In vivo HSPC gene therapy of hemoglobinopathies without drug selection of corrected cells
- 1University of Washington, Department of Medicine, Division of Medical Genetics, Box 357720, Seattle, WA 98195, USA
Abstract
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: {gamma}-globin gene addition, {gamma}-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 {beta}/{beta}-thalassemia patient were subjected to EPORW439*-mediated EPO hypersensitivity alongside a therapeutic {gamma}-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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BibTeX
@misc{velmurugan2026vivo,
title = {{In vivo HSPC gene therapy of hemoglobinopathies without drug selection of corrected cells}},
author = {A. Sakunthala Velmurugan and K. Paschoudi and J. A. Queenan and S. Gil and A. Maynard and C. E. Fontana and R. Sharma and A. Long and C. Li and H. Wang and A. K. Anderson and A. Georgakopoulou and N. Psatha and M. Giannaki and E. Vlachaki and N. Ahmed and A. A. Sousa and D. R. Liu and E. Yannaki and A. Lieber and K. V. Karuppusamy},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.15.751289},
url = {https://www.biorxiv.org/content/10.64898/2026.09.15.751289v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - In vivo HSPC gene therapy of hemoglobinopathies without drug selection of corrected cells
AU - A. Sakunthala Velmurugan
AU - K. Paschoudi
AU - J. A. Queenan
AU - S. Gil
AU - A. Maynard
AU - C. E. Fontana
AU - R. Sharma
AU - A. Long
AU - C. Li
AU - H. Wang
AU - A. K. Anderson
AU - A. Georgakopoulou
AU - N. Psatha
AU - M. Giannaki
AU - E. Vlachaki
AU - N. Ahmed
AU - A. A. Sousa
AU - D. R. Liu
AU - E. Yannaki
AU - A. Lieber
AU - K. V. Karuppusamy
PY - 2026
DA - 2026/09/15
PB - bioRxiv
DO - 10.64898/2026.09.15.751289
UR - https://www.biorxiv.org/content/10.64898/2026.09.15.751289v1
AB - 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: {gamma}-globin gene addition, {gamma}-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 {beta}/{beta}-thalassemia patient were subjected to EPORW439*-mediated EPO hypersensitivity alongside a therapeutic {gamma}-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.
ER -