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Autologous hematopoietic stem cell transplantation (ASCT) improves survival in multiple myeloma (MM). However, many individuals are unable to collect optimal CD34 + hematopoietic stem and progenitor cell (HSPC) numbers with granulocyte colony-stimulating factor (G-CSF) mobilization. Motixafortide is a novel cyclic-peptide CXCR4 inhibitor with extended in vivo activity. The GENESIS trial was a prospective, phase 3, double-blind, placebo-controlled, multicenter study with the objective of assessing the superiority of motixafortide + G-CSF over placebo + G-CSF to mobilize HSPCs for ASCT in MM. The primary endpoint was the proportion of patients collecting ≥6 × 10 6 CD34 + cells kg –1 within two apheresis procedures; the secondary endpoint was to achieve this goal in one apheresis. A total of 122 adult patients with MM undergoing ASCT were enrolled at 18 sites across five countries and randomized (2:1) to motixafortide + G-CSF or placebo + G-CSF for HSPC mobilization. Motixafortide + G-CSF enabled 92.5% to successfully meet the primary endpoint versus 26.2% with placebo + G-CSF (odds ratio (OR) 53.3, 95% confidence interval (CI) 14.12–201.33, P  < 0.0001). Motixafortide + G-CSF also enabled 88.8% to meet the secondary endpoint versus 9.5% with placebo + G-CSF (OR 118.0, 95% CI 25.36–549.35, P  < 0.0001). Motixafortide + G-CSF was safe and well tolerated, with the most common treatment-emergent adverse events observed being transient, grade 1/2 injection site reactions (pain, 50%; erythema, 27.5%; pruritis, 21.3%). In conclusion, motixafortide + G-CSF mobilized significantly greater CD34 + HSPC numbers within two apheresis procedures versus placebo + G-CSF while preferentially mobilizing increased numbers of immunophenotypically and transcriptionally primitive HSPCs. Trial Registration: ClinicalTrials.gov , NCT03246529 The phase 3 GENESIS trial reports the superiority of the novel CXCR4 inhibitor motixafortide with G-CSF in mobilizing hematopoietic progenitor cells for autologous stem cell transplantation in multiple myeloma.

Among boys with cerebral adrenoleukodystrophy, matched donors for allogeneic transplantation are available to only a few. Infusion of CD34+ autologous stem cells transduced with a lentiviral vector prevented neurologic deterioration in 15 of 17 boys with the disease.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells 1 , 2 . We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34 + hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4–1.8 copies per neutrophil) and the persistence of 16–46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients. Initial results from phase I/II lentiviral gene therapy trials provide early evidence supporting its safety and efficacy in treating patients with X-linked chronic granulomatous disease.

In β-thalassemia, either γ-globin induction to form fetal hemoglobin (α2γ2) or β-globin repair to restore adult hemoglobin (α2β2) could be therapeutic. ABE8e, a recently evolved adenine base editor variant, can achieve efficient adenine conversion, yet its application in patient-derived hematopoietic stem cells needs further exploration. Here, we purified ABE8e for ribonucleoprotein electroporation of β-thalassemia patient CD34 + hematopoietic stem and progenitor cells to introduce nucleotide substitutions that upregulate γ-globin expression in the BCL11A enhancer or in the HBG promoter. We observed highly efficient on-target adenine base edits at these two regulatory regions, resulting in robust γ-globin induction. Moreover, we developed ABE8e-SpRY, a near-PAMless ABE variant, and successfully applied ABE8e-SpRY RNP to directly correct HbE and IVS II-654 mutations in patient-derived CD34 + HSPCs. Finally, durable therapeutic editing was produced in self-renewing repopulating human HSCs as assayed in primary and secondary recipients. Together, these results support the potential of ABE-mediated base editing in HSCs to treat inherited monogenic blood disorders. Here, Liao and colleagues apply adenine base editor ABE8e and its PAM-less variant ABE8e-SpRY to β-thalassemia patient hematopoietic stem cells in the form of ribonucleoprotein complexes, resulting in efficient long-term editing and β-thalassemia alleviation.

Of 30 patients with severe sickle cell disease who were treated with gene-edited autologous hematopoietic stem and progenitor cells, 29 were free from vaso-occlusive crises for at least 12 consecutive months.

Severe hemophilia A is managed with factor VIII replacement or hemostatic products that stop or prevent bleeding. Data on gene therapy with hematopoietic stem-cell (HSC)-based expression of factor VIII for the treatment of severe hemophilia A are lacking. We conducted a single-center study involving five participants 22 to 41 years of age with severe hemophilia A without factor VIII inhibitors. Autologous HSCs were transduced with CD68-ET3-LV - a lentiviral vector including a new transgene ( ) with a myeloid-directed CD68 promoter - either without transduction enhancer (group 1) or with transduction enhancer (group 2). Transduced HSCs were transplanted into recipients after myeloablative conditioning. The treatment was assessed for safety (engraftment and regimen-related toxic effects) and efficacy (factor VIII activity and annualized bleeding rate). Participants received CD68-ET3-LV-transduced autologous CD34+ HSCs at doses of 5.0×10 to 6.1×10 per kilogram of body weight. The vector copy numbers in the final drug product were 1.0 and 0.6 copies per cell for the two participants in group 1 and 1.5, 0.6, and 2.2 copies per cell for the three participants in group 2. The duration of severe neutropenia was 7 to 11 days and of severe thrombocytopenia was 1 to 7 days. The median factor VIII activity level, measured with the use of a one-stage assay, after day 28 until the last follow-up visit was 5.2 IU per deciliter (range, 3.0 to 8.7) and 1.7 IU per deciliter (range, 1.0 to 4.0) with a peripheral-blood vector copy number of 0.2 and 0.1 copies per cell, respectively, in the two group 1 participants, and 37.1 IU per deciliter (range, 18.3 to 73.6), 19.3 IU per deciliter (range, 6.6 to 34.5), and 39.9 IU per deciliter (range, 20.6 to 55.1) with a peripheral-blood vector copy number of 4.4, 3.2, and 4.8 copies per cell, respectively, in the three group 2 participants. The annualized bleeding rate was zero for all five participants over a cumulative follow-up of 81 months (median follow-up, 14 months; range, 9 to 27). Gene therapy for hemophilia A with the use of lentiviral vector-transduced autologous HSCs resulted in stable factor VIII expression, with factor VIII activity correlating to vector copy number in the peripheral blood. (Funded by the Ministry of Science and Technology, Government of India, and others; ClinicalTrials.gov number, NCT05265767; Clinical Trials Registry-India number, CTRI/2022/03/041304.).

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia 1 , 2 , the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens 3 – 5 . Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34 + HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning. Epitope engineering of donor haematopoietic stem/progenitor cells endows haematopoietic lineages with selective resistance to CAR T cells or monoclonal antibodies, without affecting protein function or regulation, enabling the targeting of genes that are essential for leukaemia survival and reducing the risk of tumour immune escape.

Background Gastric cancer is a considerable burden for worldwide patients. And diffuse gastric cancer is the most insidious subgroup with poor survival. The phenotypic characterization of the diffuse gastric cancer cell line can be useful for gastric cancer researchers. In this article, we aimed to characterize the diffuse gastric cancer cells with MRI and transcriptomic data. We hypothesized that gene expression pattern is associated with the phenotype of the cells and that the heterogeneous enhancement pattern and the high tumorigenicity of SNU484 can be modulated by the perturbation of the highly expressed gene. Methods We evaluated the 9.4 T magnetic resonance imaging and transcriptomic data of the orthotopic mice models from diffuse gastric cancer cells such as SNU484, Hs746T, SNU668, and KATO III. We included MKN74 as an intestinal cancer control cell. After comprehensive analysis integrating MRI and transcriptomic data, we selected CD34 and validated the effect by shRNA in the BALB/c nude mice models. Results SNU484, SNU668, Hs746T, and MKN74 formed orthotopic tumors by the 5 weeks after cell injection. The diffuse phenotype was found in the SNU484 and Hs746T. SNU484 was the only tumor showing the heterogeneous enhancement pattern on T2 images with a high level of CD34 expression. Knockdown of CD34 decreased the round-void shape in the H&E staining ( P  = 0.028), the heterogeneous T2 enhancement, and orthotopic tumorigenicity (100% vs 66.7%). The RNAseq showed that the suppressed CD34 is associated with the downregulated gene-sets of the extracellular matrix remodeling. Conclusion Suppression of CD34 in the human-originated gastric cancer cell suggests that it is important for the round-void histologic shape, heterogeneous enhancement pattern on MRI, and the growth of gastric cancer cell line.

With CRISPR-Cas9, a repressor of fetal hemoglobin expression was disrupted in hematopoietic stem cells from three persons with sickle cell disease. Edited cells were reinfused. Hematopoiesis was restored.

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient’s own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders. Mucopolysaccharidosis type I (MPSI) is a lysosomal storage disease caused by insufficient iduronidase (IDUA) activity. Here, the authors use an ex vivo genome editing approach to overexpress IDUA in human hematopoietic stem and progenitor cells and show it can phenotypically correct MSPI in mouse model.