Multiplex Prime Editing and PASSIGE ^TM for Non-Viral Generation of an Allogeneic CAR-T Cell Product

While CAR-T cell therapy represents a major advance in personalized immunotherapy, the autologous nature of commercially available CAR-T products have delayed its broad application beyond a subset of hematological malignancies. In particular, manufacturing autologous CAR-T therapies is costly and time-consuming, and success relies on the fitness of a patient's cells. An allogeneic off-the-shelf CAR-T product could overcome these cell quality and quantity issues and could be accessed on-demand. However, a successful allogeneic CAR-T product will require multiplex gene knockout in addition to stable CAR integration to prevent graft-versus-host disease (GvHD) and graft rejection by the patient's immune system.

Strategies for the delivery and expression of CAR transgenes include semi-random integration using lentivirus or transposons, which risk unintended gene disruption or activation. Targeted integration can be achieved using nucleases in combination with a template for homology-directed repair (HDR), but efficiency of this approach is generally low and there are risks associated with the induction of double-strand breaks (DSBs), including p53 activation and chromothripsis. This is further complicated in a situation where multiplex editing is required, as multiple DSBs can lead to gross chromosomal rearrangements. Alternatively, base editors have been used for targeted gene disruption without the induction of DSBs via single nucleotide conversion to disrupt a splice site or introduce a single premature stop (pmSTOP) codon. However, this approach is limited to transition mutations and cannot be used to integrate large genetic cargo, which is required to generate CAR-T cells.

Prime Editing (PE) can be used for precise and programmable gene disruption via multiple strategies, including introduction of multiple pmSTOP codons, splice site disruption, frameshift mutations, or deletion of large segments of regulatory or coding sequence. Further, Prime-Assisted Site-Specific Integrase Gene Editing (PASSIGE) can be used to precisely and flexibly integrate large genetic cargo at a specific locus. Together, PE-mediated gene knock out and PASSIGE can be tailored to generate a more broadly applicable, potentially safer, and more effective CAR-T cell product.

To evaluate the efficacy of an all-PE non-viral approach to allogeneic CAR-T cell generation, PASSIGE and PE mediated gene knockout were used to generate CD19-CAR-T cells. Multiplex PE precisely disrupted expression of the endogenous T cell receptor alpha constant ( TRAC) and beta-2-microglobulin ( B2M) loci in over 90% of human T cells. Co-delivery of a non-viral DNA donor template with PASSIGE editing components resulted in targeted integration of a 3.5 kb CD19-CAR transgene expression cassette at the TRAC locus in over 60% of the T cells, with no observed impact on T cell viability, phenotype, or functionality.

CD19 CAR-T cells generated using PASSIGE show potent anti-tumor activity and cytokine production in response to CD19 ⁺ tumor cell lines in vitro and in vivo. The PASSIGE-generated CD19 CAR-T cells reduce tumor burden and prolong survival of mice bearing CD19 ⁺ tumors. These results show that a PE platform can be used to generate multiplex edited CAR-T cells without the need for viral vectors and without causing DSBs. This modular, one-step, non-viral delivery Prime Editing platform expands the applicability of T cell therapies for the treatment of tumors and immune diseases.