Dr Alyssa Cull - Centre for Blood Research at York - Kent Lab

Haematopoietic stem cell clonal dynamics following gene therapy for sickle cell disease

Gene therapy (GT) provides a potentially curative treatment option for patients with blood disorders including sickle cell disease (SCD). However, the occurrence of myeloid malignancies in GT clinical trials has raised significant concerns, with several postulated mechanisms to explain why cancers occur following therapy. Recent advances in clonal stem cell tracking strategies allow us for the first time to study unperturbed human haematopoiesis. We have developed a method that uses whole genome sequencing (WGS) to map the clonal dynamics of hematopoietic stem cells (HSCs) by employing spontaneous somatic mutations as unique clonal tags (Lee-Six et al., Nature, 2018). These tags enable the retrospective analysis of individual stem cell clones and the construction of phylogenetic trees mapping out stem cell relatedness, with mutations being acquired in a near-linear fashion over the course of an individual's life. In the context of SCD, we used this powerful approach to assess the clonal dynamics of gene-modified and unmodified HSCs in six patients with SCD at both pre- and post-GT time points. 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 haematopoiesis (DNMT3A- and EZH2-mutated clones in particular) was observed across the sampled HSC population, suggesting positive selection of mutant clones during GT. These findings highlight the importance of identifying low frequency HSCs harbouring pre-existing driver mutations that would not be detected using standard clinical sequencing panels. Future studies will define the long-term fate of these rapidly expanding mutant clones, including any contribution to expansions associated with myeloid neoplasms.