Evaluation of AAV-mediated delivery of shRNA to target basal-like breast cancer genetic vulnerabilities.

Adeno-associated viral vectors (AAV) for gene therapy applications are gaining momentum, with more therapies moving into later stages of clinical development and towards market approval, namely for cancer therapy. The development of cytotoxic vectors is often hampered by side effects arising when non-target cells are infected, and their production can be hindered by toxic effects of the transgene on the producing cell lines. In this study, we evaluated the potential of rAAV-mediated delivery of short hairpin RNAs (shRNA) to target basal-like breast cancer genetic vulnerabilities. Our results show that by optimizing the stoichiometry of the plasmids upon transfection and time of harvest, it is possible to increase the viral titers and quality. All rAAV-shRNA vectors obtained efficiently transduced the BLBC cell lines MDA-MB-468 and HCC1954. In MDA-MB-468, transduction with rAAV-shRNA vector targeting PSMA2 was associated with significant decrease in cell viability and apoptosis induction. Importantly, rAAV2-PSMA2 also slowed tumor growth in a BLBC mouse xenograft model, thus potentially representing a therapeutic strategy against this type of cancer.

Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration.

Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions. The main aim of this work was to identify the key parameters governing GaLV-pseudotyped gammaretroviral vector productivity in stable producer cells, using a retroviral vector expression cassette enabling positive (facilitating cell enrichment) and negative cell selection (allowing cell elimination). The retroviral vector contains a thymidine kinase suicide gene fused with a ouabain-resistant Na+,K+-ATPase gene, a potential safer and faster marker. The establishment of retroviral vector producer cells is traditionally performed by randomly integrating the retroviral vector expression cassette codifying the transgene. More recently, recombinase-mediated cassette exchange methodologies have been introduced to achieve targeted integration. Herein we compared random and targeted integration of the retroviral vector transgene construct. Two retroviral producer cell lines, 293 OuaS and 293 FlexOuaS, were generated by random and targeted integration, respectively, producing high titers (on the order of 107 infectious particles·ml-1). Results showed that the retroviral vector transgene cassette is the key retroviral vector component determining the viral titers notwithstanding, single-copy integration is sufficient to provide high titers. The expression levels of the three retroviral constructs (gag-pol, GaLV env, and retroviral vector transgene) were analyzed. Although gag-pol and GaLV env gene expression levels should surpass a minimal threshold, we found that relatively modest expression levels of these two expression cassettes are required. Their levels of expression should not be maximized. We concluded, to establish a high producer retroviral vector cell line only the expression level of the genomic retroviral RNA, that is, the retroviral vector transgene cassette, should be maximized, both through (1) the optimization of its design (i.e., genetic elements composition) and (2) the selection of high expressing chromosomal locus for its integration. The use of methodologies identifying and promoting integration into high-expression loci, as targeted integration or high-throughput screening are in this perspective highly valuable.