PROJECT SUMMARY/ABSTRACT
Nucleic acids (NA) have a broad and expanding therapeutic potential, but a major limiting factor remains the
difficulty to deliver these molecules. We have developed a highly scalable platform technology with the
capacity for delivering NAs for use in multiple disease systems. Our goal is to adapt this technology to deliver
small interfering RNA (siRNA) precursor form, short-hairpin (shRNA) as an NA–based therapeutic. In proof-of-
concept studies, we have established that a VLV (RNA replicon-based vector) carrying RNA encoding all three
HBV major antigens (MHBs, HBcAg and polymerase) in a single ORF (VLV-3xT2A) drives a broad multi-
specific immune response that resulted in substantial clearance of HBV in the liver. In order to achieve long
term viral suppression or complete elimination of the virus in the liver, we have targeted for disruption the
checkpoint inhibitor, programmed cell death receptor ligand 1 (PD-L1) by shRNA technology. The hypothesis is
that disruption of the PD-1/PD-L1 pathway will reinvigorate the otherwise exhausted T cell function. In
preliminary studies, we have engineered CARG-101 to incorporate one or multiple copies of shRNA against
PD-L1, and we have showed that these vectors specifically downregulate stably transfected PD-L1 in a BHK21
cell line. In this study, we will examine whether the in vivo blockade of the PD-1/PD-L1 pathway will enhance
virus-specific T cell immunity that will lead to the complete resolution of chronic infection in mice. To this end,
we will carry out the following specific aims: (1) Optimize VLV-shRNA vectors for inhibition of PD-L1; (2)
Investigate the kinetics of PD-L1/PD-L2 and PD-1 expression in naïve and chronic HBV mice; and (3)Evaluate
clearance using the AAV-HBV mouse model of HBV persistence. At the completion of Phase I, we will have:
(a) established that VLV platform can deliver NAs to cells and tissues in vivo; (b) generated novel therapeutic
that enhances the efficacy of therapeutic vaccine. A robust CD8+ T cell response via PD-1/PD-L1 pathway
blockade will accrue, thereby driving a drastic or complete clearance of HBV in the liver. The demonstrated
capacity to express NAs has broad utility for treating multiple diseases. Another compelling value proposition
of this technology lies in its potential application to chronic and intractable diseases such as HIV, HPV, TB,
malaria, and cancer which are refractory to current vaccine technologies but which may now be amenable to
VLV technology.
