Compositions and methods for inhibiting expression of apolipoprotein C-III (APOC3)

Abstract

Double-stranded ribonucleic acid (dsRNA) that targets the APOC3 gene and methods for inhibiting APOC3 expression using dsRNA are provided. [Solution] We provide a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the APOC3 gene, the dsRNA comprising a sense strand consisting of a specific nucleotide sequence and an antisense strand consisting of another specific nucleotide sequence (AD-45149.1UM).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 61 / 499,620, filed June 23, 2011, which is incorporated herein by reference in its entirety.

[0002] Array list reference This application contains a sequence listing that has been submitted electronically as a text file named 11111US_sequencelisting.txt, created on XX / XX / 201X, and having a size of X00,000 bytes. The sequence listing is incorporated by reference.

[0003] The present invention relates to double-stranded ribonucleic acid (dsRNA) that targets the APOC3 gene and methods for inhibiting APOC3 expression using this dsRNA. [Background technology]

[0004] In the United States, 30% of adults have elevated triglycerides (TG) >150 mg / dL. The prevalence of adults with severe hypertriglyceridemia (TG >500 mg / dL) is 1.7%. Current treatments include lifestyle changes (diet, exercise, and smoking cessation), prescription-grade fish oil, fibrates, and niacin.

[0005] ApoC3 is a secreted hepatic protein that has been shown to inhibit lipoprotein lipase, which hydrolyzes TGs into free fatty acids; inhibit ApoE-mediated hepatic uptake of TG-rich lipoproteins via LDLR and LRP and receptor-independent endocytosis; and promote hepatic VLDL secretion. At least one mutation in the human APOC3 gene has been associated with a favorable lipid profile (Non-Patent Document 1).

[0006] Double-stranded RNA molecules (dsRNA) have been shown to block gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). Patent document 1 (Fire et al.) discloses the use of dsRNA of at least 25 nucleotides in length to inhibit gene expression in nematodes. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, for example, Patent document 2, Waterhouse et al.; and Patent document 3, Heifetz et al.), Drosophila (see, for example, Non-Patent document 2), and mammals (see Patent document 4, Limmer; and Patent document 5, Kreutzer et al.). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 99 / 32619 Brochure [Patent Document 2] International Publication No. 99 / 53050 Brochure [Patent Document 3] International Publication No. 99 / 61631 Brochure [Patent Document 4] International Publication No. 00 / 44895 Brochure [Patent Document 5] German Patent No. 10100586.5 [Non-patent literature]

[0008] [Non-Patent Document 1] Pollin TI et al.(2008)A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection.Science.322(5908):1702-5 [Non-patent document 2] Yang, D., et al., Curr. Biol. (2000) 10:1191-1200 Summary of the Invention [Means for solving the problem]

[0009] Disclosed herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of the APOC3 gene, the dsRNA comprising a sense strand and an antisense strand, each having a length of 30 nucleotides or less, wherein the antisense strand comprises at least 15 contiguous nucleotides of an antisense sequence in Table 1, 2, 6, 7, or 10. In one embodiment, the dsRNA comprises a sense strand consisting of the nucleotide sequence SEQ ID NO: 70 and an antisense strand consisting of the nucleotide sequence SEQ ID NO: 151 (AD-45149.1UM). In another embodiment, the sense strand sequence is selected from Table 1, 2, 6, 7, or 10, and the antisense strand is selected from Table 1, 2, 6, 7, or 10.

[0010] In some embodiments, at least one nucleotide of the dsRNA is a modified nucleotide, for example, the at least one modified nucleotide is selected from the group consisting of 2'-O-methyl modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base containing nucleotides.

[0011] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide, or each strand comprises a 3' overhang of 2 nucleotides.

[0012] Any dsRNA of the present invention may further comprise a ligand, for example, a ligand conjugated to the 3' end of the sense strand of the dsRNA. In some embodiments, the dsRNA of the present invention further comprises at least one N-acetyl-galactosamine.

[0013] Additionally, the present invention provides cells comprising any of the dsRNAs of the present invention; vectors encoding at least one strand of any of the dsRNAs of the present invention, and cells comprising the vectors.

[0014] The present invention also includes a pharmaceutical composition for inhibiting the expression of the APOC3 gene, comprising any of the dsRNAs of the present invention. The pharmaceutical composition may comprise a lipid formulation, for example, a lipid formulation containing MC3.

[0015] Another aspect of the present invention is a method for inhibiting APOC3 expression in cells, the method comprises: (a) contacting cells with the APOC3 dsRNA of the present invention; and (b) maintaining the cells produced in step (a) for a sufficient time to obtain degradation of the mRNA transcript of APOC3 gene, thereby inhibiting the expression of APOC3 gene in cells.In some embodiments, APOC3 expression is inhibited by at least 30%.

[0016] A further aspect of the present invention is a method for treating a disease mediated by APOC3 expression, comprising administering a therapeutically effective amount of an APOC3 dsRNA of the present invention or a pharmaceutical composition of the present invention to a human in need of such treatment. The disease may be, for example, elevated triglyceride levels, e.g., triglyceride levels >150 mg / dL or >500 mg / dL. In some embodiments, the administration results in increased lipoprotein lipase and / or hepatic lipase activity. The dsRNA or pharmaceutical composition may be administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg. [Brief explanation of the drawings]

[0017] [Figure 1]1 is a graph showing the effects on target mRNA, triglyceride (TG), and total cholesterol levels in mice after treatment with siRNAs targeting APOC3 ("siRNA#1" and siRNA#2"). [Figure 2] The structure of GalNAc is shown. [Figure 3] 1 shows the structure of siRNA conjugated to Chol-p-(GalNAc)3 via a phosphate bond at the 3' end. [Figure 4] The structure of siRNA conjugated to LCO(GalNAc)3 ((GalNAc)3-3'-lithochol-oleoyl siRNA conjugate) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0018] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0019] The present invention provides dsRNA and methods for using the dsRNA to inhibit expression of the APOC3 gene in cells or mammals, where the dsRNA targets the APOC3 gene. The present invention also provides compositions and methods for treating mammalian pathological conditions and diseases caused by expression of the APOC3 gene. The APOC3 dsRNA directs sequence-specific degradation of APOC3 mRNA.

[0020] definition For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. If there is an apparent discrepancy between the use of a term in other parts of this specification and its definition provided in this section, the definition in this section shall control.

[0021] "G," "C," "A," and "U" generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. "T" and "dT" are used interchangeably herein to refer to deoxyribonucleotides in which the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the terms "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to modified nucleotides or surrogate replacement moieties, as described in more detail below. Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base-pairing properties of oligonucleotides containing nucleotides with such replacement moieties. For example, and without limitation, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine may be substituted with, for example, a nucleotide containing inosine within the nucleotide sequences of the present invention. Sequences containing such replacement moieties are embodiments of the present invention.

[0022] "APOC3" refers to the apolipoprotein C-III gene. According to the NCBI NLM website, apolipoprotein C-III is a very low-density lipoprotein (VLDL) protein. APOC3 inhibits lipoprotein lipase and hepatic lipase; it is thought to slow the catabolism of triglyceride-rich particles. The APOA1, APOC3, and APOA4 genes are closely related in both the rat and human genomes. The AI ​​and A-IV genes are transcribed from the same strand, while the A-1 and C-III genes are transcribed convergently. Elevated APOC-III levels lead to the development of hypertriglyceridemia. The human APOC3 mRNA sequence has GenBank accession number NM_000040.1 and is included herein as SEQ ID NO: 1. The cynomolgus monkey (Macaca fascicularis) ANGPTL3 mRNA sequence has GenBank accession number X68359.1.

[0023] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the APOC3 gene, including mRNA that is the product of RNA processing of a primary transcript.

[0024] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence designated using standard nucleotide nomenclature.

[0025] The term "complementary," as used herein, unless otherwise indicated, when describing a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a double-stranded structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under defined conditions, as would be understood by one of skill in the art.

[0026] For example, a first nucleotide sequence can be described as complementary to a second nucleotide sequence if the two sequences hybridize (e.g., anneal) under stringent hybridization conditions. Hybridization conditions include temperature, ionic strength, pH, and organic solvent concentration for the annealing and / or washing steps. The term stringent hybridization conditions refers to conditions under which a first nucleotide sequence hybridizes preferentially to its target sequence, e.g., a second nucleotide sequence, and hybridizes to a lesser extent or not at all to other sequences. Stringent hybridization conditions are sequence-dependent and vary under different environmental parameters. Generally, stringent hybridization conditions are determined by the thermal melting point (T) of a nucleotide sequence at a defined ionic strength and pH. m ) is chosen to be approximately 5°C lower than T mis the temperature (under defined ionic strength and pH) at which 50% of a first nucleotide sequence hybridizes to a perfectly matched target sequence. Extensive guidance on nucleic acid hybridization is found, for example, in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part I, chap. 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier, NY (“Tijssen”).

[0027] Other conditions can be applied, such as physiologically relevant conditions that may be encountered internally. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate use of the hybridized nucleotides.

[0028] This involves base pairing an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of the first and second nucleotide sequences. Such sequences may be referred to herein as "fully complementary" with respect to each other. However, when a first sequence is referred to herein as "substantially complementary" with respect to a second sequence, the two sequences may be perfectly complementary, or may form one or more, but not more than four, three, or two mismatched base pairs after hybridization while retaining the ability to hybridize under conditions most relevant to their end use. However, if two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered mismatches for purposes of determining complementarity. For example, for purposes described herein, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length can still be referred to as "fully complementary" if the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the sequence of the shorter oligonucleotide.

[0029] As used herein, "complementary" sequences may also include or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, so long as the above requirements related to their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairs.

[0030] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used in reference to matching bases between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as understood from the context of their use.

[0031] As used herein, a polynucleotide "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding APOC3), including the 5' UTR, open reading frame (ORF), or 3' UTR. For example, a polynucleotide is complementary to at least a portion of an APOC3 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding APOC3.

[0032] In one embodiment, the antisense strand of the dsRNA is sufficiently complementary to the target mRNA so as to result in cleavage of the target mRNA.

[0033] The term "double-stranded RNA" or "dsRNA" as used herein refers to a complex of ribonucleic acid molecules, which has a double-stranded structure and comprises two antiparallel and substantially complementary nucleic acid strands as defined above. Generally, the majority of nucleotides in each strand are ribonucleotides, but as described in detail herein, each or both strands may also comprise at least one non-ribonucleotide, such as deoxyribonucleotide and / or modified nucleotide. In addition, as used herein, "dsRNA" may also comprise chemical modifications to ribonucleotides, including corresponding modifications in many nucleotides, and also includes all types of modifications disclosed herein or known in the art. Any such modifications used in siRNA-type molecules are encompassed by "dsRNA" for the purposes of this specification and claims.

[0034] The two strands forming the double-stranded structure may be different parts of one larger RNA molecule, or may be separate RNA molecules. When the two strands are part of one larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of the corresponding other strand, the connected RNA strand is called a "hairpin loop." When the two strands are covalently connected by means other than an uninterrupted chain of nucleotides between the 3' end of one strand forming the double-stranded structure and the 5' end of the corresponding other strand, the connected structure is called a "linker." The RNA strands may have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the double strand. In addition to the double-stranded structure, the dsRNA may also contain one or more nucleotide overhangs. The term "siRNA" may also be used to refer to the above-mentioned dsRNA.

[0035] As used herein, "nucleotide overhang" refers to an unpaired nucleotide or nucleotides that protrude from the double-stranded structure of a dsRNA, such as when the 3'-end of one strand of the dsRNA extends beyond the 5'-end of the other strand, or vice versa. "Blunt" or "blunt-ended" means that there are no unpaired nucleotides at the end of the dsRNA, i.e., there are no nucleotide overhangs. A "blunt-ended" dsRNA is a dsRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule.

[0036] The term " antisense strand " refers to the strand of dsRNA that comprises a region that is substantially complementary to target sequence.The term " complementary region " used herein refers to the region on the antisense strand that is substantially complementary to sequence, for example, target sequence, as defined herein.If the complementary region is not completely complementary to target sequence, mismatch is most tolerated in terminal region, and if present, it is generally in the terminal region, for example, 6, 5, 4, 3 or 2 nucleotides from the 5' and / or 3' end.

[0037] As used herein, the term "sense strand" refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0038] The term "nucleic acid-lipid particle" as used herein includes the term "SNALP" and refers to a lipid vesicle that coats a reduced aqueous interior and contains a nucleic acid, such as dsRNA or a plasmid from which dsRNA is transcribed. Nucleic acid-lipid particles, such as SNALP, are described, for example, in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and U.S. Patent Application No. 61 / 045,228, filed April 15, 2008. These applications are incorporated herein by reference.

[0039] "Introducing into a cell," when referring to dsRNA, means facilitating uptake or absorption into the cell, as understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unassisted diffusion processes or active cellular processes, or can occur with auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; dsRNA can also be "introduced into a cell" when the cell is part of a living organism. In such cases, introduction into a cell would include delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. Introduction into a cell in vitro includes methods known in the art, such as electroporation and lipofection. Additional techniques are described herein or known in the art.

[0040] The terms "silence," "inhibit expression," "downregulate expression," "suppress expression," and the like, as they refer to the APOC3 gene herein, refer to at least partial suppression of expression of the APOC3 gene, which suppression is manifested by a reduction in the amount of mRNA that can be isolated from a first cell or group of cells that is transcribed and treated to inhibit expression of the APOC3 gene, compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been treated (control cells). The degree of inhibition is typically measured by:

number

[0041] Alternatively, the degree of inhibition can be given by the reduction of the parameter functionally related to APOC3 gene expression, for example, the amount of protein coded by APOC3 gene secreted by cell, or the reduction of the number of cells that exhibit a certain phenotype, for example, apoptosis.In principle, APOC3 gene silencing can be determined by any suitable assay in any cell that expresses target constitutively or by genetic engineering.However, when reference is required to determine whether a certain dsRNA inhibits the expression of APOC3 gene to a certain extent and therefore falls within the scope of the present invention, the assay provided in the following examples will serve as such reference.

[0042] For example, in certain cases, expression of the APOC3 gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotides featuring the invention. In some embodiments, the APOC3 gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotides featuring the invention. In some embodiments, the APOC3 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotides featuring the invention.

[0043] The terms "treat," "treatment," and the like, as used herein in the context of APOC3 expression, refer to the alleviation or alleviation of a pathological process mediated by APOC3 expression. In the context of the present invention, insofar as it relates to any of the other medical conditions cited herein below (other than a pathological process mediated by APOC3 expression), the terms "treat," "treatment," and the like, mean alleviating or alleviating at least one symptom associated with such medical condition, or slowing or reversing the progression of such condition.

[0044] As used herein, the phrase "effective amount" refers to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a pathological process mediated by APOC3 expression or an overt symptom of a pathological process mediated by APOC3 expression. The specific amount that is effective can be readily determined by an ordinary physician and may vary depending on factors known in the art, such as the type of pathological process mediated by APOC3 expression, the patient's medical history and age, the stage of the pathological process mediated by APOC3 expression, and the administration of other agents that combat the pathological process mediated by APOC3 expression.

[0045] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of dsRNA and a pharmaceutically acceptable carrier. As used herein, a "pharmacologically effective amount," a "therapeutically effective amount," or simply an "effective amount" refers to the amount of RNA that is effective to produce the intended pharmacological, therapeutic, or preventive result. For example, if a given medical treatment is effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, then a therapeutically effective amount of a drug for treating that disease or disorder is the amount necessary to achieve at least a 25% reduction in that parameter. For example, a therapeutically effective amount of a dsRNA targeting APOC3 can reduce APOC3 serum levels by at least 25%.

[0046] The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include pharmaceutically acceptable excipients, such as, but not limited to, inert diluents, tablet disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable tablet disintegrants. Binders include starch and gelatin, while lubricants, if present, will generally be magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

[0047] As used herein, a "transformed cell" is a cell into which a vector has been introduced from which a dsRNA molecule can be expressed.

[0048] double-stranded ribonucleic acid (dsRNA) As described in more detail herein, the present invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting APOC3 gene expression in cells or mammals, wherein the dsRNA comprises an antisense strand having a region of complementarity complementary to at least a portion of an mRNA formed upon expression of the APOC3 gene, the region of complementarity being less than 30 nucleotides in length, generally 19-24 nucleotides in length, and the dsRNA inhibits APOC3 gene expression by at least 30% after contact with cells expressing the APOC3 gene, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or protein-based methods such as Western blot. APOC3 gene expression may be reduced by at least 30%, as measured by the assays described in the Examples below. For example, APOC3 gene expression in cell culture, such as Hep3B cells, can be assayed by measuring APOC3 mRNA levels, for example, by bDNA or TaqMan assays, or by measuring protein levels, for example, by ELISA assays. The dsRNA of the present invention may further comprise one or more single-stranded nucleotide overhangs.

[0049] dsRNA can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc., as further described below. dsRNA contains two RNA strands sufficiently complementary to hybridize to form a double-stranded structure. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary, and generally perfectly complementary, to a target sequence derived from the sequence of the mRNA formed during expression of the APOC3 gene, while the other strand (the sense strand) contains a region complementary to the antisense strand; thus, when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure. Generally, the double-stranded structure has a length of 15 to 30, or 25 to 30, or 18 to 25, or 19 to 24, or 19 to 21, or 19, 20, or 21 base pairs. In one embodiment, the duplex has a length of 19 base pairs. In another embodiment, the duplex has a length of 21 base pairs. When two different siRNAs are used in combination, the lengths of the duplexes can be the same or different.

[0050] Each strand of the dsRNA of the present invention is generally 15 to 30, or 18 to 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In another embodiment, each strand is 25 to 30 nucleotides in length. Each strand of the duplex may have the same length or different lengths. When two different siRNAs are used in combination, the length of each strand of each siRNA may be the same or different.

[0051] The dsRNA of the invention includes dsRNA longer than 21-23 nucleotides, e.g., dsRNA of sufficient length to be processed by the RNAeIII enzyme Dicer into 21-23 base pair siRNAs, which are then incorporated into RISC. Thus, the dsRNA of the invention can have a length of at least 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or at least 100 base pairs.

[0052] The dsRNA of the present invention may include one or more single-stranded overhangs consisting of one or more nucleotides. In one embodiment, at least one end of the dsRNA may include a single-stranded nucleotide overhang of 1 to 4, typically 1 or 2, nucleotides. In another embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotide overhangs at each of the 3' and 5' ends beyond the sense strand. In a further embodiment, the sense strand of the dsRNA has 1 to 10 nucleotide overhangs at each of the 3' and 5' ends beyond the antisense strand.

[0053] dsRNAs with at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. In some embodiments, the presence of only one nucleotide overhang enhances the interference activity of dsRNA without affecting overall stability. dsRNAs with only one overhang have been shown to be particularly stable and effective in vivo and in a variety of cells, cell culture media, blood, and serum. Generally, the single-stranded overhang is located at the 3'-end of the antisense strand, or alternatively, at the 3'-end of the sense strand. dsRNAs can also have a blunt end, generally located at the 5'-end of the antisense strand. Such dsRNAs may have improved stability and inhibitory activity, allowing for administration at low dosages, i.e., less than 5 mg / kg of recipient body weight per day. Generally, the antisense strand of dsRNA has a nucleotide overhang at the 3'-end and a blunt 5'-end. In another embodiment, one or more of the nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0054] In one embodiment, the APOC3 gene is a human APOC3 gene. In certain embodiments, the sense strand of the dsRNA is one of the sense sequences in Tables 1, 2, 6, 7, 11, or 12, and the antisense strand is one of the antisense sequences in Tables 1, 2, 6, 7, 11, or 12. Alternative antisense agents that target other portions of the target sequence provided in Tables 1, 2, 6, 7, 11, or 12 can be easily determined using the target sequence and adjacent APOC3 sequences.

[0055] Those skilled in the art are well aware that dsRNAs with a double-stranded structure between 20 and 23 base pairs, particularly 21 base pairs, are recognized to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, some have found that shorter or longer dsRNAs can also be effective. In the above-described embodiments, depending on the nature of the oligonucleotide sequences provided in Tables 1, 2, 6, 7, 11, or 12, the dsRNAs characterizing the present invention may comprise at least one of the lengths described herein. It can be reasonably expected that shorter dsRNAs, having a sequence comprising one of the sequences in Tables 1, 2, 6, 7, 11, or 12 minus only a few nucleotides on one or both ends, can be similarly effective compared to the dsRNAs described above. Thus, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides from one of the sequences in Tables 1, 2, 6, 7, 11, or 12, and differences in their ability to inhibit APOC3 gene expression in the assays described herein below that are no more than 5, 10, 15, 20, 25, or 30% inhibition of dsRNAs containing the entire sequence, are contemplated by the present invention. Furthermore, dsRNAs that cleave within a desired APOC3 target sequence can be readily generated using the corresponding APOC3 antisense sequence and the complementary sense sequence.

[0056] In addition, the dsRNAs provided in Tables 1, 2, 6, 7, 11, or 12 identify sites within APOC3 that are susceptible to RNAi-based cleavage. Thus, the present invention is further characterized by dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves a message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 consecutive nucleotides from one of the sequences provided in Tables 1, 2, 6, 7, 11, or 12, which will be linked to additional nucleotide sequences taken from the region adjacent to the selected sequence within the APOC3 gene.

[0057] The cleavage of RNA target can be routinely detected by gel electrophoresis known in the art, and if necessary, by related nucleic acid hybridization techniques.The cleavage site of dsRNA on the target mRNA can generally be determined by the method known to those skilled in the art, for example, by the 5'-RACE method described in Soutschek et al., Nature; 2004, Vol.432, pp.173-178 (this paper is incorporated by reference for all purposes).

[0058] The dsRNA characterizing the present invention may contain one or more mismatches to the target sequence. In one embodiment, the dsRNA characterizing the present invention contains three or fewer mismatches. When the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the range of mismatches is not located in the center of the region of complementarity. When the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatches are limited to five nucleotides from either end, for example, 5, 4, 3, 2, or 1 nucleotide from either the 5' or 3' end of the region of complementarity. For example, in the case of a 23-nucleotide dsRNA strand complementary to a region of the APOC3 gene, the dsRNA generally does not contain any mismatches within the central 13 nucleotides. The methods described in the present invention can be used to determine whether a dsRNA containing mismatches in the target sequence is effective in inhibiting the expression of the APOC3 gene. It is important to consider the efficacy of mismatched dsRNA in inhibiting expression of the APOC3 gene, especially when a particular complementary region within the APOC3 gene is known to have polymorphic sequence variation within the population.

[0059] In another aspect, the present invention provides single-stranded antisense oligonucleotide RNAi. Antisense oligonucleotides are single-stranded oligonucleotides complementary to a sequence within a target mRNA. Antisense oligonucleotides can inhibit translation by base pairing to mRNA and stoichiometrically interfering with the translation machinery. See Dias, N. et al., (2002) Mol. Cancer Ther. 1:347-355. Antisense oligonucleotides can also inhibit target protein expression by binding to mRNA targets and promoting RNAe-H-mediated destruction of the mRNA. Single-stranded antisense RNA molecules can be about 13 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, single-stranded antisense RNA molecules can include a sequence of at least about 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the antisense sequences in Tables 1, 2, 6, 7, or 10.

[0060] qualification In yet another embodiment, dsRNA may be chemically modified to improve stability. Nucleic acids featured in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, incorporated herein by reference. Specific examples of dsRNA compounds useful in the present invention include RNAs containing modified backbones or lacking natural internucleoside linkages. As defined herein, dsRNAs with modified backbones include those that retain a phosphorus atom in the backbone and those that lack a phosphorus atom in the backbone. For purposes of this specification, and as sometimes referred to in the art, modified dsRNAs that lack a phosphorus atom in their internucleoside backbone may also be considered oligonucleosides.

[0061] Modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0062] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,4 Nos. 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is incorporated herein by reference.

[0063] Modified dsRNA backbones that do not contain internal phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, or mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0064] Representative patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,471,968; 5,472,969; 5,473,969; 5,474,969; 5,475,969; 5,476,969; 5,477,969; 5,478,969; 5,479,969; 5,480,062; 5,481,062; 5,482,063; 5,483,064; 5,484,065; 5,485,066; 5,486,067; 5,487,068; 5,488,069; 5,489,069; 5,490,069; 5,491,069; 5,492,069; 5,493,069; 5,494,069; 5,495,069; 5,496,069; 5,497,069; 5,498,069; 5,499,069; 5,500,069; 5,501,069; 5,502, Nos. 489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is incorporated herein by reference.

[0065] In other suitable dsRNA mimics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a dsRNA mimic, that has been shown to have excellent hybridization properties is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of dsRNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The acid groups are retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. Representative patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. No. 5,539,082; U.S. Pat. No. 5,714,331; and U.S. Pat. No. 5,719,262, each of which is incorporated herein by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0066] Another embodiment of the present invention is dsRNA with phosphorothioate backbone, and oligonucleosides with heteroatom backbone of the above-referenced US Patent No. 5,489,677, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- (known as methylene (methylimino) or MMI backbone), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2- (wherein natural phosphodiester backbone is represented as -OPO-CH2-) and amide backbone of the above-referenced US Patent No. 5,602,240. Also preferred are dsRNA with morpholino backbone structure of the above-referenced US Patent No. 5,034,506.

[0067] Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs contain one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) nCH3)]2, where n and m are from 1 to about 10, are particularly preferred. Other preferred dsRNAs contain one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group that improves the pharmacokinetic properties of a dsRNA, or a group that improves the pharmacodynamic properties of a dsRNA, and other substituents with similar properties. Preferred modifications include 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxy-alkoxy group. Further preferred modifications include the group O(CH2)2ON(CH3)2, also known as 2'-dimethylaminooxyethoxy, i.e., 2'-DMAOE, as described in the Examples herein below, and 2'-dimethylaminoethoxyethoxy (also known as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2, as described in the Examples herein below.

[0068] Other preferred modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the dsRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide, or in 2'-5'-linked dsRNAs and at the 5' position of the 5'-terminal nucleotide. DsRNAs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427 Nos. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the present application and each of which is incorporated herein by reference in its entirety.

[0069] dsRNA may also contain modified or substituted nucleobases (often simply referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-aminoadenine, 5-methyl-2-methyl-3-methyl-4-methyl-5-methyl-6-methyl-1-methyl-2-methyl-3-methyl-4-methyl-1-methyl-2-methyl-3 ... These include other synthetic and natural nucleobases such as 8-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-dazaadenine, and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pp. 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pp. 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds that characterize the present invention.These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, Y.S., Crooke, S.T., and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0070] Representative United States patents that teach the routine preparation of the above and other modified nucleobases include, but are not limited to, U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,300; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; and 5,484,908. Nos. 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is incorporated herein by reference, as well as U.S. Pat. No. 5,750,692, which is also incorporated herein by reference.

[0071] Conjugates Another modification of the dsRNA of the invention involves chemically linking to the dsRNA one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the dsRNA.Such moieties include, but are not limited to, cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), fatty chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., J. Am. Chem. Soc., 1994, 4:1053-1060), and the like. al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamines or polyethylene glycol chains (Manoharan et al. al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or lipid moiety such as octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0072] Not all positions in a given compound need be uniformly modified; in fact, two or more of the aforementioned modifications may be incorporated into a single compound, or even into a single nucleoside within a dsRNA. The present invention also includes dsRNA compounds that are chimeric compounds. In the context of the present invention, a "chimeric" dsRNA compound or "chimera" refers to a dsRNA compound, particularly a dsRNA that contains two or more chemically distinct regions, each of which is composed of at least one monomer unit, i.e., in the case of a dsRNA compound, a nucleotide. These dsRNAs typically contain at least one region in which the dsRNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for a target nucleic acid. Additional regions of the dsRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNAe H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNAe H results in cleavage of the RNA target, thereby greatly improving the efficiency of dsRNA inhibition of gene expression. As a result, similar results can often be obtained with shorter dsRNAs when using chimeric dsRNAs compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region.

[0073] In certain cases, dsRNA may be modified with non-ligand groups. Many non-ligand molecules have been conjugated to dsRNA to improve the activity, cellular distribution, or cellular uptake of dsRNA, and procedures for such conjugation are available in the scientific literature.Such non-ligand moieties include cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al. al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al. al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such dsRNA conjugates are listed above.A typical conjugation protocol involves synthesizing dsRNA bearing an amino linker at one or more positions of the sequence.Then, using an appropriate coupling or activating reagent, the molecule to be conjugated is reacted with the amino group.The conjugation reaction can be carried out either with the dsRNA still attached to a solid support or after cleaving the dsRNA in solution.Generally, HPLC purification of the dsRNA conjugate gives a pure conjugate.

[0074] Conjugating a ligand to dsRNA can improve its cellular absorption and targeting to specific tissues or uptake by specific cell types, such as hepatocytes. In some cases, hydrophobic ligands are conjugated to dsRNA to promote direct penetration of the cell membrane and / or uptake across hepatocytes. Alternatively, the ligand conjugated to dsRNA is a substrate for receptor-mediated endocytosis. These techniques have been used to enhance the cellular penetration of antisense oligonucleotides and dsRNA agents. For example, cholesterol has been conjugated to various antisense oligonucleotides, resulting in compounds that are substantially more active than their unconjugated analogs. See M. Manoharan, Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrenebutyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters cells via folate-receptor-mediated endocytosis. dsRNA compounds bearing folic acid will be efficiently transported into cells via folate-receptor-mediated endocytosis. Li and coworkers reported that attaching folic acid to the 3' end of an oligonucleotide increased the cellular uptake of the oligonucleotide by 8-fold. Li, S.; Deshmukh, HM; Huang, L. Pharm. Res. 1998, 15, 1540. Other ligands that have been conjugated to oligonucleotides include polyethylene glycol, carboxylate clusters, crosslinkers, porphyrin conjugates, delivery peptides, and lipids such as cholesterol and cholesterylamine. Examples of carboxylate clusters include Chol-p-(GalNAc)3 (N-acetylgalactosamine cholesterol) and LCO(GalNAc)3 (N-acetylgalactosamine-3'-lithochol-oleoyl).

[0075] Carbohydrate conjugates In some embodiments of the compositions and methods of the present invention, the dsRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated dsRNA is advantageous for in vivo nucleic acid delivery, as described herein, and the composition is suitable for in vivo therapeutic use. As used herein, "carbohydrate" refers to either a compound that is a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic), each of which is bound to an oxygen, nitrogen, or sulfur atom; or a compound that has, as part of its structure, a carbohydrate moiety composed of one or more monosaccharide units, each of which has at least six carbon atoms (which may be linear, branched, or cyclic), each of which is bound to an oxygen, nitrogen, or sulfur atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include sugars of C5 or greater (e.g., C5, C6, C7, or C8); di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0076] In one embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is a monosaccharide. [ka] and other N-acetylgalactosamines.

[0077] In another embodiment, the carbohydrate conjugate used in the compositions and methods of the present invention is: [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0078] Other exemplary carbohydrate conjugates for use in the embodiments described herein include, but are not limited to: [ka] Examples include:

[0079] When one of X or Y is an oligonucleotide, the other is hydrogen.

[0080] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands, such as those described above, including, but not limited to, a PK modulator and / or a cell-penetrating peptide.

[0081] Linker In some embodiments, the conjugates or ligands described herein may be attached to the dsRNA of the invention using a variety of linkers, which may be cleavable or non-cleavable.

[0082] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, for example, covalently bonds the two parts of a compound. Linkers are typically a direct bond, or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a group including, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, arylheteroarylalkynyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhetero(herero)cyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkene and aryl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhetero(herero)aryl (wherein one or more methylenes may be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic); where R is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker consists of about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 17, or 8 to 16 atoms.

[0083] A cleavable linking group is one that is sufficiently stable outside a cell, but is cleaved after entering a target cell to release the two moieties held together by the linker. In preferred embodiments, the cleavable linking group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or more, or at least about 100 times faster inside the target cell or under first reference conditions (which may, for example, be selected to mimic or represent intracellular conditions) than in the subject's blood or under second reference conditions (which may, for example, be selected to mimic or represent conditions found in blood or serum).

[0084] The conjugable linking group is sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include: oxidizing or reducing enzymes present in cells, or redox agents that are selective for specific substrates or have no substrate specificity, including reducing agents such as mercaptans, which can degrade redox-cleavable linking groups by reduction; esterases; agents that can create endosomes or acidic environments, for example, resulting in a pH of 5 or less; enzymes that can act as general acids and thereby hydrolyze or degrade acid-cleavable linking groups, peptidases (which may be substrate-specific), and phosphatases.

[0085] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0086] The linker may contain a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, a liver-targeting ligand may be linked to a cationic lipid via a linker containing an ester group. Because hepatocytes are rich in esterases, this linker will be cleaved more efficiently in hepatocytes than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

[0087] Linkers containing peptide bonds can be used to target cell types rich in peptidases, such as hepatocytes and synoviocytes.

[0088] In general, the suitability of a candidate cleavable binding group can be evaluated by testing the ability of a degradative agent (or degradative condition) to cleave the candidate binding group. It may also be desirable to test the ability of the candidate cleavable binding group to resist cleavage in blood or upon contact with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between first and second conditions, the first selected to be indicative of cleavage within target cells, and the second selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. This evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0089] In one embodiment, the cleavable linking group is a redox-cleavable linking group that is cleaved after reduction or oxidation. An example of a reductively cleavable linking group is a disulfide bond (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or suitable for use with, for example, a specific dsRNA moiety and a specific targeting agent, the methods described herein can be used. For example, the candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art, which mimics the cleavage rate that would be observed in cells, such as target cells. The candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In one example, the candidate compound is cleaved in blood at most about 10%. In another embodiment, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster inside cells (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0090] In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group that is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves phosphate groups within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0091] In another embodiment, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less) or by an agent, such as an enzyme, that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0092] In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved by enzymes such as intracellular esterases and amylases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0093] In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved by enzymes, such as intracellular peptidases and proteases. An example of a peptide-based cleavable linking group is a peptide bond formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give peptides and proteins. Peptide-based cleaving groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, but do not include all amide functional groups. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)— (SEQ ID NO: 13), where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0094] In one embodiment, the dsRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of dsRNA carbohydrates conjugated to linkers of the compositions and methods of the present invention include: [ka] [ka] Examples include:

[0095] When one of X or Y is an oligonucleotide, the other is hydrogen.

[0096] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0097] In one embodiment, the dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XXXI) to (XXXIV): [ka]

[0098] wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently for each occurrence represent 0 to 20, and the repeating units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C are each, independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is independently for each occurrence absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R5A , R 5B , R 5C are each independently absent for each occurrence, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., each is independently for each occurrence a monosaccharide (such as GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; R a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives, such as those of formula (XXXV), are particularly useful in conjunction with RNAi agents to inhibit expression of target genes: [ka] Formula XXXV In the formula, L 5A , L 5B and L 5C represents a monosaccharide such as a GalNAc derivative.

[0099] Examples of suitable divalent and trivalent branched linker groups for conjugation to GalNAc derivatives include, but are not limited to, the structures cited above as Formulas II_VII, XI, X, and XIII.

[0100] Representative patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. No. 4,828,979; U.S. Pat. No. 4,948,882; U.S. Pat. No. 5,218,105; U.S. Pat. No. 5,525,465; U.S. Pat. No. 5,541,313; U.S. Pat. No. 5,545,730; U.S. Pat. No. 5,552,538; U.S. Pat. No. 5,578,717; U.S. Pat. No. 5,580,731; U.S. Pat. No. 5,591,584; U.S. Pat. No. 5,109,124; U.S. Pat. No. 5,118,80 2; U.S. Patent No. 5,138,045; U.S. Patent No. 5,414,077; U.S. Patent No. 5,486,603; U.S. Patent No. 5,512,439; U.S. Patent No. 5,578,718; U.S. Patent No. 5,608,046; U.S. Patent No. 4,587,044; U.S. Patent No. 4,605,735; U.S. Patent No. 4,667,025; U.S. Patent No. 4,762,779; U.S. Patent No. 4,789,737; U.S. Patent No. 4,824,941; U.S. Patent No. 4,835,2 63; U.S. Patent No. 4,876,335; U.S. Patent No. 4,904,582; U.S. Patent No. 4,958,013; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,082,830; U.S. Patent No. 5,112,963; U.S. Patent No. 5,214,136; U.S. Patent No. 5,245,022; U.S. Patent No. 5,254,469; U.S. Patent No. 5,258,506; U.S. Patent No. 5,262, 536; U.S. Patent No. 5,272,250; U.S. Patent No. 5,292,873; U.S. Patent No. 5,317,098; U.S. Patent No. 5,371,241, U.S. Patent No. 5,391,723; U.S. Patent Nos. 5,416,203, 5,451,463; U.S. Patent No. 5,510,475; U.S. Patent No. 5,512,667; U.S. Patent No. 5,514,785; U.S. Patent No. 5,565,552; U.S. Patent No. 5,567,810; U.S. Patent No. 5,574,142;Nos. 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0101] Vector encoding dsRNA In another embodiment, APOC3 dsRNA molecules are expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. 00 / 22113; Conrad, International PCT Publication No. 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be incorporated into the host genome and inherited as transgenes. Transgenes can also be constructed to be inherited as extrachromosomal plasmids (Gassmann et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0102] Each strand of dsRNA can be transcribed by the promoter on two separate expression vectors and co-transfected into target cells.Alternatively, each of the individual strands of dsRNA can be transcribed by the promoter located on the same expression plasmid.In one embodiment, dsRNA is expressed as an inverted repeat that is connected by linker polynucleotide sequence, so that dsRNA has stem and loop structure.

[0103] Recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expression viral vectors can be constructed based on adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616; Rosenfeld et al. (1991, Science 252:431-434) and Rosenfeld et al. (1992), Cell 68:143-155); or alphavirus, as well as others known in the art.Retroviruses have been used to introduce a wide variety of genes into many different cell types, including epithelial cells, in vitro and / or in vivo (e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043). al.,1991,Proc.NatI.Acad.Sci.USA 88:8377-8381;Chowdhury et al.,1991,Science 254:1802-1805;van Beusechem.et al.,1992,Proc.Natl.Acad.Sci.USA 89:7640-19;Kay et al. al.,1992,Human Gene Therapy 3:641-647;Dai et al.,1992,Proc.Natl.Acad.Sci.USA89:10892-10895;Hwu et al. (See, e.g., Comette et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89 / 07136; PCT Application WO 89 / 02468; PCT Application WO 89 / 05345; and PCT Application WO 92 / 07573.) Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of cells can be generated by transfecting recombinant retroviral genomes into suitable packaging cell lines, such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349).Recombinant adenovirus vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rats, hamsters, dogs, and chimpanzees) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.

[0104] Any viral vector capable of accepting the coding sequence of the dsRNA molecule to be expressed can be used, such as vectors derived from adenovirus (AV); adeno-associated virus (AAV); retrovirus (e.g., lentivirus (LV), rhabdovirus, murine leukemia virus); herpesvirus, etc. The tropism of the viral vector can be altered, as appropriate, by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by replacing the viral capsid protein with a different one.

[0105] For example, lentiviral vectors featuring the present invention may be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors featuring the present invention can be made to target different cells by engineering the vector to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV2 / 2. The serotype 2 capsid gene in an AAV2 / 2 vector can be replaced with a serotype 5 capsid gene to generate an AAV2 / 5 vector. Techniques for constructing AAV vectors expressing different capsid protein serotypes are within the skill of those in the art; see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0106] The selection of a suitable recombinant viral vector for use in the present invention, the method of inserting a nucleic acid sequence that expresses dsRNA into the vector, and the method of delivering the viral vector to cells of interest are within the skill of those skilled in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis MA (1988), Biotechniques 6: 608-614; Miller AD (1990), Hum Gene Therap. 1: 5-14; Anderson WF (1998), Nature 392: 25-30; and Rubinson DA et al., Nat. Genet. 33: 401-406, the entire disclosures of which are incorporated herein by reference.

[0107] Viral vectors may be derived from AV and AAV. In one embodiment, the dsRNA featuring the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector, e.g., having either a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter.

[0108] Suitable AV vectors for expressing the dsRNA characterizing the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0109] Suitable AAV vectors for expressing the dsRNAs that characterize the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher KJ et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; International Patent Application No. 94 / 13788; and International Patent Application No. 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0110] The promoter driving dsRNA expression in either the DNA plasmids or viral vectors featured in the invention may be a eukaryotic RNA polymerase I (e.g., a ribosomal RNA promoter), an RNA polymerase II (e.g., a CMV early promoter, an actin promoter, or a U1 snRNA promoter), or generally an RNA polymerase III promoter (e.g., a U6 snRNA or 7SK RNA promoter), or a prokaryotic promoter, such as a T7 promoter, provided that the expression plasmid also encodes the T7 RNA polymerase required for transcription from the T7 promoter. Promoters can also direct transgene expression to the pancreas (see, e.g., insulin regulatory sequences for the pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

[0111] In addition, transgene expression can be precisely regulated by using inducible regulatory sequences and expression systems, such as regulatory sequences sensitive to certain physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Suitable inducible expression systems for controlling transgene expression in cells or mammals include regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (EPTG). Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the dsRNA transgene.

[0112] Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below and persist in target cells. Alternatively, viral vectors that provide transient expression of dsRNA molecules may be used. Such vectors may be administered repeatedly as needed. After expression, the dsRNA binds to the target RNA and regulates its function or expression. Delivery of dsRNA expression vectors may be systemic, such as by intravenous or intramuscular administration, or by administration to target cells explanted from the patient and then reintroduced into the patient, or by any other means capable of introducing the vector into the desired target cells.

[0113] dsRNA-expressing DNA plasmids are typically transfected into target cells as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdown targeting different regions of a single APOC3 gene or multiple APOC3 genes over a period of one week or more are also contemplated by the present invention. Successful vector introduction into host cells can be monitored using a variety of known methods. For example, transient transfection can be indicated using a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of ex vivo cells can be ensured using a marker that provides transfected cells with resistance to certain environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0114] APOC3-specific dsRNA molecules can be inserted into vectors and used as gene therapy vectors for human patients.Gene therapy vectors can be delivered to subjects by, for example, intravenous injection, local administration (see U.S. Patent No. 5,328,470) or stereotactic injection (see, for example, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).Pharmaceutical preparations of gene therapy vectors can include the gene therapy vector in an acceptable diluent, or can include a slow-release matrix in which the gene delivery vehicle is incorporated.Alternatively, when the complete gene delivery vector, such as a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0115] Pharmaceutical compositions containing dsRNA In one embodiment, the present invention provides a pharmaceutical composition comprising the dsRNA described herein and a pharmaceutically acceptable carrier.The pharmaceutical composition comprising the dsRNA is useful for treating diseases or disorders associated with the expression or activity of the APOC3 gene, such as pathological processes mediated by APOC3 expression.Such pharmaceutical compositions are formulated based on delivery mode.

[0116] The pharmaceutical compositions featured in the present invention are administered in dosages sufficient to inhibit expression of the APOC3 gene.

[0117] Generally, a suitable dosage of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day.

[0118] The subjects were 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.55 mg / kg, 0.6 mg / kg, 0.65 mg / kg, 0.7 mg / kg, 0.75 mg / kg, 0.8 mg / kg, 0.85 mg / kg, 0.9 mg / kg, 0.95 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg dsRNA, 2.6 mg / kg dsRNA, 2.7 mg / kg dsRNA, 2.8 mg / kg dsRNA, 2.9 mg / kg dsRNA, 3.0 mg / kg dsRNA, 3.1 mg / kg dsRNA, 3.2 mg / kg dsRNA, 3.3 mg / kg dsRNA, 3.4 mg / kg dsRNA, 3.5 mg / kg dsRNA, 3.6 mg / kg dsRNA, 3.7 mg / kg dsRNA, 3.8 mg / kg dsRNA, 3.9 mg / kg dsRNA, 4.0 mg / kg dsRNA, 4.1 mg / kg dsRNA, 4.2 mg / kg dsRNA, 4.3 mg / kg dsRNA, 4.4 mg / kg dsRNA, 4.5 mg / kg dsRNA, 4.6 mg / kg dsRNA, 4.7 mg / kg dsRNA, 4.8 mg / kg dsRNA, 4.9 mg / kg dsRNA, 5.0 mg / kg dsRNA, 5.1 mg / kg dsRNA, 5.2 mg / kg dsRNA, 5.3 mg / kg dsRNA, ...... 5.4 mg / kg dsRNA, 5.5 mg / kg dsRNA, 5.6 mg / kg dsRNA, 5.7 mg / kg dsRNA, 5.8 mg / kg dsRNA, 5.9 mg / kg dsRNA, 6.0 mg / kg dsRNA, 6.1 mg / kg dsRNA, 6.2 mg / kg dsRNA, 6.3 mg / kg dsRNA, 6.4mg / kg dsRNA, 6.5mg / kg dsRNA, 6.6mg / kg dsRNA, 6.7mg / kg dsRNA, 6.8mg / kg dsRNA, 6.9mg / kg dsRNA, 7.0mg / kg dsRNA, 7.1mg / kg dsRNA, 7.2mg / kg dsRNA, 7.3mg / kg dsRNA, 7.4mg / kg dsRNA, 7.5mg / kg dsRNA, 7.6mg / kg dsRNA, 7.7mg / kg dsRNA, 7.8mg / kg dsRNA, 7.9mg / kg dsRNA, 8.0mg / kg dsRNA, 8.1mg / kg dsRNA, 8.2mg / kg dsRNA, 8.3mg / kg dsRNA, 8.4mg / kg dsRNA, 8.5mg / kg dsRNA, 8.6mg / kg dsRNA, 8.7mg / kg dsRNA, 8.8mg / kg dsRNA, 8.9mg / kg dsRNA, 9.0mg / kg dsRNA, 9.1mg / kg dsRNA, 9.2mg / kg dsRNA, 9.3mg / kg dsRNA, 9.4mg / kg dsRNA, 9.5mg / kg dsRNA, 9.6mg / kg dsRNA, 9.7mg / kg dsRNA, 9.8mg / kg dsRNA, 9.9mg / kg dsRNA, 9.0mg / kg dsRNA, 10mg / kg dsRNA, 15mg / kg dsRNA, 20mg / kg dsRNA, 25mg / kg dsRNA, 30mg / kg dsRNA, 35mg / kg dsRNA, 40mg / kg dsRNA, 45mg / kg A therapeutic amount of dsRNA, such as about 50 mg / kg dsRNA, may be administered. Values ​​up to and including the cited values ​​and intermediate ranges are intended to be part of the invention.

[0119] The pharmaceutical composition may be administered once a day, or the dsRNA may be administered as two, three or more subdoses at appropriate intervals throughout the day, or even by continuous infusion or sustained-release delivery.In this case, the amount of dsRNA contained in each subdose must be correspondingly smaller to achieve the total daily dose.The dosage unit may also be formulated for delivery over several days, for example, by using a conventional sustained-release formulation that provides sustained release over a period of several days.Sustained-release formulations are well known in the art and are particularly useful for delivering drugs to specific sites, and therefore can be used with the drugs of the present invention.In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

[0120] The effect of a single dose on APOC3 levels is sustained, so that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart, or no more than 5, 6, 7, 8, 9, or 10 weeks apart.

[0121] Those skilled in the art will recognize that certain factors, including but not limited to the severity of disease or illness, previous treatment, the overall health and / or age of the subject, and other existing diseases, can affect the dosage and time required to effectively treat the subject.In addition, the treatment of a subject with a therapeutically effective amount of a composition can comprise a single treatment or a series of treatments.The effective dosage and in vivo half-life of each dsRNA encompassed by the present invention can be estimated by conventional methodology or based on the in vivo test using suitable animal models as described elsewhere herein.

[0122] Advances in mouse genetics have produced many mouse models for testing various human diseases, such as pathological processes mediated by APOC3 expression.Such models are used for in vivo testing of dsRNA and determining therapeutically effective doses.Preferred mouse model is the mouse that contains the plasmid that expresses human APOC3.Another preferred mouse model is the transgenic mouse that carries the transgene that expresses human APOC3.

[0123] Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of the compositions characterizing the invention lies within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods characterizing the invention, a therapeutically effective dose may be initially estimated from cell culture assays. A dose may also be formulated in animal models to achieve a circulating plasma concentration range of the compound, or, if appropriate, the polypeptide product of the target sequence (e.g., achieve a reduction in polypeptide concentrations), that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0124] The dsRNA characterizing the present invention may be administered in combination with other known drugs that are effective in treating pathological processes mediated by target gene expression.In either case, the administering physician can adjust the amount and timing of dsRNA administration based on the results observed using standard efficacy measures known in the art or described herein.

[0125] Administration The present invention also includes pharmaceutical compositions and formulations containing the dsRNA compounds characteristic of the present invention.The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated.Administration can be topical (including buccal and sublingual), pulmonary, for example, by inhalation or insufflation of powder or aerosol, including nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral.Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, for example, intraparenchymal, intrathecal or intraventricular administration.

[0126] The dsRNA can be delivered in a manner that targets specific tissues.

[0127] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the dsRNA characterizing the present invention is mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleyltetramethylaminopropyl DOTAP and dioleylphosphatidylethanolamine DOTMA). The dsRNA characterizing the present invention may be encapsulated in liposomes or complexed to liposomes, particularly cationic liposomes. Alternatively, the dsRNA may be complexed to lipids, particularly cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C1-10 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0128] Liposomal formulation In addition to microemulsions, there are many engineered surfactant structures that have been tested and used in drug formulations. These include monolayers, micelles, bilayers, and vesicles. Vesicles such as liposomes have attracted great interest due to the specificity and delayed action they offer in terms of drug delivery. The term "liposome" as used in the present invention refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer.

[0129] Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of fusing with the cell wall. Non-cationic liposomes do not fuse well with the cell wall but are taken up by macrophages in vivo.

[0130] To cross intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of a suitable transdermal gradient. It is therefore desirable to use liposomes that are highly deformable and can pass through such pores.

[0131] Further advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; liposomes can protect the encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Form, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

[0132] Liposomes are useful for transporting and delivering active ingredients to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, they begin to integrate with the cell membrane, and as the integration of liposomes with cells progresses, the liposome contents are released into the cells where the active agent can act.

[0133] Liposomal formulations have been the subject of extensive research as a delivery mode for many drugs. For topical administration, liposomes have increasingly been demonstrated to have several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the potential for intradermal administration of a wide variety of drugs, both hydrophilic and hydrophobic.

[0134] Several reports have detailed the ability of liposomes to deliver drugs, including high-molecular-weight DNA, into the skin. Analgesics, antibodies, hormones, and compounds containing high-molecular-weight DNA have been administered to the skin. The majority of applications have resulted in targeting of the upper epidermis.

[0135] Liposomes are divided into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged molecules to form stable complexes. The positively charged DNA / liposome complexes bind to the negatively charged cell surface and are transported into endosomes. The acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0136] pH-sensitive, negatively charged liposomes entrap DNA rather than complexing with it. Because both DNA and lipids are similarly charged, repulsion occurs rather than complexation. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0137] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic membrane-fusogenic liposomes are primarily formed from dioleylphosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Other types are formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0138] Several studies have evaluated the topical delivery of liposomal drug formulations to the skin. Application of interferon-containing liposomes to guinea pig skin resulted in a reduction in the pain of cutaneous herpes, while delivery of interferon via other means (e.g., as a solution or emulsion) was ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Furthermore, further studies tested the efficacy of interferon administered as part of a liposomal formulation against administration of interferon using an aqueous system and concluded that liposomal formulations were superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0139] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A to the dermis of mouse skin. The results showed that such nonionic liposomal systems were effective in promoting the deposition of cyclosporine A in different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4, 6, 466).

[0140] Liposomes also include "sterically stabilized" liposomes, a term used herein to refer to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in enhanced circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1or (B) those that are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without being bound by any particular theory, it is believed in the art that the enhanced circulation half-life of these sterically stabilized liposomes, at least for those containing gangliosides, sphingomyelin, or PEG-derivatized lipids, is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0141] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) reported the use of monosialoganglioside G, which improves the blood half-life of liposomes. M1 reported the ability of (1) sphingomyelin and (2) ganglioside G to bind to sphingomyelin. These findings are detailed by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both to Allen et al., report the ability of (1) sphingomyelin and (2) ganglioside G to bind to sphingomyelin. M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. WO 97 / 13499 (Lim et al.) discloses liposomes containing 1,2-sn-dimyristoylphosphatidylcholine.

[0142] Numerous liposomes containing lipids derivatized with one or more hydrophilic polymers, and methods for their preparation, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) describe liposomes containing PEG moieties and non-ionic detergents, 2C 1215Ghave described liposomes containing PEG- or PEG-stearate-derivatized phosphatidylethanolamine (PE). Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols resulted in significantly enhanced blood half-lives. Modification of synthetic phospholipids by attaching carboxyl groups of polyalkylene glycols (e.g., PEG) has been described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments showing that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate significantly increased blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended these observations to other PEG-derivatized phospholipids, such as DSPE-PEG, formed from a combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes bearing covalently bound PEG moieties on their outer surface are described in European Patent Publication No. 0445131B1 and International Publication No. WO 90 / 04384 to Fisher. Liposomal compositions containing 1 to 20 mole percent PE derivatization with PEG, and methods for their use, have been described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and EP 0496813B1). Liposomes containing a number of other lipid-polymer conjugates are described in WO 91 / 05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and WO 94 / 20073 (Zalipsky et al.), and liposomes containing PEG-modified ceramide lipids are described in WO 96 / 10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0143] Numerous liposomes containing nucleic acids are known in the art. International Publication No. 96 / 40062 to Thierry et al. discloses a method for encapsulating high molecular weight nucleic acids in liposomes. U.S. Patent No. 5,264,221 to Tagawa et al. discloses protein-bound liposomes and claims that the contents of such liposomes may contain dsRNA. U.S. Patent No. 5,665,710 to Rahman et al. describes a method for encapsulating oligodeoxynucleotides in liposomes. International Publication No. 97 / 04787 to Love et al. discloses liposomes containing dsRNA targeting the raf gene.

[0144] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive as potential drug delivery vehicles. Transfersomes may be described as lipid droplets, which are highly deformable and therefore can easily penetrate pores smaller than the droplets. Transfersomes are adaptable to the environment in which they are used, for example, self-optimizing (adapting to the shape of pores in the skin), self-repairing, often reaching their target without fragmentation, and are often self-loading. To create transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0145] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0146] If the surfactant molecule is not ionized, the surfactant is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0147] When surfactant molecule dissolves or disperses in water, if it carries negative charge, this surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylates, acyl amides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.The most important members of anionic surfactant class are alkyl sulfates and soaps.

[0148] If the surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic.Cationic surfactants include quaternary ammonium salts and ethoxylated amines.Quaternary ammonium salts are the most commonly used members of this class.

[0149] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0150] The use of surfactants in drug products, formulations and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0151] nucleic acid lipid particles In one embodiment, the APOC3 dsRNA characterizing the present invention is fully encapsulated in a lipid formulation to form, for example, SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles, including SPLP. As used herein, the term "SPLP" refers to nucleic acid-lipid particles containing plasmid DNA encapsulated within lipid vesicles. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALP and SPLP have an extended circulation life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically separated from the administration site), making them extremely useful for systemic application. SPLP includes "pSPLP," which contains an encapsulated condensing agent-nucleic acid complex, as described in PCT Publication No. WO 00 / 03683. The particles of the present invention typically have an average particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. Additionally, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication WO 96 / 40964.

[0152] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) will be within the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. In some embodiments, the lipid to dsRNA ratio may be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1.

[0153] Typically, the lipid-nucleic acid particles are suspended in a buffer solution, such as PBS, for administration. In one embodiment, the pH of the lipid-formulated dsiRNA is 6.8 to 7.8, e.g., 7.3 or 7.4. The osmolality can be, for example, 250 to 350 mOsm / kg, e.g., approximately 300, e.g., 298, 299, 300, 301, 302, 303, 304, or 305.

[0154] Examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-di Methylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR ,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200 or Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0155] Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- The lipids may be anionic or neutral lipids, including maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or mixtures thereof. When cholesterol is included, the non-cationic lipid may comprise about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0156] The conjugated lipid that inhibits particle aggregation may be, for example, a polyethylene glycol (PEG)-lipid, including but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. PEG-DAA conjugates may be, for example, PEG-dilauryloxypropyl (Ci), PEG-dimyristyloxypropyl (Ci), PEG-dipalmityloxypropyl (Ci), or PEG-distearyloxypropyl (Ci). 18Other examples of PEG conjugates include PEG-cDMA (N-[(methoxypoly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxyl(oxl)propyl-3-amine), mPEG2000-DMG (mPEG-dimyrystylglycerol (having an average molecular weight of 2,000), and PEG-C-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxyl(oxl)propyl-3-amine). The conjugated lipid that prevents particle aggregation can be 0 mol % to about 20 mol %, or about 1.0, 1.1, 1.2, 1.13, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % of the total lipid present in the particle.

[0157] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, from about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

[0158] In one embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0159] For example, lipid-siRNA particles may contain 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (mol percent), with a particle size of 63.0±20 nm and an siRNA / lipid ratio of 0.027.

[0160] In yet another embodiment, the compound 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1) can be used to prepare lipid-siRNA particles. For example, dsRNA can be formulated into a lipid formulation containing Tech-G1, distearoylphosphatidylcholine (DSPC), cholesterol, and mPEG2000-DMG in a molar ratio of 50:10:38.5:1.5, with a total lipid to siRNA ratio of 7:1 (wt:wt).

[0161] In one embodiment, lipidoid ND98·4HCl (MW1487), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01 particles). LNP01 formulations are described, for example, in WO 2008 / 042973, which is incorporated herein by reference.

[0162] Additional exemplary formulations are listed in Table A.

[0163] [Table 1]

[0164] Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in WO 2009 / 127060, filed April 15, 2009, which is incorporated herein by reference.

[0165] Formulations containing XTC are described, for example, in U.S. Provisional Patent Application Nos. 61 / 148,366, filed January 29, 2009; 61 / 156,851, filed March 2, 2009; 61 / 156,851, filed June 10, 2009; 61 / 228,373, filed July 24, 2009; 61 / 239,686, filed September 3, 2009; and International Application No. PCT / US2010 / 022614, filed January 29, 2010, which are incorporated herein by reference.

[0166] Formulations containing MC3 are described, for example, in U.S. Provisional Patent Application No. 61 / 244,834, filed September 22, 2009, U.S. Provisional Patent Application No. 61 / 185,800, filed June 10, 2009, and International Application No. PCT / US10 / 28224, filed June 10, 2010, which are incorporated herein by reference.

[0167] Formulations containing ALNY-100 are described, for example, in International Patent Application No. PCT / US09 / 63933, filed November 10, 2009, which is incorporated herein by reference.

[0168] Formulations containing C12-200 are described in U.S. Provisional Patent Application No. 61 / 175,770, filed May 5, 2009, and International Application No. PCT / US10 / 33777, filed May 5, 2010, which are incorporated herein by reference.

[0169] Formulations prepared by either standard or extrusion-free methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. The formulation should be a whitish, translucent solution without aggregates or precipitates. The particle size and particle size distribution of the lipid-nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be approximately 20-300 nm in size, e.g., 40-100 nm. The particle size distribution should be unimodal. The total siRNA concentration in the formulation and the entrapped fraction can be estimated using a dye exclusion assay. Samples of the formulated siRNA may be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes), in the presence or absence of a formulation-disrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in the formulation can be determined by comparing the signal from the surfactant-containing sample against a standard curve. The entrapped fraction is determined by subtracting the "free" siRNA content (measured by the signal in the absence of surfactant) from the total siRNA content. The percent of entrapped siRNA is typically >85%. For SNALP formulations, particle sizes are at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. Suitable ranges are typically at least about 50 nm to at least about 110 nm, at least about 60 nm to at least about 100 nm, or at least about 80 nm to at least about 90 nm.

[0170] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be desired. In some embodiments, oral formulations are those in which the dsRNA characterizing the present invention is administered with one or more permeation enhancers, surfactants, and chelating agents. Suitable surfactants include fatty acids and / or esters or their salts, bile acids and / or their salts. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as a fatty acid / salt in combination with a bile acid / salt. An exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The DsRNA characterizing the present invention may be orally delivered in the form of granules, including spray-dried particles, or complexed to form micro- or nanoparticles.dsRNA complexing agents include polyamino acids, polyimines, polyacrylates, polyalkylacrylates, polyoxetanes, polyalkylcyanoacrylates, cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch, polyalkylcyanoacrylates, DEAE-derivatized polyimines, pullulan, cellulose, and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyanoacrylate), DEAE-methacrylate, DEAE-hexylacrylate. Oral formulations for dsRNA and their formulations are described in U.S. Pat. No. 6,887,906, U.S. Patent Application Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

[0171] Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, including, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

[0172] The pharmaceutical compositions of the present invention include, but are not limited to, liquids, emulsions, and liposome-containing formulations. These compositions may be made from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. When treating liver diseases such as hepatocarcinoma, liver-targeting formulations are particularly preferred.

[0173] The pharmaceutical formulations of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing the active ingredient into association with pharmaceutical carriers or excipients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0174] The compositions of the present invention may be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain a stabilizer.

[0175] emulsion The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another in the form of small droplets, usually greater than 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, Volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing). Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems containing two immiscible liquid phases intimately mixed and dispersed with each other. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w) types. When the aqueous phase is finely divided and dispersed as minute droplets into a bulk oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely divided and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion. Emulsions may contain additional components in addition to the dispersed phase and active drug, which may be present in the aqueous phase, as a solution in the oil phase, or as a separate phase itself. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions consisting of three or more phases, such as are the cases of oil-in-water-in-oil (o / w / o) and water-in-oil-in-water (w / o / w) emulsions.Such complex formulations often offer certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o / w emulsion enclose small water droplets constitute w / o / w emulsions. Similarly, systems of oil droplets enclosed in globules of water stabilized in an oil continuous phase provide o / w / o emulsions.

[0176] Emulsions are characterized by little or no thermodynamic stability. In many cases, the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase and maintained in this form by means of emulsifiers or through the viscosity of the formulation. Either phase of an emulsion may be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Other means of stabilizing emulsions include the use of emulsifiers, which can be incorporated into either phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0177] Synthetic surfactants, also known as surface active agents, find wide application in the preparation of emulsions and are reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p. 199). Surfactants are typically amphiphilic, containing hydrophilic and hydrophobic moieties. The ratio of a surfactant's hydrophilic to hydrophobic properties, referred to as the hydrophilic / lipophilic balance (HLB), is a valuable tool in classifying formulations and selecting surfactants for formulation preparation. Surfactants can be divided into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285).

[0178] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases, such as anhydrous lanolin and hydrophilic petrolatum, possess hydrophilic properties that allow them to incorporate water to form water-in-oil emulsions while still maintaining their semisolid consistency. Finely divided solids have been used as good emulsifiers, especially in surfactant combinations and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate, and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0179] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions, including fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0180] Hydrophilic colloids, or hydrocolloids, include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse in or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around droplets of the dispersed phase and by increasing the viscosity of the external phase.

[0181] Emulsions often contain many ingredients, such as carbohydrates, proteins, sterols, and phosphatides, which can easily support the growth of microorganisms, so these preparations often incorporate preservatives. Commonly used preservatives in preparations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also usually added to emulsion preparations to prevent the preparation from deteriorating. The antioxidants used may be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, and butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0182] The application of emulsion formulations via skin, oral and parenteral routes, and their preparation methods have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199).Emulsion formulations for oral delivery are very widely used due to their ease of preparation and effectiveness in terms of absorption and bioavailability (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional formulas are among the materials that are commonly administered orally as o / w emulsions.

[0183] In one embodiment of the present invention, the composition of dsRNA and nucleic acid is formulated as microemulsion.Microemulsion can be defined as a system of water, oil and amphiphilic substance, which is a single, optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245).Typically, microemulsion is prepared by first dispersing oil in aqueous surfactant solution, and then adding a sufficient amount of a fourth component, generally a medium-chain alcohol, to form a transparent system. Thus, microemulsions are described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are usually prepared using a combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used and the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in: Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0184] The phenomenological approach of utilizing phase diagrams has been extensively studied, and those skilled in the art have gained comprehensive knowledge of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.335). Compared with traditional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that form spontaneously.

[0185] Surfactants used in preparing microemulsions, alone or in combination with cosurfactants, include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750). The cosurfactant, typically a short-chain alcohol such as ethanol, 1-propanol, or 1-butanol, serves to increase interfacial fluidity by penetrating the surfactant film, resulting in the formation of an irregular film due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG 300, PEG 400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase may include materials such as, but not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

[0186] Microemulsions are of particular interest from the standpoint of drug solubilization and improved drug absorption. Lipid-based microemulsions (both o / w and w / o) have been proposed to improve the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer the advantages of improved drug solubilization, protection of drugs from enzymatic hydrolysis, potential enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical efficacy, and reduced toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). In many cases, microemulsions can form spontaneously when the components of the microemulsion are combined at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides, or dsRNA. Microemulsions are also effective for the cohesive delivery of active ingredients in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of dsRNA and nucleic acids from the gastrointestinal tract and improved local cellular uptake of dsRNA and nucleic acids.

[0187] The microemulsions of the present invention may also contain additional components and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve formulation properties and enhance absorption of the dsRNA and nucleic acids of the present invention. The penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

[0188] penetration enhancers In one embodiment, the present invention uses various penetration enhancers to achieve the efficient delivery of nucleic acid, especially dsRNA, to animal skin.Most drugs are in both ionized and non-ionized form in solution.However, usually, only lipophilic or lipophilic drugs can easily cross cell membrane.It has been found that even non-lipophilic drugs can cross the cell membrane when the cell membrane to be crossed is treated with penetration enhancer.In addition, penetration enhancer also improves the permeability of lipophilic drugs, so as to help the dispersion of non-lipophilic drugs across cell membrane.

[0189] Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above classes of permeation enhancers is described in more detail below.

[0190] Surfactants: In the context of the present invention, surfactants (or "surface-active agents") are chemical entities that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and other liquids, thereby improving the absorption of dsRNA through mucous membranes. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43 (Takahashi et al., J.Pharm.Pharmacol., 1988, 40, 252).

[0191] Fatty acids: Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacyclopentan-2-one, acylcarnitines, acylcholines, and their C 1~10 Included are alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; ElHariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0192] Bile salts: The physiological role of bile includes promoting the distribution and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Therefore, the term "bile salts" includes any naturally occurring component of bile and any synthetic derivatives thereof. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glycolate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Swinyard, Chapter 39 In: Remington's Pharmaceuticals Sciences,18th Ed.,Gennaro,ed.,Mack Publishing Co.,Easton,Pa.,1990,pp.782-783;Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems,1990,7,1-33;Yamamoto et al.,J.Pharm.Exp.Ther.,1992,263,25;Yamashita et al. al., J. Pharm. Sci., 1990, 79, 579-583).

[0193] Chelating agent: The chelating agent used in the present invention can be defined as a compound that forms a complex with metal ions, removes the metal ions from solution, and thereby improves the absorption of dsRNA through mucous membranes.In relation to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also acting as DNase inhibitors, as DNA nucleases require divalent metal ions for catalytic activity, and therefore are most often characterized by being inhibited by chelating agents (Jarrett, J.Chromatogr., 1993,618,315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanillate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0194] Non-chelating non-surfactant: Non-chelating non-surfactant penetration enhancer compounds used herein can be defined as compounds that show little activity as chelating agent or surfactant, but still improve the absorption of dsRNA through digestive mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7,1-33).This class of penetration enhancer includes, for example, unsaturated cyclic urea, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and non-steroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J.Pharm.Pharmacol., 1987,39,621-626).

[0195] Carrier Certain compositions of the present invention also incorporate a carrier compound in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or analog thereof that may be inert (i.e., not possessing biological activity) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of biologically active nucleic acids, for example, by degrading the biologically active nucleic acid or promoting its removal from the circulation. Co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter, can substantially reduce the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoir, possibly due to competition between the carrier compound and the nucleic acid for a common receptor. For example, recovery of partial phosphorothioates in liver tissue can be reduced when they are co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0196] excipients In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected to provide the desired bulk, consistency, etc. when combined with the nucleic acids and other desired components of the pharmaceutical composition, taking into account the intended method of administration. Typical pharmaceutical carriers include, but are not limited to, binders (such as pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, maize starch, polyethylene glycol, sodium benzoate, sodium acetate, and the like); tablet disintegrants (such as starch, sodium starch glycolate, and the like); and wetting agents (such as sodium lauryl sulfate, and the like).

[0197] Suitable organic or inorganic excipients that are pharmaceutically acceptable for parenteral administration and do not adversely react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0198] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions, or solutions of nucleic acids in liquid or solid oil bases in common solvents such as alcohol. Solutions can also contain buffers, diluents, and other suitable additives. Suitable organic or inorganic excipients that are pharmaceutically acceptable for parenteral administration and do not adversely react with nucleic acids can be used.

[0199] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0200] Other components The compositions of the present invention may also contain other auxiliary components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically active ingredients, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or additional ingredients useful for physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, when added, these ingredients should not unduly interfere with the biological activity of the components of the compositions of the present invention. The formulations may be sterilized and, if desired, mixed with auxiliary agents that do not adversely interact with the nucleic acid of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances.

[0201] The aqueous suspension may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain a stabilizer.

[0202] In some embodiments, pharmaceutical compositions featuring the invention may contain (a) one or more dsRNA compounds and (b) one or more anti-cytokine biologics that function by a non-RNAi mechanism. Examples of such biologics include biologics that target IL1β (e.g., anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab, or certolizumab).

[0203] The toxicity and therapeutic efficacy of such compounds can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds with high therapeutic indices are preferred.

[0204] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of the compositions featuring the present invention generally lies within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods featuring the present invention, the therapeutically effective dose can be initially estimated by cell culture assays. Dosages can be formulated to achieve a circulating plasma concentration range of the compound, or, if appropriate, the polypeptide product of the target sequence, in animal models (e.g., achieve a reduction in polypeptide concentrations) that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as measured in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0205] In addition to the above-mentioned administration, the dsRNA characterizing the present invention can be administered in combination with other known drugs that are effective in treating the pathological process mediated by APOC3 expression.In any case, the administering physician can adjust the amount and administration time of dsRNA based on the observed results, using standard efficacy scales known in the art or as described herein.

[0206] Methods for inhibiting the expression of the APOC3 gene The present invention also provides a method for reducing and / or inhibiting APOC3 expression in a cell using a composition containing the dsRNA of the present invention and / or the iRNA of the present invention. The method includes contacting a cell with the dsRNA of the present invention and maintaining the cell for a sufficient time to allow degradation of the mRNA transcript of the APOC3 gene, thereby inhibiting expression of the APOC3 gene in the cell. The reduction in gene expression can be assessed by any method known in the art. For example, the reduction in APOC3 expression can be determined by determining the mRNA expression level of APOC3 using methods routine to those skilled in the art, such as Northern blotting or qRT-PCR, by determining the protein level of APOC3 using methods routine to those skilled in the art, such as Western blotting or immunological techniques, and / or by measuring the biological activity of APOC3, such as affecting one or more molecules related to triglyceride levels, e.g., lipoprotein lipase (LPL) and / or hepatic lipase, or by measuring triglyceride levels themselves in an in vitro environment.

[0207] In the methods of the present invention, the cells may be contacted in vitro or in vivo, i.e., the cells may be within a subject.

[0208] The cell suitable for treatment using the method of the present invention can be any cell that expresses APOC3 gene.The cell suitable for use in the method of the present invention can be mammalian cell, for example, primate cell (human cell or non-human primate cell, for example, monkey cell or chimpanzee cell), non-primate cell (cow cell, pig cell, camel cell, llama cell, horse cell, goat cell, rabbit cell, sheep cell, hamster, guinea pig cell, cat cell, dog cell, rat cell, mouse cell, lion cell, tiger cell, bear cell, or buffalo cell), bird cell (for example, duck cell or goose cell), or whale cell.In one embodiment, the cell is human cell, for example, human hepatocyte.

[0209] APOC3 expression is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% inhibited.

[0210] The in vivo method of the present invention may comprise administering to a subject a composition comprising dsRNA, wherein the dsRNA comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the APOC3 gene of the mammal to be treated.When the organism to be treated is a mammal, such as a human, the composition can be administered by any means known in the art, including but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (for example, intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration.In some embodiments, the composition is administered by intravenous infusion or injection or subcutaneous injection.

[0211] In some embodiments, administration is by depot injection. Depot injection can release dsRNA consistently over a long period of time. Therefore, depot injection can reduce the frequency of administration required to achieve desired effects, such as the desired inhibition of APOC3, or therapeutic or preventive effects. Depot injection can also provide more consistent serum concentration. Depot injection can include subcutaneous injection or intramuscular injection. In a preferred embodiment, depot injection is subcutaneous injection.

[0212] In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In some embodiments, the pump is a subcutaneously implanted osmotic pump. In another embodiment, the pump is an infusion pump. The infusion pump can be used for intravenous, subcutaneous, intraarterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In another embodiment, the pump is a surgically implanted pump that delivers dsRNA to the liver.

[0213] The mode of administration may be selected based on whether local or systemic treatment is desired and on the area to be treated. The route and site of administration may be selected to improve targeting.

[0214] In one aspect, the present invention also provides a method for inhibiting APOC3 gene expression in a mammal. The method includes administering to the mammal a composition containing dsRNA targeting the APOC3 gene in the cells of the mammal and maintaining the mammal for a sufficient period of time to result in degradation of the mRNA transcript of the APOC3 gene, thereby inhibiting APOC3 gene expression in the cells. The reduction in gene expression can be assessed by any method known in the art and described herein, such as qRT-PCR. The reduction in protein production can be assessed by any method known in the art and described herein, such as ELISA. In one embodiment, a puncture liver biopsy sample serves as tissue material for monitoring the reduction in APOC3 gene and / or protein expression. In another embodiment, the inhibition of APOC3 gene expression is monitored indirectly, for example, by measuring the expression and / or activity of genes in the APOC3 pathway. For example, the activity of lipoprotein lipase (LPL) or hepatic lipase can be monitored to determine the inhibition of APOC3 gene expression. Triglyceride levels in a sample, such as a blood or liver sample, can also be measured. Inhibition of APOC3 inhibition can also be monitored by observing the effect on clinical symptoms of high triglyceride levels, such as premature chronic heart disease (CHD), eruptive xanthomas, hepatosplenomegaly, and pancreatitis. Suitable assays are further described in the Examples section below.

[0215] The present invention further provides a method for treating a subject in need thereof, which comprises administering a therapeutically effective amount of a dsRNA of the present invention to a subject, e.g., a subject who would benefit from reduced and / or inhibited APOC3 expression, of a dsRNA targeting the APOC3 gene or a pharmaceutical composition containing a dsRNA targeting the APOC3 gene.

[0216] The dsRNA of the present invention may be administered in a "naked" form or as "free dsRNA." Naked dsRNA is administered in the absence of a pharmaceutical composition. Naked dsRNA may be in a suitable buffer solution. The buffer solution may include acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmolality of the buffer solution containing the dsRNA may be adjusted to be suitable for administration to a subject. Additional buffers are described below.

[0217] Alternatively, the dsRNA of the invention can be administered as a pharmaceutical composition, for example, as a dsRNA liposomal formulation. Additional liposomal formulations are described herein.

[0218] Subjects who would benefit from reduced and / or inhibited APOC3 gene expression include those with high triglyceride levels, e.g., TG > 150 mg / dL, or those with severe hypertriglyceridemia, e.g., TG > 500 mg / dL. In one embodiment, the subject has an APOC3 gene variant with a gain-of-function mutation. In another embodiment, the patient has mixed HTG (type V) reduced LPL activity and / or familial HTG (IV) inactivating LPL mutation and / or familial combined elevated ApoB-100 levels. In another embodiment, the subject has uncontrolled hypertriglyceridemia with acute pancreatitis, or the subject is an HIV patient undergoing treatment, or the subject has a high-fat diet (postprandial hypertriglyceridemia), metabolic syndrome, compound treatment (retinoid therapy), or insulin resistance. Treatment of subjects who would benefit from reduced and / or inhibited APOC3 gene expression includes therapeutic and prophylactic treatment.

[0219] The present invention also provides a method for using dsRNA or its pharmaceutical composition to treat subjects who will benefit from reducing and / or inhibiting APOC3 expression, for example, subjects with high triglyceride levels, in combination with other medicines and / or other therapeutic methods, for example, known medicines and / or known therapeutic methods, such as those currently used to treat high triglyceride levels.For example, in some embodiments, dsRNA targeting APOC3 is administered in combination with a drug useful for treating high triglyceride levels.For example, additional treatments and therapeutic methods suitable for treating subjects who will benefit from reducing APOC3 expression, for example, subjects with high triglyceride levels, include lifestyle and dietary changes, prescription-grade fish oil, fibrates, niacin, ApoC3 antisense, CETP inhibitors, bile acid sequestrants, nicotinic acid, HMG CoA reductase inhibitors, gemfibrozil, fenofibrate, cholesterol absorption inhibitors, neomycin, omega-3 fatty acids, etc. The dsRNA and the additional therapeutic agent and / or treatment may be administered simultaneously and / or in the same combination, e.g., parenterally, or the additional therapeutic agent may be administered as part of a separate composition or at different times, and / or by other methods known in the art or described herein.

[0220] In one embodiment, the method includes administering a composition featured herein such that expression of the target APOC3 gene is reduced for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, or 24 hours, or 28, 32, or 36 hours. In one embodiment, expression of the target APOC3 gene is reduced for an extended duration, e.g., at least about 2, 3, 4 days, or longer, e.g., about 1, 2, 3, 4 weeks, or longer.

[0221] The administration of dsRNA by the method of the present invention can reduce the severity, signs, symptoms, and / or markers of such diseases or disorders in patients with high triglyceride levels.In this context, "reduction" means a statistically significant reduction in these levels.Reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or about 100%.

[0222] The efficacy of treatment or disease prevention can be assessed by measuring, for example, disease progression, disease remission, symptom severity, quality of life, the dose of drug required to maintain treatment efficacy, the level of a disease marker, or any other measurable parameter appropriate to the given disease being treated or targeted for prevention. Monitoring the efficacy of treatment or prevention by measuring any one or any combination of these parameters is within the skill of one of ordinary skill in the art. For example, the efficacy of treating elevated triglyceride levels can be assessed by, for example, periodic measurement of serum triglyceride levels. Comparing subsequent readings with initial readings provides the physician with an indication of whether the treatment is effective. Monitoring the efficacy of treatment or prevention by measuring any one or any combination of such parameters is within the skill of one of ordinary skill in the art. In relation to administration of a dsRNA targeting APOC3 or a pharmaceutical composition thereof, "effective against" elevated triglyceride levels indicates that administration in a clinically relevant manner results in a beneficial effect, such as improvement of symptoms, cure, reduction of disease, prolongation of survival, improvement in quality of life, or other effect generally recognized as positive by a physician familiar with elevated triglyceride levels and related causes, in at least a statistically significant proportion of patients.

[0223] The effectiveness of treatment or prevention is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no worsening or otherwise expected symptoms.As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more of the measurable parameters of the disease can indicate effective treatment.The efficacy of dsRNA drugs or a given formulation of the drug can also be determined using experimental animal models for a given disease, as known in the art.When using experimental animal models, the efficacy of treatment is proven when a statistically significant reduction in markers or symptoms is observed.

[0224] Alternatively, efficacy can be measured by a reduction in the severity of disease as determined by a person skilled in the art of diagnosis based on a clinically accepted scale for assessing the severity of disease, such as the Child-Pugh score (sometimes referred to as the Child-Turcotte-Pugh score). Any positive change, e.g., resulting in a reduction in the severity of disease, as measured using an appropriate scale, indicates sufficient treatment with the dsRNA or dsRNA formulations described herein.

[0225] To the subjects, 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.07 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.55 mg / kg, 0.6 mg / kg, 0.65 mg / kg, 0.7 mg / kg, 0.75 mg / kg, 0.8 mg / kg, 0.85 mg / kg, 0.9 mg / kg, 0.95 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg dsRNA, 2.6 mg / kg dsRNA, 2.7 mg / kg dsRNA, 2.8 mg / kg dsRNA, 2.9 mg / kg dsRNA, 3.0 mg / kg dsRNA, 3.1 mg / kg dsRNA, 3.2 mg / kg dsRNA, 3.3 mg / kg dsRNA, 3.4 mg / kg dsRNA, 3.5 mg / kg dsRNA, 3.6 mg / kg dsRNA, 3.7 mg / kg dsRNA, 3.8 mg / kg dsRNA, 3.9 mg / kg dsRNA, 4.0 mg / kg dsRNA, 4.1 mg / kg dsRNA, 4.2 mg / kg dsRNA, 4.3 mg / kg dsRNA, 4.4 mg / kg dsRNA, 4.5 mg / kg dsRNA, 4.6 mg / kg dsRNA, 4.7 mg / kg dsRNA, 4.8 mg / kg dsRNA, 4.9 mg / kg dsRNA, 5.0 mg / kg dsRNA, 5.1 mg / kg dsRNA, 5.2 mg / kg dsRNA, 5.3 mg / kg dsRNA, 5.4 mg / kg dsRNA, 5.5 mg / kg dsRNA, 5.6 mg / kg dsRNA, 5.7 mg / kg dsRNA, 5.8 mg / kg dsRNA, 5.9 mg / kg dsRNA, 6.0 mg / kg dsRNA, 6.1 mg / kg dsRNA, 6.2 mg / kg dsRNA, 6.3 mg / kg dsRNA, 6.4mg / kg dsRNA, 6.5mg / kg dsRNA, 6.6mg / kg dsRNA, 6.7mg / kg dsRNA, 6.8mg / kg dsRNA, 6.9mg / kg dsRNA, 7.0mg / kg dsRNA, 7.1mg / kg dsRNA, 7.2mg / kg dsRNA, 7.3mg / kg dsRNA, 7.4mg / kg dsRNA, 7.5mg / kg dsRNA, 7.6mg / kg dsRNA, 7.7mg / kg dsRNA, 7.8mg / kg dsRNA, 7.9mg / kg dsRNA, 8.0mg / kg dsRNA, 8.1mg / kg dsRNA, 8.2mg / kg dsRNA, 8.3mg / kg dsRNA, 8.4mg / kg dsRNA, 8.5mg / kg dsRNA, 8.6mg / kg dsRNA, 8.7mg / kg dsRNA, 8.8mg / kg dsRNA, 8.9mg / kg dsRNA, 9.0mg / kg dsRNA, 9.1mg / kg dsRNA, 9.2mg / kg dsRNA, 9.3mg / kg dsRNA, 9.4mg / kg dsRNA, 9.5mg / kg dsRNA, 9.6mg / kg dsRNA, 9.7mg / kg dsRNA, 9.8mg / kg dsRNA, 9.9mg / kg dsRNA, 9.0mg / kg dsRNA, 10mg / kg dsRNA, 15mg / kg dsRNA, 20mg / kg dsRNA, 25mg / kg dsRNA, 30mg / kg dsRNA, 35mg / kg dsRNA, 40mg / kg dsRNA, 45mg / kg A therapeutic amount of dsRNA, such as about 50 mg / kg dsRNA, may be administered. Values ​​up to and including the cited values ​​and intermediate ranges are intended to be part of the invention.

[0226] In certain embodiments, for example, when the composition of the present invention contains a dsRNA and a lipid as described herein, a subject is administered about 0.01 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 5 mg / kg, or about 0.2mg / kg to about 10mg / kg, about 0.3mg / kg to about 5mg / kg, about 0.3mg / kg to about 10mg / kg, about 0.4mg / kg to about 5mg / kg, about 0.4mg / kg to about 10mg / kg, about 0.5mg / kg to about 5mg / kg, about 0.5mg / kg to about 10mg / kg, about 1mg / kg to about 5mg / kg, about 1mg / kg to about 10mg / kg, about 1.5mg / kg to about 5mg / kg, about 1.5mg / kg~about 10mg / kg, about 2mg / kg~about 2.5mg / kg, about 2mg / kg~about 10mg / kg, about 3mg / kg~about 5mg / kg, about 3mg / kg~about 10mg / kg, about 3.5mg / kg~about 5mg / kg, about 4mg / kg to about 5mg / kg, about 4.5mg / kg to about 5mg / kg, about 4mg / kg to about 10mg / kg, about 4.5mg / kg to about 10mg / kg, about 5mg / kg to about 10mg / kg A therapeutic amount of dsRNA may be administered, such as about 5.5 mg / kg to about 10 mg / kg, about 6 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mg / kg. Values ​​up to and including the recited values ​​and intermediate ranges are contemplated as part of the present invention.

[0227] For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or about 10 mg / kg. Values ​​up to and including the recited values ​​and intermediate ranges are contemplated as part of the present invention.

[0228] In another embodiment, for example, when the composition of the present invention contains a dsRNA and N-acetylgalactosamine described herein, a subject is administered about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / mg, about 1.5 to about 50 mg / kb, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, or about 7.5 to about 50 mg / kg , about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 5 0mg / kg, about 0.1 to about 45mg / kg, about 0.25 to about 45mg / kg, about 0.5 to about 45mg / kg, about 0.75 to about 45mg / kg, about 1 to about 45mg / mg, about 1.5 to about 45mg / kb, about 2 to about 45mg / kg, about 2.5 to about 45mg / kg, about 3 to about 45m g / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg, about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 ~40mg / mg, 1.5~40mg / kb, 2~40mg / kg, 2.5~40mg / kg, 3~40mg / kg, 3.5~40mg / kg, 4~40mg / kg, 4.5~40mg / kg, 5~40mg / kg, 7.5~40mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / mg, about 1.5 to about 30 mg / kb, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg , about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about Therapeutic amounts of dsRNA may be administered, such as doses of 0.1 to about 20 mg / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​up to and including the recited values ​​and intermediate ranges are contemplated as part of the present invention.

[0229] For example, the subject may be given doses of approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5 ,7.6,7.7,7.8,7.9,8,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10.5,11,11.5,12,12.5,13,13.5,14,14.5,15,15.5,16,16.5,1 Therapeutic amounts of dsRNA such as 7, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg may be administered. Values ​​up to and including the recited values ​​and intermediate ranges are contemplated as part of the invention.

[0230] dsRNA can be administered by intravenous infusion for a certain period of time, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or about 25 minutes.Administration can be repeated periodically, for example, every other week (i.e., every two weeks) for one month, two months, three months, four months or longer.After the initial treatment plan, treatment can be administered less frequently.For example, after 3 months of administration every other week, administration can be repeated once a month for six months, or one year or longer. Administration of dsRNA can, for example, increase APOC3 levels in cells, tissues, blood, urine, or other compartments of a patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, , 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more.

[0231] Before administering the full dose of dsRNA, the patient may be administered a smaller dose (e.g., a 5% infusion reaction) and monitored for adverse effects, such as allergic reactions. In another example, the patient may be observed for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-α or INF-α) levels.

[0232] Due to the inhibitory effect on APOC3 expression, the composition according to the present invention or a pharmaceutical composition prepared from said composition can improve the quality of life.

[0233] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar to or equivalent to those described herein can be used to practice or test the dsRNA and methods that characterize the present invention, and suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.In the case of conflict, the present specification, including definitions, will control.In addition, materials, methods and examples are merely illustrative and are not intended to be limiting. [Example]

[0234] Example 1 dsRNA synthesis Reagent Source If the source of a reagent is not specifically given herein, the reagents may be obtained from any supplier in molecular biology to quality / purity standards for use in molecular biology.

[0235] siRNA synthesis Single-stranded RNA was produced on a 1 μmole scale by solid-phase synthesis using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as the solid support. RNA and RNA containing 2'-O-methyl nucleotides were produced by solid-phase synthesis using the corresponding phosphoramidites and 2'-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated into selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry as described in *Current protocols in nucleic acid chemistry*, Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA. Phosphorothioate linkages were introduced by replacing the iodine oxidizer solution with a solution (1%) of Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile. Further auxiliary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

[0236] Crude oligoribonucleotides were deprotected and purified by anion-exchange HPLC according to established procedures. Yields and concentrations were determined by UV absorption of each RNA solution at 260 nm using a spectrophotometer (DU 640B, Beckman Coulter GmbH, Unterschleissheim, Germany). Equimolar solutions of complementary strands were mixed in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated to 85–90°C in a water bath for 3 min, and cooled at room temperature for 3–4 h to generate double-stranded RNA. The annealed RNA solution was stored at -20°C until use.

[0237] The nucleic acid sequences are presented below using standard nomenclature, specifically the abbreviations in Table B.

[0238] [Table 2]

[0239] Example 2: APOC3 siRNA design Transcripts siRNA design was performed to identify siRNAs targeting all human and cynomolgus monkey (Macaca fascicularis; hereafter "cyno") APOC3 transcripts annotated in the NCBI gene database (http: / / www.ncbi.nlm.nih.gov / gene / ). The following transcripts from NCBI were used for the design: human-NM_000040.1; cyno-X68359.1. All siRNA duplexes sharing 100% identity with the listed human and cyno transcripts were designed.

[0240] siRNA design, specificity and efficacy prediction siRNAs were selected based on predicted specificity, predicted potency, and GC content.

[0241] The predicted specificity of all possible 19-mers was predicted from each sequence. Candidate 19-mers lacking repeats longer than 7 nucleotides were then selected. These 171 candidate siRNAs were used in a comprehensive search against the human transcriptome (defined as the set of NM_ and XM_ records in the human NCBI Refseq set).

[0242] Scores were calculated based on the position and number of mismatches between the siRNA and any potential "off-target" transcripts, and the frequencies of heptamers and octamers derived from hexamers derived from three different seeds (positions 2-9 from the 5' end of the molecule) for each oligo were compared. According to the calculated scores, both siRNA strands were assigned to specificity categories: a score greater than 3 was considered highly specific, a score equal to 3 was considered specific, and a score between 2.2 and 2.8 was considered somewhat specific. We then selected duplexes whose antisense oligos had a total GC content of less than 70%, lacked GC at the first position, and did not match the mouse APOC3 transcript NM_023114.3.

[0243] siRNA sequence selection A total of 27 sense and 27 antisense siRNA oligos were synthesized and duplexed.

[0244] Example 3 APOC3 siRNA synthesis Synthesis of modified and unmodified ApoC3 sequences APOC3 tile sequences were synthesized on a MerMade 192 synthesizer at either a 1 or 0.2 umol scale.

[0245] Single and double strands were generated using either unmodified, 2'-O-methyl, or 2'-fluoro chemical modifications. Synthesis conditions were modified appropriately based on the nature of the chemical modification within the single strand.

[0246] Synthesis, cleavage and deprotection: Synthesis of APOC3 sequences (unmodified, 2-O-methyl or 2'-fluoro) was achieved using solid-phase oligonucleotide synthesis using phosphoramidite chemistry.

[0247] The synthesis of the above sequences was carried out at a 1 or 0.2 μm scale in 96-well plates. Unmodified and modified (2-O-methyl or 2′-fluoro) amidite solutions were prepared at 0.1 M concentrations, and ethylthiotetrazole (0.6 M in acetonitrile) was used as an activator.

[0248] The synthesized sequences were cleaved and deprotected in 96-well plates. Either aqueous ammonia or aqueous methylamine was used in the first step, and fluoride reagent was used in the second step. The crude sequences were precipitated using an acetone:ethanol (80:20) mix, and the pellet was resuspended in 0.2 M sodium acetate buffer to convert the crude single strands to their sodium salts. Samples from each sequence were analyzed by LC-MS to confirm identity, quantified by UV, and determined purity by IEX chromatography.

[0249] Purification and desalting: The APOC3 tile sequence was precipitated and purified using a Sephadex column on an AKTA Purifier system. This process was performed at ambient temperature. Sample injection and collection were performed in a 96-well (1.8 mL deep well) plate. A single peak corresponding to the full-length sequence was collected in the eluent. The desalted APOC3 sequence was analyzed for concentration (by UV measurement at A260) and purity (by ion-exchange HPLC). Complementary single strands were then combined at a 1:1 stoichiometry to form siRNA duplexes.

[0250] Tables 1 and 2 provide a first set of unmodified and modified sequences.

[0251] Example 4 In vitro screening of APOC3 siRNA Cell culture and transfection: Hep3B cells (ATCC, Manassas, VA) were grown to near confluence at 37°C in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) in a 5% CO atmosphere and then released from the plate by trypsinization. Transfection was performed by adding 14.8 μl of Opti-MEM + 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, cat# 13778-150) per well to 5 μl of siRNA duplexes per well in a 96-well plate and incubating at room temperature for 15 minutes. Approximately 2 × 10 cells were then transfected with 14.8 μl of Opti-MEM + 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, cat# 13778-150) per well and incubated at room temperature for 15 minutes. 4 Eighty microliters of complete growth medium containing Hep3B cells was added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single-dose experiments were performed at final duplex concentrations of 10 nM and 0.1 nM, and dose-response experiments were performed at final duplex concentrations of 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, and 0.00001 nM.

[0252] Total RNA isolation using DYNABEADS mRNA isolation kit (Invitrogen, part#:610-12): The cells were harvested and lysed in 150 μl of lysis / binding buffer, followed by mixing for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic particles and 80 μl of lysis / binding buffer mixture were added to a round-bottom plate and mixed for 1 minute. The magnetic particles were captured using a magnetic stand, and the supernatant was removed without disturbing the particles. After removing the supernatant, the lysed cells were added to the remaining particles and mixed for 5 minutes. After removing the supernatant, the magnetic particles were washed twice with 150 μl of wash buffer A and mixed for 1 minute. The particles were captured again, and the supernatant was removed. The particles were then washed with 150 μl of wash buffer B, captured, and the supernatant was removed. The particles were then washed with 150 μl of elution buffer, captured, and the supernatant was removed. The particles were then dried for 2 minutes. After drying, 50 μl of elution buffer was added and mixed for 5 minutes at 70°C. The particles were captured on a magnet for 5 minutes. 40 μl of the supernatant was removed and added to another 96-well plate.

[0253] cDNA synthesis using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat# 4368813): A master mix consisting of 2 μl of 10× buffer, 0.8 μl of 25× dNTPs, 2 μl of random primers, 1 μl of reverse transcriptase, 1 μl of RNAe inhibitor, and 3.2 μl of HO was added to 10 μl of total RNA per reaction. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermocycler (Hercules, CA) through the following steps: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 seconds, and a 4°C hold.

[0254] Real-time PCR: Two microliters of cDNA was added to a master mix containing 0.5 μl of GAPDH TaqMan probe (Applied Biosystems Cat# 4326317E), 0.5 μl of ApoC3 TaqMan probe (Applied Biosystems Cat# Hs00163644_m1), and 5 μl of Lightcycler 480 Probe Master Mix (Roche Cat# 04887301001) per well in a 384-well 50-well plate (Roche Cat# 04887301001). Real-time PCR was performed in an ABI7900HT Real Time PCR System (Applied Biosystems) using the ΔΔCt (RQ) assay. Unless otherwise noted in the summary table, each duplex was tested in two independent transfections, and each transfection was assayed in duplicate.

[0255] To calculate relative fold changes, real-time data were analyzed using the ΔΔCt method and normalized to assays performed with 10 nM AD-1955-transfected or mock-transfected cells. IC50s were calculated using a four-parameter fit model with XLFit and normalized to AD-1955-transfected or native cells over the same dose range, or to the lowest dose per assay.

[0256] Viability Screening Cell viability in HeLa and Hep3B cells was measured 3 and 5 days after transfection with 100, 10, 1, 0.1, 0.01, and 0.0001 nM siRNA. Cells were seeded at a density of 10,000 cells per well in 96-well plates. Each siRNA was assayed in triplicate, and data were averaged. siRNAs targeting PLK1 and AD-19200 were included as positive controls for loss of viability, and AD-1955 was included as a negative control. PLK1 and AD-19200 cause a dose-dependent loss of viability. To measure viability, 20 μl of CellTiter Blue (Promega) was added to each well of the 96-well plate after 3 and 5 days and incubated at 37°C for 2 hours. Plates were then read in a spectrophotometer (Molecular Devices) at 560Ex / 590Em. Viability was expressed as the mean value of light units from three replicate transfections + / - standard deviation. In some cases, relative viability was assessed by first averaging the three replicate transfections and then normalizing to the value obtained from the lowest dose (0.001 nM).

[0257] The results are provided in Tables 3, 4 and 5.

[0258] Example 5: In vivo testing of APOC3 in mice siRNA targeting APOC3 was administered to wild-type (5.0 mg / kg) and transgenic SREBPtg / LDLR- / -KO mice (1.0 mg / kg). Two days after administration, the mice were sacrificed and liver target mRNA, serum triglyceride, and serum total cholesterol levels were measured. A LNp11 formulation containing MC3 was used.

[0259] The results for wild-type mice are shown in Figure 1. Administration of siRNA targeting APOC3 resulted in knockdown of mRNA levels, a 50% reduction in triglycerides, and a reduction in total cholesterol in wild-type mice. Administration of siRNA targeting APOC3 resulted in an 80% reduction in triglycerides in a hyperlipidemia model mouse. Data not shown. These results indicate that APOC3 is a validated target for siRNA-based treatment of hypertriglyceridemia, including coronary heart disease (CAD) and pancreatitis.

[0260] Example 6: Synthesis and screening of modified siRNAs targeting APOC3 (second set) Additional modified APOC3 siRNAs were synthesized using the methods described above, as listed in Tables 6 and 7. The UMdTdsdT modification pattern involves the addition of dT-phosphorothioate-dT to each strand. The DECAF modification pattern is as follows: sense strand—2'O-methyl on all pyrimidines, dTsdT / dTdT overhangs; antisense strand—modified "U" at any two sites within the dinucleotide motif UU / UA / UG in the seed region (positions 2-9) + 2'O-methyl on the final three nucleotides (positions 17-19) + 2'O-methyl on all "U"s at positions 10-16; dTsdT / dTdT overhangs. The FOME modification pattern is as follows: sense strand—2'F (5' first base) followed by alternating 2'OMe; antisense strand—2'OMe (5' first base) followed by alternating 2'F.

[0261] The siRNAs listed in Tables 6 and 7 were assayed in Hep3b cells as described above, and the results are shown in Table 8.

[0262] Example 7: Synthesis and screening of modified siRNAs targeting APOC3 (third set) Additional modified APOC3 siRNAs were synthesized using the methods described above, as described in Tables 9 and 10. The siRNAs were assayed in Hep3b cells as described above, and the results are shown in Table 11.

[0263] Table 3

[0264] Table 4

[0265] Table 5

[0266] Table 6

[0267] Table 7

[0268] Table 8

[0269] Table 9

[0270] Table 10

[0271] Table 11

[0272] Table 12

[0273] Table 13

[0274] Table 14

[0275] Table 15

[0276] Table 16

[0277] Table 17

[0278] Table 18

[0279] Table 19

[0280] Table 20

[0281] Table 21

[0282] Table 22

[0283] Table 23

[0284] [Table 24]

[0285] [Table 25]

[0286] [Table 26]

[0287] [Table 27]

[0288] [Table 28]

[0289] [Table 29]

[0290] [Table 30]

[0291] [Table 31]

[0292] [Table 32]

[0293] SEQ ID NO: 1 NCBI Reference Sequence: NM_000040.1, Homo sapiens apolipoprotein C-III (APOC3), mRNA

change

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent, or a salt thereof, for inhibiting the expression of the apolipoprotein C3 (APOC3) gene in cells, The dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, The sense strand comprises at least 18 consecutive nucleotides from the nucleotide sequence 5'-GCUUAAAGGGACAGUAUU-3' of SEQ ID NO: 70, and the antisense strand comprises at least 18 consecutive nucleotides from the nucleotide sequence 5'-AAUACUGUCCCCUUUUAAGC-3' of SEQ ID NO:

151. The dsRNA agent or a salt thereof comprises at least one modified nucleotide selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, and 2'-deoxy modified nucleotides. A double-stranded ribonucleic acid (dsRNA) agent or salt thereof, wherein the dsRNA agent or salt thereof further comprises at least one phosphorothioate nucleotide interbond at the 3' end of the antisense strand.

2. The dsRNA agent or salt thereof according to claim 1, wherein the sense strand comprises the nucleotide sequence 5'-GCUUAAAGGGACAGUAUU-3' of SEQ ID NO: 70, and the antisense strand comprises the nucleotide sequence 5'-AAUACUGUCCCCUUUUAAGC-3' of SEQ ID NO:

151.

3. The dsRNA agent or salt thereof according to claim 1, wherein the sense strand comprises the nucleotide sequence 5'-UGCUUAAAGGGACAGUAU-3' of SEQ ID NO: 82, and the antisense strand comprises the nucleotide sequence 5'-AUACUGUCCCCUUUUAAGCA-3' of SEQ ID NO:

163.

4. The dsRNA agent or salt thereof according to claim 1, wherein at least one strand comprises a 3' overhang of at least one or two nucleotides.

5. The dsRNA agent or salt thereof according to claim 1, further comprising a ligand.

6. The dsRNA agent or salt thereof according to claim 5, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA.

7. The dsRNA agent or salt thereof according to claim 5, wherein the ligand is one or more N-acetyl-galactosamine (GalNAc) derivatives.

8. The dsRNA agent or salt thereof according to claim 7, wherein one or more GalNAc derivatives are bound to the dsRNA or a salt thereof via a divalent or trivalent branched linker.

9. Isolated cells containing a dsRNA agent or a salt thereof according to any one of claims 1 to 8.

10. A pharmaceutical composition for inhibiting the expression of the APOC3 gene, comprising a dsRNA agent or a salt thereof according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition according to claim 10, comprising a lipid preparation.

12. The pharmaceutical composition according to claim 11, comprising a lipid preparation containing MC3.

13. An in vitro method for inhibiting APOC3 expression in cells, wherein the method is: (a) Contacting the cells with the dsRNA agent or salt thereof according to any one of claims 1 to 8; (b) A method comprising maintaining the cells generated in step (a) for a sufficient period of time to obtain degradation of the mRNA transcript of the APOC3 gene, thereby inhibiting the expression of the APOC3 gene within the cells.

14. The method according to claim 13, wherein APOC3 expression is inhibited by at least 30%.

15. A dsRNA agent or salt thereof according to any one of claims 1 to 8, or a pharmaceutical composition according to any one of claims 10 to 12, for use in a method of treating a human subject having a disease mediated by APOC3 expression, wherein the method comprises administering a therapeutically effective amount of the dsRNA agent or salt thereof, or the pharmaceutical composition, to the subject.

16. The dsRNA agent or a salt thereof, or pharmaceutical composition according to claim 15, wherein the disease is hypertriglyceridemia.

17. The dsRNA agent or salt thereof, or pharmaceutical composition according to claim 16, wherein the human subject has a high triglyceride level.

18. The dsRNA agent or salt thereof, or pharmaceutical composition according to claim 15, wherein the human subject has a triglyceride level of >150 mg / dL or >500 mg / dL.

19. The dsRNA agent or salt thereof, or pharmaceutical composition according to claim 15, wherein administration of the dsRNA agent or salt thereof, or the pharmaceutical composition, results in an increase in lipoprotein lipase and / or hepatic lipase activity.

20. The dsRNA agent or salt thereof, or pharmaceutical composition according to claim 15, wherein the dsRNA agent or salt thereof, or the pharmaceutical composition, is for subcutaneous administration.

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