Izotova, Natalia; (2022) Immune cell tracking following hematopoietic cell and gene therapy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this project we combined standard cellular and molecular assays with a custom PCR-based technology based on high throughput sequencing to track genetically engineered cells in treated patients by means of viral integration sites (IS) analysis. We leveraged this analytical pipeline to 1) assess whether naïve T cells can still be produced for many years even in absence of any supply by multipotent progenitors in the bone marrow and 2) to investigate the origin of CAR-T cells that mediate anti-leukaemic responses or long-term immune surveillance. In a clinical trial X-linked Severe Combined Immunodeficiency patients received an infusion of autologous hematopoietic stem/progenitor cells corrected via retroviral vector encoding the interleukin-2 common cytokine receptor gamma chain. In these patients, many years after gene therapy only vector-positive T and NK cells persist while no other genetically engineered blood cell populations are detectable. By a comprehensive long-term immunophenotypic, molecular and functional characterization we demonstrated that the thymus is actively producing a new and diverse repertoire of vector-positive naïve T cells (TN). This suggests that, even if gene corrected HSC are absent, a de novo production of genetically engineered T cell is maintained by a population of gene-corrected long term lymphoid progenitors (Lt-LP). Moreover, tracking IS clonal markers overtime, we inferred that Lt-LP can support both T and NK cells production. In a separate clinical trial of CD19-CAR-T cells for the treatment of haematological malignancies, we used IS analysis to investigate the origin of short- and long-term circulating CAR-T cells that mediate early anti-leukaemic responses or long-term immune surveillance. We compared IS between the product and CAR-T cells at early/late timepoints in vivo. This analysis suggested that T memory stem cells (TSCMs) contained in the infused cell product, contributed the most to the generation of CAR-T cell clones during the peak response phase as well as to the generation of long-term persisting CAR-T cells.

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