Furin-knockdown bi-functional RNA
Inventors
Nemunaitis, John J. • Senzer, Neil • Maples, Phillip B. • Rao, Donald
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9695422-B2
Publication Date
2017-07-04
Expiration Date
Abstract
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.
Core Innovation
The invention relates to a cancer vaccine platform (FANG) that combines a GM-CSF transgene with a furin-targeted bi-functional shRNA intended to attenuate TGF-beta immunosuppression. The bi-functional shRNA is described as a furin bi-shRNA construct that links furin knockdown to reduced TGF-beta1/beta2 activity and associated immunosuppressive effects. The platform is framed as enhancing GM-CSF-mediated antigen presentation through the furin-targeting component.
The bi-functional shRNA is described as including two stem-loop modules: an siRNA-like cleavage-dependent component and a miRNA-like cleavage-independent component. The construct uses a miR-30a backbone, and it targets specific regions of furin mRNA. The specification discusses targeting of listed furin mRNA base regions, including possible 3'UTR targeting, and describes the bi-shRNA as having defined guide and passenger sequence complementarity across the two stem-loop structures.
The specification provides a rationale connecting furin knockdown to changes in TGF-beta1/beta2 signaling and to elevated GM-CSF expression as supportive observations. It also discusses clinical and manufacturing context including autologous modified tumor cell vaccines, with mention of a CMV promoter and an optional picornaviral 2A ribosomal skip peptide in the expression context. Reported patient/cell-line observations are referenced, including GM-CSF elevation and TGF-beta knockdown, to support the approach.
Claims Coverage
The partial claim set includes two independent claims directed to expression vectors encoding GM-CSF cDNA together with a bi-functional shRNA (bi-shRNA) and the bi-shRNA alone, each with two stem-loop structures whose guide sequences hybridize to specified furin mRNA base regions and whose passenger sequences have specified complementarity patterns. The inventive features are primarily defined by the vector architecture, the presence of GM-CSF for the first independent claim, and the detailed bi-shRNA guide/passenger design targeting regions relative to SEQ ID NO:2.
Expression vector encoding GM-CSF and bi-functional shRNA with two stem loops
An expression vector comprising a first nucleic acid insert encoding a Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) cDNA sequence operably linked to a promoter, and a second nucleic acid insert encoding a bi-functional short hairpin RNA (bi-shRNA) operably linked to the promoter, where the bi-shRNA comprises a first stem loop with a first guide sequence hybridizing to specified base ranges of an mRNA transcript corresponding to SEQ ID NO:2 and a first passenger sequence fully complementary to the first guide sequence, and a second stem loop with a second guide sequence hybridizing to specified base ranges of an mRNA transcript corresponding to SEQ ID NO:2 and a second passenger sequence partially complementary to the second guide sequence.
Expression vector encoding bi-functional small hairpin RNA with two stem loops
An expression vector comprising a nucleic acid insert encoding a bi-functional small hairpin RNA (bi-shRNA) operably linked to a promoter, where the bi-shRNA comprises a first stem loop with a first guide sequence capable of hybridizing to a region of an mRNA transcript corresponding to specified base ranges of SEQ ID NO:2 and a first passenger sequence fully complementary to the first guide sequence, and a second stem loop with a second guide sequence capable of hybridizing to a region of an mRNA transcript corresponding to specified base ranges of SEQ ID NO:2 and a second passenger sequence partially complementary to the second guide sequence.
Across the two independent claims, the coverage centers on expression vectors that encode a bi-shRNA having two stem-loop structures with specified guide/passenger complementarity and guide targeting to defined furin mRNA base regions relative to SEQ ID NO:2. The first independent claim additionally requires an operably linked GM-CSF cDNA insert encoding Granulocyte Macrophage Colony Stimulating Factor (GM-CSF).
Stated Advantages
Enhancement of GM-CSF-mediated antigen presentation via furin knockdown driven by the bi-shRNA component.
Attenuation of TGF-beta immunosuppression through reduced TGF-beta1/beta2 activity associated with furin targeting.
Documented Applications
FANG cancer vaccine platform context combining a GM-CSF transgene with a furin-targeted bi-functional shRNA.
Autologous modified tumor cell vaccines described in the clinical/manufacturing context.
Expression context for use with autologous or xenograft-expanded autologous/allogeneic tumor cells as described.
Interested in licensing this patent?