Base Editing of Hematopoietic Stem Cells Rescues T-Cell Development for CD3d Severe Combined Immunodeficiency

Introduction: CD3δ severe combined immunodeficiency (SCID) is a devastating inborn error of immunity caused by a homozygous mutation in the CD3D gene (c.202C>T). This defect results in the absence of CD3δ protein and early arrest of thymocyte development. Predominately found in a Mennonite population, CD3δ SCID patients present with a profound deficiency of circulating, mature αβ and γδ T cells, often leading to infant mortality. Here, we demonstrate a novel, adenine base editing (ABE) gene therapy for autologous HSCT without requiring double-stranded breaks or donor DNA templates.

Methods and Results: Preliminary data in a clonal Jurkat T-cell disease model (CD3D(C202T) Jurkat T cells) revealed 93 ± 2.3% (mean ± SD) correction of CD3D c.202C>T and restored expression of CD3 protein complex in up to 85% of cells manipulated with sgRNA + ABEmax- NRTH. Calcium flux analysis demonstrated wildtype levels of CD3/TCR signaling in CD3D(C202T) Jurkat T cells treated with ABEmax-NRTH, suggesting ABE as a highly efficient correction strategy for CD3δ SCID. We next explored the ability to base edit the pathogenic mutation in long-term, repopulating HSCs in a humanized xenograft model. Healthy human CD34+ HSPCs were transduced with a lentiviral vector (LV) expressing a CD3D cDNA containing the CD3D c.202C>T mutation. Transduced HSPCs were electroporated with mRNA encoding ABEmax-NRTH and sgRNA to edit the mutant CD3D gene and transplanted into NBSGW immunodeficient mice. Sixteen weeks after infusion, flow cytometric analysis demonstrated 96.2 ± 1.45% of hCD45+ human cells in all recipient mice bone marrow (BM). Relative abundances of human CD33+ myeloid, CD19+ B cells, CD3+ T cells, CD56+ NK cells, and CD34+ HSPC lineages were equivalent in mice that received untreated, LV-treated, or LV + ABE-treated cells, suggesting that base editing did not disrupt engraftment and HSC differentiation. Analyses of on-target gene correction in CD33+ myeloid, CD19+ B cells, CD56+ NK cells, CD34+ HSPCs, and hCD45+ whole BM isolated from recipient mice BM are ongoing and will be presented. Next, we evaluated the ability of human CD3δ SCID patient base-edited HSPCs to recapitulate healthy T-cell development. ABEmax-NRTH mRNA and sgRNA electroporated into CD34+ cells isolated from affected patient BM resulted in 71.2 ± 7.85% editing of CD3D c.202C>T. Base-edited and unedited CD3δ SCID HSPCs were aggregated with an MS5 mouse BM stromal cell line to form artificial thymic organoids (ATOs). Compared to healthy donor controls, ATOs aggregated with unedited CD3δ SCID HSPCs demonstrated undetectable or low-level CD3+TCRab+ frequencies (0.28 ± 0.23%), consistent with the clinical presentation of CD3δ SCID patients. Notably, T-cell development of base-edited CD3δ SCID HSPCs revealed striking fidelity to healthy donor HSPCs with robust rescue of the CD3+TCRαβ+ population in up to 91.4% of TCRγδ- cells. Furthermore, CD8 single-positive T cells generated from edited patient HSPCs demonstrated wildtype levels of proliferation and calcium flux in response to CD3/CD28 stimulus. CITE-seq is currently being performed on the unedited and edited patient thymocytes to assess TCR repertoire diversity and correlation of thymocyte transcriptome and protein measurements on the single-cell level and will be presented.

Conclusions: These studies demonstrate a novel base editing strategy for CD3δ SCID,capable of overcoming the arrest in thymopoiesis and restoring functional, mature T cells, in vitro. Additionally, the approach displays high rates of stable CD3D c.202C>T correction in human CD34+ HPSCs transplanted into immunodeficient animals. Altogether, these findings demonstrate preclinical efficacy of an ABE-mediated, autologous gene therapy for CD3δ SCID.

Liu:Beam Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Prime Medicine: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pairwise Plants: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Exo Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Chroma Medicine: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Resonance Medicine: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Tevard Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Voyager Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Nvelop Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Insitro: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Kohn:ImmunoVec: Consultancy; Allogene Therapeutics: Consultancy, Other: Member of Scientific Advisory Board; Pluto Immunotherapeutics: Consultancy, Other: Membership on Scientific Advisory Board; MyoGene Bio: Consultancy, Other: Membership on Scientific Advisory Board; Innoskel: Consultancy; Cimeio Therapeutics: Consultancy; TransformaTx: Consultancy.