mC*mU*

20618 2275 3488 TEG 3489 C. 36%

P.mU.

G.mU. G. A.fC.fU.fU.fU.fU. G.

A.mC.mC. A. A. A. G.fU.fC.

A. G.mU. A.Chl- A.mC*mA*mC*mU*m

20619 2275 3490 TEG 3491 C*mU* C. 28%

G.mU. G. P.mU.

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*

21381 2275 3492 Chl 3493 C. 28%

G.mU. G.

A.mC.mC. A. A. A. P.mU.

A. A.fC.fU.fU.fU.fU. G.

G*mU*mA.TEG- G.fU.fC. A.mC*

21382 2275 3494 Chl 3495 A*fC*mU*fC*mU* C. 28%

P.mU.

mG*mU*mG.mA. A.fC.fU.fU.fU.fU. G.

mC.mC.mA.mA.m G.fU.mC. A.mC*

A.mA.mG*mU*m A*mC*mU*mC*mU*

21383 2275 3496 A.TEG-Chl 3497 C. 43% mG*mU*mG.mA. P.mU.

mC.mC.mA.mA.m A.fC.fU.fU.fU.fU. G.

A.mA.mG*mU*m G.fU.fC. A.mC*

21384 2275 3498 A.TEG-Chl 3499 A*fC*mU*fC*mU* C. 50% % remaining mRNA expression (1

Oligo Start SEQ ID SEQ ID uM sd-rxRNA, Number Site NO Sense sequence NO Antisense sequence A549)

G.mU. G. P.mU.

A.mC.mC. A. A. A. A.fC.fU.fU.fU.fU. G.

A. G.mU. A.TEG- G.fU.fC. A.fC*

20392 2275 3500 Chl 3501 A*fC*fU*fC*fU* C. 28%

G.mC.

A.mC.mC.mU.mU. P.mU.fC.fU. A. G. A. A.

mU.mC.mU. A. G. A. G. G.fU. G.fC* A*

20393 2296 3502 A.TEG-Chl 3503 A* A*fC* A* U. 35%

G.mU. G.

A.mC.mC. A. A. A. P.mU.

A. A.fC.fU.fU.fU.fU. G.

G*mU*mA.Teg- G.fU.fC. A.mC*

21429 2275 3504 Chl 3505 A*fC*mU*fC*mU* C. 36%

G.mU. G. P.mU.

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*

21430 2275 3506 Chl 3507 C. 31%

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

% remaining

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

mU.mC.mC.

A.mU.mC.mU. P.mU. G.fU.fU. G.fU.

A.mC. A. A.mC. A. G. A.fU. G. G. A* A*

14412 852 3516 A.Chi 3517 A*mU*mC* A* C. 76%

P.mU.fU. G.fU. A. G.

mU.mU.mU.mC.mC A.fU. G. G. A. A.

. A.mU.mC.mU. A*mU*mC*

14413 850 3518 A.mC. A. A.Chi 3519 A*mC*mC* U. 98%

P.mA.

mC. G.mC.mC. A. A. A.fC.fC.fU.fC.fC.fU.fU.

G. G. A. G. G. G.mC. G*mU* A*

14414 944 3520 G.mU.mU.Chl 3521 G*mU* A* C. 100%

P.mU.fU.fC.fU. G.

G.mU. G. G.mU. G. A.fU.fC. A.fC.mC.

A.mU.mC. A. G. A. A.mC*mU* G*

14415 1513 3522 A.Chi 3523 G*mU* A* U. n/a mC.mU.mC.mC.mU P.mA.fC. A.fU.fU. A.

. G.mC.mU. A. G.fC. A. G. G. A. G*

14416 1572 3524 A.mU. G.mU. Chi 3525 A*mU* G*mU* G* G. 100%

P.mU. A.fU. A.fU.

A.mC.mC.mU.mC.m G.fU. G. G. A. G.

C. A.mC. A.mU. G.mU* G*mC*mC*

14417 1497 3526 A.mU. A.Chi 3527 A*mU* C. 73%

P.mU.fC.fC.fU. A. G.fU.

A. A. G.mU.mC.mC. G. G.

A.mC.mU. A. G. G. A.mC.mU. mU*mU*

14418 1533 3528 A.Chi 3529 A*mU* A* G* U. 98%

P.mU.fU.fU.fC.fU. G.

mU. G. G.mU. G. A.fU.fC. A.mC.mC.

A.mU.mC. A. G. A. A*mC*mU* G*

14419 1514 3530 A. A.Chi 3531 G*mU* A. 86%

P.mU.fU.fC.fC.fU. A.

A. G.mU.mC.mC. G.fU. G. G.

A.mC.mU. A. G. G. A.mC.mU*mU*mU*

14420 1534 3532 A. A.Chi 3533 A*mU* A* G. 99%

A.mC. G.mC.mC. A. P.mA.fC.fC.fU.fC.fC.fU.

A. G. G. A. G. fU. G. G.mC. G.mU*

14421 943 3534 G.mU. Chi 3535 A* G*mU* A*mC* U. 41% mU. A.mU.mU.mU. P.mU. A.fC. A.fC. A.

A.mU.mU. G.mU. A.fU. A. A. A.fU. A*

18570 2445 3536 G.mU. A.Chi 3537 A*fC*fU*fC* A* C. 79% mU.mU.

A.mU.mU.mU. P.mU. A.fC. A.fC. A.

A.mU.mU. G.mU. A.fU. A. A. A.fU. A*

18571 2445 3538 G.mU. A.Chi 3539 A*fC*fU*fC* A* C. 75%

A.mU. C. A. G.mU. P.mU.fU.fU.fU. A.

G.mU.mU. A. A. A. A.fC. A.fC.fU. G. A.fU*

18572 2083 3540 A.Chi 3541 G* A* A*fC*fC* A. 47% mC. A.mU.mC. A. P.mU.fU.fU.fU. A.

G.mU. G.mU.mU. A.fC. A.fC.fU. G. A.fU*

18573 2083 3542 A. A. A. A.Chi 3543 G* A* A*fC*fC* A. 17% % remaining

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

A.mU. G. P.mU.fU.fC.fC.fU.fU.

G.mC.mU.mU. A. A. A. A. G.fC.fC. A.

18574 2544 3544 G. G. A. A.Chi 3545 U*fC*fC* A*fU* G* A. 59%

G. A.mU. G. P.mU.fU.fC.fC.fU.fU.

G.mC.mU.mU. A. A. A. A. G.fC.fC. A.

18575 2544 3546 G. G. A. A.Chi 3547 U*fC*fC* A*fU* G* A. 141% mU.mU. G.mU. P.mU. A. A.fC. A. G. A.

G.mU.mU.mC.mU. A.fC. A.fC. A. A*

18576 2137 3548 G.mU.mU. A.Chi 3549 A*fC*fU*fU*fC* C. 77% mU.mU.mU. G.mU. P.mU. A. A.fC. A. G. A.

G.mU.mU.mC.mU. A.fC. A.fC. A. A*

18577 2137 3550 G.mU.mU. A.Chi 3551 A*fC*fU*fU*fC* C. 59%

A. A. A.mU. P.mU. G. G.fC. A. A. A.

A.mC.mU.mU.mU. G.fU. A.fU.fU.fU* G*

18578 2520 3552 G.mC.mC. A.Chi 3553 G*fU*fC*fU* C. 75% mC. A. A. A.mU. P.mU. G. G.fC. A. A. A.

A.mC.mU.mU.mU. G.fU. A.fU.fU.fU* G*

18579 2520 3554 G.mC.mC. A.Chi 3555 G*fU*fC*fU* C. 55% mC.mU.mU. G.mC. P.mU.fU.fU. G.fU. A.

A.mC.mU. A.mC. A. G.fU. G.fC. A. A.

18580 3183 3556 A. A.Chi 3557 G*fU*fC* A* A* A* C. 84%

A.mC.mU. mU. P.mU.fU.fU. G.fU. A.

G.mC. A.mC.mU. G.fU. G.fC. A. A.

18581 3183 3558 A.mC. A. A. A.Chi 3559 G*fU*fC* A* A* A* C. 80%

G. A. P.mU. A.fC.fU. A. A.fU.

A.mU.mU.mU. A. A.

A.mU.mU. A. G.mU. A.fU.fU.fC*fU*fU*fC*f

18582 2267 3560 A.Chi 3561 C* A* G. 82%

A. G. A. P.mU. A.fC.fU. A. A.fU.

A.mU.mU.mU. A. A.

A.mU.mU. A. G.mU. A.fU.fU.fC*fU*fU*fC*f

18583 2267 3562 A.Chi 3563 C* A* G. 67% mU.mU. G.mC. P.mU.fU.fU.fU. G.fU.

A.mC.mU. A.mC. A. A. G.fU. G.fC. A. A*

18584 3184 3564 A. A. A.Chi 3565 G*fU*fC* A* A* A. 77% mC.mU.mU. G.mC. P.mU.fU.fU.fU. G.fU.

A.mC.mU. A.mC. A. A. G.fU. G.fC. A. A*

18585 3184 3566 A. A. A.Chi 3567 G*fU*fC* A* A* A. 59%

P.mU.fC. A.fC.fC.fU.

A.mU. A. A. A.

G.fU.fU.fU.fU.

A.mC. A. G. G.mU.

A.fU*fU*fU*fU*fC*fC

G. A.Chi

18586 2493 3568 3569 * A. 84%

P.mU.fC. A.fC.fC.fU.

A. A.mU. A. A. A.

G.fU.fU.fU.fU.

A.mC. A. G. G.mU.

A.fU*fU*fU*fU*fC*fC

G. A.Chi

18587 2493 3570 3571 * A. 70%

P.mU. G.fU.fU.

G. A.mC. A. A.mC.

G.fU.fU. G.fU.fU.

A. A.mC. A. A.mC.

G.fU.fC* G*fU*fU*

A.Chi

18588 2297 3572 3573 G*fU* U. 40% % remaining

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

P.mU.fU. G.fU.fU.

A.mU. G.

A.fC. A. A. G.fC.

C.mU.mU. G.mU. A.

A.fU*fC* A*fU*fC* G*

A.mC. A. A.Chi

18589 2046 3574 3575 U. 39% mC. A. G. A. A. P.mU.fC. A.fU. G. A.

A.mC.mU.mC. G.fU.fU. fU.fC.fU. G*

18590 2531 3576 A.mU. G. A. Chi 3577 G*fC* A* A* A* G. 56%

G.mU. A.mU.mU. P.mU. G.fC. A.fU. A.

G.mC.mU. A.mU. G.fC. A. A.fU. A.fC* A*

18591 2389 3578 G.mC. A.Chi 3579 G* A* A* A* A. 64% mC.mC. A. G. A. A. P.mU. A.fU. G. A.

A.mC.mU.mC. G.fU.fU. fU.fC.fU. G.

18592 2530 3580 A.mU. A.Chi 3581 G*fC* A* A* A* G* U. 44%

P.mU. G.fC.fU.fC.

A.mC.mU.mC. A. A.

G.fU.fU. fU. G. A.

A.mC. G. A. G.mC.

G.fU*fU*fC* A* A* G*

A.Chi

18593 2562 3582 3583 U. 87%

A.mU. A.mU. G. P.mU.fU.fC.fU.fC. G.

A.mC.mC. G. A. G. G.fU.fC. A.fU. A.fU*

18594 2623 3584 A. A.Chi 3585 A* A*fU* A* A* C. 69% mC. G. A.mC. G. P.mU.fU.fC. G.fU.fU.

A.mC. A. A.mC. G. G.fU.fC. G.fU.fC.

18595 2032 3586 A. A.Chi 3587 G*fU*fC* A*fU*fC* A. 55%

G.mU. A. A. P.mU.fU.fC. A.fC.fU. G.

A.mC.mC. A. G.mU. G.fU.fU. fU. A.fC*fU*

18596 2809 3588 G. A. A.Chi 3589 A* A* A*fC* U. 58% mU.mU. G.mU.mC. P.mU.fC.fU. A. A.

A. G.mU.mU.mU. A. A.fC.fU. G. A.fC. A. A*

18597 2798 3590 G. A.Chi 3591 A* G* A* A*fC* C. 38% mU.mC. A.mU.mC. P.mU.fU. A. A.fC.

A. G.mU. A.fC.fU. G. A.fU. G. A*

18598 2081 3592 G.mU.mU. A. A.Chi 3593 A*fC*fC* A* A* G. 25%

P.mU.fC.fU.fC.

A. A.mC.mU.mC. A. G.fU.fU. fU. G. A.

A. A.mC. G. A. G. G.fU.fU*fC* A* A*

18599 2561 3594 A.Chi 3595 G*fU* U. 57%

P.mU.fU.fU. G.fU.fU.

mC. G. A.mC. A. G.fU.fU. G.fU.fC.

A.mC. A. A.mC. A. G*fU*fU* G*fU*fU*

18600 2296 3596 A. A.Chi 3597 C. 69%

P.mU.fC. A.fU.fC.

A.mC. G. A.mC. A. G.fU.fU. G.fU.fC.

A.mC. G. A.mU. G. G.fU*fC* G*fU*fC*

18601 2034 3598 A.Chi 3599 A*fU. 22%

G.mC.mU. P.mU.fU.fC.fC.fU.fU.

G.mC.mC.mU. A. A. A. G. G.fC. A. G.fC*fU*

18602 2681 3600 G. G. A. A.Chi 3601 G* A*fU* A* C. 43%

A.mU.mU.mC.mU.

A.mC. P.mU. G. A. A. A.fU.

A.mU.mU.mU.mC. G.fU. A. G. A. A.fU* A*

18603 2190 3602 A.Chi 3603 A* G* G*fC* C. 128% % remaining

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

P.mU.fU.fU.fU. A.

mC. A.mU.mC. A. A.fC. A.fC.fU. G.

G.mU. G.mU.mU. A.mU* G* A*

20604 2083 3604 A. A. A. A.Chi 3605 A*mC*mC* A. 19%

P.mU.fU.fU.fU. A.

mC. A.mU.mC. A. A.fC. A.fC.fU. G.

G.mU. G.mU.mU. A.mU* G* A*

20605 2083 3606 A. A. A. A.Chi 3607 A*fC*mC* A. 20% mC. A.mU.mC. A. P.mU. U. U. U. A. A. C.

G.mU. G.mU.mU. A. C. U. G. A.mU* G*

20606 2083 3608 A. A. A. A.Chi 3609 A* A*mC*mC* A. 82%

P.mU.fU.fU.fU. A.

mC. A.mU.mC. A. A.fC. A.fC.fU. G.

G.mU. G.mU.mU. A.fU*mG*mA*mA*fC

20607 2083 3610 A. A. A. A.Chi 3611 *fC* A. 59% mU.mC. A.mU.mC. P.mU.fU. A. A.fC.

A. G.mU. A.fC.fU. G. A.fU. G. A*

21722 2081 3612 G.mU.mU. A. A.Chi 3613 A*mC*mC* A* A* G. 34%

P.mU.fU. A. A.fC.

mU.mC. A.mU.mC. A.fC.fU. G. A.fU.

A. G.mU. G.mA*mA*mC*mC*m

21723 2081 3614 G.mU.mU. A. A.Chi 3615 A*mA* G. 53%

P.mU.fU. A. A.fC.

mU.mC. A.mU.mC. A.fC.fU. G. A.mU.

A. G.mU. G.mA*mA*mC*mC*m

21724 2081 3616 G.mU.mU. A. A.Chi 3617 A*mA* G. 48% mU.mC. A.mU.mC. P.mU.fU. A. A.fC.

A. G.mU. A.fC.fU. G. A.fU. G. A*

21725 2081 3618 G.mU.mU. A. A.Chi 3619 A*fC*fC*mA*mA* G. 45%

P.mU.fU. A. A.fC.

mU.mC. A.mU.mC. A.fC.fU. G. A.fU.

A. G.mU. G.mA*mA*fC*fC*mA*

21726 2081 3620 G.mU.mU. A. A.Chi 3621 mA* G. 54% mU.mC. A.mU.mC.

A. G.mU. P.mU.fU. A. A.fC.

G.mU.mU*mA*mA. A.fC.fU. G. A.fU. G. A*

21727 2081 3622 TEG-Chl 3623 A*fC*fC* A* A* G. 29% mU*mC* A.mU.mC.

A. G.mU. P.mU.fU. A. A.fC.

G.mU.mU*mA*mA. A.fC.fU. G. A.fU. G. A*

21728 2081 3624 TEG-Chl 3625 A*fC*fC* A* A* G. 27% mU*mC*mA.mU.m

C.mA.mG.mU.mG. P.mU.fU. A. A.fC.

mU.mU*mA*mA.TE A.fC.fU. G. A.fU. G. A*

21729 2081 3626 G-Chl 3627 A*fC*fC* A* A* G. 30% mU.mC. A.mU.mC.

A. G.mU. P.mU.fU. A. A.fC.

G.mU.mU*mA*mA. A.fC.fU. G. A.fU. G. A*

21375 2081 3628 TEG-Chl 3629 A*mC*mC* A* A* G. 29% % remaining

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

mU.mC. A.mU.mC.

A. G.mU. P.mU.fU. A. A.fC.

G.mU.mU*mA*mA. A.fC.fU. G. A.fU. G. A*

21376 2081 3630 TEG-Chl 3631 A*fC*fC*mA*mA* G. 30% mU.mC. A.mU.mC. P.mU.fU. A. A.fC.

A. G.mU. A.fC.fU. G. A.fU.

G.mU.mU*mA*mA. G.mA*mA*fC*fC*mA*

21377 2081 3632 TEG-Chl 3633 mA* G. 37% mU*mC*mA.mU.m

C.mA.mG.mU.mG. P.mU.fU. A. A.fC.

mU.mU*mA*mA.TE A.fC.fU. G. A.fU. G. A*

21378 2081 3634 G-Chl 3635 A*mC*mC* A* A* G. 32% mU*mC*mA.mU.m

C.mA.mG.mU.mG. P.mU.fU. A. A.fC.

mU.mU*mA*mA.TE A.fC.fU. G. A.fU. G. A*

21379 2081 3636 G-Chl 3637 A*fC*fC*mA*mA* G. 31% mU*mC*mA.mU.m P.mU.fU. A. A.fC.

C.mA.mG.mU.mG. A.fC.fU. G. A.fU.

mU.mU*mA*mA.TE G.mA*mA*fC*fC*mA*

21380 2081 3638 G-Chl 3639 mA* G. 39%

Table 22: Inhibition of gene expression with TGFBl sd-rxRNA sequences (Accession Number: NM_000660.3)

% remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

P.mG.fC. A.fC. G.

A. A.mC. A.mU. G. A.fU.fC. A.fU.

A.mU.mC. G.mU. G.mU.mU* G* G*

14399 2002 3650 G.mC.Chl 3651 A*mC* A* G. n/a

P.mC. G.fC. A.fC. G.

A.mC. A.mU. G. A.fU.fC. A.mU.

A.mU.mC. G.mU. G.mU*mU* G* G*

14400 2003 3652 G.mC. G.Chi 3653 A*mC* A. n/a

P.mC. A. G. G.

mC. A. G.mC. A. A. G. A.fC.fC.fU.fU.

G.mU.mC.mC.mU. G.mC.mU. G*mU*

14401 1869 3654 G.Chi 3655 A*mC*mU* G* C. 82% mC.mC. A. A.mC. P.mA.fC. G. A.fU.fC.

A.mU. G. A.mU.mC. A.fU. G.fU.mU. G. G*

14402 2000 3656 G.mU. Chi 3657 A*mC* A* G*mC* U. 66%

P.mA.fU. G.fC.

A. G.mC. G. G. A. A. G.fC.fU.fU.fC.fC.

G.mC. G.mC. G.mC.mU*mU*mC*

14403 986 3658 A.mU. Chi 3659 A*mC*mC* A. 78%

P.mA.fU. G.

G.mC. A.mU.mC. G. G.fC.fC.fU.fC. G. A.mU.

A. G. G.mC.mC. G.mC*

14404 995 3660 A.mU. Chi 3661 G*mC*mU*mU*mC* C. 79%

P.mC. A.fU. G.fU.fC. G.

G. A.mC.mU. A.fU. A.

A.mU.mC. G. A.mC. G.mU.mC*mU*mU*

14405 963 3662 A.mU. G.Chi 3663 G*mC* A* G. 80%

A.mC.mC.mU. G.mC. P.mU. A. G.fU.fC.fU.fU.

A. A. G. A.mC.mU. G.fC. A. G. G.mU* G*

14406 955 3664 A.Chi 3665 G* A*mU* A* G. 88%

P.mU.fU.fC.fU.fC.fC.

G.mC.mU.mC.mC. G.fU. G. G. A.

A.mC. G. G. A. G. A. G.mC*mU* G* A* A*

14407 1721 3666 A.Chi 3667 G* C. n/a mC. A.mC. A. G.mC. P.mU. A.fU. A.fU. A.fU.

A.mU. A.mU. A.mU. G.fC.fU. G.fU. G*fU*

18454 1246 3668 A.Chi 3669 G*fU* A*fC* U. 58% mC. A. G.mC. A.mU. P.mU. A.fU. A.fU. A.fU.

A.mU. A.mU. A.mU. A.fU. G.fC.fU. G*fU*

18455 1248 3670 A.Chi 3671 G*fU* G*fU* A. 87%

G.mU. A.mC. P.mU. A. A. G.fU.fC. A.

A.mU.mU. G. A.fU. G.fU. A.fC* A*

18456 1755 3672 A.mC.mU. mU. A.Chi 3673 G*fC*fU* G* C. 107% mU. G.mU. A.mC. P.mU. A. A. G.fU.fC. A.

A.mU.mU. G. A.fU. G.fU. A.fC* A*

18457 1755 3674 A.mC.mU. mU. A.Chi 3675 G*fC*fU* G* C. 77%

A. A.mC.mU.

A.mU.mU. P.mU. G. A. A. G.fC. A.

G.mC.mU.mU.mC. A.fU. A. G.fU.fU* G*

18458 1708 3676 A.Chi 3677 G*fU* G*fU* C. 75% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

mC. A. A.mC.mU.

A.mU.mU. P.mU. G. A. A. G.fC. A.

G.mC.mU.mU.mC. A.fU. A. G.fU.fU* G*

18459 1708 3678 A.Chi 3679 G*fU* G*fU* C. 73%

G.mC. A.mU. A.mU. P.mU. A.fC. A.fU. A.fU.

A.mU. A.mU. G.mU. A.fU. A.fU. G.fC*fU*

18460 1250 3680 A.Chi 3681 G*fU* G*fU* G. n/a mU. G.mU. A.mC. P.mU. A. G.fU.fC. A.

A.mU.mU. G. A.fU. G.fU. A.fC. A*

18461 1754 3682 A.mC.mU. A.Chi 3683 G*fC*fU* G*fC* C. 91% mC.mU. G.mU. P.mU. A. G.fU.fC. A.

A.mC. A.mU.mU. G. A.fU. G.fU. A.fC. A*

18462 1754 3684 A.mC.mU. A.Chi 3685 G*fC*fU* G*fC* C. 92%

A. G.mC. A.mU. P.mU.fC. A.fU. A.fU.

A.mU. A.mU. A.mU. A.fU. A.fU. G.fC.fU*

18463 1249 3686 G. A.Chi 3687 G*fU* G*fU* G* U. n/a mC. A. G.mC. A. P.mU. G. A. A.fU.fU.

A.mC. A. G.fU.fU. G.fC.fU. G*fU*

18464 1383 3688 A.mU.mU.mC. A.Chi 3689 A*fU*fU*fU* C. 77% mC. A.mU. A.mU. P.mU. A. A.fC. A.fU.

A.mU. A.mU. A.fU. A.fU. A.fU.

18465 1251 3690 G.mU.mU. A.Chi 3691 G*fC*fU* G*fU* G* U. 84% mU.mU. P.mU. G. A. G.fC.fU. G.

G.mC.mU.mU.mC. A. A. A. G.fC. A. A*fU* A*

18466 1713 3692 G.mC.mU.mC. A.Chi 3693 G*fU*fU* G. n/a

A.mU.mU. P.mU. G. A. G.fC.fU. G.

G.mC.mU.mU.mC. A. A. A. G.fC. A. A*fU* A*

18467 1713 3694 G.mC.mU.mC. A.Chi 3695 G*fU*fU* G. 83%

A.mC. A. G.mC. P.mU.fU. A.fU. A.fU.

A.mU. A.mU. A.mU. A.fU. G.fC.fU. G.fU*

18468 1247 3696 A. A.Chi 3697 G*fU* G*fU* A* C. 96%

A.mU.mU. P.mU. A. G.fC.fU. G. A.

G.mC.mU.mU.mC. A. A. G.fC. A. A.fU* A*

18469 1712 3698 G.mC.mU. A.Chi 3699 G*fU*fU* G* G. 90% mU. A.mU.mU. P.mU. A. G.fC.fU. G. A.

G.mC.mU.mU.mC. A. A. G.fC. A. A.fU* A*

18470 1712 3700 G.mC.mU. A.Chi 3701 G*fU*fU* G* G. 98% mC. A. A. P.mU.fU. G.fC.fU.fU. G.

G.mU.mU.mC. A. A. A. A.fC.fU.fU. G*fU*fC*

18471 1212 3702 G.mC. A. A.Chi 3703 A*fU* A* G. n/a mC. A. G. A. G.mU. P.mU. G.fU. G.fU. G.fU.

A.mC. A.mC. A.mC. A.fC.fU.fC.fU. G*

18472 1222 3704 A.Chi 3705 C*fU*fU* G* A* A. 45%

A.mC. A.mC. A.mC. P.mU.fU. A.fU. G.fC.fU.

A. G.mC. A.mU. A. G.fU. G.fU. G.fU*

18473 1228 3706 A.Chi 3707 A*fC*fU*fC*fU* G. 36% mC. A. G.mC. A.mU. P.mU. A.fU. A.fU. A.fU.

A.mU. A.mU. A.mU. A.fU. G.fC.fU. G*fU*

18474 1233 3708 A.Chi 3709 G*fU* G*fU* A. 68% mU.mC. A. A. G.mC. P.mU.fU. A.fC.fU.fC.fU.

A. G. A. G.mU. A. G.fC.fU.fU. G. A*

18475 1218 3710 A.Chi 3711 A*fC*fU*fU* G* U. 64% % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

A. G.mC. A.mU. P.mU.fC. A.fU. A.fU.

A.mU. A.mU. A.mU. A.fU. A.fU. G.fC.fU*

18476 1235 3712 G. A.Chi 3713 G*fU* G*fU* G* U. 78%

P.mU.fU. G.fU. G.fU.

A. G. A. G.mU. A.mC. G.fU. A.fC.fU.fC.fU*

18477 1225 3714 A.mC. A.mC. A. A.Chi 3715 G*fC*fU*fU* G* A. 92%

P.mU.fU. G.fU.

A.fC.fU.fC.fU.

A. A. G.mC. A. G. A. G.fC.fU.fU* G* A*

18478 1221 3716 G.mU. A.mC. A. A.Chi 3717 A*fC*fU* U. 103% mU.mU.mC. A. A.mC. P.mU.fU. G. A.fU. G.fU.

A.mC. A.mU.mC. A. G.fU.fU. G. A. A* G* A*

18479 1244 3718 A.Chi 3719 A*fC* A* U. 84%

P.mU. G.fU. G.fU.

A. G.mC. A. G. A. A.fC.fU.fC.fU.

G.mU. A.mC. A.mC. G.fC.fU*fU* G* A*

18480 1224 3720 A.Chi 3721 A*fC* U. 37%

A.mU. A.mU. A.mU. P.mU. A. A. G. A. A.fC.

G.mU.mU.mC.mU.m A.fU. A.fU. A.fU* A*fU*

18481 1242 3722 U. A.Chi 3723 G*fC*fU* G. 62%

G. A.mC. A. A. P.mU.fC.fU.fU. G. A.

G.mU.mU.mC. A. A. A.fC.fU.fU. G.fU.fC*

18482 1213 3724 G. A.Chi 3725 A*fU* A* G* A* U. 47% mU.mU. A. A. A. G. P.mU.fC.fU.fC.fC.

A.mU. G. G. A. G. A.fU.fC.fU.fU.fU. A.

18483 1760 3726 A.Chi 3727 A*fU* G* G* G* G* C. 69% mC.mU. A.mU. G. P.mU. A. A.fC.fU.fU.

A.mC. A. A. G.fU.fC. A.fU. A. G*

18484 1211 3728 G.mU.mU. A.Chi 3729 A*fU*fU*fU*fC* G. n/a mC. A. A.mC. G. A. A. P.mU.fU. A. G.

A.mU.mC.mU. A. A.fU.fU.fU.fC. G.fU.fU.

19411 1212 3730 A.Chi 3731 G*fU* G* G* G*fU*fU. 52% mU. A.mU. G. A.mC. P.mU. G. A. A.fC.fU.fU.

A. A. G.mU.mU.mC. G.fU.fC. A.fU. A* G*

19412 1222 3732 A.Chi 3733 A*fU*fU*fU*fC. 51%

A. A. G.mU.mU.mC. P.mU.fC.fU. G.fC.fU.fU.

A. A. G.mC. A. G. G. A. A.fC.fU.fU*

19413 1228 3734 A.Chi 3735 G*fU*fC* A*fU* A. n/a

P.mU. G.fU.

A.fC.fU.fC.fU.

mC. A. A. G.mC. A. G. G.fC.fU.fU. G* A*

19414 1233 3736 A. G.mU. A.mC. A.Chi 3737 A*fC*fU*fU* G. 41%

P.mU.fU.fU. G.fU.fC.

A. A.mU.mC.mU. A.fU. A. G.

A.mU. G. A.mC. A. A. A.fU.fU*fU*fC*

19415 1218 3738 A.Chi 3739 G*fU*fU* G. 104% mC. A.mC. A.mC. A. P.mU. A.fU. A.fU.

G.mC. A.mU. A.mU. G.fC.fU. G.fU. G.fU.

19416 1244 3740 A.Chi 3741 G*fU* A*fC*fU*fC*fU. 31%

G. A. A. A.mU. P.mU.fU.fU. G.fC.fU.

A.mU. A. G.mC. A. A. A.fU. A.fU.fU.fU.fC*fU*

19417 655 3742 A.Chi 3743 G* G*fU* A* G. n/a % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

G. A. P.mU.fC.fU. G. G.fU. A.

A.mC.mU.mC.mU. G. A. G.fU.fU.fC*fU*

19418 644 3744 A.mC.mC. A. G. A.Chi 3745 A*fC* G*fU* G. n/a

P.mU.fC. A.fU.fU.

G.mC. A. A. A. G. A.fU.fC.fU.fU.fU.

A.mU. A. A.mU. G. G.fC*fU* G*fU*fC* A*

19419 819 3746 A.Chi 3747 C. n/a

A. A.mC.mU.mC.mU. P.mU.fU.fC.fU. G. G.fU.

A.mC.mC. A. G. A. A. G. A. G.fU.fU*fC*fU*

19420 645 3748 A.Chi 3749 A*fC* G* U. n/a

P.mU.fU.fU.fC.fU. G.

A.mC.mU.mC.mU. G.fU. A. G. A.

A.mC.mC. A. G. A. A. G.fU*fU*fC*fU* A*fC*

19421 646 3750 A.Chi 3751 G. n/a

P.mU.fU.

A.fU.fC.fU.fU.fU.

A.mC. A. G.mC. A. A. G.fC.fU. G.fU*fC* A*fC*

19422 816 3752 A. G. A.mU. A. A.Chi 3753 A* A* G. n/a mC. A. A.mU.mC.mU. P.mU.fU. G.fU.fC. A.fU.

A.mU. G. A.mC. A. A. G. A.fU.fU. G*fC*

19423 495 3754 A.Chi 3755 G*fU*fU* G* U. n/a

A. G. A.mU.mU.mC. P.mU.fU. G. A.fC.fU.fU.

A. A. G.mU.mC. A. G. A. A.fU.fC.fU*fC*fU*

19424 614 3756 A.Chi 3757 G*fC* A* G. n/a mC.mU. G.mU. G. G. P.mU. G.fU.fU.

A. G.mC. A. A.mC. G.fC.fU. fC.fC. A.fC. A.

19425 627 3758 A.Chi 3759 G*fU*fU* G* A*fC* U. n/a mU. G. A.mC. A. P.mU.fU.fC.fU.fU.fU.

G.mC. A. A. A. G. A. G.fC.fU. G.fU.fC. A*fC*

19426 814 3760 A.Chi 3761 A* A* G* A* G. n/a

P.mU.fU. G.

G.fU.fU. fU.fU. G.fU.fC.

A.mU. G. A.mC. A. A. A.fU* A* G* A*fU*fU*

19427 501 3762 A. A.mC.mC. A. A.Chi 3763 G. n/a

G. A. G. P.mU. G. A.fC.fU.fU. G.

A.mU.mU.mC. A. A. A. A.fU.fC.fU.fC*fU*

19428 613 3764 G.mU.mC. A.Chi 3765 G*fC* A* G* G. n/a

P.mU. A.fU. A.fU.

mC. A.mC. A.mC. A. G.fC.fU. G.fU. G.fU.

G.mC. A.mU. A.mU. G*mU*

21240 1244 3766 A.Chi 3767 A*mC*mU*mC* U. 0.875

P.mU. A.fU. A.fU.

mC. A.mC. A.mC. A. G.fC.fU. G.fU. G.fU.

G.mC. A.mU. A.mU. G*mU*mA*mC*mU*m

21241 1244 3768 A.Chi 3769 C* U. 0.88

P.mU. A.fU. A.fU.

mC. A.mC. A.mC. A. G.fC.fU. G.fU.

G.mC. A.mU. A.mU. G.fU.mG*mU*mA*mC*

21242 1244 3770 A.Chi 3771 mU*mC* U. 0.635 % remaining

Oligo Start SEQ ID SEQ ID expression Number Site NO Sense sequence NO Antisense sequence (l uM A549)

P.mU. A.fU. A.fU.

mC. A.mC. A.mC. A. G.fC.fU. G.fU.

G.mC. A.mU. A.mU. G.fU.mG*fU*mA*fC*m

21243 1244 3772 A.Chi 3773 U*fC* U. 0.32

P.mU. A.fU. A.fU.

mC. A.mC. A.mC. A. G.fC.fU. G.fU. G.fU.

G.mC. A.mU. A.mU. G*fU* A*fC*mU*mC*

21244 1244 3774 A.Chi 3775 U. 0.36 mC. A.mC. A.mC. A. P.mU. A.fU. A.fU.

G.mC. A.mU. G.fC.fU. G.fU. G.fU.

21245 1244 3776 A*mU*mA.TEG-Chl 3777 G*fU* A*fC*fU*fC*fU. 0.265 mC*mA*mC. A.mC. P.mU. A.fU. A.fU.

A. G.mC. A.mU. G.fC.fU. G.fU. G.fU.

21246 1244 3778 A*mU*mA.TEG-Chl 3779 G*fU* A*fC*fU*fC*fU. 0.334 mC*mA*mC.mA.mC. P.mU. A.fU. A.fU.

mA.mG.mC.mA.mU. G.fC.fU. G.fU. G.fU.

21247 1244 3780 mA*mU*mA.TEG-Chl 3781 G*fU* A*fC*fU*fC*fU. 0.29 mA. G.

A.mU.mU.mC. A. A. P.mU.fU. G. A.fC.fU.fU.

G.mU.mC*mA*mA.T G. A. A.fU.fC.fU*fC*fU*

21248 614 3782 EG-Chl 3783 G*fC*fU* U. n/a

P.mU. A.fU. A.fU.

mC. A.mC. A.mC. A. G.fC.fU. G.fU. G.mU.

G.mC. A.mU. A.mU. G*mU*

20608 1244 3784 A.Chi 3785 A*mC*mU*mC* U. 79%

P.mU. A.fU. A.fU.

mC. A.mC. A.mC. A. G.fC.fU. G.fU. G.mU.

G.mC. A.mU. A.mU. G*fU* A*mC*fU*mC*

20609 1244 3786 A.Chi 3787 U. 60% mC. A.mC. A.mC. A. P.mU. A. U. A. U. G. C.

G.mC. A.mU. A.mU. U. G. U. G.mU. G*mU*

20610 1244 3788 A.Chi 3789 A*mC*mU*mC* U. 93%

P.mU. A.fU. A.fU.

mC. A.mC. A.mC. A. G.fC.fU. G.fU.

G.mC. A.mU. A.mU. G.mU.mG*mU*mA*mC

20611 1244 3790 A.Chi 3791 *mU*mC* U. n/a

P.mU. A.fU. A.fU.

mC*mA*mC.mA.mC. G.fC.fU. G.fU.

mA.mG.mC.mA.mU. G.fU.mG*fU*mA*fC*m

21374 614 3792 mA*mU*mA.TEG-Chl 3793 U*fC* U. 24% Table 23: CB1 sequences

Ref SEQ ID

Pos SEQ ID NO 19-mer Sense Seq NO 25-mer Sense Seq w/A @ 25

AUCAUGGUGUAUGAUGUC CUUGCAAUCAUGGUGUAUGAUGUC

1393 3880 U 3881 A

CAAUCAUGGUGUAUGAUG UGCUUGCAAUCAUGGUGUAUGAUG

1391 3882 U 3883 A

1125 3884 UGAAACCUACCUGAUGUUC 3885 CAUUGAUGAAACCUACCUGAUGUUA

1090 3886 CAAUCUGUUUGCUCAGACA 3887 AAACUGCAAUCUGUUUGCUCAGACA

1089 3888 GCAAUCUGUUUGCUCAGAC 3889 GAAACUGCAAUCUGUUUGCUCAGAA

1088 3890 UGCAAUCUGUUUGCUCAGA 3891 AGAAACUGCAAUCUGUUUGCUCAGA

1087 3892 CUGCAAUCUGUUUGCUCAG 3893 GAGAAACUGCAAUCUGUUUGCUCAA

UGGUGUAUGAUGUCUUUG CAAUCAUGGUGUAUGAUGUCUUUG

1397 3894 G 3895 A

AUGGUGUAUGAUGUCUUU GCAAUCAUGGUGUAUGAUGUCUUU

1396 3896 G 3897 A

1120 3898 AUUGAUGAAACCUACCUGA 3899 CCACACAUUGAUGAAACCUACCUGA

1118 3900 ACAUUGAUGAAACCUACCU 3901 UCCCACACAUUGAUGAAACCUACCA

1117 3902 CACAUUGAUGAAACCUACC 3903 UUCCCACACAUUGAUGAAACCUACA

1116 3904 ACACAUUGAUGAAACCUAC 3905 U U UCCCACACAU UG AU GAAACCU AA

UACCUGAUGUUCUGGAUC

1132 3906 G 3907 GAAACCUACCUGAUGUUCUGGAUCA

845 3908 UGUUCCACCGCAAAGAUAG 3909 UCCACGUGUUCCACCGCAAAGAUAA

844 3910 GUGUUCCACCGCAAAGAUA 3911 UUCCACGUGUUCCACCGCAAAGAUA

573 3912 CUUCAAGGAGAAUGAGGAG 3913 CUCGUCCUUCAAGGAGAAUGAGGAA

572 3914 CCUUCAAGGAGAAUGAGGA 3915 UCUCGUCCUUCAAGGAGAAUGAGGA

571 3916 UCCUUCAAGGAGAAUGAGG 3917 CUCUCGUCCUUCAAGGAGAAUGAGA

GUUUGCAUUCUGCAGUAUGCUCUG

1449 3918 AUUCUGCAGUAUGCUCUGC 3919 A

UGUUUGCAUUCUGCAGUAUGCUCU

1448 3920 CAUUCUGCAGUAUGCUCUG 3921 A

GUGUUUGCAUUCUGCAGUAUGCUC

1447 3922 GCAUUCUGCAGUAUGCUCU 3923 A

1253 3924 AGAGCAUCAUCAUCCACAC 3925 CCCAGAAGAGCAUCAUCAUCCACAA

1252 3926 AAGAGCAUCAUCAUCCACA 3927 ACCCAGAAGAGCAUCAUCAUCCACA

1247 3928 CCCAGAAGAGCAUCAUCAU 3929 GUGGCACCCAGAAGAGCAUCAUCAA

1246 3930 ACCCAGAAGAGCAUCAUCA 3931 CG U G G CACCCAG AAG AG CAU CAU CA

AGGUUAUGAAGUCGAUCCUAGAUG

311 3932 UGAAGUCGAUCCUAGAUGG 3933 A

GAGGUUAUGAAGUCGAUCCUAGAU

310 3934 AUGAAGUCGAUCCUAGAUG 3935 A

1249 3936 CAGAAGAGCAUCAUCAUCC 3937 GGCACCCAGAAGAGCAUCAUCAUCA

585 3938 UGAGGAGAACAUCCAGUGU 3939 GGAGAAUGAGGAGAACAUCCAGUGA

583 3940 AAUGAGGAGAACAUCCAGU 3941 AAGGAGAAUGAGGAGAACAUCCAGA

581 3942 AGAAUGAGGAGAACAUCCA 3943 UCAAGGAGAAUGAGGAGAACAUCCA

580 3944 GAGAAUGAGGAGAACAUCC 3945 UUCAAGGAGAAUGAGGAGAACAUCA

579 3946 GGAGAAUGAGGAGAACAUC 3947 CUUCAAGGAGAAUGAGGAGAACAUA

578 3948 AGGAGAAUGAGGAGAACAU 3949 CCUUCAAGGAGAAUGAGGAGAACAA

577 3950 AAGGAGAAUGAGGAGAACA 3951 UCCUUCAAGGAGAAUGAGGAGAACA

UUCAAGGAGAAUGAGGAG

574 3952 A 3953 UCGUCCUUCAAGGAGAAUGAGGAGA

1257 3954 CAUCAUCAUCCACACGUCU 3955 GAAGAGCAUCAUCAUCCACACGUCA

1255 3956 AGCAUCAUCAUCCACACGU 3957 CAGAAGAGCAUCAUCAUCCACACGA

1682 3958 AGGUAACCAUGUCUGUGUC 3959 UUGCCAAGGUAACCAUGUCUGUGUA

1681 3960 AAGGUAACCAUGUCUGUGU 3961 AUUGCCAAGGUAACCAUGUCUGUGA Ref SEQ ID

Pos SEQ ID NO 19-mer Sense Seq NO 25-mer Sense Seq w/A @ 25

1680 3962 CAAGGUAACCAUGUCUGUG 3963 GAUUGCCAAGGUAACCAUGUCUGUA

AUGCUCUGAGGAGUAAGG

1499 3964 A 3965 UCAUCUAUGCUCUGAGGAGUAAGGA

UAUGCUCUGAGGAGUAAG

1498 3966 G 3967 AUCAUCUAUGCUCUGAGGAGUAAGA

1497 3968 CUAUGCUCUGAGGAGUAAG 3969 CAUCAUCUAUGCUCUGAGGAGUAAA

1496 3970 UCUAUGCUCUGAGGAGUAA 3971 CCAUCAUCUAUGCUCUGAGGAGUAA

UUGCAAUCAUGGUGUAUG CUCUGCUUGCAAUCAUGGUGUAUG

1388 3972 A 3973 A

CUUGCAAUCAUGGUGUAU

1387 3974 G 3975 CCUCUGCUUGCAAUCAUGGUGUAUA

GCUUGCAAUCAUGGUGUA

1386 3976 U 3977 CCCUCUGCUUGCAAUCAUGGUGUAA

UGCUUGCAAUCAUGGUGU

1385 3978 A 3979 GCCCUCUGCUUGCAAUCAUGGUGUA

CUGCUUGCAAUCAUGGUG

1384 3980 U 3981 GGCCCUCUGCUUGCAAUCAUGGUGA

UCUGCUUGCAAUCAUGGU

1383 3982 G 3983 GGGCCCUCUGCUUGCAAUCAUGGUA

1382 3984 CUCUGCUUGCAAUCAUGGU 3985 GGGGCCCUCUGCUUGCAAUCAUGGA

1314 3986 CCGCAUGGACAUUAGGUUA 3987 CCAAGCCCGCAUGGACAUUAGGUUA

CUGUUUGCUCAGACAUUU

1094 3988 U 3989 UGCAAUCUGUUUGCUCAGACAUUUA

UCUGUUUGCUCAGACAUU

1093 3990 U 3991 CUGCAAUCUGUUUGCUCAGACAUUA

1083 3992 GAAACUGCAAUCUGUUUGC 3993 CUGCGAGAAACUGCAAUCUGUUUGA

1082 3994 AGAAACUGCAAUCUGUUUG 3995 ACUGCGAGAAACUGCAAUCUGUUUA

1080 3996 CGAGAAACUGCAAUCUGUU 3997 GAACUGCGAGAAACUGCAAUCUGUA

323 3998 UAGAUGGCCUUGCAGAUAC 3999 CGAUCCUAGAUGGCCUUGCAGAUAA

322 4000 CUAGAUGGCCUUGCAGAUA 4001 UCGAUCCUAGAUGGCCUUGCAGAUA

CGUGUAUGCGUACAUGUA GUUCAUCGUGUAUGCGUACAUGUA

1179 4002 U 4003 A

UCGUGUAUGCGUACAUGU UGUUCAUCGUGUAUGCGUACAUGU

1178 4004 A 4005 A

AUCGUGUAUGCGUACAUG CUGUUCAUCGUGUAUGCGUACAUG

1177 4006 U 4007 A

1320 4008 GGACAUUAGGUUAGCCAAG 4009 CCGCAUGGACAUUAGGUUAGCCAAA

1319 4010 UGGACAUUAGGUUAGCCAA 4011 CCCGCAUGGACAUUAGGUUAGCCAA

1318 4012 AUGGACAUUAGGUUAGCCA 4013 GCCCGCAUGGACAUUAGGUUAGCCA

1317 4014 CAUGGACAUUAGGUUAGCC 4015 AGCCCGCAUGGACAUUAGGUUAGCA

1316 4016 GCAUGGACAUUAGGUUAGC 4017 AAGCCCGCAUGGACAUUAGGUUAGA

1315 4018 CGCAUGGACAUUAGGUUAG 4019 CAAGCCCGCAUGGACAUUAGGUUAA

1415 4020 GGAAGAUGAACAAGCUCAU 4021 UCUUUGGGAAGAUGAACAAGCUCAA

552 4022 UUACAACAAGUCUCUCUCG 4023 AGAAUUUUACAACAAGUCUCUCUCA

551 4024 UUUACAACAAGUCUCUCUC 4025 CAGAAUUUUACAACAAGUCUCUCUA

550 4026 UUUUACAACAAGUCUCUCU 4027 ACAGAAUU U U ACAACAAG U C U CU CA

476 4028 GUCCCUUCCAAGAGAAGAU 4029 GGGGAAGUCCCUUCCAAGAGAAGAA

474 4030 AAG UCCCU UCCAAGAG AAG 4031 UAGGGGAAGUCCCUUCCAAGAGAAA

473 4032 GAAGUCCCUUCCAAGAGAA 4033 UUAGGGGAAGUCCCUUCCAAGAGAA

GGCGUUUUGCCUGAUGUGGACCAU

1020 4034 UUGCCUGAUGUGGACCAUA 4035 A Ref SEQ ID

Pos SEQ ID NO 19-mer Sense Seq NO 25-mer Sense Seq w/A @ 25

UUUGCCUGAUGUGGACCA UGGCGUUUUGCCUGAUGUGGACCA

1019 4036 U 4037 A

UUUUGCCUGAUGUGGACC GUGGCGUUUUGCCUGAUGUGGACC

1018 4038 A 4039 A

GUGUGGGGAGAACUUCAUGGACAU

606 4040 GGAGAACUUCAUGGACAUA 4041 A

1568 4042 AGCCUCUGGAUAACAGCAU 4043 CUGCGCAGCCUCUGGAUAACAGCAA

UCUGUUCAUCGUGUAUGC

1170 4044 G 4045 ACUGCUUCUGUUCAUCGUGUAUGCA

UUCUGUUCAUCGUGUAUG UACUGCUUCUGUUCAUCGUGUAUG

1169 4046 C 4047 A

CUUCUGUUCAUCGUGUAU GUACUGCUUCUGUUCAUCGUGUAU

1168 4048 G 4049 A

1421 4050 UGAACAAGCUCAUUAAGAC 4051 GGAAGAUGAACAAGCUCAUUAAGAA

1420 4052 AUGAACAAGCUCAUUAAGA 4053 GGGAAGAUGAACAAGCUCAUUAAGA

1419 4054 GAUGAACAAGCUCAUUAAG 4055 UGGGAAGAUGAACAAGCUCAUUAAA

1418 4056 AGAUGAACAAGCUCAUUAA 4057 UUGGGAAGAUGAACAAGCUCAUUAA

1417 4058 AAGAUGAACAAGCUCAUUA 4059 UUUGGGAAGAUGAACAAGCUCAUUA

UGUUCAUCGUGUAUGCGU UGCUUCUGUUCAUCGUGUAUGCGU

1172 4060 A 4061 A

1078 4062 UGCGAGAAACUGCAAUCUG 4063 UGGAACUGCGAGAAACUGCAAUCUA

825 4064 CAGCUUCAUUGACUUCCAC 4065 UGUCUACAGCUUCAUUGACUUCCAA

824 4066 ACAGCUUCAUUGACUUCCA 4067 UUGUCUACAGCUUCAUUGACUUCCA

823 4068 UACAGCUUCAUUGACUUCC 4069 UUUGUCUACAGCUUCAUUGACUUCA

UUUUUGUCUACAGCUUCAUUGACU

821 4070 UCUACAGCUUCAUUGACUU 4071 A

820 4072 GUCUACAGCUUCAUUGACU 4073 AUUUUUGUCUACAGCUUCAUUGACA

612 4074 CUUCAUGGACAUAGAGUGU 4075 GGAGAACUUCAUGGACAUAGAGUGA

611 4076 ACUUCAUGGACAUAGAGUG 4077 GGGAGAACUUCAUGGACAUAGAGUA

610 4078 AACUUCAUGGACAUAGAGU 4079 GGGGAGAACUUCAUGGACAUAGAGA

549 4080 AUUUUACAACAAGUCUCUC 4081 UACAGAAUUUUACAACAAGUCUCUA

547 4082 GAAUUUUACAACAAGUCUC 4083 AU U ACAGAAU U U U ACAACAAG UCU A

UCUGUUCAUCGUGUAUGCGUACAU

1176 4084 CAUCGUGUAUGCGUACAUG 4085 A

UUCUGUUCAUCGUGUAUGCGUACA

1175 4086 UCAUCGUGUAUGCGUACAU 4087 A

1174 4088 UUCAUCGUGUAUGCGUACA 4089 CUUCUGUUCAUCGUGUAUGCGUACA

GUUCAUCGUGUAUGCGUA GCUUCUGUUCAUCGUGUAUGCGUA

1173 4090 C 4091 A

CUGUUCAUCGUGUAUGCG CUGCUUCUGUUCAUCGUGUAUGCG

1171 4092 U 4093 A

609 4094 GAACUUCAUGGACAUAGAG 4095 UGGGGAGAACUUCAUGGACAUAGAA

608 4096 AGAACUUCAUGGACAUAGA 4097 GUGGGGAGAACUUCAUGGACAUAGA

UGUGGGGAGAACUUCAUGGACAUA

607 4098 GAGAACUUCAUGGACAUAG 4099 A

1322 4100 ACAU U AGG U U AGCCAAG AC 4101 GCAUGGACAUUAGGUUAGCCAAGAA

1321 4102 GACAUUAGGUUAGCCAAGA 4103 CGCAUGGACAUUAGGUUAGCCAAGA

1027 4104 AUGUGGACCAUAGCCAUUG 4105 UGCCUGAUGUGGACCAUAGCCAUUA

545 4106 CAG AAU U U U ACAACAAG UC 4107 ACAU U ACAGAAU U U U ACAACAAG U A

532 4108 CAGGUGAACAUUACAGAAU 4109 GCAGACCAGGUGAACAUUACAGAAA

GAGUGUCAUUUUUGUCUACAGCUU

813 4110 CAUUUUUGUCUACAGCUUC 4111 A Ref SEQ ID

Pos SEQ ID NO 19-mer Sense Seq NO 25-mer Sense Seq w/A @ 25

UCAUUUUUGUCUACAGCU GGAGUGUCAUUUUUGUCUACAGCU

812 4112 U 4113 A

GUCAUUUUUGUCUACAGC GGGAGUGUCAUUUUUGUCUACAGC

811 4114 U 4115 A

GUGUCAUUUUUGUCUACA UGGGGAGUGUCAUUUUUGUCUACA

809 4116 G 4117 A

AGUGUCAUUUUUGUCUAC CUGGGGAGUGUCAUUUUUGUCUAC

808 4118 A 4119 A

569 4120 CGUCCUUCAAGGAGAAUGA 4121 CUCUCUCGUCCUUCAAGGAGAAUGA

568 4122 UCGUCCUUCAAGGAGAAUG 4123 UCUCUCUCGUCCUUCAAGGAGAAUA

UUUGCAUUCUGCAGUAUG ACGGUGUUUGCAUUCUGCAGUAUG

1444 4124 C 4125 A

GUUUGCAUUCUGCAGUAU GACGGUGUUUGCAUUCUGCAGUAU

1443 4126 G 4127 A

GGUGUUUGCAUUCUGCAGUAUGCU

1446 4128 UGCAUUCUGCAGUAUGCUC 4129 A

UUGCAUUCUGCAGUAUGC CGGUGUUUGCAUUCUGCAGUAUGC

1445 4130 U 4131 A

UGUUUGCAUUCUGCAGUA AGACGGUGUUUGCAUUCUGCAGUA

1442 4132 U 4133 A

1677 4134 UGCCAAGGUAACCAUGUCU 4135 CAAGAUUGCCAAGGUAACCAUGUCA

1676 4136 UUGCCAAGGUAACCAUGUC 4137 UCAAGAUUGCCAAGGUAACCAUGUA

1675 4138 AU U GCCAAGG U AACCAUG U 4139 GUCAAGAUUGCCAAGGUAACCAUGA

1603 4140 CU G CACAAACACG CAAACA 4141 G ACU G CCU GC ACAAACACG CAAACA

1110 4142 UUUCCCACACAUUGAUGAA 4143 AGACAUUUUCCCACACAUUGAUGAA

1109 4144 UUUUCCCACACAUUGAUGA 4145 CAGACAUUUUCCCACACAUUGAUGA

1108 4146 AUUUUCCCACACAUUGAUG 4147 UCAGACAUUUUCCCACACAUUGAUA

1605 4148 GCACAAACACGCAAACAAU 4149 CUGCCUGCACAAACACGCAAACAAA

1604 4150 U GC ACAAACACG CAAACAA 4151 ACUGCCUGCACAAACACGCAAACAA

1671 4152 CAAGAUUGCCAAGGUAACC 4153 CACGGUCAAGAUUGCCAAGGUAACA

1670 4154 UCAAGAU U GCCAAGG UAAC 4155 GCACGGUCAAGAUUGCCAAGGUAAA

1669 4156 GUCAAGAUUGCCAAGGUAA 4157 AGCACGGUCAAGAUUGCCAAGGUAA

628 4158 UGUUUCAUGGUCCUGAACC 4159 AUAGAGUGUUUCAUGGUCCUGAACA

1115 4160 CACACAU UG AU GAAACCU A 4161 UUUUCCCACACAUUGAUGAAACCUA

1114 4162 CCACACAUUGAUGAAACCU 4163 AUUUUCCCACACAUUGAU GAAACCA

1113 4164 CCCACACAUUGAUGAAACC 4165 CAUUUUCCCACACAUUGAUGAAACA

1112 4166 UCCCACACAUUGAUGAAAC 4167 ACAUUUUCCCACACAUUGAUGAAAA

1111 4168 UUCCCACACAUUGAUGAAA 4169 GACAUUUUCCCACACAUUGAUGAAA

AGUGUCAUUUUUGUCUACAGCUUC

814 4170 AUUUUUGUCUACAGCUUCA 4171 A

1659 4172 CAAGAGCACGGUCAAGAUU 4173 CUGCAUCAAGAGCACGGUCAAGAUA

1657 4174 AUCAAGAGCACGGUCAAGA 4175 AGCUGCAUCAAGAGCACGGUCAAGA

GCUUCUGUUCAUCGUGUA

1167 4176 U 4177 CGUACUGCUUCUGUUCAUCGUGUAA

UGCUUCUGUUCAUCGUGU GCGUACUGCUUCUGUUCAUCGUGU

1166 4178 A 4179 A

1668 4180 GGUCAAGAUUGCCAAGGUA 4181 GAGCACGGUCAAGAUUGCCAAGGUA

819 4182 UGUCUACAGCUUCAUUGAC 4183 CAUUUUUGUCUACAGCUUCAUUGAA

UCAUUUUUGUCUACAGCUUCAUUG

818 4184 UUGUCUACAGCUUCAUUGA 4185 A

UUUGUCUACAGCUUCAUU GUCAUUUUUGUCUACAGCUUCAUU

817 4186 G 4187 A Ref SEQ ID

Pos SEQ ID NO 19-mer Sense Seq NO 25-mer Sense Seq w/A @ 25

UUUUGUCUACAGCUUCAU UGUCAUUUUUGUCUACAGCUUCAU

816 4188 U 4189 A

1543 4190 UUUCCCUCUUGUGAAGGCA 4191 AGCAUGUUUCCCUCUUGUGAAGGCA

1660 4192 AAGAGCACGGUCAAGAUUG 4193 UGCAUCAAGAGCACGGUCAAGAUUA

1030 4194 UGGACCAUAGCCAUUGUGA 4195 CUGAUGUGGACCAUAGCCAUUGUGA

531 4196 CCAGG UG AACAU U ACAGAA 4197 AGCAGACCAGGUGAACAUUACAGAA

1259 4198 UCAUCAUCCACACGUCUGA 4199 AGAGCAUCAUCAUCCACACGUCUGA

1258 4200 AUCAUCAUCCACACGUCUG 4201 AAGAGCAUCAUCAUCCACACGUCUA

Table 24: Summary of CTGF Leads

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 : UU AG A A AGGUGC A A AC A AGG) ; 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

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 4320; and lead 21234 corresponds to SEQ ID NOs 4321 and 4322. Table 26: Summar of hTGFBl Leads

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

Table 27: Summary of hTGFB2 Leads

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 September 22, 2009, and entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS" and PCT Publication No. WO2009/102427 (Application No. PCT/US2009/000852), filed on February 11, 2009, and entitled, "MODIFIED RNAI POLYNUCLEOTIDES AND USES

THEREOF."

What is claimed is:

Claims

1. 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.

2. 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.

3. 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.

4. 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.

5. The dsRNA of any one of claims 1-4, wherein the dsRNA is directed against CTGF.

6. The dsRNA of claim 5, wherein 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.

7. The dsRNA of claim 5, wherein 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.

8. The dsRNA of any one of claims 5-7, wherein 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.

9. The dsRNA of any one of claims 5-8, wherein 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.

10. The dsRNA of any one of claims 5-9, wherein the sense strand comprises SEQ ID NO:2463 and the antisense strand comprises SEQ ID NO:2464.

11. The dsRNA of claim 10, wherein the sense strand comprises SEQ ID NO: 3429 and the antisense strand comprises SEQ ID NO: 3430.

12. The dsRNA of any one of claims 5-9, wherein the sense strand comprises SEQ ID NO:2443 and the antisense strand comprises SEQ ID NO:4203.

13. The dsRNA of claim 12, wherein the sense strand comprises SEQ ID

NO:3445 and the antisense strand comprises SEQ ID NO:3446.

14. The dsRNA of any one of claims 5-9, wherein the sense strand comprises SEQ ID NO:2459 and the antisense strand comprises SEQ ID NO:2460.

15. The dsRNA of claim 14, wherein the sense strand comprises SEQ ID NO:3493 and the antisense strand comprises SEQ ID NO:3494.

16. The dsRNA of any one of claims 5-9, wherein the sense strand comprises SEQ ID NO:2465 and the antisense strand comprises SEQ ID NO:2466.

17. The dsRNA of claim 16, wherein the sense strand comprises SEQ ID NO:3469 and the antisense strand comprises SEQ ID NO:3470.

18. The dsRNA of claim 7, wherein 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.

19. The dsRNA of claim 18, wherein the sense strand comprises a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849.

20. The dsRNA of any one of claims 1-19, wherein the dsRNA is

hydrophobic ally modified.

21. The dsRNA of claim 20, wherein the dsRNA is linked to a hydrophobic conjugate.

22. A composition comprising the dsRNA of any one of claims 1-21.

23. The composition of claim 22, wherein the composition comprises dsRNA directed against genes encoding for more than one protein.

24. The composition of claim 22 or 23 wherein the composition is formulated for delivery to the skin.

25. The composition of claim 24, wherein the composition is in a neutral formulation.

26. The composition of claim 24 or 25, wherein the composition is formulated for topical delivery.

27. The composition of claim 24 or 25, wherein the composition is formulated for intradermal injection.

28. A method comprising delivering the dsRNA of any one of claims 1-21 or the composition of any one of claims 22-27 to the skin of a subject in need thereof.

29. 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 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.

30. 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 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.

31. 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 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.

32. 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 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.

33. The method of any one of claims 29-32, wherein the method is a method for treating compromised skin.

34. The method of any one of claims 29-32, wherein the method is a method for treating or preventing a fibrotic disorder.

35. The method of any one of claims 29-34, wherein the dsRNA is administered via intradermal injection.

36. The method of any one of claims 29-34, wherein the dsRNA is administered locally to the skin.

37. The method of any one of claims 29-36, wherein two or more nucleic acid molecules are administered simultaneously or sequentially.

38. The method of claim 37, wherein one or more of the dsRNAs is

hydrophobic ally modified.

39. The method of claim 38, wherein one or more of the dsRNAs is linked to a hydrophobic conjugate.

40. The method of any one of claims 29-39, wherein the dsRNA is directed against CTGF.

41. The method of claim 40, wherein 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.

42. The method of claim 40, wherein 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.

43. The method of any one of claims 40-42, wherein 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.

44. The method of any one of claims 40-43, wherein 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.

45. The method of any one of claims 40-44, wherein the sense strand comprises

SEQ ID NO:2463 and the antisense strand comprises SEQ ID NO:2464.

46. The method of claim 45, wherein the sense strand comprises SEQ ID NO: 3429 and the antisense strand comprises SEQ ID NO: 3430.

47. The method of any one of claims 40-44, wherein the sense strand comprises SEQ ID NO:2443 and the antisense strand comprises SEQ ID NO:4203.

48. The dsRNA of claim 47, wherein the sense strand comprises SEQ ID NO: 3445 and the antisense strand comprises SEQ ID NO: 3446.

49. The method of any one of claims 40-44, wherein the sense strand comprises SEQ ID NO:2459 and the antisense strand comprises SEQ ID NO:2460.

50. The method of claim 49, wherein the sense strand comprises SEQ ID NO:3493 and the antisense strand comprises SEQ ID NO:3494.

51. The method of any one of claims 40-44, wherein the sense strand comprises

SEQ ID NO:2465 and the antisense strand comprises SEQ ID NO:2466.

52. The method of claim 51, wherein the sense strand comprises SEQ ID NO: 3469 and the antisense strand comprises SEQ ID NO: 3470.

53. The method of claim 40, wherein 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.

54. The method of claim 53, wherein the sense strand comprises a sequence selected from the group consisting of: SEQ ID NOs: 1835, 1847, 1848 and 1849.

55. The method of claim 34, wherein 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.