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US 8,299,045 B2Granted
Adenoviral VA1 Pol III expression system for RNAi expression
AssigneeCity of Hope
InventorsJohn J. Rossi, Nan-Sook Lee
Life of this patent
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Abstract
An adenoviral VA1 Pol III expression system for RNAi expression is provided.
Claims12
A method for expressing an interfering RNA molecule in a mammalian cell, comprising:
- (a) introducing into a mammalian cell an expression cassette comprising an adenoviral VA1 gene and a nucleic acid encoding an interfering RNA (RNAi) precursor molecule, wherein the adenoviral VA1 gene comprises the adenoviral VA1 promoter and a coding sequence for the VA1 RNA, wherein the nucleic acid is inserted at a BstEII site within a non-essential stem region of the adenoviral VA1 coding sequence, wherein the nucleic acid encoding the RNAi precursor molecule encodes a hairpin siRNA (shRNA) or a precursor microRNA (precursor miRNA);
- (b) maintaining the mammalian cell produced in step (a) under conditions and for a time sufficient to express the VA1 RNA containing the RNAi precursor molecule and to cleave the RNAi precursor molecule out of the VA1 RNA;
- and
- (c) maintaining the mammalian cell produced in step (b) under conditions and for a time sufficient for Dicer to cleave the RNAi precursor molecule to produce an interfering RNA molecule, wherein the RNA interfering molecule is an siRNA or a miRNA.
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A method for producing a double stranded RNA precursor molecule in a mammalian cell, comprising:
- (a) introducing a vector into a mammalian cell, wherein the vector comprises an expression cassette comprising an adenoviral VA1 gene and a nucleic acid encoding an interfering RNA (RNAi) precursor molecule, wherein the adenoviral VA1 gene comprises the adenoviral VA1 promoter and a coding sequence for the VA1 RNA, wherein the nucleic acid is inserted at a BstEII site within a non-essential stem region of the adenoviral VA1 coding sequence, wherein the nucleic acid encoding the RNAi precursor molecule encodes a hairpin siRNA (shRNA) or a precursor microRNA (precursor miRNA);
- (b) maintaining the mammalian cell produced in step (a) under conditions and for a time sufficient to express the VA1 RNA containing the RNAi precursor molecule, wherein the RNAi precursor molecule is inactive in the VA1 RNA;
- and
- (c) maintaining the mammalian cell produced in step (b) under conditions and for a time sufficient to cleave the RNAi precursor molecule out of the VA1 RNA, wherein the cleaved RNAi precursor molecule is a double stranded RNA molecule and is a substrate for Dicer.
1 more element
Dependencies are inferred from the claim text.