Methods for producing interfering RNA molecules in mammalian cells and therapeutic uses for such molecules

摘要

Methods for producing interfering RNA molecules in mammalian cells are provided. Therapeutic uses for the expressed molecules, including inhibiting expression of HIV, are also provided.

主权项

1. A method for testing the expression and function of a small interfering RNA (siRNA) molecule, comprising:
co-introducing into a mammalian cell (a) one or more vectors comprising (i) a first cassette comprising an RNA pol III promoter operatively linked to a first nucleic acid encoding a sense strand of a double stranded siRNA molecule and (ii) a second cassette comprising an RNA pol III promoter operatively linked to a second nucleic acid encoding an antisense strand of the double stranded siRNA molecule, wherein the first nucleic acid comprises a DNA sequence encoding the sense strand positioned at the first nucleotide of the transcript of the RNA pol III promoter and the second nucleic acid comprises a DNA sequence encoding the antisense strand positioned at the first nucleotide of the transcript of the RNA pol III promoter, wherein the first cassette and the second cassette are in a same vector or are in separate vectors and wherein the double stranded siRNA molecule is specific for a target sequence in a target gene, and (b) a vector comprising the target gene and a suitable second promoter; growing the mammalian cell under conditions suitable for expression of the nucleic acid sequences and the target gene; determining if the double stranded siRNA molecule is expressed and if expression of the target gene is reduced, whereby the expression and function of the siRNA molecule is tested.