Furin-knockdown and GM-CSF-augmented (FANG) cancer vaccine
Compositions and methods for cancer treatment are disclosed herein. More specifically, the present invention describes an autologous cancer vaccine genetically modified for Furin knockdown and GM-CSF expression. The vaccine described herein attenuates the immunosuppressive activity of TGF-β through the use of bi-functional shRNAs to knock down the expression of furin in cancer cells, and to augment tumor antigen expression, presentation, and processing through expression of the GM-CSF transgene.
1. A method of manufacturing a bi-shRNA furin /GMCSF cancer vaccine, comprising:
(a) forming a suspension of tumor cells;
(b) transfecting the tumor cells with a bi-shRNA furin /GMCSF expression vector plasmid comprising
(i) a first insert comprising a nucleic acid sequence encoding a human Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) cDNA; and
(ii) a second insert comprising a nucleic acid sequence encoding a bi-functional short hairpin RNA (bi-shRNA) capable of hybridizing to a furin mRNA transcript;
(c) harvesting the transfected tumor cells; and
(d) freezing the transfected tumor cells.
2. The method of claim 1 , further comprising harvesting a tumor comprising tumor cells from an individual and placing the tumor in an antibiotic solution in a sterile container prior to the formation of the tumor cell suspension.
3. The method of claim 1 , wherein the tumor cell suspension is formed by enzymatic dissection, mechanical disaggregation, or any combination thereof.
4. The method of claim 1 , wherein the tumor cells are transfected by electroporation with the expression vector.
5. The method of claim 1 , further comprising incubating the transfected tumor cells overnight prior to harvesting.
6. The method of claim 1 , further comprising rendering the transfected tumor cells proliferation-incompetent prior to freezing.
7. The method of claim 6 , wherein the transfected tumor cells are rendered proliferation-incompetent by irradiation.
8. The method of claim 6 , wherein the transfected tumor cells are rendered proliferation-incompetent by X-ray irradiation.
9. The method of claim 1 , wherein the transfected tumor cells are enumerated and aliquoted prior to freezing.
10. The method of claim 1 , wherein the tumor cells are derived from a melanoma, a non-small-cell lung cancer, a gall bladder cancer, a colorectal cancer, a breast cancer, a ovarian cancer, a liver cancer, or a Ewing's sarcoma.
11. The method of claim 1 , further comprising incubating the transfected tumor cells with γIFN after transfection.
12. The method of claim 11 , wherein the transfected tumor cells are incubated with about 100 U/ml of γIFN for 48 hours or about 500 U/ml of γIFN for 24 hours.
13. The method of claim 1 , wherein the first insert is operably linked to a promoter.
14. The method of claim 13 , wherein the second insert is operably linked to the promoter.
15. The method of claim 13 , wherein the promoter is a CMV mammalian promoter and the expression vector further comprises a CMV IE 5′ UTR enhancer sequence and a CMV IE Intron A sequence.
16. The method of claim 1 , wherein the expression vector further comprises a picornaviral 2A ribosomal skip peptide sequence between the first and the second nucleic acid inserts.
17. The method of claim 1 , wherein the bi-shRNA is capable of hybridizing within the 3′ UTR region of the furin mRNA transcript.
18. The method of claim 1 , wherein second insert comprises:
(a) a first stem loop structure comprising
(i) a first guide sequence capable of hybridizing to a furin mRNA transcript; and
(ii) a first passenger sequence fully complementary to the first guide strand; and
(b) a second stem loop structure comprising
(i) a second guide sequence capable of hybridizing to a furin mRNA transcript; and
(ii) a second passenger sequence partially complementary to the second guide strand.
19. The method of claim 18 , wherein the second passenger sequence has a three basepair mismatch with the second guide sequence at positions 9 to 11 of the second passenger strand.
20. The method of claim 19 , wherein the second insert comprises a nucleic acid sequence encoding a bi-functional short hairpin RNA (bi-shRNA) according to SEQ ID NO: 2.