WO2025217569A1 - Conjugated chemically-modified dsrna during ex vivo perfusion of organs - Google Patents

Abstract

This disclosure relates to methods of ex vivo perfusion of an organ with a double strand RNA (dsRNA) comprising a functional moiety. Also provided are systems of ex vivo perfusion of an organ and ex vivo perfusion solutions containing said dsRNA.

Description

CONJUGATED CHEMICALLY-MODIFIED DSRNA DURING EX VIVO PERFUSION OF ORGANS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 63/632,779, filed April 11, 2024, the entire disclosure of which is hereby incorporated herein by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under GM131839, AI153612, and OD020012 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

This disclosure relates to conjugated, chemically-modified double-stranded RNA (dsRNA) for use in ex vivo machine perfusion of organs.

BACKGROUND

Organ transplantation, such as heart and lung, is the only cure for end-stage disease, but few of the waitlisted patients receive transplants due to a shortage of donor organs. Moreover, many transplanted organs fail to function adequately after implantation. Severe dysfunction of the organs post-transplant, termed primary graft dysfunction (PGD), may result in the need for lifesaving mechanical circulatory support devices, such as extracorporeal membrane oxygenation (ECMO). PGD is mainly due to ischemia during procurement and the subsequent damage after reperfusion, also known as ischemia-reperfusion injury (IRI). Given the risk of PGD and its associated morbidity and mortality, transplant programs only consider organs with excellent baseline function for transplant (only about 34% of offered hearts). Strategies enabling the use of discarded organs would greatly reduce the waitlist for said organs. Currently, no clinical therapy exists to attenuate IRI during transplantation. Normothermic ex vivo perfusion (EVP) has recently been used to preserve and evaluate organs from donors in geographically distant regions or in cases of donation after circulatory death (DCD).

However, there exists a need to protect transplantable organs during EVP. SUMMARY

The disclosure provides methods and systems for the ex vivo prefusion of donor organs with a double stranded RNA (dsRNA) linked to at least one functional moiety to modulate the expression of a target gene in said donor organ. The methods advantageously lead to the rapid accumulation of the dsRNA in the perfused organ, allowing for rapid target gene modulation to ensure the donor organ does not remain outside of a body for extended periods of time.

In some aspects, the disclosure provides a method of modulating the expression of a target gene in an organ during ex vivo perfusion of the organ, the method comprising perfusing the organ ex vivo with a perfusion solution comprising a double stranded RNA (dsRNA) comprising an antisense strand with complementarity to the target gene and a sense strand, and wherein the dsRNA is linked to at least one functional moiety.

In some embodiments, the functional moiety comprises a hydrophobic moiety.

In some embodiments, the functional moiety comprises an N- acetylgalactosamine (GalNAc) moiety.

In some embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof.

In some embodiments, the fatty acid selected from the group consisting of Docosanoic acid (DCA), Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA).

In some embodiments, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA).

In some embodiments, the functional moiety is linked to the antisense strand and/or sense strand by a linker.

In some embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker comprises a phosphodiester linkage, a disulfide linkage, an acid-labile linkage, a photocleavable linkage, or a dTdT dinucleotide with phosphodiester internucleotide linkages.

In some embodiments, the acid-labile linkage comprises a P -thiopropionate linkage or a carboxy dimethylmaleic anhydride (CDM) linkage.

In some embodiments, the linker comprises a divalent or trivalent linker.

In some embodiments, the divalent or trivalent linker is selected from the group consisting of wherein n is 1, 2, 3, 4, or 5.

In some embodiments, when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative.

In some embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of

; and wherein X is 0, S or BH3.

In some embodiments, the antisense strand is about 15 nucleotides to 25 nucleotides in length. In some embodiments, the sense strand is about 15 nucleotides to 25 nucleotides in length. In some embodiments, the antisense strand is 20 nucleotides in length. In some embodiments, the antisense strand is 21 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length. In some embodiments, the sense strand is 15 nucleotides in length. In some embodiments, the sense strand is 16 nucleotides in length. In some embodiments, the sense strand is 18 nucleotides in length. In some embodiments, the sense strand is 20 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length.

In some embodiments, the dsRNA comprises a double-stranded region of 15 base pairs to 20 base pairs. In some embodiments, the dsRNA comprises a doublestranded region of 15 base pairs. In some embodiments, the dsRNA comprises a doublestranded region of 16 base pairs. In some embodiments, the dsRNA comprises a doublestranded region of 18 base pairs. In some embodiments, the dsRNA comprises a doublestranded region of 20 base pairs. In some embodiments, the dsRNA comprises a doublestranded region of 21 base pairs.

In some embodiments, the dsRNA comprises a blunt-end.

In some embodiments, the dsRNA comprises at least one single stranded nucleotide overhang.

In some embodiments, the dsRNA comprises about a 2-nucleotide to 5- nucleotide single stranded nucleotide overhang.

In some embodiments, the dsRNA comprises 2-nucleotide single stranded nucleotide overhang.

In some embodiments, the dsRNA comprises 5-nucleotide single stranded nucleotide overhang.

In some embodiments, the dsRNA comprises naturally occurring nucleotides.

In some embodiments, the dsRNA comprises at least one modified nucleotide.

In some embodiments, the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nonnatural base comprising nucleotide, or a mixture thereof.

In some embodiments, the dsRNA comprises at least one modified internucleotide linkage.

In some embodiments, the modified internucleotide linkage comprises a phosphorothioate intemucleotide linkage.

In some embodiments, the dsRNA comprises 4-16 phosphorothioate internucleotide linkages. In some embodiments, the dsRNA comprises 4-13 phosphorothioate intemucleotide linkages. In some embodiments, the dsRNA comprises 8 or 13 phosphorothioate intemucleotide linkages.

In some embodiments, the dsRNA comprises at least one modified intemucleotide linkage of Formula I:

(i); wherein:

B is a base pairing moiety;

W is selected from the group consisting of O, OCH2, OCH, CH2, and CH;

X is selected from the group consisting of halo, hydroxy, and C1-6 alkoxy;

Y is selected from the group consisting of O , OH, OR, NH , NH2, S", and SH;

Z is selected from the group consisting of O and CH2;

R is a protecting group; and = is an optional double bond.

In some embodiments, the dsRNA comprises at least 80% chemically modified nucleotides.

In some embodiments, the dsRNA is fully chemically modified. In some embodiments, the antisense strand comprises a 5’ phosphate, a 5’- alkyl phosphonate, a 5’ alkylene phosphonate, or a 5’ alkenyl phosphonate.

In some embodiments, the antisense strand comprises a 5’ vinyl phosphonate.

In some embodiments, the target gene is an inflammatory gene, a cell death gene, an endothelial cell adhesion gene, or a major histocompatibility complex (MHC) gene.

In some embodiments, the inflammatory gene is selected from the group consisting of JAK1, TNFa, IFNy, IFNy receptor, IL-ip, IL-6, HMGB1, CXCL9, CXCL10, and CXCL11.

In some embodiments, the cell death gene is selected from the group consisting of FAS, TNFR1, and TP53.

In some embodiments, the endothelial cell adhesion gene is selected from the group consisting of E-Selectin, P-Selectin, ICAM1, ICAM2, and VCAM1.

In some embodiments, the MHC gene is a human leukocyte antigen (HLA) gene or P2 microglobulin (B2M).

In some embodiments, expression of the target gene is reduced.

In some embodiments, modulating the expression of the target gene results in one or more of a reduced inflammatory response in the organ, a reduction in ischemia-reperfusion injury (IRI) in the organ, and a reduced immune rejection response in the organ.

In some embodiments, the organ is perfused with a perfusion solution.

In some embodiments, the perfusion solution comprises the dsRNA.

In some embodiments, the perfusion solution comprises one or more of a nutrient, a buffer, a vasodilator, an endothelial stabilizer, a preservative, and whole blood.

In some embodiments, the ex vivo perfusion is normothermic ex vivo perfusion.

In some embodiments, the organ is selected from the group consisting of a heart, a lung, a liver, and a kidney.

In some embodiments, the organ is a human organ or porcine organ.

In some embodiments, the organ is perfused under ex vivo perfusion for about 30 minutes to about 16 hours (e.g., about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, or about 16 hours).

In some embodiments, the organ is perfused under ex vivo perfusion for about 1 hour to about 4 hours.

In some embodiments, the organ is from a donor subject.

In one aspect, the disclosure provides a method of transplanting an organ into a subject, the method comprising: i) obtaining a donor organ; ii) perfusing the donor organ to modulate the expression of a target gene in the donor organ according to the method described herein; and iii) transplanting the perfused donor organ in the subject.

In one aspect, the disclosure provides a system comprising an ex vivo perfusion device, an organ subject to ex vivo perfusion, and a double stranded RNA (dsRNA) comprising an antisense strand with complementarity to a target gene and a sense strand, wherein the dsRNA is linked to at least one functional moiety.

In some embodiments, the ex vivo perfusion device comprises perfusion circuit comprising: a pump, a gas exchanger, and a heating subsystem.

In some embodiments, the pump is configured to perfuse the organ with a perfusion solution.

In some embodiments, the heating subsystem is configured to maintain the temperature of the perfusion solution at a normothermic temperature.

In some embodiments, the oligonucleotide is present in the perfusion solution.

In some embodiments, the functional moiety comprises a hydrophobic moiety.

In some embodiments, the functional moiety comprises an N- acetylgalactosamine (GalNAc) moiety.

In some embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof.

In some embodiments, the fatty acid selected from the group consisting of Docosanoic acid (DCA), Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA).

In some embodiments, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA).

In one aspect, the disclosure provides an isolated ex vivo perfusion solution comprising: i) a double stranded RNA (dsRNA) comprising an antisense strand with complementarity to a target gene and a sense strand, wherein the dsRNA is linked to at least one functional moiety; and ii) one or more of a nutrient, a buffer, a vasodilator, an endothelial stabilizer, a preservative, a colloid, a hormone, a steroid, and whole blood.

In some embodiments, the isolated ex vivo perfusion solution further comprises one or more of magnesium sulfate anhydrate, at least one of a phosphodiesterase inhibitor, and a nitrate.

In certain embodiments, the perfusion solution is blood (e.g., donated blood and/or oxygenated blood).

In certain embodiments, the perfusion solution is STEEN solution™. In certain embodiments, the perfusion solution comprises serum albumin, dextran, and electrolytes. In certain embodiments, the perfusion solution comprises one or more of sodium, potassium, magnesium, calcium, dextran, glucose, phosphate, and serum albumin.

In certain embodiments, the perfusion solution comprises sodium at about 50- 150 mmol/L (e.g., 86 mmol/L), potassium at about 1-15 mmol/L (e.g., 4.6 mmol/L), magnesium at about 0.1-5 mmol/L (e.g., 0.8 mmol/L), calcium at about 0.5-10 mmol/L (e.g., 1.5 mmol/L), dextran at about 1-50 g/L (e.g., 5 g/L), glucose at about 1-50 mmol /L (e.g., 11 mmol/L), phosphate at about 0.5-10 mmol/L (e.g., 1.2 mmol/L), and serum at about 10-100 g/L (e.g., 70 g/L).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Fig. 1 depicts fluorescent images of porcine heart left ventricle (LV) and right ventricle (RV) biopsies during ex vivo perfusion of a heart. The left panel of each image depicts nuclei staining while the right panel depicts Cy3 imaging from Cy3 -labeled siRNA having a DCA functional moiety. Tissue was taken once an hour for six hours from the outer epicardium.

Fig. 2 depicts relative JAK1 mRNA levels in porcine heart LV and RV biopsies during ex vivo perfusion of a heart.

Fig. 3 depicts mean fluorescence intensity (MFI) in rat heart ventricles from imaging of Cy3-labeled siRNA having a DCA functional moiety (DCA-Jakl) following 6 hours of rat heart ex vivo perfusion under normothermic conditions.

Fig. 4 depicts several biochemical parameters of the rat ex vivo perfused heart over time. Perfusate pH, Co2 (mmHg), 02 (mmHg), lactate (mmol/L), calcium (mmol/L), potassium (mmol/L), chloride (mmol/L), glucose (mmol/L), and pressure (mmHg) were measured. Heart rate, heart weight, and percent change of heart weight were also measured. Data was collected from rat hearts administered a DCA-conjugated Jakl -targeting siRNA, a DCA-conjugated no treatment control siRNA, an unconjugated Jakl -targeting siRNA, and an unconjugated no treatment control siRNA.

Fig. 5 depicts relative JAK1 mRNA levels in rat heart samples after 6 hours of ex vivo perfusion of the heart. mRNA expression analyzed by bDNA QuantiGene Assay with 2x2mm terminal punches from right and left ventricles after 6 hours ex vivo perfusion. Data was normalized to Hprt.

Fig. 6A depicts MFI from flow cytometry for various cell types from rat heart ventricular punches from imaging of Cy3 -labeled siRNA having a DCA functional moiety (DCA-Jakl) following 6 hours of rat heart ex vivo perfusion under normothermic conditions. Cells detecting were all living cells, immune cells, macrophages, endothelial cells cardiomyocytes, stromal cells, and smooth muscle cells. Fig. 6B depicts MFI (top) and percent Cy3 (bottom) in various cell types from rat heart ventricular punches from imaging of Cy3 -labeled siRNA having a DCA functional moiety (DCA-Jakl) following 6 hours of rat heart ex vivo perfusion under normothermic conditions. Fig. 6C depicts MFI of heart ventricles on top and stemloop qPCR quantification of antisense strands on the bottom. The MFI images were scored by 2 individuals in blinded fashion, with averaged scores per heart shown. Fig. 6D depicts antisense strand accumulation as determined by stem-loop qPCR at several listed concentrations after 6 hours or 14 hours of perfusion of the rat heart. Fig. 7A depicts fluorescent images of human heart biopsies during ex vivo perfusion of a heart. The left panel of each image depicts nuclei staining while the right panel depicts Cy3 imaging from Cy3-labeled siRNA having a DCA functional moiety. Tissue was taken once an hour for six hours from the outer epicardium. Fig. 7B depicts antisense strand accumulation as determined by stem-loop qPCR at 3 or 6 hours of perfusion of the human heart. Fig. 7C depicts antisense strand accumulation as determined by stem-loop qPCR in a porcine heart after 4 hours of perfusion. Distribution of DCA conjugated Jakl targeting siRNA was determined in the heart recipient pig in non-heart tissues.

Fig. 8 depicts fluorescent images of porcine lung biopsies during a split lung ex vivo perfusion of lungs. One lung of the pair of porcine lungs was ex vivo perfused with a control perfusion solution and the other lung was ex vivo perfused with the perfusion solution containing 1 pMy3-labeled siRNA having a DCA functional moiety. Biopsies were taken a time 0, 2 hours, 4 hours, and 6 hours.

Fig. 9 depicts relative porcine JAK1 mRNA levels in porcine lung samples after 0, 2, and 4 hours of ex vivo perfusion of the lung. mRNA expression analyzed by bDNA QuantiGene Assay with 2x2mm terminal punches from right and left lungs were taken.

DETAILED DESCRIPTION

Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

So that the invention may be more readily understood, certain terms are first defined.

By “alteration” is meant a change (increase or decrease) in the expression levels of a gene, mRNA or polypeptide as detected by standard art known methods such as those described herein. As used herein, an increase or decrease includes a 10% change in expression levels, a 25% change, a 40% change, or a 50% or greater change in expression levels. In certain embodiments, an increase or decrease is a change in expression levels of between about 30% and about 50% or between about 30% and about 40%. “Alteration” can also indicate a change (increase or decrease) in the biological activity of any of the mRNAs or polypeptides of the invention (As used herein, an increase or decrease includes a 10% change in biological activity, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in biological activity. In certain preferred embodiments, an increase or decrease is a change in expression levels of between about 30% and about 50% or between about 30% and about 40%.

By “therapeutic amount” is meant an amount that when administered to ##.

By “subject” is meant a mammal, including, but not limited to, a human or nonhuman mammal, such as non-human primates or other animals such as, e.g., bovine, equine, canine, ovine, feline, murine and the like.

The term “nucleoside” refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. Additional exemplary nucleosides include inosine, 1 -methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and 2,2N,N-dimethylguanosine (also referred to as “rare” nucleosides). The term “nucleotide” refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester linkage between 5' and 3' carbon atoms.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” is single- stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”) (also referred to in the art as “short interfering RNAs”) refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference. Preferably, a siRNA comprises between about 15-30 nucleotides or nucleotide analogs, more preferably between about 16-25 nucleotides (or nucleotide analogs), even more preferably between about 18-23 nucleotides (or nucleotide analogs), and even more preferably between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term “short” siRNA refers to a siRNA comprising about 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA.

The term “nucleotide analog” or “altered nucleotide” or “modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. Examples of positions of the nucleotide which may be derivatized include the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8- bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotide analogs such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotides. For example the 2' OH-group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, COOR, or OR, wherein R is substituted or unsubstituted Ci-Ce alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions which allow the nucleotide to perform its intended function such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2): 117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. l l(2):77-85, and U.S. Pat. No. 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) preferably decrease the rate of hydrolysis of, for example, polynucleotides comprising said analogs in vivo or in vitro.

The term “oligonucleotide” refers to a short polymer of nucleotides and/or nucleotide analogs. The term “RNA analog” refers to an polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA. As discussed above, the oligonucleotides may be linked with linkages which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages. For example, the nucleotides of the analog may comprise methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, and/or phosphorothioate linkages. Preferred RNA analogues include sugar- and/or backbone-modified ribonucleotides and/or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). An RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate (mediates) RNA interference.

As used herein, the term “RNA interference” (“RNAi”) refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.

An RNAi agent, e.g., an RNA silencing agent, having a strand which is “sequence sufficiently complementary to a target mRNA sequence to direct targetspecific RNA interference (RNAi)” means that the strand has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.

As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) refers to RNA molecules which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

As used herein, the term “RNA silencing” refers to a group of sequence-specific regulatory mechanisms (e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression) mediated by RNA molecules which result in the inhibition or “silencing” of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

The term “discriminatory RNA silencing” refers to the ability of an RNA molecule to substantially inhibit the expression of a “first” or “target” polynucleotide sequence while not substantially inhibiting the expression of a “second” or “non-target polynucleotide sequence,” e.g., when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene, while the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele, while the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is the DNA sequence encoding the regulatory region (e.g. promoter or enhancer elements) of a target gene. In other embodiments, the target polynucleotide sequence is a target mRNA encoded by a target gene.

The term “zzz vitro" has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term “zzz vivo" also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism.

As used herein, the term “transgene” refers to any nucleic acid molecule, which is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from the cell. Such a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. The term “transgene” also means a nucleic acid molecule that includes one or more selected nucleic acid sequences, e.g., DNAs, that encode one or more engineered RNA precursors, to be expressed in a transgenic organism, e.g., animal, which is partly or entirely heterologous, i.e., foreign, to the transgenic animal, or homologous to an endogenous gene of the transgenic animal, but which is designed to be inserted into the animal’s genome at a location which differs from that of the natural gene. A transgene includes one or more promoters and any other DNA, such as introns, necessary for expression of the selected nucleic acid sequence, all operably linked to the selected sequence, and may include an enhancer sequence. A gene “involved” in a disease or disorder includes a gene, the normal or aberrant expression or function of which effects or causes the disease or disorder or at least one symptom of said disease or disorder.

As used herein, the term “target gene” is a gene whose expression is to be substantially inhibited or “silenced.” This silencing can be achieved by RNA silencing, e.g., by cleaving the mRNA of the target gene or translational repression of the target gene. The term “non-target gene” is a gene whose expression is not to be substantially silenced. In one embodiment, the polynucleotide sequences of the target and non-target gene (e.g. mRNA encoded by the target and non-target genes) can differ by one or more nucleotides, e.g., at an intronic region. In another embodiment, the target and non- target genes can differ by one or more polymorphisms (e.g., Single Nucleotide Polymorphisms or SNPs). In another embodiment, the target and non-target genes can share less than 100% sequence identity. In another embodiment, the non-target gene may be a homologue (e.g. an orthologue or paralogue) of the target gene.

A “target allele” is an allele (e.g., a SNP allele) whose expression is to be selectively inhibited or “silenced.” This silencing can be achieved by RNA silencing, e.g., by cleaving the mRNA of the target gene or target allele by a siRNA. The term “non-target allele” is a allele whose expression is not to be substantially silenced. In certain embodiments, the target and non-target alleles can correspond to the same target gene. In other embodiments, the target allele corresponds to, or is associated with, a target gene, and the non-target allele corresponds to, or is associated with, a non-target gene. In one embodiment, the polynucleotide sequences of the target and non-target alleles can differ by one or more nucleotides. In another embodiment, the target and non-target alleles can differ by one or more allelic polymorphisms (e.g., one or more SNPs). In another embodiment, the target and non-target alleles can share less than 100% sequence identity.

The term “polymorphism” as used herein, refers to a variation (e.g., one or more deletions, insertions, or substitutions) in a gene sequence that is identified or detected when the same gene sequence from different sources or subjects (but from the same organism) are compared. For example, a polymorphism can be identified when the same gene sequence from different subjects are compared. Identification of such polymorphisms is routine in the art, the methodologies being similar to those used to detect, for example, breast cancer point mutations. Identification can be made, for example, from DNA extracted from a subject's lymphocytes, followed by amplification of polymorphic regions using specific primers to said polymorphic region. Alternatively, the polymorphism can be identified when two alleles of the same gene are compared. In particular embodiments, the polymorphism is a single nucleotide polymorphism (SNP).

A variation in sequence between two alleles of the same gene within an organism is referred to herein as an “allelic polymorphism.” In certain embodiments, the allelic polymorphism corresponds to a SNP allele. For example, the allelic polymorphism may comprise a single nucleotide variation between the two alleles of a SNP. The polymorphism can be at a nucleotide within a coding region but, due to the degeneracy of the genetic code, no change in amino acid sequence is encoded. Alternatively, polymorphic sequences can encode a different amino acid at a particular position, but the change in the amino acid does not affect protein function. Polymorphic regions can also be found in non-encoding regions of the gene. In exemplary embodiments, the polymorphism is found in a coding region of the gene or in an untranslated region (e.g., a 5' UTR or 3' UTR) of the gene.

As used herein, the term “RNA silencing agent” refers to an RNA which is capable of inhibiting or “silencing” the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of a mRNA molecule through a post- transcriptional silencing mechanism. RNA silencing agents include small (< 50 b.p.), noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, and dual-function oligonucleotides as well as precursors thereof. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.

As used herein, the term “rare nucleotide” refers to a naturally occurring nucleotide that occurs infrequently, including naturally occurring deoxyribonucleotides or ribonucleotides that occur infrequently, e.g., a naturally occurring ribonucleotide that is not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1 -methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, ²N-methylguanosine and ^2,2N,N-dimethylguanosine. The term “engineered,” as in an engineered RNA precursor, or an engineered nucleic acid molecule, indicates that the precursor or molecule is not found in nature, in that all or a portion of the nucleic acid sequence of the precursor or molecule is created or selected by a human. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by mechanisms within a cell. Thus, an RNA precursor produced within a cell from a transgene that includes an engineered nucleic acid molecule is an engineered RNA precursor.

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.

As used herein, the term “dual functional oligonucleotide” refers to a RNA silencing agent having the formula T-L-p, wherein T is an mRNA targeting moiety, L is a linking moiety, and p is a miRNA recruiting moiety. As used herein, the terms “mRNA targeting moiety,” “targeting moiety,” “mRNA targeting portion” or “targeting portion” refer to a domain, portion or region of the dual functional oligonucleotide having sufficient size and sufficient complementarity to a portion or region of an mRNA chosen or targeted for silencing (i.e., the moiety has a sequence sufficient to capture the target mRNA). As used herein, the term “linking moiety” or “linking portion” refers to a domain, portion or region of the RNA-silencing agent which covalently joins or links the mRNA.

As used herein, the term “antisense strand” of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of the gene targeted for silencing. The antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or complementarity sufficient to trigger translational repression of the desired target mRNA. The term “sense strand” or “second strand” of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is complementary to the antisense strand or first strand. Antisense and sense strands can also be referred to as first or second strands, the first or second strand having complementarity to the target sequence and the respective second or first strand having complementarity to said first or second strand. miRNA duplex intermediates or siRNA-like duplexes include a miRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a miRNA* strand having sufficient complementarity to form a duplex with the miRNA strand.

As used herein, the term “guide strand” refers to a strand of an RNA silencing agent, e.g., an antisense strand of an siRNA duplex or siRNA sequence, that enters into the RISC complex and directs cleavage of the target mRNA.

As used herein, the term “asymmetry,” as in the asymmetry of the duplex region of an RNA silencing agent (e.g., the stem of an shRNA), refers to an inequality of bond strength or base pairing strength between the termini of the RNA silencing agent (e.g., between terminal nucleotides on a first strand or stem portion and terminal nucleotides on an opposing second strand or stem portion), such that the 5' end of one strand of the duplex is more frequently in a transient unpaired, e.g., single-stranded, state than the 5' end of the complementary strand. This structural difference determines that one strand of the duplex is preferentially incorporated into a RISC complex. The strand whose 5' end is less tightly paired to the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

As used herein, the term “bond strength” or “base pair strength” refers to the strength of the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., an siRNA duplex), due primarily to H-bonding, van der Waals interactions, and the like between said nucleotides (or nucleotide analogs).

As used herein, the “5' end,” as in the 5' end of an antisense strand, refers to the 5' terminal nucleotides, e.g., between one and about 5 nucleotides at the 5' terminus of the antisense strand. As used herein, the “3' end,” as in the 3' end of a sense strand, refers to the region, e.g., a region of between one and about 5 nucleotides, that is complementary to the nucleotides of the 5' end of the complementary antisense strand. As used herein the term “destabilizing nucleotide” refers to a first nucleotide or nucleotide analog capable of forming a base pair with second nucleotide or nucleotide analog such that the base pair is of lower bond strength than a conventional base pair (i.e., Watson-Crick base pair). In certain embodiments, the destabilizing nucleotide is capable of forming a mismatch base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide is capable of forming a wobble base pair with the second nucleotide. In yet other embodiments, the destabilizing nucleotide is capable of forming an ambiguous base pair with the second nucleotide.

As used herein, the term “base pair” refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of a RNA silencing agent and a target mRNA sequence), due primarily to H-bonding, van der Waals interactions, and the like between said nucleotides (or nucleotide analogs). As used herein, the term “bond strength” or "base pair strength" refers to the strength of the base pair.

As used herein, the term “mismatched base pair” refers to a base pair consisting of non- complementary or non-Watson-Crick base pairs, for example, not normal complementary G:C, A:T or A:U base pairs. As used herein the term “ambiguous base pair” (also known as a non-discriminatory base pair) refers to a base pair formed by a universal nucleotide.

As used herein, term “universal nucleotide” (also known as a “neutral nucleotide”) include those nucleotides (e.g. certain destabilizing nucleotides) having a base (a “universal base” or “neutral base”) that does not significantly discriminate between bases on a complementary polynucleotide when forming a base pair. Universal nucleotides are predominantly hydrophobic molecules that can pack efficiently into antiparallel duplex nucleic acids (e.g., double-stranded DNA or RNA) due to stacking interactions. The base portions of universal nucleotides typically comprise a nitrogen-containing aromatic heterocyclic moiety.

As used herein, the terms “sufficient complementarity” or “sufficient degree of complementarity” mean that the RNA silencing agent has a sequence (e.g. in the antisense strand, mRNA targeting moiety or miRNA recruiting moiety) which is sufficient to bind the desired target RNA, respectively, and to trigger the RNA silencing of the target mRNA. As used herein, the term “translational repression” refers to a selective inhibition of mRNA translation. Natural translational repression proceeds via miRNAs cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur naturally or can be initiated by the hand of man, for example, to silence the expression of target genes.

Various methodologies of the instant invention include step that involves comparing a value, level, feature, characteristic, property, etc. to a “suitable control,” referred to interchangeably herein as an “appropriate control.” A “suitable control” or “appropriate control” is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNA silencing agent of the invention into a cell or organism. In another embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a “suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and example are illustrative only and not intended to be limiting.

In some embodiments, the RNA silencing agents of the invention are designed to target intronic regions in mRNA molecules encoding one or more proteins. Various aspects of the invention are described in further detail in the following subsections.

I. siRNA Design

In some embodiments, siRNAs are designed as follows. First, a portion of the target gene, e.g., one or more of the target sequences, is selected of a target gene. Cleavage of mRNA at these sites should eliminate translation of corresponding soluble protein. Sense strands were designed based on the target sequence. Preferably, the portion (and corresponding sense strand) includes about 30 to 35 nucleotides, e.g., 30, 31, 32, 33, 34 or 35 nucleotides. More preferably, the portion (and corresponding sense strand) includes 21, 22 or 23 nucleotides. The skilled artisan will appreciate, however, that siRNAs having a length of less than 19 nucleotides or greater than 25 nucleotides can also function to mediate RNAi. Accordingly, siRNAs of such length are also within the scope of the instant invention provided that they retain the ability to mediate RNAi. Longer RNAi agents have been demonstrated to elicit an interferon or PKR response in certain mammalian cells which may be undesirable. Preferably, the RNAi agents of the invention do not elicit a PKR response (i.e., are of a sufficiently short length). However, longer RNAi agents may be useful, for example, in cell types incapable of generating a PRK response or in situations where the PKR response has been down-regulated or dampened by alternative means.

The sense strand sequence is designed such that the target sequence is essentially in the middle of the strand. Moving the target sequence to an off-center position may, in some instances, reduce efficiency of cleavage by the siRNA. Such compositions, i.e., less efficient compositions, may be desirable for use if off-silencing of the wild-type mRNA is detected.

The antisense strand is routinely the same length as the sense strand and includes complementary nucleotides. In one embodiment, the strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands comprise align or anneal such that 1-, 2- or 3 -nucleotide overhangs are generated, i.e., the 3' end of the sense strand extends 1, 2 or 3 nucleotides further than the 5' end of the antisense strand and/or the 3' end of the antisense strand extends 1, 2 or 3 nucleotides further than the 5' end of the sense strand. Overhangs can comprise (or consist of) nucleotides corresponding to the target gene sequence (or complement thereof). Alternatively, overhangs can comprise (or consist of) deoxyribonucleotides, for example dTs, or nucleotide analogs, or other suitable non-nucleotide material.

To facilitate entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pair strength between the 5' end of the sense strand and 3' end of the antisense strand can be altered, e.g., lessened or reduced, as described in detail in U.S. Patent Nos. 7,459,547, 7,772,203 and 7,732,593, entitled “Methods and Compositions for Controlling Efficacy of RNA Silencing” (filed Jun. 2, 2003) and U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705, entitled “Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi” (filed Jun. 2, 2003), the contents of which are incorporated in their entirety by this reference. In one embodiment of these aspects of the invention, the base-pair strength is less due to fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the second or sense strand than between the 3' end of the first or antisense strand and the 5' end of the second or sense strand. In another embodiment, the base pair strength is less due to at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In certain exemplary embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In another embodiment, the base pair strength is less due to at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the second or sense strand. In another embodiment, the base pair strength is less due to at least one base pair comprising a rare nucleotide, e.g., inosine (I). In certain exemplary embodiments, the base pair is selected from the group consisting of an I: A, I:U and I:C. In yet another embodiment, the base pair strength is less due to at least one base pair comprising a modified nucleotide. In certain exemplary embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6- diamino-G, and 2,6-diamino-A.

The design of siRNAs suitable for targeting the target sequences is described in detail below. siRNAs can be designed according to the above exemplary teachings for any other target sequences found in the gene. Moreover, the technology is applicable to targeting any other target sequences, e.g., non-disease causing target sequences.

To validate the effectiveness by which siRNAs destroy mRNAs, the siRNA can be incubated with cDNA in a Drosophila- & Q in vitro mRNA expression system. Radiolabeled with ³²P, newly synthesized mRNAs are detected autoradiographically on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include omission of siRNA. Alternatively, control siRNAs are selected having the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.

Sites of siRNA-mRNA complementation are selected which result in optimal mRNA specificity and maximal mRNA cleavage.

II. RNAi Agents

The present invention includes siRNA molecules designed, for example, as described above. The siRNA molecules of the invention can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from e.g., shRNA, or by using recombinant human DICER enzyme, to cleave in vitro transcribed dsRNA templates into pools of 20-, 21- or 23-bp duplex RNA mediating RNAi. The siRNA molecules can be designed using any method known in the art.

In one aspect, instead of the RNAi agent being an interfering ribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAi agent can encode an interfering ribonucleic acid, e.g., an shRNA, as described above. In other words, the RNAi agent can be a transcriptional template of the interfering ribonucleic acid. Thus, RNAi agents of the present invention can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4- 5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3' UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21- 23 nucleotides (Brummelkamp et al., 2002; Lee et al., 2002, Supra, Miyagishi et al., 2002; Paddison et al., 2002, supra; Paul et al., 2002, supra; Sui et al., 2002 supra; Yu et al., 2002, supra. More information about shRNA design and use can be found on the internet at the following addresses: katandin.cshl.org:9331/RNAi/docs/BseRI- BamHI_Strategy.pdf and katandin.cshl.org: 933 l/RNAi/docs/Web_version_of_ PCR strategy 1.pdf).

Expression constructs of the present invention include any construct suitable for use in the appropriate expression system and include, but are not limited to, retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs can include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or Hl RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct. (Tuschl, T., 2002, Supra).

Synthetic siRNAs can be delivered into cells by methods known in the art, including cationic liposome transfection and electroporation. To obtain longer term suppression of the target genes and to facilitate delivery under certain circumstances, one or more siRNA can be expressed within cells from recombinant DNA constructs. Such methods for expressing siRNA duplexes within cells from recombinant DNA constructs to allow longer-term target gene suppression in cells are known in the art, including mammalian Pol III promoter systems (e.g., Hl or U6/snRNA promoter systems (Tuschl, T., 2002, supra) capable of expressing functional double-stranded siRNAs; (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002), supra; Sui et al., 2002, supra). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence. The siRNA is complementary to the sequence of the target gene in 5'-3' and 3'-5' orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by Hl or U6 snRNA promoter and expressed in cells, can inhibit target gene expression (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002), supra; Sui et al., 2002, supra). Constructs containing siRNA sequence under the control of T7 promoter also make functional siRNAs when cotransfected into the cells with a vector expressing T7 RNA polymerase (Jacque et al., 2002, supra). A single construct may contain multiple sequences coding for siRNAs, such as multiple regions of the target gene, targeting the same gene or multiple genes, and can be driven, for example, by separate PolIII promoter sites.

Animal cells express a range of noncoding RNAs of approximately 22 nucleotides termed micro RNA (miRNAs) which can regulate gene expression at the post transcriptional or translational level during animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop, probably by Dicer, an RNase Ill-type enzyme, or a homolog thereof. By substituting the stem sequences of the miRNA precursor with sequence complementary to the target mRNA, a vector construct that expresses the engineered precursor can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zeng et al., 2002, supra). When expressed by DNA vectors containing polymerase III promoters, micro-RNA designed hairpins can silence gene expression (McManus et al., 2002, supra). MicroRNAs targeting polymorphisms may also be useful for blocking translation of mutant proteins, in the absence of siRNA- mediated gene-silencing. Such applications may be useful in situations, for example, where a designed siRNA caused off-target silencing of wild type protein.

Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al., 2002, supra). Infection of HeLa cells by these recombinant adenoviruses allows for diminished endogenous target gene expression. Injection of the recombinant adenovirus vectors into transgenic mice expressing the target genes of the siRNA results in in vivo reduction of target gene expression. Id. In an animal model, whole-embryo electroporation can efficiently deliver synthetic siRNA into postimplantation mouse embryos (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be accomplished by “high-pressure” delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA containing solution into animal via the tail vein (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis et al., 2002. Nanoparticles and liposomes can also be used to deliver siRNA into animals. In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs into cells, e.g., neural cells (e.g., brain cells) (US Patent Applications 2014/0296486, 2010/0186103, 2008/0269149, 2006/0078542 and 2005/0220766).

The nucleic acid compositions of the invention include both unmodified siRNAs and modified siRNAs as known in the art, such as crosslinked siRNA derivatives or derivatives having non nucleotide moieties linked, for example to their 3' or 5' ends. Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

Engineered RNA precursors, introduced into cells or whole organisms as described herein, will lead to the production of a desired siRNA molecule. Such an siRNA molecule will then associate with endogenous protein components of the RNAi pathway to bind to and target a specific mRNA sequence for cleavage and destruction. In this fashion, the mRNA to be targeted by the siRNA generated from the engineered RNA precursor will be depleted from the cell or organism, leading to a decrease in the concentration of the protein encoded by that mRNA in the cell or organism. The RNA precursors are typically nucleic acid molecules that individually encode either one strand of a dsRNA or encode the entire nucleotide sequence of an RNA hairpin loop structure.

The nucleic acid compositions of the invention can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability and/or half-life. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99- 112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).

The nucleic acid molecules of the present invention can also be labeled using any method known in the art. For instance, the nucleic acid compositions can be labeled with a fluorophore, e.g., Cy3, fluorescein, or rhodamine. The labeling can be carried out using a kit, e.g., the SILENCER™ siRNA labeling kit (Ambion). Additionally, the siRNA can be radiolabeled, e.g., using ³H, ³²P or other appropriate isotope.

Moreover, because RNAi is believed to progress via at least one single-stranded RNA intermediate, the skilled artisan will appreciate that ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also be designed (e.g., for chemical synthesis) generated (e.g., enzymatically generated) or expressed (e.g., from a vector or plasmid) as described herein and utilized according to the claimed methodologies. Moreover, in invertebrates, RNAi can be triggered effectively by long dsRNAs (e.g., dsRNAs about 100-1000 nucleotides in length, preferably about 200-500, for example, about 250, 300, 350, 400 or 450 nucleotides in length) acting as effectors of RNAi. (Brondani et al., Proc Natl Acad Sci USA. 2001 Dec. 4; 98(25): 14428-33. Epub 2001 Nov. 27.)

III. RNA Silencing Agents

The present invention features RNA silencing agents against target genes (e.g., siRNA and shRNAs), methods of making said RNA silencing agents, and methods (e.g., research and/or therapeutic methods) for using said improved RNA silencing agents (or portions thereof) for RNA silencing of one or more proteins encoded by the target genes. The RNA silencing agents comprise an antisense strand (or portions thereof), wherein the antisense strand has sufficient complementary to a heterozygous single nucleotide polymorphism to mediate an RNA-mediated silencing mechanism (e.g. RNAi).

The target gene of the RNA silencing agents (e.g., siRNA) is any one of a inflammatory gene, a cell death gene, an endothelial cell adhesion gene, or a major histocompatibility complex (MHC) gene. Modulating (e.g., reducing) the expression of these target genes may be useful during normothermic ex vivo perfusion of an organ to maintain organ function prior to transplantation in a host. Moreover, silencing of MHC genes during normothermic ex vivo perfusion of an organ may reduce the risk of organ rejection and reduce graft vs. host disease in the recipient of the organ after transplantation. In certain embodiments, the inflammatory gene is selected from the group consisting of JAK1, TNFa, IFNy, IFNy receptor, IL-ip, IL-6, HMGB1, CXCL9, CXCL10, and CXCL11.

In certain embodiments, the cell death gene is selected from the group consisting of FAS, TNFR1, and TP53.

In certain embodiments, the endothelial cell adhesion gene is selected from the group consisting of E-Selectin, P-Selectin, ICAM1, ICAM2, and VCAM1.

In certain embodiments, the MHC gene is a human leukocyte antigen (HLA) gene or P2 microglobulin (B2M).

In certain embodiments, modulating the expression of the target gene results in one or more of a reduced inflammatory response in the organ, a reduction in ischemiareperfusion injury (IRI) in the organ, and a reduced immune rejection response in the organ. a) Design of siRNA Molecules

An siRNA molecule of the invention is a duplex consisting of a sense strand and complementary antisense strand, the antisense strand having sufficient complementary to an mRNA to mediate RNAi. Preferably, the siRNA molecule has a length from about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). More preferably, the siRNA molecule has a length from about 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is sufficiently complementary to a target region. Preferably, the strands are aligned such that there are at least 1, 2, or 3 bases at the end of the strands which do not align (i.e., for which no complementary bases occur in the opposing strand) such that an overhang of 1, 2 or 3 residues occurs at one or both ends of the duplex when strands are annealed. Preferably, the siRNA molecule has a length from about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). More preferably, the siRNA molecule has a length from about 16-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially complementary to a target sequence, and the other strand is identical or substantially identical to the first strand. Generally, siRNAs can be designed by using any method known in the art, for instance, by using the following protocol:

1. The siRNA should be specific for a target sequence, e.g., a target sequence. In one embodiment, a target sequence is found in a soluble mRNA, but not in the full- length mRNA. In another embodiment, a target sequence is found in both a soluble mRNA and the full-length mRNA. In another embodiment, a target sequence is found in the full-length mRNA. The first strand should be complementary to the target sequence, and the other strand is substantially complementary to the first strand. In one embodiment, the target sequence is encoded in an intronic region of one or more soluble mRNA sequences. Exemplary target sequences correspond to one or more intronic regions of a target gene. Cleavage of mRNA at these sites should eliminate translation of corresponding soluble protein but not of the full-length protein. Target sequences from other regions of the gene are also suitable for targeting. A sense strand is designed based on the target sequence. Further, siRNAs with lower G/C content (35-55%) may be more active than those with G/C content higher than 55%. Thus in one embodiment, the invention includes nucleic acid molecules having 35-55% G/C content.

2. The sense strand of the siRNA is designed based on the sequence of the selected target site. Preferably the sense strand includes about 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25 nucleotides. More preferably, the sense strand includes 21, 22 or 23 nucleotides. In some embodiments, the sense strand includes 16 nucleotides. In some embodiments, the sense strand includes 17 nucleotides. In some embodiments, the sense strand includes 18 nucleotides. In some embodiments, the sense strand includes 19 nucleotides. In some embodiments, the sense strand includes 20 nucleotides. In some embodiments, the sense strand includes 21 nucleotides. In some embodiments, the sense strand includes 22 nucleotides. In some embodiments, the sense strand includes 23 nucleotides. The skilled artisan will appreciate, however, that siRNAs having a length of less than 19 nucleotides or greater than 25 nucleotides can also function to mediate RNAi. Accordingly, siRNAs of such length are also within the scope of the instant invention provided that they retain the ability to mediate RNAi. Longer RNA silencing agents have been demonstrated to elicit an interferon or Protein Kinase R (PKR) response in certain mammalian cells which may be undesirable. Preferably the RNA silencing agents of the invention do not elicit a PKR response (i.e., are of a sufficiently short length). However, longer RNA silencing agents may be useful, for example, in cell types incapable of generating a PRK response or in situations where the PKR response has been down-regulated or dampened by alternative means.

The siRNA molecules of the invention have sufficient complementarity with the target sequence such that the siRNA can mediate RNAi. In general, siRNA containing nucleotide sequences sufficiently identical to a target sequence portion of the target gene to effect RISC-mediated cleavage of the target gene are preferred. Accordingly, in a preferred embodiment, the sense strand of the siRNA is designed have to have a sequence sufficiently identical to a portion of the target. For example, the sense strand may have 100% identity to the target site. However, 100% identity is not required. Greater than 80% identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity, between the sense strand and the target RNA sequence is preferred. The invention has the advantage of being able to tolerate certain sequence variations to enhance efficiency and specificity of RNAi. In one embodiment, the sense strand has 4, 3, 2, 1, or 0 mismatched nucleotide(s) with a target region, such as a target region that differs by at least one base pair between a soluble and a full-length allele, e.g., a target region comprising the gain-of-function mutation, and the other strand is identical or substantially identical to the first strand. Moreover, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides may also be effective for mediating RNAi. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.

Sequence identity may be 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). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local 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.

In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A preferred, non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

3. The antisense or guide strand of the siRNA is routinely the same length as the sense strand and includes complementary nucleotides. In some embodiments, the antisense or guide strand is longer than the sense strand. In some embodiments, the antisense or guide strand is shorter than the sense strand. In some embodiments, the antisense or guide strand includes about 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25 nucleotides. In some embodiments, the antisense or guide strand includes 21, 22 or 23 nucleotides. In some embodiments, the antisense or guide strand includes 16 nucleotides. In some embodiments, the antisense or guide strand includes 17 nucleotides. In some embodiments, the antisense or guide strand includes 18 nucleotides. In some embodiments, the antisense or guide strand includes 19 nucleotides. In some embodiments, the antisense or guide strand includes 20 nucleotides. In some embodiments, the antisense or guide strand includes 21 nucleotides. In some embodiments, the antisense or guide strand includes 22 nucleotides. In some embodiments, the antisense or guide strand includes 23 nucleotides.

In one embodiment, the guide and sense strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands of the siRNA can be paired in such a way as to have a 3' overhang of 1 to 4, e.g., 2, nucleotides. In some embodiments, the 3’ overhang is 1 nucleotide. In some embodiments, the 3’ overhang is 2 nucleotides. In some embodiments, the 3’ overhang is 3 nucleotides. In some embodiments, the 3’ overhang is 4 nucleotides. In some embodiments, the 3’ overhang is 5 nucleotides. Overhangs can comprise (or consist of) nucleotides corresponding to the target gene sequence (or complement thereof). Alternatively, overhangs can comprise (or consist of) deoxyribonucleotides, for example dTs, or nucleotide analogs, or other suitable non-nucleotide material. Thus in another embodiment, the nucleic acid molecules may have a 3' overhang of 2 nucleotides, such as TT. The overhanging nucleotides may be either RNA or DNA. As noted above, it is desirable to choose a target region wherein the mutant:wild type mismatch is a purine:purine mismatch.

4. Using any method known in the art, compare the potential targets to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. One such method for such sequence homology searches is known as BLAST, which is available at National Center for Biotechnology Information website.

5. Select one or more sequences that meet your criteria for evaluation.

Further general information about the design and use of siRNA may be found in “The siRNA User Guide,” available at The Max-Plank-Institut fur Biophysikalishe Chemi e website.

Alternatively, the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with the target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C or 70 °C hybridization for 12-16 hours; followed by washing). Additional preferred hybridization conditions include hybridization at 70 °C in IxSSC or 50 °C in IxSSC, 50% formamide followed by washing at 70 °C in 0.3xSSC or hybridization at 70 °C in 4xSSC or 50 °C in 4xSSC, 50% formamide followed by washing at 67 °C in IxSSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5- 10 °C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C)=81.5+16.6(log 10[Na+])+0.41(% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for lxSSC=0.165 M). Additional 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.

Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. Such negative controls may be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.

6. To validate the effectiveness by which siRNAs destroy target mRNAs, the siRNA may be incubated with target cDNA in a Drosophila- & Q in vitro mRNA expression system. Radiolabeled with ³²P, newly synthesized target mRNAs are detected autoradiographically on an agarose gel. The presence of cleaved target mRNA indicates mRNA nuclease activity. Suitable controls include omission of siRNA and use of non-target cDNA. Alternatively, control siRNAs are selected having the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence. siRNAs may be designed to target any of the target sequences described supra. Said siRNAs comprise an antisense strand which is sufficiently complementary with the target sequence to mediate silencing of the target sequence. In certain embodiments, the RNA silencing agent is a siRNA.

Sites of siRNA-mRNA complementation are selected which result in optimal mRNA specificity and maximal mRNA cleavage. b) siRNA-Like Molecules siRNA-like molecules of the invention have a sequence (i.e., have a strand having a sequence) that is “sufficiently complementary” to a target sequence of a mRNA to direct gene silencing either by RNAi or translational repression. siRNA-like molecules are designed in the same way as siRNA molecules, but the degree of sequence identity between the sense strand and target RNA approximates that observed between an miRNA and its target. In general, as the degree of sequence identity between a miRNA sequence and the corresponding target gene sequence is decreased, the tendency to mediate post-transcriptional gene silencing by translational repression rather than RNAi is increased. Therefore, in an alternative embodiment, where post- transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity with one or more short sequences (complementarity sites) dispersed within the target mRNA (e.g. within the 3'-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003). Since the mechanism of translational repression is cooperative, multiple complementarity sites (e.g., 2, 3, 4, 5 or 6) may be targeted in certain embodiments.

The capacity of a siRNA-like duplex to mediate RNAi or translational repression may be predicted by the distribution of non-identical nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent at the site of complementarity. In one embodiment, where gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central portion of the complementarity site so that duplex formed by the miRNA guide strand and the target mRNA contains a central “bulge” (Doench J G et al., Genes & Dev., 2003). In another embodiment 2, 3, 4, 5 or 6 contiguous or non-contiguous nonidentical nucleotides are introduced. The non-identical nucleotide may be selected such that it forms a wobble base pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a further preferred embodiment, the “bulge” is centered at nucleotide positions 12 and 13 from the 5' end of the miRNA molecule. c) Short Hairpin RNA (shRNA) Molecules

In certain featured embodiments, the instant invention provides shRNAs capable of mediating RNA silencing of an target sequence with enhanced selectivity. In contrast to siRNAs, shRNAs mimic the natural precursors of micro RNAs (miRNAs) and enter at the top of the gene silencing pathway. For this reason, shRNAs are believed to mediate gene silencing more efficiently by being fed through the entire natural gene silencing pathway. 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 miRNA precursors (pre-miRNA) have a single strand that forms a duplex stem including two portions that are generally complementary, and a loop, that connects the two portions of the stem. In typical pre-miRNAs, the stem includes one or more bulges, e.g., extra nucleotides that create a single nucleotide “loop” in one portion of the stem, and/or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other. Short hairpin RNAs, or engineered RNA precursors, of the invention are artificial constructs based on these naturally occurring pre-miRNAs, but which are engineered to deliver desired RNA silencing agents (e.g., siRNAs of the invention). By substituting the stem sequences of the pre-miRNA with sequence complementary to the target mRNA, a shRNA is formed. The shRNA is processed by the entire gene silencing pathway of the cell, thereby efficiently mediating RNAi.

The requisite elements of a shRNA molecule include a first portion and a second portion, having sufficient complementarity to anneal or hybridize to form a duplex or double-stranded stem portion. The two portions need not be fully or perfectly complementary. The first and second “stem” portions are connected by a portion having a sequence that has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as a “loop” portion in the shRNA molecule. The shRNA molecules are processed to generate siRNAs. shRNAs can also include one or more bulges, i.e., extra nucleotides that create a small nucleotide "loop" in a portion of the stem, for example a one-, two- or three- nucleotide loop. The stem portions can be the same length, or one portion can include an overhang of, for example, 1-5 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. Such Us are notably encoded by thymidines (Ts) in the shRNA-encoding DNA which signal the termination of transcription.

In shRNAs (or engineered precursor RNAs) of the instant invention, one portion of the duplex stem is a nucleic acid sequence that is complementary (or anti-sense) to the target sequence. Preferably, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of said target RNA via RNA interference (RNAi). Thus, engineered RNA precursors include a duplex stem with two portions and a loop connecting the two stem portions. The antisense portion can be on the 5' or 3' end of the stem. The stem portions of a shRNA are preferably about 15 to about 50 nucleotides in length. Preferably the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In preferred embodiments, the length of the stem portions should be 21 nucleotides or greater. When used in mammalian cells, the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses like the interferon pathway. In non-mammalian cells, the stem can be longer than 30 nucleotides. In fact, the stem can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA). In fact, a stem portion can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA).

The two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem. Thus, the two portions can be, but need not be, fully or perfectly complementary. In addition, the two stem portions can be the same length, or one portion can include an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. The loop in the shRNAs or engineered RNA precursors may differ from natural pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop in the shRNAs or engineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length.

The loop in the shRNAs or engineered RNA precursors may differ from natural pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop portion in the shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length. A preferred loop consists of or comprises a “tetraloop” sequences. Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA, where N is any nucleotide and R is a purine nucleotide, GGGG, and uuuu.

In certain embodiments, shRNAs of the invention include the sequences of a desired siRNA molecule described supra. In other embodiments, the sequence of the antisense portion of a shRNA can be designed essentially as described above or generally by selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from within the target RNA, for example, from a region 100 to 200 or 300 nucleotides upstream or downstream of the start of translation. In general, the sequence can be selected from any portion of the target RNA (e.g., mRNA) including an intronic region, the 5' UTR (untranslated region), coding sequence, or 3' UTR, provided said portion is distant from the site of the gain-of-function mutation. This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected to be UU. This 21 or so nucleotide sequence is used to create one portion of a duplex stem in the shRNA. This sequence can replace a stem portion of a wild-type pre-miRNA sequence, e.g., enzymatically, or is included in a complete sequence that is synthesized. For example, one can synthesize DNA oligonucleotides that encode the entire stem-loop engineered RNA precursor, or that encode just the portion to be inserted into the duplex stem of the precursor, and using restriction enzymes to build the engineered RNA precursor construct, e.g., from a wild-type pre-miRNA. Engineered RNA precursors include in the duplex stem the 21-22 or so nucleotide sequences of the siRNA or siRNA-like duplex desired to be produced in vivo. Thus, the stem portion of the engineered RNA precursor includes at least 18 or 19 nucleotide pairs corresponding to the sequence of an exonic portion of the gene whose expression is to be reduced or inhibited. The two 3' nucleotides flanking this region of the stem are chosen so as to maximize the production of the siRNA from the engineered RNA precursor and to maximize the efficacy of the resulting siRNA in targeting the corresponding mRNA for translational repression or destruction by RNAi in vivo and in vitro.

In certain embodiments, shRNAs of the invention include miRNA sequences, optionally end-modified miRNA sequences, to enhance entry into RISC. The miRNA sequence can be similar or identical to that of any naturally occurring miRNA (see e.g. The miRNA Registry; Griffiths- Jones S, Nuc. Acids Res., 2004). Over one thousand natural miRNAs have been identified to date and together they are thought to comprise about 1% of all predicted genes in the genome. Many natural miRNAs are clustered together in the introns of pre-mRNAs and can be identified in silico using homologybased searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g. MiRScan, MiRSeeker) that predict the capability of a candidate miRNA gene to form the stem loop structure of a pri-mRNA (Grad et al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai E C et al., Genome Bio., 2003). An online registry provides a searchable database of all published miRNA sequences (The miRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other natural miRNAs from humans and certain model organisms including Drosophila melanogaster , Caenorhabditis elegans. zebrafish, Arabidopsis thalania. Mus musculus, and Rattus norvegicus as described in International PCT Publication No. WO 03/029459.

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 (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can exist transiently in vivo as a doublestranded duplex, but only one strand is taken up by the RISC complex to direct gene silencing. Certain miRNAs, e.g., plant miRNAs, have perfect or near-perfect complementarity to their target mRNAs and, hence, direct cleavage of the target mRNAs. Other miRNAs have less than perfect complementarity to their target mRNAs and, hence, direct translational repression of the target mRNAs. The degree of complementarity between an miRNA and its target mRNA is believed to determine its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA is predictive of a cleavage mechanism (Yekta et al., Science, 2004), whereas less than perfect complementarity is predictive of a translational repression mechanism. In particular embodiments, the miRNA sequence is that of a naturally-occurring miRNA sequence, the aberrant expression or activity of which is correlated with an miRNA disorder. d) Dual Functional Oligonucleotide Tethers

In other embodiments, the RNA silencing agents of the present invention include dual functional oligonucleotide tethers useful for the intercellular recruitment of a miRNA. Animal cells express a range of miRNAs, noncoding RNAs of approximately 22 nucleotides which can regulate gene expression at the post transcriptional or translational level. By binding a miRNA bound to RISC and recruiting it to a target mRNA, a dual functional oligonucleotide tether can repress the expression of genes involved e.g., in the arteriosclerotic process. The use of oligonucleotide tethers offer several advantages over existing techniques to repress the expression of a particular gene. First, the methods described herein allow an endogenous molecule (often present in abundance), an miRNA, to mediate RNA silencing. Accordingly, the methods described herein obviate the need to introduce foreign molecules (e.g., siRNAs) to mediate RNA silencing. Second, the RNA- silencing agents and, in particular, the linking moiety (e.g., oligonucleotides such as the 2'-O-methyl oligonucleotide), can be made stable and resistant to nuclease activity. As a result, the tethers of the present invention can be designed for direct delivery, obviating the need for indirect delivery (e.g. viral) of a precursor molecule or plasmid designed to make the desired agent within the cell. Third, tethers and their respective moieties, can be designed to conform to specific mRNA sites and specific miRNAs. The designs can be cell and gene product specific. Fourth, the methods disclosed herein leave the mRNA intact, allowing one skilled in the art to block protein synthesis in short pulses using the cell's own machinery. As a result, these methods of RNA silencing are highly regulatable.

The dual functional oligonucleotide tethers (“tethers”) of the invention are designed such that they recruit miRNAs (e.g., endogenous cellular miRNAs) to a target mRNA so as to induce the modulation of a gene of interest. In preferred embodiments, the tethers have the formula T-L-p, wherein T is an mRNA targeting moiety, L is a linking moiety, and p is an miRNA recruiting moiety. Any one or more moiety may be double stranded. Preferably, however, each moiety is single stranded.

Moieties within the tethers can be arranged or linked (in the 5' to 3' direction) as depicted in the formula T-L-p (i.e., the 3' end of the targeting moiety linked to the 5' end of the linking moiety and the 3' end of the linking moiety linked to the 5' end of the miRNA recruiting moiety). Alternatively, the moieties can be arranged or linked in the tether as follows: p-T-L (i.e., the 3' end of the miRNA recruiting moiety linked to the 5' end of the linking moiety and the 3' end of the linking moiety linked to the 5' end of the targeting moiety).

The mRNA targeting moiety, as described above, is capable of capturing a specific target mRNA. According to the invention, expression of the target mRNA is undesirable, and, thus, translational repression of the mRNA is desired. The mRNA targeting moiety should be of sufficient size to effectively bind the target mRNA. The length of the targeting moiety will vary greatly depending, in part, on the length of the target mRNA and the degree of complementarity between the target mRNA and the targeting moiety. In various embodiments, the targeting moiety is less than about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides in length. In a particular embodiment, the targeting moiety is about 15 to about 25 nucleotides in length.

The miRNA recruiting moiety, as described above, is capable of associating with a miRNA. According to the invention, the miRNA may be any miRNA capable of repressing the target mRNA. Mammals are reported to have over 250 endogenous miRNAs (Lagos-Quintana et al. (2002) Current Biol. 12:735-739; Lagos-Quintana et al. (2001) Science 294:858-862; and Lim et al. (2003) Science 299:1540). In various embodiments, the miRNA may be any art-recognized miRNA.

The linking moiety is any agent capable of linking the targeting moieties such that the activity of the targeting moieties is maintained. Linking moieties are preferably oligonucleotide moieties comprising a sufficient number of nucleotides such that the targeting agents can sufficiently interact with their respective targets. Linking moieties have little or no sequence homology with cellular mRNA or miRNA sequences. Exemplary linking moieties include one or more 2'-O-methylnucleotides, e.g., 2'-0- methyladenosine, 2'-O-methylthymidine, 2'-O-methylguanosine or 2'-O-methyluridine.

IV. Modified RNA Silencing Agents

In certain aspects of the invention, an RNA silencing agent (or any portion thereof) of the invention as described supra may be modified such that the activity of the agent is further improved. For example, the RNA silencing agents described in herein may be modified with any of the modifications described infra. The modifications can, in part, serve to further enhance target discrimination, to enhance stability of the agent (e.g., to prevent degradation), to promote cellular uptake, to enhance the target efficiency, to improve efficacy in binding (e.g., to the targets), to improve patient tolerance to the agent, and/or to reduce toxicity.

In certain embodiments, siRNA compounds are provided having one or any combination of the following properties: (1) fully chemically-stabilized (i.e., no unmodified 2’-OH residues); (2) asymmetry; (3) 11-16 base pair duplexes; and (4) single-stranded, fully phosphorothioated tails of 5-8 bases. The number of phosphorothioate modifications is varied from 6 to 17 total in different embodiments.

Certain compounds of the invention having the structural properties described above and herein may be referred to as “hsiRNA-ASP” (hydrophobically-modified, small interfering RNA, featuring an advanced stabilization pattern). In addition, this hsiRNA-ASP pattern showed a dramatically improved distribution through the brain, spinal cord, delivery to liver, placenta, kidney, spleen and several other tissues, making them accessible for therapeutic intervention. The compounds of the invention can be described in the following aspects and embodiments.

In an embodiment of the first aspect of the disclosure, the nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 20 from the 5’ end of the antisense strand are not 2’-methoxy-ribonucleotides and the nucleotides at positions 4, 6, 8, 10, and 14 from the 5’ end of the sense strand are not 2’-methoxy-ribonucleotides.

In an embodiment of the first aspect of the disclosure, the antisense strand is 21 nucleotides in length and the sense strand is 16 nucleotides in length.

1) Modifications to Enhance Target Discrimination

In certain embodiments, the RNA silencing agents of the invention may be substituted with a destabilizing nucleotide to enhance single nucleotide target discrimination (see U.S. application Ser. No. 11/698,689, filed Jan. 25, 2007 and U.S. Provisional Application No. 60/762,225 filed Jan. 25, 2006, both of which are incorporated herein by reference). Such a modification may be sufficient to abolish the specificity of the RNA silencing agent for a non-target mRNA (e.g. wild-type mRNA), without appreciably affecting the specificity of the RNA silencing agent for a target mRNA (e.g. gain-of-function mutant mRNA).

In preferred embodiments, the RNA silencing agents of the invention are modified by the introduction of at least one universal nucleotide in the antisense strand thereof. Universal nucleotides comprise base portions that are capable of base pairing indiscriminately with any of the four conventional nucleotide bases (e.g. A, G, C, U). A universal nucleotide is preferred because it has relatively minor effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotide include those having an inosine base portion or an inosine analog base portion selected from the group consisting of deoxyinosine (e.g. 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine, 2'-O- methoxyethyl-inosine, and 2'-OMe-inosine. In particularly preferred embodiments, the universal nucleotide is an inosine residue or a naturally occurring analog thereof.

In certain embodiments, the RNA silencing agents of the invention are modified by the introduction of at least one destabilizing nucleotide within 5 nucleotides from a specificity-determining nucleotide (i.e., the nucleotide which recognizes the disease- related polymorphism). For example, the destabilizing nucleotide may be introduced at a position that is within 5, 4, 3, 2, or 1 nucleotide(s) from a specificity-determining nucleotide. In exemplary embodiments, the destabilizing nucleotide is introduced at a position which is 3 nucleotides from the specificity-determining nucleotide (i.e., such that there are 2 stabilizing nucleotides between the destablilizing nucleotide and the specificity-determining nucleotide). In RNA silencing agents having two strands or strand portions (e.g. siRNAs and shRNAs), the destabilizing nucleotide may be introduced in the strand or strand portion that does not contain the specificitydetermining nucleotide. In preferred embodiments, the destabilizing nucleotide is introduced in the same strand or strand portion that contains the specificity-determining nucleotide.

2) Modifications to Enhance Efficacy and Specificity

In certain embodiments, the RNA silencing agents of the invention may be altered to facilitate enhanced efficacy and specificity in mediating RNAi according to asymmetry design rules (see U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such alterations facilitate entry of the antisense strand of the siRNA (e.g., a siRNA designed using the methods of the invention or an siRNA produced from a shRNA) into RISC in favor of the sense strand, such that the antisense strand preferentially guides cleavage or translational repression of a target mRNA, and thus increasing or improving the efficiency of target cleavage and silencing. Preferably the asymmetry of an RNA silencing agent is enhanced by lessening the base pair strength between the antisense strand 5' end (AS 5') and the sense strand 3' end (S 3') of the RNA silencing agent relative to the bond strength or base pair strength between the antisense strand 3' end (AS 3') and the sense strand 5' end (S '5) of said RNA silencing agent.

In one embodiment, the asymmetry of an RNA silencing agent of the invention may be enhanced such that there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion than between the 3' end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. Preferably, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In another embodiment, the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one base pair comprising a rare nucleotide, e.g., inosine (I). Preferably, the base pair is selected from the group consisting of an I:A, I:U and I:C. In yet another embodiment, the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one base pair comprising a modified nucleotide. In preferred embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6- diamino-G, and 2,6-diamino-A.

3) RNA Silencing Agents with Enhanced Stability

The RNA silencing agents of the present invention can be modified to improve stability in serum or in growth medium for cell cultures. In order to enhance the stability, the 3'-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNA interference.

In a preferred aspect, the invention features RNA silencing agents that include first and second strands wherein the second strand and/or first strand is modified by the substitution of internal nucleotides with modified nucleotides, such that in vivo stability is enhanced as compared to a corresponding unmodified RNA silencing agent. As defined herein, an “internal” nucleotide is one occurring at any position other than the 5' end or 3' end of nucleic acid molecule, polynucleotide or oligonucleotide. An internal nucleotide can be within a single-stranded molecule or within a strand of a duplex or double-stranded molecule. In one embodiment, the sense strand and/or antisense strand is modified by the substitution of at least one internal nucleotide. In another embodiment, the sense strand and/or antisense strand is modified by the substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In another embodiment, the sense strand and/or antisense strand is modified by the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides. In yet another embodiment, the sense strand and/or antisense strand is modified by the substitution of all of the internal nucleotides.

In some embodiments, the sense strand is modified by the substitution of at least 50% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 55% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 60% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 65% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 70% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 75% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 80% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 85% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 90% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 95% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 96% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 97% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 98% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of at least 99% of the internal nucleotides. In some embodiments, the sense strand is modified by the substitution of 100% of the internal nucleotides.

In some embodiments, the antisense strand is modified by the substitution of at least 50% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 55% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 60% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 65% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 70% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 75% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 80% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 85% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 90% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 95% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 96% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 97% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 98% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of at least 99% of the internal nucleotides. In some embodiments, the antisense strand is modified by the substitution of 100% of the internal nucleotides.

In a preferred embodiment of the present invention, the RNA silencing agents may contain at least one modified nucleotide analogue. The one or more nucleotide analogues may be located at positions where the target-specific silencing activity, e.g., the RNAi mediating activity or translational repression activity is not substantially effected, e.g., in a region at the 5'-end and/or the 3'-end of the siRNA molecule. Particularly, the ends may be stabilized by incorporating modified nucleotide analogues.

Exemplary nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In exemplary sugar- modified ribonucleotides, the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is Ci-Ce alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

In particular embodiments, the modifications are 2'-fluoro, 2'-amino and/or 2'- thio modifications. Particularly preferred modifications include 2'-fluoro-cytidine, 2'- fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino- uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, and/or 5-amino-allyl-uridine. In a particular embodiment, the 2'-fluoro ribonucleotides are every uridine and cytidine. Additional exemplary modifications include 5-bromo- uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino- butyryl-pyrene-uridine, 5-fluoro-cytidine, and 5-fluoro-uridine. 2'-deoxy-nucleotides and 2'-0me nucleotides can also be used within modified RNA-silencing agents of the instant invention. Additional modified residues include, deoxy-abasic, inosine, N3- methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin. In a particularly preferred embodiment, the 2' moiety is a methyl group such that the linking moiety is a 2'-O-methyl oligonucleotide.

In an exemplary embodiment, the RNA silencing agent of the invention comprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modified nucleotides that resist nuclease activities (are highly stable) and possess single nucleotide discrimination for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21 :74-81). These molecules have 2'-O,4'-C-ethylene-bridged nucleic acids, with possible modifications such as 2'-deoxy-2"-fluorouridine. Moreover, LNAs increase the specificity of oligonucleotides by constraining the sugar moiety into the 3'- endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of the oligonucleotide by as much as 10 °C per base.

In another exemplary embodiment, the RNA silencing agent of the invention comprises Peptide Nucleic Acids (PNAs). PNAs comprise modified nucleotides in which the sugar-phosphate portion of the nucleotide is replaced with a neutral 2-amino ethylglycine moiety capable of forming a polyamide backbone which is highly resistant to nuclease digestion and imparts improved binding specificity to the molecule (Nielsen, et al., Science, (2001), 254: 1497-1500).

Also preferred are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined. In other embodiments, cross-linking can be employed to alter the pharmacokinetics of the RNA silencing agent, for example, to increase half-life in the body. Thus, the invention includes RNA silencing agents having two complementary strands of nucleic acid, wherein the two strands are crosslinked. The invention also includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 3' terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like). Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

Other exemplary modifications include: (a) 2' modification, e.g., provision of a 2' OMe moiety on a U in a sense or antisense strand, but especially on a sense strand, and/or a 2' F moiety on a U in a sense or antisense strand, but especially on a sense strand, and/or a 2' OMe moiety in a 3' overhang, e.g., at the 3' terminus (3' terminus means at the 3' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context) and/or a 2' F moiety; (b) modification of the backbone, e.g., with the replacement of an 0 with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; e.g., with the replacement of a P with an S; (c) replacement of the U with a C5 amino linker; (d) replacement of an A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and (d) modification at the 2', 6', 7', or 8' position. Exemplary embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications. Yet other exemplary modifications include the use of a methylated P in a 3' overhang, e.g., at the 3' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3' overhang, e.g., at the 3' terminus; modification with a 3' alkyl; modification with an abasic pyrrolidone in a 3' overhang, e.g., at the 3' terminus; modification with naproxen, ibuprofen, or other moi eties which inhibit degradation at the 3' terminus. Heavily modified RNA silencing agents

In certain embodiments, the RNA silencing agent comprises at least 80% chemically modified nucleotides. In certain embodiments, the RNA silencing agent is fully chemically modified, i.e., 100% of the nucleotides are chemically modified.

In certain embodiments, the RNA silencing agent is 2’-O-methyl rich, i.e., comprises greater than 50% 2’-O-methyl content. In certain embodiments, the RNA silencing agent comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2’-O-methyl nucleotide content. In certain embodiments, the RNA silencing agent comprises at least about 70% 2’-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent comprises between about 70% and about 90% 2’-O-methyl nucleotide modifications.

In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and sense strand. In some embodiments, the antisense strand comprises at least about 50% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises greater than about 50% 2’-O-methyl nucleotide modifications (e.g., 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%, or 90% 2’-O-methyl nucleotide modifications). In some embodiments, the antisense strand comprises greater than 50% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises greater than 60% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 50% 2’-O- methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 55% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 60% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 65% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 70% 2’-O- methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 75% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 80% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 85% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 90% 2’-O- methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 95% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 99% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 100% 2’-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises at least about 70% 2’-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises between about 70% and about 90% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 70% to about 90% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 50% to about 100% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 50% to about 90% 2’-O- methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 50% to about 80% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 50% to about 75% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 50% to about 70% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 50% to about 65% 2’-O-methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 50% to about 60% 2’-O- methyl nucleotide modifications. In some embodiments, the antisense strand comprises about 50% to about 55% 2’-O-methyl nucleotide modifications.

In some embodiments, the sense strand comprises at least about 60% 2’-O- methyl nucleotide modifications. In some embodiments, the sense strand comprises greater than 60% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least about 70% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises 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%, or 100% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 60% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 65% 2’-O- methyl nucleotide modifications. In some embodiments, the sense strand comprises about 70% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 75% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 80% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 85% 2’-O- methyl nucleotide modifications. In some embodiments, the sense strand comprises about 90% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 99% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 100% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between about 70% and about 90% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between 100% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 70% to about 90% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 60% to about 100% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 60% to about 95% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 60% to about 90% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 60% to about 85% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 60% to about 80% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 60% to about 75% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 60% to about 70% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 65% to about 90% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 65% to about 85% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 65% to about 80% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 65% to about 75% 2’-O-methyl nucleotide modifications. In some embodiments, the sense strand comprises about 65% to about 70% 2’-O-methyl nucleotide modifications.

2’-O-methyl rich RNA silencing agents and specific chemical modification patterns are further described in U.S. Patent No. l l,279,930B2 and US2021/0115442A1, each of which is incorporated herein by reference.

Internucleotide linkage modifications

In certain embodiments, at least one internucleotide linkage, intersubunit linkage, or nucleotide backbone is modified in the RNA silencing agent. In certain embodiments, all of the internucleotide linkages in the RNA silencing agent are modified. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate intemucleotide linkage. In certain embodiments, the RNA silencing agent comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 phosphorothioate intemucleotide linkages. In certain embodiments, the RNA silencing agent comprises 4-16 phosphorothioate intemucleotide linkages. In certain embodiments, the RNA silencing agent comprises 8-13 phosphorothioate intemucleotide linkages. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and a sense strand, each comprising a 5’ end and a 3’ end. In certain embodiments, the nucleotides at positions 1 and 2 from the 5’ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 3’ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 5’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-8 from the 3’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, or 1-8 from the 3’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-7 from the 3’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages.

In one aspect, the disclosure provides a modified oligonucleotide (e.g., a dsRNA of the disclosure), said oligonucleotide having a 5’ end, a 3’ end, that is complementary to a target, wherein the oligonucleotide comprises a sense and antisense strand, and at least one modified intersubunit linkage of Formula (I): (i); wherein:

B is a base pairing moiety;

W is selected from the group consisting of O, OCH2, OCH, CH2, and CH;

X is selected from the group consisting of halo, hydroxy, and C1-6 alkoxy;

Y is selected from the group consisting of O , OH, OR, NH , NH2, S", and SH;

Z is selected from the group consisting of O and CH2;

R is a protecting group; and = is an optional double bond.

In an embodiment of Formula (I), when W is CH, = is a double bond.

In an embodiment of Formula (I), when W selected from the group consisting of O, OCH2, OCH, CH2, = is a single bond.

In an embodiment of Formula (I), when Y is O , either Z or W is not O.

In an embodiment of Formula (I), Z is CH2 and W is CH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (II):

In an embodiment of Formula (I), Z is CH2 and W is O. In another embodiment, wherein the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (III):

In an embodiment of Formula (I), Z is O and W is CH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (IV):

In an embodiment of Formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula V:

(V).

In an embodiment of Formula (I), Z is O and W is OCH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VI:

In an embodiment of Formula (I), Z is CH2 and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VII:

In an embodiment of Formula (I), the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

In an embodiment, the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5’ end, a 3’ end, that is complementary to a target, wherein the siRNA comprises a sense and antisense strand, and at least one modified intersubunit linkage of any one or more of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), or Formula (VII).

Modified intersubunit linkages are further described in WO 2020/198509 and WO 2021/195533, each of which is incorporated herein by reference.

4) Conjugated Functional Moieties

In other embodiments, RNA silencing agents may be modified with one or more functional moieties. A functional moiety is a molecule that confers one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moieties enhance cellular uptake by target cells (e.g., neuronal cells). Thus, the disclosure includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 5’ and/or 3' terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1- 3): 137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, poly cations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).

In a certain embodiment, the functional moiety is a hydrophobic moiety. In a certain embodiment, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In a certain embodiment, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA). In a certain embodiment, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EP A), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA). In a certain embodiment, the vitamin selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof. In a certain embodiment, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

In a certain embodiment, an RNA silencing agent of disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand that includes a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of an siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyl oxy hexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

In certain embodiments, the functional moieties may comprise one or more ligands tethered to an RNA silencing agent to improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism. Ligands and associated modifications can also increase sequence specificity and consequently decrease off-site targeting. A tethered ligand can include one or more modified bases or sugars that can function as intercalators. These can be located in an internal region, such as in a bulge of RNA silencing agent/target duplex. The intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking capabilities, and can include systems with 2, 3, or 4 fused rings. The universal bases described herein can be included on a ligand. In one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid. The cleaving group can be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. The metal ion chelating group can include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu(III). In some embodiments, a peptide ligand can be tethered to a RNA silencing agent to promote cleavage of the target RNA, e.g., at the bulge region. For example, l,8-dimethyl-l,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage. A tethered ligand can be an aminoglycoside ligand, which can cause an RNA silencing agent to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C- acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can increase sequence specificity. For example, neomycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity. An acridine analog, neo-5- acridine, has an increased affinity for the HIV Rev-response element (RRE). In some embodiments, the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an RNA silencing agent. In a guanidinoglycoside, the amine group on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog can enhance cell permeability of an RNA silencing agent. A tethered ligand can be a poly-arginine peptide, peptoid or peptidomimetic, which can enhance the cellular uptake of an oligonucleotide agent.

Exemplary ligands are coupled, either directly or indirectly, via an intervening tether, to a ligand-conjugated carrier. In certain embodiments, the coupling is through a covalent bond. In certain embodiments, the ligand is attached to the carrier via an intervening tether. In certain embodiments, a ligand alters the distribution, targeting or lifetime of an RNA silencing agent into which it is incorporated. In certain embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.

Exemplary ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified RNA silencing agent, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides. Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resi stance conferring moi eties; and natural or unusual nucleobases. General examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epi friedel anol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid, or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L- aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L- lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N- isopropyl acrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptidepolyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a poly amine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine (GalNAc) or derivatives thereof, N- acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B 12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines and substituted acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lys tripeptide, aminoglycosides, guanidium aminoglycodies, artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (and thio analogs thereof), cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., Cio, Cn, C12, C13, C14, C15, Ci6, C17, Cis, C19, or C20 fatty acids) and ethers thereof, e.g., Cio, Cn, C12, C13, C14, C15, Ci6, C17, Cis, C19, or C20 alkyl; e.g., 1,3-bis- O(hexadecyl)glycerol, l,3-bis-O(octaadecyl)glycerol), geranyl oxy hexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridineimidazole conjugates, Eu³⁺ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.

In certain embodiments, the GalNAc is represented by the formula below:

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-kB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the RNA silencing agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The ligand can increase the uptake of the RNA silencing agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFD), interleukin- 1 beta, or gamma interferon. In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can bind a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA. A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney. In a certain embodiment, the lipid based ligand binds HSA. A lipid- based ligand can bind HSA with a sufficient affinity such that the conjugate will be distributed to a non-kidney tissue. However, it is contemplated that the affinity not be so strong that the HSA-ligand binding cannot be reversed. In another embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These can be useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, such as a helical cellpermeation agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent can be an alpha-helical agent, which may have a lipophilic and a lipophobic phase. The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to oligonucleotide agents can affect pharmacokinetic distribution of the RNA silencing agent, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. The peptide moiety can be an L-peptide or D-peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptidomimetic tethered to an RNA silencing agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine- aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

In certain embodiments, the functional moiety is linked to the 5’ end and/or 3’ end of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and/or 3’ end of an antisense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and/or 3’ end of a sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 3’ end of a sense strand of the RNA silencing agent of the disclosure.

In certain embodiments, the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and/or sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3’ end of a sense strand by a linker. In certain embodiments, the linker is a cleavable linker. In certain embodiments, the cleavable linker comprises a phosphodiester linkage, a disulfide linkage, an acid-labile linkage, or a photocleavable linkage.

In certain embodiments, the cleavable linker comprises a dTdT dinucleotide with phosphodiester internucleotide linkages.

In certain embodiments, the acid-labile linkage comprises a P-thiopropionate linkage or a carboxy dimethylmaleic anhydride (CDM) linkage.

In certain embodiments, the linker comprises a divalent or trivalent linker. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. In certain embodiments, the divalent or trivalent linker is selected from:

In certain embodiments, the linker further comprises a phosphodiester or phosphodiester derivative. In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of

(Zc2); ; and wherein X is O, S or BH3.

In certain embodiments, the functional moiety PC-DCA with a C7 amino linker is represented by: corresponds to the 3’ end of the sense strand.

The various functional moieties of the disclosure and means to conjugate them to RNA silencing agents are described in further detail in W02017/030973A1 and WO2018/031933A2, incorporated herein by reference.

Ex Vivo Organ Perfusion

The instant disclosure is directed to the use of oligonucleotides linked to functional moieties to improve the survival and function of organs during ex vivo perfusion.

As used herein, the term “ex vivo perfusion” refers to the perfusion of an organ that has been extracted from a donor subject, such as a human or pig donor. Perfusion is often achieved with a device that is capable of circulating a perfusion solution through the organ. The device includes several components, which include, but are not limited to, a pump that moves the perfusion solution through the organ. The pump may be capable of moving the perfusion solution at variable flow rates and pressure. The device may also include a reservoir that contains the perfusion solution. The device may also include a gas delivery component (i.e., gas exchanger) that regulates gas levels in the perfusion solution, such as O2, CO2, and N2. The device may also include a temperature regulating component (i.e., a heating subsystem) to regulate the temperature of the perfusion solution.

Any known perfusion system may be employed in the methods described herein. Exemplary perfusion stems include TransMedics Organ Care Systems™, such as OCS Lung (described in https://www.transmedics.com/ocs-lung/), OCS Heart (described in https://www.transmedics.com/ocs-heart/), and OCS Liver (described in https://www.transmedics.com/ocs-liver/), incorporated herein by reference.

Ex vivo lung perfusion procedures and systems are described in further detail in Langmuur et al. (Multimed Man Cardiothorac Surg. 2023 Apr 3:2023), incorporated herein by reference.

In some embodiments, the ex vivo perfusion described herein is normothermic ex vivo perfusion. Normothermic ex vivo perfusion refers to ex vivo perfusion that occurs at normal body temperatures from which the donor organ was obtained. In some embodiments, the normothermic ex vivo perfusion occurs at about 25° C to about 37° C. In some embodiments, the normothermic ex vivo perfusion occurs at about 37° C.

In some embodiments, the organ subjected to ex vivo perfusion is selected from the group consisting of a heart, a lung, a liver, and a kidney. In some embodiments, the organ is a human organ or porcine organ.

As used herein, the term “perfusion solution” refers to the solution that is moved through the organ subject to ex vivo perfusion. The perfusion solution may be designed to mimic physiological conditions that the organ would be subjected to in a donor or host body. The perfusion solution comprises one or more of a nutrient, a buffer, a vasodilator, an endothelial stabilizer, a colloid, a hormone, a steroid, a preservative, an antimicrobial and/or antifungal agent, and whole blood.

The nutrient may include, but not limited to, a carbohydrate, such as glucose, sodium chloride, potassium chloride, and a multi-vitamin including fat-soluble and water-soluble vitamins. The colloid may include, but not limited to, a dextran. The hormone may include, but not limited to, insulin. The steroid may include, but not limited to, methylprednisolone. The buffer may include, but not limited to, disodium phosphate anhydrate, monopotassium phosphate and sodium bicarbonate. The vasodilator may include, but not limited to, milrinone, nitroglycerin and magnesium sulfate anhydrate. The antimicrobial or antifungal agent may include, but not limited to, cefazolin, ciprofloxacin, and voriconazole. In certain embodiments, the perfusion solution is blood (e.g., donated blood and/or oxygenated blood).

In certain embodiments, the perfusion solution is STEEN solution™. In certain embodiments, the perfusion solution comprises serum albumin, dextran, and electrolytes. In certain embodiments, the perfusion solution comprises one or more of sodium, potassium, magnesium, calcium, dextran, glucose, phosphate, and serum albumin.

In certain embodiments, the perfusion solution comprises sodium at about 50- 150 mmol/L (e.g., 86 mmol/L), potassium at about 1-15 mmol/L (e.g., 4.6 mmol/L), magnesium at about 0.1-5 mmol/L (e.g., 0.8 mmol/L), calcium at about 0.5-10 mmol/L (e.g., 1.5 mmol/L), dextran at about 1-50 g/L (e.g., 5 g/L), glucose at about 1-50 mmol /L (e.g., 11 mmol/L), phosphate at about 0.5-10 mmol/L (e.g., 1.2 mmol/L), and serum at about 10-100 g/L (e.g., 70 g/L).

In certain embodiments, the perfusion solution is KPS-1® solution for kidney perfusion. The KPS-1 solution is described in further detail in https://www. organrecovery. com/preservation-solutions/kps-1 -kidney -perfusion-solution/, incorporated herein by reference.

The oligonucleotide of the disclosure (e.g., dsRNA) is present in the perfusion solution. The concentration of the oligonucleotide in the perfusion solution is a concentration necessary to modulate the expression of the target gene of said oligonucleotide to elicit a desired effect in the organ. Such an effect includes, but is not limited to, a reduced inflammatory response in the organ, a reduction in ischemiareperfusion injury (IRI) in the organ, and a reduced immune rejection response in the organ. In some embodiments, the concentration of the oligonucleotide (e.g., dsRNA) in the perfusion solution is about 0.01 pM, about 0.05 pM, about 0.1 pM, about 0.5 pM, about 1.0 pM, about 1.5 pM, about 2.0 pM, about 2.5 pM, about 3.0 pM, about 3.5 pM, about 4.0 pM, about 4.5 pM, about 5.0 pM, about 10 pM, about 20 pM, about 30 pM, about 40 pM, about 50 pM, about 60 pM, about 70 pM, about 80 pM, about 90 pM, or about 100 pM.

It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

EXAMPLES

Example 1. Ex vivo perfusion of hearts with functional moiety conjugated dsRNA

The ability to silence a target gene with a functional moiety conjugated dsRNA during normothermic ex vivo perfusion of a heart was assessed.

In a first example, a porcine heart was subjected to normothermic ex vivo perfusion. Briefly, the heart was connected to a perfusion system containing a pulsatile pump, an oxygenator, and a reservoir containing a perfusion solution. The perfusion solution was 2 L of Steen solution containing 1.0 pM of a fluorescently labeled Cy3 siRNA targeting porcine, human, and rodent JAK1. The siRNA used is depicted below:

“V” corresponds to vinyl phosphonate; “m” corresponds to a 2’-O-methy modification; “f” corresponds to a 2’ -fluoro modification; “#” corresponds to a phosphorothioate intemucleotide linkage; “dT” corresponds to a thymine DNA nucleotide; “DCA” corresponds to a docosanoic acid functional moiety; “CyMN3” corresponds to the Cy3 fluorescent dye.

For perfusion, ventricular biopsies (i.e., left ventricle (LV) and right ventricle (RV) biopsies) from the outer epicardium and perfusate samples were taken every hour for 6 hours. As shown in Fig. 1, at baseline there is no Cy3 signal, but over time, the Cy3 signal from the DCA-siRNA appears, as early as 1 hour after normothermic perfusion. Moreover, the DCA-siRNA broadly distributes throughout the left and right ventricle and includes endothelial cell uptake.

As shown in Fig. 2, relative Jakl expression was measured at the different time points and different heart locations (LV and RV). Jakl, which is a proinflammatory marker, was found to steadily rise during the ex vivo perfusion process.

In a second example, a rat heart was subjected to normothermic ex vivo perfusion. As with the porcine heart, the distribution of Cy3-labeled siRNA having a DCA functional moiety (DCA-Jakl) was measured. As shown in Fig. 3, the mean fluorescence intensity (MFI) in rat heart ventricles from Cy3-labeled siRNA having a DCA functional moiety (DCA-Jakl) following 6 hours of rat heart ex vivo perfusion under normothermic conditions was measured. The MFI of DCA-Jakl was substantially higher than control, indicating the robust uptake of the siRNA in the rat heart.

Several biochemical parameters of the rat ex vivo perfused heart were monitored over time as well. Perfusate pH, CO2 (mmHg), O2 (mmHg), lactate (mmol/L), calcium (mmol/L), potassium (mmol/L), chloride (mmol/L), glucose (mmol/L), and pressure (mmHg) were measured. Heart rate, heart weight, and percent change of heart weight were also measured. Data was collected from rat hearts administered a DCA-conjugated Jakl -targeting siRNA, a DCA-conjugated no treatment control siRNA, an unconjugated Jakl -targeting siRNA, and an unconjugated no treatment control siRNA. As shown in Fig- 4, all of the measured parameters remained steady in both the Jakl siRNA treatment and non-target treatment groups.

The relative JAK1 mRNA level in rat heart samples after 6 hours of ex vivo perfusion of the heart was also measured. mRNA expression was analyzed by bDNA QuantiGene Assay with 2x2mm terminal punches from right and left ventricles after 6 hours ex vivo perfusion. Data was normalized to Hprt. As shown in Fig. 5, the conjugated Jakl siRNA was able to reduce Jakl mRNA levels after 6 hours while the untreated and DCA-non-target control failed to suppress Jakl mRNA in the same manner. The results indicate the use of a functional moiety conjugated siRNA during ex vivo perfusion is able to silence a target gene, even in the relatively short time period of 6 hours. The cell types that were taking up DCA-conjugated Jakl siRNA were also determined. After 6 hours of normothermic perfusion (37° C), rat heart was isolated, bisected coronally, and 4 or 8mm punches were taken from ventricles and stored in either Miltenyi MACs or Cryostor tissue storage solutions for downstream flow cytometry analysis of cell-type specific Cy3 uptake.

As shown in Fig. 6A, the DCA-conjugated siRNA showed higher levels in several cell types of the heart compared to unconjugated control (UNC). Cells detected were all living cells, immune cells, macrophages, endothelial cells cardiomyocytes, stromal cells, and smooth muscle cells. A similar experiment was performed, using 1 pM siRNA in the perfusion system for 6 hours. As shown in Fig. 6B, there was a significant increase in MFI in the DCA-conjugated siRNA treatment group compared to UNC. The DCA-conjugated siRNA had a 1.9x increase in bulk immune cells, 1.7x increase in macrophages, 1.5x increase in cardiomyocytes, 1.8x increase in endothelial cells, 1.7x increase smooth muscle cells, and a 1.8x increase in stromal cells, compared to UNC, based on MFI. A similar observation was found when measuring the percent of Cy3 positive cells.

MFI in heart ventricles was also determined. As shown in Fig. 6C, there was a dramatic increase in MFI in rat ventricles with the DCA-conjugated siRNA compared to UNC. This correlated with an increase in antisense strand accumulation in the ventricles.

Notably, there was a dose-dependent effect of the siRNA in the perfusion assay. As shown in Fig. 6D, antisense strand accumulation increased as the concentration of the DCA-conjugated siRNA increased, with a significant jump in accumulation at 2 pM siRNA. Moreover, a longer duration of perfusion led to increased antisense accumulation.

In a third example, a human heart was subjected to normothermic ex vivo perfusion for 6 hours with 1 pM of Cy3-labeled siRNA having a DCA functional moiety (DCA-Jakl). As with the porcine heart, the distribution of Cy3-labeled siRNA having a DCA functional moiety (DCA-Jakl) was measured. As shown in Fig. 7A, the Cy3-labeled siRNA having a DCA functional moiety (DCA-Jakl) was taken up by various human heart tissues.

Separately, antisense strand accumulation was determined in the human heart after 3 hours and 6 hours of perfusion with 1 pM of the DCA-conjugated siRNA. Samples were taken from both the left and right ventricle. As shown in Fig. 7B, antisense strand accumulation was observed after just 3 hours of perfusion. The short duration of time to get substantial accumulation is an important factor when an organ cannot remain outside of the body for long periods of time.

The above data demonstrates that a DCA-conjugated siRNA can rapidly accumulate in an ex vivo perfused organ. Next, an ex vivo perfused organ was tested post transplantation. A donor pig heart was harvested and subjected to 4 hours of ex vivo prefusion with a DCA-conjugated siRNA. Following perfusion, the heart was transplanted into a recipient pig. Blood was collected from the recipient pig at the time of transplantation as well as 10 minutes, 1 hour, 2 hours, 3 hours, and 4 hours post transplantation. Following sacrifice of the recipient pig, tissue samples were taken from the left and right ventricle of the donor heart, apex of the donor heart, septum of the donor heart, liver, lungs, kidney, and spleen. From these tissue samples, antisense strand accumulation was determined using stem-loop qPCR. As shown in Fig. 7C, the DCA-conjugated siRNA remained accumulated in the donor heart up to 4 hours post transplantation. Importantly, other tissues, such as liver, kidney, spleen, and lung, did not show accumulation, demonstrating that the ex vivo perfusion method with a DCA- conjugated siRNA is highly organ specific. This an important safety consideration showing that the perfused organ does not significantly leech out the DCA-conjugated siRNA into off-target tissues at least up to 4 hours.

Example 2. Ex vivo perfusion of lungs with functional moiety conjugated dsRNA

The utility of conjugated siRNA in lung normothermic ex vivo perfusion was also tested. A split lung perfusion system was employed in which one lung of the pair of porcine lungs was ex vivo perfused with a control perfusion solution and the other lung was ex vivo perfused with the perfusion solution containing 1 pM Cy3-labeled siRNA having a DCA functional moiety. A Steen perfusate solution was employed.

As shown in Fig. 8, the DCA-conjugated Jakl siRNA was robustly taken up by the treated lung compared to the control lung. Uptake was observed at 2-, 4-, and 6- hours perfusion.

As shown in Fig. 9, levels of JAK1 mRNA increased in the control lung over time, while the DCA-conjugated JAK1 siRNA successfully suppressed AJK1 expression during the short 4-hour perfusion time point. Equivalents

The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

Claims

Claims What is claimed:

1. A method of modulating the expression of a target gene in an organ during ex vivo perfusion of the organ, the method comprising perfusing the organ ex vivo with a perfusion solution comprising a double stranded RNA (dsRNA) comprising an antisense strand with complementarity to the target gene and a sense strand, and wherein the dsRNA is linked to at least one functional moiety.

2. The method of claim 1, wherein the functional moiety comprises a hydrophobic moiety.

3. The method of claim 1, wherein the functional moiety comprises an N- acetylgalactosamine (GalNAc) moiety.

4. The method of claim 3, wherein the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof.

5. The method of claim 4, wherein the fatty acid selected from the group consisting of Docosanoic acid (DC A), Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA).

6. The method of claim 4, wherein the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA).

7. The method of any one of claims 1-6, wherein the functional moiety is linked to the antisense strand and/or sense strand by a linker.

8. The method of claim 7, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

9. The method of claim 7 or 8, wherein the linker is a cleavable linker.

10. The method of claim 9, wherein the cleavable linker comprises a phosphodiester linkage, a disulfide linkage, an acid-labile linkage, a photocleavable linkage, or a dTdT dinucleotide with phosphodiester intemucleotide linkages.

11. The method of claim 10, wherein the acid-labile linkage comprises a P- thiopropi onate linkage or a carboxy dimethylmaleic anhydride (CDM) linkage.

12. The method of any one of claims 7-11, wherein the linker comprises a divalent or trivalent linker.

13. The method of claim 12, wherein the divalent or trivalent linker is selected from the group consisting of: wherein n is 1, 2, 3, 4, or 5.

14. The method of claim 13, wherein when the linker is a trivalent linker, the linker further links a phosphodi ester or phosphodiester derivative.

15. The method of claim 14, wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of:

(Zcl);

; and

(Zc3)

HO^_p„O

©x'

(Zc4) wherein X is O, S or BH3.

16. The method of any one of claims 1-15, wherein the antisense strand is about

15 nucleotides to 25 nucleotides in length.

17. The method of any one of claims 1-16, wherein the sense strand is about 15 nucleotides to 25 nucleotides in length.

18. The method of any one of claims 1-17, wherein the antisense strand is 20 nucleotides in length.

19. The method of any one of claims 1-17, wherein the antisense strand is 21 nucleotides in length.

20. The method of any one of claims 1-17, wherein the antisense strand is 22 nucleotides in length.

21. The dsRNA of any one of claims 1-20, wherein the sense strand is 15 nucleotides in length.

22. The method of any one of claims 1-20, wherein the sense strand is 16 nucleotides in length.

23. The method of any one of claims 1-20, wherein the sense strand is 18 nucleotides in length.

24. The method of any one of claims 1-20, wherein the sense strand is 20 nucleotides in length or 21 nucleotides in length.

25. The method of any one of claims 1-24, comprising a double-stranded region of 15 base pairs to 20 base pairs.

26. The method of any one of claims 1-25, comprising a double-stranded region of 15 base pairs.

27. The method of any one of claims 1-25, comprising a double-stranded region of 16 base pairs.

28. The method of any one of claims 1-25, comprising a double-stranded region of 18 base pairs.

29. The method of any one of claims 1-25, comprising a double-stranded region of 20 base pairs or 21 base pairs.

30. The method of any one of claims 1-29, wherein said dsRNA comprises a blunt-end.

31. The method of any one of claims 1-30, wherein said dsRNA comprises at least one single stranded nucleotide overhang.

32. The method of claim 31, wherein said dsRNA comprises about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang.

33. The method of claim 31, wherein said dsRNA comprises 2-nucleotide single stranded nucleotide overhang.

34. The method of claim 31, wherein said dsRNA comprises 5-nucleotide single stranded nucleotide overhang.

35. The method of any one of claims 1-34, wherein said dsRNA comprises naturally occurring nucleotides.

36. The method of any one of claims 1-34, wherein said dsRNA comprises at least one modified nucleotide.

37. The method of claim 36, wherein said modified nucleotide comprises a 2'-O- methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy- modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, or a mixture thereof.

38. The method of any one of claims 1-37, wherein said dsRNA comprises at least one modified internucleotide linkage.

39. The method of claim 38, wherein said modified intemucleotide linkage comprises a phosphorothioate intemucleotide linkage.

40. The method of any one of claims 1-39, comprising 4-16 phosphorothioate intemucleotide linkages.

41. The method of any one of claims 1-39, comprising 4-13 phosphorothioate intemucleotide linkages, optionally wherein the dsRNA comprises 8 or 13 phosphorothioate intemucleotide linkages.

42. The method of any one of claims 1-38, wherein said dsRNA comprises at least one modified intemucleotide linkage of Formula I:

wherein:

B is a base pairing moiety;

W is selected from the group consisting of O, OCH2, OCH, CH2, and CH;

X is selected from the group consisting of halo, hydroxy, and C1-6 alkoxy;

Y is selected from the group consisting of O , OH, OR, NH , NH2, S", and SH;

Z is selected from the group consisting of O and CH2;

R is a protecting group; and = is an optional double bond.

43. The method of any one of claims 1-42, wherein said dsRNA comprises at least 80% chemically modified nucleotides.

44. The method of any one of claims 1-43, wherein said dsRNA is fully chemically modified.

45. The method of any one of claims 1-44, wherein the antisense strand comprises a 5’ phosphate, a 5 ’-alkyl phosphonate, a 5’ alkylene phosphonate, or a 5’ alkenyl phosphonate.

46. The method of claim 45, wherein the antisense strand comprises a 5’ vinyl phosphonate.

47. The method of any one of claims 1-46, wherein the target gene is an inflammatory gene, a cell death gene, an endothelial cell adhesion gene, or a major histocompatibility complex (MHC) gene.

48. The method of claim 47, wherein the inflammatory gene is selected from the group consisting of JAK1, TNFa, ZFNy, ZFNy receptor, IL-ip, IL-6, HMGB1, CXCL9, CXCL10, and CXCL11.

49. The method of claim 47, wherein the cell death gene is selected from the group consisting of FAS, TNFR1, and TP53.

50. The method of claim 47, wherein the endothelial cell adhesion gene is selected from the group consisting of E-Selectin, P-Selectin, ICAM1, ICAM2, and VCAM1.

51. The method of claim 47, wherein the MHC gene is a human leukocyte antigen (HLA) gene or P2 microglobulin (B2M).

52. The method of any one of claims 1-51, wherein expression of the target gene is reduced.

53. The method of any one of claims 1-52, wherein modulating the expression of the target gene results in one or more of a reduced inflammatory response in the organ, a reduction in ischemia-reperfusion injury (IRI) in the organ, and a reduced immune rejection response in the organ.

54. The method of any one of claims 1-53, wherein the perfusion solution comprises one or more of a nutrient, a buffer, a vasodilator, an endothelial stabilizer, a preservative, and whole blood.

55. The method of any one of claims 1-54, wherein the ex vivo perfusion is normothermic ex vivo perfusion.

56. The method of any one of claims 1-55, wherein the organ is selected from the group consisting of a heart, a lung, a liver, and a kidney.

57. The method of any one of claims 1-56, wherein the organ is a human organ or porcine organ.

58. The method of any one of claims 1-57, wherein the organ is perfused under ex vivo perfusion for about 30 minutes to about 16 hours.

59. The method of any one of claims 1-58, wherein the organ is perfused under ex vivo perfusion for about 1 hour to about 4 hours.

60. The method of any one of claims 1-59, wherein the perfusing comprising pumping the perfusion solution through the organ.

61. The method of any one of claims 1-60, wherein the perfusion solution comprises serum albumin, dextran, and electrolytes.

62. The method of any one of claims 1-61, wherein the perfusion solution comprises one or more of sodium, potassium, magnesium, calcium, dextran, glucose, phosphate, and serum albumin.

63. The method of any one of claims 1-63, wherein the organ is from a donor subject.

64. A method of transplanting an organ into a subject in need thereof, the method comprising: i) obtaining a donor organ; ii) perfusing the donor organ to modulate the expression of a target gene in the donor organ according to the method of any one of claims 1-63; iii) transplanting the perfused donor organ in the subject.

65. A system comprising an ex vivo perfusion device, an organ subject to ex vivo perfusion, and a double stranded RNA (dsRNA) comprising an antisense strand with complementarity to a target gene and a sense strand, wherein the dsRNA is linked to at least one functional moiety.

66. The system of claim 65, wherein the ex vivo perfusion device comprises perfusion circuit comprising: a pump, a gas exchanger, and a heating subsystem.

67. The system of claim 66, wherein the pump is configured to perfuse the organ with a perfusion solution.

68. The system of claim 66, wherein the heating subsystem is configured to maintain the temperature of the perfusion solution at a normothermic temperature.

69. The system of claim 67 or 68, wherein the dsRNA is present in the perfusion solution.

70. The system of any one of claims 65-69, wherein the functional moiety comprises a hydrophobic moiety.

71. The system of any one of claims 65-70, wherein the functional moiety comprises an N-acetylgalactosamine (GalNAc) moiety.

72. The system of claim 70, wherein the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof.

73. The system of claim 72, wherein the fatty acid selected from the group consisting of Docosanoic acid (DC A), Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EP A).

74. The system of claim 72 wherein the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA).