Clonal selection of hematopoietic stem cells after gene therapy for sickle cell disease.

Acknowledgements: This work was supported by the Bill and Melinda Gates Foundation (INV-002189 and INV-038816, D.G.K.). M.S.C. was supported by a Wellcome Clinical PhD Fellowship. Work conducted by D.A.W. and colleagues was supported by National Institutes of Health grant R01HL (137848) and NHLBI Cure Sickle grant (OT2HL 154815). Investigators at the Sanger Institute were supported by a core grant from the Wellcome Trust. This research was funded in part by the Wellcome Trust (206194 and 108413/A/15/D). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. J.N. is supported by a Cancer Research UK Fellowship. Work in the D.G.K. laboratory is supported by a European Research Council Starting Grant (ERC-2016-STG-715371) and a Cancer Research UK Programme Foundation Award (DCRPGF\100008). The authors thank the IT Support team of the Cancer, Ageing and Somatic mutation programme at the Sanger Institute for their support and V. Sankaran and L. Naldini for helpful discussion. The authors also thank the patients for donating the samples that have been used in this study.Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT clinical trials has prompted concern, with several postulated mechanisms. Here, we used whole-genome sequencing to track hematopoietic stem cells (HSCs) from six patients with SCD at pre- and post-GT time points to map the somatic mutation and clonal landscape of gene-modified and unmodified HSCs. Pre-GT, phylogenetic trees were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified among gene-modified or unmodified cells; however, an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed in both genetically modified and unmodified cells, suggesting positive selection of mutant clones during GT. This work sheds light on HSC clonal dynamics and the mutational landscape after GT in SCD, highlighting the enhanced fitness of some HSCs harboring pre-existing driver mutations. Future studies should define the long-term fate of mutant clones, including any contribution to expansions associated with myeloid neoplasms

Clonal selection of hematopoietic stem cells after gene therapy for sickle cell disease

Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT clinical trials has prompted concern, with several postulated mechanisms. Here, we used whole-genome sequencing to track hematopoietic stem cells (HSCs) from six patients with SCD at pre- and post-GT time points to map the somatic mutation and clonal landscape of gene-modified and unmodified HSCs. Pre-GT, phylogenetic trees were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified among gene-modified or unmodified cells; however, an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed in both genetically modified and unmodified cells, suggesting positive selection of mutant clones during GT. This work sheds light on HSC clonal dynamics and the mutational landscape after GT in SCD, highlighting the enhanced fitness of some HSCs harboring pre-existing driver mutations. Future studies should define the long-term fate of mutant clones, including any contribution to expansions associated with myeloid neoplasms

Clonal selection of hematopoietic stem cells after gene therapy for sickle cell disease.

Acknowledgements: This work was supported by the Bill and Melinda Gates Foundation (INV-002189 and INV-038816, D.G.K.). M.S.C. was supported by a Wellcome Clinical PhD Fellowship. Work conducted by D.A.W. and colleagues was supported by National Institutes of Health grant R01HL (137848) and NHLBI Cure Sickle grant (OT2HL 154815). Investigators at the Sanger Institute were supported by a core grant from the Wellcome Trust. This research was funded in part by the Wellcome Trust (206194 and 108413/A/15/D). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. J.N. is supported by a Cancer Research UK Fellowship. Work in the D.G.K. laboratory is supported by a European Research Council Starting Grant (ERC-2016-STG-715371) and a Cancer Research UK Programme Foundation Award (DCRPGF\100008). The authors thank the IT Support team of the Cancer, Ageing and Somatic mutation programme at the Sanger Institute for their support and V. Sankaran and L. Naldini for helpful discussion. The authors also thank the patients for donating the samples that have been used in this study.Gene therapy (GT) provides a potentially curative treatment option for patients with sickle cell disease (SCD); however, the occurrence of myeloid malignancies in GT clinical trials has prompted concern, with several postulated mechanisms. Here, we used whole-genome sequencing to track hematopoietic stem cells (HSCs) from six patients with SCD at pre- and post-GT time points to map the somatic mutation and clonal landscape of gene-modified and unmodified HSCs. Pre-GT, phylogenetic trees were highly polyclonal and mutation burdens per cell were elevated in some, but not all, patients. Post-GT, no clonal expansions were identified among gene-modified or unmodified cells; however, an increased frequency of potential driver mutations associated with myeloid neoplasms or clonal hematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed in both genetically modified and unmodified cells, suggesting positive selection of mutant clones during GT. This work sheds light on HSC clonal dynamics and the mutational landscape after GT in SCD, highlighting the enhanced fitness of some HSCs harboring pre-existing driver mutations. Future studies should define the long-term fate of mutant clones, including any contribution to expansions associated with myeloid neoplasms

Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease

BACKGROUND: Sickle cell disease is characterized by hemolytic anemia, pain, and progressive organ damage. A high level of erythrocyte fetal hemoglobin (HbF) comprising alpha- and gamma-globins may ameliorate these manifestations by mitigating sickle hemoglobin polymerization and erythrocyte sickling. BCL11A is a repressor of gamma-globin expression and HbF production in adult erythrocytes. Its down-regulation is a promising therapeutic strategy for induction of HbF.METHODS: We enrolled patients with sickle cell disease in a single-center, open-label pilot study. The investigational therapy involved infusion of autologous CD34+ cells transduced with the BCH-BB694 lentiviral vector, which encodes a short hairpin RNA (shRNA) targeting BCL11A mRNA embedded in a microRNA (shmiR), allowing erythroid lineage-specific knockdown. Patients were assessed for primary end points of engraftment and safety and for hematologic and clinical responses to treatment.RESULTS: As of October 2020, six patients had been followed for at least 6 months after receiving BCH-BB694 gene therapy; median follow-up was 18 months (range, 7 to 29). All patients had engraftment, and adverse events were consistent with effects of the preparative chemotherapy. All the patients who could be fully evaluated achieved robust and stable HbF induction (percentage HbF/(F+S) at most recent follow-up, 20.4 to 41.5%), with HbF broadly distributed in red cells (F-cells 58.9 to 93.6% of untransfused red cells) and HbF per F-cell of 9.0 to 18.6 pg per cell. Clinical manifestations of sickle cell disease were reduced or absent during the follow-up period.CONCLUSIONS: This study validates BCL11A inhibition as an effective target for HbF induction and provides preliminary evidence that shmiR-based gene knockdown offers a favorable risk-benefit profile in sickle cell disease. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656)