bioRxiv : the preprint server for biologySep 24, 2026

Gene therapy for blood disorders using stem cells without drug-based selection of fixed cells

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Sep 25, 2026

Abstract

Over 70% of erythroid cells showed positive hemoglobin F after treatment involving an EPOR variant.

In vivo editing of hematopoietic stem/progenitor cells (HSPCs) was conducted using a nonpathogenic EPOR variant to enhance erythropoietin sensitivity.
The combination of EPOR editing with three therapeutic approaches aimed to correct hemoglobinopathies, including gene addition and mutation correction.
In vitro studies indicated that the EPORW439* variant provided a significant growth advantage to genetically modified erythroid progenitors.
Mice treated with CD34+ cells from a beta-thalassemia patient exhibited a notable reduction in disease symptoms alongside increased HbF-positive erythroid cells.
Therapeutic effects, such as decreased oxidative stress and iron deposition, were observed following a single intravenous administration of the vectors.

Simplified

In vivo hematopoietic stem/progenitor cell (HSPC) gene therapy remains limited by low gene-editing efficiency and a lack of clinically applicable selection strategies to enrich therapeutically corrected progeny. We used in vivo base and prime editing to introduce a nonpathogenic EPOR variant into HSPCs, conferring erythropoietin hypersensitivity and promoting preferential expansion of gene-corrected erythroid cells. EPOR editing was combined with three therapeutic approaches for the correction of hemoglobinopathies: γ-globin gene addition, γ-globin reactivation, or correction of the sickle cell disease mutation. Tropism-modified helper-dependent adenoviral vectors (HDAd6/3+) targeting HSPCs were used to simultaneously deliver the EPOR-editing machinery and the corresponding therapeutic components. In vitro studies in an erythroid progenitor cell line and primary CD34+ cells demonstrated that the EPORW439 variant conferred a strong proliferative advantage to therapeutically modified erythroid progenitors. Mice humanized with CD34+ cells from a β⁰/β⁰-thalassemia patient were subjected to EPORW439-mediated EPO hypersensitivity alongside a therapeutic γ-globin transgene which resulted in >70% HbF-positive erythroid cells and substantial reversion of the disease-associated phenotype, including reduced oxidative stress, near-complete elimination of splenic iron deposition, and reduced splenomegaly. Importantly, these effects were achieved after simple intravenous administration of the vectors following HSPC mobilization and cytokine prophylaxis, without subsequent pharmacologic selection. Together, these findings establish a strategy to amplify the therapeutic benefit of otherwise limited in vivo HSPC gene editing for hemoglobinopathies.

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Gene therapy for blood disorders using stem cells without drug-based selection of fixed cells

Abstract

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