Adaptive Routes of Hematopoietic Stem Cell Differentiation to Disease Conditions and Age in Gene Therapy Patients

In lentiviral vector-based Hematopoietic Stem Progenitor Cell (HSPC) gene therapy (GT), transplantation of genetically modified HSPCs can result in the full reconstitution of the patients’ hematopoietic system and provide a therapeutic benefit in a variety of genetic diseases. However, it is still unclear how the underlying genetic disease and other factors may impact the reconstitution process, lineage specification, and patients’ safety.

Here we analyzed the clonal repertoire and dynamics of hematopoietic reconstitution by studying >4 million vector integration sites (IS), a surrogate of clonal identity, from purified myeloid, lymphoid, erythroid cell lineages and HSPCs from 53 patients treated by lentiviral-based HSPC-GT up to 8 years of follow-up, 29 patients for metachromatic leukodystrophy (MLD) - a neurodegenerative lysosomal storage disorder, 15 patients for Wiskott-Aldrich syndrome (WAS) - an immunodeficiency with thrombocytopenia, and 9 patients for β-thalassemia (β-Thal) - a hemoglobinopathy.

This analysis showed that patients had a polyclonal repertoire without signs of insertional mutagenesis. We next dissected the hematopoietic reconstitution over time in terms of HSPC numbers, output and commitment of HSPCs toward the different lineages. We estimated that the number of engrafted and active HSPCs in patients over time ranged from 770 to 35,000 cells, remaining stable long-term. The estimates were based on the Chao1 model, a well-established capture-recapture statistics developed in ecology. A large fraction of clones estimated in the early phase were exhausted within 12 months and were replaced by a smaller, yet substantial, number of long-term IS in stable and sustained steady-state hematopoiesis. We did not observe any statistical difference of the fraction of HSPCs actively contributing to hematopoiesis at steady state across the 3 trials, ranging from 0.0007% to 0.0274%. Unsupervised clustering analysis revealed a positive correlation between the clonal diversity and the total dose (CD34+ cells 10e6/kg) of infused HSPCs, and between in vivo HSPC size and the patient’s age.

About 50% of hematopoietic clones had multilineage potential in all disease conditions, while the remaining clones showed a preferential lineage output and long-term linage commitment which was specific to the disease condition: MLD patients showed a long-term commitment towards myeloid lineages, while patients treated for WAS had a preferential output and commitment towards lymphoid lineages and β-Thal for erythroid lineages, especially in adult patients.

Our results showed for the first time that the long-term output, as well as the lineage commitment of HSPCs, is strongly modulated by the patients’ genetic background to better compensate for the demands posed by the specific clinical condition