Claims for Patent: 9,695,422

Summary for Patent: 9,695,422

Title:	Furin-knockdown bi-functional RNA
Abstract:	Compositions and methods to attenuate the immunosuppressive activity of TGF-.beta. 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.
Inventor(s):	Nemunaitis; John J. (Cedar Hill, TX), Senzer; Neil (Delray Beach, FL), Maples; Phillip B. (Pilot Point, TX), Rao; Donald (Dallas, TX)
Assignee:	GRADALIS, INC. (Dallas, TX)
Application Number:	14/844,912
Patent Claims:	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.times.10.sup.7 cells to about 2.5.times.10.sup.7 cells. 15. The method of claim 6, wherein the composition further comprises a therapeutically effective dose of .gamma.IFN (gamma interferon). 16. The method of claim 15, wherein the therapeutically effective dose of .gamma.IFN is about 50 .mu.g/m.sup.2 or about 100 .mu.g/m.sup.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.

Details for Patent 9,695,422

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2029-12-23
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2029-12-23
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2029-12-23