Optimization of multiplex crispr-cas9 editing of human primary t cells using lipid nanoparticles (lnps) and subsequent off-target evaluation

Background & Aim CRISPR/Cas9 has emerged as a dynamic tool for gene editing of human primary T cells. Previously, we demonstrated a novel lipid nanoparticle (LNP) reagent for engineering of gene-edited CAR T cells with high cell viabilities and potency, even after multiple genetic manipulations. Here, we extend this work by optimizing multi-gene knockouts and assessing potential off-target editing effects in T cells, with RNAs delivered either by electroporation or LNPs. Further, through extensive T cell media evaluation, we show the critical nature of cell culture conditions for efficient LNP-mediated transfections. Methods, Results & Conclusion: Methods LNPs encapsulating wild type or high fidelity S.p. Cas9 mRNA and various TRAC and CD52 targeted guide RNAs (sgRNAs) were produced using the scalable NxGenTM mixing platform. Alongside, electroporation was performed to deliver equivalent cargoes. Primary T cells were cultured, activated, and expanded in different commercial media T cell media in static culture. Gene expression and cell viability were measured using flow cytometry. Multi-target performance of CRISPR-Cas9 editing was evaluated through rhAmpSeq-based targeted next-generation sequencing (NGS) and indels analyzed for with CRISPRAltRations. Results TCR or CD52 targeted Cas9 mRNA-LNP addition or electroporation yielded high single and double knockout efficiencies. We tested a range of sgRNA targets, wild-type and high-fidelity Cas9 mRNAs, and determined off-target editing. We achieved 90% ±2.6% double edited T cells (TCR–/CD52–). Similar results were obtained when comparing different LNP batch sizes (micro to milligram RNA) and cell culture vessels (0.1 to 45 million cells), demonstrating scalability of both LNPs and cell treatment. Editing efficiency and potency was affected by the type of culture media. LNP delivery to T cells for gene editing was optimized in two commercially available T cell culture media yielding similar EC50 values (0.3 to 0.5 µg/million cells). Conclusions The results from this study further support the utility RNA-LNPs for the genetic engineering of primary T cells. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins for future cell therapies. These LNPs can be easily manufactured from small-scale screening of RNA libraries to rapid scale-up for clinical translation.