Furin-knockdown bi-functional RNA
Compositions and methods to attenuate the immunosuppressive activity of TGF-β through the use of bi-functional shRNAs is 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 Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) cDNA sequence; and
a second nucleic acid insert operably linked to the promoter, wherein the second insert encodes a bi-functional short hairpin RNA (bi-shRNA), wherein the bi-shRNA comprises:
(a) a first stem loop structure comprising
(i) a first guide sequence capable of hybridizing to a region of a mRNA transcript corresponding to base sequences 300-318, 731-740, 1967-1991, 2425-2444, 2827-2851, or 2834-2852 of SEQ ID NO:2; and
(ii) a first passenger sequence fully complementary to the first guide sequence; and
(b) a second stem loop structure comprising
(i) a second guide sequence capable of hybridizing to a region of an mRNA transcript corresponding to base sequences 300-318, 731-740, 1967-1991, 2425-2444, 2827-2851,or 2834-2852 of SEQ ID NO:2; and
(ii) a second passenger sequence partially complementary to the second guide sequence.
2. The expression vector of claim 1 , further comprising a nucleic acid sequence encoding a picornaviral 2A ribosomal skip peptide between the first and the second nucleic acid inserts.
3. The expression vector of claim 1 , wherein the promoter is a cytomegalovirus (CMV) mammalian promoter.
4. The expression vector of claim 3 , further comprising a CMV intermediate-early (IE) 5′ UTR enhancer sequence and a CMV IE Intron A sequence.
5. The expression vector of claim 1 , wherein the bi-shRNA targets a sequence within the 3′ UTR region of a furin mRNA transcript.
6. A method of enhancing an immune response in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of cells transfected with the expression vector of claim 1 .
7. The method of claim 6 , wherein the GM-CSF is human.
8. The method of claim 6 , wherein the expression vector further comprises a nucleic acid encoding a picornaviral 2A ribosomal skip peptide between the first and the second nucleic acid inserts.
9. The method of claim 6 , where the promoter is a CMV mammalian promoter, and wherein the expression vector further comprises a CMV enhancer sequence and a CMV intron sequence.
10. The method of claim 6 , wherein the region targeted by the bi-shRNA is within the 3′ UTR region sequence of a furin mRNA transcript.
11. The method of claim 6 , wherein the region targeted by the bi-shRNA is within the coding region of a furin mRNA transcript.
12. The method of claim 6 , wherein the cells are autologous tumor cells, xenograft expanded autologous tumor cells, or allogeneic tumor cells.
13. The method of claim 6 , wherein the individual has a cancer.
14. The method of claim 6 , wherein the therapeutically effective amount of cells is about 1×10 7 cells to about 2.5×10 7 cells.
15. The method of claim 6 , wherein the composition further comprises a therapeutically effective dose of γIFN (gamma interferon).
16. The method of claim 15 , wherein the therapeutically effective dose of γIFN is about 50 μg/m 2 or about 100 μg/m 2 .
17. An expression vector comprising a nucleic acid insert encoding a bi-functional small hairpin RNA (bi-shRNA) operably linked to a promoter, wherein the bi-shRNA comprises:
(a) a first stem loop structure comprising
(i) a first guide sequence capable of hybridizing to a region of a mRNA transcript corresponding to base sequences 300-318, 731-740, 1967-1991, 2425-2444, 2827-2851, or 2834-2852 of SEQ ID NO:2; and
(ii) a first passenger sequence fully complementary to the first guide sequence; and
(b) a second stem loop structure comprising
(i) a second guide sequence capable of hybridizing to a region of an mRNA transcript corresponding to base sequences 300-318, 731-740, 1967-1991, 2425-2444, 2827-2851, or 2834-2852 of SEQ ID NO:2; and
(ii) a second passenger sequence partially complementary to the second guide sequence.
18. The expression vector claim 1 , wherein the bi-shRNA targets a sequence within the coding region of a furin mRNA transcript.