5' antisense DNA modifications to enhance RNA interference activity

Abstract

Provided herein according to some embodiments are double-stranded RNA molecules comprising a sense strand and an antisense strand with two or more dT overhangs on a 5' end of the antisense strand, and compositions comprising the double-stranded RNAs. Methods of enhancing RNA interference activity at a target are provided, comprising delivering a double-stranded RNA to the target. Methods of treating a disorder or disease are also provided.

Description

5' ANTISENSE DNA MODIFICATIONS TO ENHANCERNA INTERFERENCE ACTIVITYSTATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 509,756, filed June 22, 2023, the entire contents of which are incorporated by reference herein.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0002] A Sequence Listing in XML format, entitled 5470-947WO_ST26.xml, 197,017 bytes in size, generated on June 11, 2024, and filed herewith, is hereby incorporated by reference in its entirety for its disclosures.FIELD OF THE INVENTION

[0003] The invention relates to 5' antisense DNA modifications and their use for enhanced RNA interference activity for inhibiting expression of one or more target genes.BACKGROUND

[0004] RNA interference (RNAi)-based therapies present a unique alternative strategy for targeting “undruggable” proteins like MYC and KRAS and operate through the RNA-induced silencing complex (RISC). Briefly, cytosolic RISC unwinds delivered dsRNA and loads the antisense strand to recognize and cleave complementary mRNA sequences. Although RNAi is sequence-specific and potent, up until recently it has faced several clinical obstacles, including in vivo instability, lack of tissue-specific delivery, off-target silencing effects, and immunogenicity. However, recent innovations in the RNAi therapeutics field have led to the adoption of receptortargeting ligands conjugated to fully chemically modified siRNAs. These advances have helped overcome many physiological barriers, leading to several FDA-approved RNAi-based drugs that silence mRNA targets in the liver.

[0005] Given the opportunities for RNAi therapeutics, enhancements to RNAi activity for inhibiting expression of one or more target genes would be advantageous.SUMMARY OF THE INVENTION

[0006] The present invention is based on the development of double-stranded RNA (dsRNA) with DNA-based modifications at the 5' end of the antisense strand of the dsRNAs, which can be used in RNA interference at a target gene. The addition of these modifications can regulate steady-state levels of mRNAs to thereby inhibit transcription of a target gene more potently than dsRNAs that do not have these modifications. These constructs provide remarkable inhibition of gene expression of target genes and are unexpectedly superior to dsRNAs without the 5' modifications. Accordingly, one aspect of the invention relates to a dsRNA molecule comprising a sense strand and an antisense strand, the antisense strand comprising a nucleotide overhang of two or more deoxythymidines on the 5' end.

[0007] In an aspect, one, two, or more of the dsRNA molecules target to different genes or different portions of the same gene are provided in a composition, which may optionally comprise a nanoparticle.

[0008] In another aspect, a pharmaceutical composition comprising the dsRNA of the present invention and / or the composition of the invention and a pharmaceutically acceptable carrier are provided.

[0009] In a further aspect, a method for reducing the expression of a target gene in a cell is provided, comprising contacting the cell with a dsRNA, a composition, and / or a pharmaceutical composition of the present invention, thereby reducing the expression of the target gene in the cell.

[0010] In an additional aspect, a method for delivering dsRNA to a target in a subject is provided, comprising administering a dsRNA, a composition, and / or a pharmaceutical composition of the present invention to the subject.

[0011] In an aspect, a method of treating cancer in a subject in need thereof is provided, the method comprising delivering to the subject a dsRNA, a composition, and / or a pharmaceutical composition of the present invention, thereby treating cancer in the subject.

[0012] These and other aspects of the invention are set forth in more detail in the description of the invention below.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1. Schematic showing exemplary siRNA with IdT, 2dT, 3dT, and 4dT overhangs at the 5' end of the antisense strand of a KRAS-targeting siRNA.

[0014] FIGS. 2A-2B. Representative dose-response curves (FIG. 2A) and relative ED50s (effective dose for 50% of the population; FIG. 2B) of KRAS-Firefly luciferase reporter expression in A-431 KRAS-knockout cells treated with the control (Neg Ctrl), Kseq2 (KRAS- targeting siRNA) with Hi20Me modifications, Kseq2 IdT, Kseq2 2dT, Kseq2 3dT, and Kseq2 4dT siRNAs for 144 hours at doses ranging from 20 nM to 0.01 nM. All Firefly-luciferase luminescence values were normalized with Renilla luciferase luminescence and expressed as a percentage. Error bars represent SEM (standard error of the mean). Relative potency for each siRNA is calculated as GI50 (concentration for 50% of maximal inhibition of cell proliferation) of Kseq2 -treated cells divided by GI50 of siRNA-treated cells.

[0015] FIGS. 3A-3B. Representative dose-response curves (FIG. 3A) and relative ED50s (FIG. 3B) of KRAS-Firefly luciferase reporter expression in A-431 KRAS-knockout cells treated with the control (Neg Ctrl), Kseq2 with Hi20Me modifications, Kseq3 (KRAS-targeting siRNA) with Hi20Me modifications, Kseq3 2dT, and Kseq3 3dT for 144 hours at doses ranging from 20 nM to 0.01 nM. All Firefly-luciferase luminescence values were normalized with Renilla luciferase luminescence and expressed as a percentage. Error bars represent SEM. Relative potency for each siRNA is calculated as GI50 of Kseq3 -treated cells divided by GI50 of siRNA-treated cells.

[0016] FIGS. 4A-4B. Representative dose-response curves (FIG. 4A) and relative ED50s (FIG. 4B) of LN2A cells treated with the control (Neg Ctrl), Mseq2 (MYC-targeting siRNA) with Hi20Me modifications, Mseq2 2dT, and Mseq2 3dT for 144 hours at doses ranging from 20 nM to 0.01 nM. All Firefly-luciferase luminescence values were normalized with Renilla luciferase luminescence and expressed as a percentage. Error bars represent SEM. Relative potency for each siRNA is calculated as GI50 of Mseq2 -treated cells divided by GI50 of siRNA-treated cells.

[0017] FIG. 5A-5B. Representative dose-response curves (FIG. 5A) and relative ED50s (FIG. 5B) of MIA-PaCa2 cells treated with the control (Neg Ctrl), Mseq2 with Hi20Me modifications, Mseq2 2dT, and Mseq2 3dT for 144 hours at doses ranging from 20 nM to 0.01 nM. All Fireflyluciferase luminescence values were normalized with Renilla luciferase luminescence and expressed as a percentage. Error bars represent SEM. Relative potency for each siRNA is calculated as GI50 of Mseq2 -treated cells divided by GI50 of siRNA-treated cells.

[0018] FIGS. 6A-6C. Representative dose-response curves of KRAS-Firefly luciferase reporter in either wild-type (FIG. 6A) or G12V mutant (FIG. 6B) in A-431 KRAS-knockout cells treated with a control (Neg Ctrl), Kseq2 Hi20Me, V4-G12V Hi20Me (mutant KRAS-specific-targetingsiRNA), V4-G12V Hi20Me 2dT, or V4-G12V Hi20Me 3dT siRNAs for 72 hours at doses ranging from 40 nMto 0.018 nM. All Firefly-luciferase luminescence values were normalized with Renilla luciferase luminescence and expressed as a percentage. Error bars represent SEM. Relative potency (FIG. 6C) for each siRNA is calculated as GI50 of V4-G12V-treated cells divided by GI50 of siRNA-treated cells. Only relative potency values for the G12V-KRAS-luciferase-treated cells are shown.

[0019] FIGS. 7A-7B. Representative dose-response curves for MIA-PaCa2 (FIG. 7A) and A427 (FIG. 7B) cells treated for six days at doses ranging from 20 nM to 0.01 nM with a control siRNA (Neg Ctrl), Mseq2 Hi20Me, Mseq2 2dT, Mseq2 3dT, Mseq2 dTG (perfectly complementary to target mRNA by 2 nucleotides), or Mseq2 dTGA (perfectly complementary to target mRNA by 3 nucleotides). Error bars represent SEM.

[0020] FIG. 8. Representative dose-response curves of KRAS-Firefly luciferase expression in A- 431 KRAS-knockout cells treated with a control (Neg Ctrl), Kseq2 (KRAS-targeting siRNA) Hi20Me, Kseq2 2dT, Kseq2 3dT, Kseq2 dGT (perfectly complementary to target mRNA by 2 nucleotides), or Kseq2 dGTG (perfectly complementary to target mRNA by 3 nucleotides) siRNAs. All Firefly-luciferase luminescence values were normalized with Renilla luciferase luminescence and expressed as a percentage. Error bars represent SEM.

[0021] FIGS. 9A-9B. Tumor growth curves showing average fold change (FIG. 9A) and tumor volume (FIG. 9B) of subcutaneous H727 tumors over 7 days (n=10 for all treatment groups). Each group was treated with a ligand-conjugated negative control siRNA or a ligand-conjugated siRNA targeting KRAS G12V mutant with 3dT overhangs. Error bars represent SEM and one-tailed unpaired t-test was used for statistical comparisons. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

[0022] FIG. 10. Graph showing change in luminescence following treatment of subcutaneous A431 tumors containing a KRAS G12V-luciferase reporter with phosphate-buffered saline (PBS), ligand-conjugated KRAS G12V mutant-targeting siRNA, or ligand-conjugated KRAS G12V mutant-targeting siRNA with 3dT overhangs for 9 days (n=5-7 mice per treatment). Luminescence was measured at each time point following injection of 100 uL of D-Luciferin and normalized to tumor volume. Error bars represent SEM and two-tailed unpaired t-test was used for statistical comparison. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.DETAILED DESCRIPTION

[0023] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In addition, any references cited herein are incorporated by reference in their entireties.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0025] Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.831 and established usage.

[0026] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).

[0027] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0028] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0029] To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0030] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0031] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0032] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0033] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0034] The term “consists essentially of’ (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and / or C-terminal ends of the recited sequence or additional nucleotides on the 5' and / or 3' ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered. The total of ten or less additional amino acids or nucleotides includes the total number of additional amino acids or nucleotides on both ends added together. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activities / properties (e.g., chaperone and / or isomerase activity) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0035] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold.

[0036] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reductionresults in little or essentially no detectible activity (at most, an insignificant amount, e. ., less than about 10% or even 5%).

[0037] A “therapeutically effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a “therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g, in the case of cancer, reduction in tumor burden, prevention of further tumor growth, prevention of metastasis, or increase in survival time). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0038] “ Treat,” “treating,” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject; it is intended that the severity of the subject's condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered, e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0039] ‘ ‘Prevent” or “preventing” or “prevention” refer to prevention or delay of the onset of the disorder and / or a decrease in the severity of the disorder in a subject relative to the severity that would develop in the absence of the methods of the invention. The prevention can be complete, e.g., the total absence of cancer in a subject. The prevention can also be partial, such that the occurrence or severity of cancer in a subject is less than that which would have occurred without the present invention.

[0040] As used herein, “nucleic acid,” “nucleotide sequence,” and “polynucleotide" are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain. The nucleic acid can be double-stranded or single-stranded. Where singlestranded, the nucleic acid can be a sense strand or an antisense strand. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of this invention. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'- hydroxy in the ribose sugar group of the RNA can also be made.

[0041] An “isolated polynucleotide” is a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' noncoding (e.g., promoter) sequences that are immediately contiguous to a coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced byPCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence. An isolated polynucleotide that includes a gene is not a fragment of a chromosome that includes such gene, but rather includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the chromosome.

[0042] The term “isolated” can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and / or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an “isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. “Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.

[0043] An “isolated cell” refers to a cell that is separated from other components with which it is normally associated in its natural state. For example, an isolated cell can be a cell in culture medium and / or a cell in a pharmaceutically acceptable carrier of this invention. Thus, an isolated cell can be delivered to and / or introduced into a subject. In some embodiments, an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then returned to the subject.

[0044] The term “fragment,” as applied to a polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, and / or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.

[0045] The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence andcomprising, consisting essentially of, and / or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention.

[0046] A “vector” is any nucleic acid molecule for the cloning of and / or transfer of a nucleic acid into a cell. A vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence. A “replicon” can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both viral and nonviral (e.g, plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and / or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. For example, the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini. Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and / or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and / or selection of host cells that incorporate and express the proteins encoded by the marker. A “recombinant” vector refers to a viral or non-viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences.

[0047] Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, and / or adenovirus vectors. Non-viral vectors include, but are notlimited to, plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and / or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).

[0048] Vectors may be introduced into the desired cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, diethylaniinoethyl (DEAE)- dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu et al. (1992) J. Biol. Chem. 267:963; Wu et al. (1988) J. Biol. Chem. 263: 14621; Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0049] In some embodiments, a polynucleotide of this invention can be delivered to a cell in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a nucleotide sequence of this invention (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413; Mackey et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8027; Ulmer et al. (1993) Science 259: 1745). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner et al. (1989) Science 337:387). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95 / 18863 and WO 96 / 17823, and in U.S. Patent No. 5,459,127. The use of lipofection to introduce exogenous nucleotide sequences into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:8027). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules can be coupled to liposomes chemically.

[0050] In various embodiments, other molecules can be used for facilitating delivery of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO 95 / 21931), peptides derived from nucleic acid binding proteins (e.g., WO 96 / 25508), and / or a cationic polymer (e.g., WO 95 / 21931).

[0051] It is also possible to introduce a vector in vivo as naked nucleic acid (see U.S. Patent Nos. 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated nucleic acid delivery approaches can also be used (Curiel et al. (1992) Hum. Gene Ther. 3: 147; Wu et al. (1987) J. Biol. Chem. 262:4429).

[0052] As used herein, the terms “protein” and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0053] A “fusion protein” is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame. Illustrative fusion polypeptides include fusions of a polypeptide of the invention (or a fragment thereof) to all or a portion of glutathione-S-transferase, maltose-binding protein, or a reporter protein (e. ., Green Fluorescent Protein, [3-glucuronidase, 0-galactosidase, luciferase, etc.), hemagglutinin, c-Myc, FLAG® epitope, etc.

[0054] By the term “express” or “expression” of a polynucleotide coding sequence, it is meant that the sequence is transcribed, and optionally, translated. Typically, according to the present invention, expression of a coding sequence of the invention will result in production of the polypeptide of the invention. The entire expressed polypeptide or fragment can also function in intact cells without purification.

[0055] As used herein, the term “over-expression” or “over-expressing” refers to increased levels of a polypeptide being produced and / or increased time of expression (e.g., constitutively expressed) compared to a wild-type cell.

[0056] As used herein, the term “gene” refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and 5' and 3' untranslated regions). A gene may be “isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in chemically synthesizing the nucleic acid.

[0057] As used herein, “complementary” polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. For example, the sequence “A-G-T” binds to the complementary sequence “T-C-A ” It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.

[0058] The terms “complementary” or “complementarity,” as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity between two single-stranded molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the singlestranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0059] As used herein, the terms “substantially complementary” or “partially complementary” mean that two nucleic acid sequences are complementary at least about 60%, 70%, 80%, or 90% of their nucleotides. In some embodiments, the two nucleic acid sequences can be complementary at least at 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of their nucleotides. The terms “substantially complementary” and “partially complementary” can also mean that two nucleic acid sequences can hybridize under high stringency conditions and such conditions are well known in the art.

[0060] As used herein, “heterologous” refers to a nucleic acid sequence that either originates from another species or is from the same species or organism but is modified from either its original form or the form primarily expressed in the cell. Thus, a nucleotide sequence derived from an organism or species different from that of the cell into which the nucleotide sequence is introduced, is heterologous with respect to that cell and the cell’s descendants. In addition, a heterologous nucleotide sequence includes a nucleotide sequence derived from and inserted into the same natural, original cell type, but which is present in a non-natural state, e.g., a different copy number, and / or under the control of different regulatory sequences than that found in nature.

[0061] As used herein, the terms “contacting,” “introducing,” and “administering” are used interchangeably, and refer to a process by which dsRNA of the present invention or a nucleic acidmolecule encoding a dsRNA of this invention is delivered to a cell, in order to inhibit or alter or modify expression of a target gene. The dsRNA may be administered in a number of ways, including, but not limited to, direct introduction into a cell (i.e., intracellularly) and / or extracellular introduction into a cavity, interstitial space, or into the circulation of the organism.

[0062] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous (i.v.), subcutaneous (s.c.), transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. “Concurrent administration,” “administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.

[0063] “Introducing” in the context of a cell or organism means presenting the nucleic acid molecule to the organism and / or cell in such a manner that the nucleic acid molecule gains access to the interior of a cell. Where more than one nucleic acid molecule is to be introduced these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the sameor different nucleic acid constructs. Accordingly, these polynucleotides can be introduced into cells in a single transformation event or in separate transformation events. Thus, the terms “transformation” and “transfection” as used herein refer to the introduction of a heterologous nucleic acid into a cell. Transformation or transfection of a cell may be stable or transient.

[0064] “ Transient transformation” or “transient transfection” in the context of a polynucleotide means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.

[0065] By “stably introducing” or “stably introduced” in the context of a polynucleotide introduced into a cell, it is intended that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.

[0066] “Stable transformation” or “stable transfection” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell. As such, the integrated nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. “Genome” as used herein includes the nuclear and mitochondrial genome, and therefore includes integration of the nucleic acid into, for example, the mitochondrial genome. Stable transformation or stable transfection as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome.

[0067] Transient transformation may be detected by, for example, an enzyme-linked immunosorbent assay (ELISA) or western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism. Stable transformation of a cell can be detected by, for example, a northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0068] Embodiments of the invention are directed to expression cassettes designed to express the nucleic acids of the present invention. As used herein, “expression cassette” means a nucleic acid molecule having at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable interaction with the nucleotide sequences for the dsRNAs of the invention are provided in expression cassettes for expression in an organism or cell.

[0069] As used herein, the term “promoter” refers to a region of a nucleotide sequence that incorporates the necessary signals for the efficient expression of a coding sequence. This may include sequences to which an RNA polymerase binds but is not limited to such sequences and can include regions to which other regulatory proteins bind together with regions involved in the control of protein translation and can also include coding sequences.

[0070] Furthermore, a “promoter” of this invention is a promoter capable of initiating transcription in a cell of an organism. Such promoters include those that drive expression of a nucleotide sequence constitutively, those that drive expression when induced, and those that drive expression in a tissue- or developmentally-specific manner, as these various types of promoters are known in the art.

[0071] For purposes of the invention, the regulatory regions (z.c., promoters, transcriptional regulatory regions, and translational termination regions) can be native / analogous to the organism or cell and / or the regulatory regions can be native / analogous to the other regulatory regions. Alternatively, the regulatory regions may be heterologous to the organism or cell and / or to each other (i.e., the regulatory regions). Thus, for example, a promoter can be heterologous when it is operably linked to a polynucleotide from a species different from the species from which the polynucleotide was derived. Alternatively, a promoter can also be heterologous to a selected nucleotide sequence if the promoter is from the same / analogous species from which the polynucleotide is derived, but one or both (i.e., promoter and polynucleotide) are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

[0072] The choice of promoters to be used depends upon several factors, including, but not limited to, cell- or tissue-specific expression, desired expression level, efficiency, inducibility and selectability. For example, where expression in a specific tissue or organ is desired, a tissuespecific promoter can be used. In contrast, where expression in response to a stimulus is desired,an inducible promoter can be used. Where continuous expression is desired throughout the cells of an organism, a constitutive promoter can be used. It is a routine matter for one of skill in the art to modulate the expression of a nucleotide sequence by appropriately selecting and positioning promoters and other regulatory regions relative to that sequence.

[0073] In addition to the promoters described above, the expression cassette also can include other regulatory sequences. As used herein, “regulatory sequences” means nucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, enhancers, introns, translation leader sequences and polyadenylation signal sequences.

[0074] The expression cassette also can optionally include a transcriptional and / or translational termination region (z.e., termination region) that is functional in the organism. A variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the transgene and correct mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the host, or any combination thereof).

[0075] A sequence encoding a signal sequence can be operably linked to nucleic acids of the present invention to direct the nucleotide sequence into a cellular compartment. In this manner, the expression cassette will comprise a nucleotide sequence encoding the dsRNA operably linked to a nucleic acid sequence for the signal sequence. The signal sequence may be operably linked at the 5' or 3' end of the dsRNA.

[0076] Regardless of the type of regulatory sequence(s) used, they can be operably linked to the nucleotide sequence of the dsRNA. As used herein, “operably linked” means that elements of a nucleic acid construct such as an expression cassette are configured so as to perform their usual function. Thus, regulatory or control sequences (e.g., promoters) operably linked to a nucleotide sequence of interest are capable of effecting expression of the nucleotide sequence of interest. The control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promotersequence can still be considered “operably linked” to the coding sequence. A nucleotide sequence of the present invention (i.e., a dsRNA) can be operably linked to a regulatory sequence, thereby allowing its expression in a cell and / or subject.

[0077] The expression cassette also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed organism or cell. As used herein, “selectable marker” means a nucleic acid that when expressed imparts a distinct phenotype to the organism or cell expressing the marker and thus allows such transformed organisms or cells to be distinguished from those that do not have the marker. Such a nucleic acid may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening. Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0078] In some embodiments of the present invention, the expression cassette can comprise an expression control sequence operatively linked to a nucleotide sequence that is a template for one or both strands of the dsRNA. In further embodiments, a promoter can flank either end of the template nucleotide sequence, wherein the promoters drive expression of each individual DNA strand, thereby generating two complementary (or substantially complementary) RNAs that hybridize and form the dsRNA. In alternative embodiments, the nucleotide sequence is transcribed into both strands of the dsRNA on one transcription unit, wherein the sense strand is transcribed from the 5' end of the transcription unit and the antisense strand is transcribed from the 3' end, wherein the two strands are separated by about 3 to about 500 base pairs, and wherein after transcription, the RNA transcript folds on itself to form a short hairpin RNA (shRNA) molecule.

[0079] As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin & Griffin, eds.) Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology (von Heinje, ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov& Devereux, eds.) Stockton Press, New York (1991).

[0080] As used herein, the term “substantially identical” or “corresponding to” means that two nucleic acid sequences have at least 60%, 70%, 80% or 90% sequence identity. In some embodiments, the two nucleic acid sequences can have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity.

[0081] An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.

[0082] As used herein, the term “percent sequence identity” or “percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). In some embodiments, “percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.

[0083] Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, MA). Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this invention “percent identity” may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0084] The percent of sequence identity can be determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc, Madison, WI). “Gap” utilizes the algorithm of Needleman and Wunsch (Needleman & Wunsch(1970) J Mol. Biol. 48:443-453) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. “BestFit” performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith & Waterman (1981) Adv. Appl. Math. 2:482-489, Smith et al. (1983) Nucleic Acids Res. 11 :2205-2220).

[0085] Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers (Bishop, ed., Academic Press, San Diego (1994)), and Carillo & Lipton (1988) Applied Math 48: 1073). More particularly, preferred computer programs for determining sequence identity include but are not limited to the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, MD; see BLAST Manual (Altschul et al. (1990) J. Mol. Biol. 215:403-410); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and, for polynucleotide sequence BLASTN can be used to determine sequence identity.

[0086] As used herein, “RNAi” or “RNA interference” refers to the process of sequence-specific post-transcriptional gene silencing, mediated by double- stranded RNA (dsRNA). As used herein, “dsRNA” refers to RNA that is partially or completely double-stranded. Double-stranded RNA is also referred to as small interfering RNA (siRNA), small interfering nucleic acid (siNA), microRNA (miRNA), and the like. In the RNAi process, dsRNA comprising a first (antisense) strand that is complementary to a portion of a target gene and a second (sense) strand that is fully or partially complementary to the first antisense strand is introduced into an organism. After introduction into the organism, the target gene-specific dsRNA is processed into relatively small fragments (siRNAs) and can subsequently become distributed throughout the organism, leading to a phenotype that, over the period of a generation, may come to closely resemble the phenotype arising from a complete or partial deletion of the target gene.

[0087] MicroRNAs (miRNAs) are non -protein coding RNAs, generally of between about 18 to about 25 nucleotides in length. These miRNAs direct cleavage in trans of target transcripts, negatively regulating the expression of genes involved in various regulation and development pathways (Bartel (2004) Cell 116:281-297; Zhang et al. (2006) Dev. Biol. 289:3-16). As such, miRNAs have been shown to be involved in different aspects of growth and development as wellas in signal transduction and protein degradation. Since the first miRNAs were discovered in plants (Reinhart et al. (2002) Genes Dev. 16: 1616-1626, Park et al. (2002) Curr. Biol. 12:1484-1495) many hundreds have been identified. Many microRNA genes (MIR genes) have been identified and made publicly available in a database (miRBase; microrna.sanger.ac.uk / sequences). miRNAs are also described in U.S. Patent Publications 2005 / 0120415 and 2005 / 144669A1, the entire contents of which are incorporated by reference herein.

[0088] Genes encoding miRNAs yield primary miRNAs (termed a “pri-miRNA”) of 70 to 300 bp in length that can form imperfect stem-loop structures. A single pri-miRNA may contain from one to several miRNA precursors. In animals, pri-miRNAs are processed in the nucleus into shorter hairpin RNAs of about 65 nt (pre-miRNAs) by the RNaselll enzyme Drosha and its cofactor DGCR8 / Pasha. The pre-miRNA is then exported to the cytoplasm, where it is further processed by another RNaselll enzyme, Dicer, releasing a miRNA / miRNA* duplex of about 22 nt in size. Many reviews on microRNA biogenesis and function are available, for example, see, Bartel (2004) Cell 116:281-297, Murchison et al. (2004) Curr. Opin. Cell Biol. 16:223-229, Dugas et al. (2004) Curr. Opin. Plant Biol. 7:512-520, and Kim (2005) Nature Rev. Mol. Cell Biol. 6:376-385.

[0089] A “subject” may be any vertebrate organism in various embodiments. A subject may be an individual to whom an agent is administered, e.g., for experimental, diagnostic, and / or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal, e.g., a human, non-human primate, lagomorph (e.g., rabbit), or rodent (e.g., mouse, rat). In some embodiments a human subject is a neonate, child, adult or geriatric subject. In some embodiments a human subject is at least 50, 60, 70, 80, or 90 years old.RNA Molecules

[0090] The invention relates to a RNA molecule that is a dsRNA molecule, e.g., a siRNA molecule, comprising a sense strand and an antisense strand with a nucleotide overhang of two or more deoxythymidines (dTs) on the 5' end of the antisense strand. dsRNA with a nucleotide overhang of two or more dTs on the 5' end of the antisense strand is surprisingly effective at inhibiting gene expression and is unexpectedly far more potent against target genes relative to dsRNAs without a 5' antisense modification of two or more dTs. The modified dsRNA advantageously provides a modification that allows tailoring of dsRNAs designed for inhibiting expression of a broad range of target genes to achieve enhanced RNA interference activity.

[0091] The dsRNA of the present invention can comprise an overhang of two or more dTs on the 5' end of the antisense strand, for example, 2 dTs, 3 dTs, 4 dTs, 5 dTs, 6 dTs, 7 dTs, or 8 dTs. In some embodiments, the 5' end of the antisense strand of the dsRNA is modified to comprise between 2 dTs and 8 dTs, between 2 dTs and 6 dTs, between 2 dTs and 4 dTs, or 3 dTs or 4 dTs.

[0092] The dsRNA may have an overall length of about 14 to 40 nucleotides, e.g., about 14 to 35 nucleotides, e.g., about 30 to 40 nucleotides, e.g., about 17 to 25 nucleotides, e.g., about 19 to 23 nucleotides, e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides or any value or range therein.

[0093] One aspect of the invention relates to a dsRNA molecule of the invention, wherein the molecule comprises as least one chemical modification. In some embodiments, the dsRNA comprises 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides on each strand modified with a 2'-fluoro group. In some embodiments, the dsRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more nucleotides on each strand modified with a 2'-O-methyl group. In some embodiments, the molecule is fully chemically modified. The term “fully chemically-modified” means that every nucleotide in the molecule contains a chemical modification. In some embodiments, each nucleotide in the molecule is modified with a 2'-O-methyl group or a 2'-fluoro group. In some embodiments, a modified dsRNA with 20%, 30%, 40%, 50%, 60%, or 70% or more of the nucleotides with 2'-O-methyl modifications (Hi2OMe), may confer improvements in metabolic stability within the endosomal compartment.

[0094] In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 of the nucleotide linkages in the dsRNA molecule are chemically modified. In some embodiments, the dsRNA molecule comprises at least one phosphorothioate linkage. In some embodiments, the sense strand and / or the antisense strand comprise two or more phosphorothioate modifications. In some embodiments, the dsRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 phosphorothioate linkages. In some embodiments, a phosphorothioate linkage is placed between the last 2 nucleotides of each strand of the molecule on the 3' end, the 5' end, or both the 3' end and the 5' end of both the sense strand and the antisense strand. In embodiments, the phosphorothioate modifications are provided on the nucleotide linkages of the dsRNA on the 5' end of the antisense strand but not the dT overhangs.

[0095] A dsRNA of the present invention is capable of binding to a mRNA of a target gene and inhibiting expression of that target gene. The target gene may be any gene of interest for which inhibition of expression is desired. In some embodiments, the target gene is one being studied for research purposes. In other embodiments, the target gene is one that is over-expressed in a disease state or otherwise associated with a disease state, and inhibition of expression is for therapeutic or prophylactic purposes. In some embodiments, the target gene is one that is expressed by a pathogen, e.g., a bacteria, fungus, parasite, or virus, e.g., a coronavirus such as SARS-CoV-2. In some embodiments, the target gene is one over-expressed in or associated with a cancer. In a particular embodiment, the dsRNA targets c-Myc. In one embodiment, the dsRNA targets KRAS; in some embodiments, the dsRNA is specific for a KRAS comprising a G12 mutant, optionally a G12V mutant.

[0096] In particular embodiments, the present invention provides dsRNA containing a nucleotide sequence that is fully complementary to a region of the target gene for inhibition. However, it is to be understood that 100% complementarity between the antisense strand of the dsRNA molecule and the target sequence is not required to practice the present invention. Thus, sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence may also be effective for inhibition.

[0097] In some embodiments, the dsRNA may be covalently or non-covalently bound to another molecule, e.g., a linker (e.g., for binding to a carrier) or a ligand (e.g., for targeting the molecule to a specific cell or binding site).

[0098] In some embodiments, the dsRNA molecule comprises, consists essentially of, or consists of a sequence at least 90% identical to any one of SEQ ID NOS:3, 4, 7, 8, 9, 10, 13, 14, 17 or 18, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

[0099] One aspect of the invention relates to a dsRNA, e.g., an dsRNA molecule, comprising an antisense strand and a sense strand, wherein the nucleotide sequence of the antisense strand is complementary to a region of a nucleotide sequence of a c-Myc gene, the region consisting essentially of about 18 to about 25 consecutive nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, 25 consecutive nucleotides, or any value or range therein); wherein the dsRNA molecule inhibits expression of a c-Myc gene. The dsRNA molecules provide decreased expression of c-Myc in a cell as compared to cells without the RNA molecules (e.g., a control cell or nontransformed cell).The dsRNA molecules comprising two or more dTs on the 5' end of the antisense strand provide decreased expression of c-Myc in a cell as compared to cells with dsRNA molecules without an overhang of two or more dTs on the 5' end of the antisense strand. In some embodiments, expression of c-Myc is inhibited by 1.5-fold, 2-fold, or 3-fold, or more relative to an dsRNA without two or more dTs on the 5' end of the antisense strand.

[0100] One aspect of the invention relates to a dsRNA, e.g., an siRNA molecule, comprising an antisense strand and a sense strand, wherein the nucleotide sequence of the antisense strand is complementary to a region of a nucleotide sequence of a KRAS gene, the region consisting essentially of about 18 to about 25 consecutive nucleotides (e.g, 18, 19, 20, 21, 22, 23, 24, 25 consecutive nucleotides, or any value or range therein); wherein the dsRNA molecule inhibits expression of a KRAS gene. The dsRNA provides decreased expression of KRAS in a cell as compared to cells without the RNA molecules (e.g., a control cell or nontransformed cell). The dsRNA molecules comprising two or more dTs on the 5' end of the antisense strand provide decreased expression of KRAS in a cell as compared to cells with dsRNA molecules without an overhang of two or more dTs on the 5' end of the antisense strand. In some embodiments, expression of KRAS is inhibited by 1.5-fold, 2-fold, or 3-fold, or more relative to an dsRNA without two or more dTs on the 5' end of the antisense strand. In some embodiments, the dsRNA comprising a G12 (Gly 12), e.g., G12V (Gly 12 to Vai substitution), mutant-specific KRAS antisense strand with two or more dTs on the 5' end has increased potency in a dose-dependent manner while maintaining wild-type KRAS sparing relative to dsRNA without two or more dTs on the 5' end of the antisense strand.

[0101] In some embodiments, the nucleotide sequence of the sense strand comprises a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of any of SEQ ID NOS:1, 5, 11, or 15, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or identical to the nucleotide sequence of any of SEQ ID NOS:1, 5, 11, or 15. In some embodiments, the nucleotide sequence of the sense strand comprises, consists essentially of, or consists of the nucleotide sequence of any of SEQ ID NOS:1, 5, 11, or 15.

[0102] In some embodiments, the nucleotide sequence of the antisense strand comprises a nucleotide sequence that is at least about 80% identical to the nucleotide sequence of any of SEQ ID NOS:3, 4, 7, 8, 9, 10, 13, 14, 17 or 18, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to the nucleotide sequence of any of SEQ IDNOS:3, 4, 7, 8, 9, 10, 13, 14, 17 or 18 In some embodiments, the nucleotide sequence of the antisense strand comprises, consists essentially of, or consists of the nucleotide sequence of any of SEQ ID NOS:3, 4, 7, 8, 9, 10, 13, 14, 17 or 18

[0103] In some embodiments, a dsRNA of the invention comprises a sense strand and an antisense strand, wherein the dsRNA molecule comprises one of the following pairs of sequences: sense strand of SEQ ID NO:1 and antisense strand of SEQ ID NO:3; sense strand of SEQ ID NO:1 and antisense strand of SEQ ID NO:4 ; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NO:6 ; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NO :7; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NO:8; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NO:9; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NQ:10; sense strand of SEQ ID NO: 11 and antisense strand of SEQ ID NO: 12; sense strand of SEQ ID NO: 11 and antisense strand of SEQ ID NO: 13; sense strand of SEQ ID NO: 11 and antisense strand of SEQ ID NO: 14; sense strand of SEQ ID NO: 15 and antisense strand of SEQ ID NO: 16; sense strand of SEQ ID NO: 15 and antisense strand of SEQ ID NO: 17; or sense strand of SEQ ID NO:15 and antisense strand of SEQ ID NO:18.

[0104] In some embodiments of this invention, the sense strand of the dsRNA molecule can be fully complementary to the antisense strand or the sense strand can be substantially complementary or partially complementary to the antisense strand. By substantially or partially complementary is meant that the sense strand and the antisense strand can be mismatched at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide pairings. Such mismatches can be introduced into the sense strand sequence, e.g., near the 3' end, to enhance processing of the dsRNA molecule by Dicer, to duplicate a pattern of mismatches in a dsRNA molecule inserted into a chimeric nucleic acid molecule or artificial microRNA precursor molecule of this invention, and the like, as would be known to one of skill in the art. Such modification will weaken the base pairing at one end of the duplex and generate strand asymmetry, therefore enhancing the chance of the antisense strand, instead of the sense strand, being processed and silencing the intended gene (Geng & Ding (2008) Acta Pharmacol. Sin. 29:211-216; Schwarz et al. (2003) Cell 115:199-208).

[0105] The dsRNA molecule of the invention may be in the form of any type of RNA interference molecule known in the art. In some embodiments, the dsRNA molecule is a small interfering RNA (siRNA) molecule. In other embodiments, the dsRNA molecule is a short hairpin RNA (shRNA) molecule. In other embodiments, the dsRNA molecule is part of a microRNA precursor molecule.

[0106] Exemplary methods of making dsRNA molecules are described in International Publication No. WO 2022 / 271624, in particular at paragraphs

[0153] -

[0154] , incorporated herein by reference.

[0107] The invention further relates to a nucleic acid construct comprising the RNA molecule of the invention. The invention further relates to a nucleic acid construct encoding the RNA molecule of the invention and a nucleic acid construct comprising the nucleic acid molecule encoding the RNA molecule. In each of these embodiments, the nucleic acid construct may be a vector or a plasmid, e.g., an expression vector.

[0108] An embodiment of the invention relates to a composition comprising the dsRNA molecule or nucleic acid construct of the invention and other components, .e.g., a suitable carrier. In some embodiments, the composition comprises two or more of the dsRNAs, or nucleic acid constructs of the invention, wherein each comprise a different antisense strand, each of which comprises an overhang of two or more dTs on the 5' end of the antisense strand. In some embodiments, the composition is a pharmaceutical composition comprising the dsRNA, or nucleic acid construct of the invention and a pharmaceutically acceptable carrier.

[0109] It is understood that the compositions of this invention can comprise, consist essentially of, or consist of any of the dsRNAs or nucleic acid constructs of the invention in any combination and in any ratio relative to one another. In some aspects of the invention, the composition or pharmaceutical composition further comprises additional components that enhance the delivery of the dsRNA e.g., by enhancing the stability of the dsRNA.

[0110] In some embodiments, the composition comprises an additional component that may be a particle, e.g., a microparticle or nanoparticle. In some embodiments, the particle is a lipid particle, e.g., a liposome, e.g., a microliposome or a nanoliposome. The liposome, microliposome, or nanoliposome may contain any components known in the art to be suitable for preparing liposomes. In some embodiments, the liposome comprises l,2-dioleoyl-sn-glycero-3- phosphatidylcholine (DOPC). Liposomes may be prepared by methods known in the art, e.g., as described in Peco et al. (2014) Mol. Cancer Ther. 13:2876, incorporated by reference herein in its entirety. In some embodiments, the RNA molecule is formed into a stable nucleic acid lipid particle (SNALP), e.g., using particles such as those provided by Arbutus Biopharma (Doylestown, PA). In certain embodiments, the lipid particle comprises, consists essentially of, or consists of cholesterol, l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), PEG-cDMA (3- [(methoxypoly(ethylene glycol)2000)carbamoyl]-l ,2-dimyrestyloxypropylamine) or PEG-cDSA (3- / V-(- rn ethoxy polyethylene glycol)2000)carbamoyl-l,2-distearyloxy-propylamine), and DLinDMA ( l ,2-dilinoleyloxy-3-( / V, / V-dimethyl)aminopropane) (see Judge et al. (2009) J. Clin. Invest. 119:661). In some embodiments, the lipid particle comprises two or more of the dsRNA of the present invention. In some embodiments, the additional component is a targeted delivery moiety to which the dsRNA molecule(s), or nucleic acid construct(s) are covalently or noncovalently conjugated, e.g., ligands, aptamers, or monoclonal antibodies.[0U1] The present invention encompasses cells comprising the RNA molecules and / or nucleic acid constructs of the invention. Thus, in some embodiments, the present invention provides a transformed cell comprising a RNA molecule and / or a nucleic acid construct and / or a composition of this invention, wherein the transformed cell has reduced expression of the target gene as compared to a control cell.Methods

[0112] Various methods are provided herein, employing the nucleic acid molecules, nucleic acid constructs, and / or compositions of this invention. Thus, in an embodiment, the present invention provides a method of reducing expression of a target gene in a cell, the method comprising contacting the cell with the dsRNA, nucleic acid construct, composition, and / or pharmaceutical composition of the invention, thereby reducing the expression of the target gene in the cell. In an aspect, the dsRNA reduces expression of the target gene more than a dsRNA without two or more dTs on 5' end of its antisense strand.

[0113] The present invention further provides a method of delivering dsRNA to a target in a subject, comprising administering the dsRNA, nucleic acid construct, composition, and / or pharmaceutical composition of the invention to the subject. In some embodiments, the subject has a disease or disorder, for example cancer.

[0114] Also provided herein is a method of treating cancer in a subject in need thereof, comprising administering the dsRNA, nucleic acid construct, composition, and / or pharmaceutical composition of the invention to the subject, thereby treating the cancer in the subject. In some embodiments, the dsRNA is specific for an oncogene or other gene associated with cancer. In some embodiments, the dsRNA is specific for a KRAS gene or a c-Myc gene. In an embodiment, the dsRNA is specific for a KRAS comprising a G12 mutation, optionally G12V. Accordingly, a cancer comprising overexpression of a human c-Myc gene (a cancer, e.g. , a tumor, in which one or more cells over-expressthe c-Myc gene) is an exemplary cancer to be treated. Cancers comprising a KRASG12are also cancers to be treated by the dsRNA molecules and compositions of the present invention.

[0115] In one embodiment of each method, the subject may be one that has been diagnosed with a disorder, e.g., cancer. In another embodiment, the subject may be one that is at risk of developing a disorder, e.g., cancer (e.g., predisposed due to hereditary factors, smoking, viral infection, exposure to carcinogens, etc.). In a further embodiment, the subject may be one that is infected with a pathogen. In a further embodiment, the subject may be one that has been identified as overexpressing a target gene.

[0116] The dsRNA, nucleic acid construct, composition, and / or pharmaceutical composition of the invention can be delivered directly into a cell by any method known in the art, e.g., by transfection or microinjection, e.g., as part of a composition comprising lipid particles. In other embodiments, the dsRNA can be delivered to a subject in the form of polynucleotides encoding the RNA to provide expression of the dsRNA within the cells of the subject. Those skilled in the art will appreciate that the isolated polynucleotides encoding the RNAs of the invention will typically be associated with appropriate expression control sequences, e.g., transcription / translation control signals and polyadenylation signals.

[0117] It will further be appreciated that a variety of promoter / enhancer elements can be used depending on the level and tissue-specific expression desired. The promoter can be constitutive or inducible, depending on the pattern of expression desired. The promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced. The promoter is chosen so that it will function in the target cell(s) of interest.

[0118] To illustrate, the RNA coding sequence can be operatively associated with a cytomegalovirus (CMV) major immediate-early promoter, an albumin promoter, an Elongation Factor 1-oc (EFl-a) promoter, a phosphoglycerate kinase 1 (PGK) promoter, a retroviral MFG promoter, or a Rous sarcoma virus (RSV) promoter.

[0119] Inducible promoter / enhancer elements include hormone-inducible and metal -inducible elements, and other promoters regulated by exogenously supplied compounds, including without limitation, the zinc-inducible metallothionein (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (see WO 98 / 10088); the ecdysone insect promoter (No et al. (1996) Proc. Natl. Acad. Set. USA 93:3346);the tetracycline-repressible system (Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547); the tetracycline-inducible system (Gossen etal. (1995) Science 268: 1766; see also Harvey etal. (1998) Curr. Opin. Chem. Biol. 2:512); the RU486-inducible system (Wang et al. (1997) Nat. Biotech. 15:239; Wang et al. (1997) Gene Ther. 4:432); and the rapamycin-inducible system (Magari et al. (1997) Clin. Invest. 100:2865).

[0120] Other tissue-specific promoters or regulatory promoters include, but are not limited to, promoters that typically confer tissue-specificity in neurons. These include, but are not limited to, promoters for synapsin 1, tubulin al, platelet-derived growth factor B -chain, tyrosine hydroxylase, neuron-specific enolase, and neurofilaments. Skeletal muscle cell promoters include, but are not limited to, promoters for [Lactin, Pituitary homeobox 3 (Pitx3), creatine kinase, and myosin light chain. Cardiac muscle cell promoters include, but are not limited to, promoters for cardiac actin, cardiac troponin T, troponin C, myosin light chain-2, and a-myosin heavy chain. Islet (beta) cell promoters include, but are not limited to, glucokinase, gastrin, insulin, and islet amyloid polypeptide.

[0121] Moreover, specific initiation signals are generally required for efficient translation of inserted RNA coding sequences. These translational control sequences, which can include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

[0122] The isolated nucleic acid encoding the dsRNA can be incorporated into an expression vector. Expression vectors compatible with various host cells are well known in the art and contain suitable elements fortranscription and translation of nucleic acids. Typically, an expression vector contains an “expression cassette,” which includes, in the 5' to 3' direction, a promoter, a coding sequence encoding a dsRNA operatively associated with the promoter, and, optionally, a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylase.

[0123] Non-limiting examples of animal and mammalian promoters known in the art include, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the El A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EFl) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin(Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, a-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), and pathogenesis and / or disease-related promoters. In addition, any of these expression sequences of this invention can be modified by addition of enhancer and / or regulatory sequences and the like.

[0124] Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor I (EFl) enhancer, yeast enhancers, viral gene enhancers, and the like.

[0125] Termination control regions, i.e., terminator or polyadenylation sequences, may be derived from various genes native to the preferred hosts. In some embodiments of the invention, the termination control region may comprise or be derived from a synthetic sequence, a synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.

[0126] It will be apparent to those skilled in the art that any suitable vector can be used to deliver the polynucleotide to a cell or subject. The vector can be delivered to cells in vivo. In other embodiments, the vector can be delivered to cells ex vivo, and then cells containing the vector are delivered to the subject. The choice of delivery vector can be made based on a number of factors known in the art, including age and species of the target host, in vitro versus in vivo delivery, level and persistence of expression desired, intended purpose (e.g., for therapy or screening), the target cell or organ, route of delivery, size of the isolated polynucleotide, safety concerns, and the like.

[0127] Suitable vectors include, but are not limited to, plasmid vectors, viral vectors (e.g., retrovirus, alphavirus, vaccinia virus, adenovirus, adeno-associated virus and other parvoviruses, lentivirus, poxvirus, or herpes simplex virus), lipid vectors, poly-lysine vectors, synthetic polyamino polymer vectors, and the like.

[0128] Any viral vector that is known in the art can be used in the present invention. Protocols for producing recombinant viral vectors and for using viral vectors for nucleic acid delivery can be found in Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York) and other standard laboratory manuals (e.g., Vectors for Gene Therapy. In: Current Protocols in Human Genetics. John Wiley and Sons, Inc., 1997).

[0129] Non-viral transfer methods can also be employed. Many non-viral methods of nucleic acid transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In particular embodiments, non-viral nucleic acid delivery systems rely on endocytic pathways for the uptake of the nucleic acid molecule by the targeted cell. Exemplary nucleic acid delivery systems of this type include liposomal-derived systems, polylysine conjugates, and artificial viral envelopes.

[0130] In particular embodiments, plasmid vectors are used in the practice of the present invention. For example, naked plasmids can be introduced into muscle cells by injection into the tissue. Expression can extend over many months, although the number of positive cells is typically low (Wolff et al. (1989) Science 247:247). Cationic lipids have been demonstrated to aid in introduction of nucleic acids into some cells in culture (Feigner & Ringold (1989) Nature 337:387). Injection of cationic lipid plasmid DNA complexes into the circulation of mice has been shown to result in expression of the DNA in lung (Brigham et al. (1989) Am. J. Med. Sci. 298:278). One advantage of plasmid DNA is that it can be introduced into non-replicating cells.

[0131] In a representative embodiment, a nucleic acid molecule (e.g., a plasmid) can be entrapped in a lipid particle bearing positive charges on its surface and, optionally, tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547; PCT publication WO 91 / 06309; Japanese patent application 1047381; European patent publication EP-A-43075).

[0132] Liposomes that consist of amphiphilic cationic molecules are useful as non-viral vectors for nucleic acid delivery in vitro and in vivo (reviewed in Crystal (1995) Science 270:404; Blaese et al. (1995) Cancer Gene Ther. 2:291; Behr et al. (1994) Bioconjugate Chem. 5:382; Remy et al. (1994) Bioconjugate Chem. 5:647; Gao et al. (1995) Gene Therapy 2:710). The positively charged liposomes are believed to complex with negatively charged nucleic acids via electrostatic interactions to form lipidmucleic acid complexes. The lipidmucleic acid complexes have several advantages as nucleic acid transfer vectors. Unlike viral vectors, the lipidmucleic acid complexes can be used to transfer expression cassettes of essentially unlimited size. Since the complexes lack proteins, they can evoke fewer immunogenic and inflammatory responses. Moreover, they cannot replicate or recombine to form an infectious agent and have low integration frequency. A number of publications have demonstrated that amphiphilic cationic lipids can mediate nucleic aciddelivery in vivo and in vitro (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413; Loeffler et al. (1993) Meth. Enzymol. 217:599; Feigner et al. (1994) J. Biol. Chem. 269:2550).

[0133] Several groups have reported the use of amphiphilic cationic lipidmucleic acid complexes for in vivo transfection both in animals and in humans (reviewed in Gao et al. (1995) Gene Therapy 2:710; Zhu et al. (1993) Science 261 :209; Thierry et al. (1995) Proc. Natl. Acad. Sci. USA 92:9742). U.S. Patent No. 6,410,049 describes a method of preparing cationic lipidmucleic acid complexes that have a prolonged shelf-life.

[0134] Nuclear localization signals (NLSs) can also be used to enhance the targeting of the dsRNA or expression vector into the proximity of the nucleus and / or its entry into the nucleus. Such nuclear localization signals can be protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS. These nuclear localization signals interact with a variety of nuclear transport factors such as the NLS receptor (karyopherin alpha) which then interacts with karyopherin beta. An exemplary application comprises use of NLS peptides on the surface of a nanoparticle allowing for localization of the dsRNA.

[0135] Expression vectors can be designed for expression of dsRNA in prokaryotic or eukaryotic cells. For example, dsRNA can be expressed in bacterial cells such as E. coli, insect cells (e.g., the baculovirus expression system), yeast cells, plant cells or mammalian cells. Some suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Examples of bacterial vectors include, but are not limited to, pQE70, pQE60, pQE-9 (Qiagen), pBS, pDlO, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia). Examples of vectors for expression in the yeast S. cerevisiae include pYepSecl (Baldari et al. (1987) £ALBO J. 6:229), pMFa (Kurjan & Herskowitz (1982) Cell 30:933), pJRY88 (Schultz et al. (1987) Gene 54: 113), and pYES2 (Invitrogen Corporation, San Diego, CA). Nonlimiting examples of baculovirus vectors available for expression of nucleic acids to produce proteins in cultured insect cells e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell. Biol. 3:2156) and the pVL series (Lucklow & Summers (1989) Virology 170:31).

[0136] Examples of mammalian expression vectors include pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187). When used in mammalian cells, the expression vector’s control functions are often provided by viral regulatory elements. Forexample, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.

[0137] Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, adenovirus, geminivirus, and caulimovirus vectors. Non-limiting examples of non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and / or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).

[0138] In addition to the regulatory control sequences discussed above, the recombinant expression vector can contain additional nucleotide sequences. For example, the recombinant expression vector can encode a selectable marker gene to identify host cells that have incorporated the vector.

[0139] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acids (e.g., DNA and RNA) into a host cell, including, but are not limited to, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine™-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989), and other laboratory manuals.

[0140] If stable integration is desired, often only a small fraction of cells (in particular, mammalian cells) integrate the foreign DNA into their genome. In order to identify and select integrants, a nucleic acid that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the nucleic acid of interest. Preferred selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as thatcomprising the nucleic acid of interest or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0141] The dsRNA molecule of the invention is administered directly to the subject. Generally, the compounds of the invention will be suspended in a pharmaceutically-acceptable carrier (e.g., physiological saline) and administered orally, topically, or by intravenous infusion, or injected subcutaneously, intramuscularly, intracranially, intrathecally, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily. They are preferably delivered directly to the site of the disease or disorder, such as the lung, intestine, or pancreas. The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Suitable dosages are in the range of 0.01 pg / kg to 100.0 pg / kg (e.g., 0.01, 0.05, 0.1, 0.5, or 1 to 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 pg / kg, or any value or range therein). Wide variations in the needed dosage are to be expected in view of the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection (e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150- or more fold). Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple. Encapsulation of the inhibitor in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.

[0142] According to certain embodiments, the dsRNA, can be targeted to specific cells or tissues in vivo. Targeting delivery vehicles, including liposomes and viral vector systems are known in the art. For example, a liposome can be directed to a particular target cell or tissue by using a targeting agent, such as an antibody, soluble receptor or ligand, incorporated with the liposome, to target a particular cell or tissue to which the targeting molecule can bind. Targeting liposomes are described, for example, in Ho et al. (1986) Biochemistry 25:5500; Ho et al. (1987) J. Biol. Chem. 262: 13979; Ho et al. (1987) J. Biol. Chem. 262: 13973; and U.S. Patent No. 4,957,735 to Huang et al., each of which is incorporated herein by reference in its entirety). Enveloped viral vectors can be modified to deliver a nucleic acid molecule to a target cell by modifying or substituting an envelope protein such that the virus infects a specific cell type. In adenoviral vectors, the geneencoding the attachment fibers can be modified to encode a protein domain that binds to a cellspecific receptor. Herpesvirus vectors naturally target the cells of the central and peripheral nervous system. Alternatively, the route of administration can be used to target a specific cell or tissue. For example, intracoronary administration of an adenoviral vector has been shown to be effective for the delivery of a gene to cardiac myocytes (Maurice et al. (1999) J. Clin. Invest. 104:21). Intravenous delivery of cholesterol-containing cationic liposomes has been shown to preferentially target pulmonary tissues (Liu et al. (1997) Nature Biotechnol. 15: 167), and effectively mediate transfer and expression of genes in vivo. Other examples of successful targeted in vivo delivery of nucleic acid molecules are known in the art. Finally, a recombinant nucleic acid molecule can be selectively (i.e., preferentially, substantially exclusively) expressed in a target cell by selecting a transcription control sequence, and preferably, a promoter, which is selectively induced in the target cell and remains substantially inactive in non-target cells.

[0143] The dsRNA of the present invention can optionally be delivered in conjunction with other therapeutic agents. The additional therapeutic agents can be delivered concurrently with the dsRNA of the invention. As used herein, the word “concurrently” means sufficiently close in time to produce a combined effect (that is, concurrently can be simultaneously, or it can be two or more events occurring within a short time period before or after each other). In one embodiment, the dsRNA, molecule of the invention are administered in conjunction with agents useful for treating cancer, such as: (1) vinca alkaloids (e.g., vinblastine, vincristine); (2) epipodophyllotoxins (e.g., etoposide and teniposide); (3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); (4) enzymes (e.g., L-asparaginase); (5) biological response modifiers (e.g., interferon-alfa); (6) platinum coordinating complexes (e.g., cisplatin and carboplatin); (7) anthracenediones (e.g., mitoxantrone); (8) substituted ureas (e.g., hydroxyurea); (9) methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MIH)); (10) adrenocortical suppressants (e.g., mitotane (o,p'-DDD) and aminoglutethimide); (11) adrenocorticosteroids (e.g., prednisone); (12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); (13) estrogens (e.g., diethyl stilbestrol and ethinyl estradiol); (14) antiestrogens (e.g., tamoxifen); (15) androgens (e.g., testosterone propionate and fluoxymesterone); (16) antiandrogens (e.g, flutamide): and (17) gonadotropin-releasing hormone analogs (e.g., leuprolide). In another embodiment, the compounds of the invention areadministered in conjunction with anti-angiogenesis agents, such as antibodies to vascular endothelial growth factor (VEGF) (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS)) and other promoters of angiogenesis e.g., bFGF, angiopoietin-1), antibodies to alpha-v / beta-3 vascular integrin (e.g., VITAXIN), angiostatin, endostatin, dalteparin, ABT-510 (CAS No. 251579-55-2), CNGRC peptide TNF alpha conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation (Abraxane), soy isoflavone (Genistein), tamoxifen citrate, thalidomide, ADH-1 (EXHERIN), AG-013736 (CAS No. 319460-85-0), AMG- 706 (CAS No. 453562-69-1), AZD2171 (CAS No. 288383-20-0), sorafenib tosylate, BMS-582664 (CAS No. 649735-63-7), CHIR-265 (CAS No. 927880-90-8), pazopanib, PI-88 (CAS No. 1020105-89-8), vatalanib, everolimus, suramin, sunitinib malate, XL184 (CAS No. 849217-68-1), ZD6474 (CAS No. 443913-73-3), ATN-161 (CAS No. 262438-43-7), cilenigtide, and celecoxib, or any combination thereof.

[0144] The term “cancer,” as used herein, refers to any benign or malignant abnormal growth of cells. Examples include, without limitation, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma. In some embodiments, the cancer is selected from the group of tumor-forming cancers.Pharmaceutical compositions

[0145] As a further aspect, the invention provides pharmaceutical formulations and methods of administering the same to achieve any of the therapeutic effects (e.g., treatment of cancer) discussed above. The pharmaceutical formulation may comprise any of the reagents discussed above in a pharmaceutically acceptable carrier.

[0146] By “pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects such as toxicity.

[0147] The formulations of the invention can optionally comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.

[0148] The dsRNA of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA). In the manufacture of a pharmaceutical formulation according to the invention, the dsRNA (including the physiologically acceptable salts thereof) is typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and is preferably formulated with the dsRNA as a unit-dose formulation, for example, a tablet, which can contain from 0.01% or 0.5% to 95% or 99% by weight of the dsRNA. One or more dsRNAs can be incorporated in the formulations of the invention, which can be prepared by any of the well-known techniques of pharmacy.

[0149] A further aspect of the invention is a method of treating subjects in vivo, comprising administering to a subject a pharmaceutical composition comprising a dsRNA of the invention in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount. Administration of the dsRNA of the present invention to a human subject or an animal in need thereof can be by any means known in the art for administering compounds.

[0150] Non-limiting examples of formulations of the invention include those suitable for oral, rectal, buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular including skeletal muscle, cardiac muscle, diaphragm muscle and smooth muscle, intradermal, intravenous, intraperitoneal), topical (i.e., both skin and mucosal surfaces, including airway surfaces), intranasal, transdermal, intraarticular, intracranial, intrathecal, and inhalation administration, administration to the liver by intraportal delivery, as well as direct organ injection (e.g., into the liver, into a limb, into the brain or spinal cord for delivery to the central nervous system, into thepancreas, or into a tumor or the tissue surrounding a tumor). The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular compound which is being used. In some embodiments, it may be desirable to deliver the formulation locally to avoid any side effects associated with systemic administration. For example, local administration can be accomplished by direct injection at the desired treatment site, by introduction intravenously at a site near a desired treatment site (e.g., into a vessel that feeds a treatment site). In some embodiments, the formulation can be delivered locally to ischemic tissue. In certain embodiments, the formulation can be a slow release formulation, c. ., in the form of a slow release depot.

[0151] For injection, the carrier will typically be a liquid, such as sterile pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or CREMOPHOR EL® (BASF, Parsippany, NJ). For other methods of administration, the carrier can be either solid or liquid.

[0152] For oral administration, the compound can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Compounds can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that can be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or fdm coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric- coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

[0153] Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

[0154] Formulations of the present invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can include suspending agents and thickening agents. The formulations can be presented in unit / dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for- inj ection immediately prior to use.

[0155] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, in one aspect of the present invention, there is provided an injectable, stable, sterile composition comprising a compound of the invention, in a unit dosage form in a sealed container. The compound or salt is provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection thereof into a subject. The unit dosage form typically comprises from about 10 mg to about 10 grams (e.g., 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, or 10 g) of the compound or salt. When the compound or salt is substantially water-insoluble, a sufficient amount of emulsifying agent which is pharmaceutically acceptable can be employed in sufficient quantity to emulsify the compound or salt in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0156] Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by admixing the compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

[0157] Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which can be used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

[0158] Formulations suitable for transdermal administration can be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis(see, for example, Tyle (1986) Pharm. Res. 3:318) and typically take the form of an optionally buffered aqueous solution of the compound. Suitable formulations comprise citrate or bis\tris buffer (pH 6) or ethanol / water and contain from 0.1 M to 0.2 M of the compound.

[0159] The compound can alternatively be formulated for nasal administration or otherwise administered to the lungs of a subject by any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the compound, which the subject inhales. The respirable particles can be liquid or solid. The term “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets, as can be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition suspended in air or other carrier gas, which can be delivered by insufflation from an inhaler device, for example. See Ganderton & Jones (1987) Drug Delivery to the Respiratory Tract, Ellis Horwood; Gonda (1990) Crit. Rev. Therap. Drug Carrier Systems 6:273-313; Raeburn et al. (1992) J. Pharmacol. Toxicol. Meth. 27: 143. Aerosols of liquid particles comprising the compound can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e. ., U.S. Patent No. 4,501,729. Aerosols of solid particles comprising the compound can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.

[0160] Alternatively, one can administer the compound in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0161] Further, the present invention provides liposomal formulations of the compounds disclosed herein and salts thereof. The technology for forming liposomal suspensions is well known in the art. When the compound or salt thereof is an aqueous-soluble salt, using conventional liposome technology, the same can be incorporated into lipid vesicles. In such an instance, due to the water solubility of the compound or salt, the compound or salt will be substantially entrained within the hydrophilic center or core of the liposomes. The lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free. When the compound or salt of interest is water-insoluble, again employing conventional liposome formation technology, the salt can be substantially entrained within the hydrophobic lipid bilayer which forms the structure of the liposome. In either instance, the liposomes which are produced can be reduced in size, as through the use of standard sonication and homogenization techniques.

[0162] The liposomal formulations containing the compounds disclosed herein or salts thereof, can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.

[0163] In the case of water-insoluble compounds, a pharmaceutical composition can be prepared containing the water-insoluble compound, such as for example, in an aqueous base emulsion. In such an instance, the composition will contain a sufficient amount of pharmaceutically acceptable emulsifying agent to emulsify the desired amount of the compound. Particularly useful emulsifying agents include phosphatidylcholines and lecithin.

[0164] In particular embodiments, the compound is administered to the subject in a therapeutically effective amount, as that term is defined above. Dosages of pharmaceutically active compounds can be determined by methods known in the art, see, e.g., Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA). The therapeutically effective dosage of any specific compound will vary somewhat from compound to compound, and patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.001 mg / kg to about 50 mg / kg (e.g., 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, 40 or 50 mg / kg, or any value or range therein) will have therapeutic efficacy, with all weights being calculated based upon the weight of the compound, including the cases where a salt is employed. Toxicity concerns at the higher level can restrict intravenous dosages to a lower level such as up to about 10 mg / kg, with all weights being calculated based upon the weight of the compound, including the cases where a salt is employed. A dosage from about 10 mg / kg to about 50 mg / kg can be employed for oral administration. Typically, a dosage from about 0.5 mg / kg to 5 mg / kg can be employed for intramuscular injection. Particular dosages are about 1 pmol / kg to 50 pmol / kg, and more particularly to about 22 pmol / kg and to 33 pmol / kg of the compound for intravenous or oral administration, respectively.

[0165] In particular embodiments of the invention, more than one administration (e.g., two, three, four, or more administrations) can be employed over a variety of time intervals (e.g. , hourly, daily, weekly, monthly, etc.) to achieve therapeutic effects.

[0166] The present invention finds use in veterinary and medical applications. Suitable subjects include both avians and mammals, with mammals being preferred. The term “avian” as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, and pheasants. The term “mammal” as used herein includes, but is not limited to, humans, bovines, ovines, caprines,equines, felines, canines, lagomorphs, etc. Human subjects include neonates, infants, juveniles, and adults. In other embodiments, the subject is an animal model of cancer. In certain embodiments, the subject has or is at risk for cancer.

[0167] The following examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the present invention. As will be understood by one skilled in the art, there are several embodiments and elements for each aspect of the claimed invention, and all combinations of different elements are hereby anticipated, so the specific combinations exemplified herein are not to be construed as limitations in the scope of the invention as claimed. If specific elements are removed or added to the group of elements available in a combination, then the group of elements is to be construed as having incorporated such a change.

[0168] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.EXAMPLESExample 1.

[0169] As described herein, dsRNA with overhangs at the 5' end of the antisense strand provides surprisingly enhanced RNA interference activity relative to dsRNA without the overhang on the 5' antisense strand. FIG. 1 and Table 1 show exemplary siRNA with IdT, 2dT, 3dT, and 4dT overhangs of an exemplary siRNA, Kseq2, a pan KRAS-targeting siRNA. Exemplary modifications to nucleotides in the dsRNA, including 2'0-methyl, 2'-fluoro and phosphorothioates, are indicated in FIG. 1 and Table 1. Unexpectedly, it was found that Kseq2 with 5'-dT overhangs of 2 or more dTs on the antisense strand performed better than Kseq2 alone or with a single dT overhang, with increasing potency directly correlated with the addition of each dT (e.g., 2dT > IdT, 3dT > 2dT), although the increased potency plateaued at 3 dT (3dT « 4dT). The dT overhangs at the 5' end of the antisense strand of an example siRNA, Kseq2, increase its potency in a dosedependent manner with 1 dT, 2 dT, 3 dT and 4 dT overhangs (FIGS. 2A-2B). Another exemplary dsRNA, Kseq3, also shows 2 dT and 3 dT overhangs at the 5' end of the Kseq3 antisense strand, which increase potency in a dose-dependent manner (FIGS. 3A-3B).

[0170] dT overhangs at the 5' end of an example c-Myc dsRNA was also explored, with 2 dT and 3dT overhangs on the example Mseq2 antisense strand increasing potency in the LN2A cell line (FIGS. 4A-4B) and the human pancreatic ductal adenocarcinoma cell line MIA-PaCa2 (FIGS. 5A-5B)

[0171] dsRNAs with a G12V-mutant-specific KRAS antisense strand with 2dT or 3dT overhangs at the 5' end were investigated in A-431 KRAS-knockout cells. A-431 KRAS-knockout cells were treated with a negative control, Kseq2 Hi20Me (pan KRAS siRNA), V4-G12V Hi20Me (mutantspecific siRNA), V4-G12V Hi20Me 2dT, or V4-G12V Hi2OMe 3dT siRNAs. The results in FIGS. 6A-6C indicate that dT overhangs at the 5' end of a G12V mutant-specific KRAS antisense strand increase its potency in a dose-dependent manner while maintaining wild-type sparing properties.

[0172] Notably, dT overhangs at the 5' end of the Mseq2 antisense strand increase its potency more than shifting the seed region of Mseq2 with perfectly complementary overhangs in MIA- PaCa2KRASGi2c (FIG. 7A) and A427KRASGI2D (FIG. 7B) cells. Similarly, dT overhangs at the 5' end of the Kseq2 antisense strand increase its potency more than shifting the seed region of Kseq2 with perfectly complementary overhangs (FIG. 8).

[0173] In vivo analysis was conducted to determine the antitumor effect of dT overhangs at the 5' end of dsRNA. The results of this analysis indicated that 3dT overhangs at the 5' end of the conjugated KRAS G12V-targeting antisense strand reduced tumor burden in mice after 7 days of treatment (FIGS. 9A-9B). In addition, 3dT overhangs at the 5' end of the conjugated G12V antisense strand increases its stability in vivo (FIG. 10).Table 1. dsRNA sequencesm = 2'-0-methyl on sugar moieties; 2fl = 2'-fluoro on sugar moieties; * = phosphorothioate in between nucleotides; T = deoxy-Thymine

[0174] The foregoing examples are illustrative of the present invention and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A double-stranded RNA (dsRNA) molecule that inhibits the expression of a target gene, the dsRNA comprising a sense strand and an antisense strand, the antisense strand comprising a S' nucleotide overhang of two or more dTs.

2. The dsRNA of claim 1, wherein the overhang comprises between 2 and 8 dTs, optionally between 2 and 4 dTs.

3. The dsRNA molecule of claim 1 or 2, which is about 14 to 40 nucleotides in length.

4. The dsRNA of any one of claims 1-3, wherein the molecule comprises at least one chemical modification.

5. The dsRNA of any one of claims 1-4, wherein the sense strand and / or the antisense strand comprise two or more phosphorothioate modifications.

6. The dsRNA of any one of claims 1-5, wherein one or more nucleotides in the sense strand and / or antisense strand comprise one or more of 2'-O-methyl and / or 2'-fluoro modifications.

7. The dsRNA of any one of claims 1-6, further comprising a targeting ligand, antibody, or aptamer conjugated to the dsRNA.

8. The dsRNA of any one of claims 1-7, wherein the dsRNA binds to c-Myc or KRAS.

9. The dsRNA of claim 8, wherein the dsRNA binds to a KRAS G12 mutant, optionally a G12V KRAS mutant.

10. The dsRNA of any one of claims 1-9, comprising a sense strand and an antisense strand, wherein the dsRNA molecule comprises one of the following pairs of sequences:sense strand of SEQ ID NO:1 and antisense strand of SEQ ID NO:3; sense strand of SEQ ID NO:1 and antisense strand of SEQ ID NO:4; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NO:6; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NO:7; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NO:8; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NO:9; sense strand of SEQ ID NO:5 and antisense strand of SEQ ID NO:10; sense strand of SEQ ID NO: 11 and antisense strand of SEQ ID NO: 12; sense strand of SEQ ID NO:11 and antisense strand of SEQ ID NO:13; sense strand of SEQ ID NO: 11 and antisense strand of SEQ ID NO: 14; sense strand of SEQ ID NO: 15 and antisense strand of SEQ ID NO: 16; sense strand of SEQ ID NO: 15 and antisense strand of SEQ ID NO: 17; or sense strand of SEQ ID NO: 15 and antisense strand of SEQ ID NO: 18.

11. A composition comprising the dsRNA molecule of any one of claims 1-10.

12. A composition comprising two or more of the dsRNA molecules of any one of claims 1- 10 in any combination, wherein the two or more dsRNA molecules each comprise a different sequence.

13. The composition of claim 11 or 12, further comprising a nanoparticle.

14. A pharmaceutical composition comprising the dsRNA of any one of claims 1-10 and / or the composition of any one of claims 11-13, and a pharmaceutically acceptable carrier.

15. A method for reducing the expression of a target gene in a cell, comprising contacting the cell with the dsRNA of any one of claims 1-10, the composition of any one of claims 11-13, and / or the pharmaceutical composition of claim 14, thereby reducing the expression of the target gene in the cell.

16. The method of claim 15, wherein the dsRNA reduces expression of the target gene more than a dsRNA without two or more dTs on 5' end of its antisense strand.

17. A method for delivering dsRNA to a target in a subject, comprising administering the dsRNA of any one of claims 1-10, the composition of any one of claims 11-13, and / or the pharmaceutical composition of claim 14 to the subject.

18. The method of claim 17, wherein the subject has a disease or disorder.

19. The method of claim 18, wherein the disease or disorder is cancer.

20. A method of treating cancer in a subject in need thereof, the method comprising delivering to the subject a therapeutically effective amount of the dsRNA of any one of claims 1-10, the composition of any one of claims 11-13, and / or the pharmaceutical composition of claim 14, thereby treating cancer in the subject.

21. The method of claim 20, wherein the delivery is systemic delivery.

22. The method of claim 20 or 21, wherein the dsRNA is specific for a KRAS or a c-Myc gene.

23. The method of claim 22, wherein the dsRNA is specific for a KRAS comprising a G12 mutant, optionally G12V KRAS mutant.

Images