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.

Implementation of therapeutic genome editing requires a potent, versatile, and transient delivery system to enable safe and effective in vivo applications. Here, we report on an optimized virus-like particle (VLP) platform for protein-based delivery of Cas9 ribonucleoproteins and Cas9-derived base editors and prime editors, termed LV-VLP-MA, that enables flexible editor deployment. By systematically engineering a panel of truncated Gag-Cas9 fusion variants, we identify a minimal MA-Cas9 configuration that maximizes editor packaging while effectively preserving efficient particle production and functional delivery. Systematic refinement of VLP production parameters enhances particle yield, supporting robust editing activity across diverse genomic targets. Importantly, systemic administration of LV-VLP-MA mediates efficient in vivo editing of the Pcsk9 locus with functional target suppression, establishing proof-of-concept for therapeutic application. Together, these results define a programmable, modular VLP-based platform that combines potency, flexibility, and transient delivery to expand the scope of in vivo genome engineering for therapeutic development.

Traditional retroviral gene transfer protocols for the genetic modification of hematopoietic stem and progenitor cells (HSPC) include a multiday ex vivo culture period, which can negatively affect the biology of the cells and is costly. As an alternative approach, we outline here a method for gene transfer into quiescent human HSPCs using lentiviral (LV) vectors and gamma-retroviral virus-like particles (eVLP). To achieve this, we use LV vectors and eVLPs pseudotyped with the modified baboon endogenous retroviral glycoprotein BaEVRLess, which targets the neutral amino acid transporters ASCT1 and ASCT2 on HSPCs. This envelope enables immediate transduction of freshly isolated or thawed, unstimulated HSPCs with higher gene transfer efficiencies than those obtained with VSVg-pseudotyped LV vectors and enables the transplantation of transduced HSPCs within less than 24 hr of cell isolation. These protocols provide detailed guidance on the production and titration of BaEVRLess-pseudotyped LV vectors and eVLPs and the cultivation and transduction of quiescent HSPCs, and highlights critical steps and potential pitfalls of these processes. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Generation of BaEVRLess-pseudotyped lentiviral vectors Alternate Protocol: Generation of BaEVRLess-pseudotyped virus-like particles Support Protocol: Preparation and testing of polyethyleneimine solution for DNA transfection Basic Protocol 2: Transduction of quiescent human CD34+ hematopoietic stem and progenitor cells.

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure disorder associated with myelodysplastic syndrome and acute myeloid leukemia predisposition. Nearly all patients carry a hypomorphic SBDS c.258+2T>C mutation, which produces aberrant mRNA splicing, reduces gene expression, and impairs ribosome biogenesis. Prime editing (PE) is a CRISPR-based genome editing method that enables versatile installation of short sequence changes templated by a prime editor guide RNA (pegRNA). PE has minimal off-target potential and designs that evade mismatch repair (MMR) bypass potentially genotoxic double strand break intermediates. Here, we investigate PE to correct the recurrent SBDS mutation in hematopoietic stem cells (HSCs). Using K562 cells engineered to carry SBDS c.258+2T>C, we observed that correcting the mutation alone (using a +3C>T pegRNA) was relatively inefficient, with 5.3% prime edits. We evaluated pegRNAs that introduced additional intronic substitutions predicted to evade MMR and preserve splicing and identified a pegRNA that when employed as PE2-type editing (without a second gRNA nicking the opposite strand) achieved 45.2% precise edits, resulting in efficient rescue of full-length SBDS transcript expression. Compared to PE3-type editing, PE2-type showed a similar editing efficiency but a markedly improved precise edit-to-indel ratio (120:1 vs. 18:1). Mouse embryonic fibroblasts (MEFs) engineered to carry a humanized intron 2 segment containing the c.258+2T>C mutation in the cognate mouse Sbds locus (c.199+2T>C) recapitulate the splicing defect (Peters et al. ASH 2022). PE resulted in efficient editing (68%), rescue of SBDS mRNA and protein expression, and normalization of the polysome profile including the 60S:80S ratio and translation rate as assessed by OP-Puro incorporation in Sbds c.199+2T>C homozygous MEFs and in SDS patient-derived fibroblasts. In 3 healthy donor CD34⁺ HSPCs, PE achieved 29±4% editing efficiency at the SBDSP1 pseudogene, which harbors the c.258+2T>C SBDS sequence, with undetectable editing of the nonmutant SBDS gene, consistent with the exquisite precision of PE. Peripheral blood CD34+ HSPCs were mobilized from a patient with SDS due to compound heterozygous SBDS c.258+2T>C and c.183_184del TAinsCT (p.K62*) mutations. After mobilization with G-CSF and plerixafor, 28 CD34+ HSPCs per mcL were mobilized and 3 blood volumes were processed by apheresis yielding 2.3x106 CD34+ HSPCs per kg. The patient harbored 2 somatic clones carrying TP53 mutations: c.524G>A (3.4% VAF) and c.535C>T (3.8% VAF). Ex vivo PE with PEmax-La mRNA and synthetic pegRNA electroporation produced 30% prime edits of SBDS. Using the same number of starting HSPCs allocated to each condition, untreated control and PE treated SDS patient cells were infused to NBSGW mice. Peripheral blood analysis showed increased human chimerism at 9 and 17 weeks in PE treated compared to untreated controls. In bone marrow, human chimerism was 14% in unedited recipients compared to 52% in PE-treated recipients. The edit frequency in PE-treated recipients increased from 30% in the infused HSPCs to 78% in the engrafted BM cells. Single cell RNA sequencing analysis showed that PE treatment rescues multilineage hematopoiesis and reverses ribosomal biogenesis and TP53 intrinsic apoptotic signaling gene expression signatures. Secondary transplantation showed a 12.4-fold increase in long-term repopulation capacity after PE treatment. SBDS edit frequency was 90% in engrafted secondary recipients. We further evaluated the impact of PE on the somatic TP53 mutant clones. In the controls, TP53 mutation frequency remained stable at 14% after engraftment compared to 15% in input HSPCs. In contrast, after PE treatment, the TP53 mutation frequency decreased from 19% in input HSPCs to 5% in engrafting cells. Colony-forming unit (CFU) analysis from engrafting HSPCs showed that 3 of 15 colonies lacking SBDS editing had a TP53 mutation (20%), while only 1 of 87 colonies with SBDS mutation correction showed a TP53 mutation (1.1%), suggesting that SBDS mutation correction counter-selects for engraftment of TP53 mutant HSPCs. Together, these results demonstrate a near-universal and efficient PE approach with high product purity that corrects the recurrent SBDS c.258+2T>C mutation and restores hematopoietic engraftment function in SDS patient HSPCs.

Gene and cell therapies pose safety concerns due to potential insertional mutagenesis by viral vectors. We introduce MELISSA, a regression-based statistical framework for analyzing Integration Site (IS) data to assess insertional mutagenesis risk, by estimating and comparing gene-specific integration rates and their impact on clone fitness. We characterized the IS profile of a lentiviral vector on Mesenchymal Stem Cells (MSCs) and compared it with that of Hematopoietic Stem and Progenitor Cells (HSPCs). We applied MELISSA to published IS data from patients enrolled in gene therapy clinical trials, successfully identifying both known and novel genes that drive changes in clone growth through vector integration. MELISSA offers a quantitative tool to bridge the gap between IS data and safety and efficacy evaluation, facilitating the generation of comprehensive data packages supporting Investigational New Drug (IND) and Biologics License (BLA) applications and the development of safe and effective gene and cell therapies.

In the original publication [...].

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.

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.

Introduction. Chimeric antigen receptor T cells (CAR-T), bispecific and antibody-drug conjugates are promising adoptive immunotherapies that can overcome the limitations of conventional cancer treatments and have demonstrated striking efficacy when targeting dispensable lineage antigens (Ag), e.g. CD19 for B-ALL. Nonetheless, the absence of safely actionable tumor-restricted markers hampers their application to other hematological malignancies, such as acute myeloid leukemia (AML). Since AML shares most surface markers with normal hematopoietic stem/progenitor cells (HSPC) or differentiated myeloid cells, on-target/off-tumor toxicities would result in myeloid aplasia and impairment of hematopoietic reconstitution. Furthermore, due to AML intra-tumoral heterogeneity, targeting more than one Ag may be required, exacerbating the risk of overlapping toxicity. Despite this, AML immunotherapies are currently under development, but their role will likely be restricted to bridge treatment before allogeneic HSPC transplantation (HSCT), decreasing the chances of AML eradication. Removal of targeted Ag through CRISPR-Cas KO from donor HSPCs used in HSCT has recently been proposed, but this can only be applied to genes dispensable for hematopoietic function. However, targeting irrelevant genes may facilitate tumor escape through Ag downregulation. Here, we show that precise editing of the targeted epitope within FLT3, KIT (CD117) and IL3RA (CD123) in HSPCs results in loss of Ab binding without KO, preserving physiologic protein expression, regulation, and intracellular signaling. Critically, this strategy enables targeting one or more genes fundamental for leukemia survival, resulting in potent anti-leukemia efficacy with minimal on-target/off-tumor toxicity. Methods. Through epitope-mapping, we identified substitutions in the FLT3, KIT and CD123 extracellular-domains that avoid detection by therapeutic Abs. We validated the functionality of mutated receptors (ligand affinity, western-blot, proliferation, RNAseq, phospho-proteomic MS) and their resistance to on-target killing (mAb-affinity, CAR-T co-culture). We optimized a base-editing protocol to introduce these mutations in CD34+HSPCs and developed advanced in vivo models with co-engraftment of healthy HSPCs, patient-derived AML xenografts (PDX) and CAR-T to assess selective elimination of leukemia and protection of healthy hematopoiesis. Results. To develop our approach, we selected mAbs under development for AML therapy: clone 4G8 (FLT3), Fab-79D (KIT) and 7G3 (CD123). To identify residues involved in mAb binding, we designed Sleeping Beauty epitope mapping libraries. We found that single amino-acid substitutions can disrupt therapeutic mAb binding despite preserved surface receptor. Since epitope engineering can be achieved by point mutations, we reasoned that base editing (BE) could be a suitable and safer option compared to homology directed repair (HDR). By electroporating sgRNA+ABE variants, we achieved successful epitope editing with minimal toxicity. By using fluorescent FLT3L, SCF and IL-3, we confirmed preserved ligand binding to edited receptors. Activation of downstream signaling was confirmed by western blot. By performing in vitro killing assays, we found that, while cells expressing WT FLT3, KIT or CD123 were eliminated, those expressing epitope-edited variants were resistant to CAR-mediated killing and survived up to experiment termination without eliciting T cell activation and degranulation. To introduce our variants into human HSPCs, we optimized a BE protocol on mobilized peripheral blood-derived CD34+ cells, achieving up to 86%, 78% and 78% efficiency for FLT3, KIT and CD123, respectively. Contrary to previous observations with HDR editing, BE efficiencies were similar in bulk and primitive, HSC-enriched subsets (CD90+45RA-) with no skewing of stem phenotype. BE HSPCs were resistant to CAR or mAb-mediated killing in vitro. To confirm the safety of our approach, we compared FLT3, CD123, KIT edited to AAVS1 control HSPCs and found no differences in proliferative response, transcriptional changes (RNAseq), phospho-proteomic profile and colony-forming capacity. Xenotransplantation of BE HSPCs in NBSGW mice showed preserved repopulation and multilineage differentiation capacity, both in primary and secondary recipients. To assess if FLT3 CAR can eliminate AML while sparing FLT3-edited hematopoiesis, we sequentially engrafted NBSGW mice with HSPCs and human PDX cells. We observed a significant increase in the percentage of FLT3 edited cells in CAR-treated mice and relative depletion of CD19+ subsets (pre-B, pro-B), granulocytes, granulo-mono progenitors (GMP) and lymphoid-primed multipotent progenitors (LMPP) only in the AAVS1-BE group, while mice engrafted with FLT3-BE HPSC were protected. Concomitantly, mice treated with 4G8-CAR achieved complete AML eradication. CAR-T exposed to FLT3-BE hematopoiesis displayed lower PD-1 expression compared to AAVS1-BE. As done for FLT3, we transplanted CD123-BE HSPCs and confirmed multilineage repopulation comparable to controls. Mice treated with CD123 CAR-T showed eradication of AML cells and concomitant protection of epitope-edited myeloid lineages, including granulocytes, DCs and HSPCs. To test whether our approach allows multiplexing, we tested combinations of FLT3, KIT and CD123 editing, which provided additive protection from triple-specific CAR-T cells in vitro. Furthermore, combined dual FLT3+CD123 BE could protect hematopoietic lineages in vivo when mice were treated with FLT3+CD123 CAR-T, which in turn were able to eradicate PDXs resistant to FLT3-targeting alone. Discussion. Our studies provide proof of concept that tumor-associated Ags shared by normal tissue can be safely targeted by precisely modifying the epitope recognized by adoptive immunotherapies in healthy cells, endowing them with selective resistance and generating an artificial leukemia-restricted Ag. The innovative tools developed in this work can increase the therapeutic index of AML immunotherapies and enable long-term anti-leukemia maintenance. By restricting on-target activity to leukemia cells, epitope-editing can reduce the Ag burden to which CAR-T are exposed, decreasing undesired CAR-T stimulation, cytokine secretion and exhaustion. Epitope editing can easily be multiplexed to enable combination therapies while avoiding overlapping toxicities, further enhancing the chances for tumor eradication. Finally, epitope editing may be exploited to improve non-genotoxic conditioning for autologous gene therapy or HSCT, either alone or in combination with other therapeutic targets, to avoid depletion of transplanted cells and achieve in vivo selection of genome-engineered cells. Conclusion. We believe that epitope-engineering of HSPCs is a novel and highly promising technology that can enable safer and more effective immunotherapies when on-target/off-tumor toxicities are the key limiting factor to successful clinical translation. Citation Format: Gabriele Casirati, Andrea Cosentino, Adele Mucci, Mohammed S. Mahmoud, Iratxe Ugarte Zabala, Jing Zeng, Scott B. Ficarro, Denise Klatt, Christian Brendel, Alessandro Rambaldi, Jerome Ritz, Jarrod A. Marto, Danilo Pellin, Daniel E. Bauer, Scott A. Armstrong, Pietro Genovese. Epitope editing enables targeted immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr NG05.

Hematopoietic stem cell transplantation with lentiviral vector (LVV) transduced autologous cells has proven an effective therapeutic strategy for sickle cell disease (SCD). However, ex vivo culture or proliferative stress associated with in vivo reconstitution may amplify any underlying genetic risk of leukemia. We aimed to minimize culture-induced stress and reduce genomic damage during ex vivo culture, enhance stem cell fitness and reconstitution of SCD CD34+ cells transduced with BCL11A shmiR-encoding LVV currently in clinical trials (NCT NCT03282656). UM171, a pyrimidoindole derivative can expand normal hematopoietic stem cells (HSCs) during in vitro culture and has been shown to be safe and effective in clinical trials using umbilical cord blood (NCT02668315). We examined the effect of UM171 during ex vivo LVV transduction of SCD HSCs. Culture of SCD CD34+ HSCs with UM171 during transduction reduced DNA damage and reactive oxygen species (ROS), decreased apoptosis, and was associated with increased numbers of immunophenotypically defined long-term HSCs. UM171 increased the engraftment of LVV transduced human HSCs in immunodeficient mice and barcode tracing revealed increased clonal diversity of engrafting cells. In competitive transplantation assays, analysis of BM showed that cells transduced in the presence of UM171 consistently outcompeted those transduced under control conditions. In summary, exposure of SCD peripheral blood CD34+ cells to UM171 during LVV transduction enhances stem cell fitness. These findings suggest manufacturing of genetically modified HSCs in the presence of UM171 may improve efficacy, safety and sustainability of gene therapy utilizing ex vivo approaches.

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high -titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and ef fi cient gene transfer to human CD34 + hematopoietic stem and progenitor cells (HSPCs). This LVV contains locus control region cores expressing an anti-sickling b AS3 -globin gene and two microRNAadapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbA AS3 + HbF), while concurrently decreasing sickle hemoglobin (HbS). The decrease in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34 + cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo . With high titer and enhanced transduction of HSPC at a low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to decrease costs and help improve accessibility to gene therapy for SCD.

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.