Hematopoietic stem cell (HSC) gene therapy has demonstrated the potential to become the treatment of choice for many monogenic diseases, as reported by the recent success of several clinical trials. An autologous gene therapy approach offers several advantages over allogeneic HSC transplantation (HSCT), overcoming the immunological risks of graft-vs-host disease and graft rejection and relieving the need for post-transplant immunosuppressive regimens, thus significantly reducing the morbidity and mortality of the transplant procedure. However, the random integration pattern of currently used gene transfer vectors still poses the risk of insertional mutagenesis and ectopic/unregulated expression of the therapeutic transgene. The recent development of gene editing technologies, which enable precise correction of a locus of interest, has the potential to overcome these issues and thus drastically change the scenario of genomic manipulation for HSC therapies. When working in the group of Luigi Naldini, San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), we have originally shown that editing in primitive HSC is constrained by gene transfer efficiency and limited proficiency of homology-directed repair, likely due to HSC quiescence and low expression of the DNA repair machinery. We could overcome these barriers at least in part by culturing HSC in conditions that induce cell activation while preserving long-term repopulation capacity and by tailoring the delivery vehicles for the editing machinery to achieve a transient but robust spike of expression with limited toxicity. By this strategy, we provided for the first time evidence of targeted gene correction of SCID-X1 causing mutations on the IL2RG gene in HSC collected from an affected patient (Genovese et al., Nature 2014). To support the rationale for further developing this gene correction strategy as compared to other available approaches, we took advantage of a humanized SCID-X1 mouse model. Using this setting, we unraveled unexpected safety concerns related to the transplantation of low cellular amounts into a SCID-X1 host, developed a new genetic engineering protocol for mouse HSCs, and explored the use of biological conditioning in the context of primary immunodeficiencies (Schiroli et al., Science Translational Medicine 2017).
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One of the major advantages of in situ gene correction strategies is the possibility of restoring both the function and the expression control of the affected gene (Ferrari et al., Frontiers in Genome Editing 2021). This issue becomes even more relevant when the affected gene is directly active in cell differentiation and proliferation because its ectopic or constitutive expression may trigger uncontrolled cell expansion with a significant risk of oncogenic transformation, such as in the case of the CD40LG gene in the X-linked hyper-IgM syndrome (HIGM1).
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Since in the HIGM1 the genetic defect is not lethal to T cells, we compared the therapeutic potential of adoptive T-cell transfer and HSC transplantation with increasing fraction of functional cells into the HIGM1 mouse model and showed comparable and substantial benefits for both approaches. We then developed a conditional “one-size-fits-all” strategy for the safe correction of CD40LG in both human T cells and HSPCs and reached unprecedented efficiencies that match or surpass the therapeutic threshold predicted in the mouse model. In order to increase the total amount and relative proportion of functional cells in the therapeutic product, we aimed to enrich for corrected cells in vitro by transcriptionally coupling the corrective cDNA to a clinically compatible, optimized version of the truncated EGFR gene. This “purifying” selection strategy allows enrichment, tracking, and depletion, in case of adverse events, of edited cells with a clinical-grade monoclonal antibody and, surprisingly, also increased CD40L expression, reaching, for the first time in the field, physiologic levels of expression of a cDNA-edited gene. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cells ahead of HSC gene therapy because of easier translation, lower safety concerns, and potentially comparable clinical benefits (Vavassori, Mercuri et al., EMBO Molecular Medicine 2021). This strategy is currently in advanced phases of manufacturing development, and a first-of-this-kind trial for this disease is expected to be opened at SR-Tiget in a few years.
Mutations of the RAG1 gene are among the most frequent causes of immunodeficiency in humans, ranging from severe combined immunodeficiency (SCID) to autoimmunity. The mainstay of treatment for patients with severe forms of RAG1 deficiency is allogeneic HSC transplantation, but often, it results in a high rate of graft failure and incomplete T and B cell immune reconstitution. While these observations indicate the need to develop novel forms of definitive cure, preclinical models of lentivirus-based gene therapy aimed at delivering a normal copy of the RAG1 cDNA in Rag1-/- mice have resulted in incomplete immune reconstitution due to suboptimal and dysregulated transgene expression. The RAG1 gene is tightly regulated in lymphocyte precursors, and its inappropriate expression may lead to double-strand breaks and chromosomal translocations or may result in the development of autoimmune reactions, as in Omenn syndrome. Thus, in collaboration with the group of Anna Villa, San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), and Luigi Notarangelo, NIAID, we aim to address this unmet medical need and develop an effective novel treatment directed at restoring both function and expression control of the RAG1 gene in autologous patient-derived HSC (Castiello et al., Science Translational Medicine 2024).