540. Vectofusin-1 Significantly Enhances Transduction of Ex-Vivo Activated T Cells With RD114-TR-Pseudotyped Lentiviral Vectors Derived from the RD3-MolPack Stable Packaging Cells

Abstract

Cell therapy with T cells genetically modified with lentiviral vectors (LVs) carrying therapeutic genes is growing as a promising tool for tumor treatment. Noticeably, the clinical production of these cells relies on the availability of either LVs, whose production is cumbersome from a safety, cost and reproducibility standpoint, or transduction enhancers to get optimal transduction rates. Development of LV packaging cells represents therefore an obliged goal in LV-based gene therapy, allowing reduction of the manufacturing cost and increasing the overall quality of the vectors. Similarly, the identification of the optimal transduction conditions is crucial. We investigated the efficiency of the recently described transduction enhancer Vectofusin-1® in promoting transduction of human ex-vivo activated T cells with a RD114-TR-pseudotyped SIN-GFP LV derived from MolMed's proprietary RD3-MolPack-SIN-GFP producer cells. Vectofusin-1® is an histidine-rich cationic amphipathic peptide that has been recently shown to increase LV gene transfer in hematopoietic stem cells without inducing toxicity through improvement of adhesion and fusion of LVs with the cellular membrane. Vectofusin-1® is easily synthetized, purified, and can be added to the viral supernatant without a pre-coating step, thus requiring less overall manipulation. RD3-MolPack-SIN-GFP was generated exploiting our RD-MolPack technology which is based on the RD114-TR envelope glycoprotein that, in contrast to the most utilized VSV-G envelope, permits generation of constitutive packaging cells for the production of SIN-LV. We selected a producer clone growing continuously in disposable two-compartment bioreactor for longer than 3 months and releasing LV with a titer, on average, of 1-5 × 106 TU/ml. Here, we show that the addition of Vectofusin-1® significantly improved RD3-MolPack-SIN-GFP LV transduction rate, without spinoculation, of either CD3/CD28 or OKT-3 activated T cells (70% GFP+) compared to the absence of adjuvant (6% GFP+) at MOI 1. The analysis of the vector copy number (VCN) integration confirmed these results (VCN=4 in the presence of Vectofusin-1® vs VCN=0.09 in the absence of Vectofusin-1®, respectively). When Vectofusin-1® was compared with Retronectin® and polybrene a similar effect was registered. No major differences in the immune phenotype and expansion rate were observed between cells transduced with Vectofusin-1® and cells transduced without any adjuvant. In summary, we conclude that combination of SIN-LV produced by RD3-MolPack cells and Vectofusin-1® might be considered for clinical manufacturing of LV-transduced T cells.