Improved Therapeutic Efficacy of Cancer Gene Therapy using Retroviral Replicating Vectors Designed for Multiple Transgene Transduction

Retroviral Replicating Vectors (RRVs) carrying the human codon optimized yeast cytosine deaminase (yCD2) suicide gene are currently in Phase I/II clinical trials for treatment of high-grade glioma. These vectors are highly tumor specific due to their intrinsic inability to infect non-dividing cells, and their restriction by the innate immune system, which is often suppressed in the tumor microenvironment. Currently, this system relies on delivery of a single transgene, yCD2, to the tumor mass. However, the current ‘gold standard’ in medical care is to treat cancer with a multi-drug regimen to combat the heterogeneous nature of tumors. As RRV therapy progresses, it must also address the issue of heterogeneity and seek to deliver more therapeutics to the cancer. These studies explore the possibility of using RRVs armed with multiple transgenes to improve the therapeutic efficacy of RRV-mediated cancer gene therapy.We examined the use of independent intrinsic promoter to drive RRV transgene expression in multiple cell lines. As transgene space in limited in RRVs, a smaller promoter was studied. Super Core Promoter (SCP1) was found to have varying degrees of protein expression across different cell lines. As certain immune factors are confined to a therapeutic window, low expression of transgene by SCP1 was used in U87 cells to express cytokine mGM-CSF along with yCD2. Protein levels of yCD2 dropped, but the transgene still remained functional, while levels of mGM-CSF remained within the therapeutic range.The MLV 4070A envelope, suffers from “superinfection resistance”, a phenomenon in which a virus is unable to infect cells that have already been infected by a virus expressing the same envelope protein. By switching out the envelope of RRV for different viral envelopes, the efficiency of cotransduction was studied across multiple cell lines. Results showed that cotransduction efficiencies varied between cell lines, potentially as a function of receptor availability or intrinsic viral immunity. Based on results from these studies, prime candidates for cotransduction of U87 cells were established. Simultaneous cotransduction using two RRVs, expressing the noninterfering 4070A (AC3) and gibbon ape leukemia virus (GALV) envelopes, resulted in increased transgene expression from AC3-RRV, whereas transgene expression from GALV-RRV decreased slightly when compared to transduction with a single RRV.In order to increase transgene expression of larger transgenes without compromising transgene stability, P2A was attached to the end of 4070A and GALV envelope proteins. P2A proved to increase protein expression in most cell lines compared to SCP1, however, the vector copy number per cell decreased. GALV pseudotyped RRV with P2A was then used to cotransduce cells in combination with AC3-yCD2. Expression of the yCD2 transgene increased, while expression of the GALV-RRV transgene decreased slightly. Functional assays revealed that cotransduction with two RRVs resulted in increased cell death, except at the highest concentrations of ganciclovir. Thus use of pseudotyped RRVs to deliver multiple transgenes has the potential to increase the therapeutic efficacy of RRV-mediated gene therapy.