Co-delivery of lentiviral vectors and Cas9-containing virus-like particles enables rapid, scalable manufacture of gene-edited CAR T cells.

Chimeric antigen receptor T cell immunotherapies are transforming therapies for hematological malignancies and solid tumors and can be enhanced by targeted gene knockout. Here, we report lentiviral-based virus-like particles that package and deliver Cas9 ribonucleoproteins to primary human T cells. Using distinct pseudotyping strategies for virus-like particles and for lentiviral or γ-retroviral vectors, we achieved chimeric antigen receptor expression and targeted gene disruption. Under optimized transduction conditions, more than 50% of T cells expressed a chimeric antigen receptor by flow cytometry, with vector copy numbers exceeding two. Editing efficiencies were above 70% at three different target loci tested: T cell receptor α constant chain, β2-microglobulin, and DNA methyltransferase 3α. When the editing efficiency of virus-like particles was directly compared to electroporation, electroporation achieved a higher editing efficiency (99% versus 70%-90%). However, virus-like particle treatment resulted in twice as many cells being recovered compared with electroporation with a 10% increase in cell viability. Furthermore, off-target editing in virus-like particle-treated cells was reduced compared to ribonucleoprotein electroporated cells. These results support the feasibility of using virus-like particle-mediated delivery of Cas9 ribonucleoprotein to disrupt genes of interest, enabling a more scalable and cost-effective process for generating T cell immunotherapies.