Furin-knockdown and GM-CSF-augmented (FANG) cancer vaccine
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.
1. An autologous cell vaccine comprising:
a bishRNAfurin/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 wherein the shRNA incorporates cleavage dependent siRNA and cleavage independent miRNA motifs, wherein the shRNA comprises SEQ ID NO:2; 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 a nucleotide sequence encoding a picornaviral 2A ribosomal skip peptide sequence is intercalated between the first and the second nucleic acid inserts.
4. The composition of claim 1 , wherein the promoter is a CMV mammalian promoter.
5. The composition of claim 4 , wherein the CMV mammalian promoter contains a CMV IE 5′ UTR enhancer sequence and a CMV IE Intron A.
6. An autologous furin-knockdown and Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) augmented (FANG) cancer vaccine composition comprising:
an autologous cancer cell transfected with a vector comprising:
a first nucleic acid insert operably linked to a promoter, wherein the first insert encodes the GM-CSF cDNA; 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 a region of a mRNA transcript encoding furin, thereby inhibiting furin expression via RNA interference wherein the shRNA incorporates cleavage dependent siRNA and cleavage independent miRNA motifs of SEQ ID NO: 2.
7. An autologous cell vaccine composition for cancer treatment by inhibition of furin expression via RNA interference comprising:
a bishRNAfurin/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 (shRNAfurin) providing a single targeted site for both a cleavage and a sequestering mechanism of RNA interference, wherein the bi-functional shRNAfurin 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, wherein the shRNA comprises SEQ ID NO: 2, thereby inhibiting furin expression via RNA interference; and
one or more vaccine adjuvants.