Recombinant suicide gene delivery vectors manufactured in the presence of specific small RNA is therapeutic in a syngeneic model of T-cell lymphoma

Background & Aim The availability of adequate quantity of recombinant Adeno-associated virus (AAV) vectors with a high-potency during suicide gene therapy has been a major impediment in its clinical translation. Several studies have highlighted the rate-limiting role of viral capsid post-translational modifications (PTMs) such as phosphorylation for a successful gene transfer. We hypothesized that inclusion of microRNA (miR-431, miR-636) during AAV vector packaging that not only binds to AAV inverted terminal repeats but is also known to downregulate cellular kinases can be a useful strategy. Methods, Results & Conclusion miR-431 or miR-636 plasmids were incorporated in an equimolar ratio to the conventional triple transfection protocol that utilizes AAV- transgene, pHelper and AAV-rep/cap plasmids in a producer cell line. A global transcriptome profiling demonstrated a significant down regulation(2.1-3.1 fold) of mitogen activated protein kinase (MAPK) signalling pathways in the presence of both microRNAs. We then employed this quadruple transfection protocol to generate single-stranded(ss) or self-complementary(sc) AAV2-EGFP vectors. Our data demonstrated that the transduction efficiency of microRNA based vectors is enhanced by 1.5-8 fold in multiple cell lines of hepatic/retinal origin. Subsequently, we confirmed the efficacy of this novel packaging protocol with an alternate AAV serotype. A scAAV6 vector encoding a inducible caspase 9 suicide (iCasp9) gene was packaged in the presence of microRNAs and evaluated in a syngeneic murine lymphoma . This model was generated by s.c administration of 2 × 106 EL4 cells in C57B6/J mice. All the animals (n=5) in the control group succumbed by day18 after a tumor challenge. In contrast, the survival rate was higher (∼60-80%) in vector treated mice (n= 5 animals/group; total=15). Furthermore, mice that received suicide gene therapy with AAV6-iCasp9 packaged in the presence of miR-636 had a considerable reduction in tumor volume(∼2.2-fold, p *NK, SC, HS contributed equally to this study.