Allele-specific silencing of disease genes

A mammalian cell comprising an expression cassette encoding an isolated first strand of RNA of 15 to 30 nucleotides in length, and an isolated second strand of RNA of 15 to 30 nucleotides in length, wherein the first strand comprises a sequence that is complementary to at least 15 contiguous nucleotides of a targeted gene of interest, wherein at least 12 nucleotides of the first and second strands are complementary to each other and form a small interfering RNA (siRNA) duplex under physiological conditions, and wherein the siRNA silences only one allele of the targeted gene in the cell.

An isolated RNA duplex comprising a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides complementary to mutant TorsinA transcript encoded by SEQ ID NO:55, and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand.

An RNA duplex comprising a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides complementary to mutant Ataxin-3 transcript encoded by SEQ ID NO:8, and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand.

An RNA duplex comprising a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides complementary to mutant Tau transcript encoded by SEQ ID NO:39 (siA9/C12), and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand.

A method of performing allele-specific gene silencing in a mammal comprising administering to the mammal an isolated first strand of RNA of 15 to 30 nucleotides in length, and an isolated second strand of RNA of 15 to 30 nucleotides in length, wherein the first strand comprises a sequence that is complementary to at least 15 contiguous nucleotides of a targeted gene of interest, wherein at least 12 nucleotides of the first and second strands are complementary to each other and form a small interfering RNA (siRNA) duplex under physiological conditions, and wherein the siRNA silences only one allele of the targeted gene in the mammal.

A method of substantially silencing a targeted allele while allowing substantially continued expression of a wild-type allele comprising contacting a cell with an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding a small interfering RNA molecule (siRNA) targeted against the targeted allele, wherein expression from the targeted allele is substantially silenced but wherein expression of the wild-type allele is not substantially silenced.

A method of treating dominantly inherited disease in an allele-specific manner comprising administering to a patient in need thereof an expression cassette, wherein the expression cassette comprises a nucleic acid sequence encoding a small interfering RNA molecule (siRNA) targeted against a targeted allele, wherein expression from the targeted allele is substantially silenced but wherein expression of the wild-type allele is not substantially silenced.

A method of performing allele-specific gene silencing comprising administering an expression cassette comprising a pol II promoter operably-linked to at least one strand of a nucleic acid encoding a small interfering RNA molecule (siRNA) targeted against a gene of interest, wherein the siRNA silences only one allele of a gene.

A method of performing allele-specific gene silencing in a mammal comprising administering to the mammal a vector comprising an expression cassette, wherein the expression cassette comprises a nucleic acid encoding at least one strand a small interfering RNA molecule (siRNA) targeted against a gene of interest, wherein the siRNA silences only one allele of a gene.

A method of screening of allele-specific siRNA duplexes comprising

• (a) contacting a cell containing a predetermined mutant allele with an siRNA with a known sequence,
• (b) contacting a cell containing a wild-type allele with an siRNA with a known sequence, and
• (c) determining if the mutant allele is substantially silenced while the wild-type allele retains substantially normal activity.

A method of screening of allele-specific siRNA duplexes comprising

• (a) contacting a cell containing a predetermined mutant allele and a wild-type allele with an siRNA with a known sequence, and
• (b) determining if the mutant allele is substantially silenced while the wild-type allele retains substantially normal activity.

A method for determining the function of an allele comprising:

• (a) contacting a cell containing a predetermined allele with an siRNA with a known sequence, and
• (b) determining if the function of the allele is substantially modified.

A method for determining the function of an allele comprising:

• (a) contacting a cell containing a predetermined mutant allele and a wild-type allele with an siRNA with a known sequence, and
• (b) determining if the function of the allele is substantially modified while the wild-type allele retains substantially normal function.