Restoration of the immune system with base editing and non-genotoxic conditioning in a Rag2 point-mutant mouse model.

Transplantation of donor hematopoietic stem and progenitor cells (HSPCs) is a well-established curative treatment for various blood and immune diseases, including severe combined immunodeficiency (SCID). However, it comes with significant toxicities, including graft-versus-host disease (GvHD) and tissue damage resulting from the use of genotoxic chemotherapy-containing conditioning regimens. Autologous transplantation using gene-modified HSPCs eliminates GvHD but currently still relies on genotoxic conditioning. Further, gene modification of HSPCs has commonly utilized integrating viruses, which carry the risk of oncogenesis. The ideal therapy would eliminate the risks associated with current hematopoietic stem cell (HSC) gene-modification and conditioning approaches. Here, we combined base editors (BEs), engineered virus-like particles (eVLPs), and non-genotoxic alphaCD117 antibody-drug conjugate (ADC) conditioning to explore optimal curative treatment of SCID. We generated a Rag2 SCID mouse model with a single point mutation (pm) and corresponding BE. Rag2(pm/pm) HSPCs were corrected using SpCas9NG-ABE-eVLPs without off-target effects being detected. Even in settings of low editing, transplantation of BE-corrected HSPCs into alphaCD117-ADC-conditioned mice led to efficient immune cell production in peripheral blood with normal B cell progenitors in the bone marrow. Combining alphaCD117-ADC conditioning with transplantation of HSPCs that were base edited using eVLPs successfully reversed the SCID phenotype in mice, showcasing a significant advancement in reducing treatment-related toxicities while enabling disease correction.