Methods and compositions for inhibiting the function of polynucleotide sequences
A therapeutic composition for inhibiting the function of a target polynucleotide sequence in a mammalian cell includes an agent that provides to a mammalian cell an at least partially double-stranded RNA molecule comprising a polynucleotide sequence of at least about 200 nucleotides in length, said polynucleotide sequence being substantially homologous to a target polynucleotide sequence. This RNA molecule desirably does not produce a functional protein. The agents useful in the composition can be RNA molecules made by enzymatic synthetic methods or chemical synthetic methods in vitro; or made in recombinant cultures of microorganisms and isolated therefrom, or alternatively, can be capable of generating the desired RNA molecule in vivo after delivery to the mammalian cell. In methods of treatment of prophylaxis of virus infections, other pathogenic infections or certain cancers, these compositions are administered in amounts effective to reduce or inhibit the function of the target polynucleotide sequence, which can be of pathognic origin or produced in response to a tumor or other cancer, among other sources.
1 - 67 . (canceled)
68 . A method for producing a double stranded interfering RNA molecule in a mammalian cell, comprising:
a) inserting DNA sequences encoding a sense strand and an antisense strand of the RNA molecule into a vector comprising an RNA pol III promoter; and
b) introducing the vector into a mammalian cell so that the RNA molecule can be expressed.
69 . The method of claim 68 , wherein the RNA molecule is a double stranded RNA molecule of between 11 to 30 nucleotides.
70 . The method of claim 68 , wherein the mammalian cell is a human cell.
71 . The method of claim 68 , wherein the DNA sequences are inserted into separate vectors.
72 . The method of claim 68 , wherein the DNA sequences are inserted into the same vector.
73 . The method of claim 68 , wherein the vector is a plasmid vector.
74 . The method of claim 68 , wherein the vector is a viral vector.
75 . The method of claim 68 , wherein the vector is introduced into the mammalian cell in vitro.
76 . The method of claim 68 , wherein the vector is introduced into the mammalian cell in vivo.
77 . A method for inhibiting the expression of a target gene, comprising:
introducing one or more vectors into a mammalian cell, wherein the one or more vectors comprise a suitable promoter and DNA sequences encoding a sense strand and an antisense strand of a double stranded, RNA molecule of between 11 to 30 nucleotides, so that the RNA molecule of between 11 to 30 nucleotides can be expressed and initiate RNA interference of expression of a target gene in the mammalian cell, thereby inhibiting expression of the target gene.
78 . The method of claim 77 , wherein the mammalian cell is a human cell.
79 . The method of claim 77 , wherein the DNA sequences are in separate vectors.
80 . The method of claim 77 , wherein the DNA sequences are ln the same vector.
81 . The method of claim 80 , wherein the vector is a plasmid vector.
82 . The method of claim 80 , wherein the vector is a viral vector.
83 . The method of claim 81 , wherein the promoter is an RNA pol III promoter.
84 . The method of claim 77 , wherein the target gene is an HIV target gene.
85 . The method of claim 84 , wherein the HIV is HIV-1.
86 . The method of claim 78 , wherein the mammalian cell is an, HIV-infected human cell.