Epitope Editing Combined with Extended Schedule Anti-KIT Antibody Treatment Allows Immune-Based In Vivo Selection of Multiplex Genome-Engineered Cells

Hematopoietic stem/progenitor cell (HSPC) transplantation (HSCT) offers curative options for conditions for which the substitution of host hematopoiesis with gene-corrected stem cells can halt the pathogenic process. Yet, short and long-term effects of genotoxic conditioning remain a barrier to a wider application of HSCT and gene therapies. Whereas monoclonal antibodies (mAbs) targeting HSPCs-expressed markers, such as c-KIT, have been proposed as an alternative to chemo/radiotherapy, their pharmacokinetics hamper safe and effective clinical use due to risk of on-target depletion of transplanted HSPCs, leading to increased risk of graft failure or incomplete myeloablation. We previously demonstrated that precise editing of the targeted epitope in HSPCs can endow hematopoietic cells with selective resistance to mAbs or CAR-T cells without affecting stem cell functionality. To this end, we identified amino-acid changes in the extracellular domain of c-KIT tyrosine kinase receptor that abrogate the binding of a therapeutic mAb (Fab79D) without affecting surface expression, ligand affinity and downstream signaling. We exploited adenine base editing (BE) to efficiently (~80%) introduce these mutations in CD34+ HSPCs, which displayed preserved long-term repopulation and multilineage differentiation capacity in vivo. As BE can introduce precise base changes without the need for DNA double strand breaks nor template donors, it is suitable for combinatorial approaches with additional therapeutic genome modifications, such as the disruption of BCL11A erythroid enhancer to enforce fetal hemoglobin (HbF) expression, a well-characterized approach to provide therapeutic benefit for patients affected by sickle cell disease. We multiplexed KIT and sgRNAs targeting the +55 and +58 BCL11A DNAse hypersensitivity sites (DHS) and obtained BE efficiencies comparable to single edits, while achieving upregulation of HbF proportional to the number of disrupted WGATAR motifs (up to 80%). Critically, by single-cell sorting we observed high degree of biallelic co-editing, with 60% HSPCs bearing biallelic KIT edits and at least 3 disrupted WGATAR motifs. This feature translated to the possibility to achieve dose-dependent in vitro co-selection of multiplex-BE CD34+ HSPCs when treated with anti-KIT mAb, as Fab79D inhibits receptor signaling through impairment of KIT dimerization. We next assessed the possibility to select for multiplex edited cells in vivo by performing a competitive transplant with co-injection of multiplex KIT+BCL11A BE and AAVS1 BE CD34+ cells in NBSGW mice. We observed progressive enrichment of both KIT and BCL11A edits (~2x) in mice treated with a standard mAb regimen – similar to a conventional dose-dense HSCT conditioning – when compared to controls. We speculated that optimized mAb schedules with extended exposure to KIT blocking mAb may provide superior results thanks to protracted survival advantage of edited cells. We performed competitive transplants with lentiviral-encoded fluorescent marking of the KIT+BCL11A BE (mTagBFP+) and AAVS1 BE cells (mNeonGreen+), to enable precise tracking of HSPCs and their progeny by high dimensional cytometry. We treated mice with either dose-dense or extended Ab regimens (6 doses, either every 5 or 10 days), with the same cumulative dose. Extended regimens produced markedly improved in vivo selection with near complete replacement (80-100%) within progenitor and myeloid/erythroid compartments, which translated into higher therapeutic gene editing and enhanced HbF induction. The selection w/in each subpopulation was proportional to KIT surface levels and half-life. The same approach was applied to hematopoietic replacement, where mice were pre-engrafted with non-edited HSPCs were then treated with KIT Ab regimen and transplanted with a second donor’s BE HSPCs, which showed superior engraftment and gene marking as compared to controls. As epitope editing eliminates limitations due to Ab on-target toxicity, we can envision innovative, non-genotoxic gene therapy transplant regimens to achieve therapeutic levels of corrected HSPCs with substantial advantages over conventional alternatives in terms of safety and tolerability. Extended conditioning schedules with prolonged exposure to mAbs may provide novel avenues to further reduce toxicity and hematopoietic aplasia while preserving stem cell depleting capacity.