IntroductionSTING-associated vasculopathy with onset in infancy (SAVI) is a rare, severe type I interferonopathy caused by gain-of-function mutations in STING1, leading to early-onset systemic inflammation, cutaneous vasculopathy, and life-threatening interstitial lung disease. Current treatments attenuate downstream inflammation without fully addressing the pathogenic driver and carry significant adverse effects. Recently, gene editing has emerged as a paradigm-shifting approach for IEI, with substantial potential for gain-of-function disorders that require allele-specific correction. We present a highly efficient and specific base- and prime-editing strategy that supports both ex vivo and in vivo therapeutic applications.ResultsK562 were transduced to generate stable STING1-WT and STING1-V155M lines (K562wt, K562mut). 8 sgRNAs were screened for base editing across PAM-flexible nucleases (SpCas9-NG, SpRY) in K562mut, and sgRNA2 and sgRNA5 were selected based on on-target editing. Given the high bystander activity observed with sgRNA5, we leveraged a previously developed library-derived in silico TadA design tool to identify >15 variants for testing. The TadA variant prioritized after experimental screening (var_17) reduced bystander editing (p<0.05). To overcome bystander constraints, PEGsm was selected for prime editing after comprehensive screening. Top-performing guides and selected editor variants were delivered by electroporation as in vitro–transcribed mRNA, achieving high on-target editing (90.10% ± 2.73%) for sgRNA5 and PEGsm (PE2 and PE3) and 53.48% ± 2.02% for sgRNA2. To functionally assess editing, interferon-stimulated gene transcripts (IFIT1, ISG15, and IFI44L) were quantified by droplet digital PCR (ddPCR) before and after 2′3′-cGAMP stimulation. ISG15 and IFIT1 were significantly reduced in unstimulated K562mut-edited cells (sgRNA2, PEGsm) versus K562mut, while after stimulation, K562wt and K562mut-edited reached the K562mut plateau as expected. In healthy donor hematopoietic stem and progenitor cells (HSPCs), a surrogate sgRNA was used to benchmark editing efficiency within the target window.Surrogate sgRNA achieved a mean bystander editing rate of 75.0% ± 4.4%. Finally, peripheral blood mononuclear cells (PBMCs) were reprogrammed into patient-derived induced pluripotent stem cells (iPSCs) using non-integrating Sendai vectors. iPSCs were prime edited to generate fully corrected isogenic single-cell–derived clones.ConclusionWe developed a comprehensive, bespoke base- and prime-editing platform for SAVI, enabling efficient and specific STING1 variant correction with functional normalization of interferon-stimulated genes (ISGs) expression, paving the way for future therapeutic applications. We also generated multiple fully corrected isogenic iPSC clones to support mechanistic studies of SAVI pathogenesis.Figure 1.Engineering of a STING1-V155M K562 model and functional assessment of bespoke base and prime editing. (A) Bidirectional third-generation lentiviral (LV) vector used to generate stable K562wt and K562mut lines expressing STING1-WT or STING1-V155M together with an EGFRt surface marker. (B) In vitro transcription (IVT) mRNA electroporation of top guide–editor pairs yields high A-to-G editing in K562mut, including base editing with sgRNA2/sgRNA5, a bystander-reducing TadA variant (var17) with sgRNA5, and prime editing with PEGsm in PE2 and PE3 configurations; editing was quantified by Sanger sequencing (EditR). (C) Functional readout by ddPCR showing IFIT1 and ISG15 expression (normalized to HPRT1) in K562mut across editing conditions and after STING inhibition (H-151). Statistics were computed by one-way ANOVA with multiple comparisons correction; significance is indicated, and where not otherwise specified, **** denotes p < 0.0001.

Midena et al.1 employ a nanoengineered 3D “nichoid” substrate that mechanically supports CD34+ hematopoietic stem and progenitor cells (HSPCs) during ex vivo manipulation, reducing culture-associated stress and improving engraftment and polyclonal output after gene editing or lentiviral gene addition. The work spotlights mechanobiology as a manufacturing lever for improving HSPC gene therapies.

Targeted genomic integration of gene-sized cassettes into hematopoietic stem and progenitor cells (HSPCs) for genetic disease treatment is constrained by the low efficiency of homology-directed repair (HDR) and frequent unintended genetic changes at the editing site. Here, to overcome these challenges, we introduce selection by means of artificial transactivators (SMArT), which transiently implements AND reporter gates to achieve templated integration of a functional cassette at the target site. HDR-edited HSPCs were enriched to 80-100% purity through transient selector expression, whereas cells carrying undesired and potentially genotoxic on-target edits were preferentially depleted. Xenotransplantation of SMArT-enriched HSPCs in immunodeficient mice resulted in fully HDR-edited human grafts with the selector no longer detectable. SMArT strategies were implemented through clinically compliant manufacturing and selectors. They support both safe harbor integration and gene correction, can preserve physiological transcriptional regulation and are portable across loci also with polyfunctional editors. Overall, SMArT strategies may broaden the therapeutic applicability of gene-sized editing while reducing its genotoxic burden.

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 therapies1-4. Although monoclonal antibodies targeting KIT have been proposed as alternatives to chemotherapy or radiotherapy5-7, 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.

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.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment landscape of hematologic malignancies, delivering durable remissions in diseases previously associated with poor outcomes. However, translating this success to solid tumors has proven challenging due to antigen heterogeneity, limited tumor infiltration, immunosuppressive tumor microenvironments, and progressive T-cell exhaustion. In response, next-generation CAR T-cell platforms have emerged that integrate advances in receptor architecture, intracellular signaling, and programmable control systems to enhance specificity, persistence, and safety. This review comprehensively examines recent innovations in CAR T-cell engineering, including optimization of extracellular binding domains, hinge and transmembrane modifications, fine-tuning of intracellular signaling motifs, and the incorporation of alternative protein scaffolds. We discuss logic-gated strategies such as synNotch receptors, inducible ON-switch CARs, inhibitory CARs, and modular adaptor systems that enable context-dependent activation and reduce off-tumor toxicity. In parallel, we explore approaches aimed at overcoming T-cell dysfunction through intrinsic checkpoint rewiring, cytokine armoring, and epigenetic reprogramming to sustain antitumor activity in hostile microenvironments. The development of allogeneic and off-the-shelf CAR T-cell products derived from healthy donors, induced pluripotent stem cells, natural killer cells, γδ T cells, and macrophages is also reviewed, highlighting strategies to mitigate graft-versus-host disease and host immune rejection while enabling scalable manufacturing. Finally, we address current translational bottlenecks related to immunogenicity, regulatory complexity, and production logistics, and outline future directions for integrating Boolean logic circuits, safety switches, and automated GMP-compliant processes. Collectively, these advances position next-generation CAR T-cell therapies as programmable and adaptable immunotherapeutic platforms with the potential to extend durable clinical benefit beyond hematologic cancers into solid tumors.

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.

Severe congenital neutropenia (SCN) is a life-threatening disorder of neutrophil production most frequently caused by dominant mutations in the ELANE gene. ELANE pathogenic mutations lead to the production of mutant neutrophil elastase (NE) proteins that can disrupt NE folding and trafficking, which triggers the unfolded protein response (UPR) leading to cellular apoptosis and neutrophil maturation arrest at the promyelocyte-to-myelocyte stage of neutrophil differentiation. Previously, we and others have demonstrated that introducing indels in ELANE via Cas9-mediated gene editing to activate nonsense-mediated decay (NMD) can restore normal neutrophil development, serving as a potential universal therapeutic strategy. However, nuclease editing is associated with drawbacks such as p53-dependent DNA damage responses, unintended large deletions or rearrangements resulting from double-strand breaks (DSBs), and limited multiplex potential. Here, we report efficient, potent, and specific ELANE base and prime editing (BE and PE) approaches as less genotoxic alternatives. We designed a gRNA for adenine base editing (ABE) targeting a splice site in ELANE at the intron 2–exon 3 splice acceptor site and a pegRNA targeting exon 2 to introduce premature termination codons. In CD34⁺ HSPCs, ELANE editing with ABE8e yielded 94% A>G conversion at target position A8 with 43% bystander editing at A10, while PE with PEmax-La:ELANE-pegRNA without a nicking sgRNA (PE2-type) achieved 68% precise prime edits. To model SCN in vitro, we sequentially introduced a pathogenic ELANE exon 5 mutation via Cas9 editing and therapeutic edits with Cas9, ABE8e, or PEmax-La in CD34⁺ HSPCs. Both BE and PE restored neutrophil maturation arrest by inducing NMD and reducing ELANE mRNA expression. To test the therapeutic potential for SCN, mobilized CD34⁺ HSPCs from an ELANE p.Ser126Leu mutant patient were edited ex vivo with either nuclease, BE or PE targeting ELANE (as well as mock and neutral locus edited controls). We transferred the edited cells to NBSGW mice for in vivo engraftment and multilineage differentiation. After 16 weeks, high bone marrow human chimerism (>90%) was maintained across all groups. Granulocyte engraftment was significantly improved with ELANE editing (62% for nuclease, 62% for BE and 56% for PE) versus mock (12%) and safe locus controls (9%). We performed a competitive transplant with ABE8e:ELANE and neutral locus edited cells. A ratio of 20:80 edited:control HSPCs produced similar ELANE edits in neutrophils as fully undiluted edited HSPCs, while edits in B cells were reduced as expected by dilution, indicating selective advantage for edited cells to bypass neutrophil maturation defect. Single cell RNA sequencing of control and ELANE edited cells showed an ER stress gene expression signature and neutrophil differentiation block only in unedited cells. Using in silico CRISPRmeand cell-based GUIDE-seq off-target nomination, we verified a single ABE8e:ELANE off-target site with 18% ± 2% editing. Previously, RNA-DNA hybrid gRNAs were described as a strategy to reduce nuclease off-target editing. We screened 37 hybrid gRNAs and found several that discriminated off-target editing. In CD34+ HSPCs, the unmodified gRNA produced 97% on-target and 30% off-target editing, while a hybrid DNA-RNA sgRNA produced 86% on-target and 0.2% off-target editing, not significantly different from an unedited control sample. For prime editing, we did not detect off-target edits in HSPCs after PEmax-La:ELANE pegRNA editing even though the same spacer produced a frequent off-target (22% edits) in a Cas9:gRNA RNP context. To evaluate whether neutrophil functions are preserved following ELANE base or prime editing, we isolated the engrafted mature human neutrophils from NBSGW mice. ELANE edited neutrophils displayed comparable oxidase activity, phagocytic activity, neutrophil extracellular trap formation, and E. coli killing capacity when compared to unedited control neutrophils, indicating preserved neutrophil function of edited cells. These results demonstrate highly efficient near-universal BE and PE approaches to ELANE gene editing of CD34+ HSPCs that lack detectable off-target effects, correct neutrophil maturation arrest, and preserve neutrophil function and HSPC engraftment for potential therapy of ELANE-mutant SCN.

BackgroundIn adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells.MethodsWe simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCRED) and permanently disrupted LAG3, TIM-3 or 2B4 genes (IRKO) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCRED-IRKO and IR competent (TCRED-IRCOMP) cells were tested in short-term co-culture assays and under a chronic stimulation setting in vitro. Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models.ResultsWe show that upon chronic stimulation, TCRED-IRKO cells are superior to TCRED-IRCOMP cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge in vivo. Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells.ConclusionThese results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.

The baboon endogenous retrovirus (BaEV) glycoprotein is superior to the commonly used vesicular stomatitis virus glycoprotein (VSVg) for retroviral gene transfer into resting hematopoietic stem cells and lymphocyte populations. The derivative BaEVRLess (lacking the R domain) produces higher viral titers compared with wild-type BaEV, but vector production is impaired by syncytia formation and cell death of the HEK293T cells due to the high fusogenic activity of the glycoprotein. This lowers viral titers, leads to increased batch-to-batch variability, and impedes the establishment of stable packaging cell lines essential for the economical production of viral supernatants. Here, we show that knockout of the entry receptor ASCT2 in HEK293T producer cells eliminates syncytia formation, resulting in a 2-fold increase in viral titers, reduced toxicity of viral supernatants, and enables the generation of stable packaging cell lines. In successive steps, we stably integrated BaEVRLess and a-retroviral a.Gag/Pol expression cassettes and isolated clones supporting titers up to 108 to 109 infectious particles/mL, a 10-fold increase in concentrated viral titers. The additional overexpression of CD47 and knockout of b 2-micro- globulin in the packaging cell line are tailored for future use in in vivo gene therapy applications by reducing non-specific uptake by macrophages and the immunogenicity of viral particles.