Epitope editing of KIT enables antibody-based, non-genotoxic conditioning that selectively enriches therapeutic BCL11A-edited haematopoietic stem/progenitor cells, supports durable engraftment, preserves clonal diversity and enhances induction of fetal haemoglobin, a therapeutic approach for conditions such as sickle cell disease and β-thalassemia.
Abstract
The short-term and long-term effects of genotoxic pre-transplant conditioning remain barriers to the broader application of haematopoietic stem/progenitor cell (HSPC) transplantation and gene therapies1, 2, 3–4. Although monoclonal antibodies targeting KIT have been proposed as alternatives to chemotherapy or radiotherapy5, 6–7, their pharmacokinetics hinder clinical applications owing to the risk of depleting transplanted HSPCs. Here, to address this issue, we identified amino acid changes in the extracellular domain of KIT that disrupt the binding of two therapeutic monoclonal antibodies8,9, which impair stem cell factor (SCF)-mediated signalling without affecting KIT expression or functionality. We exploited adenine base editing10 or prime editing11 to efficiently introduce these mutations in HSPCs and combined them with the disruption of the BCL11A erythroid enhancer to promote expression of fetal haemoglobin (HbF)12,13, a therapeutic approach for several haemoglobinopathies. This strategy enables in vivo co-selection of gene-engineered cells to reach the threshold required to provide therapeutic benefit in patients affected by sickle cell disease and β-thalassaemia. We show progressive enrichment of KIT plus BCL11A multiplex-edited haematopoiesis under selective pressure with KIT monoclonal antibody, in vitro and in vivo. We report that extended treatment with anti-KIT regimens leads to superior in vivo enrichment while avoiding clonal selection, as assessed by a lentiviral barcoded library. Finally, by overcoming the limitations of monoclonal antibody pharmacokinetics, epitope editing enables novel haematopoietic replacement regimens that are not limited by on-target graft elimination, allowing prolonged immune-based conditioning that maximizes haematopoietic niche clearance without chemo-radiotherapy or monoclonal antibody wash-out. Epitope editing of KIT enables antibody-based, non-genotoxic conditioning that selectively enriches therapeutic BCL11A-edited haematopoietic stem/progenitor cells, supports durable engraftment, preserves clonal diversity and enhances induction of fetal haemoglobin, a therapeutic approach for conditions such as sickle cell disease and β-thalassemia.
Targeted immunotherapies have transformed the treatment of hematologic malignancies, yet their clinical utility is often constrained by on-target, off-tumor toxicity arising from shared antigen expression between malignant cells and essential healthy tissues. An early approach to mitigate this limitation involved the knockout (KO) of the target antigen in donor hematopoietic stem and progenitor cells (HSPCs). However, this strategy is restricted to markers that are dispensable for normal hematopoietic function. Epitope engineering has emerged as an alternative paradigm to decouple therapeutic susceptibility from physiological function by modifying the target antigen on healthy cells while preserving biological activity. In this review, we discuss recent advances in base and prime editing approaches used for epitope editing. We examine recent preclinical and emerging translational studies of this strategy in both malignant and non-malignant contexts. Finally, we discuss challenges related to editing efficiency, off-target effects, delivery strategies, and long-term safety in hematopoietic stem cells. Collectively, epitope engineering of hematopoietic stem cells represents a versatile platform to expand the therapeutic window of precision immunotherapies and may enable safer, more effective combinatorial treatment strategies for both non-malignant and malignant hematologic conditions.
Joanne Baek, G. Casirati, Pietro Genovese et al.· Blood Advances· 0 citations
The 30-year evolution of autologous gene therapy as a vital alternative to allogeneic hematopoietic stem cell transplantation for IEIs is explored, tracing the transition from early gamma-retroviral gene addition - which successfully restored immunity in severe combined immunodeficiency but carried high risks of insertional mutagenesis and leukemogenesis - to the adoption of safer self-inactivating lentiviral vectors.
Jasmeen Dara, Claire Booth· Expert Opinion on Biological...· 0 citations
The results suggest that this approach may overcome the reliance on busulfan or other myeloablative conditioning regimens with their associated morbidities, and by enabling toxin-free conditioning and in vivo selection of edited cells, may facilitate clinical implementation of these highly valuable genetic therapies.
Romina Marone, Rosalba Lepore, K. Paschoudi et al.· bioRxiv· 0 citations
Recent developments and refinements in gene transfer and editing technologies for HSPCs are reviewed, while also discussing the critical limitations and hurdles to clinical translation, as recently presented at the New Investigator Committee Gene Therapy webinar.
Kohei Shiroshita, A. Stolz, C. Malouf et al.· Experimental Hematology· 0 citations
Current cell and gene therapies lack clinically practical mechanisms to selectively promote or suppress therapeutic cells in vivo, a limitation that is particularly acute in patients requiring ongoing immunosuppression. This includes gene therapy for immune dysregulation syndromes, and antigen-specific or chimeric antigen receptor (CAR) T-cell therapy for patients requiring immunosuppression (e.g., transplant recipients), where both pathogenic and therapeutic cells may be suppressed. Here, we develop a multiplex prime-editing platform that converts commonly used immunosuppressive drugs into tools for in vivo control of T-cell therapies via defined, pathway-specific drug resistance. Focusing initially on gene therapy, prime editing efficiently edited loci of multiple pathogenic variants associated with immune dysregulation in primary human T-cells and corrected the HAVCR2 driver mutation in T-cells from multiple patients with subcutaneous panniculitis-like T-cell lymphoma (SPTCL). Comprehensive genomic, transcriptional, immunophenotypic, and clonal analyses demonstrated minimal off-target perturbation. Multiplexed gene correction and drug-resistance editing of T-cells from patients with SPTCL enabled selective in vivo expansion of corrected cells under immunosuppressive pressure in humanized mouse models and exhibited retained sensitivity to alternative agents permitting rapid in vivo suppression. Extending this approach, prime edited, drug-resistant antigen-specific and CAR T-cells retained effector function despite pharmacologic immunosuppression, demonstrating the generalizability of this platform to diverse cellular therapies. Together, these findings establish multiplex prime editing as a promising preclinical framework for generating drug-controllable T-cell therapies, enabling selective in vivo modulation in settings where immunosuppression cannot be withdrawn.
E. Bandala-Sanchez, Emma V. Petley, K. Ramsay et al.· Blood· 0 citations
This review highlights key engineering strategies enabling in vivo CAR T-cell generation, summarizes emerging clinical research and development, and discusses future opportunities for expanding in vivo CAR T-cell therapies as scalable immunotherapy platforms.
Janani Gopalakrishnan, B. Rathod, Sachin Puri· International Immunopharmaco...· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
