Abstract
Abstract The successes of chimeric antigen receptor (CAR) T cells in treating blood cancers have highlighted the cell therapy era. However, the difficulty of delivering molecules into immune cells has been an obstacle to more rapid advancement. Here we present an innovative large-scale Lentivirus (LV) production system as a solution to lower the cost and time of viral production. On the other hand, the next generation cell therapy will rely heavily on gene editing, especially in a safer non-viral integration manner. We have demonstrated that our novel non-viral all-in-one electroporation method provides high efficiency of gene knock-in in primary T cells. The new LV production system was developed for the clinical grade production of lentiviral vectors (LVVs) on a large-scale serum-free suspension platform. This technology employs a newly developed propriety set of GMP reagents comprising of culture media, suspension cells, transfection reagent and boosting enhancers. The system is able to deliver greater than 1 × 108 (TU/mL) functional titer with un-concentrated LVVs. For CRISPR/Cas9 gene editing in primary T cells, we were able to reach more than 90% knockout efficiency for most genes we tested, including T cell receptor (TCR), with Cas9 RNP electroporation using Neon Transfection System. More importantly, gene knock-in efficiency can be reached to greater than 30% with all-in-one electroporation, which delivers Cas9 RNP and donor DNA in one reaction using our newly developed electroporation buffer. Moreover, TCR knockout in addition to a knock-in at another locus can also be done in a single electroporation using our all-in-one method, which is ideal for developing next generation CAR and TCR-T cells.
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