Furin-knockdown and gm-csf-augmented (fang) cancer vaccine

Abstract

Compositions and methods for cancer treatment are discloses 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.

What is claimed is: 
     
         1 . An autologous cell vaccine comprising:
 a bishRNA furin /GMCSF expression vector plasmid, wherein the vector plasmid comprises   a first nucleic acid insert operably linked to a promoter, wherein the first insert encodes a Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) cDNA;   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 a region of a mRNA transcript encoding furin, thereby inhibiting furin expression via RNA interference; and   one or more optional vaccine adjuvants.   
     
     
         2 . The composition of  claim 1 , wherein the GM-CSF is human. 
     
     
         3 . The composition of  claim 1 , wherein the shRNA incorporates siRNA (cleavage dependent) and miRNA (cleavage-independent) motifs. 
     
     
         4 . The composition of  claim 1 , wherein the shRNA is both the cleavage dependent and cleavage independent inhibitor of furin expression. 
     
     
         5 . The composition of  claim 1 , wherein the shRNA is further defined as a bi-functional shRNA. 
     
     
         6 . The composition of  claim 1 , wherein a picornaviral 2A ribosomal skip peptide is intercalated between the first and the second nucleic acid inserts. 
     
     
         7 . The composition of  claim 1 , wherein the promoter is a CMV mammalian promoter. 
     
     
         8 . The composition of  claim 7 , wherein the CMV mammalian promoter contains a CMV IE 5′ UTR enhancer sequence and a CMV IE Intron A. 
     
     
         9 . The composition of  claim 1 , wherein the region targeted by the shRNA is the coding region of the furin mRNA transcript. 
     
     
         10 . An autologous furin-knockdown and Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) augmented (FANG) cancer vaccine composition comprising:
 a bishRNA furin /GMCSF expression vector plasmid, wherein the vector plasmid comprises   a first nucleic acid insert operably linked to a promoter, wherein the first insert encodes the GM-CSF cDNA;   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 a region of a mRNA transcript encoding furin, thereby inhibiting furin expression via RNA interference; and   one or more optional vaccine adjuvants.   
     
     
         11 . The composition of  claim 10 , wherein the composition is used to prevent, treat, and/or ameliorate the symptoms of a cancer, wherein the cancer is selected from the group consisting of melanoma, non-small-cell lung cancer, gall bladder cancer, colorectal cancer, breast cancer, ovarian, liver cancer, liver cancer metastases, and Ewing's sarcoma. 
     
     
         12 . The composition of  claim 10 , wherein the region targeted by the shRNA is the coding region of the furin mRNA transcript. 
     
     
         13 . An autologous cell vaccine composition for cancer treatment by inhibition of furin expression via RNA interference comprising:
 a bishRNA furin /GMCSF expression vector plasmid, wherein the vector plasmid comprises   a first nucleic acid insert operably linked to a promoter, wherein the first insert encodes a Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) cDNA;   a second nucleic acid insert operably linked to the promoter, wherein the second insert encodes one or more bi-functional short hairpin RNAs (shRNA furin ) providing a single targeted site for both a cleavage and a sequestering mechanism of RNA interference, wherein the bi-functional shRNA furin  comprises a first stem-loop structure comprising a complete complementary guide strand and a passenger strand and a second stem-loop structure comprising one or more basepair mismatches of the passenger strand capable of hybridizing to a region of a mRNA transcript encoding furin, thereby inhibiting furin expression via RNA interference; and   one or more optional vaccine adjuvants.   
     
     
         14 . The composition of  claim 13 , wherein the second stem-loop structure comprises three base pair mismatches. 
     
     
         15 . The composition of  claim 13 , wherein the basepair mismatches are at positions 9 to 11 of the passenger strand. 
     
     
         16 . The composition of  claim 13 , wherein the region targeted by the shRNA is the coding region of the furin mRNA transcript. 
     
     
         17 . An autologous cell vaccine composition for cancer treatment by inhibition of furin expression via RNA interference comprising:
 a bishRNA furin /GMCSF expression vector plasmid, wherein the vector plasmid comprises   a first nucleic acid insert operably linked to a promoter, wherein the first insert encodes a Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) cDNA;   a second nucleic acid insert operably linked to the promoter, wherein the second insert encodes one or more bi-functional short hairpin RNAs (shRNA furin ) providing a single targeted site for both a cleavage and a sequestering mechanism of RNA interference, wherein the bi-functional shRNA furin  comprises a first stem-loop structure comprising a complete complementary guide strand and a passenger strand and a second stem-loop structure comprising one or more basepair mismatches of the guide strand capable of hybridizing to a region of a mRNA transcript encoding furin, thereby inhibiting furin expression via RNA interference; and   one or more optional vaccine adjuvants.   
     
     
         18 . The composition of  claim 17 , wherein the second stem-loop structure comprises three base pair mismatches. 
     
     
         19 . The composition of  claim 17 , wherein the basepair mismatches are at positions 9 to 11 of the guide strand. 
     
     
         20 . The composition of  claim 17 , wherein the region targeted by the shRNA is the coding region of the furin mRNA transcript.