Lentiviral vectors (LVs) hold great potential as gene delivery vehicles. However, the manufacturing and purification of these vectors still present major challenges, mainly because of the low stability of the virus, essentially due to the fragility of the membrane envelope. The main goal of this work was the establishment of a fast, scalable, and robust downstream protocol for LVs, combining microfiltration, anion-exchange, and ultrafiltration membrane technologies toward maximization of infectious LVs recovery. CIM(®) (Convective Interaction Media) monolithic columns with diethylaminoethanol (DEAE) anion exchangers were used for the purification of clarified LV supernatants, allowing infectious vector recoveries of 80%, which is 10% higher than the values currently reported in the literature. These recoveries, combined with the results obtained after optimization of the remaining downstream purification steps, resulted in overall infectious LV yields of 36%. Moreover, the inclusion of a Benzonase step allowed a removal of approximately 99% of DNA impurities. The entire downstream processing strategy herein described was conceived based on disposable and easily scalable technologies. Overall, CIM DEAE columns have shown to be a good alternative for the purification of LVs, since they allow faster processing of the viral bulks and enhanced preservation of virus biological activity, consequently, increasing infectious vector recoveries.
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 10 infectious particles·ml). 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.
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