Methods and compositions for the inhibition of IRF4

Abstract

The present invention relates to the inhibition of the expression of interferon regulatory factor-4 (IRF4) using a multivalent combination of RNA interference, chemically modified oligonucleotides, and / or chimeric siRNAs. The present invention further relates to methods of treating IRF4-related diseases such as multiple myeloma.

Description

Technical Field

[0001] [Priority Claim] This application claims the benefit of U.S. Provisional Application No. 63 / 354,049, filed on Jun. 21, 2022, under 35 U.S.C. § 119(e), the entire content of which is hereby incorporated by reference in its entirety.

[0002] [Statement Regarding Electronic Filing of Sequence Listing] An XML-formatted sequence listing named 5470-932WO_ST26.xml (size: 397,082 bytes, created on Jun. 21, 2023) is filed herewith and incorporated by reference in its entirety as disclosure herein.

[0003] [Technical Field] The present invention relates to the inhibition of the expression of interferon regulatory factor-4 (IRF4) using a multivalent combination of RNA interference, chemically modified oligonucleotides, and / or chimeric siRNAs. The present invention further relates to methods of treating IRF4-related diseases such as multiple myeloma.

Background Art

[0004] Multiple myeloma (MM) is a cancer of plasma cells that causes many problems, including kidney failure, fractures, low blood cell counts, and infections. The annual incidence in the United States is approximately 30,000 new cases, and more than 10,000 people die (Surveillance, Epidemiology, and End Results (SEER) Program; SEER*Stat Database: Populations - Total U.S. (1969-2020) <Katrina / Rita Adjustment> - Linked To County Attributes - Total U.S., 1969-2020 Counties, National Cancer Institute, DCCPS, Surveillance Research Program, published in January 2022). MM is mainly treated with systemic therapies; currently, FDA-approved treatment classes / approaches include proteasome inhibitors, immunomodulatory agents, monoclonal antibodies, stem cell transplantation, and more recently chimeric antigen receptor T cells (CAR-T), among others. Despite these diverse options, MM remains incurable and ultimately becomes treatment-resistant. Patients generally progress over several years through many treatment lines, and MM becomes increasingly resistant to available drugs with each new treatment line. With this resistance, MM becomes more difficult to control and causes more organ damage. Combined with the accumulation of treatment-related toxicities, almost all MM patients ultimately reach a point where further systemic therapy is futile, with any treatment having a low likelihood of providing clinically meaningful disease control and a high risk of serious toxicities.Due to the progress of MM treatment, this time point has been delayed in the majority of patients, and although the prognosis has improved more than ever, the fact that almost all MM patients will ultimately die from MM, rather than from other causes, emphasizes that the need for additional effective drugs to control MM and potentially cure it in the future remains unmet (Binder et al., Mortality trends in multiple myeloma after the introduction of novel therapies in the United States. Leukemia. 2022;36(3):801-808).

[0005] Interferon regulatory factor-4 (IRF4) is an important transcription factor for the regulation of plasma cells and affects areas such as differentiation, proliferation, immunoglobulin class switching, metabolism, and immune activity. IRF4 is often overexpressed in malignant (i.e., progressed to MM) plasma cells, and preclinical models have shown that suppressing IRF4 reduces the in vitro survival rate of MM cells (Agnarelli et al., IRF4 in multiple myeloma - Biology, disease and therapeutic target. Leuk Res. 2018; 72:52-58.). Despite this promise, IRF4 has not been fully evaluated as a therapeutic target until now.

[0006] The present invention overcomes the drawbacks in the art by providing compositions and methods for specifically inhibiting IRF4 using RNA interference and through the combination of dual silencing of IRF4 and c-Myc.

Summary of the Invention

[0007] The present invention is based on the identification of RNA molecules that inhibit the expression of the IRF4 sequence. Accordingly, one aspect of the present invention is a double-stranded RNA molecule comprising an antisense strand and a sense strand, wherein the nucleotide sequence of the antisense strand is complementary to a region of the nucleotide sequence of the human IRF4 gene, the region consisting essentially of about 18 to about 25 contiguous nucleotides; and the double-stranded RNA molecule is related to a double-stranded RNA molecule that inhibits the expression of the human IRF4 gene. Recent studies have revealed that IRF4 and c-Myc form a positive regulatory loop, suggesting that the ability to co-silence both targets may have additive and / or synergistic anti-cancer effects. In one embodiment, multiple siRNAs are used to inhibit both the IRF4 and c-Myc genes simultaneously.

[0008] Another aspect of the present invention relates to a composition (e.g., a pharmaceutical composition) comprising one or more RNA molecules of the present invention.

[0009] A further aspect of the present invention relates to a method of inhibiting the expression of the human IRF4 gene, the method comprising contacting the cell with an RNA molecule of the present invention, thereby inhibiting the expression of the human IRF4 gene in the cell.

[0010] An additional aspect of the present invention relates to a method of treating cancer in a subject in need thereof, the cancer expressing or overexpressing the human IRF4 gene, the method comprising delivering an RNA molecule of the present invention to the subject, thereby treating the cancer in the subject.

[0011] Another aspect of the present invention relates to inhibiting the intracellular expression of the human IRF4 gene using an RNA molecule of the present invention and treating cancer in a subject in need thereof, the cancer comprising overexpression of the human c-Myc gene.

[0012] Another aspect of the present invention relates to an siRNA molecule that targets native human IRF4 mRNA, the siRNA molecule comprising at least one chemical modification, and the siRNA molecule comprising one of the following sequence pairs: The sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2; The sense strand of SEQ ID NO: 3 and the antisense strand of SEQ ID NO: 4; The sense strand of SEQ ID NO: 5 and the antisense strand of SEQ ID NO: 6; The sense strand of SEQ ID NO: 7 and the antisense strand of SEQ ID NO: 8; The sense strand of SEQ ID NO: 9 and the antisense strand of SEQ ID NO: 10; The sense strand of SEQ ID NO: 11 and the antisense strand of SEQ ID NO: 12; The sense strand of SEQ ID NO: 13 and the antisense strand of SEQ ID NO: 14; The sense strand of SEQ ID NO: 15 and the antisense strand of SEQ ID NO: 16; The sense strand of SEQ ID NO: 17 and the antisense strand of SEQ ID NO: 18; The sense strand of SEQ ID NO: 19 and the antisense strand of SEQ ID NO: 20; The sense strand of SEQ ID NO: 21 and the antisense strand of SEQ ID NO: 22; The sense strand of SEQ ID NO: 23 and the antisense strand of SEQ ID NO: 24; Or A sequence having at least 90% identity with these.

[0013] Another aspect of the present invention relates to a composition (e.g., a pharmaceutical composition) comprising one or more siRNA molecules of the present invention.

[0014] A further aspect of the present invention relates to a method of inhibiting the intracellular expression of the human IRF4 gene, said method comprising contacting said cells with one or more siRNA molecules of the present invention, thereby inhibiting the intracellular expression of said human IRF4 gene.

[0015] An additional aspect of the present invention relates to a method of treating cancer in a subject in need thereof, said cancer expressing or overexpressing the human IRF4 gene, said method comprising delivering an siRNA molecule of the present invention to said subject, thereby treating cancer in said subject.

[0016] Another aspect of the present invention relates to using the siRNA molecules of the present invention to inhibit the intracellular expression of the human IRF4 gene and treat cancer in a subject in need thereof, wherein the cancer expresses or overexpresses the human IRF4 gene.

[0017] Another aspect of the present invention is a double-stranded RNA molecule, wherein the first RNA molecule comprises an antisense strand and a sense strand targeting the human IRF4 gene, and the second RNA molecule comprises an antisense strand and a sense strand targeting the human c-Myc gene, The nucleotide sequence of the first RNA molecule has an antisense strand complementary to a region of the nucleotide sequence of the human IRF4 gene, the region consisting essentially of about 18 to about 25 consecutive nucleotides; the double-stranded RNA molecule inhibits the expression of the human IRF4 gene, and The nucleotide sequence of the second RNA molecule has an antisense strand complementary to a region of the nucleotide sequence of the human c-Myc gene, the region consisting essentially of about 18 to about 25 consecutive nucleotides; the double-stranded RNA molecule inhibits the expression of the human c-Myc gene, relates to a double-stranded RNA molecule.

[0018] Another aspect of the present invention relates to a pair of siRNA molecules, the pair of siRNA molecules comprising a first siRNA molecule targeting native human IRF4 mRNA, the siRNA molecule comprising at least one chemical modification, and the siRNA molecule comprising one of the following sequence pairs: The sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2; The sense strand of SEQ ID NO: 3 and the antisense strand of SEQ ID NO: 4; The sense strand of SEQ ID NO: 5 and the antisense strand of SEQ ID NO: 6; The sense strand of SEQ ID NO: 7 and the antisense strand of SEQ ID NO: 8; The sense strand of SEQ ID NO: 9 and the antisense strand of SEQ ID NO: 10; The sense strand of SEQ ID NO: 11 and the antisense strand of SEQ ID NO: 12; Sense strand of SEQ ID NO: 13 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: 17 and antisense strand of SEQ ID NO: 18; Sense strand of SEQ ID NO: 19 and antisense strand of SEQ ID NO: 20; Sense strand of SEQ ID NO: 21 and antisense strand of SEQ ID NO: 22; Sense strand of SEQ ID NO: 23 and antisense strand of SEQ ID NO: 24; Or Sequences having at least 90% identity with these; And a second siRNA molecule targeting the native human c-Myc sequence, said siRNA molecule comprising at least one chemical modification. In some embodiments, said second siRNA molecule comprises one of the following sequence pairs: Sense strand of SEQ ID NO: 48 and antisense strand of SEQ ID NO: 49; Sense strand of SEQ ID NO: 50 and antisense strand of SEQ ID NO: 51; Sense strand of SEQ ID NO: 52 and antisense strand of SEQ ID NO: 53; Sense strand of SEQ ID NO: 54 and antisense strand of SEQ ID NO: 55; Sense strand of SEQ ID NO: 56 and antisense strand of SEQ ID NO: 57; Sense strand of SEQ ID NO: 58 and antisense strand of SEQ ID NO: 59; Sense strand of SEQ ID NO: 60 and antisense strand of SEQ ID NO: 61; Sense strand of SEQ ID NO: 62 and antisense strand of SEQ ID NO: 63; Sense strand of SEQ ID NO: 64 and antisense strand of SEQ ID NO: 65; Sense strand of SEQ ID NO: 66 and antisense strand of SEQ ID NO: 67; Sense strand of SEQ ID NO: 68 and antisense strand of SEQ ID NO: 69; Sense strand of SEQ ID NO: 70 and antisense strand of SEQ ID NO: 71; Sense strand of SEQ ID NO: 72 and antisense strand of SEQ ID NO: 73; Sense strand of SEQ ID NO: 74 and antisense strand of SEQ ID NO: 75; The sense strand of SEQ ID NO: 76 and the antisense strand of SEQ ID NO: 77; or The sense strand of SEQ ID NO: 78 and the antisense strand of SEQ ID NO: 79; or A sequence having at least 90% identity therewith.

[0019] Another aspect of the present invention relates to chimeric multivalent siRNA molecules that target one or more genes. In one embodiment, the chimeric siRNA molecule comprises a first siRNA molecule that targets native human IRF4 mRNA (the siRNA molecule comprises at least one chemical modification), and a second siRNA molecule that targets native human c-Myc mRNA (the siRNA molecule comprises at least one chemical modification), wherein the first siRNA molecule is attached to the second siRNA molecule by a linker region to form the chimeric siRNA molecule.

[0020] Another aspect of the present invention relates to a composition (e.g., a pharmaceutical composition) comprising one or more siRNA molecules of the present invention.

[0021] A further aspect of the present invention relates to a method of inhibiting the intracellular expression of the human IRF4 gene and the human c-Myc gene, the method comprising contacting the cells with the siRNA molecule of the present invention, thereby inhibiting the intracellular expression of the human IRF4 gene and the human c-Myc gene.

[0022] An additional aspect of the present invention relates to a method of treating cancer in a subject in need thereof, the cancer expressing or overexpressing the human IRF4 gene, the method comprising delivering the siRNA molecule of the present invention to the subject, thereby treating the cancer in the subject.

[0023] Another aspect of the present invention relates to using the siRNA molecules of the present invention to inhibit the intracellular expression of the human IRF4 gene and treat cancer in a subject in need thereof, wherein the cancer expresses or overexpresses the human IRF4 gene.

[0024] These and other aspects of the present invention are described in more detail in the following description of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

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Mode for Carrying Out the Invention

[0026] The present invention will be described in more detail below with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. However, the present invention can be implemented in various forms and should not be construed as limited to the embodiments shown 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.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0028] Nucleotide sequences are presented herein by single strands only, from left to right in the 5' to 3' direction, unless otherwise specified. Nucleotides and amino acids are represented herein in the format 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 CFR § 1.822 and established usage.

[0029] Unless otherwise indicated, standard methods known to those skilled in the art can be used for cloning genes, amplifying and detecting nucleic acids, etc. These techniques are known to those skilled in the art. See, for example, Green 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., New York).

[0030] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0031] Furthermore, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted.

[0032] For example, when it is described herein that a complex comprises components A, B, and C, it is specifically intended that any one of A, B, or C, or a combination thereof, may be omitted and excluded, either alone or in any combination.

[0033] [Definitions] As used in the specification and claims of the present invention, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0034] 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 absence of combinations when interpreted alternatively ("or").

[0035] The term "about", as used herein when referring to a measurable value such as, for example, the amount of a polypeptide, dosage, time, temperature, enzyme activity, or other biological activity, means an inclusion of variations of ±10%, ±5%, ±1%, ±0.5%, and even ±0.1% of the specified amount.

[0036] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be construed to include the recited materials or steps and those that do not materially affect the basic and novel characteristics (s) of the claimed invention. Thus, the term "consisting essentially of" as used herein should not be construed as equivalent to "comprising".

[0037] When the term "consisting essentially of" (and grammatical variations) is applied to a polynucleotide sequence of the present invention, it means a polynucleotide consisting of both the recited sequence (e.g., SEQ ID NO) and a total of 10 or fewer nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) added to the 5' end and / or 3' end of the recited sequence, where the function of the polynucleotide is not materially altered. The total of 10 or fewer additional nucleotides includes the total number of additional nucleotides added to both ends. The term "materially altered", when applied to a polynucleotide of the present invention, refers to an increase or decrease of at least about 50% or more in the ability to inhibit the expression of the target mRNA, as compared to the expression level of a polynucleotide consisting of the recited sequence (consisting of).

[0038] The terms "enhance" or "increase" refer to an increase of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, and even 15-fold in a particular parameter.

[0039] As used herein, the terms "inhibit" or "reduce", or grammatical variations thereof, refer to a decrease or reduction of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, or more, at a particular level or activity. In specific embodiments, as a result of the inhibition or reduction, the detectable activity is little or essentially none (at most trace amounts, e.g., about 10%, or even less than 5%).

[0040] As used herein, a "therapeutically effective" amount is an amount that confers some improvement or benefit to a subject. In other words, a "therapeutically effective" amount is an amount that results in some alleviation, mitigation, or reduction in at least one clinical symptom in the subject (e.g., in the case of cancer, a decrease in tumor burden, prevention of further tumor growth, prevention of metastasis, or an increase in survival time). One of ordinary skill in the art will understand that the therapeutic effect need not be complete or curative as long as some benefit is conferred to the subject.

[0041] The terms "treat", "treating", or "treatment of" are intended to mean that the severity of a subject's condition is decreased and / or at least partially improved or modified, and that some alleviation, mitigation, or reduction is achieved in at least one clinical symptom.

[0042] "Prevent" or "preventing" or "prevention" refers to the prevention or delay of the onset of a disorder and / or a decrease in the severity of a disorder in a subject as compared to the severity that would occur if the methods of the present invention were not used. The prevention may be complete, e.g., where there is no cancer in the subject. The prevention may also be partial, e.g., where the occurrence or severity of cancer in the subject is lower than what would occur without the present invention.

[0043] As used herein, the terms "nucleic acid," "nucleotide sequence," and "polynucleotide" are used interchangeably and include 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 strand of nucleotides, regardless of the length of the strand. The nucleic acid can be double-stranded or single-stranded. In the case of a single-stranded nucleic acid, it can be a sense strand or an antisense strand. Nucleic acids 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 with altered base pairing ability or improved resistance to nucleases. The present invention further provides nucleic acids that are complements of the nucleic acids, nucleotide sequences, or polynucleotides of the present invention, which can be either perfect or partial complements. When dsRNA is synthetically produced, unusual bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind to RNA with high affinity and be potent antisense inhibitors of gene expression. Other modifications, such as modifications to the phosphodiester backbone or the 2'-hydroxy group of the ribose sugar of RNA, can also be made.

[0044] "Isolated polynucleotide" refers to a nucleotide sequence (such as DNA or RNA) that is not directly adjacent to the nucleotide sequences that are directly adjacent in the natural genome of the organism from which it is derived (the sequences adjacent to the 5' end and the sequences adjacent to the 3' end). Thus, in one embodiment, an isolated nucleic acid comprises a part or all of a 5' non-coding (such as a promoter) sequence that is directly adjacent to a coding sequence. Thus, this term includes, for example, recombinant DNA incorporated into a vector, an autonomously replicating plasmid or virus, or in the genomic DNA of a prokaryote or eukaryote, or recombinant DNA existing as another molecule independent of other sequences (such as a cDNA or genomic DNA fragment generated by PCR or restriction endonuclease treatment). This also includes recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence. An isolated polynucleotide containing a gene contains the coding region and regulatory regions related to the gene, but not the additional genes that naturally exist on the chromosome, and is not a fragment of the chromosome containing the gene.

[0045] The term "isolated" can refer to a nucleic acid, nucleotide sequence, or polypeptide that is substantially free of cellular components, viral components, and / or culture medium (when generated by recombinant DNA technology), or chemical precursors or other chemicals (when chemically synthesized). Also, an "isolated fragment" is a fragment of a nucleic acid, nucleotide sequence, or polypeptide that does not naturally exist as a fragment and cannot be found in the natural state. "Isolated" does not mean that the preparation is technically pure (homogeneous), but is pure enough that the polypeptide or nucleic acid can be supplied in a form that can be used for the intended purpose.

[0046] "Isolated cell" refers to a cell that has been separated from other components with which it is normally associated in its natural state. For example, an isolated cell can be a cell in a culture medium and / or a cell in a pharmaceutically acceptable carrier of the present 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, manipulated ex vivo as described herein, and then returned to the subject.

[0047] The term "fragment", when applied to a polynucleotide, means a nucleotide sequence that is shortened in length compared to a reference nucleic acid or nucleotide sequence, and that contains, consists essentially of, and / or consists of a nucleotide sequence of consecutive nucleotides that is identical or nearly identical (e.g., having 90%, 92%, 95%, 98%, 99% identity) to the reference nucleic acid or nucleotide sequence. Such nucleic acid fragments according to the present invention can, where appropriate, be contained within a larger polynucleotide of which they are a component. In some embodiments, such fragments can contain, consist essentially of, and / or consist of an oligonucleotide 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 present invention.

[0048] The term "fragment", when applied to a polypeptide, means an amino acid sequence that is shortened in length as compared to a reference polypeptide or amino acid sequence and that comprises, consists essentially of, and / or consists of a contiguous amino acid sequence that is identical or substantially identical (e.g., having 90%, 92%, 95%, 98%, 99% identity) to the reference polypeptide or amino acid sequence. Such polypeptide fragments according to the invention can, where appropriate, be included within a larger polypeptide of which they are a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of a peptide 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 contiguous amino acids of a polypeptide or amino acid sequence according to the invention.

[0049] "Vector" refers to any nucleic acid molecule for cloning nucleic acids and / or for transferring nucleic acids into cells. A vector can be a replicon that can add another nucleotide sequence and enable replication of the added nucleotide sequence. "Replicon" refers to 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., can replicate under its own control. The term "vector" includes both viral and non-viral (e.g., plasmid) nucleic acid molecules for introducing nucleic acids into cells in vitro, ex vivo, and / or in vivo. Numerous vectors known in the art can be used, for example, for manipulating nucleic acids and incorporating response elements and promoters into genes. For example, inserting a nucleic acid fragment corresponding to a response element and a promoter into an appropriate vector can be achieved by ligating the appropriate nucleic acid fragment into a selected vector having complementary sticky ends. Alternatively, the ends of the nucleic acid molecule may be enzymatically modified, or any site may be generated by ligating a nucleotide sequence (linker) to the nucleic acid ends. Such vectors can be modified to contain a sequence encoding a selectable marker for selecting cells containing the vector and / or cells in which the nucleic acid of the vector has been integrated into the cell genome. Such markers can be used to identify and / or select host cells that incorporate and express the protein encoded by the marker. "Recombinant" vector refers to a viral or non-viral vector containing one or more heterologous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five, or more heterologous nucleotide sequences.

[0050] Viral vectors have been used in a variety of applications for gene delivery in cells and living animals. Viral vectors that can be used include, but are not limited to, the following: retroviruses, lentiviruses, adeno-associated viruses, poxviruses, alphaviruses, baculoviruses, vaccinia viruses, herpesviruses, Epstein-Barr viruses, and / or adenoviral vectors. Non-viral vectors include, but are not limited to, the following: plasmids, liposomes, charged lipids (lipofectamine), nucleic acid-protein complexes, and biopolymers. In addition to the nucleic acid of interest, the vector may also contain one or more regulatory regions and / or selectable markers that are useful for selecting, measuring, and monitoring the results of nucleic acid transfer (such as delivery to a specific tissue, duration of expression, etc.).

[0051] Vectors can be introduced into the desired cells by methods known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, for example, Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).

[0052] In some embodiments, the polynucleotides of the invention can be delivered to cells in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties and risks encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of the nucleotide sequences of the invention (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987); Mackey, et al., Proc. Natl. Acad. Sci. U.S.A. 85:8027 (1988); and Ulmer et al., Science 259:1745 (1993)). The use of cationic lipids can promote encapsulation of negatively charged nucleic acids and also promote fusion with negatively charged cell membranes (Felgner et al., Science 337:387 (1989)). Lipid compounds and compositions particularly useful for nucleic acid delivery are described in International Patent Publications WO95 / 18863 and WO96 / 17823, and in U.S. Patent No. 5,459,127. There are certain practical advantages to using lipofection to introduce exogenous nucleotide sequences into specific organs in vivo. Targeting liposomes to specific cells is one example of an advantage. It is clearly particularly preferred to direct transfection to specific cell types in tissues having cell heterogeneity such as the pancreas, liver, kidney, and brain. Lipids can be chemically conjugated to other molecules for targeting purposes (Mackey, et al., 1988, supra). Target peptides (e.g., hormones or neurotransmitters), and proteins (such as antibodies), or non-peptide molecules can be chemically conjugated to liposomes.

[0053] In various embodiments, other molecules can be used to facilitate delivery of nucleic acids in vivo. For example, cationic oligopeptides (e.g., WO95 / 21931), peptides derived from nucleic acid binding proteins (e.g., WO96 / 25508), and / or cationic polymers (e.g., WO95 / 21931).

[0054] It is also possible to introduce the vector as naked nucleic acid in vivo (see U.S. Pat. Nos. 5,693,622, 5,589,466, and 5,580,859). Receptor-mediated nucleic acid delivery methods may also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).

[0055] As used herein, the terms "protein" and "polypeptide" are used interchangeably and, unless otherwise indicated, include both peptides and proteins.

[0056] A "fusion protein" is a polypeptide that is produced when two (or more) different nucleotide sequences encoding different polypeptides that are not fused in nature, or fragments thereof, are fused within the correct translational reading frame. Exemplary fusion polypeptides include fusions of a polypeptide of the invention (or a fragment thereof) with all or a portion of any of the following: glutathione S-transferase, maltose binding protein, or a reporter protein (e.g., green fluorescent protein, β-glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc.

[0057] The term "express" or "expression" of a coding sequence of a polynucleotide means that the sequence is transcribed and optionally translated. Typically, according to the present invention, as a result of expressing the coding sequence of the present invention, the polypeptide of the present invention is produced. The whole or a fragment of the expressed polypeptide may also function in the cell as it is without purification.

[0058] As used herein, the terms "over-expression" or "over-expressing" refer to an increase in the level at which a polypeptide is produced and / or an increase in the time of expression (e.g., when constitutively expressed) as compared to wild-type cells.

[0059] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, etc. A gene may or may not be capable of being used to produce a functional protein. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, 5′ and 3′ untranslated regions). When a gene is "isolated", it means a nucleic acid that does not substantially or essentially contain components normally found in association with that nucleic acid in its natural state. Such components include other cellular materials, culture media from recombinant products, and / or various chemicals used in the chemical synthesis of the nucleic acid.

[0060] As used herein, "complementary" polynucleotides are those capable of base pairing according to the standard Watson-Crick base complementarity rules. Specifically, a purine forms a base pair with a pyrimidine, guanine pairs with cytosine (G:C), and in the case of DNA, adenine pairs with thymine (A:T), or in the case of RNA, adenine pairs with uracil (A:U). For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". It is understood that two polynucleotides can hybridize to each other if each has at least one region that is substantially complementary to the other, even if they are not completely complementary to each other.

[0061] As used herein, the terms "complementary" or "complementarity" refer to the natural binding of polynucleotides by base pairing under permissive salt and temperature conditions. Complementarity between two single-stranded molecules may be "partial," in which case only some of the nucleotides bind, or "complete," in which case there is complete complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between the nucleic acid strands.

[0062] As used herein, the terms "substantially complementary" or "partially complementary" mean that two nucleic acid sequences are complementary at at least about 50%, 60%, 70%, 80%, or 90% of their nucleotides. In some embodiments, two nucleic acid sequences can be complementary at at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of their nucleotides. The terms "substantially complementary" and "partially complementary" may also mean that two nucleic acid sequences can hybridize under high stringency conditions, such conditions being well known in the art.

[0063] As used herein, "heterologous" refers to a nucleotide sequence that is derived from a different species or that is derived from the same species or organism but has been modified from its original form or the form in which it is predominantly expressed in the cell. Thus, a nucleotide sequence that is derived from a different organism or species than the cell into which the nucleotide sequence has been introduced is heterologous with respect to that cell and the progeny of that cell. In addition, heterologous nucleotide sequences include nucleotide sequences that are derived from the same native cell type and have been inserted but are present in an unnatural state, for example, nucleotide sequences that are under the control of regulatory sequences that are different in copy number and / or different from those found in nature.

[0064] As used herein, the terms "contacting," "introducing," and "administering" are used interchangeably and refer to the process of delivering a dsRNA of the invention or a nucleic acid molecule encoding the dsRNA of the invention to a cell and inhibiting, altering, or modifying the expression of a target gene. The dsRNA can be administered in a variety of forms including, but not limited to: direct introduction into a cell (i.e., intracellularly), and / or extracellular introduction into a cavity, the interstitial space, or the circulatory system of a living body.

[0065] "Introducing" means presenting a nucleic acid molecule to an organism and / or cell in a form that allows the nucleic acid molecule to access the interior of the cell, in the context of a cell or organism. When multiple nucleic acid molecules are introduced, these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid constructs, or as separate polynucleotides or nucleic acid constructs, and can be arranged on the same or different nucleic acid constructs. Thus, these polynucleotides can be introduced into a cell by a single transformation event or by separate transformation events. Thus, the term "transformation" as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell can be stable or transient.

[0066] "Transient transformation" or "transient transfection" means, in the context of a polynucleotide, that the polynucleotide is introduced into a cell but is not integrated into the genome of the cell.

[0067] As used herein, "stably introducing" or "stably introduced" in the context of a polynucleotide introduced into a cell is intended to mean that the introduced polynucleotide is stably integrated into the genome of the cell, such that the cell is stably transformed with the polynucleotide.

[0068] As used herein, "stable transformation" or "stable transfection" means that a nucleic acid molecule is introduced into a cell and integrated into the genome of the cell. Thus, the integrated nucleic acid molecule can be inherited by its progeny, more specifically by progeny of multiple successive generations. As used herein, "genome" includes the nuclear genome and the mitochondrial genome, and thus includes the integration of a nucleic acid into, for example, the mitochondrial genome. Stable transformation or stable transfection as used herein can also refer to a transgene maintained extrachromosomally, for example, as a minichromosome.

[0069] Transient transformation or transient transfection can be detected, for example, by enzyme-linked immunosorbent assay (ELISA) or Western blot. These can detect the presence of peptides or polypeptides encoded by one or more transgenes introduced into an organism. Stable transformation or stable transfection of cells can be detected, for example, by Southern blot hybridization assay on the genomic DNA of cells using a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of a transgene introduced into the organism. Stable transformation or stable transfection of cells can be detected, for example, by Northern blot hybridization assay on the RNA of cells using a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of a transgene introduced into the organism. Stable transformation of cells can also be detected, for example, by polymerase chain reaction (PCR) or other amplification reactions well known in the art. At this time, a specific primer sequence that hybridizes to the target sequence(s) of the transgene is used to amplify the transgene sequence and detect it according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0070] Embodiments of the present 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 one control sequence operably linked to a nucleotide sequence of interest. Thus, for example, a promoter that operably interacts with the nucleotide sequence for the siRNA of the present invention is provided within the expression cassette for expression in an organism or cell.

[0071] As used herein, the term "promoter" refers to a region of a nucleotide sequence that incorporates signals necessary for efficient expression of a coding sequence. This may include, but is not limited to, a sequence to which RNA polymerase binds, and may include regions to which other regulatory proteins bind, as well as regions involved in the control of protein translation, and may also include a coding sequence.

[0072] Furthermore, the "promoter" of the present invention is a promoter capable of initiating transcription within a cell of an organism. Such promoters include those that constitutively drive the expression of a nucleotide sequence, those that drive expression when induced, and those that drive expression in a tissue-specific or development-stage-specific manner, and these various types of promoters are known in the art.

[0073] For the purposes of the present invention, the regulatory regions (i.e., promoters, transcriptional regulatory regions, and translation termination regions) may be native / analogous to the organism or cell, and / or the regulatory regions may be native / analogous to other regulatory regions. Alternatively, the regulatory regions may be heterologous to the organism or cell, and / or to each other (i.e., between regulatory regions). Thus, for example, a promoter may be heterologous, which is the case when the promoter is operably linked to a polynucleotide from a species different from the species from which the polynucleotide is derived. Alternatively, a promoter may also be heterologous to a selected nucleotide sequence, for example, when the species from which the polynucleotide is derived and its promoter are from the same / analogous species, but one or both (i.e., the promoter and the polynucleotide) have been substantially modified from their original form and / or genomic location, or when the promoter is not the native promoter for the polynucleotide to which it is operably linked.

[0074] The selection of the promoter to be used depends on several factors including, but not limited to, the following: cell-specific or tissue-specific expression, desired expression level, efficiency, inducibility, and selectivity. For example, if expression in a particular tissue or organ is desired, a tissue-specific promoter may be used. In contrast, if expression in response to a stimulus is desired, an inducible promoter may be used. If constitutive expression throughout the cells of an organism is desired, a constitutive promoter may be used. It is routine work for those skilled in the art to regulate the expression of a nucleotide sequence by appropriately selecting and arranging the promoter and other regulatory regions relative to its sequence.

[0075] In addition to the promoters described above, the expression cassette may also contain other regulatory sequences. As used herein, "regulatory sequences" means nucleotide sequences located upstream (5' untranslated sequences), within, or downstream (3' untranslated sequences) of a coding sequence and that affect 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.

[0076] The expression cassette may also contain transcription and / or translation termination regions (i.e., termination regions) that function within the organism. A variety of transcription terminators are available for use in the expression cassette and are responsible for termination of transcription beyond the transgene and accurate mRNA polyadenylation. The termination region may be originally present in the transcription initiation region, originally present in the nucleotide sequence of interest operably linked thereto, originally present in the host, or may be derived from a heterologous source (i.e., foreign or heterologous to the promoter, nucleotide sequence of interest, host, or any combination thereof).

[0077] The signal sequence can be operably linked to the nucleic acid of the present invention to direct the nucleotide sequence into an intracellular compartment. Thus, the expression cassette comprises a nucleotide sequence encoding siRNA operably linked to a nucleic acid sequence for the signal sequence. The signal sequence can be operably linked to the N-terminus or C-terminus of the siRNA.

[0078] Regardless of the type of regulatory sequence(s) used, they can be operably linked to the nucleotide sequence of the siRNA. As used herein, "operably linked" means that the elements of a nucleic acid construct, such as an expression cassette, are configured so as to perform their normal functions. Thus, a regulatory or control sequence (e.g., a promoter) operably linked to a nucleotide sequence of interest enables the expression of the nucleotide sequence of interest. The control sequence need not be adjacent to the nucleotide sequence of interest so long as it functions to direct its expression. Thus, for example, intervening untranslated but transcribed sequences can be present between a promoter and a coding sequence, and the promoter sequence is still considered to be "operably linked" to the coding sequence. The nucleotide sequences of the present invention (i.e., siRNA) can be expressed in cells and / or subjects by being operably linked to a regulatory sequence.

[0079] The expression cassette may also contain a nucleotide sequence for a selectable marker that can be used to select the transformed organism or cell. As used herein, "selectable marker" means a nucleic acid that, when expressed, confers a distinct phenotype on the organism or cell expressing the marker, such that the transformed organism or cell can be distinguished from those without the marker. Such nucleic acids can encode either a selectable marker or a screenable marker, depending on whether the marker confers a property that can be selected by chemical means, such as the use of a selective agent (e.g., an antibiotic), or is simply a property that can be identified through tests such as observation or screening. Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0080] In some embodiments of the invention, the expression cassette may contain an expression control sequence operably linked to a nucleotide sequence that is a template for one or both strands of the dsRNA. In further embodiments, a promoter can flank each end of the template nucleotide sequence, and the promoter drives the expression of the individual DNA strands to produce two complementary (or substantially complementary) RNAs that hybridize to form dsRNA. In alternative embodiments, the nucleotide sequence is transcribed into both strands of the dsRNA in one transcription unit, with the sense strand transcribed from the 5' end of the transcription unit and the antisense strand transcribed from the 3' end, and the two strands are separated by about 3 to about 500 base pairs, and after transcription, the RNA transcript undergoes self-folding to form a short hairpin RNA (shRNA) molecule.

[0081] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout an alignment window of components (e.g., nucleotides or amino acids). "Identity" can be readily calculated by known methods including, but not limited to, those described below: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0082] As used herein, the terms "substantially identical" or "corresponding to" mean 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% sequence identity.

[0083] The "identity fraction" of an aligned segment of a test array and a reference array is the value obtained by dividing the number of identical components shared by the two aligned arrays by the total number of components of the reference array segment (i.e., the entire reference array or a more narrowly defined portion of the reference array).

[0084] As used herein, the terms "percent sequence identity" or "percent identity" refer to the percentage of nucleotides that match when the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) is compared to a test ("subject") polynucleotide molecule (or its complementary strand) and the two sequences are optimally aligned (where appropriate nucleotide insertions, deletions, or gaps total less than 20% of the reference sequence over the comparison window). In some embodiments, "percent identity" may refer to the percentage of matching amino acids in an amino acid sequence.

[0085] Optimal alignment of sequences to align comparison windows is well known to those skilled in the art and can be performed by tools such as: the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally, computer implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA, which are available as part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, Massachusetts). The percent sequence identity is expressed as the value obtained by multiplying the identity rate by 100. Comparison of one or more polynucleotide sequences can be performed against the full-length polynucleotide sequence or a portion thereof, or against a longer polynucleotide sequence. For the purposes of the present invention, the "percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0086] The percent sequence identity can be determined using the "BestFit" or "Gap" programs of the Sequence Analysis Software Package (trademark) (version 10; Genetics Computer Group, Inc., Madison, Wisconsin). "Gap" utilizes the Needleman and Wunsch algorithm (Needleman and Wunsch, J Mol. Biol. 48:443-453, 1970) to find an alignment of two sequences, maximizing the number of matches and minimizing the number of gaps. "BestFit" uses the Smith and Waterman local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482-489, 1981, Smith et al., Nucleic Acids Res. 11:2205-2220, 1983) to perform an optimal alignment of the most similar segment between two sequences and inserts gaps to maximize the number of matches.

[0087] Useful methods for determining sequence identity are also described below: Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo, H., and Lipton, D., (Applied Math 48:1073(1988)). More specifically, 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 the National Center for Biotechnology Information (NCBI) at the National Library of Medicine, National Institutes of Health (Bethesda, Maryland 20894); see BLAST Manual, Altschul et al., NCBI, NLM, NIH; (Altschul et al., J. Mol. Biol. 215:403-410 (1990)); for versions of the BLAST program 2.0 and above, introduction of gaps (deletions and insertions) into the alignment is possible; for peptide sequences, BLASTX can be used to determine sequence identity; and for polynucleotide sequences, BLASTN can be used to determine sequence identity.

[0088] 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), etc. In the RNAi process, dsRNA containing a first (antisense) strand complementary to a portion of the 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 (siRNA), which are then distributed throughout the organism and, over generations, can come to exhibit a phenotype very similar to that resulting from complete or partial deletion of the target gene.

[0089] MicroRNAs (miRNAs) are typically non-protein-coding RNAs that are approximately 18 to approximately 25 nucleotides in length. These miRNAs induce trans cleavage of target transcripts and negatively regulate the expression of genes involved in various regulatory and developmental pathways (Bartel, Cell, 116:281-297 (2004); Zhang et al. Dev. Biol. 289:3-16 (2006)). Thus, miRNAs have been shown to be involved in various aspects of growth and development, as well as in signaling and proteolysis. Since the first miRNA was discovered in plants (Reinhart et al. Genes Dev. 16:1616-1626 (2002), Park et al. Curr. Biol. 12:1484-1495 (2002)), hundreds have been identified. Many microRNA genes (MIR genes) have been identified and are publicly available in databases (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 herein by reference.

[0090] Genes encoding miRNAs produce primary miRNAs (referred to as "pri-miRNAs") that are 70 - 300 bp (base pairs) in length and can form an imperfect stem-loop structure. One pri-miRNA can contain one or more miRNA precursors. In animals, pri-miRNAs are processed in the nucleus by the RNaseIII enzyme Drosha and its cofactor DGCR8 / Pasha into shorter hairpin RNAs (pre-miRNAs) of approximately 65 nt (nucleotides). The pre-miRNAs are then transported to the cytoplasm where they are further processed by another RNaseIII enzyme, Dicer, to release an miRNA / miRNA* duplex of approximately 22 nt in size. Many reviews on miRNA biogenesis and function are available, see for example: Bartel Cell 116:281 - 297 (2004), Murchison et al. Curr. Opin. Cell Biol. 16:223 - 229 (2004), Dugas et al. Curr. Opin. Plant Biol. 7:512 - 520 (2004), and Kim Nature Rev. Mol. Cell Biol. 6:376 - 385 (2005).

[0091] [RNA molecule] The present invention presents an alternative therapeutic approach by targeting IRF4 at the transcriptional level. The present invention consists of dsRNA molecules (e.g., short interfering RNAs (siRNAs)) that can bind complementarily to IRF4 messenger RNA and inhibit the transcription of oncogenes, inducing gene knockdown and subsequent apoptosis in cancer cells. Conventional siRNAs have faced clinical limitations due to lack of tissue specificity, rapid degradation in vivo, and immune activation. However, the synthetic siRNAs herein contain novel chemical modifications that confer drug-like properties, protecting them from degradation in vivo and eliminating the need for nanocarriers or other delivery systems.

[0092] Accordingly, one aspect of the present invention relates to a double-stranded RNA molecule comprising an antisense strand and a sense strand, wherein the nucleotide sequence of the antisense strand is complementary to a region of the nucleotide sequence of the human IRF4 gene, said region consisting essentially of, or consisting of, about 18 to about 25 consecutive nucleotides; and said double-stranded RNA molecule inhibits the expression of the human IRF4 gene. The RNA molecule reduces the expression of IRF4 in cells as compared to cells that do not have the RNA molecule (e.g., control cells or non-transformed cells). In some embodiments, the expression of IRF4 is inhibited by at least about 50%, e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0093] The double-stranded RNA molecule can comprise, consist essentially of, or consist of about 18 to about 25 nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25, or any range thereof). Additional nucleotides can be added to the 3′ end, 5′ end, or both the 3′ and 5′ ends to facilitate manipulation of the RNA molecule, but they do not materially affect the basic properties or functions of the double-stranded RNA molecule in RNA interference (RNAi). Additionally, one or two nucleotides can be deleted from either or both ends of any of the sequences disclosed herein, and they also do not materially affect the basic properties or functions of the double-stranded RNA molecule in RNAi. As used herein, the term "materially affect" refers to a change in the ability to inhibit the expression of the protein encoded by the mRNA of no more than about 50%, e.g., no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less. Such additional nucleotides can be nucleotides that extend the complementarity of the antisense strand along the target sequence, and / or such nucleotides can be nucleotides that facilitate manipulation of the RNA molecule or the nucleic acid molecule encoding the RNA molecule, which is known to those of skill in the art. For example, a TT overhang sequence can be present at the 3′ end, which is used to stabilize the siRNA duplex but does not affect the specificity of the siRNA.

[0094] The dsRNA of the present invention may contain single-stranded overhang sequences at one or both ends. The double-stranded structure can be formed by one self-complementary RNA strand (i.e., one that forms a hairpin loop) or two complementary RNA strands. The formation of the RNA double-strand can be initiated intracellularly or extracellularly. When the dsRNA of the present invention forms a hairpin loop, it may contain an intron and / or a nucleotide spacer (a continuous portion of nucleotides between complementary RNA strands) to stabilize the hairpin sequence intracellularly. The RNA can be introduced in an amount such that at least one copy can be delivered per cell. By increasing the dose of the double-stranded substance, more effective inhibition can be obtained.

[0095] In a specific embodiment, the present invention provides a double-stranded RNA containing a nucleotide sequence that is completely complementary to the region of the target gene to be inhibited. However, it should be understood that 100% complementarity between the antisense strand of the double-stranded RNA molecule and the target sequence is not essential for practicing the present invention. Thus, sequence variations predicted to result from genetic mutations, strain polymorphisms, or evolutionary divergence may be tolerated. RNA sequences having insertions, deletions, and single nucleotide mutations relative to the target sequence may also be effective for inhibition.

[0096] In some embodiments, the nucleotide sequences of the sense strand and the antisense strand have at least about 80% identity with any of the nucleotide sequences of SEQ ID NOs: 1-24, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity with any of the nucleotide sequences of SEQ ID NOs: 1-24, and include nucleotide sequences. In some embodiments, the nucleotide sequences of the sense strand and the antisense strand of the double-stranded RNA include, and consist essentially of, one of the following sequence pairs. The sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 2; The sense strand of SEQ ID NO: 3 and the antisense strand of SEQ ID NO: 4; The sense strand of SEQ ID NO: 5 and the antisense strand of SEQ ID NO: 6; Sense strand of SEQ ID NO:7 and antisense strand of SEQ ID NO:8; Sense strand of SEQ ID NO:9 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:13 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:17 and antisense strand of SEQ ID NO:18; Sense strand of SEQ ID NO:19 and antisense strand of SEQ ID NO:20; Sense strand of SEQ ID NO:21 and antisense strand of SEQ ID NO:22; Sense strand of SEQ ID NO:23 and antisense strand of SEQ ID NO:24; Or a sequence having at least 80% identity with these, for example, a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity with these.

[0097] In some embodiments, one or both of the sense strand and the antisense strand contain a TT overhang sequence or other dinucleotide overhang sequence at the 3′ end.

[0098] In some embodiments of the present invention, the sense strand of the double-stranded RNA molecule may be completely complementary to the antisense strand, or the sense strand may be substantially or partially complementary to the antisense strand. Substantially or partially complementary means that the sense strand and the antisense strand may differ by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide pairs. Such mismatches may be introduced into the sense strand sequence, for example, near the 3′ end, which, as is known to those skilled in the art, promotes the processing of double-stranded RNA molecules by Dicer, replicates the pattern of mismatches in siRNA molecules inserted into the chimeric nucleic acid molecules or artificial microRNA precursor molecules of the present invention, and for similar purposes. Such modifications weaken base pairing at one end of the double strand and generate strand asymmetry, thereby promoting the processing of the antisense strand rather than the sense strand and enhancing the opportunity to silence the target gene (Geng and Ding “Double-mismatched siRNAs enhance selective gene silencing of a mutant ALS-causing Allele1” Acta Pharmacol. Sin. 29:211-216 (2008); Schwarz et al. “Asymmetry in the assembly of the RNAi enzyme complex” Cell 115:199-208 (2003)).

[0099] The double-stranded RNA molecule of the present invention can be in the form of any type of RNA interference molecule known in the art. In some embodiments, the double-stranded RNA molecule is a small interfering RNA (siRNA) molecule. In other embodiments, the double-stranded RNA molecule is a short hairpin RNA (shRNA) molecule. In other embodiments, the double-stranded RNA molecule is part of a microRNA precursor molecule.

[0100] [Chemically Modified siRNA] One aspect of the present invention relates to siRNA molecules that target IRF4 mRNA, wherein the siRNA comprises at least one chemical modification. In some embodiments, the siRNA molecule is fully chemically-modified. The term "fully chemically-modified" means that all nucleotides of the siRNA contain a chemical modification. In some embodiments, each nucleotide within the siRNA molecule is modified with a 2'-O-methyl group or a 2'-fluoro group.

[0101] In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotide linkages within the siRNA are chemically modified. In some embodiments, the siRNA comprises at least one phosphorothioate linkage. In some embodiments, the siRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 phosphorothioate linkages. In some embodiments, the siRNA comprises all phosphorothioate linkages.

[0102] In certain embodiments, the siRNA molecule comprising at least one chemical modification comprises a sense strand and an antisense strand, and the siRNA molecule comprises one of the following sequence pairs: The sense strand of SEQ ID NO: 25 and the antisense strand of SEQ ID NO: 26; The sense strand of SEQ ID NO: 27 and the antisense strand of SEQ ID NO: 28; The sense strand of SEQ ID NO: 29 and the antisense strand of SEQ ID NO: 30; The sense strand of SEQ ID NO: 31 and the antisense strand of SEQ ID NO: 32; The sense strand of SEQ ID NO: 33 and the antisense strand of SEQ ID NO: 34; The sense strand of SEQ ID NO: 35 and the antisense strand of SEQ ID NO: 36; The sense strand of SEQ ID NO: 37 and the antisense strand of SEQ ID NO: 38; Sense strand of SEQ ID NO: 39 and antisense strand of SEQ ID NO: 40; Sense strand of SEQ ID NO: 41 and antisense strand of SEQ ID NO: 42; or Sense strand of SEQ ID NO: 43 and antisense strand of SEQ ID NO: 44.

[0103] In some embodiments, the siRNA molecule can be combined in tandem with a second siRNA molecule (e.g., one that targets human c-Myc mRNA) (e.g., non-covalently, or covalently or non-covalently bound and co-administered). In some embodiments, the siRNA molecule that targets human c-Myc mRNA is partially or fully chemically modified. In some embodiments, the c-Myc siRNA comprises a sense strand and an antisense strand, and the siRNA molecule comprises one of the sequence pairs listed in Tables 1 and 2, or a sequence having at least 90% identity thereto, e.g., having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0104] JPEG2025524423000002.jpg141166

[0105] JPEG2025524423000003.jpg201166

[0106] In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotide linkages within the c-Myc targeting siRNA are chemically modified. In some embodiments, the c-Myc targeting siRNA comprises at least one phosphorothioate linkage. In some embodiments, the c-Myc targeting siRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 phosphorothioate linkages. In some embodiments, the targeting siRNA comprises all phosphorothioate linkages.

[0107] Double-stranded RNA molecules or chemically modified siRNA molecules can be constructed using chemical synthesis and enzymatic ligation reactions by procedures known in the art. For example, double-stranded RNA or chemically modified siRNA molecules can be chemically synthesized using natural nucleotides or various modified nucleotides designed to improve the biological stability of the molecule or to improve the physical stability of the double-stranded formed between the double-stranded RNA or chemically modified siRNA molecule and the target nucleotide sequence. For example, phosphorothioate derivatives and acridine-substituted nucleotides can be used. Examples of modified nucleotides that can be used to generate double-stranded RNA or chemically modified siRNA molecules include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueuosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouridine, β-D-mannosylqueuosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouridine, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, double-stranded RNA can be generated using an expression vector in which the nucleic acid encoding the double-stranded RNA has been cloned.

[0108] The double-stranded RNA or chemically modified siRNA molecule may further comprise a nucleotide sequence, wherein at least one or all of the internucleotide bridging phosphate residues are modified phosphates, such as methylphosphonate, methylphosphonothioate, phosphoromorpholidate, phosphoropiperazidate, and phosphoramidate. For example, all or every other internucleotide bridging phosphate residue may be modified as described above. In another non-limiting example, the double-stranded RNA or chemically modified siRNA molecule is a nucleotide sequence in which at least one (or all) of the nucleotides contain a 2'-lower alkyl moiety (e.g., C1-C4, straight or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl). In another example, one or more nucleotides may be 2'-fluoronucleotides, 2'-O-methyl nucleotides, or locked nucleic acid nucleotides. For example, all or every other nucleotide may be modified as described above. See also, Furdon et al., Nucleic Acids Res. 17:9193 (1989); Agrawal et al., Proc. Natl. Acad. Sci. USA 87:1401 (1990); Baker et al., Nucleic Acids Res. 18:3537 (1990); Sproat et al., Nucleic Acids Res. 17:3373 (1989); Walder and Walder, Proc. Natl. Acad. Sci. USA 85:5011 (1988); which are hereby incorporated by reference in their entirety herein as teachings of methods for making polynucleotide molecules including those containing modified nucleobases.

[0109] The present invention further relates to an RNA molecule that is a multivalent chimeric double-stranded RNA or a chemically modified siRNA molecule. In some embodiments, the multivalent chimeric siRNA molecule targets multiple genes (e.g., 2, 3, 4, 5, or more genes), and each siRNA is linked by a metabolically labile linker. In some embodiments, the multivalent chimera includes at least one siRNA that targets IRF4 and at least one siRNA that targets other genes. In some embodiments, the multivalent chimera can be used to deliver two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) siRNAs that target the same gene. The chimera may be formed from two or more double-stranded RNA or chemically modified siRNA molecules linked by a metabolically labile nucleotide linker (e.g., phosphodiester thymine, phosphodiester adenine, phosphodiester TCA linker, etc.), and includes, for example, 2 or more nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides, such as 2 to 6 or 3 to 5 nucleotides.

[0110] In some embodiments, the multivalent chimeric molecule includes a first siRNA and a second siRNA linked by a phosphodiester linker region. In some embodiments, the first and second siRNAs are in the same direction relative to each other (e.g., from 5' to 3'), such as a tandem chimeric molecule. In some embodiments, the first and second siRNAs are in opposite directions relative to each other (e.g., one from 5' to 3' and the other from 3' to 5'), such as an inverse chimeric molecule. In some embodiments, the multivalent chimeric molecule includes a first strand that includes one strand of each of the first siRNA and the second siRNA linked by a linker, a first complementary strand that is at least partially complementary to the strand of the first siRNA, and a second complementary strand that is at least partially complementary to the strand of the second siRNA.

[0111] In certain embodiments, the chimeric molecule comprises an IRF4 siRNA molecule that targets human IRF4 mRNA, a c-Myc siRNA molecule that targets human c-Myc mRNA, and a phosphodiester linker region that links the IRF4 siRNA and the c-Myc siRNA. The siRNA can be configured in a tandem or reverse format.

[0112] In some embodiments, the siRNA comprises, consists essentially of, or consists of a sequence having at least 90% identity with one of SEQ ID NOs: 51-53, for example, having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0113] In some embodiments, the siRNA multivalent chimeric molecule has one or more chemical modifications. In some embodiments, the siRNA multivalent chimeric molecule is fully chemically modified. In some embodiments, each nucleotide within the siRNA multivalent chimeric molecule is modified with a 2'-O-methyl group or a 2'-fluoro group. In some embodiments, the siRNA multivalent chimeric molecule siRNA molecule comprises at least one phosphorothioate bond.

[0114] In some embodiments, the siRNA multivalent chimeric molecule comprises a single strand comprising the sequence of SEQ ID NO: 45, a first complementary strand comprising SEQ ID NO: 46, and a second complementary strand comprising SEQ ID NO: 47.

[0115] The present invention further relates to nucleic acid constructs comprising the RNA molecules of the present invention. The present invention further relates to nucleic acid constructs encoding the RNA molecules of the present invention, and nucleic acid constructs comprising nucleic acid molecules encoding the RNA molecules. In each of these embodiments, the nucleic acid construct can be a vector or plasmid, such as an expression vector.

[0116] Another aspect of the present invention relates to a composition comprising an RNA molecule, a chemically modified siRNA molecule, or a nucleic acid construct of the present invention and another component (e.g., a suitable carrier). In some embodiments, the composition comprises two or more RNA molecules, chemically modified siRNA molecules, or nucleic acid constructs of the present invention, and the two or more RNA molecules or chemically modified siRNA molecules each comprise a different antisense strand. In certain embodiments, the two or more RNA molecules are present on the same nucleic acid construct, on different nucleic acid constructs, or any combination thereof. In some embodiments, the composition is a pharmaceutical composition comprising an RNA molecule(s), a chemically modified siRNA molecule(s), or a nucleic acid construct(s) of the present invention and a pharmaceutically acceptable carrier.

[0117] It is understood that the compositions of the present invention can comprise, consist essentially of, or consist of any combination and in any ratio to each other of any of an RNA molecule, a chemically modified siRNA molecule, and a nucleic acid construct. Further, "two or more" means two, three, four, five, six, seven, eight, nine, ten, etc., up to the total number of RNA molecules, chemically modified siRNA molecules, and nucleic acid constructs of the present invention.

[0118] In some embodiments of the present invention, the composition or pharmaceutical composition further comprises an additional component that facilitates delivery of the RNA molecule(s), chemically modified siRNA molecule(s), or nucleic acid construct(s) of the present invention to a subject (e.g., by enhancing the stability of the RNA molecule(s), chemically modified siRNA molecule(s), or nucleic acid construct(s)). In some embodiments, the additional component can 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 multilamellar liposome or unilamellar liposome). The liposome, multilamellar liposome, or unilamellar liposome can contain any components known in the art suitable for preparing liposomes. In some embodiments, the liposome comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Liposomes can be prepared by methods known in the art (e.g., as described in Pecot et al., Mol. Cancer Ther. 13:2876 (2014), which is incorporated herein by reference in its entirety). In some embodiments, the RNA molecule can be formed within stable nucleic acid lipid particles (SNALPs) (e.g., using particles such as those provided by Arbutus Biopharma (Doylestown, Pennsylvania)). In certain embodiments, the lipid particle comprises, consists essentially of, or consists of: cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), PEG-cDMA or PEG-cDSA, and 1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA) (see Judge et al., J. Clin. Invest. 119:661 (2009)). In some embodiments, the lipid particle comprises two or more RNA molecules or chemically modified siRNA molecules of the present invention.In some embodiments, the additional component is a target delivery site to which an RNA molecule(s), chemically modified siRNA molecule(s), or nucleic acid construct(s) is covalently or non-covalently conjugated, e.g., a ligand, aptamer, or monoclonal antibody.

[0119] The present invention encompasses cells comprising the RNA molecules and / or nucleic acid constructs of the present invention. Thus, in some embodiments, the present invention provides a transformed or transfected cell comprising the RNA molecule and / or nucleic acid construct and / or composition of the present invention, wherein the expression of IRF4 and / or c-Myc is decreased in the transformed or transfected cell as compared to a control cell.

[0120] [Method] Various methods of using the nucleic acid molecules, nucleic acid constructs, and / or compositions of the present invention are provided herein. Thus, in one aspect, the present invention provides a method of inhibiting the expression of the human IRF4 gene, the method comprising contacting a cell with an RNA molecule, chemically modified siRNA molecule, nucleic acid construct, composition, and / or pharmaceutical composition of the present invention, thereby inhibiting the expression of the human IRF4 gene in the cell.

[0121] Also provided herein is a method of treating cancer in a subject in need thereof, wherein the cancer expresses or overexpresses the human IRF4 gene, the method comprising delivering to the subject an RNA molecule, chemically modified siRNA molecule, nucleic acid construct, composition, and / or pharmaceutical composition of the present invention, thereby treating the cancer in the subject. A cancer that expresses or overexpresses the human IRF4 gene is a cancer (e.g., a tumor) in which one or more cells express or overexpress the IRF4 gene. In some embodiments, the cancer also expresses or overexpresses the human c-Myc gene.

[0122] In one embodiment of each of these aspects, the subject can be a subject diagnosed with cancer. In another embodiment, the subject can be a subject at risk of developing cancer (e.g., having a predisposition due to genetic factors, smoking, viral infection, exposure to chemicals, etc.). In a further embodiment, the subject may be a subject identified as expressing or overexpressing the IRF4 gene, and may or may not be diagnosed with cancer. The cancer can be multiple myeloma or other IRF4-related diseases (i.e., diseases caused by the expression or mutation of IRF4).

[0123] The double-stranded RNA or chemically modified siRNA molecule of the present invention can be directly delivered into cells by any method known in the art, such as by transfection or microinjection, for example as part of a composition containing lipid particles. In other embodiments, the double-stranded RNA can be delivered in the form of a polynucleotide encoding the RNA to a subject to generate the expression of the double-stranded RNA in the cells of the subject. Those skilled in the art will understand that the isolated polynucleotide encoding the RNA of the present invention will typically be associated with appropriate expression control sequences, such as transcription / translation control signals and polyadenylation signals.

[0124] Furthermore, it will be understood that various promoter / enhancer elements can be used depending on the desired expression level and tissue-specific expression. The promoter can be constitutive or inducible depending on the desired expression pattern. The promoter can be endogenous or exogenous and can be a natural or synthetic sequence. Exogenous means that the transcription start region is not present in the wild-type host into which it is introduced. The promoter is selected to function within the target cell(s) of interest.

[0125] As an example, an RNA coding sequence can be operably associated with: a cytomegalovirus (CMV) major immediate early promoter, an albumin promoter, an elongation factor 1-α (EF1-α) promoter, a PγK promoter, an MFG promoter, or a Rous sarcoma virus promoter.

[0126] Inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements, and other promoters regulated by exogenously supplied compounds, including but not limited to: the zinc-inducible metallothionein (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (see WO98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA 93:3346 (1996)); the tetracycline repression system (Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547 (1992)); the tetracycline-inducible system (see also Gossen et al., Science 268:1766 (1995); Harvey et al., Curr. Opin. Chem. Biol. 2:512 (1998)); the RU486-inducible system (Wang et al., Nat. Biotech. 15:239 (1997); Wang et al., Gene Ther., 4:432 (1997)); and the rapamycin-inducible system (Magari et al., J. Clin. Invest. 100:2865 (1997)).

[0127] Other tissue-specific promoters or regulatory promoters typically include, but are not limited to, promoters that confer tissue specificity to nerve cells. These include, but are not limited to, the promoters of synapsin 1, tubulin α1, platelet-derived growth factor B chain, tyrosine hydroxylase, neuron-specific enolase, and neurofilament. Skeletal muscle cell promoters include, but are not limited to, the promoters of β-actin, Pitx3, creatine kinase, and myosin light chain. Cardiac muscle cell promoters include, but are not limited to, the promoters of cardiac actin, cardiac troponin T, troponin C, myosin light chain 2, and α-myosin heavy chain. Pancreatic islet (β) cell promoters include, but are not limited to, the promoters of glucokinase, gastrin, insulin, and pancreatic islet amyloid polypeptide.

[0128] Furthermore, efficient translation of the inserted RNA coding sequence generally requires specific initiation signals. These translation control sequences may include the ATG start codon and adjacent sequences and may be of various origins, both natural and synthetic.

[0129] Isolated nucleic acids encoding double-stranded RNA can be incorporated into an expression vector. Expression vectors compatible with various host cells are well known in the art and contain elements suitable for transcription and translation of nucleic acids. Typically, an expression vector contains an "expression cassette" that, in the 5' to 3' direction, includes a promoter, a coding sequence encoding double-stranded RNA operably linked to the promoter, and a termination sequence that may include a termination signal for RNA polymerase and a polyadenylation signal for polyadenylase.

[0130] 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 promoter of the E1A or major late promoter (MLP) gene of adenovirus (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, the baculovirus IE1 promoter, the elongation factor 1α (EF1) promoter, the phosphoglycerate kinase (PGK) promoter, the ubiquitin (Ubc) promoter, the albumin promoter, the mouse metallothionein-L promoter and regulatory sequences of transcriptional control regions, ubiquitous promoters (HPRT, vimentin, α-actin, tubulin, etc.), promoters of intermediate filaments (desmin, neurofilament, keratin, GFAP, etc.), promoters of therapeutic genes (such as MDR, CFTR, or factor VIII type), and pathogenic and / or disease-related promoters. In addition, any of these expression sequences of the present invention can be modified by addition of enhancers and / or regulatory sequences, etc.

[0131] Enhancers that can be used in embodiments of the present invention include, but are not limited to: the SV40 enhancer, the cytomegalovirus (CMV) enhancer, the elongation factor I (EF1) enhancer, yeast enhancers, viral gene enhancers, etc.

[0132] Termination control regions, i.e., terminators or polyadenylation sequences, can be derived from various genes specific to the preferred host. In some embodiments of the present invention, the termination control region can include or be derived from: synthetic sequences, synthetic polyadenylation signals, the SV40 late polyadenylation signal, the SV40 polyadenylation signal, the bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, etc.

[0133] It will be apparent to those skilled in the art that any suitable vector can be used to deliver polynucleotides to cells or a subject. The vector can be delivered in vivo to cells. In other embodiments, the vector may be delivered ex vivo to cells, and then the cells containing the vector are delivered to the subject. The choice of delivery vector can be made based on many factors known in the art, including: the age and species of the target host, comparison of in vitro and in vivo delivery, desired expression level and persistence, intended purpose (e.g., for therapy or screening), target cell or organ, delivery route, size of the isolated polynucleotide, safety concerns, etc.

[0134] Suitable vectors include, but are not limited to: plasmid vectors, viral vectors (e.g., retroviruses, alphaviruses; vaccinia virus; adenoviruses, adeno-associated viruses, and other parvoviruses, lentiviruses, poxviruses, or herpes simplex viruses), lipid vectors, polylysine vectors, synthetic polyaminopolymer vectors, etc.

[0135] Any viral vector known in the art can be used in the present invention. Protocols for generating recombinant viral vectors and for using viral vectors for nucleic acid delivery are described below: 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).

[0136] Non-viral delivery methods can also be used. Many non-viral methods of nucleic acid delivery rely on normal mechanisms used by mammalian cells for macromolecule uptake and intracellular transport. In specific embodiments, non-viral nucleic acid delivery systems rely on the endocytosis pathway for uptake of nucleic acid molecules by target cells. Examples of this type of nucleic acid delivery system include systems derived from liposomes, polylysine conjugates, and artificial virus envelopes.

[0137] In specific 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 tissue. Expression can last for several months, but the number of positive cells is usually low (Wolff et al., Science 247:247 (1989)). Cationic lipids have been shown to assist in the introduction of nucleic acids into cultured cells (Felgner and Ringold, Nature 337:387 (1989)). Injection of cationic lipid plasmid DNA complexes into the circulation system of mice has been shown to result in DNA expression in the lungs (Brigham et al., Am. J. Med. Sci. 298:278 (1989)). One advantage of plasmid DNA is that it can also be introduced into non-proliferating cells.

[0138] In a representative embodiment, a nucleic acid molecule (e.g., a plasmid) may be encapsulated in lipid particles having a positive charge on the surface and optionally tagged with an antibody to a cell surface antigen of the target tissue (Mizuno et al., No Shinkei Geka 20:547 (1992); PCT Publication WO91 / 06309; Japanese Patent Application No. 1047381; and European Patent Publication EP-A-43075).

[0139] Liposomes composed of amphiphilic cationic molecules are useful as non-viral vectors for nucleic acid delivery in vitro and in vivo (reviewed in Crystal, Science 270:404 (1995); Blaese et al., Cancer Gene Ther. 2:291 (1995); Behr et al., Bioconjugate Chem. 5:382 (1994); Remy et al., Bioconjugate Chem. 5:647 (1994); and Gao et al., Gene Therapy 2:710 (1995)). Positively charged liposomes are thought to complex with negatively charged nucleic acids via electrostatic interactions to form lipid: nucleic acid complexes. Lipid: nucleic acid complexes have several advantages as nucleic acid delivery vectors. Unlike viral vectors, lipid: nucleic acid complexes can be used to deliver expression cassettes of essentially unlimited size. Since the complexes lack proteins, they are less likely to cause immunogenic and inflammatory reactions. Furthermore, they cannot replicate or recombine to form infectious agents and have a low integration frequency. Many publications have shown that amphiphilic cationic lipids can mediate nucleic acid delivery in vivo and in vitro (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987); Loeffler et al., Meth. Enzymol. 217:599 (1993); Felgner et al., J. Biol. Chem. 269:2550 (1994)).

[0140] The use of amphiphilic cationic lipid:nucleic acid complexes for in vivo transfection in both animals and humans has been reported by multiple groups (reviewed in Gao et al., Gene Therapy 2:710 (1995); Zhu et al., Science 261:209 (1993); and Thierry et al., Proc. Natl. Acad. Sci. USA 92:9742 (1995)). U.S. Patent No. 6,410,049 describes a method for preparing cationic lipid:nucleic acid complexes with extended shelf life.

[0141] Nuclear localization signals can also be used to facilitate the targeting of double-stranded RNA or an expression vector to the vicinity of the nucleus and / or its entry into the nucleus. Such nuclear localization signals can be proteins or peptides such as the SV40 large T antigen NLS or nucleoplasmin NLS. These nuclear localization signals interact with various nuclear transport factors such as the NLS receptor (karyopherin α) and then interact with karyopherin β.

[0142] Expression vectors can be designed to express double-stranded RNA in prokaryotic or eukaryotic cells. For example, double-stranded RNA can be expressed in bacterial cells such as Escherichia coli (E. coli), insect cells (e.g., baculovirus expression systems), yeast cells, plant cells, or mammalian cells. Some suitable host cells are described in Goeddel, Gene Expression Technology: Further discussed in Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Examples of bacterial vectors include, but are not limited to: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, 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 yeast S. cerevisiae include: pYepSecl (Baldari et al., EMBO J. 6:229 (1987)), pMFa (Kurjan and Herskowitz, Cell 30:933 (1982)), pJRY88 (Schultz et al., Gene 54:113 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Non-limiting examples of baculovirus vectors that can be used to express nucleic acids and produce proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell. Biol. 3:2156 (1983)) and the pVL series (Lucklow and Summers Virology 170:31 (1989)).

[0143] Examples of mammalian expression vectors include the following: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, PBPV, pMSG, PSVL (Pharmacia), pCDM8 (Seed, Nature 329:840 (1987)), and pMT2PC (Kaufman et al., EMBO J. 6:187 (1987)). When used in mammalian cells, the control functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus type 2, cytomegalovirus, and simian virus 40.

[0144] Viral vectors have been used in a variety of applications for gene delivery in cell and in vivo animal subjects. Viral vectors that can be used include, but are not limited to, the following: retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpesvirus, Epstein–Barr virus, adenovirus, geminivirus, and calimovirus vectors. Non-limiting examples of non-viral vectors include the following: plasmid, liposome, charged lipid (Lipofectamine), nucleic acid–protein complex, and biopolymer. In addition to the nucleic acid of interest, the vector may also contain one or more regulatory regions and / or selectable markers useful for selecting, measuring, and monitoring the results of nucleic acid transfer (such as delivery to a specific tissue, duration of expression, etc.).

[0145] In addition to the regulatory control sequences described above, the recombinant expression vector may contain additional nucleotide sequences. For example, the recombinant expression vector may encode a selectable marker gene for identifying host cells into which the vector has been incorporated.

[0146] 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 various techniques recognized in the art for introducing foreign nucleic acids (e.g., DNA and RNA) into host cells, including but not limited to: coprecipitation with calcium phosphate or calcium chloride, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, sonication of cells, gene bombardment using high-velocity microprojectiles, and virus-mediated transfection. Suitable methods for transforming or transfecting host cells are described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989) and other laboratory manuals.

[0147] When stable integration is desired, often only a small fraction of cells (specifically mammalian cells) will integrate foreign DNA into their genome. To identify and select for integrants, a nucleic acid encoding a selectable marker (e.g., resistance to an antibiotic) can be introduced into the host cell along with the nucleic acid of interest. Preferred selectable markers include those conferring resistance to agents such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell in the same vector as the vector containing the nucleic acid of interest or in 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 survive while other cells die).

[0148] In one embodiment, the double-stranded RNA or chemically modified siRNA molecule of the invention is administered directly to a subject. Generally, the compounds of the invention are suspended in a pharmaceutically acceptable carrier (e.g., physiological saline) and administered by oral, topical, or intravenous injection, or injected subcutaneously, intramuscularly, intracranially, intrathecally, intraperitoneally, rectally, vaginally, nasally, intragastrically, intratracheally, or into the lung. These are preferably delivered directly to the site of the disease or disorder, such as the lung, intestine, or pancreas. The required dosage depends on the following: the choice of administration route; the nature of the formulation; the nature of the patient's disease; the size, weight, body surface area, age, and gender of the subject; other drugs being administered; and the judgment of the attending physician. Suitable dosages range from 0.01 to 100.0 μg / kg. Considering that the efficiency varies among different administration routes for the required dosage, significant variations are expected. For example, oral administration is expected to require a higher (e.g., 2, 3, 4, 6, 8, 10, 20, 50, 100, 150, or more times) dosage than administration by intravenous injection. These variations in dosage levels can be adjusted using standard empirical routines for optimization well understood in the art. Administration can be single or multiple. Encapsulating the inhibitor in a suitable delivery medium (e.g., polymeric microparticles or implantable devices) can improve delivery efficiency, especially for oral administration.

[0149] According to certain embodiments, double-stranded RNA or chemically modified siRNA molecules can target specific cells or tissues in vivo. Targeted delivery vehicles, including liposome and viral vector systems, are known in the art. For example, liposomes can target specific cells or tissues to which the targeting molecule can bind by using targeting agents such as antibodies, soluble receptors, or ligands incorporated into the liposome that are directed towards the specific target cell or tissue. Targeted liposomes are described, for example, in: Ho et al., Biochemistry 25:5500 (1986); Ho et al., J. Biol. Chem. 262:13979 (1987); Ho et al., J. Biol. Chem. 262:13973 (1987); and U.S. Patent No. 4,957,735 by Huang et al. (each of which is incorporated herein by reference in its entirety). Enveloped viral vectors can be modified to deliver nucleic acid molecules to target cells by modifying or replacing the envelope protein such that the virus infects a specific cell type. In adenoviral vectors, the gene encoding the attachment fiber can be modified to encode a protein domain that binds to a cell-specific receptor. Herpes viral vectors inherently target cells of the central and peripheral nervous systems. Alternatively, the route of administration can be used to target specific cells or tissues. For example, intracoronary administration of adenoviral vectors has been shown to be effective for gene delivery to cardiomyocytes (Maurice et al., J. Clin. Invest. 104:21 (1999)). Also, intravenous delivery of cholesterol-containing cationic liposomes preferentially targets lung tissue (Liu et al., Nature Biotechnol. 15:167 (1997)) and has been shown to effectively mediate gene transfer and expression in vivo. Other examples of successful in vivo targeted delivery of nucleic acid molecules are also known in the art.Finally, the recombinant nucleic acid molecule can be selectively (i.e., preferentially, substantially exclusively) expressed in target cells by selecting transcriptional control sequences, preferably promoters, which are selectively induced in the target cells and remain substantially inactive in non-target cells.

[0150] The double-stranded RNA or chemically modified siRNA molecules of the present invention may be delivered in combination with other therapeutic agents. The additional therapeutic agent may be delivered concurrently with the double-stranded RNA or chemically modified siRNA molecules of the present invention. As used herein, the term "concurrently" means close enough in time to produce a combined effect (i.e., concurrently may mean simultaneously or two or more events may occur in succession within a short period of time). In one embodiment, the double-stranded RNA or chemically modified siRNA molecules of the present invention are administered in combination with agents useful for, for example, the following cancer treatments: 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 (mitomycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-α); 6) platinum coordination 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) adrenocortical steroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol 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 are administered in combination with, for example, the following anti-angiogenic agents: antibodies against VEGF (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS)) and other angiogenesis promoting factors (e.g., bFGF, angiopoietin-1), antibodies against αv / β3 vascular integrin (e.g., VITAXIN), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNFα conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation (Abraxane), soy isoflavones (genistein), tamoxifen citrate, thalidomide, ADH-1 (EXHERIN), AG-013736, AMG-706, AZD2171, sorafenib tosylate, BMS-582664, CHIR-265, pazopanib, PI-88, batatinib, everolimus, sunitinib, sunitinib malate, XL184, ZD6474, ATN-161, cilenigtide, and celecoxib, or any combination thereof.

[0151] The term "cancer", as used herein, refers to any abnormal proliferation of benign or malignant cells. Examples include, but are not limited to: breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain tumor, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck tumor, breast tumor, ovarian tumor, lung tumor, small cell lung tumor, Wilms tumor, cervical tumor, testicular tumor, bladder tumor, pancreatic tumor, stomach tumor, colon tumor, prostate tumor, urogenital tumor, thyroid tumor, esophageal tumor, myeloma, multiple myeloma, adrenal tumor, renal cell tumor, endometrial tumor, adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid tumor, choriocarcinoma, fungating polypoid tumor, hypercalcemia of malignancy, cervical dysplasia, 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 thrombocythemia, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteosarcoma, primary macroglobulinemia, and retinoblastoma. In some embodiments, the cancer is selected from the group of neoplastic cancers.

[0152] [Pharmaceutical composition] In a further aspect, the present invention provides a pharmaceutical formulation and a method of administering the same for achieving any of the above-described therapeutic effects (e.g., treatment of cancer). The pharmaceutical formulation may contain any of the above-described reagents in a pharmaceutically acceptable carrier.

[0153] "Pharmaceutically acceptable" means 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.

[0154] The formulations of the present invention may include pharmaceutical substances, pharmaceutical materials, carriers, adjuvants, dispersants, diluents, and the like.

[0155] The double-stranded RNA, chemically modified siRNA molecule, or nucleic acid composition of the present invention can be formulated for administration in a pharmaceutical carrier according to known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9th Ed. 1995). In the manufacture of pharmaceutical formulations according to the present invention, the double-stranded RNA or chemically modified siRNA molecule (including its physiologically acceptable salts) is typically admixed with, among other things, an acceptable carrier. The carrier can be solid or liquid, or both, and is preferably formulated with the double-stranded RNA or chemically modified siRNA molecule as a unit dosage formulation, e.g., as a tablet containing from 0.01% or 0.5% to 95% or 99% by weight of the double-stranded RNA or chemically modified siRNA molecule. One or more double-stranded RNA or chemically modified siRNA molecules may be incorporated into the formulations of the present invention, and these can be prepared by any of the techniques well known in pharmacy.

[0156] A further aspect of the present invention is a method of treating a subject in vivo, comprising administering to the subject a pharmaceutical composition comprising the double-stranded RNA or chemically modified siRNA molecule of the present invention in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount. Administration of the double-stranded RNA or chemically modified siRNA molecule of the present invention to a human or animal subject in need thereof can be effected by any means known in the art for administering a compound.

[0157] Non-limiting examples of formulations of the present invention include those suitable for the following: oral, rectal, buccal (e.g., sublingual), vaginal, parenteral (e.g., subcutaneous, intramuscular (including skeletal muscle, cardiac muscle, diaphragmatic muscle, and smooth muscle), intradermal, intravenous, intraperitoneal), topical (i.e., including skin and mucosal surfaces, airway surfaces), intranasal, transdermal, intra-articular, intracranial, intrathecal, and inhalation administration, administration to the liver by portal vein delivery, and 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 the pancreas, or into a tumor or peritumoral tissue). In any case, the most appropriate route depends on the nature and severity of the condition being treated and the nature of the specific compound 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 achieved by direct injection into the desired treatment site, by intravenous introduction into a site close to the desired treatment site (e.g., into a blood vessel supplying the treatment site). In some embodiments, the formulation can be delivered locally to ischemic tissue. In one embodiment, the formulation can be a sustained-release formulation (e.g., in the form of a sustained-release depot).

[0158] In the case of injection, the carrier is typically a liquid, such as pyrogen-free water, pyrogen-free phosphate buffered saline, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, NJ). In the case of other administration methods, the carrier can be either solid or liquid.

[0159] In the case of 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. The compound can be encapsulated within gelatin capsules together with inert ingredients such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, and powdered carriers. Examples of additional inert ingredients that can be added to provide desired color, taste, stability, buffering capacity, dispersibility, or other known desired properties include red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink, and the like. Similar diluents can be used in the manufacture of compressed tablets. Both tablets and capsules can be manufactured as sustained-release products that release the drug continuously over several hours. Compressed tablets can be sugar-coated or film-coated to mask unpleasant tastes and protect the tablets from the atmosphere, or enteric-coated to selectively disintegrate within the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring components to enhance patient acceptability.

[0160] Formulations suitable for buccal (sublingual) administration include lozenges containing the compound in a flavored base (usually sucrose and acacia or tragacanth); and pastilles containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

[0161] Formulations of the invention suitable for parenteral administration include sterile aqueous and non-aqueous injection solutions of the compound, and these preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacteriostats, and solutes that render the preparation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions can contain suspending and thickening agents. The formulations can be provided in single-dose or multi-dose containers (e.g., sealed ampules and vials) and can be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier (e.g., a saline solution or water for injection) immediately prior to use.

[0162] Interim injection solutions and suspensions can be prepared from sterile powders, granules, and tablets as described above. For example, in one aspect of the present invention, an injectable and stable sterile composition containing the compound of the present invention in a unit dosage form in a sealed container is provided. The compound or salt is provided in the form of a lyophilized product that can be reconstituted using a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form typically contains from about 10 mg to about 10 g of the compound or salt. If the compound or salt is substantially water-insoluble, a sufficient amount of a pharmaceutically acceptable emulsifier can be used in an amount sufficient to emulsify the compound or salt in an aqueous carrier. An example of a useful emulsifier is phosphatidylcholine.

[0163] Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These can be prepared by mixing the compound with one or more conventional solid carriers (e.g., cocoa butter) and then shaping the resulting mixture.

[0164] Formulations suitable for topical application to the skin preferably take the form of ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils. Carriers that can be used include petrolatum, lanolin, polyethylene glycol, alcohol, transdermal absorption promoters, and combinations of two or more thereof.

[0165] Formulations suitable for transdermal administration can be presented as individual patches adapted to remain in intimate contact with the recipient's epidermis for an extended period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, e.g., Tyle, Pharm. Res. 3:318 (1986)) and typically take the form of an aqueous solution of the compound, optionally buffered. Suitable formulations contain citric acid or Bis-Tris buffer (pH 6) or ethanol / water and contain 0.1 - 0.2 M of the compound.

[0166] The compound may alternatively be formulated for nasal administration or administered to the lungs of a subject by any suitable means (e.g., by aerosol suspension of inhalable particles containing the compound which the subject inhales). The inhalable particles can be liquid or solid. The term "aerosol" includes any gas-borne suspension phase that is inhalable into the bronchus or nasal cavity. Specifically, aerosols include gas-borne suspensions of droplets that can be generated in a metered dose inhaler or nebulizer or in a mist spray. Aerosols also include dry powder compositions suspended in air or other carrier gas that can be delivered, for example, by the airstream from an inhalation device. See Ganderton & Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313; and Raeburn et al., J. Pharmacol. Toxicol. Meth. 27:143 (1992). Aerosols of liquid particles containing the compound can be generated by any suitable means, such as a pressure-driven aerosol nebulizer or ultrasonic nebulizer, as known to those of skill in the art. See, for example, U.S. Patent No. 4,501,729. Similarly, aerosols of solid particles containing the compound can be generated using any solid particle pharmaceutical aerosol generator by techniques known in the pharmaceutical art.

[0167] Alternatively, the compound can be administered locally rather than systemically, for example, in the form of a depot or sustained release formulation.

[0168] Furthermore, the present invention provides liposomal formulations of the compounds and salts thereof disclosed herein. Techniques for forming liposomal suspensions are well known in the art. When the compound or its salt is a water-soluble salt, by using conventional liposome techniques, these can be incorporated into lipid vesicles. In such cases, due to the water solubility of the compound or salt, the compound or salt is substantially loaded within the hydrophilic center or core of the liposome. The lipid layer used may be any conventional composition, with or without cholesterol. Even when the compound or salt of interest is water-insoluble, by using conventional liposome-forming techniques, the salt can be substantially loaded within the hydrophobic lipid bilayer that forms the structure of the liposome. In either case, the resulting liposomes can be reduced in size by using standard sonication and homogenization techniques.

[0169] The liposomal formulation containing the compound or its salt disclosed herein can be lyophilized to produce a lyophilized product, which can be reconstituted with a pharmaceutically acceptable carrier such as water to regenerate the liposomal suspension.

[0170] In the case of water-insoluble compounds, for example, a pharmaceutical composition containing the water-insoluble compound can be prepared in an aqueous base emulsion. In such cases, the composition contains a pharmaceutically acceptable emulsifier in an amount sufficient to emulsify the desired amount of the compound. Particularly useful emulsifiers include phosphatidylcholine and lecithin.

[0171] In a specific embodiment, the compound is administered to a subject in a therapeutically effective amount (as defined above). The dosage of the pharmaceutically active compound can be determined by methods known in the art. See, for example, Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa). The therapeutically effective dosage of a particular compound varies somewhat for each compound and for each patient, depending on the patient's condition and the route of delivery. Generally speaking, dosages of about 0.001 to about 50 mg / kg have a therapeutic effect, and all weights are calculated based on the weight of the compound, including when salts are used. Due to concerns about toxicity at higher levels, intravenous administration can be limited to lower levels, for example up to about 10 mg / kg, and all weights are calculated based on the weight of the compound, including when salts are used. For oral administration, dosages of about 10 mg / kg to about 50 mg / kg can be used. Typically, for intramuscular injection, dosages of about 0.5 mg / kg to 5 mg / kg can be used. The specific dosage is about 1 μmol / kg to 50 μmol / kg, and more specifically, the compound is up to about 22 μmol / kg for intravenous administration and up to 33 μmol / kg for oral administration.

[0172] In a specific embodiment of the present invention, to achieve a therapeutic effect, multiple administrations (e.g., 2, 3, 4, or more administrations) can be used at various time intervals (e.g., hourly, daily, weekly, monthly, etc.).

[0173] The present invention is used in veterinary and medical applications. Suitable subjects include both avians and mammals, with mammals being preferred. As used herein, the term "avian" includes, but is not limited to, chickens, ducks, geese, quails, turkeys, and pheasants. As used herein, the term "mammal" includes, but is not limited to, humans, cows, sheep, goats, horses, cats, dogs, rabbits, etc. Human subjects include neonates, infants, children, and adults. In other embodiments, the subject is a cancer animal model. In certain embodiments, the subject has cancer or is at risk of cancer.

[0174] The following examples are not intended to limit the scope of the claims of the present invention, but rather are intended to illustrate certain embodiments. It is intended that any variations of the exemplified methods that occur to those skilled in the art be included within the scope of the present invention. As will be understood by those skilled in the art, there are multiple embodiments and elements for each aspect of the claimed invention, and all combinations of different elements are contemplated herein, so the specific combinations exemplified herein should not be construed as limiting the scope of the claimed invention. When specific elements are removed from or added to a group of combinable elements, that group of elements should be construed as incorporating such changes.

Examples

[0175] [Example 1: Design of IRF4 siRNA] IRF4 is often overexpressed in malignant (i.e., MM - progressed) plasma cells, and pre - clinical models have shown that suppressing IRF4 reduces the in - vitro survival rate of MM cells (Figure 1). The heatmap at the bottom of Figure 1 shows Demeter2 - dependent data (the more negative the value, the higher the dependence), revealing that MM cells are often highly dependent on IRF4. To develop a therapeutic agent that inhibits IRF4 expression, the complete sequence of IRF4 (ORF + UTR) was input into multiple online databases to design siRNAs. However, there was little overlap between the results obtained from different databases. Therefore, a scoring system / ranking variable was created that included the following variables: GC content (30 - 55%), the low level of BLAST hits, appearance in multiple databases, and homology with the mouse IRF4 sequence. Twelve siRNAs with a maximum total score > 6 were selected. The selected siRNAs target both the ORF and UTR sequences (Figure 2). The sequences are shown in Table 3.

[0176] JPEG2025524423000004.jpg180166

[0177] Twelve siRNAs were tested for their ability to inhibit IRF4 expression in RPMI 8226 myeloma cells. As a result, it was shown that almost all siRNAs could inhibit IRF4 expression (Figure 3). Furthermore, it was evaluated whether the selected fully chemically modified siRNAs could inhibit the expression of both IRF4 and c - Myc over time (Figure 4). A decrease in the protein levels of IRF4 and c - Myc was detected by immunoblotting (Figure 5).

[0178] [Example 2: Synthetic chemically modified siRNA reduces c - IRF4 protein expression] Seven out of 12 IRF4 siRNAs were selected and fully chemically modified using the Hi2F pattern. This pattern consists of an approximate 50 / 50 mixture of 2′-fluoro (2′F) and 2′-O-methyl (2′OMe) ribose modifications (Figure 6). The sequences are disclosed in Table 4. Hi2F siRNA was able to decrease the mRNA levels of IRF4 and c-Myc over time in RPMI-8229 cells (Figure 7), which is consistent with IRF4 having a feed-forward regulatory loop on c-Myc expression. Furthermore, siRNAs against IRF4, c-Myc, or their combination decreased the mRNA levels of IRF4 and c-Myc (Figure 8). The main data of this experiment are shown in Figure 9. Collectively, these data indicate that multiple effective chemically modified IRF4 siRNAs with the Hi2F pattern were identified.

[0179] JPEG2025524423000005.jpg219166

[0180] Similar experiments using KMS-11 myeloma cells showed that siRNAs against IRF4 and c-Myc decreased the mRNA levels of IRF4 and c-Myc over time (Figure 10). This decrease was reflected in a time-dependent decrease in protein levels (Figure 11). These data indicate that IRF4 siRNA works in multiple MM cell lines.

[0181] The effect of IRF4 siRNA on cell viability was tested in RPMI-8226 cells. As a result, it was shown that the cell viability on day 5 decreased in a dose-dependent manner (Figure 12). c-Myc siRNA also showed a similar effect on cell viability (Figure 13). A similar decrease in cell viability by IRF4 siRNA was also seen in KMS-11 cells (Figure 14).

[0182] Three out of 12 IRF4 siRNAs were selected and fully chemically modified using the HiOMe pattern. This pattern consists of a majority of 2′OMe ribose modifications and minimal 2′F modifications, aiming to improve stability in vivo and avoid nuclease degradation (Figure 15). The sequences are disclosed in Table 4. The HiOMe-modified siRNAs had a similar effect on the siRNA effect as the Hi2F modification (Figure 16). This similarity was also seen in protein expression (Figures 17 and 18) and cell viability (Figures 19 and 20, and Table 5). These data indicate that the more stabilized HiOMe pattern still has a high level of activity in the silencing of IRF4 at the transcript and protein levels, and in the inhibition of cell viability.

[0183] JPEG2025524423000006.jpg60166

[0184] Using apoptosis and cell proliferation assays in RPMI-8226 cells, IRF4 siR10-HiOMe was shown to cause cytotoxicity (Figures 21 and 22). Similarly, IRF4 siR10 Hi2F and HiOMe siRNAs increased cytotoxicity and decreased viability in the spheroids of RPMI-8226 cells (Figure 23). IRF4 siR10-HiOMe also inhibited agarose colony formation in RPMI-8226 cells (Figure 24). These data indicate that the IRF4 siR10 Hi2F and HiOMe compositions have potent activity in inhibiting the MM phenotype.

[0185] [Example 3: Inverted chimeras combining c-Myc and IRF4-targeting siRNAs] An inverted chimera was designed in which Myc HiOMe siRNA and IRF4 siR10 HiOMe siRNA were separated and linked by a cleavable DNA bridge (Figures 26 and Table 6). This chimera had a significantly improved effect on the viability of RPMI-8226 cells compared to the individual siRNAs (Figures 26 and Table 7), GI 50resulted in a significant leftward shift and showed a substantial improvement in potency. This chimera induced cytotoxicity in RPMI-8226 cells (Figure 27). Collectively, the inverted chimeras targeting both Myc and IRF4 have synergistic activity in the targeted MM cells.

[0186] JPEG2025524423000007.jpg108166

[0187] JPEG2025524423000008.jpg36166

[0188] All publications, patents, and patent applications are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0189] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but it will be apparent that certain changes and modifications may be practiced within the scope of the foregoing embodiments and the appended claims listing.

Claims

1. A siRNA molecule that targets human interferon regulator-4 (IRF4) mRNA, The siRNA molecule contains at least one chemical modification, and The aforementioned siRNA molecule is one of the following sequence pairs: The sense chain of sequence number 1 and the antisense chain of sequence number 2; The sense strand of sequence number 3 and the antisense strand of sequence number 4; The sense strand of sequence number 5 and the antisense strand of sequence number 6; The sense strand of sequence number 7 and the antisense strand of sequence number 8; The sense strand of sequence number 9 and the antisense strand of sequence number 10; The sense strand of sequence number 11 and the antisense strand of sequence number 12; The sense strand of sequence number 13 and the antisense strand of sequence number 14; The sense strand of sequence number 15 and the antisense strand of sequence number 16; The sense strand of sequence number 17 and the antisense strand of sequence number 18; The sense strand of sequence number 19 and the antisense strand of sequence number 20; The sense chain of sequence number 21 and the antisense chain of sequence number 22; The sense strand of sequence number 23 and the antisense strand of sequence number 24; or Sequences having at least 90% identity with these including, siRNA molecule.

2. The siRNA molecule described in claim 1, Completely chemically modified, siRNA molecule.

3. The siRNA molecule described in claim 2, Each nucleotide within the siRNA molecule is modified with a 2'-O-methyl group or a 2'-fluoro group. siRNA molecule.

4. The siRNA molecule described in claim 1, The siRNA molecule contains at least one phosphorothioate bond. siRNA molecule.

5. The siRNA molecule described in claim 4, The aforementioned siRNA molecule is one of the following sequence pairs: The sense strand of sequence number 25 and the antisense strand of sequence number 26; The sense strand of sequence number 27 and the antisense strand of sequence number 28; The sense strand of sequence number 29 and the antisense strand of sequence number 30; The sense strand of sequence number 31 and the antisense strand of sequence number 32; The sense chain of sequence number 33 and the antisense chain of sequence number 34; The sense strand of sequence number 35 and the antisense strand of sequence number 36; The sense strand of sequence number 37 and the antisense strand of sequence number 38; The sense strand of sequence number 39 and the antisense strand of sequence number 40; The sense strand of sequence number 41 and the antisense strand of sequence number 42; or The sense strand of sequence number 43 and the antisense strand of sequence number 44; including, siRNA molecule.

6. A composition comprising the siRNA described in claim 1.

7. A composition comprising two or more siRNAs described in claim 1 in any combination, The two or more siRNAs each contain different sequences, composition.

8. A composition according to claim 6 or 7, Further containing nanoparticles, composition.

9. The composition according to claim 8, The aforementioned nanoparticles are nanoliposomes. composition.

10. A pharmaceutical composition comprising the siRNA described in claim 1 and / or the composition described in claim 6, and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition according to claim 10, The siRNA is conjugated to a ligand, antibody, or aptamer. Pharmaceutical composition.

12. A pharmaceutical composition according to claim 10 for inhibiting intracellular expression of the human IRF4 gene.

13. A pharmaceutical composition according to claim 10 for treating cancer in a subject requiring it, The aforementioned cancer expresses the human IRF4 gene, Pharmaceutical composition.

14. A pharmaceutical composition according to claim 13, The aforementioned cancer is multiple myeloma or other IRF4-related disease. Pharmaceutical composition.

15. A pharmaceutical composition according to claim 13, It is delivered to the whole body. Pharmaceutical composition.

16. A pair of siRNA molecules comprising a c-Myc siRNA molecule targeting human c-Myc mRNA and an IRF4 siRNA molecule targeting human IRF4 mRNA, The siRNA molecule contains at least one chemical modification, and The aforementioned IRF4 siRNA molecule is one of the following sequence pairs: The sense chain of sequence number 1 and the antisense chain of sequence number 2; The sense strand of sequence number 3 and the antisense strand of sequence number 4; The sense strand of sequence number 5 and the antisense strand of sequence number 6; The sense strand of sequence number 7 and the antisense strand of sequence number 8; The sense strand of sequence number 9 and the antisense strand of sequence number 10; The sense strand of sequence number 11 and the antisense strand of sequence number 12; The sense strand of sequence number 13 and the antisense strand of sequence number 14; The sense strand of sequence number 15 and the antisense strand of sequence number 16; The sense strand of sequence number 17 and the antisense strand of sequence number 18; The sense strand of sequence number 19 and the antisense strand of sequence number 20; The sense chain of sequence number 21 and the antisense chain of sequence number 22; The sense strand of sequence number 23 and the antisense strand of sequence number 24; or Sequences having at least 90% identity with these including, A pair of siRNA molecules.

17. A pair of siRNA molecules according to claim 16, Completely chemically modified, A pair of siRNA molecules.

18. A pair of siRNA molecules according to claim 17, Each nucleotide within the siRNA molecule is modified with a 2'-O-methyl group or a 2'-fluoro group. A pair of siRNA molecules.

19. A pair of siRNA molecules according to claim 16, The siRNA molecule contains at least one phosphorothioate bond. A pair of siRNA molecules.

20. A pair of siRNA molecules according to claim 19, The aforementioned IRF4 siRNA molecule is one of the following sequence pairs: The sense strand of sequence number 25 and the antisense strand of sequence number 26; The sense strand of sequence number 27 and the antisense strand of sequence number 28; The sense strand of sequence number 29 and the antisense strand of sequence number 30; The sense strand of sequence number 31 and the antisense strand of sequence number 32; The sense chain of sequence number 33 and the antisense chain of sequence number 34; The sense strand of sequence number 35 and the antisense strand of sequence number 36; The sense strand of sequence number 37 and the antisense strand of sequence number 38; The sense strand of sequence number 39 and the antisense strand of sequence number 40; The sense strand of sequence number 41 and the antisense strand of sequence number 42; or The sense strand of sequence number 43 and the antisense strand of sequence number 44; including, A pair of siRNA molecules.

21. A pair of siRNA molecules according to claim 19, The aforementioned c-Myc siRNA molecule is one of the following sequence pairs: The sense strand of sequence number 48 and the antisense strand of sequence number 49; The sense strand of sequence number 50 and the antisense strand of sequence number 51; The sense strand of sequence number 52 and the antisense strand of sequence number 53; The sense strand of sequence number 54 and the antisense strand of sequence number 55; The sense strand of sequence number 56 and the antisense strand of sequence number 57; The sense strand of sequence number 58 and the antisense strand of sequence number 59; The sense strand of sequence number 60 and the antisense strand of sequence number 61; The sense strand of sequence number 62 and the antisense strand of sequence number 63; The sense strand of sequence number 64 and the antisense strand of sequence number 65; The sense strand of sequence number 66 and the antisense strand of sequence number 67; The sense strand of sequence number 68 and the antisense strand of sequence number 69; The sense strand of sequence number 70 and the antisense strand of sequence number 71; The sense strand of sequence number 72 and the antisense strand of sequence number 73; The sense strand of sequence number 74 and the antisense strand of sequence number 75; The sense strand of SEQ ID NO: 76 and the antisense strand of SEQ ID NO: 77; or The sense strand of sequence number 78 and the antisense strand of sequence number 79; including, A pair of siRNA molecules.

22. A composition comprising the siRNA molecule pair described in claim 16.

23. A composition comprising, in any combination, at least one c-Myc siRNA molecule targeting human c-Myc and two or more IRF4 siRNA molecules targeting human IRF4, as described in claim 16, The two or more IRF4 siRNAs each contain different sequences. composition.

24. A composition according to claim 22 or 23, Further containing nanoparticles, composition.

25. The composition according to claim 24, The aforementioned nanoparticles are nanoliposomes. composition.

26. A pharmaceutical composition comprising an siRNA molecule pair as described in claim 16 and / or the composition as described in claim 22, and a pharmaceutically acceptable carrier.

27. A pharmaceutical composition according to claim 26, At least one siRNA is conjugated to a ligand, antibody, or aptamer. Pharmaceutical composition.

28. A pharmaceutical composition according to claim 26 for inhibiting intracellular expression of the human IRF4 gene.

29. A pharmaceutical composition according to claim 26 for treating cancer in a subject requiring it, The aforementioned cancer expresses the human IRF4 gene, Pharmaceutical composition.

30. A pharmaceutical composition according to claim 29, The aforementioned cancer is multiple myeloma. Pharmaceutical composition.

31. A pharmaceutical composition according to claim 29, It is delivered to the whole body. Pharmaceutical composition.

32. It is a siRNA polyvalent chimeric molecule: IRF4 siRNA molecules that target human IRF4 mRNA; A c-Myc siRNA molecule that targets human c-Myc mRNA; and A phosphodiester linker region that links the IRF4 siRNA and the c-Myc siRNA; including, siRNA polyvalent chimeric molecule.

33. A siRNA polyvalent chimeric molecule as described in claim 32, The IRF4 siRNA and the c-Myc siRNA are oriented in the same direction relative to each other. siRNA polyvalent chimeric molecule.

34. A siRNA polyvalent chimeric molecule as described in claim 32, The IRF4 siRNA and the c-Myc siRNA are in opposite directions relative to each other. siRNA polyvalent chimeric molecule.

35. A siRNA polyvalent chimeric molecule as described in claim 32, The siRNA polyvalent chimeric molecule has one or more chemical modifications. siRNA polyvalent chimeric molecule.

36. A siRNA polyvalent chimeric molecule as described in claim 35, The IRF4 siRNA and the c-Myc siRNA are completely chemically modified. siRNA polyvalent chimeric molecule.

37. A siRNA polyvalent chimeric molecule as described in claim 36, Each nucleotide in the IRF4 siRNA and the c-Myc siRNA is modified with a 2'-O-methyl group or a 2'-fluoro group. siRNA polyvalent chimeric molecule.

38. A siRNA polyvalent chimeric molecule as described in claim 32, The siRNA molecule contains at least one phosphorothioate bond. siRNA polyvalent chimeric molecule.

39. A siRNA polyvalent chimeric molecule as described in claim 38, The siRNA polyvalent chimeric molecule comprises a single strand containing the sequence of SEQ ID NO: 45, a first complementary strand containing SEQ ID NO: 46, and a second complementary strand containing SEQ ID NO:

47. siRNA polyvalent chimeric molecule.

40. A composition comprising the siRNA described in claim 32.

41. The composition according to claim 40, Further containing nanoparticles, composition.

42. The composition according to claim 41, The aforementioned nanoparticles are nanoliposomes. composition.

43. A pharmaceutical composition comprising the siRNA described in claim 32 and / or the composition described in claim 40, and a pharmaceutically acceptable carrier.

44. A pharmaceutical composition according to claim 43, The siRNA is conjugated to a ligand, antibody, or aptamer. Pharmaceutical composition.

45. A pharmaceutical composition according to claim 43 for inhibiting intracellular expression of the human IRF4 gene.

46. A pharmaceutical composition according to claim 43 for treating cancer in a subject requiring it, The aforementioned cancer expresses the human IRF4 gene, Pharmaceutical composition.

47. A pharmaceutical composition according to claim 46, The aforementioned cancer is multiple myeloma. Pharmaceutical composition.

48. A pharmaceutical composition according to claim 46, It is delivered to the whole body. Pharmaceutical composition.

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