Base Editing Hematopoietic Stem/Progenitor Cell Gene Therapy for Treatment of X-Linked Severe Combined Immunodeficiency

Gene editing of hematopoietic stem cells: applications and advances.

Gene therapy approaches for inborn errors of immunity: from bench to bedside.

INTRODUCTION Inborn errors of immunity (IEI) are rare genetic defects that disrupt immune function, often resulting in life-threatening infections, malignancies, and immune dysregulation. Allogeneic hematopoietic stem cell transplantation (HSCT), a curative option for some diagnoses, is limited by donor availability and risks of graft-versus-host disease. This review explores the 30-year evolution of autologous gene therapy as a vital alternative to allogeneic hematopoietic stem cell transplantation for IEIs. AREAS COVERED Literature search using PubMed for gene therapy for IEI in the last 20 years. We trace the transition from early gamma-retroviral gene addition - which successfully restored immunity in severe combined immunodeficiency (SCID) but carried high risks of insertional mutagenesis and leukemogenesis - to the adoption of safer self-inactivating lentiviral vectors. The field is rapidly advancing beyond viral gene addition toward highly precise gene editing technologies, including CRISPR/Cas9, and base/prime editing, which offer targeted correction with minimized genotoxicity. EXPERT OPINION Recent milestones in diseases like Wiskott-Aldrich syndrome (WAS) and chronic granulomatous disease (CGD) highlight enormous scientific success, yet significant barriers to accessibility, manufacturing, and affordability remain. Overcoming this requires innovative regulatory frameworks and collaborative funding models. Streamlining development and ensuring equitable access are essential next steps to establishing gene therapy as a safe alternative.

Autologous Ex Vivo Lentiviral Gene Therapy for Severe Leukocyte Adhesion Deficiency-I Achieves Durable Immune Reconstitution and Reduction of Infection-Related Morbidity: Updated 3.5-5.5-Year Results from a Phase I/II Study

Severe leukocyte adhesion deficiency-I (LAD-I) is caused by biallelic deleterious variants in ITGB2, resulting in <2% CD18 expression and impaired leukocyte extravasation, particularly neutrophils. Affected infants and children experience recurrent, life-threatening bacterial and fungal infections, poor wound healing, and significant mortality in the absence of allogeneic hematopoietic stem cell transplantation (alloHSCT). Although alloHSCT can be curative, many patients lack suitable donors and remain at risk for graft-versus-host disease (GvHD), graft failure, and transplant-related morbidity and mortality. Treatment with a durable autologous hematopoietic stem cell (HSC) gene therapy was developed to address these limitations. To evaluate the long-term safety and efficacy of RP-L201 (marnetegragene autotemcel), an autologous CD34+ HSC gene therapy using a Chim-CD18-WPRE lentiviral vector incorporating ITGB2, we investigated restoration of CD18 expression, normalization of neutrophil function, and reduction in infection-related morbidity in children with severe LAD-I. Children ≥3 months old with severe LAD-I were enrolled in the phase I/II study (NCT03812263). CD34+ cells obtained via G-CSF/plerixafor mobilization and apheresis were transduced ex vivo with RP-L201 and infused following therapeutic drug-monitored myeloablative conditioning. Endpoints included survival without alloHSCT, restoration of peripheral blood (PB) polymorphonuclear (PMN) CD18 expression, peripheral blood mononuclear cell (PBMC) vector copy number (VCN), integration site analysis, resolution of baseline leukocytosis, and incidence of infection-related hospitalizations. All treated patients entered the long-term follow-up (LTFU) study (NCT06282432). Nine patients (age 9.8-117.4 months at infusion) received RP-L201 and were followed for a median (range) of 50.92 (42.6-67.9) months as of the June 18, 2025, LTFU data cut. AlloHSCT-free survival was 100% with no cases of graft failure. PBMC VCN/cell rose to 0.42-2.4 at month 3 and remained stable through month 12 (mean 1.73) with persistent long-term VCN levels across months 42-60. All patients achieved sustained PB PMN CD18 expression within or approaching the target levels, maintained through >3.5 years post-infusion. Longer-term results demonstrate markedly lower annualized incidences of prespecified serious infections, infection-related hospitalizations, and prolonged infection-related hospitalizations after RP-L201 treatment relative to pre-treatment incidences. No new skin or oral infections occurred in the LTFU period. RP-L201 was well-tolerated; no RP-L201-related adverse events occurred. Integration site analyses demonstrated polyclonal integration patterns without dominant or expanding clones. RP-L201 conferred durable engraftment, sustained CD18 restoration, and significant reductions in infection-related morbidity in nine children with severe LAD-I, with at least 3.5 years of follow-up. All nine patients are alive without alloHSCT. There were no RP-L201-related adverse events. These updated results support autologous HSC gene therapy as an effective alternative to alloHSCT with a favorable risk-benefit profile for severe LAD-I.

Development of a Protective Cellular Therapy to Prevent Graft Versus Host Disease Post Allogenic Hematopoietic Stem Cell Transplant 2331939

Graft-versus-host disease (GVHD) remains a primary cause of morbidity and mortality post-allogeneic hematopoietic stem cell transplantation (allo-HSCT), driven by donor T cells’ dysregulated inflammation against host tissues. This limits survival and underscores the need for therapies that curb GVHD while preserving graft-versus-tumor (GVT) effects. Innate-like T cell subsets, including MAIT, NKT, and Vγ2 γδ T cells correlate with milder GVHD. These share the BTB-ZF transcription factor PLZF (Zbtb16), a master regulator of innate-like programming. Building on our finding that ectopic PLZF imparts innate-like traits to conventional αβ T cells, we tested if PLZF-overexpressing donor T cells suppress GVHD in vivo. In a B6→BALB/c acute GVHD model, lethally irradiated BALB/c recipients received bone marrow plus purified CD4+/CD8+ T cells from C57BL/6 donors, transduced with retroviral PLZF or empty vector (EV) control (n = 15/group). Mice were monitored daily for survival, clinical scores, and weight loss. In mouse models of acute GVHD (B6→BALB/c), adoptive transfer of retrovirally transduced, PLZF-overexpressing conventional T cells markedly delayed disease onset and enhanced survival relative to empty vector-transduced controls. Mechanistically, PLZF-overexpressing conventional T cell promoted expansion of regulatory T cell (Treg) populations, thereby driving a phenotypic shift toward innate-like suppression of alloimmunity. These results affirm PLZF overexpression as a viable, programmable approach for GVHD prophylaxis, poised for clinical advancement. Through innate-like T cell reprogramming, this strategy holds promise for revolutionizing allo-HSCT by decoupling immune tolerance from antitumor immunity. Current investigations are evaluating PLZF-overexpressing conventional T cell preservation of graft versus tumor (GVT) activity against leukemia and extending these findings to humanized models. NJ PHORCE Transplantation Immunology (TRAN)

CD117 epitope-shielded hematopoietic stem cell transplantation with toxin-free conditioning and in vivo selection ameliorates a β-thalassemia model

Clinical evidence demonstrates that ex vivo gene therapy and genome engineering of hematopoietic stem and progenitor cells (HSPCs) could represent one-time cures. However, while genome editing itself has become increasingly efficient and precise, the toxic conditioning required for hematopoietic stem cell transplantation remains a major barrier to broad clinical implementation of these otherwise curative therapies. In particular, the use of busulfan for myeloablative conditioning constitutes a major safety concern. While preclinical studies established CD117 as a promising target for antigen-specific therapy, clinical translation faced setbacks balancing efficacy and safety. To overcome current limitations, we generated a new CD117-blocking monoclonal antibody (CIM058) and demonstrate its potency to block wild-type HSPCs. To enable long-term blockade of host HSPCs even after transplantation, we used prime editing to engineer CIM058-resistant human CD34+ HSPCs. When combined, CIM058 and the epitope engineered CD34+ HSPCs ameliorated disease phenotype in a β-thalassemia model. Our 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.

Functional restoration of immune defects in STAT1 gain-of-function disease following stem cell gene editing.

Germline gain-of-function (GOF) mutations in the signal transducer and activator of transcription 1 (STAT1) gene cause a dominantly inherited inborn error of immunity (IEI) characterized by chronic mucocutaneous candidiasis, autoimmunity, severe opportunistic infections and an increased risk of malignancy. Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is curative but is associated with increased risk of morbidity and mortality in STAT1 GOF patients compared to other IEI. To develop a curative, autologous alternative to HSCT, we evaluated gene editing strategies in STAT1 GOF model cell lines, primary T cells, and patient-derived HSCs. Universal and mutation-specific strategies using CRISPR/Cas-mediated homology-directed repair (HDR) were limited by low efficacy (<25%), poor viability, and a lack of allele-specificity. In contrast, adenine base editing corrected the recurrent and highly pathogenic p.T385M mutation with upwards of 90% efficiency in patient T cells and HSCs without significant unintended on- or off-target genomic aberrations. Gene editing functionally restored total STAT1 expression (p<0.0217), STAT1 phosphorylation (p<0.0056), interferon-stimulated gene expression (OAS1; p=0.0005) and improved IL-17 production (p<0.0001). Edited HSCs retained multilineage differentiation capacity and sustained engraftment with persistence of the corrected allele at 16 weeks in humanized immunodeficient mice. These data demonstrate efficient and precise correction of STAT1 GOF mutations by base editing, with maintenance of the correction through long-term engraftment in vivo. This represents the first application of gene editing to correct a dominant gain-of-function mutation causing immunodeficiency, with potential applicability to other genetic disorders associated with heterozygous and gain-of-function mutations.