Enhancing the Safety and Efficiency of Retroviral Replicating Vectors for Cancer Gene Therapy
An improved Retroviral Replicating Vector (RRV), Toca 511, encoding an optimized yeast cytosine deaminase prodrug activator gene, is currently under investigation for recurrent high-grade glioma in Phase I/II clinical trials. RRVs exhibit inherent tumor selectivity, due to their intrinsic inability to enter post-mitotic cells, and unlike replication defective vectors, are capable of efficient gene transfer to tumors, as the virus continues to spread through the tumor mass after initial infection1. However, tumor transduction may still be limited by cellular anti-viral immune factors. Moreover, concerns remain about the use of RRVs for gene therapy given the possibility that viral integration might lead to off-target effects, or genotoxicity, in non-cancerous cells. Therefore, we investigated mechanisms to improve both the safety and efficiency of RRV-mediated gene therapy.
Enhancing the tumor specificity of RRVs
We examined whether the incorporation of miRNA target sequences (miRTs) into the RRV genome could restrict viral replication in a tissue-specific manner. Accordingly, we developed novel RRVs with one to four copies of the target sequence for human miR142-3p, a lymphohematopoietic-specific miRNA, inserted into the ψ packaging signal, the 3' LTR and/or the transgene cassette of the RRV. We showed that incorporation of the miR142-3p target sequence (miRT142) significantly reduced viral spread to hematopoietic tissues, without inhibiting intratumoral replication or cytotoxic effect. However, the genomic location of the miRT affected viral replication kinetics, transgene stability, and miRT sequence integrity, in some cases. Therefore careful placement of miRTs is necessary to preserve efficient viral replication and genomic stability in non-restricted tissues, while maximizing suppression of RRV replication in lymphohematopoietic cells. MicroRNA-based restriction can effectively suppress RRV replication in a tissue-specific manner, and this approach may add an additional safeguard to gene therapy applications.
Improving the efficiency of RRV-mediated gene transfer
To explore the effect of cellular factors on RRV replication, we monitored RRV spread in a panel of primary human glioblastoma (glioma) cell lines, and quantified the expression, in these same cells, of various cellular factors that may play a role in RRV replication. We confirmed the finding, by another group, that tetherin (BST2, CD317) is strongly overexpressed in glioma cells, and identified an additional antiviral factor, protein kinase R, whose expression may also be dysregulated. We demonstrated that tetherin expression is correlated with slower RRV spread and reduced viral titers in the supernatants of infected cells, and suggest a possible antitumor effect of tetherin knockdown in glioma. Our results can be extended to future studies of the effect of tetherin on RRV replication or glioma tumor progression, or may be used to inform either the development of novel RRVs with enhanced infectivity, or the stratification of patients in clinical trials.