WO2012027713A2 - Compositions and methods for inhibition of snca - Google Patents

Compositions and methods for inhibition of snca Download PDF

Info

Publication number
WO2012027713A2
WO2012027713A2 PCT/US2011/049427 US2011049427W WO2012027713A2 WO 2012027713 A2 WO2012027713 A2 WO 2012027713A2 US 2011049427 W US2011049427 W US 2011049427W WO 2012027713 A2 WO2012027713 A2 WO 2012027713A2
Authority
WO
WIPO (PCT)
Prior art keywords
dsrna
snca
ad
sense
antis
Prior art date
Application number
PCT/US2011/049427
Other languages
French (fr)
Other versions
WO2012027713A3 (en
Inventor
Gregory Hinkle
David Bumcrot
Original Assignee
Alnylam Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US37743710P priority Critical
Priority to US61/377,437 priority
Application filed by Alnylam Pharmaceuticals, Inc. filed Critical Alnylam Pharmaceuticals, Inc.
Publication of WO2012027713A2 publication Critical patent/WO2012027713A2/en
Publication of WO2012027713A3 publication Critical patent/WO2012027713A3/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/14Type of nucleic acid interfering N.A.
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/31Chemical structure of the backbone
    • C12N2310/315Phosphorothioates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/34Spatial arrangement of the modifications
    • C12N2310/344Position-specific modifications, e.g. on every purine, at the 3'-end
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/34Spatial arrangement of the modifications
    • C12N2310/346Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) targeting alpha- synuclein (SNCA) gene expression and methods of using the dsRNA to inhibit expression of SNCA.

Description

COMPOSITIONS AND METHODS FOR INHIBITION OF SNCA

Cross Reference to Related Applications

This application claims the benefit of U.S. Provisional Application No. 61/377,437, filed August 26, 2010, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Field of the Invention

The invention relates to compositions and methods for treating neurodegenerative diseases, and more particularly to the downregulation of the alpha-synuclein gene for the treatment of synucleinopathies. Background of the Invention

Expression of the alpha-synuclein (SNCA) gene produces the protein alpha-synuclein. Mutations in the SNCA gene and SNCA gene multiplications have been linked to familial Parkinson's disease. Parkinson's disease patients demonstrate alpha-synuclein protein aggregates in the brain. Similar aggregates are observed in patients diagnosed with sporadic Parkinson's disease, Alzheimer's disease, multiple system atrophy, and Lewy body dementia.

Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al ; and WO 99/61631, Heifetz et al), Drosophila (see, e.g., Yang, D., et al, Curr. Biol. (2000) 10: 1 191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al).

Disclosure of dsRNA targeting SNCA can be found in the following published references, patents, and patent applications:

U.S. Pat. No. 7,579,458;

Lewis et al, In vivo silencing of alpha-synuclein using naked siRNA, Mol.

Neurodegener. 2008 Nov 1; 3: 19;

Sapru et al, Silencing of human alpha-synuclein in vitro and in rat brain using lentiviral- mediated RNAi, Exp Neurol. 2006 Apr;198(2):382-90.;

Manfredsson et al, RNA knockdown as a potential therapeutic strategy in Parkinson's disease, Gene Ther. 2006 Mar; 13(6):517-24.;

U.S. Ser. No. 12/334,080; U.S. Ser. No. 12/725,337;

U.S. Pat. No. 7,595,306;

U.S. Ser. No. 10/991,286;

U.S. Ser. No. 12/416, 140;

PCT/US04/18271

Summary of the Invention

Disclosed herein are dsRNAs targeted to the alpha-synuclein (SNCA) gene for inhibiting expression of SNCA in a cell. Also disclosed are methods of using the SNCA dsRNA for siRNA inhibition of SNCA expression and treatment of disease associated with expression and/or over expression of SNCA, e.g., neurodegenerative disorders.

In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of SNCA, comprising a sense strand and an antisense strand comprising a region of complementarity to an mRNA encoding SNCA, wherein each strand is at least 15 nucleotides in length, and wherein the sense strand and antisense strand comprise at least 15 contiguous nucleotides of sequences selected from Tables 2, 3, 4, or 6. In a related embodiment, the sense strand comprises SEQ ID NO: l, 17, 27, 41, 63, 75, 79, 85, 107, 1 13, 119, 123, 127, 135, 147, 155, 157, 161, 165, or 173 and/or the antisense strand comprises SEQ ID NO:2, 18, 28, 42, 64, 76, 80, 86, 108, 1 14, 120, 124, 128, 136, 148, 156, 158, 162, 166, or 174. In another related embodiment, the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO: 157. In yet another related embodiment, the antisense strand is complementary to at least the first 1 1 nucleotides of SEQ ID NO: 158. In yet another related embodiment, the sense strand sequence comprises SEQ ID NO: 157 and the antisense strand sequence comprises SEQ ID NO: 158.

In yet another embodiment, the invention provides a dsRNA for inhibiting expression of alpha-synuclein SNCA, comprising a sense strand and an antisense strand comprising a region of complementarity to an mRNA encoding SNCA, wherein each strand is at least 15 nucleotides in length, wherein the sense strand comprises SEQ ID NO:553, 569, 579, 593, 615, 627, 631, 637, 659, 665, 671, 675, 679, 687, 699, 707, 709, 713, 717, or 725; and/or the antisense strand comprises SEQ ID NO:554, 570, 580, 594, 616, 628, 632, 638, 660, 666, 672, 676, 680, 688, 700, 708, 710, 714, 718, or 726. In yet another embodiment, the sense strand sequence comprises SEQ ID NO: 707 and the antisense strand sequence comprises SEQ ID NO: 708.

In yet another embodiment, the invention provides a dsRNA selected from the group consisting of: AD-21752, AD-21760, AD-21765, AD-21772, AD-21781, AD-21786, AD-21788, AD-21790, AD-21801, AD-21804, AD-21806, AD-21808, AD-21810, AD-21813, AD-21817, AD-21821, AD-21822, AD-21824, AD-21826, and AD-21830. In a related embodiment, the dsRNA is AD-21822.

In yet another embodiment, the invention provides a dsRNA for inhibiting expression of SNCA, comprising a sense strand and an antisense strand comprising a region of

complementarity to an mR A encoding SNCA, wherein each strand is at least 15 nucleotides in length, and wherein the sense strand and antisense strand comprise at least 15 contiguous nucleotides of sequences selected from Tables 2, 3, 4, or 6, and wherein the SNCA is either human SNCA encoded by NM_007308.1 or monkey SNCA encoded by XM 001095402.1. In a related embodiment, the region of complementarity is between 19 and 21 nucleotides in length. In another related related embodiment, the region of complementarity is 19 nucleotides in length.

In another embodiment of the dsRNA for inhibiting expression of SNCA described in the Summary above, at least one strand of the dsRNA comprises a 3' overhang of at least 1 nucleotide. In another embodiment, the dsRNA comprises a nucleotide overhang having 1 to 4 nucleotides. In yet another embodiment, each strand comprises a 3 ' overhang consisting of 2 nucleotides. In yet another embodiment, each strand comprises a 3 ' overhang consisting of dTsdT.

In yet another embodiment, the dsRNA for inhibiting expression of SNCA comprises at least one modified nucleotide. In a related embodiment, the modified nucleotide is selected from the group of: a 2'-0-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In another related embodiment, said modified nucleotide is chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In yet another related embodiment, the dsRNA comprises at least one 2'-0-methyl modified nucleotide and at least one 2'-deoxythymidine-3'-phosphate nucleotide comprising a 5'-phosphorothioate group. In yet another related embodiment, the sense strand of the modified dsRNA comprises all 2'-0-methyl modified pyrimidines and the antisense strand comprises 2'-0-methyl modified pyrimidines when the pyrimidine is adjacent to A; and wherein each strand comprises dTdT at the 3 ' end. In yet another related embodiment, the sense strand of the modified dsRNA comprises all 2'-0-methyl modified pyrimidines and the antisense strand comprises 2'-0-methyl modified pyrimidines when the pyrimidine is adjacent to A; and wherein each strand comprises dTsdT at the 3' end. In yet another related embodiment, the sense strand of the modified dsRNA comprises all 2'-0-methyl modified pyrimidines and the antisense strand comprises 2'-0-methyl modified pyrimidines when a) the pyrimidine is adjacent to A, or b) the pyrimidine is a uracil adjacent to a U or a G; and wherein each strand comprises dTsdT at the 3' end.

In yet another embodiment, the dsRNA for inhibiting expression of SNCA further comprising a ligand. In a related embodiment, the ligand is conjugated to the 3 '-end of the sense strand of the dsRNA.

In yet another embodiment, the invention provides a composition for inhibiting expression of a SNCA gene comprising one of the dsRNAs for inhibitting expression of SNCA described above and a pharmaceutical formulation. In a related embodiment, the pharmaceutical formulation is a lipid formulation. In another related embodiment, the pharmaceutical formulation is a LNP formulation, a LNP01 formulation, a XTC-SNALP formulation, a SNALP formulation, or a LNP 11 formulation.

In another embodiment, the invention provides a dsRNA composition described above for inhibiting expression of a SNCA gene, wherein upon contact with a cell expressing the SNCA gene, the dsRNA inhibits expression of the SNCA gene by at least 40% compared to a cell not so contacted. In another embodiment, the dsRNA has an IC50 of 10 pM or less. In another embodiment, the invention provides a dsRNA composition described above for inhibiting expression of a SNCA gene, wherein administration of 0.1 nM of the dsRNA to a NCI-H460 cell or a BE(2)M17 cell results in greater than 50% inhibition of SNCA mRNA expression as measured by a real time PCR assay or administration of 10 nM of the dsRNA to a NCI-H460 cell or a BE(2)M17 cell results in greater than 66% inhibition of SNCA mRNA expression as measured by a real time PCR assay.

In another embodiment, the invention provides an isolated cell containing one of the dsRNAs described above for inhibiting expression of a SCNA gene. In yet another embodiment, the invention provides a vector comprising a nucleotide sequence that encodes the nucleic acid sequence of at least one strand of a dsRNA described above. In a related embodiment, the invention provides a cell comprising this vector.

In yet another embodiment, the invention provides a method of inhibiting SNCA expression in a cell, the method comprising: (a) introducing into the cell a dsRNA comprising a sense strand and an antisense strand comprising a region of complementarity to an mRNA encoding SNCA, wherein each strand is at least 15 nucleotides in length, and wherein the sense strand and antisense strand comprise at least 15 contiguous nucleotides of sequences selected from Tables 2, 3, 4, or 6; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a SNCA gene, thereby inhibiting expression of the SNCA gene in the cell. In a related embodiment, expression is inhibited by at least 20%, 40%, 60%, or at least 80%. In another related embodiment, the dsRNA is a dsRNA for inhibiting SNCA expression described above. In yet another related embodiment, the invention provides a method treating a disorder mediated by SNCA expression, comprising administering to a human in need of such treatment a therapeutically effective amount of a dsRNA for inhibiting SNCA expression described above. In yet another embodiment, the invention provides a method of treating, preventing or managing a neurodegenerative disorder comprising administering to a patient in need of such treatment, prevention or management a therapeutically or

prophylactically effective amount of a dsRNA for inhibiting SNCA described above. In a related embodiment, the neurodegenerative disorder is a synucleinopathy. In another related embodiment, the neurodegenerative disorder is Parkinson's disease. In yet another related embodiment, the neurodegenerative disorder is Alzheimer's disease, multiple system atrophy, or Lewy body dementia.

In yet another embodiment, the invention provides a method of treating a human comprising: identifying a human diagnosed as having a neurodegenerative disorder or at risk for developing the neurodegenerative disorder and administering a dsRNA for inhibiting SNCA described above. In a related embodiment, the method comprises administering an additional composition. In yet another related embodiment, the additional composition is a second dsRNA.

In a further embodiment, the invention provides a method of inhibiting SNCA expression in the brain comprising administering an effective amount of a dsRNA described above into the brain of a subject to obtain suppression of expression of SNCA mRNA, thereby inhibiting SNCA expression in the brain. In a related embodiment, the administration is by infusion. In other related embodiments, the dsRNA comprises a sense strand of SEQ ID NO: 709 or 679 and/or an antisense strand of SEQ ID NO: 710 or 680. In further related embodiments, the dsRNA is AD- 21822 or AD-21810. In another related embodiment, the administration of the dsRNA results in greater than 40%, 50%, 60%, 70%, 80%, or 90% inhibition of SNCA mRNA expression. In additional related embodiments, the administration of the dsRNA results in greater than 65% inhibition of SNCA mRNA expression at the end of infusion, greater than 70% inhibition of SNCA mRNA expression one week after infusion, greater than 40% inhibition of SNCA mRNA expression two weeks after infusion, or greater than 50% inhibition of SNCA mRNA expression four weeks after infusion. In other related embodiments, the administration of the dsRNA results in greater than 50%, 60%, 70%, or 80% inhibition of SNCA mRNA expression after 7 or 28 days of infusion. Additional embodiments of the invention are set forth in the following description and in the claims.

Description of the Drawings

Not applicable. Detailed Description of the Invention

The invention provides dsRNAs and methods of using the dsRNAs for inhibiting the expression of alpha-synuclein (SNCA). The invention also provides compositions and methods for treating pathological conditions and diseases, such as neurodegenerative diseases in a mammal caused by the over-expression of SNCA. A dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

The dsRNAs of the compositions featured herein include an antisense strand having a region which is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and is complementary to at least part of an mRNA transcript of a SNCA gene. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in pathologies associated with SNCA expression in mammals. Very low dosages of SNCA dsRNAs in particular can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a SNCA gene. Using cell-based assays, the present inventors demonstrate that dsRNAs targeting SNCA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a SNCA gene. Thus, methods and compositions including these dsRNAs are useful for treating pathological processes that can be mediated by down regulating SNCA over-expression, such as, e.g., treatment of neurodegenerative diseases.

The following detailed description discloses how to make and use the compositions containing dsRNAs to inhibit the expression of a SNCA gene, as well as compositions (e.g., pharmaceutical compositions) and methods for treating diseases and disorders caused by the expression of this gene.

Accordingly, in some aspects, pharmaceutical compositions containing a SNCA dsRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of a SNCA gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of a SNCA gene are featured in the invention.

As used herein, disorders associated with SNCA expression refer to any biological or pathological state that (1) is mediated in part by the presence of SNCA protein and (2) whose outcome can be affected by reducing the level of SNCA protein present. Specific disorders associated with SNCA expression are noted below. Because dsRNA mediated silencing can persist for several days after administering the dsRNA composition, in many instances, it is possible to administer the composition with a frequency once per day or less, or, for some instances, only once for the entire therapeutic regimen.

Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

"G," "C," "A" and "U" each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. "T" and "dT" are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g.,

deoxyribothymine. However, it will be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.

As used herein "SNCA" refers to a SNCA mRNA, protein, peptide, or polypeptide. The term "SNCA" is also known in the art as alpha-synuclein.

The sequence of a human SNCA mRNA transcript can be found at NM_007308.1. The sequence of rhesus monkey SNCA mRNA can be found at XM 001095402.1.

As used herein "target" or "target gene" refers to a SNCA gene.

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

As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. As used herein, and unless otherwise indicated, the term "complementary," when used to describe 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 duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as "fully complementary" for the purposes described herein.

"Complementary" sequences, as used herein, may also include, or be formed entirely from, non- Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non- Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.

The terms "complementary," "fully complementary" and "substantially complementary" herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is "substantially complementary to at least part of a messenger R A (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., a target gene, e.g., an mRNA encoding SNCA) including a 5 ' UTR, an open reading frame (ORF), or a 3 ' UTR. For example, a polynucleotide is complementary to at least a part of a SNCA mRNA if the sequence is substantially complementary to a non- interrupted portion of an mRNA encoding SNCA.

The term "double-stranded RNA" or "dsRNA," as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, "dsRNA" may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such

modifications, as used in an siRNA type molecule, are encompassed by "dsRNA" for the purposes of this specification and claims

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3 '-end of one strand and the 5 '-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a "hairpin loop." Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3'- end of one strand and the 5 '-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a "linker." The RNA strands may have the same or a 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 that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. The term "siRNA" is also used herein to refer to a dsRNA as described above.

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

The term "antisense strand" refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and/or 3 ' terminus.

The term "sense strand," as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA agent or a plasmid from which an iRNA agent is transcribed. SNALP are described, e.g., in U.S. Patent Application Publication Nos. 2006/0240093, 2007/0135372, and USSN 61/045,228 filed on April 15, 2008. These applications are hereby incorporated by reference.

"Introducing into a cell," when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be "introduced into a cell," wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.

The terms "silence," "inhibit the expression of," "down-regulate the expression of," "suppress the expression of and the like, in as far as they refer to a target gene, herein refer to the at least partial suppression of the expression of a SNCA gene, as manifested by a reduction of the amount of mRNA which may be isolated or detected from a first cell or group of cells in which a SNCA gene is transcribed and which has or have been treated such that the expression of a SNCA gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of (mRNA in control cells) - (mRNA in treated cells) , ..„ .

— - · 100%

(mRNA in control cells)

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to SNCA gene transcription, e.g., the amount of protein encoded by a SNCA gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g., apoptosis. In principle, SNCA gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a SNCA gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of a SNCA gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a SNCA gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a SNCA gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide featured in the invention.

As used herein in the context of SNCA expression, the terms "treat," "treatment," and the like, refer to relief from or alleviation of pathological processes mediated by SNCA expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by SNCA expression), the terms "treat," "treatment," and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.

As used herein, the term "SNCA-mediated condition or disease" and related terms and phrases refer to a condition or disorder characterized by inappropriate, e.g. , greater than normal, SNCA activity. Inappropriate SNCA functional activity might arise as the result of SNCA expression in cells which normally do not express SNCA, or increased SNCA expression (leading to, e.g., neurodegenerative disease). A SNCA-mediated condition or disease may be completely or partially mediated by inappropriate SNCA functional activity. However, a SNCA- mediated condition or disease is one in which modulation of SNCA results in some effect on the underlying condition or disorder (e.g., a SNCA inhibitor results in some improvement in patient well-being in at least some patients).

As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a pathological processes mediated by SNCA expression, such as a neurodegenerative disorder such as synucleinopathy (e.g., Parkinson's disease) or an overt symptom of pathological processes mediated by SNCA expression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by SNCA expression, the patient's history and age, the stage of pathological processes mediated by SNCA expression, and the administration of other anti-pathological processes mediated by SNCA expression agents.

As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein,

"pharmacologically effective amount," "therapeutically effective amount" or simply "effective amount" refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.

The term "pharmaceutically acceptable carrier" refers to a carrier for administration of 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 medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, 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 disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

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

Alp ha-synuclein

The alpha-synuclein protein (SNCA) is primarily found in the cytoplasm, but has also been localized to the nucleus. In dopaminergic neurons, alpha-synuclein is membrane bound. The protein is a soluble monomer normally localized at the presynaptic region of axons. The protein can form filamentous aggregates that are the major component of intracellular inclusions in neurodegenerative synucleinopathies.

The alpha-synuclein protein is associated with a number of diseases characterized by synucleinopathies. Three point mutations (A53T, A30P and E46K), and SNCA duplication and triplication events are linked to autosomal dominant Parkinson's disease (familial Parkinson's disease, also called FPD). The A53T and A30P mutations cause configuration changes in the SNCA protein that promote in vitro protofibril formation. The triplication event results in a twofold overexpression of SNCA protein. Alpha-synuclein is a major fibrillar component of Lewy bodies, the cytoplasmic inclusions that are characteristic of FPD and idiopathic Parkinson's disease, and the substantia nigra of a Parkinson's disease brain is characterized by fibrillar alpha- synuclein. In Alzheimer's patients, SNCA peptides are a major component of amyloid plaques in the brains of patients with Alzheimer's disease.

Aggregation of alpha-synuclein in the cytoplasm of cells can be caused by a number of mechanisms, including overexpression of the protein, inhibition of protein degradation, or a mutation that affects the structure of the protein, resulting in an increased tendency of the protein to self-associate.

An SNCA gene product can be a target for treatment methods of neurodegenerative diseases such as Parkinson's disease. The treatment methods can include targeting of an SNCA nucleic acid with a dsRNA. Alternatively, or additionally, an antisense RNA can be used to inhibit gene expression, or an antibody or small molecule can be used to target an SNCA nucleic acid. In general, an antisense RNA, anti-SNCA antibody, or small molecule can be used in place of a dsRNA, e.g., by any of the methods or compositions described herein. A combination of therapies to downregulate SNCA expression and activity can also be used.

Sequencing of the SNCA gene has revealed common variants including a dinucleotide repeat sequence (REPl) within the promoter. REP l varies in length across populations, and certain allelic variants are associated with an increased risk for Parkinson's disease (Kruger et ah, Ann Neurol. 45:61 1-7, 1999). The SNCA gene REP l locus is necessary for normal gene expression (Touchman et ah, Genome Res. 1 1 :78-86, 2001). SNCA gene expression levels among the different REPl alleles varied significantly over a 3 -fold range, suggesting that the association of specific genotypes with an increased risk for Parkinson's disease may be a consequence of SNCA gene over-expression (Chiba-Falek and Nussbaum, Hum Mol. Genet. 10:3101-9, 2001). Functional analysis of intra-allelic variation at the SNCA gene REPl locus implied that overall length of the allele plays the main role in transcriptional regulation;

sequence heterogeneity is unlikely to confound genetic association studies based on alleles defined by length (Chiba-Falek et al, Hum Genet. 113:426-31, 2003). The recent discovery of SNCA gene triplication as a rare cause of Parkinson's disease is consistent with the observation that polymorphism within the gene promoter confers susceptibility via the same mechanism of gene over-expression (Singleton et al, Science 302:841, 2003).

Three splice variants of SNCA have been identified. The full-length 140 amino acid protein is the most abundant form. A 128 amino acid form lacks exon 3, and a 112 amino acid form lacks exon 5. An iRNA featured in the invention can target any isoform of SNCA. An iRNA can target a common exon (e.g., exon 2, 4, 6, or 7) to effectively target all known isoforms. A dsRNA can target a splice junction or an alternatively spliced exon to target specific isoforms. For example, to target the 112 amino acid isoform, a dsRNA can target an mRNA sequence that overlaps the exon 4/exon 6 splice junction. To target the 128 amino acid protein isoform, a dsRNA can target an mRNA sequence that overlaps the exon 2/exon 4 junction.

Double-stranded ribonucleic acid (dsRNA)

As described in more detail herein, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a SNCA gene in a cell or mammal, where the dsRNA includes a sense strand having a first sequence and an antisense strand comprising a second sequence complementary to mRNA encoding SNCA, wherein said first sequence is complementary to said second sequence at a region of complementarity and wherein each strand is 15 to 30 base pairs in length. In some embodiments, the dsRNA of the invention inhibits the expression of said SNCA gene by at least 40% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of a SNCA gene can be reduced by at least 30% when measured by an assay as described in the Examples below. For example, expression of a SNCA gene in cell culture, such as in HepB3 cells, can be assayed by measuring SNCA mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by ELISA assay.

The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. The dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure.

One strand of the dsRNA (the antisense strand) includes a region of complementarity that is complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a target gene, the other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. The region of complementarity is generally at least 15 nucleotides in length, or between 19 and 21 nucleotides in length, or 19, 20, or 21 nucleotides in length. In some embodiments the region of complementarity includes at least 15 contiguous nucleotides of one of the antisense sequences listed in Tables 2, 3, or 4. In other embodiments the region of complementarity includes one of the antisense sequences listed in Tables 2, 3, or 4.

Generally, the duplex structure is between 15 and 30, or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different dsRNAs are used in combination, the duplex lengths can be identical or can differ.

Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is 25-30 nucleotides in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ.

The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, or 1, 2, 3, or 4 nucleotides. In another embodiment, the overhang includes dTdT. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3' end and the 5' end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3' end and the 5' end over the antisense strand.

A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3 '-terminal end of the antisense strand or, alternatively, at the 3 '-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5 '-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., 5 mg/kg body weight of the recipient or less per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3 '-end, and the 5 '-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In one embodiment, a SNCA gene is a human SNCA gene, e.g., the sequence identified by GenBank accession number NM_007308.1. In another embodiment, a SNCA gene is a rhesus monkey SNCA gene, e.g., the sequence identified by GenBank accession number

XM_001095402.1.

In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Tables 3-6, and the antisense strand is one of the antisense sequences of Tables 3-6.

Alternative antisense agents that target elsewhere in the target sequence provided in Tables 3-6 can readily be determined using the target sequence and the flanking SNCA sequence.

The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al, EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 3-6, the dsRNAs featured in the invention can include at least one strand of a length described therein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Tables 3-6 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, 21, or 22, or more contiguous nucleotides from one of the sequences of Tables 3-6, and differing in their ability to inhibit the expression of a SNCA gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30 % inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired SNCA target sequence can readily be made using the corresponding SNCA antisense sequence and a complementary sense sequence.

In addition, the dsRNAs provided in Tables 3-6 identify a site in a SNCA that is susceptible to RNAi based cleavage. As such, the present invention further features 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 the 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 contiguous nucleotides from one of the sequences provided in Tables 3-6 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a SNCA gene.

Additional dsRNA of the invention include those that cleave a target mRNA at the same location as a dsRNA described in any of the tables. In general, a RISC complex will cleave a target mRNA between the nucleotides complementary to nucleotides 10 and 11 of the antisense strand of a dsRNA, e.g., siRNA, of the invention. Cleavage sites can be assayed using, e.g., a 5' RACE assay.

For example, the duplex AD-21752 includes the sense and antisense strands below.

Treatment of a cell with this duplex results in cleavage of human SNCA mRNA at the nucleotides complementary to nucleotides 10 and 11 of the antisense strand, e.g., nucleotides 333 and 334. Therefore, also included in the invention are those dsRNA that cleave at that location.

The dsRNA featured in the invention can contain one or more mismatches to the target sequence. In one embodiment, the dsRNA featured in the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of

complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 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, for a 23 nucleotide dsRNA strand which is complementary to a region of a target gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a target gene.

Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a target gene is important, especially if the particular region of complementarity in a target gene is known to have polymorphic sequence variation within the population.

Modifications

In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids featured in the invention may 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.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Specific examples of dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Modified dsRNA backbones include, for example, phosphorothioates, chiral

phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and

aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates,

thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the 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.

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; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519, 126;

5,536,821; 5,541,316; 5,550, 11 1 ; 5,563,253; 5,571,799; 5,587,361 ; and 5,625,050, each of which is herein incorporated by reference.

Modified dsR A backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or ore or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

Representative U.S. 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;

5,489,677; 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 herein

incorporated by reference.

In other suitable dsRNA mimetics, both the sugar and the intemucleoside 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 mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of a dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.

5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et ah, Science, 1991, 254, 1497-1500.

Other embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular— CH2~NH-CH2— , -CH2— N(CH3)-0~CH2~ [known as a methylene (methylimino) or MMI backbone],— CH2— O— N(CH3)-CH2-, -CH2-N(CH3)--N(CH3)--CH2- and -N(CH3)-CH2-CH2-[wherein the native phosphodiester backbone is represented as— O— P-O— CH2— ] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N- alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted CI to CIO alkyl or C2 to CIO alkenyl and alkynyl. Particularly preferred are 0[(CH2)nO]mCH3, 0(CH2)nO CH3, 0(CH2)nNH2, 0(CH2)n CH3, 0(CH2)nONH2, and 0(CH2)nON[(CH2)n CH3)]2, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2' position: CI to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, S CH3, OCN, CI, Br, CN, CF3, OCF3, SO CH3, S02 CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,

polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a dsRNA, or a group for improving the pharmacodynamic properties of a dsRNA, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-0— CH2 CH20 CH3, also known as 2'-0- (2-methoxyethyl) or 2'-MOE) (Martin et al, Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-0- CH2-0- CH2-N(CH2)2, also described in examples herein below.

Other preferred modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may 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 the 5' position of 5' terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. 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; 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 instant application, and each of which is herein incorporated by reference in its entirety.

A dsRNA may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. 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 other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8- substituted adenines and guanines, 5-halo, particularly 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-daazaadenine and 3-deazaguanine and 3-deazaadenine.

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et ah, Angewandte Chemie,

International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°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, even more particularly when combined with 2'-0- methoxy ethyl sugar modifications. Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5, 134,066; 5,175,273; 5,367,066; 5,432,272; 5,457, 187; 5,459,255; 5,484,908; 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 herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

Conjugates

Another modification of the dsRNAs featured in the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem. Let, 1994, 4: 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al, Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J, 1991, 10: 1 11 1-11 18; Kabanov et al, FEBS Lett, 1990, 259:327-330; Svinarchuk et al, Biochimie, 1993, 75:49-54), a phospholipid, e.g., di- hexadecyl-rac-glycerol or triethyl-ammonium l,2-di-0-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al, Tetrahedron Lett, 1995, 36:3651-3654; Shea et al, Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264:229- 237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923-937).

Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;

5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109, 124; 5,1 18,802; 5,138,045;

5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;

4,762,779; 4,789,737; 4,824,941 ; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;

5,1 12,963; 5,214, 136; 5,082,830; 5,1 12,963; 5,214, 136; 5,245,022; 5,254,469; 5,258,506;

5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;

5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574, 142; 5,585,481 ; 5,587,371 ;

5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference. It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within a dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. "Chimeric" dsRNA compounds or

"chimeras," in the context of this invention, are dsRNA compounds, particularly dsR As, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al, Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al, Bioorg. Med. Chem. Lett., 1994, 4: 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al, Bioorg. Med. Chem. Let, 1993, 3:2765), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J., 1991, 10: 1 11 ; Kabanov et al, FEBS Lett, 1990, 259:327; Svinarchuk et al. , Biochimie, 1993, 75:49), a phospholipid, e.g., 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), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.

Vector encoded dsRNAs

In another aspect, dsRNA molecules of the invention are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al, TIG. (1996), 12:5-10; Skillern, A., et al. , International PCT Publication No. WO 00/221 13, Conrad, International PCT Publication No. WO 00/221 14, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al, Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, 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 variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, 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 87:61416145; Huber et al, 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al, 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al, 1991, Science 254: 1802-1805; van Beusechem. et al, 1992, Proc. Nad. Acad. Sci. USA

89:7640-19 ; Kay et al, 1992, Human Gene Therapy 3 :641-647; Dai et al, 1992, Proc.

Natl.Acad. Sci. USA 89: 10892-10895; Hwu et al, 1993, J. Immunol. 150:4104-41 15; U.S. Patent No. 4,868,1 16; U.S. Patent 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 a cell can be produced by transfecting the recombinant retroviral genome 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 adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al, 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.

For example, lentiviral vectors featured in the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors featured in the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1 : 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A ei al, Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.

Viral vectors can be derived from AV and AAV. In one embodiment, the dsRNA featured in the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H 1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the dsRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

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

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

In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al, 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D 1 - thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single target gene or multiple target genes over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Target gene specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

Pharmaceutical compositions containing dsRNA

In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a SNCA gene, such as pathological processes mediated by SNCA expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.

The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of SNCA genes. In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams siRNA per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg, , 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 2 mg/kg, 3.0 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.

In one embodiment, the dosage is between 0.01 and 0.2 mg/kg. For example, the dsRNA can be administered at a dose of 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 , 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or 0.20 mg/kg.

The dsRNA can be administered at a dose of 0.03 mg/kg.

The dsRNA can be administered at a dose of 0 to 1 mg/kg, 1 to 2 mg/kg, 2 to 3 mg/kg, 3 to 4 mg/kg. 4 to 5 mg/kg, 5 to 6 mg/kg, 6 to 7 mg/kg, 7 to 8 mg/kg, 8 to 9 mg/kg, or 9 to 10 mg/kg.

The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The effect of a single dose on SNCA levels is long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals, or at not more than 5, 6, 7, 8, 9, or 10 week intervals.

The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by SNCA expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human SNCA. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human SNCA.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) 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 more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

The dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Administration

The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration.

The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).

The present invention includes pharmaceutical compositions that can be delivered by injection directly into the brain. The injection can be by stereotactic injection into a particular region of the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus), or the dsRNA can be delivered into multiple regions of the central nervous system (e.g., into multiple regions of the brain, and/or into the spinal cord). The dsRNA can also be delivered into diffuse regions of the brain (e.g., diffuse delivery to the cortex of the brain).

In one embodiment, a dsRNA targeting SNCA can be delivered by way of a cannula or other delivery device having one end implanted in a tissue, e.g., the brain, e.g., the substantia nigra, cortex, hippocampus, striatum, corpus callosum or globus pallidus of the brain. The cannula can be connected to a reservoir of the dsRNA composition. The flow or delivery can be mediated by a pump, e.g., an osmotic pump or minipump, such as an Alzet pump (Durect,

Cupertino, CA). In one embodiment, a pump and reservoir are implanted in an area distant from the tissue, e.g., in the abdomen, and delivery is effected by a conduit leading from the pump or reservoir to the site of release. Infusion of the dsRNA composition into the brain can be over several hours or for several days, e.g., for 1, 2, 3, 5, or 7 days or more. Devices for delivery to the brain are described, for example, in U.S. Patent Nos. 6,093, 180, and 5,814,014.

Pharmaceutical compositions and formulations for topical administration may 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 dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to 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, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

Liposomal formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter 50 nm or less, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

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

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act. Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side- effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high- molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al, Biochem. Biophys. Res. Commun, 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. 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 exogenous gene was detected in the target cells (Zhou et al, Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

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

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl

dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al.

S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle- forming lipid portion of the liposome (A) comprises one or more glycolipids, such as

monosialoganglioside GMI, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a 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).

Various liposomes comprising one or more glycolipids are known in the art.

Papahadjopoulos et al (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of

monosialoganglioside GMI, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al, disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GMI or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543, 152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2Cm5G, that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B l and WO 90/04384 to Fisher. Liposome compositions containing 1- 20 mole percent of PE derivatized with PEG, and methods of use thereof, are 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 European Patent No. EP 0 496 813 Bl). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising 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.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self- repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it 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. In general their HLB values range from 2 to about 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. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

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

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.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). Nucleic acid lipid particles

In one embodiment, a SNCA dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non- cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include "pSPLP," which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter 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, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid- lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid- lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1 : 1 to about 50: 1, from about 1 : 1 to about 25: 1, from about 3 : 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1. In some embodiments the lipid to dsRNA ratio can 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.

In general, the lipid-nucleic acid particle is suspended in a buffer, e.g., PBS, for administration. In one embodiment, the pH of the lipid formulated siRNA is between 6.8 and 7.8, e.g., 7.3 or 7.4. The osmolality can be, e.g., between 250 and 350 mOsm/kg, e.g., around 300, e.g., 298, 299, 300, 301, 302, 303, 304, or 305.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3- dioleoyloxy)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), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2- Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), l,2-Dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleyoxy-3-morpholinopropane (DLin- MA), l,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3- dimethylaminopropane (DLin-S-DMA), l-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-( - methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanedio (DOAP), l,2-Dilinoleyloxo-3-(2-N,N- dimethylamino)ethoxypropane (DLin-EG-DMA), l,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,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][l,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), l, l'-(2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethylazanediyl)didodecan-2-ol (Tech Gl), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC),

dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),

dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG- dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (CI 6), or a PEG- distearyloxypropyl

(Cis). Other examples of PEG conjugates include PEG-cDMA (N-[(methoxy poly(ethylene glycol)2000)carbamyl]-l,2-dimyristyloxlpropyl-3 -amine), mPEG2000-DMG (mPEG- dimyrystylglycerol (with an average molecular weight of 2,000) and PEG-C-DOMG (R-3-[(co- methoxy-poly(ethylene glycol)2000)carbamoyl)]-l,2-dimyristyloxlpropyl-3-amine). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 1.1., 1.2, .13, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In one embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane is described in United States provisional patent application number 61/107,998 filed on October 23, 2008, which is herein incorporated by reference.

For example, the lipid-siRNA particle can include 40% 2, 2-dilinoleyl-4- dimethylaminoethyl-[l,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0 ± 20 nm and a 0.027 siRNA/Lipid Ratio.

In still another embodiment, the compound l,l'-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin- 1 - yl)ethylazanediyl)didodecan-2-ol (Tech Gl) can be used to prepare lipid-siRNA particles. For example, the dsRNA can be formulated in a lipid formulation comprising Tech-Gl, distearoyl phosphatidylcholine (DSPC), cholesterol and mPEG2000-DMG at a molar ratio of

50: 10:38.5: 1.5 at a total lipid to siR A ratio of 7: 1 (w wt). LNP01

In one embodiment, the lipidoid ND98-4HC1 (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid- siRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48: 10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

Figure imgf000039_0001

Formula 1

LNPOl formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-siRNA formulations are as follows:

Figure imgf000039_0002
(3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- ALNlOO/DSPC/Cholesterol/PEG-DMG

LNP10 octadeca-9, 12-dienyl)tetrahydro-3 aH- 50/10/38.5/1.5

cyclopentafd] [ 1 ,3 ]dioxol-5 -amine (ALN 100) Lipid: siRNA 10: 1

(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMG

LNP11 tetraen- 19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5

(MC3) Lipid: siRNA 10: 1

l,l'-(2-(4-(2-((2-(bis(2-

LNP12 hydroxydodecyl)amino)ethyl)(2- C 12-200/DSPC/Cholesterol/PEG-DMG

hydroxydodecyl)amino)ethyl)piperazin- 1 - 50/10/38.5/1.5

yl)ethylazanediyl)didodecan-2-ol (C12-200) Lipid: siRNA 10: 1

XTC/DSPC/Chol/PEG-DMG

LNP13 XTC 50/10/38.5/1.5

Lipid:siRNA: 33: 1

MC3/DSPC/Chol/PEG-DMG

LNP14 MC3 40/15/40/5

Lipid:siRNA: 11 : 1

MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-

LNP15 MC3 DSG

50/10/35/4.5/0.5

Lipid:siRNA: 11 : 1

MC3/DSPC/Chol/PEG-DMG

LNP16 MC3 50/10/38.5/1.5

Lipid:siRNA: 7:1

MC3/DSPC/Chol/PEG-DSG

LNP17 MC3 50/10/38.5/1.5

Lipid:siRNA: 10: 1

MC3/DSPC/Chol/PEG-DMG

LNP18 MC3 50/10/38.5/1.5

Lipid:siRNA: 12: 1

MC3/DSPC/Chol/PEG-DMG

LNP19 MC3 50/10/35/5

Lipid:siRNA: 8:1

MC3/DSPC/Chol/PEG-DPG

LNP20 MC3 50/10/38.5/1.5

Lipid:siRNA: 10: 1

C 12-200/DSPC/Chol/PEG-DSG

LNP21 C12-200 50/10/38.5/1.5

Lipid:siRNA: 7:1

XTC/DSPC/Chol/PEG-DSG

LNP22 XTC 50/10/38.5/1.5

Lipid:siRNA: 10: 1

D SP C : distearoy lphosphatidy lcholine

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of

2000)

PEG-DSG: PEG-distyryl glycerol (CI 8-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-l,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)

SNALP (l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed April 15, 2009, which is hereby incorporated by reference.

LNP09 formulations and XTC comprising formulations are described, e.g., in U.S.

Provisional Serial No. 61/239,686, filed September 3, 2009, and International patent application no. PCT/US 10/22614, filed January 29, 2010, which are hereby incorporated by reference.

LNP 11 formulations and MC3 comprising formulations are described, e.g., in U.S.

Provisional Serial No. 61/244,834, filed September 22, 2009, and U.S. Provisional Serial No. 61/185,800, filed June 10, 2009, which are hereby incorporated by reference.

ALN100, i.e., ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on November 10, 2009, which is hereby incorporated by reference.

LNP 12 formulations and C12-200 comprising formulations are described, e.g., in U.S.

Provisional Serial No. 61/175,770, filed May 5, 2009, and International Application No.

PCT/USlO/33777, filed May 5, 2010, which are hereby incorporated by reference.

Formulations prepared by either the standard or extrusion- free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment.

Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can 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 the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the "free" siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For SNALP formulation, the particle size is 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 1 10 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 1 10 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm. Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxy cholic 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, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether,

polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates;

polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE- derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE- hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Patent 6,887,906, U.S. Publication No. 20030027780, and U.S. Patent No.

6,747,014, each of which is incorporated herein by reference.

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 such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

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

The compositions of the present invention may be formulated into any of many possible dosage forms such as, 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 which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μιη in diameter (Idson, in Pharmaceutical Dosage Forms,

Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et ah, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water- in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into 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 phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in- water- in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide 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 a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion- style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be 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, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in

Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).

Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified 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, N.Y., volume 1, p. 285). Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include 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, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion

formulations include methyl paraben, propyl paraben, quaternary ammonium salts,

benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.

Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral, and parenteral routes and methods for their manufacture have been reviewed in the scientific literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile 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, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in- water (o/w) type is dependent on the properties of the oil and surfactant used and on 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).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water- insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously. Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310),

hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et ah, Pharmaceutical Research, 1994, 1 1, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol, 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant- induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity

(Constantinides et al, Pharmaceutical Research, 1994, 1 1, 1385; Ho et al, J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the

gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsR As and nucleic acids of the present invention. 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 has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non- lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e. , 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 mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or "surface-active agents") are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. 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).

Fatty acids: Various fatty acids and their derivatives which 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 - dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, Ci_io alkyl esters thereof (e.g. , methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (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; El Hariri et al , J. Pharm. Pharmacol, 1992, 44, 651 -654). Bile salts: The physiological role of bile includes the facilitation of dispersion 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. Thus the term "bile salts" includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), 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, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 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, J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5- methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al, J. Control Rel, 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7,

1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1- alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al, J. Pharm. Pharmacol, 1987, 39, 621-626).

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered 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, 1 15- 12 l j Takakura ei fl/., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

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, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously 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, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible,

pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.

However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include

(a) one or more dsRNA compounds and (b) one or more anti-cytokine biologic agents which function by a non-RNAi mechanism. Examples of such biologies include, biologies that target IL 1 β (e.g. , anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab, or certolizumab). Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining 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 and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) 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 more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by SNCA expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

Evaluation of candidate dsRNA agents

A candidate dsRNA agent can be evaluated for its ability to down-regulate SNCA gene expression. For example, a candidate dsRNA agent can be provided, and contacted with a cell that expresses the SNCA gene. The level of SNCA gene expression prior to and following contact with the candidate dsRNA agent can then be compared. The SNCA target gene can be an endogenous or exogenous gene within the cell. If it is determined that the amount of RNA or protein expressed from the SNCA gene is lower following contact with the dsRNA agent, then it can be concluded that the dsRNA agent downregulates SNCA gene expression. The level of SNCA RNA or protein in the cell can be determined by any method desired. For example, the level of SNCA RNA can be determined by Northern blot analysis, reverse transcription coupled with polymerase chain reaction (RT-PCR), or R Ase protection assay. The level of protein can be determined by, for example, Western blot analysis.

The dsR A agent can be tested in an in vitro or/and in an in vivo system. For example, the target gene or a fragment thereof can be fused to a reporter gene on a plasmid. The plasmid can be transfected into a cell with a candidate dsRNA agent. The efficacy of the dsRNA agent can be evaluated by monitoring expression of the reporter gene. The reporter gene can be monitored in vivo, such as by fluorescence or in situ hybridization. Exemplary fluorescent reporter genes include but are not limited to green fluorescent protein and luciferase. Expression of the reporter gene can also be monitored by Northern blot, RT-PCR, RNAse-protection assay, or Western blot analysis as described above.

Efficacy of a dsRNA agent can be tested in a mammalian cell line (e.g. , a mammalian neural cell line), such as a human neuroblastoma cell line. For example, cell lines useful for testing efficacy of a dsRNA agent are those with a neuronal phenotype (neuroblastomas, neuronally differentiated phaeochromocytomas and primary neuronal cultures) or non-neuronal cell lines (e.g., kidney, muscle or ovarian cells). Neuroblastoma cell lines include BE(2)-M17, SH-SY5Y (both human) and N2a (mouse). BE(2)-M17 cells biochemically mimic dopaminergic neurons of the human brain affected by alpha-synucleinopathies.

Controls include:

(1) testing the efficacy and specificity of a dsRNA by assaying for a decrease in expression of the target gene by, for example, comparison to expression of an endogenous or exogenous off-target RNA or protein; and

(2) testing specificity of the effect on target gene expression by administering a

"nonfunctional" dsRNA agent.

Nonfunctional control dsRNA agents can:

(a) target a gene not expressed in the cell;

(b) be of nonsensical sequence (e.g., a scrambled version of the test dsRNA); or

(c) have a sequence complementary to the target gene, but be known by previous experiments to lack an ability to silence gene expression.

Assays include time course experiments to monitor stability and duration of silencing effect by a dsRNA agent and monitoring in dividing versus nondividing cells. Presumably in dividing cells, the dsRNA is diluted out over time, thus decreasing the duration of the silencing effect. The implication is that dosage will have to be adjusted in vivo, and/or a dsRNA agent will have to be administered more frequently to maintain the silencing effect. To monitor nondividing cells, cells can be arrested by serum withdrawal. Neurons are post-mitotic cells, and thus neural cells are aptly suited for assaying the stability of dsRNA agents, such as an anti-SNCA dsRNA agent, for use in therapeutic compositions for the treatment of disorders of the nervous system, e.g., neurodegenerative disorders.

A candidate dsRNA agent can also be evaluated for cross-species reactivity. For example, cell lines derived from different species (e.g., mouse vs. human) or in biological samples (e.g., serum or tissue extracts) isolated from different species can be transfected with a target dsRNA agent and a candidate dsRNA agent. The efficacy of the dsRNA agent can be determined for the cell from the different species.

In another aspect, the invention features a method of evaluating an agent, e.g., an agent of a type described herein, such as a dsRNA agent having an antisense strand shown in Tables 2, 3, or 4, and a sense strand shown in Tables 2, 3, or 4, dsRNA for the ability to inhibit SNCA expression, e.g., an agent that targets an SNCA or SNCA nucleic acid. The method includes: providing a candidate agent and determining, e.g., by the use of one or more of the test systems described herein, if said candidate agent modulates, e.g., inhibits, SNCA expression.

In one embodiment the method includes evaluating the agent in a first test system; and, if a predetermined level of modulation is seen, evaluating the candidate in a second, e.g., a different, test system. In one embodiment the second test system includes administering the candidate agent to an animal and evaluating the effect of the candidate agent on SNCA expression in the animal. In certain embodiments, two test systems are used and the first is a high-throughput system. For example, in such embodiments the first or initial test is used to screen at least 100, 1,000, or 10,000 times more agents than is the second test, e.g., an animal system.

A test system can include: contacting the candidate agent with a target molecule, e.g., an SNCA nucleic acid, e.g., an RNA, such as in vitro, and determining if there is an interaction, e.g., binding of the candidate agent to the target, or modifying the target, e.g., by making or breaking a covalent bond in the target. Modification is correlated with the ability to modulate SNCA expression. The test system can include contacting the candidate agent with a cell and evaluating modulation of SNCA expression. For example, this can include contacting the candidate agent with a cell capable of expressing SNCA or SNCA RNA (from an endogenous gene or from an exogenous construct) and evaluating the level of SNCA or SNCA RNA. In another embodiment, the test system can include contacting the candidate agent with a cell that expresses an RNA or protein from an SNCA control region (e.g., an SNCA control region) linked to a heterologous sequence, e.g., a marker protein, e.g., a fluorescent protein such as GFP, which construct can be either chromosomal or episomal, and determining the effect on RNA or protein levels. The test system can also include contacting the candidate agent, in vitro, with a tissue sample, e.g., a brain tissue sample, e.g., a slice or section, an optical tissue sample, or other sample which includes neural tissue, and evaluating the level of SNCA or SNCA RNA. The test system can include administering the candidate agent, in vivo, to an animal, and evaluating the level of SNCA or SNCA RNA. In any of these the effect of the candidate agent on SNCA expression can include comparing SNCA gene expression with a predetermined standard, such as a control, e.g., an untreated cell, tissue or animal. SNCA gene expression can be compared, e.g., before and after contacting with the candidate agent. The method allows determining whether the dsRNA is useful for inhibiting SNCA gene expression.

In one embodiment, SNCA gene expression can be evaluated by a method to examine SNCA RNA levels (e.g., Northern blot analysis, RT-PCR, or RNAse protection assay) or SNCA protein levels (e.g., Western blot).

In one embodiment, a second test is performed by administering the agent to an animal, e.g., a mammal, such as a mouse, rat, rabbit, human, or non-human primate, and the animal is monitored for an effect of the agent. For example, a tissue of the animal, such as, a brain tissue or ocular tissue, is examined for an effect of the agent on SNCA expression. The tissue can be examined for the presence of SNCA RNA and/or protein, for example. In one embodiment, the animal is observed to monitor an improvement or stabilization of a cognitive symptom. The agent can be administered to the animal by any method, e.g., orally, or by intrathecal or parenchymal injection, such as by stereoscopic injection into the brain. In some embodiments, the agent is administered to the substantia nigra, hippocampus or cortex of the brain.

In one embodiment, the invention features a method of evaluating a dsRNA, e.g., a dsRNA described herein, that targets an SNCA nucleic acid. The method includes providing a dsRNA that targets an SNCA nucleic acid (e.g., an SNCA RNA); contacting the dsRNA with a cell containing, and capable of expressing, an SNCA gene; and evaluating the effect of the dsRNA on SNCA expression, e.g., by comparing SNCA gene expression with a control, e.g., in the cell. SNCA gene expression can be compared, e.g., before and after contacting the dsRNA with the cell. The method allows determining whether the dsRNA is useful for inhibiting SNCA gene expression. For example, the dsRNA can be determined to be useful for inhibiting SNCA gene expression if the dsRNA reduces expression by a predetermined amount, e.g., by 10, 25, 50, 75, or 90%, e.g., as compared with a predetermined reference value, e.g., as compared with the amount of SNCA RNA or protein prior to contacting the dsRNA with the cell. The SNCA gene can be endogenously or exogenously expressed. In vivo Testing

A dsRNA agent identified as being capable of inhibiting SNCA gene expression can be tested for functionality in vivo in an animal model (e.g., in a mammal, such as in mouse or rat). For example, the dsRNA agent can be administered to an animal, and the dsRNA agent evaluated with respect to its biodistribution, stability, and its ability to inhibit SNCA gene expression.

The dsRNA agent can be administered directly to the target tissue, such as by injection, or the dsRNA agent can be administered to the animal model in the same manner that it would be administered to a human. For example, the dsRNA agent can be injected directly into a target region of the brain (e.g., into the cortex, the substantia nigra, the globus pallidus, or the hippocampus), and after a period of time, the brain can be harvested and tissue slices examined for distribution of the agent.

The dsRNA agent can also be evaluated for its intracellular distribution. The evaluation can include determining whether the dsRNA agent was taken up into the cell. The evaluation can also include determining the stability (e.g., the half-life) of the dsRNA agent. Evaluation of a dsRNA agent in vivo can be facilitated by use of a dsRNA agent conjugated to a traceable

32 33 3 marker (e.g., a fluorescent marker such as fluorescein; a radioactive label, such as P, P, or H; gold particles; or antigen particles for immunohistochemistry).

A dsRNA agent useful for monitoring biodistribution can lack gene silencing activity in vivo. For example, the dsRNA agent can target a gene not present in the animal (e.g., a dsRNA agent injected into mouse can target luciferase), or a dsRNA agent can have a non-sense sequence, which does not target any gene, e.g., any endogenous gene).

Localization/biodistribution of the dsRNA can be monitored by a traceable label attached to the dsRNA agent, such as a traceable agent described above.

The dsRNA agent can be evaluated with respect to its ability to down regulate SNCA expression. Levels of SNCA expression in vivo can be measured, for example, by in situ hybridization, or by the isolation of RNA from tissue prior to and following exposure to the dsRNA agent. SNCA RNA can be detected by any desired method, including but not limited to RT-PCR, Northern blot, or RNAase protection assay. Alternatively, or additionally, SNCA gene expression can be monitored by performing Western blot analysis on tissue extracts treated with the anti-SNCA dsRNA agent.

An anti-SNCA dsRNA agent can be tested in a mouse model for Parkinson's disease, such as a mouse carrying a wildtype copy of the human SNCA gene (Masliah et al, Science 287: 1265-1269, 2000) or in mouse carrying a mutant human SNCA (Richfield et al, Exp. Neurol. 175: 35-48, 2002; Giasson et al, Neuron 34: 521-533, 2002; Lee et al, Proc Natl Acad. Sci. 99: 8968-8973, 2002). The mutant mouse can carry a human SNCA gene that expresses an A53T, A3 OP, or E46K mutation. A treated mouse model can be observed for a decrease in symptoms associated with Parkinson's disease.

Methods for treating diseases caused by expression of a SNCA gene

The invention relates in particular to the use of a dsRNA targeting SNCA and compositions containing at least one such dsRNA for the treatment of a SNCA-mediated disorder or disease. For example, a dsRNA targeting a SNCA gene can be useful for the treatment of neurodegenerative diseases, such as a synucleinopathy (e.g., Parkinson's disease), that have either an activating mutation of SNCA and/or are the result of overexpression of SNCA. The method includes administering a pharmaceutical composition featured in the invention to the patient (e.g., human), such that expression of the SNCA gene is silenced.

Because of their high specificity, the dsRNAs described herein specifically target mRNAs of the SNCA gene.

As used herein, the term "SNCA-mediated condition or disease" and related terms and phrases refer to a condition or disorder characterized by unwanted or inappropriate, e.g., abnormal SNCA activity. Inappropriate SNCA functional activity might arise as the result of SNCA expression in cells which normally do not express SNCA, increased SNCA expression and/or activity (leading to, e.g., neurodegenerative disease, or increased susceptibility to disease). A SNCA-mediated condition or disease may be completely or partially mediated by inappropriate SNCA functional activity which may result by way of inappropriate activation of SNCA. Regardless, a SNCA-mediated condition or disease is one in which modulation of SNCA via RNA interference results in some effect on the underlying condition or disorder (e.g., a SNCA inhibitor results in some improvement in patient well-being in at least some patients).

The dsRNA molecules featured herein may, therefore, be used to treat neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, multiple system atrophy, and Lewy body dementia. The dsRNA molecules featured herein are also useful for the treatment of a retinal disorder, e.g., a retinopathy.

In one aspect of the invention, methods are provided for treating a disorder mediated by SNCA expression by administering to a human in need of such treatment a therapeutically effective amount of any of the dsRNA of the invention. Also included is a method of treating, preventing or managing a neurodegenerative disorder in which a patient in need of such treatment, prevention or management is administered a therapeutically or prophylactically effective amount of a dsRNA of the invention. The method of treatment can include

administering an additional composition, e.g., a second dsRNA.

It has been discovered that a single administration can provide prolonged silencing. Thus, in another embodiment, a dose of SNCA dsRNA is administered to a patient and the dose is sufficient to downregulate SNCA mRNA or protein levels to a state that is less than or equal to 20% of pretreatment levels (or levels that would be seen in the absence of treatment) for at least 5, 10, or 15 days post-treatment; less than or equal to 40% of pretreatment levels (or levels that would be seen in the absence of treatment) for at least 5, 10, or 15 days post-treatment; less than or equal to 60% of pretreatment levels (or levels that would be seen in the absence of treatment) for at least 5, 10, 15, or 20 days post-treatment; or less than or equal to 80% of pretreatment levels (or levels that would be seen in the absence of treatment) for at least 5, 10, 15, 20, or 25 days post-treatment.

In one embodiment, a first dose of SNCA dsRNA is administered, and no subsequent dose of SNCA dsRNA is administered for at least 5, 10, 15, 20 or 30 days after the first dose. In another embodiment, a subsequent dose is administered but not until at least 5, 10, 15, 20, or 30 days have elapsed since the first dose.

In another embodiment, a patient continues to receive at least one other therapeutic treatment for the synucleinopathy while receiving treatment with SNCA dsRNA. For example, a patient with Parkinson's disease can continue to receive administration of agent for alleviating symptoms, a neuroprotective agent (e.g., for slowing or halting disease progression), or a restorative agent (e.g., for reversing the disease process). Symptomatic therapies include the drugs carbidopa/levodopa, entacapone, tolcapone, pramipexole, ropinerole, pergolide, bromocriptine, selegeline, amantadine, and several anticholingergic agents. Deep brain stimulation surgery as well as stereotactic brain lesioning may also provide symptomatic relief. Neuroprotective therapies include, for example, carbidopa/levodopa, selegeline, vitamin E, amantadine, pramipexole, ropinerole, coenzyme Q10, and GDNF. Restorative therapies can include, for example, surgical transplantation of stem cells.

In another aspect, the invention features a method of providing instructions, e.g., to a healthcare provider or a patient on the administration of SNCA dsRNA. The method includes: providing instructions to administer to the patient a dose of SNCA dsRNA in a treatment regimen described herein, e.g., a dose followed by at least 21 days within a subsequent dose of SNCA dsRNA.

In another aspect, the invention features a method of selecting or treating a patient in need of SNCA dsRNA to treat a disorder described herein. The method includes selecting a patient on the basis of the patient being in need of decreased SNCA RNA for at least 5, 10, 15, 20 or 30 days, and optionally administering the drug to the patient.

In another aspect, the invention features a method of reducing the amount of SNCA or SNCA RNA in a cell of a subject (e.g., a mammalian subject, such as a human). The method includes contacting the cell with an agent that inhibits the expression of SNCA, e.g., a dsRNA described herein, e.g., in Tables 2, 3, or 4. The inhibition can be effected at any level, e.g., at the level of transcription, the level of translation, or post-translationally.

The methods and compositions featured in the invention, e.g., the methods and dsRNA compositions to treat the neurodegenerative disorders described herein, can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

Treatment of Parkinson's disease

Any patient having Parkinson's disease (or any other alpha-synuclein related disorder), is a candidate for treatment with a method or composition described herein. Typically, the patient is not terminally ill (e.g., the patient has life expectancy of two years or more), and has not reached end-stage Parkinson's disease (i.e., Hoehn and Yahr stage 5).

Presymptomatic subjects can also be candidates for treatment with an anti-SNCA agent, e.g., an anti-SNCA dsRNA described herein, e.g., in Tables 2, 3, or 4. In one embodiment, a presymptomatic candidate is identified by either or both of risk-factor profiling and functional neuroimaging (e.g., by fluorodopa and positron emission tomography). For example, the candidate can be identified by risk- factor profiling followed by functional neuroimaging.

Individuals having any genotype are candidates for treatment. In some embodiments the patient will carry a particular genetic mutation that places the patient at increased risk for developing Parkinson's disease. For example, an individual carrying an SNCA gene

multiplication, e.g., an SNCA gene duplication or triplication is at increased risk for developing Parkinson's disease and is a candidate for treatment with the dsRNA. In addition, a gain-of- function mutation in SNCA can increase an individual's risk for developing Parkinson's disease. An individual carrying an SNCA REP1 genotype (e.g., a REP 1 "+1 allele" heterozygous or homozygous genotype) can be a candidate for such treatment. An individual homozygous for the REPl+1 allele overexpresses SNCA. An individual carrying a mutation in the UCHL-1, parkin, or SNCA gene is at increased risk for Parkinson's disease and can be a candidate for treatment with an anti-SNCA dsRNA. Particularly, a mutation in the UCHL-1 or parkin gene will cause a decrease in gene or protein activity. An individual carrying a Tau genotype (e.g. , a mutation in the Tau gene) or a Tau haplotype, such as the HI haplotype is also at risk for developing Parkinson's disease. Other genetic risk factors include mutations in the MAPT, DJl, PTNKl, and NURR1 genes, and polymorphism in several genes including the SNCA, parkin, MAPT, and NAT2 genes.

Non-genetic (e.g., environmental) risk factors for Parkinson's disease include age (e.g., over age 30, 35, 40, 45, or 50 years), gender (men are generally have a higher risk than women), pesticide exposure, heavy metal exposure, and head trauma. In general, exogenous and endogenous factors that disrupt the ubiquitin proteasomal pathway or more specifically inhibit the proteasome, or which disrupt mitochondrial function, or which yield oxidative stress, or which promote the aggregation and fibrillization of alpha-synuclein, can increase the risk of an individual for developing Parkinson's disease, and can contribute to the pathogenesis of

Parkinson's disease.

In one embodiment, a dsRNA can be used to target wildtype SNCA in subjects with Parkinson's disease.

Treatment of other SNCA-mediated disorders

Any disease characterized by a synucleinopathy can be treated with an inhibitory agent described herein (e.g., an agent that targets SNCA), including Lewy body dementia, Multiple System Atrophy, and Alzheimer's disease. Individuals having any genotype are candidates for treatment. In some embodiments, the patient will carry a particular genetic mutation that places them at increased risk for developing a synucleinopathy.

In one embodiment, a dsRNA, e.g., a dsRNA described in herein, e.g., in Tables 2, 3, or 4 can be used to target wildtype SNCA in subjects with a neurodegenerative disorder.

An individual can develop a synucleinopathy as a result of certain environmental factors. For example, oxidative stress, certain pesticides (e.g., 24D and agent orange), bacterial infection, and head trauma have been linked to an increase in the risk of developing Parkinson's disease, and can be determining factors for determining the risk of an individual for synucleinopathies. These factors (and others disclosed herein) can be considered when evaluating the risk profile of a candidate subject for anti-SNCA therapy.

In addition to their presence in the brain, alpha-synuclein polypeptides have been found in ocular tissues, including the retina and optic nerve. Accordingly, the compositions and methods described herein are suitable for treating synucleinopathies of the eye or ocular tissues, including but not limited to retinopathies.

Thus, in another aspect, the invention features a method of treating a subject by administering an agent which inhibits the expression of SNCA in the eye or in ocular tissue, e.g., a dsRNA described herein, e.g., in Tables 2, 3, or 4. In one embodiment, the subject is a mammal, such as a human, e.g., a subject diagnosed as having, or at risk for developing a synucleinopathy of the eye, e.g., a retinopathy. The inhibition can be effected at any level, e.g., at the level of transcription, the level of translation, or post-translationally.

Owing to the inhibitory effects on SNCA expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.

The invention further relates to the use of a dsRNA or a pharmaceutical composition thereof, e.g., for treating a SNCA mediated disorder or disease, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. In certain examples, a dsRNA targeting SNCA can be administered in combination with an agent for alleviating symptoms, a neuroprotective agent (e.g., for slowing or halting disease progression), or a restorative agent (e.g., for reversing the disease process).

Symptomatic therapies include the drugs carbidopa/levodopa, entacapone, tolcapone, pramipexole, ropinerole, pergolide, bromocriptine, selegeline, amantadine, and several anticholingergic agents. Deep brain stimulation surgery as well as stereotactic brain lesioning may also provide symptomatic relief. Neuroprotective therapies include, for example, carbidopa/levodopa, selegeline, vitamin E, amantadine, pramipexole, ropinerole, coenzyme Q10, and GDNF. Restorative therapies can include, for example, surgical transplantation of stem cells.

The dsRNA and an additional therapeutic agent can be administered in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

The invention features a method of administering a dsRNA targeting SNCA to a patient having a disease or disorder mediated by SNCA expression, such as a neurodegenerative disease, e.g., Parkinson's disease. Patients can be administered a therapeutic amount of dsRNA, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dsRNA can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the dsRNA can reduce SNCA levels in the blood or urine of the patient by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80 % or 90% or more. Before administration of a full dose of the dsRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction.

Many SNCA-associated diseases and disorders are hereditary. Therefore, a patient in need of a SNCA dsRNA can be identified by taking a family history. A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a SNCA dsRNA. A DNA test may also be performed on the patient to identify a mutation in the SNCA gene, before a SNCA dsRNA is administered to the patient.

Methods for inhibiting expression of a SNCA gene

In yet another aspect, the invention provides a method for inhibiting the expression of a

SNCA gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target SNCA gene is reduced or silenced. Because of their high specificity, the dsRNAs featured in the invention specifically target RNAs (primary or processed) of the target SNCA gene. Compositions and methods for inhibiting the A dsRNA of the invention, upon contact with a cell expressing said SNCA, inhibits expression of said SNCA gene by at least 40% compared to a cell not so contacted. In some embodiments, a dsRNA of the invention has a pM IC50, e.g., an IC50 of 10 pM or less.

Another aspect of the invention is method of inhibiting SNCA expression in a cell, the method including introducing into the cell any of the dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of a SNCA gene, thereby inhibiting expression of the SNCA gene in the cell. In some embodiments, expression is inhibited by at least 20%, 40%, 60%, or at least 80%.

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

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

Other embodiments are, for example, in the claims.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et ah, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and . Kaplan eds., Academic Press, Inc.); Remington's

Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B(1992).

Example 1. dsRNA synthesis

Source of reagents

Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Conjugates

For the synthesis of 3 '-cholesterol-conjugated siRNAs (herein referred to as -Chol-3'), an appropriately modified solid support is used for RNA synthesis. The modified solid support is prepared as follows:

Diethyl-2-azabutane- 1 ,4-dicarboxylate AA

Figure imgf000063_0001

A 4.7 M aqueous solution of sodium hydroxide (50 mL) is added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) isis added and the mixture is stirred at room temperature until completion of the reaction is ascertained by TLC. After 19 h the solution is partitioned with dichloromethane (3 x 100 mL). The organic layer is dried with anhydrous sodium sulfate, filtered and evaporated. The residue is distilled to afford AA (28.8 g, 61%). 3- {Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]- amino} -propionic acid ethyl ester AB

Figure imgf000064_0001

AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) is dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) is added to the solution at 0°C. It is then followed by the addition of diethyl-azabutane-l,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution is brought to room temperature and stirred further for 6 h. Completion of the reaction is ascertained by TLC. The reaction mixture is concentrated under vacuum and ethyl acetate is added to precipitate diisopropyl urea. The suspension is filtered. The filtrate is washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer is dried over sodium sulfate and concentrated to give the crude product which is purified by column chromatography (50 % EtOAC/hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC

Figure imgf000064_0002

3- {Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]- amino} -propionic acid ethyl ester AB (11.5 g, 21.3 mmol) is dissolved in 20% piperidine in dimethylformamide at 0°C. The solution is continued stirring for 1 h. The reaction mixture is concentrated under vacuum, water is added to the residue, and the product is extracted with ethyl acetate. The crude product is purified by conversion into its hydrochloride salt.

3-({6-[17-(l,5-Dimethyl-hexyl)-10, 13-dimethyl-2,3,4,7,8,9, 10, l 1, 12, 13, 14, 15, 16, 17- tetradecahydro-lH-cyclopenta[a]phenanthren-3-yloxycarbonylamino]- hexanoyl} ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD

Figure imgf000065_0001

AD

The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]- propionic acid ethyl ester AC (4.7 g, 14.8 mmol) is taken up in dichloromethane. The suspension is cooled to 0°C on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) is added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) is added. The reaction mixture is stirred overnight. The reaction mixture is diluted with dichloromethane and washed with 10% hydrochloric acid. The product is purified by flash chromatography (10.3 g, 92%).

l- {6-[17-(l,5-Dimethyl-hexyl)-10, 13-dimethyl-2,3,4,7,8,9, 10, l l, 12, 13, 14, 15, 16, 17- tetradecahydro- 1 H-cyclopenta[a] phenanthren-3 -yloxycarbonylamino]-hexanoyl} -4-oxo- pyrrolidine-3-carboxylic acid ethyl ester AE

Figure imgf000065_0002

AE

Potassium t-butoxide (1.1 g, 9.8 mmol) is slurried in 30 mL of dry toluene. The mixture is cooled to 0°C on ice and 5 g (6.6 mmol) of diester AD is added slowly with stirring within 20 mins. The temperature is kept below 5°C during the addition. The stirring is continued for 30 mins at 0°C and 1 mL of glacial acetic acid is added, immediately followed by 4 g of

NaH2P04-H20 in 40 mL of water The resultant mixture is extracted twice with 100 mL of dichloromethane each and the combined organic extracts are washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue is dissolved in 60 mL of toluene, cooled to 0°C and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts are adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which are combined, dried and evaporated to dryness. The residue is purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-l-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5- dimethyl-hexyl)-10, 13-dimethyl-2,3,4,7,8,9, 10, l l, 12,13, 14, 15, 16, 17-tetradecahydro-lH- cyclopenta[a]phenanthr n-3-yl ester AF

Figure imgf000066_0001

AF

Methanol (2 mL) is added dropwise over a period of 1 h to a refluxing mixture of b- ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring is continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HC1 (12.5 mL) is added, the mixture is extracted with ethylacetate (3 x 40 mL). The combined ethylacetate layer is dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which is purified by column chromatography (10% MeOH/CHCL;) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-l-yl}-6- oxo-hexyl)-carbamic acid 17-( 1 ,5-dimethyl-hexyl)- 10, 13 -dimethyl-

2,3,4,7,8,9, 10 -tetradecahydro-lH-cyclopenta[a]phenanthren-3-yl ester AG

Figure imgf000066_0002
Diol AF (1.25 gm 1.994 mmol) is dried by evaporating with pyridine (2 x 5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4'-dimethoxytritylchloride (0.724 g, 2.13 mmol) are added with stirring. The reaction is carried out at room temperature overnight. The reaction is quenched by the addition of methanol. The reaction mixture is concentrated under vacuum and to the residue dichloromethane (50 mL) is added. The organic layer is washed with 1M aqueous sodium bicarbonate. The organic layer is dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine is removed by evaporating with toluene. The crude product is purified by column chromatography (2% MeOH/chloroform, Rf = 0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).

Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-l-{6-[17-(l,5- dimethyl-hexyl)-l 0, 13 -dimethyl 2,3,4,7,8,9, 10, 11, 12,13, 14, 15, 16, 17-tetradecahydro-lH cyclopenta a]phenanthren-3-yloxycarbonylamino]-hexanoyl} -pyrrolidin-3-yl) ester AH

Figure imgf000067_0001

AH

Compound AG (1.0 g, 1.05 mmol) is mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C overnight. The mixture is dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) is added and the solution is stirred at room temperature under argon atmosphere for 16 h. It is then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt%, 30 mL) and water (2 X 20 mL). The organic phase is dried over anhydrous sodium sulfate and concentrated to dryness. The residue is used as such for the next step.

Cholesterol derivatized CPG AI

Figure imgf000068_0001

AI

Succinate AH (0.254 g, 0.242 mmol) is dissolved in a mixture of

dichloromethane/acetonitrile (3 :2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2'-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in

acetonitrile/dichloroethane (3 : 1, 1.25 mL) are added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) is added. The reaction mixture turned bright orange in color. The solution is agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) is added. The suspension is agitated for 2 h. The CPG is filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups are masked using acetic anhydride/pyridine. The achieved loading of the CPG is measured by taking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5'-12-dodecanoic acid bisdecylamide group (herein referred to as "5'-C32-") or a 5'-cholesteryl derivative group (herein referred to as "5'-Chol-") is performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step is performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5'-end of the nucleic acid oligomer.

Nucleic acid sequences are represented herein using standard nomenclature, and specifically the abbreviations of Table 1. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds.

Table 1 : Abbreviations of nucleoside monomers used in nucleic acid sequence representation.

Abbreviation Nucleoside(s)

A Adenosine

C Cytidine

G Guanos ine

U Uridine

N any nucleotide (G, A, C, U, or dT) Abbreviation Nucleoside(s)

a 2'-0-methyladenosine

c 2'-0-methylcytidine

G 2'-0-methylguanosine

U 2'-0-methyluridine

dT 2'-deoxythymidine

sT; sdT 2'-deoxy-thymidine-5'phosphate-phosphorothioate

s a phosphorothioate linkage

Example 2. siRNA Design and Synthesis

Transcripts

Oligonucleotide design was carried out to identify siRNAs targeting the gene "synuclein- alpha (non A4 component of amyloid precursor)" from human (NCBI symbol SNCA) and the orthologous sequence from rhesus monkey (Macaca mulatto) to ensure that the selected siRNAs were species cross-reactive. The design process used the SNCA transcripts NM_007308.1 from human (NCBI Geneld 6622) and XM_001095402.1 from rhesus (NCBI Geneld 706985) from the NCBI Refseq collection as follows:

Figure imgf000069_0001
siRNA Design and Specificity Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. The SNCA siRNAs were used in a comprehensive search against the human and rhesus

transcriptomes (defined as the set of NM_ and XM_ records within the NCBI Refseq set) using the FASTA algorithm. The Python script OfftargetFasta.py' was then used to parse the alignments and generate a score based on the position and number of mismatches between the siRNA and any potential Off-target' transcript. The off-target score is weighted to emphasize differences in the 'seed' region of siRNAs, in positions 2-9 from the 5' end of the molecule. The off-target score is calculated as follows: mismatches between the oligo and the transcript are given penalties. A mismatch in the seed region in positions 2-9 of the oligo is given a penalty of 2.8; mismatches in the putative cleavage sites 10 and 1 1 are given a penalty of 1.2, and all other mismatches a penalty of 1. The off-target score for each oligo-transcript pair is then calculated by summing the mismatch penalties. The lowest off-target score from all the oligo-transcript pairs is then determined and used in subsequent sorting of oligos. Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderate specific. In picking which oligos to synthesize, we sorted from high to low by the off-target score of the antisense strand and took the 92 best (lowest off-target score) oligo pairs.

Table 2 provides the sequences of 92 sense and antisense strands, 19 nucloetides each. Table 2 also provides the sequences of each strand including a 2 nculeotide "overhange" of NN. Table 3 provides sequences of the of 92 sense and antisense strands including dTdsdT

overhangs.

Table 2: SNCA sense and antisense strand sequences and corresponding SNCA sense and antisense strand sequences with 2 base overhangs

Numbering for target sequence is based on human NM_007308.1.

Start of Sequence with 3'

target Unmodified sequence SEQ ID dinucleotide overhangs SEQ ID

Strand sequence (5' to 3') NO: (5' to 3' ) NO: sense 325 GUAUCAAGACUACGAACCU 1 GUAUCAAGACUACGAACCUNN 185 antis 325 AGGUUCGUAGUCUUGAUAC 2 AGGUUCGUAGUCUUGAUACNN 186 sense 625 CUAAGUGACUACCACUUAU 3 CUAAGUGACUACCACUUAUNN 187 antis 625 AUAAGUGGUAGUCACUUAG 4 AUAAGUGGUAGUCACUUAGNN 188 sense 321 AAGGGUAUCAAGACUACGA 5 AAGGGUAUCAAGACUACGANN 189 antis 321 UCGUAGUCUUGAUACCCUU 6 UCGUAGUCUUGAUACCCUUNN 190 sense 326 UAUCAAGACUACGAACCUG 7 UAUCAAGACUACGAACCUGNN 191 antis 326 CAGGUUCGUAGUCUUGAUA 8 CAGGUUCGUAGUCUUGAUANN 192 sense 200 GUGACAAAUGUUGGAGGAG 9 GUGACAAAUGUUGGAGGAGNN 193 antis 200 CUCCUCCAACAUUUGUCAC 10 CUCCUCCAACAUUUGUCACNN 194 sense 327 AUCAAGACUACGAACCUGA 11 AUCAAGACUACGAACCUGANN 195 antis 327 UCAGGUUCGUAGUCUUGAU 12 UCAGGUUCGUAGUCUUGAUNN 196 sense 426 GUGCCCAGUCAUGACAUUU 13 GUGCCCAGUCAUGACAUUUNN 197 antis 426 AAAUGUCAUGACUGGGCAC 14 AAAUGUCAUGACUGGGCACNN 198 sense 731 UUAAUGAUACUGUCUAAGA 15 UUAAUGAUACUGUCUAAGANN 199 antis 731 UCUUAGACAGUAUCAUUAA 16 UCUUAGACAGUAUCAUUAANN 200 sense 429 CCCAGUCAUGACAUUUCUC 17 CCCAGUCAUGACAUUUCUCNN 201 antis 429 GAGAAAUGUCAUGACUGGG 18 GAGAAAUGUCAUGACUGGGNN 202 sense 543 AAGUGAAUACAUGGUAGCA 19 AAGUGAAUACAUGGUAGCANN 203 antis 543 UGCUACCAUGUAUUCACUU 20 UGCUACCAUGUAUUCACUUNN 204 sense 802 AGCAUGAAACUAUGCACCU 21 AGCAUGAAACUAUGCACCUNN 205 antis 802 AGGUGCAUAGUUUCAUGCU 22 AGGUGCAUAGUUUCAUGCUNN 206 sense 803 GCAUGAAACUAUGCACCUA 23 GCAUGAAACUAUGCACCUANN 207 antis 803 UAGGUGCAUAGUUUCAUGC 24 UAGGUGCAUAGUUUCAUGCNN 208 sense 410 AGUGCUCAGUUCCAAUGUG 25 AGUGCUCAGUUCCAAUGUGNN 209 antis 410 CACAUUGGAACUGAGCACU 26 CACAUUGGAACUGAGCACUNN 210 sense 518 GCAUUUCGGUGCUUCCCUU 27 GCAUUUCGGUGCUUCCCUUNN 211 antis 518 AAGGGAAGCACCGAAAUGC 28 AAGGGAAGCACCGAAAUGCNN 212 sense 798 UGUGAGCAUGAAACUAUGC 29 UGUGAGCAUGAAACUAUGCNN 213 antis 798 GCAUAGUUUCAUGCUCACA 30 GCAUAGUUUCAUGCUCACANN 214 sense 318 AGGAAGGGUAUCAAGACUA 31 AGGAAGGGUAUCAAGACUANN 215 antis 318 UAGUCUUGAUACCCUUCCU 32 UAGUCUUGAUACCCUUCCUNN 216 sense 537 UCACUGAAGUGAAUACAUG 33 UCACUGAAGUGAAUACAUGNN 217 antis 537 CAUGUAUUCACUUCAGUGA 34 CAUGUAUUCACUUCAGUGANN 218 Start of Sequence with 3 ' target Unmodified sequence SEQ ID dinucleotide overhangs SEQ ID

Strand sequence (5' to 3') NO: (5 ' to 3 ' ) NO: sense 201 UGACAAAUGUUGGAGGAGC 35 UGACAAAUGUUGGAGGAGCNN 21 9 antis 201 GCUCCUCCAACAUUUGUCA 36 GCUCCUCCAACAUUUGUCANN 220 sense 544 AGUGAAUACAUGGUAGCAG 37 AGUGAAUACAUGGUAGCAGNN 221 antis 544 CUGCUACCAUGUAUUCACU 38 CUGCUACCAUGUAUUCACUNN 222 sense 548 AAUACAUGGUAGCAGGGUC 39 AAUACAUGGUAGCAGGGUCNN 223 antis 548 GACCCUGCUACCAUGUAUU 40 GACCCUGCUACCAUGUAUUNN 224 sense 482 AGCAGUGAUUGAAGUAUCU 41 AGCAGUGAUUGAAGUAUCUNN 225 antis 482 AGAUACUUCAAUCACUGCU 42 AGAUACUUCAAUCACUGCUNN 22 6 sense 221 GUGGUGACGGGUGUGACAG 43 GUGGUGACGGGUGUGACAGNN 227 antis 221 CUGUCACACCCGUCACCAC 44 CUGUCACACCCGUCACCACNN 228 sense 328 UCAAGACUACGAACCUGAA 45 UCAAGACUACGAACCUGAANN 22 9 antis 328 UUCAGGUUCGUAGUCUUGA 46 UUCAGGUUCGUAGUCUUGANN 230 sense 942 CAUCUCACUUUAAUAAUAA 47 CAUCUCACUUUAAUAAUAANN 231 antis 942 UUAUUAUUAAAGUGAGAUG 48 UUAUUAUUAAAGUGAGAUGNN 232 sense 227 ACGGGUGUGACAGCAGUAG 49 ACGGGUGUGACAGCAGUAGNN 233 antis 227 CUACUGCUGUCACACCCGU 50 CUACUGCUGUCACACCCGUNN 234 sense 320 GAAGGGUAUCAAGACUACG 51 GAAGGGUAUCAAGACUACGNN 235 antis 320 CGUAGUCUUGAUACCCUUC 52 CGUAGUCUUGAUACCCUUCNN 23 6 sense 683 AGUUGUUAGUGAUUUGCUA 53 AGUUGUUAGUGAUUUGCUANN 237 antis 683 UAGCAAAUCACUAACAACU 54 UAGCAAAUCACUAACAACUNN 238 sense 374 UCUUGAGAUCUGCUGACAG 55 UCUUGAGAUCUGCUGACAGNN 23 9 antis 374 CUGUCAGCAGAUCUCAAGA 56 CUGUCAGCAGAUCUCAAGANN 240 sense 358 AUCUUUGCUCCCAGUUUCU 57 AUCUUUGCUCCCAGUUUCUNN 241 antis 358 AGAAACUGGGAGCAAAGAU 58 AGAAACUGGGAGCAAAGAUNN 242 sense 552 CAUGGUAGCAGGGUCUUUG 59 CAUGGUAGCAGGGUCUUUGNN 243 antis 552 CAAAGACCCUGCUACCAUG 60 CAAAGACCCUGCUACCAUGNN 244 sense 639 CUUAUUUCUAAAUCCUCAC 61 CUUAUUUCUAAAUCCUCACNN 245 antis 639 GUGAGGAUUUAGAAAUAAG 62 GUGAGGAUUUAGAAAUAAGNN 24 6 sense 425 UGUGCCCAGUCAUGACAUU 63 UGUGCCCAGUCAUGACAUUNN 247 antis 425 AAUGUCAUGACUGGGCACA 64 AAUGUCAUGACUGGGCACANN 248 sense 675 UGUUCAGAAGUUGUUAGUG 65 UGUUCAGAAGUUGUUAGUGNN 24 9 antis 675 CACUAACAACUUCUGAACA 66 CACUAACAACUUCUGAACANN 250 sense 329 CAAGACUACGAACCUGAAG 67 CAAGACUACGAACCUGAAGNN 251 antis 329 CUUCAGGUUCGUAGUCUUG 68 CUUCAGGUUCGUAGUCUUGNN 252 sense 809 AACUAUGCACCUAUAAAUA 69 AACUAUGCACCUAUAAAUANN 253 antis 809 UAUUUAUAGGUGCAUAGUU 70 UAUUUAUAGGUGCAUAGUUNN 254 sense 794 AAUAUGUGAGCAUGAAACU 71 AAUAUGUGAGCAUGAAACUNN 255 antis 794 AGUUUCAUGCUCACAUAUU 72 AGUUUCAUGCUCACAUAUUNN 25 6 sense 487 UGAUUGAAGUAUCUGUACC 73 UGAUUGAAGUAUCUGUACCNN 257 antis 487 GGUACAGAUACUUCAAUCA 74 GGUACAGAUACUUCAAUCANN 258 sense 538 CACUGAAGUGAAUACAUGG 75 CACUGAAGUGAAUACAUGGNN 25 9 antis 538 CCAUGUAUUCACUUCAGUG 76 CCAUGUAUUCACUUCAGUGNN 2 60 sense 534 CUUUCACUGAAGUGAAUAC 77 CUUUCACUGAAGUGAAUACNN 2 61 antis 534 GUAUUCACUUCAGUGAAAG 78 GUAUUCACUUCAGUGAAAGNN 2 62 sense 427 UGCCCAGUCAUGACAUUUC 79 UGCCCAGUCAUGACAUUUCNN 2 63 antis 427 GAAAUGUCAUGACUGGGCA 80 GAAAUGUCAUGACUGGGCANN 2 64 sense 567 UUUGUGUGCUGUGGAUUUU 81 UUUGUGUGCUGUGGAUUUUNN 2 65 antis 567 AAAAUCCACAGCACACAAA 82 AAAAUCCACAGCACACAAANN 2 66 sense 806 UGAAACUAUGCACCUAUAA 83 UGAAACUAUGCACCUAUAANN 2 67 Start of Sequence with 3 ' target Unmodified sequence SEQ ID dinucleotide overhangs SEQ ID

Strand sequence (5' to 3') NO: (5 ' to 3 ' ) NO: antis 806 UUAUAGGUGCAUAGUUUCA 84 UUAUAGGUGCAUAGUUUCANN 2 68 sense 738 UACUGUCUAAGAAUAAUGA 85 UACUGUCUAAGAAUAAUGANN 2 69 antis 738 UCAUUAUUCUUAGACAGUA 86 UCAUUAUUCUUAGACAGUANN 270 sense 876 UUGUAUAUAAAUGGUGAGA 87 UUGUAUAUAAAUGGUGAGANN 271 antis 876 UCUCACCAUUUAUAUACAA 88 UCUCACCAUUUAUAUACAANN 272 sense 322 AGGGUAUCAAGACUACGAA 89 AGGGUAUCAAGACUACGAANN 273 antis 322 UUCGUAGUCUUGAUACCCU 90 UUCGUAGUCUUGAUACCCUNN 274 sense 324 GGUAUCAAGACUACGAACC 91 GGUAUC AAG ACUAC GAAC CNN 275 antis 324 GGUUCGUAGUCUUGAUACC 92 GGUUCGUAGUCUUGAUACCNN 27 6 sense 334 CUACGAACCUGAAGCCUAA 93 CUACGAACCUGAAGCCUAANN 277 antis 334 UUAGGCUUCAGGUUCGUAG 94 UUAGGCUUCAGGUUCGUAGNN 278 sense 884 AAAUGGUGAGAAUUAAAAU 95 AAAUGGUGAGAAUUAAAAUNN 27 9 antis 884 AUUUUAAUUCUCACCAUUU 96 AUUUUAAUUCUCACCAUUUNN 280 sense 553 AUGGUAGCAGGGUCUUUGU 97 AUGGUAGCAGGGUCUUUGUNN 281 antis 553 ACAAAGACCCUGCUACCAU 98 ACAAAGACCCUGCUACCAUNN 282 sense 323 GGGUAUCAAGACUACGAAC 99 GGGUAUCAAGACUACGAACNN 283 antis 323 GUUCGUAGUCUUGAUACCC 100 GUUCGUAGUCUUGAUACCCNN 284 sense 620 AACACCUAAGUGACUACCA 101 AACACCUAAGUGACUACCANN 285 antis 620 UGGUAGUCACUUAGGUGUU 102 UGGUAGUCACUUAGGUGUUNN 28 6 sense 619 AA AC AC CUAAGUGACUAC C 103 AAACACCUAAGUGACUACCNN 287 antis 619 GGUAGUCACUUAGGUGUUU 104 GGUAGUCACUUAGGUGUUUNN 288 sense 682 AAGUUGUUAGUGAUUUGCU 105 AAGUUGUUAGUGAUUUGCUNN 28 9 antis 682 AGCAAAUCACUAACAACUU 106 AGCAAAUCACUAACAACUUNN 2 90 sense 732 UAAUGAUACUGUCUAAGAA 107 UAAUGAUACUGUCUAAGAANN 2 91 antis 732 UUCUUAGACAGUAUCAUUA 108 UUCUUAGACAGUAUCAUUANN 2 92 sense 515 UCAGCAUUUCGGUGCUUCC 109 UCAGCAUUUCGGUGCUUCCNN 2 93 antis 515 GGAAGCACCGAAAUGCUGA 110 GGAAGCACCGAAAUGCUGANN 2 94 sense 737 AUACUGUCUAAGAAUAAUG 111 AUACUGUCUAAGAAUAAUGNN 2 95 antis 737 CAUUAUUCUUAGACAGUAU 112 CAUUAUUCUUAGACAGUAUNN 2 96 sense 804 CAUGAAACUAUGCACCUAU 113 CAUGAAACUAUGCACCUAUNN 2 97 antis 804 AUAGGUGCAUAGUUUCAUG 114 AUAGGUGCAUAGUUUCAUGNN 2 98 sense 554 UGGUAGCAGGGUCUUUGUG 115 UGGUAGCAGGGUCUUUGUGNN 2 99 antis 554 CACAAAGACCCUGCUACCA 116 CACAAAGACCCUGCUACCANN 300 sense 943 AUCUCACUUUAAUAAUAAA 117 AUCUCACUUUAAUAAUAAANN 301 antis 943 UUUAUUAUUAAAGUGAGAU 118 UUUAUUAUUAAAGUGAGAUNN 302 sense 674 UUGUUCAGAAGUUGUUAGU 119 UUGUUCAGAAGUUGUUAGUNN 303 antis 674 ACUAACAACUUCUGAACAA 120 ACUAACAACUUCUGAACAANN 304 sense 428 GCCCAGUCAUGACAUUUCU 121 GCCCAGUCAUGACAUUUCUNN 305 antis 428 AGAAAUGUCAUGACUGGGC 122 AGAAAUGUCAUGACUGGGCNN 30 6 sense 580 GAUUUUGUGGCUUCAAUCU 123 GAUUUUGUGGCUUCAAUCUNN 307 antis 580 AGAUUGAAGCCACAAAAUC 124 AGAUUGAAGCCACAAAAUCNN 308 sense 813 AUGCACCUAUAAAUACUAA 125 AUGCACCUAUAAAUACUAANN 30 9 antis 813 UUAGUAUUUAUAGGUGCAU 126 UUAGUAUUUAUAGGUGCAUNN 310 sense 621 ACACCUAAGUGACUACCAC 127 ACACCUAAGUGACUACCACNN 311 antis 621 GUGGUAGUCACUUAGGUGU 128 GUGGUAGUCACUUAGGUGUNN 312 sense 936 UUAUCCCAUCUCACUUUAA 129 UUAUCCCAUCUCACUUUAANN 313 antis 936 UUAAAGUGAGAUGGGAUAA 130 UUAAAGUGAGAUGGGAUAANN 314 sense 349 CUAAGAAAUAUCUUUGCUC 131 CUAAGAAAUAUCUUUGCUCNN 315 antis 349 GAGCAAAGAUAUUUCUUAG 132 GAGCAAAGAUAUUUCUUAGNN 31 6 Start of Sequence with 3 ' target Unmodified sequence SEQ ID dinucleotide overhangs SEQ ID

Strand sequence (5' to 3') NO: (5 ' to 3 ' ) NO: sense 433 GUCAUGACAUUUCUCAAAG 133 GUCAUGACAUUUCUCAAAGNN 317 antis 433 CUUUGAGAAAUGUCAUGAC 134 CUUUGAGAAAUGUCAUGACNN 318 sense 350 UAAGAAAUAUCUUUGCUCC 135 UAAGAAAUAUCUUUGCUCCNN 31 9 antis 350 GGAGCAAAGAUAUUUCUUA 136 GGAGCAAAGAUAUUUCUUANN 320 sense 1036 AAGAAGGAGGAAUUUUAGA 137 AAGAAGGAGGAAUUUUAGANN 321 antis 1036 UCUAAAAUUCCUCCUUCUU 138 UCUAAAAUUCCUCCUUCUUNN 322 sense 424 AUGUGCCCAGUCAUGACAU 139 AUGUGCCCAGUCAUGACAUNN 323 antis 424 AUGUCAUGACUGGGCACAU 140 AUGUCAUGACUGGGCACAUNN 324 sense 673 GUUGUUCAGAAGUUGUUAG 141 GUUGUUCAGAAGUUGUUAGNN 325 antis 673 CUAACAACUUCUGAACAAC 142 CUAACAACUUCUGAACAACNN 32 6 sense 640 UUAUUUCUAAAUCCUCACU 143 UUAUUUCUAAAUCCUCACUNN 327 antis 640 AGUGAGGAUUUAGAAAUAA 144 AGUGAGGAUUUAGAAAUAANN 328 sense 638 ACUUAUUUCUAAAUCCUCA 145 ACUUAUUUCUAAAUCCUCANN 32 9 antis 638 UGAGGAUUUAGAAAUAAGU 146 UGAGGAUUUAGAAAUAAGUNN 330 sense 720 UUAGGUGUCUUUUAAUGAU 147 UUAGGUGUCUUUUAAUGAUNN 331 antis 720 AUCAUUAAAAGACACCUAA 148 AUCAUUAAAAGACACCUAANN 332 sense 353 GAAAUAUCUUUGCUCCCAG 149 GAAAUAUCUUUGCUCCCAGNN 333 antis 353 CUGGGAGCAAAGAUAUUUC 150 CUGGGAGCAAAGAUAUUUCNN 334 sense 357 UAUCUUUGCUCCCAGUUUC 151 UAUCUUUGCUCCCAGUUUCNN 335 antis 357 GAAACUGGGAGCAAAGAUA 152 GAAACUGGGAGCAAAGAUANN 33 6 sense 421 CCAAUGUGCCCAGUCAUGA 153 CCAAUGUGCCCAGUCAUGANN 337 antis 421 UCAUGACUGGGCACAUUGG 154 UCAUGACUGGGCACAUUGGNN 338 sense 404 UGUACAAGUGCUCAGUUCC 155 UGUACAAGUGCUCAGUUCCNN 33 9 antis 404 GGAACUGAGCACUUGUACA 156 GGAACUGAGCACUUGUACANN 340 sense 405 GUACAAGUGCUCAGUUCCA 157 GUACAAGUGCUCAGUUCCANN 341 antis 405 UGGAACUGAGCACUUGUAC 158 UGGAACUGAGCACUUGUACNN 342 sense 721 UAGGUGUCUUUUAAUGAUA 159 UAGGUGUCUUUUAAUGAUANN 343 antis 721 UAUCAUUAAAAGACACCUA 160 UAUCAUUAAAAGACACCUANN 344 sense 345 AAGCCUAAGAAAUAUCUUU 161 AAGCCUAAGAAAUAUCUUUNN 345 antis 345 AAAGAUAUUUCUUAGGCUU 162 AAAGAUAUUUCUUAGGCUUNN 34 6 sense 412 UGCUCAGUUCCAAUGUGCC 163 UGCUCAGUUCCAAUGUGCCNN 347 antis 412 GGCACAUUGGAACUGAGCA 164 GGCACAUUGGAACUGAGCANN 348 sense 418 GUUCCAAUGUGCCCAGUCA 165 GUUCCAAUGUGCCCAGUCANN 34 9 antis 418 UGACUGGGCACAUUGGAAC 166 UGACUGGGCACAUUGGAACNN 350 sense 684 GUUGUUAGUGAUUUGCUAU 167 GUUGUUAGUGAUUUGCUAUNN 351 antis 684 AUAGCAAAUCACUAACAAC 168 AUAGCAAAUCACUAACAACNN 352 sense 516 CAGCAUUUCGGUGCUUCCC 169 CAGCAUUUCGGUGCUUCCCNN 353 antis 516 GGGAAGCACCGAAAUGCUG 170 GGGAAGCACCGAAAUGCUGNN 354 sense 941 CCAUCUCACUUUAAUAAUA 171 CCAUCUCACUUUAAUAAUANN 355 antis 941 UAUUAUUAAAGUGAGAUGG 172 UAUUAUUAAAGUGAGAUGGNN 35 6 sense 566 CUUUGUGUGCUGUGGAUUU 173 CUUUGUGUGCUGUGGAUUUNN 357 antis 566 AAAUCCACAGCACACAAAG 174 AAAUCCACAGCACACAAAGNN 358 sense 226 GACGGGUGUGACAGCAGUA 175 GACGGGUGUGACAGCAGUANN 35 9 antis 226 UACUGCUGUCACACCCGUC 176 UACUGCUGUCACACCCGUCNN 3 60 sense 346 AGCCUAAGAAAUAUCUUUG 177 AGCCUAAGAAAUAUCUUUGNN 3 61 antis 346 CAAAGAUAUUUCUUAGGCU 178 CAAAGAUAUUUCUUAGGCUNN 3 62 sense 414 CUCAGUUCCAAUGUGCCCA 179 CUCAGUUCCAAUGUGCCCANN 3 63 antis 414 UGGGCACAUUGGAACUGAG 180 UGGGCACAUUGGAACUGAGNN 3 64 sense 722 AGGUGUCUUUUAAUGAUAC 181 AGGUGUCUUUUAAUGAUACNN 3 65 Start of Sequence with 3 ' target Unmodified sequence SEQ ID dinucleotide overhangs SEQ ID

Strand sequence (5' to 3') NO: (5 ' to 3 ' ) NO: antis 722 GUAUCAUUAAAAGACACCU 182 GUAUCAUUAAAAGACACCUNN 3 66 sense 330 AAGACUACGAACCUGAAGC 183 AAGACUACGAACCUGAAGCNN 3 67 antis 330 GCUUCAGGUUCGUAGUCUU 184 GCUUCAGGUUCGUAGUCUUNN 3 68

Table 3 : SNCA sense and antisense strand sequences with dTsdT overhangs

Numbering for target sequence is based on human NM_007308.1.

Start of

target Sequence with 3 'dTsdT overhangs SEQ

Strand sequence (5' to 3') ID NO: sense 325 GUAUCAAGACUACGAACCUdTsdT 369 antis 325 AGGUUCGUAGUCUUGAUACdTsdT 370 sense 625 CUAAGUGACUACCACUUAUdTsdT 371 antis 625 AUAAGUGGUAGUCACUUAGdTsdT 372 sense 321 AAGGGUAUCAAGACUACGAdTsdT 373 antis 321 UCGUAGUCUUGAUACCCUUdTsdT 374 sense 326 UAUCAAGACUACGAACCUGdTsdT 375 antis 326 CAGGUUCGUAGUCUUGAUAdTsdT 376 sense 200 GUGACAAAUGUUGGAGGAGdTsdT 377 antis 200 CUCCUCCAACAUUUGUCACdTsdT 378 sense 327 AUCAAGACUACGAACCUGAdTsdT 379 antis 327 UCAGGUUCGUAGUCUUGAUdTsdT 380 sense 426 GUGCCCAGUCAUGACAUUUdTsdT 381 antis 426 AAAUGUCAUGACUGGGCACdTsdT 382 sense 731 UUAAUGAUACUGUCUAAGAdTsdT 383 antis 731 UCUUAGACAGUAUCAUUAAdTsdT 384 sense 429 CCCAGUCAUGACAUUUCUCdTsdT 385 antis 429 GAGAAAUGUCAUGACUGGGdTsdT 386 sense 543 AAGUGAAUACAUGGUAGCAdTsdT 387 antis 543 UGCUACCAUGUAUUCACUUdTsdT 388 sense 802 AGCAUGAAACUAUGCACCUdTsdT 389 antis 802 AGGUGCAUAGUUUCAUGCUdTsdT 390 sense 803 GCAUGAAACUAUGCACCUAdTsdT 391 antis 803 UAGGUGCAUAGUUUCAUGCdTsdT 392 sense 410 AGUGCUCAGUUCCAAUGUGdTsdT 393 antis 410 CACAUUGGAACUGAGCACUdTsdT 394 sense 518 GCAUUUCGGUGCUUCCCUUdTsdT 395 antis 518 AAGGGAAGCACCGAAAUGCdTsdT 396 sense 798 UGUGAGCAUGAAACUAUGCdTsdT 397 antis 798 GCAUAGUUUCAUGCUCACAdTsdT 398 sense 318 AGGAAGGGUAUCAAGACUAdTsdT 399 antis 318 UAGUCUUGAUACCCUUCCUdTsdT 400 sense 537 UCACUGAAGUGAAUACAUGdTsdT 401 antis 537 CAUGUAUUCACUUCAGUGAdTsdT 402 sense 201 UGACAAAUGUUGGAGGAGCdTsdT 403 antis 201 GCUCCUCCAACAUUUGUCAdTsdT 404 sense 544 AGUGAAUACAUGGUAGCAGdTsdT 405 antis 544 CUGCUACCAUGUAUUCACUdTsdT 406 sense 548 AAUACAUGGUAGCAGGGUCdTsdT 407 Start of

target Sequence with 3'dTsdT overhangs SEQ

Strand sequence (5' to 3') ID NO: antis 548 GACCCUGCUACCAUGUAUUdTsdT 408 sense 482 AGCAGUGAUUGAAGUAUCUdTsdT 409 antis 482 AGAUACUUCAAUCACUGCUdTsdT 410 sense 221 GUGGUGACGGGUGUGACAGdTsdT 411 antis 221 CUGUCACACCCGUCACCACdTsdT 412 sense 328 UCAAGACUACGAACCUGAAdTsdT 413 antis 328 UUCAGGUUCGUAGUCUUGAdTsdT 414 sense 942 CAUCUCACUUUAAUAAUAAdTsdT 415 antis 942 UUAUUAUUAAAGUGAGAUGdTsdT 416 sense 227 ACGGGUGUGACAGCAGUAGdTsdT 417 antis 227 CUACUGCUGUCACACCCGUdTsdT 418 sense 320 GAAGGGUAUCAAGACUACGdTsdT 419 antis 320 CGUAGUCUUGAUACCCUUCdTsdT 420 sense 683 AGUUGUUAGUGAUUUGCUAdTsdT 421 antis 683 UAGCAAAUCACUAACAACUdTsdT 422 sense 374 UCUUGAGAUCUGCUGACAGdTsdT 423 antis 374 CUGUCAGCAGAUCUCAAGAdTsdT 424 sense 358 AUCUUUGCUCCCAGUUUCUdTsdT 425 antis 358 AGAAACUGGGAGCAAAGAUdTsdT 426 sense 552 CAUGGUAGCAGGGUCUUUGdTsdT 427 antis 552 CAAAGACCCUGCUACCAUGdTsdT 428 sense 639 CUUAUUUCUAAAUCCUCACdTsdT 429 antis 639 GUGAGGAUUUAGAAAUAAGdTsdT 430 sense 425 UGUGCCCAGUCAUGACAUUdTsdT 431 antis 425 AAUGUCAUGACUGGGCACAdTsdT 432 sense 675 UGUUCAGAAGUUGUUAGUGdTsdT 433 antis 675 CACUAACAACUUCUGAACAdTsdT 434 sense 329 CAAGACUACGAACCUGAAGdTsdT 435 antis 329 CUUCAGGUUCGUAGUCUUGdTsdT 436 sense 809 AACUAUGCACCUAUAAAUAdTsdT 437 antis 809 UAUUUAUAGGUGCAUAGUUdTsdT 438 sense 794 AAUAUGUGAGCAUGAAACUdTsdT 439 antis 794 AGUUUCAUGCUCACAUAUUdTsdT 440 sense 487 UGAUUGAAGUAUCUGUACCdTsdT 441 antis 487 GGUACAGAUACUUCAAUCAdTsdT 442 sense 538 CACUGAAGUGAAUACAUGGdTsdT 443 antis 538 CCAUGUAUUCACUUCAGUGdTsdT 444 sense 534 CUUUCACUGAAGUGAAUACdTsdT 445 antis 534 GUAUUCACUUCAGUGAAAGdTsdT 446 sense 427 UGCCCAGUCAUGACAUUUCdTsdT 447 antis 427 GAAAUGUCAUGACUGGGCAdTsdT 448 sense 567 UUUGUGUGCUGUGGAUUUUdTsdT 449 antis 567 AAAAUCCACAGCACACAAAdTsdT 450 sense 806 UGAAACUAUGCACCUAUAAdTsdT 451 antis 806 UUAUAGGUGCAUAGUUUCAdTsdT 452 sense 738 UACUGUCUAAGAAUAAUGAdTsdT 453 antis 738 UCAUUAUUCUUAGACAGUAdTsdT 454 sense 876 UUGUAUAUAAAUGGUGAGAdTsdT 455 antis 876 UCUCACCAUUUAUAUACAAdTsdT 456 Start of

target Sequence with 3'dTsdT overhangs SEQ

Strand sequence (5' to 3') ID NO: sense 322 AGGGUAUCAAGACUACGAAdTsdT 457 antis 322 UUCGUAGUCUUGAUACCCUdTsdT 458 sense 324 GGUAUCAAGACUACGAACCdTsdT 459 antis 324 GGUUCGUAGUCUUGAUACCdTsdT 460 sense 334 CUACGAACCUGAAGCCUAAdTsdT 461 antis 334 UUAGGCUUCAGGUUCGUAGdTsdT 462 sense 884 AAAUGGUGAGAAUUAAAAUdTsdT 463 antis 884 AUUUUAAUUCUCACCAUUUdTsdT 464 sense 553 AUGGUAGCAGGGUCUUUGUdTsdT 465 antis 553 ACAAAGACCCUGCUACCAUdTsdT 466 sense 323 GGGUAUCAAGACUACGAACdTsdT 467 antis 323 GUUCGUAGUCUUGAUACCCdTsdT 468 sense 620 AACACCUAAGUGACUACCAdTsdT 469 antis 620 UGGUAGUCACUUAGGUGUUdTsdT 470 sense 619 AAACACCUAAGUGACUACCdTsdT 471 antis 619 GGUAGUCACUUAGGUGUUUdTsdT 472 sense 682 AAGUUGUUAGUGAUUUGCUdTsdT 473 antis 682 AGCAAAUCACUAACAACUUdTsdT 474 sense 732 UAAUGAUACUGUCUAAGAAdTsdT 475 antis 732 UUCUUAGACAGUAUCAUUAdTsdT 476 sense 515 UCAGCAUUUCGGUGCUUCCdTsdT 477 antis 515 GGAAGCACCGAAAUGCUGAdTsdT 478 sense 737 AUACUGUCUAAGAAUAAUGdTsdT 479 antis 737 CAUUAUUCUUAGACAGUAUdTsdT 480 sense 804 CAUGAAACUAUGCACCUAUdTsdT 481 antis 804 AUAGGUGCAUAGUUUCAUGdTsdT 482 sense 554 UGGUAGCAGGGUCUUUGUGdTsdT 483 antis 554 CACAAAGACCCUGCUACCAdTsdT 484 sense 943 AUCUCACUUUAAUAAUAAAdTsdT 485 antis 943 UUUAUUAUUAAAGUGAGAUdTsdT 486 sense 674 UUGUUCAGAAGUUGUUAGUdTsdT 487 antis 674 ACUAACAACUUCUGAACAAdTsdT 488 sense 428 GCCCAGUCAUGACAUUUCUdTsdT 489 antis 428 AGAAAUGUCAUGACUGGGCdTsdT 490 sense 580 GAUUUUGUGGCUUCAAUCUdTsdT 491 antis 580 AGAUUGAAGCCACAAAAUCdTsdT 492 sense 813 AUGCACCUAUAAAUACUAAdTsdT 493 antis 813 UUAGUAUUUAUAGGUGCAUdTsdT 494 sense 621 ACACCUAAGUGACUACCACdTsdT 495 antis 621 GUGGUAGUCACUUAGGUGUdTsdT 496 sense 936 UUAUCCCAUCUCACUUUAAdTsdT 497 antis 936 UUAAAGUGAGAUGGGAUAAdTsdT 498 sense 349 CUAAGAAAUAUCUUUGCUCdTsdT 499 antis 349 GAGCAAAGAUAUUUCUUAGdTsdT 500 sense 433 GUCAUGACAUUUCUCAAAGdTsdT 501 antis 433 CUUUGAGAAAUGUCAUGACdTsdT 502 sense 350 UAAGAAAUAUCUUUGCUCCdTsdT 503 antis 350 GGAGCAAAGAUAUUUCUUAdTsdT 504 sense 1036 AAGAAGGAGGAAUUUUAGAdTsdT 505 Start of

target Sequence with 3'dTsdT overhangs SEQ

Strand sequence (5' to 3') ID NO: antis 1036 UCUAAAAUUCCUCCUUCUUdTsdT 506 sense 424 AUGUGCCCAGUCAUGACAUdTsdT 507 antis 424 AUGUCAUGACUGGGCACAUdTsdT 508 sense 673 GUUGUUCAGAAGUUGUUAGdTsdT 509 antis 673 CUAACAACUUCUGAACAACdTsdT 510 sense 640 UUAUUUCUAAAUCCUCACUdTsdT 511 antis 640 AGUGAGGAUUUAGAAAUAAdTsdT 512 sense 638 ACUUAUUUCUAAAUCCUCAdTsdT 513 antis 638 UGAGGAUUUAGAAAUAAGUdTsdT 514 sense 720 UUAGGUGUCUUUUAAUGAUdTsdT 515 antis 720 AUCAUUAAAAGACACCUAAdTsdT 516 sense 353 GAAAUAUCUUUGCUCCCAGdTsdT 517 antis 353 CUGGGAGCAAAGAUAUUUCdTsdT 518 sense 357 UAUCUUUGCUCCCAGUUUCdTsdT 519 antis 357 GAAACUGGGAGCAAAGAUAdTsdT 520 sense 421 CCAAUGUGCCCAGUCAUGAdTsdT 521 antis 421 UCAUGACUGGGCACAUUGGdTsdT 522 sense 404 UGUACAAGUGCUCAGUUCCdTsdT 523 antis 404 GGAACUGAGCACUUGUACAdTsdT 524 sense 405 GUACAAGUGCUCAGUUCCAdTsdT 525 antis 405 UGGAACUGAGCACUUGUACdTsdT 526 sense 721 UAGGUGUCUUUUAAUGAUAdTsdT 527 antis 721 UAUCAUUAAAAGACACCUAdTsdT 528 sense 345 AAGCCUAAGAAAUAUCUUUdTsdT 529 antis 345 AAAGAUAUUUCUUAGGCUUdTsdT 530 sense 412 UGCUCAGUUCCAAUGUGCCdTsdT 531 antis 412 GGCACAUUGGAACUGAGCAdTsdT 532 sense 418 GUUCCAAUGUGCCCAGUCAdTsdT 533 antis 418 UGACUGGGCACAUUGGAACdTsdT 534 sense 684 GUUGUUAGUGAUUUGCUAUdTsdT 535 antis 684 AUAGCAAAUCACUAACAACdTsdT 536 sense 516 CAGCAUUUCGGUGCUUCCCdTsdT 537 antis 516 GGGAAGCACCGAAAUGCUGdTsdT 538 sense 941 CCAUCUCACUUUAAUAAUAdTsdT 539 antis 941 UAUUAUUAAAGUGAGAUGGdTsdT 540 sense 566 CUUUGUGUGCUGUGGAUUUdTsdT 541 antis 566 AAAUCCACAGCACACAAAGdTsdT 542 sense 226 GACGGGUGUGACAGCAGUAdTsdT 543 antis 226 UACUGCUGUCACACCCGUCdTsdT 544 sense 346 AGCCUAAGAAAUAUCUUUGdTsdT 545 antis 346 CAAAGAUAUUUCUUAGGCUdTsdT 546 sense 414 CUCAGUUCCAAUGUGCCCAdTsdT 547 antis 414 UGGGCACAUUGGAACUGAGdTsdT 548 sense 722 AGGUGUCUUUUAAUGAUACdTsdT 549 antis 722 GUAUCAUUAAAAGACACCUdTsdT 550 sense 330 AAGACUACGAACCUGAAGCdTsdT 551 antis 330 GCUUCAGGUUCGUAGUCUUdTsdT 552 siRNA sequence selection

A total of 92 sense and 92 antisense human SNCA derived siRNA oligos were synthesized. The nucleotides were modified as described below. The sense and their respective antisense oligos were annealed into duplexes.

Table 4 provides the sequences and modifications of the sense and antisense strands of the 92 synthesized duplexes.

Synthesis of dsRNA

The sense and antisense strands of the dsRNA duplexes were synthesized on a MerMade 192 synthesizer at 1 μιηοΐ scale. The strands were modified as follows and as described in Table 4. In the sense strand, all pyrimidines (U, C) were replaced with corresponding 2'-0-Methyl bases (2' O-Methyl C and 2'-0-Methyl U). In the antisense strand, pyrimidines adjacent to (towards the 5' position) a ribo A nucleoside were replaced with their corresponding 2-O-Methyl nucleosides. A two base dTsdT extension at the 3 ' end of both sense and antisense sequences was introduced. The sequence file was converted to a text file to make it compatible for loading in the MerMade 192 synthesis software.

The synthesis of each strand of the dsRNA used solid supported oligonucleotide synthesis using phosphoramidite chemistry.

Synthesis was performed at 1 μιηοΐ scale in 96-well plates. The amidite solutions were prepared at 0.1 M concentration and ethyl thio tetrazole (0.6 M in acetonitrile) was used as an activator. The synthesized sequences were cleaved and deprotected in 96-well plates, using methylamine in the first step and triethylamine 3HF in the second step. The crude sequences thus obtained were precipitated using acetone: ethanol (80:20) mix and the pellet were re-suspended in 0.5 M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS and the resulting mass data confirmed the identity of the sequences. UV detection was used for sample quantitation. A selected set of samples were also analyzed for purity by IEX chromatography.

All sequences were purified on an AKTA explorer purification system using a Source 15Q column. A column temperature of 65°C was maintained during purification. Sample injection and collection was performed in 96-well (1.8 mL -deep well) plates. A single peak corresponding to the full length sequence was collected in the eluent.

The purified sequences were desalted on a Sephadex G25 column using an AKTA purifier. The desalted sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). For the preparation of duplexes, equimolar amounts of sense and antisense strand were heated in the required buffer (e.g., lx PBS) at 95°C for 2-5 minutes and slowly cooled to room temperature. Integrity of the duplex was confirmed by HPLC analysis.

Table 4: Chemically modified SNCA sense and antisense strand sequences with dTsdT overhangs

Numbering for target sequence is based on human NM_007308.1. Modifications: sense strand - all pyrimidines (U, C) are 2'OMe; antisense strand - pyrimidines adjacent to A (UA, CA) are 2'OMe; 3' end is thio (dTsdT). Residues in lower case indicate chemical modification.

Start of SEQ

Duplex target Sequence with 3 'dTsdT overhangs ID No. Strand sequence (5' to 3') NO:

AD-21752 sense 325 GuAucAAGAcuAcGAAccudTsdT 553

antis 325 AGGUUCGuAGUCUUGAuACdTsdT 554

AD-21753 sense 625 cuAAGuGAcuAccAcuuAudTsdT 555

antis 625 AuAAGUGGuAGUcACUuAGdTsdT 55 6

AD-21754 sense 321 AAGGGuAucAAGAcuAcGAdTsdT 557

antis 321 UCGuAGUCUUGAuACCCUUdTsdT 558

AD-21755 sense 326 uAucAAGAcuAcGAAccuGdTsdT 55 9

antis 326 cAGGUUCGuAGUCUUGAuAdTsdT 5 60

AD-21756 sense 200 GuGAcAAAuGuuGGAGGAGdTsdT 5 61

antis 200 CUCCUCcAAcAUUUGUcACdTsdT 5 62

AD-21757 sense 327 AucAAGAcuAcGAAccuGAdTsdT 5 63

antis 327 UcAGGUUCGuAGUCUUGAUdTsdT 5 64

AD-21758 sense 426 GuGcccAGucAuGAcAuuudTsdT 5 65

antis 426 AAAUGUcAUGACUGGGcACdTsdT 5 66

AD-21759 sense 731 uuAAuGAuAcuGucuAAGAdTsdT 5 67

antis 731 UCUuAGAcAGuAUcAUuAAdTsdT 5 68

AD-21760 sense 429 cccAGucAuGAcAuuucucdTsdT 5 69

antis 429 GAGAAAUGUcAUGACUGGGdTsdT 570

AD-21761 sense 543 AAGuGAAuAcAuGGuAGcAdTsdT 571

antis 543 UGCuACcAUGuAUUcACUUdTsdT 572

AD-21762 sense 802 AGcAuGAAAcuAuGcAccudTsdT 573

antis 802 AGGUGcAuAGUUUcAUGCUdTsdT 574

AD-21763 sense 803 GcAuGAAAcuAuGcAccuAdTsdT 575

antis 803 uAGGUGcAuAGUUUcAUGCdTsdT 57 6

AD-21764 sense 410 AGuGcucAGuuccAAuGuGdTsdT 577

antis 410 cAcAUUGGAACUGAGcACUdTsdT 578

AD-21765 sense 518 GcAuuucGGuGcuucccuudTsdT 57 9

antis 518 AAGGGAAGcACCGAAAUGCdTsdT 580

AD-21766 sense 798 uGuGAGcAuGAAAcuAuGcdTsdT 581

antis 798 GcAuAGUUUcAUGCUcAcAdTsdT 582

AD-21767 sense 318 AGGAAGGGuAucAAGAcuAdTsdT 583

antis 318 uAGUCUUGAuACCCUUCCUdTsdT 584

AD-21768 sense 537 ucAcuGAAGuGAAuAcAuGdTsdT 585

antis 537 cAUGuAUUcACUUcAGUGAdTsdT 58 6

AD-21769 sense 201 uGAcAAAuGuuGGAGGAGcdTsdT 587

antis 201 GCUCCUCcAAcAUUUGUcAdTsdT 588

AD-21770 sense 544 AGuGAAuAcAuGGuAGcAGdTsdT 58 9 Start of SEQ

Duplex target Sequence with 3'dTsdT overhangs ID No. Strand sequence (5' to 3') NO:

antis 544 CUGCuACcAUGuAUUcACUdTsdT 5 90

AD-21771 sense 548 AAuAcAuGGuAGcAGGGucdTsdT 5 91

antis 548 GACCCUGCuACcAUGuAUUdTsdT 5 92

AD-21772 sense 482 AGcAGuGAuuGAAGuAucudTsdT 5 93

antis 482 AGAuACUUcAAUcACUGCUdTsdT 5 94

AD-21773 sense 221 GuGGuGAcGGGuGuGAcAGdTsdT 5 95

antis 221 CUGUcAcACCCGUcACcACdTsdT 5 96

AD-21774 sense 328 ucAAGAcuAcGAAccuGAAdTsdT 5 97

antis 328 UUcAGGUUCGiiAGUCUUGAdTsdT 5 98

AD-21775 sense 942 cAucucAcuuuAAiiAAiiAAdTsdT 5 99

antis 942 UuAUiiAUiiAAAGUGAGAUGdTsdT 600

AD-21776 sense 227 AcGGGuGiiGAcAGcAGiiAGdTsdT 601

antis 227 CuACUGCUGUcAcACCCGUdTsdT 602

AD-21777 sense 320 GAAGGGuAucAAGAciiAcGdTsdT 603

antis 320 CGuAGUCUUGAuACCCUUCdTsdT 604

AD-21778 sense 683 AGuuGuuAGuGAiiiiiiGcuAdTsdT 605

antis 683 uAGcAAAUcACiiAAcAACUdTsdT 60 6

AD-21779 sense 374 uciiiiGAGAiiciiGciiGAcAGdTsdT 607

antis 374 CUGUcAGcAGAUCUcAAGAdTsdT 608

AD-22077 sense 358 AucuuuGcucccAGiiiiiiciidTsdT 60 9

antis 358 AGAAACUGGGAGcAAAGAUdTsdT 610

AD-21780 sense 552 cAuGGiiAGcAGGGiiciiiiiiGdTsdT 611

antis 552 cAAAGACCCUGCuACcAUGdTsdT 612

AD-22078 sense 639 cuuAuuiicuAAAiicciicAcdTsdT 613

antis 639 GUGAGGAUUuAGAAAuAAGdTsdT 614

AD-21781 sense 425 uGuGcccAGucAuGAcAuudTsdT 615

antis 425 AAUGUcAUGACUGGGcAcAdTsdT 61 6

AD-21782 sense 675 uGuucAGAAGuuGuuAGuGdTsdT 617

antis 675 cACuAAcAACUUCUGAAcAdTsdT 618

AD-21783 sense 329 cAAGAcuAcGAAccuGAAGdTsdT 61 9

antis 329 CUUcAGGUUCGuAGUCUUGdTsdT 620

AD-22079 sense 809 AAcuAuGcAccuAuAAAuAdTsdT 621

antis 809 uAUUuAuAGGUGcAuAGUUdTsdT 622

AD-21784 sense 794 AAuAuGuGAGcAuGAAAcudTsdT 623

antis 794 AGUUUcAUGCUcAcAuAUUdTsdT 624

AD-21785 sense 487 uGAuuGAAGuAucuGuAccdTsdT 625

antis 487 GGuAcAGAuACUUcAAUcAdTsdT 62 6

AD-21786 sense 538 cAcuGAAGuGAAuAcAuGGdTsdT 627

antis 538 CcAUGuAUUcACUUcAGUGdTsdT 628

AD-21787 sense 534 cuuucAcuGAAGuGAAuAcdTsdT 62 9

antis 534 GuAUUcACUUcAGUGAAAGdTsdT 630

AD-21788 sense 427 uGcccAGucAuGAcAuuucdTsdT 631

antis 427 GAAAUGUcAUGACUGGGcAdTsdT 632

AD-22080 sense 567 uuuGuGuGcuGuGGAuuuudTsdT 633

antis 567 AAAAUCcAcAGcAcAcAAAdTsdT 634

AD-21789 sense 806 uGAAAcuAuGcAccuAuAAdTsdT 635

antis 806 UuAuAGGUGcAuAGUUUcAdTsdT 63 6

AD-21790 sense 738 uAcuGucuAAGAAuAAuGAdTsdT 637

antis 738 UcAUuAUUCUuAGAcAGuAdTsdT 638 Start of SEQ

Duplex target Sequence with 3'dTsdT overhangs ID No. Strand sequence (5' to 3') NO:

AD-21791 sense 876 uuGuAuAuAAAuGGuGAGAdTsdT 63 9

antis 876 UCUcACcAUUuAuAuAcAAdTsdT 640

AD-21792 sense 322 AGGGuAucAAGAcuAcGAAdTsdT 641

antis 322 UUCGuAGUCUUGAuACCCUdTsdT 642

AD-21793 sense 324 GGuAucAAGAcuAcGAAccdTsdT 643

antis 324 GGUUCGuAGUCUUGAuACCdTsdT 644

AD-21794 sense 334 cuAcGAAccuGAAGccuAAdTsdT 645

antis 334 UuAGGCUUcAGGUUCGuAGdTsdT 64 6

AD-21795 sense 884 AAAuGGuGAGAAuuAAAAudTsdT 647

antis 884 AUUUuAAUUCUcACcAUUUdTsdT 648

AD-21796 sense 553 AuGGuAGcAGGGucuuuGudTsdT 64 9

antis 553 AcAAAGACCCUGCuACcAUdTsdT 650

AD-21797 sense 323 GGGuAucAAGAcuAcGAAcdTsdT 651

antis 323 GUUCGuAGUCUUGAuACCCdTsdT 652

AD-21798 sense 620 AAcAccuAAGuGAcuAccAdTsdT 653

antis 620 UGGuAGUcACUuAGGUGUUdTsdT 654

AD-21799 sense 619 AAAcAccuAAGuGAcuAccdTsdT 655

antis 619 GGuAGUcACUuAGGUGUUUdTsdT 65 6

AD-21800 sense 682 AAGuuGuuAGuGAuuuGcudTsdT 657

antis 682 AGcAAAUcACuAAcAACUUdTsdT 658

AD-21801 sense 732 uAAuGAuAcuGucuAAGAAdTsdT 65 9

antis 732 UUCUuAGAcAGuAUcAUuAdTsdT 660

AD-21802 sense 515 ucAGcAuuucGGuGcuuccdTsdT 661

antis 515 GGAAGcACCGAAAUGCUGAdTsdT 662

AD-21803 sense 737 AuAcuGucuAAGAAuAAuGdTsdT 663

antis 737 cAUuAUUCUuAGAcAGuAUdTsdT 664

AD-21804 sense 804 cAuGAAAcuAuGcAccuAudTsdT 665

antis 804 AuAGGUGcAuAGUUUcAUGdTsdT 666

AD-21805 sense 554 uGGuAGcAGGGucuuuGuGdTsdT 667

antis 554 cAcAAAGACCCUGCuACcAdTsdT 668

AD-22081 sense 943 AucucAcuuuAAuAAuAAAdTsdT 669

antis 943 UUuAUuAUuAAAGUGAGAUdTsdT 670

AD-21806 sense 674 uuGuucAGAAGuuGuuAGudTsdT 671

antis 674 ACuAAcAACUUCUGAAcAAdTsdT 672

AD-21807 sense 428 GcccAGucAuGAcAuuucudTsdT 673

antis 428 AGAAAUGUcAUGACUGGGCdTsdT 674

AD-21808 sense 580 GAuuuuGuGGcuucAAucudTsdT 675

antis 580 AGAUUGAAGCcAcAAAAUCdTsdT 67 6

AD-21809 sense 813 AuGcAccuAuAAAuAcuAAdTsdT 677

antis 813 UuAGuAUUuAuAGGUGcAUdTsdT 678

AD-21810 sense 621 AcAccuAAGuGAcuAccAcdTsdT 67 9

antis 621 GUGGuAGUcACUuAGGUGUdTsdT 680

AD-22472 sense 936 uuAucccAucucAcuuuAAdTsdT 681

antis 936 UiiAAAGUGAGAUGGGAiiAAdTsdT 682

AD-21811 sense 349 ciiAAGAAAuAucuuuGcucdTsdT 683

antis 349 GAGcAAAGAuAUUUCUiiAGdTsdT 684

AD-21812 sense 433 GucAuGAcAuuucucAAAGdTsdT 685

antis 433 CUUUGAGAAAUGUcAUGACdTsdT 68 6

AD-21813 sense 350 iiAAGAAAuAucuuuGcuccdTsdT 687 Start of SEQ

Duplex target Sequence with 3'dTsdT overhangs ID No. Strand sequence (5' to 3') NO:

antis 350 GGAGcAAAGAuAUUUCUuAdTsdT 688

AD-21814 sense 1036 AAGAAGGAGGAAuuuuAGAdTsdT 68 9

antis 1036 UCuAAAAUUCCUCCUUCUUdTsdT 690

AD-21815 sense 424 AuGuGcccAGucAuGAcAudTsdT 691

antis 424 AUGUcAUGACUGGGcAcAUdTsdT 692

AD-21816 sense 673 GuuGuucAGAAGuuGuuAGdTsdT 693

antis 673 CuAAcAACUUCUGAAcAACdTsdT 694

* sense 640 uuAuuucuAAAuccucAcudTsdT 695 antis 640 AGUGAGGAUUiiAGAAAiiAAdTsdT 696

AD-22082 sense 638 AcuuAiiiiiicuAAAiicciicAdTsdT 697

antis 638 UGAGGAUUiiAGAAAiiAAGUdTsdT 698

AD-21817 sense 720 uuAGGuGucuuiiuAAiiGAiidTsdT 699

antis 720 AUcAUuAAAAGAcACCiiAAdTsdT 700

AD-21818 sense 353 GAAAuAuciiiiiiGciicccAGdTsdT 701

antis 353 CUGGGAGcAAAGAuAUUUCdTsdT 702

AD-21819 sense 357 uAuciiiiiiGciicccAGiiiiiicdTsdT 703

antis 357 GAAACUGGGAGcAAAGAuAdTsdT 704

AD-21820 sense 421 ccAAiiGiiGcccAGiicAiiGAdTsdT 705

antis 421 UcAUGACUGGGcAcAUUGGdTsdT 70 6

AD-21821 sense 404 iiGuAcAAGiiGciicAGiiiiccdTsdT 707

antis 404 GGAACUGAGcACUUGuAcAdTsdT 708

AD-21822 sense 405 GuAcAAGiiGciicAGiiiiccAdTsdT 70 9

antis 405 UGGAACUGAGcACUUGuACdTsdT 710

AD-21823 sense 721 uAGGuGucuuuiiAAuGAuAdTsdT 711

antis 721 uAUcAUuAAAAGAcACCiiAdTsdT 712

AD-21824 sense 345 AAGccuAAGAAAuAuciiiiiidTsdT 713

antis 345 AAAGAiiAUUUCUuAGGCUUdTsdT 714

AD-21825 sense 412 iiGciicAGiiiiccAAiiGiiGccdTsdT 715

antis 412 GGcAcAUUGGAACUGAGcAdTsdT 71 6

AD-21826 sense 418 GuuccAAuGuGcccAGucAdTsdT 717

antis 418 UGACUGGGcAcAUUGGAACdTsdT 718

AD-21827 sense 684 GuuGuuAGuGAuiiiiGcuAiidTsdT 71 9

antis 684 AuAGcAAAUcACiiAAcAACdTsdT 720

AD-21828 sense 516 cAGcAuuucGGuGcuucccdTsdT 721

antis 516 GGGAAGcACCGAAAUGCUGdTsdT 722

AD-21829 sense 941 ccAucucAcuuiiAAiiAAiiAdTsdT 723

antis 941 iiAUuAUiiAAAGUGAGAUGGdTsdT 724

AD-21830 sense 566 cuuuGuGuGcuGuGGAuuudTsdT 725

antis 566 AAAUCcAcAGcAcAcAAAGdTsdT 72 6

AD-21831 sense 226 GAcGGGuGuGAcAGcAGuAdTsdT 727

antis 226 uACUGCUGUcAcACCCGUCdTsdT 728

AD-21832 sense 346 AGccuAAGAAAuAiiciiiiiiGdTsdT 72 9

antis 346 cAAAGAuAUUUCUiiAGGCUdTsdT 730

AD-21833 sense 414 cucAGuuccAAuGuGcccAdTsdT 731

antis 414 UGGGcAcAUUGGAACUGAGdTsdT 732

AD-21834 sense 722 AGGuGucuuuuAAuGAiiAcdTsdT 733

antis 722 GuAUcAUiiAAAAGAcACCUdTsdT 734

AD-21835 sense 330 AAGAcuAcGAAccuGAAGcdTsdT 735

antis 330 GCUUcAGGUUCGuAGUCUUdTsdT 73 6 Example 3. In vitro screening of siRNAs for mRNA suppression

The duplexes described in Table 4 were assayed for R A suppression in tissue culture cells.

Cell culture and transfections;

NCI-H460 (ATCC, Manassas, VA) or BE(2)M17 cells were grown to near confluence at

37°C in an atmosphere of 5% C02 in RPMI (ATCC) supplemented with 10% FBS,

streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization.

Reverse transfection was carried out by adding 5 μΐ of Opti-MEM to 5 μΐ of siRNA duplexes per well into a 96-well plate along with 10 μΐ of Opti-MEM plus 0.2 μΐ of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad CA. cat # 13778-150) and incubated at room temperature for 15 minutes. 80 μΐ of complete growth media without antibiotic containing 2.0 xlO4 Hela cells were then added. Cells were incubated for 24 hours prior to RNA purification.

Total RNA isolation using MagMAX-96 Total RNA Isolation Kit (Applied Biosvstem, Forer City CA, part #: AMI 830):

Cells were harvested and lysed in 140 μΐ of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (with constant mixing speed throughout the process). 20 μΐ of magnetic beads and Lysis/Binding Enhancer mixture were added into cell- lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μΐ Wash Solution 2 (ethanol added), captured and supernatant was removed. 50 μΐ of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μΐ of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μΐ Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA was eluted with 50 μΐ of water.

cDNA synthesis using ABI High capacity cDNA reverse transcription kit (Applied Biosvstems. Foster City. CA. Cat. No. 4368813):

A master mix of 2 μΐ 10X Buffer, 0.8 μΐ 25X dNTPs, 2 μΐ Random primers, 1 μΐ Reverse Transcriptase, 1 μΐ RNase inhibitor and 3.2 μΐ of H2O per reaction were added into 10 μΐ total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) through the following steps: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 seconds, and hold at 4°C.

Real time PCR:

2 μΐ of cDNA were added to a master mix containing 0.5 μΐ GAPDH TaqMan Probe (Applied Biosystems, Cat. No. 4326317E), 0.5 μΐ synuclein TaqMan probe (Applied

Biosystems, Cat. No. Hs00240906) and 5 μΐ Roche Probes Master Mix (Roche, Cat. No.

04887301001) in a total of 10 μΐ per well in a LightCycler 480 384 well plate (Roche, Cat. No. 0472974001). Real time PCR was done in a LightCycler 480 Real Time PCR machine (Roche) using the AACt assay. Each duplex was tested in two independent transfections and each transfection was assayed in duplicate.

Real-time data were analyzed using the AACt method. Each sample was normalized to GAPDH expression and knockdown was assessed relative to cells transfected with the non- targeting duplex AD- 1955.

Results

Experiments were performed at 0.1 nM and 10 nM final duplex concentrations for single dose screens with 79 synuclein duplexes. described in Table 4. Table 5 shows the results of the single dose in vitro siRNA screen. After making the determinations reported in Table 5, it was determined that certain of the oligonucleotides were subject to concentration artifacts now thought to arise from incomplete thawing of plates containing the oligonucleotides. Subsequent analysis of two large datasets (unrelated to SNCA) indicated that any such concentration artifacts would not alter the assay results more than that which can be accounted for by normal experimental variability. Moreover, these concentration artifacts are unlikely to substantially affect our conclusions about lead selection. Table 5: Fraction of target remaining following treatment with 0.1 nM and 10 nM duplex in the NC -H460 and BE(2)M17 cell lines

Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001

A subset of 20 duplexes that showed robust silencing in the 10 nM and 0.1 nM screens were assayed over a range of concentrations from 100 nM to 10 fM using 10 fold serial dilutions to determine their IC50 in BE(2)M17 cells. IC50s were defined using a 4 parameter fit model in XLfit. Table 6 shows the average IC50s for this subset of duplexes.

Table 6: Average IC50s for a subset of svnuclein duplexes

Figure imgf000086_0002
Duplex ID Average IC50 in [pM]

AD-21765 19.5

AD-21772 3.4

AD-21781 22.4

AD-21786 41.2

AD-21788 15.2

AD-21790 6.7

AD-21801 1.8

AD-21804 9.3

AD-21806 10.4

AD-21808 28.1

AD-21810 17.3

AD-21813 47.3

AD-21817 15.4

AD-21821 11.8

AD-21822 3.4

AD-21824 19.8

AD-21826 30.8

AD-21830 11.9

Example 4; Silencing of human SNCA by intraparenchymal infusion in transgenic mice

To determine the ability of several of the msot potent SNCA siRNAs (AD21822, AD- 21830, AD-21772, AD-2801, and AD-21810) to reduce levels of human SNCA siRNA in vivo, infusion studies were conducted in transgenic mice. A transgenic mouse strain (Mayo, unpublished) was generated using a P 1 artificial chromosome (PAC) comprising the entire human SNCA locus. The expression pattern of human SNCA in this transgenic mouse strain mirrors that of endogenous mouse SNCA. To assess target silencing, solutions of siRNA (30 mg/ml in PBS) were infused via osmotic minipumps (Alzet Corp.) at a flow rate of 0.25 microliters per hour directly into the CA1 region of the hippocampus (a site of high SNCA expression) in one hemisphere of the brain. Controls included an siRNA targeting Luciferase (AD- 1955) and PBS infusion. After 15 days, brains were dissected and 2 mm thick coronal sections encompassing the infusion site were obtained. Tissue punches (1.2 mm diameter) were taken from the hippocampus at the infusion site. Total RNA was isolated and used to generate cDNA. Levels of human SNCA and mouse GAPDH were measured by Taqman® analysis (Applied Biosystems, Inc.). Levels of SNCA were normalized to GAPDH for each tissue punch, and group mean values were normalized to the PBS infused animals. As shown in Table 7, infusion with AD-21830, AD21822, AD-21772, AD-2801 and AD-21810 reduced human SNCA expression by 76%, 85%, 90%, 76% and 59%, respectively, compared to PBS infusion.

Table 7: Levels of SNCA relative to GAPDH.

Figure imgf000088_0001

Example 5; Silencing of mouse SNCA by intraparenchymal infusion

To determine the ability of a potent, rodent cross-reactive SNCA siRNA (AD21822) to reduce the level of mouse SNCA siRNA in vivo, infusion studies were conducted in C57/B1 mice. To assess target silencing, solutions of siRNA (30 mg/ml in PBS) were infused via osmotic minipumps (Alzet Corp.) at a flow rate of 0.25 microliters per hour directly into the CA1 region of the hippocampus (a site of high SNCA expression) in one hemisphere of the brain. Control animals were infused with PBS. After 14 days, brains were dissected and 1 mm thick coronal sections centered on the infusion site and 1 mm posterior to the infusion site were obtained. Tissue pieces encompassing the hippocampus were dissected from the infused side of each thick section. Total RNA was isolated and used to generate cDNA. Levels of mouse SNCA and mouse GAPDH were measured by Taqman® analysis (Applied Biosystems, Inc.). Levels of SNCA were normalized to GAPDH for each tissue piece, and group mean values were normalized to the PBS samples taken at the infusion site. As shown in Table 8, infusion with AD21822 reduced mouse SNCA expression by 95% at the infusion site, and by 57% at a distance of 1 mm posterior to the infusion site.

Table 8: Levels of SNCA relative to GAPDH.

Figure imgf000088_0002
AD-21822, infusion site 6 5% 3%

PBS, 1 mm posterior 5 115% 6%

AD-21822, 1 mm posterior 6 43% 28%

Example 6. Silencing of rat SNCA by intraparenchymal infusion

To determine the ability of two potent rodent cross-reactive SNCA siRNAs (AD21822 and AD21810) to reduce the level of rat SNCA mRNA in vivo, infusion studies were conducted in Sprague-Dawley rats. To assess target silencing, solutions of siRNA (15 mg/ml in PBS) were infused via osmotic minipumps (Alzet Corp.) at a flow rate of 1 microliter per hour directly into the substantia nigra in one hemisphere of the brain. Control animals were infused with an siRNA targeting Luciferase (AD1955) or PBS. After 7 days of infusion, brains were dissected and 1 mm thick coronal sections centered on the infusion site, 1 mm anterior to the infusion site and 1mm posterior to the infusion site were obtained. Tissue pieces encompassing the substantia nigra were dissected from the infused side of each section. Total RNA was isolated and used to generate cDNA. Levels of rat SNCA and rat GAPDH were measured by Taqman® analysis (Applied Biosystems, Inc.). Levels of SNCA were normalized to GAPDH for each tissue piece, and group mean values were normalized to the PBS samples taken at the infusion site. Levels of rat Tyrosine Hydroxylase (TH) mRNA, a marker of dopaminergic neurons in the substantia nigra, were also measured by Taqman® and normalized to GAPDH as described for SNCA. Infusion with AD21822 reduced rat SNCA expression by 50% at the infusion site, by 57% at a distance of 1 mm anterior to the infusion site and by 59% at a distance of 1 mm posterior to the infusion site relative to the PBS group (infusion site). Infusion with AD21810 reduced rat SNCA expression by 90% at the infusion site, by 84% at a distance of 1 mm anterior to the infusion site and by 92% at a distance of 1 mm posterior to the infusion site relative to the PBS group (infusion site).

To explore duration of target inhibition, the two potent SNCA siRNAs (AD21822 and AD21810) were infused via osmotic minipumps (Alzet Corp.) at a flow rate of 0.5 microliters per hour into the substantia nigra in one hemisphere of the brain of Sprague-Dawley rats for 7 days. The concentration of siRNA in the pumps was 8 mg/ml. Control animals received PBS. Pumps were disconnected at the end of the infusion period. Groups of animals were sacrificed at the end of infusion, as well as 1 week, 2 weeks and 4 weeks after the end of infusion. Brain fragments were collected as described above. Measurement of rat SNCA (normalized to rat GAPDH) in the fragments nearest the infusion site demonstrated suppression of expression by 70% (AD21810) and 78% (AD21822) at the end of infusion; 72% (AD21810) and 88% (AD21822) one week after the end of infusion; 43% (AD21810) and 79% (AD21822) two weeks after the end of infusion; and 56% (AD21810) and 65% (AD21822) four weeks after the end of infusion (compared to PBS infused animals).

To further study the potency of one of the siRNAs, AD21822 was infused at a rate of 0.25 ul per hour for 7 or 28 days into the substantia nigra of one hemisphere of the brain of Sprague- Dawley rats at siRNA concentrations of 2, 1 and 0.3 mg/ml. Control animals received PBS. At the end of infusion, brain fragments encompassing the infusion site were collected and analyzed as described above. After 7 days of infusion, rat SNCA levels (normalized to rat GAPDH) were reduced by 80%, 54%, and 55% at 2 mg/ml, 1 mg/ml and 0.3 mg/ml, respectively. After 28 days of infusion, rat SNCA levels (normalized to rat GAPDH) were reduced by 82%, 69%, and 54% at 2 mg/ml, 1 mg/ml and 0.3 mg/ml, respectively.

Example 7. Inhibition of SNCA in humans

A human subject is treated with a dsRNA targeted to a SNCA gene to inhibit expression of the SNCA gene to treat a condition.

A subject in need of treatment is selected or identified. The subject can have a neurodegenerative disease, e.g., Parkinson's disease, sporadic Parkinson's disease, Alzeimer's disease, multiple system atrophy, Lewy body dementia, or any other disease associated with SNCA expression.

The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.

At time zero, a suitable first dose of an anti-SNCA siRNA is administered to the subject. The dsRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated by any suitable means, including, e.g., a reduction in disease symptoms. This measurement can be accompanied by a measurement of SNCA expression in said subject, and/or the products of the successful siRNA- targeting of SNCA mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.

After treatment, the symptoms of subject's neurodegenerative disease are reduced or arrested relative to the symptoms existing prior to the treatment, or relative to those measured in a similarly afflicted but untreated subject.

Example 8. Method of treating a patient diagnosed with a synucleinopathy

A patient diagnosed with a synucleinopathy can be administered a pharmaceutical composition containing a dsRNA that targets the SNCA gene. The composition can be delivered directly to the brain by a device that includes an osmotic pump and mini-cannula and is bilaterally implanted into the patient.

Prior to implantation of the device, the patient receives an MRI with stereotactic frame. A computer-guided trajectory is used for delivery of the cannula to the brain. The mini-pump device is implanted into the abdomen, and then the patient is hospitalized for 2-3 days to monitor for hemorrhaging.

Approximately two weeks post-implantation of the pump, the patient can receive an MRI to check the implanted device. If the human is healing well, and no complications have occurred as a result of implanting the device, then the anti-SNCA composition can be infused into the pump, and into the cannula. A test dose of the anti-SNCA agent can be administered prior to the initiation of the therapeutic regimen.

MRIs taken at 3 months, six months, and one year following the initial treatment can be used to monitor the condition of the device, and the reaction of the patient to the device and treatment with the dsRNA. Clinicians should watch for the development of edema and an inflammatory response. Following the one-year anniversary of the initiation of the treatment, MRIs can be performed as needed.

The patient can be monitored for an improvement or stabilization in disease symptoms throughout the course of the therapy. Monitoring can include serial clinical assessments and functional neuroimaging, e.g., by MRI.

Example 9. Human SNCA mRNA sequence

Shown below is the sequence of the full length mRNA of human SNCA (transcript variant NACP 1 12; GenBank Access. No. NM_007308.1 (NCBI Geneld 6622); SEQ ID

NO:737).

1 gaattcatta gccatggatg tattcatgaa aggactttca aaggccaagg agggagttgt 61 ggctgctgct gagaaaacca aacagggtgt ggcagaagca gcaggaaaga caaaagaggg

121 tgttctctat gtaggctcca aaaccaagga gggagtggtg catggtgtgg caacagtggc 181 tgagaagacc aaagagcaag tgacaaatgt tggaggagca gtggtgacgg gtgtgacagc 241 agtagcccag aagacagtgg agggagcagg gagcattgca gcagccactg gctttgtcaa 301 aaaggaccag ttgggcaagg aagggtatca agactacgaa cctgaagcct aagaaatatc 361 tttgctccca gtttcttgag atctgctgac agatgttcca tcctgtacaa gtgctcagtt

421 ccaatgtgcc cagtcatgac atttctcaaa gtttttacag tgtatctcga agtcttccat 481 cagcagtgat tgaagtatct gtacctgccc ccactcagca tttcggtgct tccctttcac 541 tgaagtgaat acatggtagc agggtctttg tgtgctgtgg attttgtggc ttcaatctac 601 gatgttaaaa caaattaaaa acacctaagt gactaccact tatttctaaa tcctcactat 661 ttttttgttg ctgttgttca gaagttgtta gtgatttgct atcatatatt ataagatttt 721 taggtgtctt ttaatgatac tgtctaagaa taatgacgta ttgtgaaatt tgttaatata 781 tataatactt aaaaatatgt gagcatgaaa ctatgcacct ataaatacta aatatgaaat 841 tttaccattt tgcgatgtgt tttattcact tgtgtttgta tataaatggt gagaattaaa 901 ataaaacgtt atctcattgc aaaaatattt tatttttatc ccatctcact ttaataataa 961 aaatcatgct tataagcaac atgaattaag aactgacaca aaggacaaaa atataaagtt

1021 attaatagcc atttgaagaa ggaggaattt tagaagaggt agagaaaatg gaacattaac

1081 cctacactcg gaattc

Example 10. Rhesus monkey SNCA mRNA sequence

Shown below is the sequence of the predicted full length mRNA of rhesus monkey SNCA (isoform NACP140); GenBank Access. No. XM_001095402.1 (NCBI Geneld 706985); SEQ ID NO:738).

1 atgcgagggc aaagtgctct cggcgcgccc ccactttccc gccttgcgcg gccaggcagg

61 cggctggaat tggtggttca ccctgcgccc cctgccccat ccccatccga gataggcaac

121 gaagagcacg ctgcagggaa agcagcgagc gctgggaggg gagcgtggag aggcgctgac

181 aaatcagcgg tgggggcgga gagccgagga gaaggagaag gaggaggacg aggaggagga

241 ggacggcgac gaccagaagg ggcccgagag agggggcgag cgaccgagcg ccgcgacgcg

301 ggagtgaggt gcgtgcgggc tgcagcgcag accccggccc ggcccctccg agagcgtcct

361 gagcgctccc tcacgccttc ccttcaaacc ttctgccttt ttctccatcc tcgtgagcgg

421 agaactggga gtggccattc gacgacaggc tccaaaacca aggagggagt ggtgcacggt

481 gtggcaacag tggctgagaa gaccaaagag caagtgacaa atgttggagg agcggtggtg

541 acgggtgtga cagcagtagc ccagaagaca gtggagggag cagggagcat tgcagcagcc

601 actggcttca tcaaaaagga ccagttgggc aagaatgaag aaggagcccc acaggaagga

661 attctacaag atatgcctgt ggatcctgac aatgaggctt atgaaatgcc ttctgaggaa

721 gggtatcaag actacgaacc tgaagcctaa gaaatatctt tgctcccagt ttcttgagat

781 ctgctgacag acgttccatc ttgtacaagt gctcagttcc aatgtgccca gtcatgacat

841 ttctcaaagt ttttacagta tattttgaag tcttccatca gcagtgattg aagtatctgt

901 acctgccccc attcagcatt tcggtgcttc cctttcactg aagtgaatac atggtagcag

961 ggtctttgtg tgctgtggat tttgtggctt caatctatga tgttaaaaca atttaaaaac

1021 acctaagtga ctaccactta tttctaaatc ctcactattt ttttgttgct gttgttcaga

1081 agttgttagt gatttgctat cgtatattat aagattttta ggtgtctttt aatgatactg

1141 tctaagaata atgatgtatt gtgaaatttg ttaatatata taatacttaa aaatatgtga

1201 gcatgaaact atgcacctat aaatactaac tatgaaattt taccgttttg tgatgtgttt

1261 tattaacttg tgtttgtata taaatggtga gaattaaaat aaaatgtcgt ctcattgcaa

1321 acaaaatttt atttttatcc catctcactt taataataaa aatcttgctt ataagcaaca

1381 tgcattgaga actgacacaa tggacataaa gttattaata ggcatttgaa gaaggaggaa 1441 ttttagaaga ggtagagaaa atgaacatta accctacact gggaattccc tgaagcagca

1501 ctgccagaag tgtgttttgt ggtgccttaa gtggctgtga ttaaaaaaaa aaaaaagtgg

1561 gctccaggga acgaagcagt gtaaaagatg attttgacta catcctcctt agagatccat

1621 gagacacttt agcacatatt agcacattca aggctctgag acaatgtggt taacttagtt

1681 taactcagca gtccccacta aaaaaaaaaa aatcatcaaa aattctctct ctctattcct

1741 ttttctctcg ctcccctttt ttccaggaaa tgcctttaaa cacctttggg aactatcagg

1801 atcaccttaa agaagatcag ttctccagac tgataaaaat ttcatgatct cttttaaatg

1861 ttgccaaata tatgaattct aggatttttc cttgggaaag gtttttctct ttcagggaag

1921 atctattaac tccccatggg tgctgaaaat aaacttgatg gtgaaaaatt ctatataaat

1981 taatttaaaa tttttttggt ttctcttttt aattattctg gggcatagtc atttttaaaa

2041 gtcactagta gaaagtataa tttcaagaca gaatattcta gacatgctag cagtttatat

2101 gtattcatga gtaatgtgat atatattggg cactggtgag gcaggaagga ggaatgagtg

2161 actataagga tggttaccat agaaacttcc ttttttacct

Other embodiments are in the claims.

Claims

CLAIMS We claim:
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of alpha-synuclein (SNCA), comprising a sense strand and an antisense strand comprising a region of
complementarity to an mRNA encoding SNCA, wherein each strand is at least 15 nucleotides in length, and wherein the sense strand and antisense strand comprise at least 15 contiguous nucleotides of sequences selected from Tables 2, 3, 4, or 6.
2. The dsRNA of claim 1, wherein the sense strand comprises SEQ ID NO: l, 17, 27, 41, 63, 75, 79, 85, 107, 113, 119, 123, 127, 135, 147, 155, 157, 161, 165, or 173 and/or the antisense strand comprises SEQ ID NO:2, 18, 28, 42, 64, 76, 80, 86, 108, 114, 120, 124, 128, 136, 148, 156, 158, 162, 166, or 174.
3. The dsRNA of claim 1, wherein the antisense strand is complementary to at least 15 contiguous nucleotides of SEQ ID NO: 157.
4. The dsRNA of claim 1 , wherein the antisense strand is complementary to at least the first 11 nucleotides of SEQ ID NO: 158.
5. The dsRNA of claim 1, wherein the sense strand sequence comprises SEQ ID NO: 157 and the antisense strand sequence comprises SEQ ID NO: 158.
6. The dsRNA of claim 1, wherein the sense strand comprises SEQ ID NO:553, 569, 579, 593, 615, 627, 631, 637, 659, 665, 671, 675, 679, 687, 699, 707, 709, 713, 717, or 725; and/or the antisense strand comprises SEQ ID NO:554, 570, 580, 594, 616, 628, 632, 638, 660, 666, 672, 676, 680, 688, 700, 708, 710, 714, 718, or 726.
7. The dsRNA of claim 1, wherein the sense strand sequence comprises SEQ ID NO:707 and the antisense strand sequence comprises SEQ ID NO: 708.
8. The dsRNA of claim 1, wherein the dsRNA is selected from the group consisting of: AD- 21752, AD-21760, AD-21765, AD-21772, AD-21781, AD-21786, AD-21788, AD-21790, AD- 21801, AD-21804, AD-21806, AD-21808, AD-21810, AD-21813, AD-21817, AD-21821, AD-
21822, AD-21824, AD-21826, and AD-21830.
9. The dsRNA of claim 1, wherein the dsRNA is AD-21822.
10. The dsRNA of claim 1, wherein the SNCA is human SNCA encoded by NM_007308.1.
11. The dsRNA of claim 1, wherein the SNCA is monkey SNCA encoded by
XM 001095402.1.
12. The dsRNA of claim 1, wherein the region of complementarity is between 19 and 21 nucleotides in length.
13. The dsRNA of claim 1, wherein the region of complementarity is 19 nucleotides in length.
14. The dsRNA of claim 1, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.
15. The dsRNA of claim 1, wherein the dsRNA comprises a nucleotide overhang having 1 to 4 nucleotides.
16. The dsRNA of claim 1, wherein each strand comprises a 3' overhang consisting of 2 nucleotides.
17. The dsRNA of claim 1, wherein each strand comprises a 3' overhang consisting of dTsdT.
18. The dsRNA of claim 1, wherein said dsRNA comprises at least one modified nucleotide.
19. The dsRNA of claim 18, wherein said modified nucleotide is selected from the group of: a 2'-0-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
20. The dsRNA of claim 18, wherein said modified nucleotide is chosen from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
21. The dsRNA of claim 18, comprising at least one 2'-0-methyl modified nucleotide and at least one 2'-deoxythymidine-3'-phosphate nucleotide comprising a 5'-phosphorothioate group.
22. The dsRNA of claim 18, wherein the sense strand comprises all 2'-0-methyl modified pyrimidines and the antisense strand comprises 2'-0-methyl modified pyrimidines when the pyrimidine is adjacent to A; and wherein each strand comprises dTdT at the 3' end.
23. The dsRNA of claim 18, wherein the sense strand comprises all 2'-0-methyl modified pyrimidines and the antisense strand comprises 2'-0-methyl modified pyrimidines when the pyrimidine is adjacent to A; and wherein each strand comprises dTsdT at the 3' end.
24. The dsRNA of claim 18, wherein the sense strand comprises all 2'-0-methyl modified pyrimidines and the antisense strand comprises 2'-0-methyl modified pyrimidines when a) the pyrimidine is adjacent to A, or b) the pyrimidine is a uracil adjacent to a U or a G; and wherein each strand comprises dTsdT at the 3 ' end.
25. The dsRNA of claim 1, further comprising a ligand.
26. The dsRNA of claim 26, wherein the ligand is conjugated to the 3'-end of the sense strand of the dsRNA.
27. A composition for inhibiting expression of a SNCA gene comprising the dsRNA of claim 1 and a pharmaceutical formulation.
28. The composition of claim 27, wherein the pharmaceutical formulation is a lipid formulation.
29. The composition of claim 27, wherein the pharmaceutical formulation is a LNP formulation, a LNPO 1 formulation, a XTC-SNALP formulation, a SNALP formulation, or a LNP 11 formulation.
30. An isolated cell containing the dsRNA of claim 1.
31. A vector comprising a nucleotide sequence that encodes at least one strand of the dsRNA of claim 1.
32. A cell comprising the vector of claim 31.
33. The dsRNA of claim 1, wherein said dsRNA, upon contact with a cell expressing said SNCA, inhibits expression of said SNCA gene by at least 40% compared to a cell not so contacted.
34. The dsRNA of claim 1, wherein the dsRNA has an IC50 of 10 pM or less.
35. The dsRNA of claim 1, wherein administration of 0.1 nM of the dsRNA to a NCI-H460 cell or a BE(2)M17 cell results in greater than 50% inhibition of SNCA mRNA expression as measured by a real time PCR assay or administration of 10 nM of the dsRNA to a NCI-H460 cell or a BE(2)M17 cell results in greater than 66% inhibition of SNCA mRNA expression as measured by a real time PCR assay.
36. A method of inhibiting SNCA expression in a cell, the method comprising:
(a) introducing into the cell the dsRNA of claim 1 ; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a SNCA gene, thereby inhibiting expression of the SNCA gene in the cell.
37. The method of claim 36, wherein expression is inhibited by at least 20%, 40%, 60%, or at least 80%.
38. A method of treating a disorder mediated by SNCA expression comprising administering to a human in need of such treatment a therapeutically effective amount of the dsRNA of claim 1.
39. A method of treating, preventing or managing a neurodegenerative disorder comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a dsRNA of claim 1.
40. The method of claim 39, wherein the neurodegenerative disorder is a synucleinopathy.
41. The method of claim 39, wherein the neurodegenerative disorder is Parkinson's disease.
42. The method of claim 39, wherein the neurodegenerative disorder is Alzheimer's disease, multiple system atrophy, or Lewy body dementia.
43. A method of treating a human comprising: identifying a human diagnosed as having a neurodegenerative disorder or at risk for developing the neurodegenerative disorder and administering the dsRNA of claim 1.
44. The method of claim 43, further comprising administering an additional composition.
45. The method of claim 43, further comprising administering a second dsRNA.
46. A method of inhibiting SNCA expression in the brain, the method comprising administering an effective amount of the dsRNA of claim 1 into the brain of a subject to obtain suppression of expression of SNCA mRNA, thereby inhibiting SNCA expression in the brain.
47. The method of claim 46, wherein the administration is by infusion.
48. The method of claim 46, wherein the dsRNA comprises a sense strand of SEQ ID NO: 709 or 679 and/or an antisense strand of SEQ ID NO: 710 or 680.
49. The method of claim 46, wherein the dsRNA is AD-21822.
50. The method of claim 46, wherein the dsRNA is AD-21810.
51. The method of claim 46, wherein the administration of the dsRNA results in greater than 40%, 50%, 60%, 70%, 80%, or 90% inhibition of SNCA mRNA expression.
52. The method of claim 47, wherein the administration of the dsRNA results in greater than 65% inhibition of SNCA mRNA expression at the end of infusion, greater than 70% inhibition of SNCA mRNA expression one week after infusion, greater than 40% inhibition of SNCA mRNA expression two weeks after infusion, or greater than 50% inhibition of SNCA mRNA expression four weeks after infusion.
53. The method of claim 47, wherein the administration of the dsRNA results in greater than 50%, 60%, 70%, or 80% inhibition of SNCA mRNA expression after 7 or 28 days of infusion.
PCT/US2011/049427 2010-08-26 2011-08-26 Compositions and methods for inhibition of snca WO2012027713A2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US37743710P true 2010-08-26 2010-08-26
US61/377,437 2010-08-26

Publications (2)

Publication Number Publication Date
WO2012027713A2 true WO2012027713A2 (en) 2012-03-01
WO2012027713A3 WO2012027713A3 (en) 2014-05-30

Family

ID=45724098

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/049427 WO2012027713A2 (en) 2010-08-26 2011-08-26 Compositions and methods for inhibition of snca

Country Status (1)

Country Link
WO (1) WO2012027713A2 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014064257A1 (en) 2012-10-26 2014-05-01 Nlife Therapeutics, S.L. Compositions and methods for the treatment of parkinson disease by the selective delivery of oligonucleotide molecules to specific neuron types
WO2019070891A1 (en) * 2017-10-03 2019-04-11 Prevail Therapeutics, Inc. Gene therapies for lysosomal disorders
WO2019138057A1 (en) 2018-01-12 2019-07-18 Roche Innovation Center Copenhagen A/S Alpha-synuclein antisense oligonucleotides and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090099069A1 (en) * 2004-12-01 2009-04-16 Whitehead Institute For Biomedical Research Modulators of alpha-synuclein toxicity
US7579458B2 (en) * 2002-11-14 2009-08-25 Dharmacon, Inc. siRNA targeting synuclein, alpha (SNCA-1)
US20090269744A1 (en) * 2005-10-28 2009-10-29 Exonhit Therapeutics Sa Cancer detection method
US20100204306A1 (en) * 2007-12-14 2010-08-12 Alnylam Pharmaceuticals, Inc. Method of Treating Neurodegenerative Disease

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7579458B2 (en) * 2002-11-14 2009-08-25 Dharmacon, Inc. siRNA targeting synuclein, alpha (SNCA-1)
US20090099069A1 (en) * 2004-12-01 2009-04-16 Whitehead Institute For Biomedical Research Modulators of alpha-synuclein toxicity
US20090269744A1 (en) * 2005-10-28 2009-10-29 Exonhit Therapeutics Sa Cancer detection method
US20100204306A1 (en) * 2007-12-14 2010-08-12 Alnylam Pharmaceuticals, Inc. Method of Treating Neurodegenerative Disease

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SORTIRIOU ET AL.: 'A single nucleotide polymorphism in the 3'UTR of the SNCA gene encoding alpha-synuclein is a new potential susceptibiity locus for parkinson disease.' NEUROSCI LETT vol. 461, no. 2, 16 September 2009, pages 196 - 201 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014064257A1 (en) 2012-10-26 2014-05-01 Nlife Therapeutics, S.L. Compositions and methods for the treatment of parkinson disease by the selective delivery of oligonucleotide molecules to specific neuron types
US9084825B2 (en) 2012-10-26 2015-07-21 Nlife Therapeutics, S.L. Compositions and methods for the treatment of parkinson disease by the selective delivery of oligonucleotide molecules to specific neuron types
JP2015536927A (en) * 2012-10-26 2015-12-24 エヌライフ、セラピューティックス、ソシエダッド、リミターダNlife Therapeutics, S.L. Compositions and methods for the treatment of Parkinson's disease by selective delivery of oligonucleotides molecule into specific neuronal types
US10125363B2 (en) 2012-10-26 2018-11-13 Nlife Therapeutics, S.L. Compositions and methods for the treatment of parkinson disease by the selective delivery of oligonucleotide molecules to specific neuron types
WO2019070891A1 (en) * 2017-10-03 2019-04-11 Prevail Therapeutics, Inc. Gene therapies for lysosomal disorders
WO2019138057A1 (en) 2018-01-12 2019-07-18 Roche Innovation Center Copenhagen A/S Alpha-synuclein antisense oligonucleotides and uses thereof

Also Published As

Publication number Publication date
WO2012027713A3 (en) 2014-05-30

Similar Documents

Publication Publication Date Title
AU2010235872B2 (en) Compositions and methods for inhibiting expression of Eg5 gene
KR101624869B1 (en) Compositions and methods for inhibiting expression of transthyretin
US20100010066A1 (en) Optimized Methods For Delivery Of DSRNA Targeting The PCSK9 Gene
JP5530933B2 (en) Compositions for the vii factor gene expression inhibition and methods
JP5855462B2 (en) Method for inhibiting dsRNA compositions and expression targeted to GNAQ
AU2009221775B2 (en) Compositions and methods for inhibiting expression of Eg5 and VEGF genes
EP2723865B1 (en) METHODS FOR DETERMINING ACTIVITY OF RNAi IN A SUBJECT
US7875711B2 (en) Compositions and methods for inhibiting expression of XBP-1 gene
JP5860029B2 (en) siRNA therapy for transthyretin (TTR) associated ocular amyloidosis
JP5723378B2 (en) Lipid formulation compositions and methods for inhibiting transthyretin (ttr)
RU2631805C2 (en) Compositions and methods for apolipoprotein c-iii (apoc3) gene expression inhibition
JP6430559B2 (en) Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof
US10246708B2 (en) Dosages and methods for delivering lipid formulated nucleic acid molecules
US10208307B2 (en) Transthyretin (TTR) iRNA compositions and methods of use thereof for treating or preventing TTR-associated diseases
EP2970974B1 (en) Complement component c5 irna compositions and methods of use thereof
RU2691580C2 (en) I-rna compositions for serpin and methods of use thereof
JP5529142B2 (en) Lipid formulations compositions and methods for inhibiting the expression of serum amyloid a gene
US10231988B2 (en) Patatin-like phospholipase domain containing 3 (PNPLA3) iRNA compositions and methods of use thereof
US20180201929A1 (en) INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN, ACID LABILE SUBUNIT (IGFALS) AND INSULIN-LIKE GROWTH FACTOR 1 (IGF-1) iRNA COMPOSITIONS AND METHODS OF USE THEREOF
US8114984B2 (en) RNAi modulation of Aha and therapeutic uses thereof
US20130289094A1 (en) Compositions and Methods for Inhibition of PCSK9 Genes
JP6422854B2 (en) Compositions and methods for inhibiting the expression of Alas1 gene
JP6495250B2 (en) SERPINA1 iRNA compositions and methods of use thereof
US9187746B2 (en) Dual targeting siRNA agents
WO2016209862A1 (en) Glucokinase (gck) irna compositions and methods of use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11820754

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 11820754

Country of ref document: EP

Kind code of ref document: A2