Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo

Abstract

Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand () gene with a median survival of 25 years, potentially treatable with CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of in edited healthy donor and patient cells recapitulating the physiological regulation. We then documented the preserved fitness of edited cells by xenotransplantation into immunodeficient mice. Finally, we transitioned to large-scale manufacturing, and developed a panel of quality control assays. Overall, our GMP-compliant process takes long-range gene editing one step closer to clinical application with a reassuring safety profile.This original article describes the development of a scalable GMP-compliant process for CD4+ T-cell gene correction in X-linked hyper-IgM syndrome caused by CD40LG mutations. Starting from a research-grade editing protocol, the authors optimized CD4+ cell selection, gene editing, enrichment of corrected cells and expansion using an integrase-defective lentiviral vector as donor template. The process preserved stem and central memory phenotypes, restored physiologically regulated CD40LG expression and function in healthy donor and patient cells, and showed preserved fitness of edited cells in a xenotransplantation model. The study also developed a quality-control panel, bringing long-range gene editing of CD4+ T cells closer to clinical translation for Hyper-IgM1.