Furin-knockdown bi-functional RNA
Compositions and methods to attenuate the immunosuppressive activity of TGF-β through the use of bi-functional shRNAs was substituted therefor described herein. The bi-functional shRNAs of the present invention knocks down the expression of furin in cancer cells to augment tumor antigen expression, presentation, and processing through expression of the GM-CSF transgene.
1. An expression vector comprising:
a first nucleic acid insert operably linked to a promoter, wherein the first insert encodes a human Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) cDNA; and
a second nucleic acid insert operably linked to the promoter, wherein the second insert encodes one or more bifunctional short hairpin RNAs (shRNA) capable of hybridizing to one of more regions of a mRNA transcript encoding furin, wherein at least one of the regions is selected from base sequences 300-318, 731-740, 1967-1991, 2425-2444, 2827-2851 or 2834-2852 of SEQ ID NO:2, thereby inhibiting furin expression via RNA interference, wherein each bifunctional short hairpin RNA comprises a first stem-loop structure that comprises an siRNA component and a second stem-loop structure that comprises a miRNA component and wherein the shRNA incorporates siRNA (cleavage dependent) and miRNA (cleavage-independent) motifs.
2. The expression vector of claim 1 , wherein a nucleotide sequence encoding a picornaviral 2A ribosomal skip peptide sequence is intercalated between the first and the second nucleic acid inserts.
3. The expression vector of claim 1 , wherein the promoter is a CMV mammalian promoter.
4. The expression vector of claim 3 , wherein the CMV mammalian promoter contains a CMV IE 5′ UTR enhancer sequence and a CMV IE Intron A.
5. An expression vector comprising a bifunctional small hairpin construct specific for knockdown of furin, wherein the expression vector plasmid further comprises: a nucleic acid insert operably linked to a promoter, wherein the insert encodes one or more short hairpin RNAs (shRNA) capable of hybridizing to a region of a mRNA transcript encoding furin selected from at least one of base sequences 300-318, 731-740, 1967-1991, 2425-2444, 2827-2851 or 2834-2852 of SEQ ID NO:2, thereby inhibiting furin expression via RNA interference, wherein each bifunctional short hairpin RNA comprises a first stem-loop structure that comprises an siRNA component and a second stem-loop structure that comprises a miRNA component.
6. The expression vector of claim 5 , wherein the siRNA component functions in a cleavage-dependent manner and the miRNA component functions in a cleavage-independent manner.
7. A composition for treating cancer comprising a therapeutically effective amount of cells transfected with an expression vector comprising a first nucleic acid insert operably linked to a promoter, wherein the first insert encodes GM-C SF and a second nucleic acid insert operably linked to the promoter, wherein the second insert encodes one or more short hairpin RNAs (shRNA) capable of hybridizing to one or more regions of a mRNA transcript encoding furin, wherein at least one of the regions is selected from base sequences 300-318, 731-740, 1967-1991, 2425-2444, 2827-2851 or 2834-2852 of SEQ ID NO:2, thereby inhibiting furin expression via RNA interference, wherein each bifunctional short hairpin RNA comprises a first stem-loop structure that comprises an siRNA component and a second stem-loop structure that comprises a miRNA component.
8. The composition of claim 7 , wherein the GM-CSF is human.
9. The composition of claim 7 , wherein the siRNA component functions in a cleavage-dependent manner and the miRNA component functions in a cleavage-independent manner.
10. The composition of claim 7 , wherein a nucleotide sequence encoding a picornaviral 2A ribosomal skip peptide sequence is intercalated between the first and the second nucleic acid inserts.
11. The composition of claim 7 , where the promoter is a CMV mammalian promoter, enhancer and intron.
12. The composition of claim 7 , wherein the cells are xenograft expanded tumor cells.
13. The composition of claim 7 , wherein the composition comprises 1×10 7 cells to 2.5×10 7 cells.
14. The composition of claim 7 , wherein the composition further comprises a therapeutically effective dose of γIFN (gamma interferon).
15. The composition of claim 14 , wherein the therapeutically effective dose of γIFN is 50 or 100 μg/m 2 .