Development, Optimization and Preclinical Modeling of Hematopoietic Stem Cell Gene Editing for the Treatment of RAG1 Immunodeficiency

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PROJECT SUMMARY Hematopoietic Stem/Progenitor Cells (HSPC) gene therapy has provided clinical benefits in several patients affected by a variety of genetic diseases, some of which already reached market authorization for selected indications. However, the use of semi-randomly integrating vectors poses the risk of insertional mutagenesis and ectopic/unregulated transgene expression. These issues become even more relevant when the affected gene needs to be highly express to exert its function and when its activity directly impacts genome stability, such as the case for Recombination-Activating 1 (RAG1) gene. RAG1 is express in a high but tightly regulated manner in differentiating lymphocyte precursors, where it directs the VDJ recombination process required for assembly the T- and B-cell receptors, and its inactivating mutations are one of the most frequent causes of severe- combined immunodeficiency (SCID). While the high risk of genomic damage due to unregulated RAG1 expression has so far hampered the use of viral vectors to treat RAG1 deficiencies, there is a need to develop novel and effective therapeutic approaches, especially for patients who lacks a compatible HSPC donor or are not eligible for allogeneic transplant. The long-term goal of our proposal is to address this unmet medical need and develop an effective novel treatment directed at restoring both function and expression control of the RAG1 gene on autologous patient derived HSC. Our central hypothesis is that gene repair strategies that preserve physiologic expression control represent a safe and effective approach for treating RAG1 deficiencies. We reported that by tailoring culture conditions and gene delivery vehicles, it is possible to partially overcome the biologic barriers that constrain gene editing in the most primitive and clinically relevant HSPC subsets (Genovese, Nature 2014; Schiroli, Science-Translational-Medicine 2017). Within this project we will capitalize our previous achievements to i) directly fix RAG1 mutations, ii) improve efficiency of current HSPC gene editing protocols and iii) investigate non-genotoxic conditioning on suitable mouse models. Functional correction of the engineered RAG1 gene will be stringently assessed on patient derived cells, by exploiting state-of-the-art in vitro T cell differentiation assay and in vivo xenotransplantation experiments. We will take advantage of our recently optimized gene editing procedure and barcoding technology (BAR-seq, Ferrari et al, Nat. Biotech. 2020) to maximize editing efficiency while reducing cellular toxicity on the treated HSPC, thus increasing the yield of long- term engrafting lymphoid cells. To support the rational for clinical testing, we will assess correction of the disease phenotype by limiting amounts of functional HSPC in two RAG1 murine models and test efficacy of emerging immunotoxin conditioning regimens to reduce transplant toxicity and increase lymphoid reconstitution. Overall, this project will contribute to the development of an innovative treatment approach for RAG1 deficiencies and position homology-based gene editing as a standard for precise HSC engineering, providing for safer and more efficacious therapeutic strategies with broad applicability in hematology.