Therapeutic gene editing of t cells corrects ctla4 insufficiency

Background: Heterozygous mutations in CTLA4 result in an inborn error of immunity (IEI) (also known as primary immunodeficiency) with a severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic stem cell transplantation. The mutational landscape and requirement for tight regulation of CTLA4 make viral gene addition approaches unappealing. Gene editing strategies permit alteration of CTLA4 while retaining the endogenous gene control machinery. Aims: We set out to devise a CRISPR/Cas9/AAV6 gene editing strategy to correct CTLA4 insufficiency in T cells. Methods: We designed several homology directed repair (HDR) editing strategies that would correct the genetic defect. We first assessed correction of an individual point mutation. We then evaluated several universal strategies that enable correction of most disease-causing mutations with a single edit; the first that inserts the CTLA4 cDNA in exon 1, and a second that inserts the CTLA4 cDNA at the 3’end of the first intron of CTLA4. All AAV6 HDR donor templates included a GFP reporter gene to enable easy identification of the edited cells. Results: Superior editing efficiencies were obtained with the intronic approach compared to the other editing strategies and this strategy was then further evaluated. CTLA4 function and expression kinetics were assessed following editing using flow cytometry-based assays. Functional studies using CTLA4 transendocytosis (TE) assays, demonstrated restoration of CD80 and CD86 internalization in the edited CD4+ T cells. Following gene editing, transgene expression kinetics were comparable to healthy control CD4+ T cells. Gene editing of T cells isolated from patients with CTLA4 insufficiency restored CTLA4 expression and rescued transendocytosis of CD80 and CD86 in vitro. Using a similar approach, gene corrected T cells from CTLA4-/- mice engrafted in immunodeficient mice at clinically relevant frequencies and tail vein bleeds were performed 1, 3 and 4 weeks post adoptive transfer. In the mice which received the GFP+ edited cells, a stable population of GFP+ cells was detectable at all timepoints demonstrating in vivo persistence as well as genetic stability. All mice were sacrificed 4 weeks after cell transfer. To assess lymphoproliferation, the cellularity of peripheral lymph nodes and spleen weight were analyzed. Spleen and lymph node size, lymph node cell counts, and spleen weight were all significantly lower in mice which received the edited cells (n=5) compared to non-edited controls (n=5) and there was no significant difference in these parameters between mice who had received edited CTLA4-/- T cells and those who received WT T cells (n=4). Analysis of lymph node and spleen cells confirmed persistence of CTLA4 expression in the edited (GFP+) cells and revealed that a higher proportion of Treg than Tconv had been successfully edited. Summary/Conclusion: Together these data demonstrated that CTLA4 edited T cells survived in vivo, expressed CTLA4 and were able to control the clinical phenotype of CTLA4 insufficiency, providing a powerful proof-of-principle of our T cell GT approach. Our data provide proof-of-concept that gene editing can restore CTLA4 function in T cells demonstrating the potential of this approach to treat CTLA4 insufficiency. A similar approach could be used in other IEIs that are caused by multiple heterozygous mutations.