Highly Efficient & Specific Repair of MAGT1 Mutation in Xmen Patient T Cells and Hematopoietic Stem Cells

Introduction: X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN) is a congenital glycosylation disorder with combined immune deficiency due to Magnesium Transporter 1 (MAGT1) mutations. Defective glycosylation of Natural Killer Group 2D (NKG2D), an activating receptor expressed on CD8 and NK cells critical for cytotoxicity, results in EBV lymphoproliferative disease and an inverted CD4:CD8 ratio in many XMEN patients. Oral magnesium supplementation for XMEN patients was not helpful, and hematopoietic stem cell transplants (HSCT) are complicated by high mortality. CRISPR-Cas9-AAV inserts MAGT1 specifically at endogenous locus but relies on double strand DNA breaks and adeno-associated virus donor that triggers DNA damage response. To address this, we evaluated base editing approach to repair a MAGT1 mutation that affects multiple patients with XMEN disease. We observed highly efficient mutation repair of XMEN T cells (75.5%) and hematopoietic stem cells (71.9%) to restore MAGT1 function and persistence of gene correction following transplant into immunodeficient mice.

Materials and methods: Peripheral blood hematopoietic stem cells (HSPCs, CD34+) and mononuclear cells (PBMC) were collected from XMEN patients and healthy donors (HD) after G-CSF and plerixafor mobilization. PBMCs were stimulated (anti-CD3/anti-CD28, IL-2 (100 IU/mL), in ImmunoCult™-XF T cell expansion medium for 5-7 days prior to editing. HSPCs were cultured in StemSpan™ SFEM II Stem Cell Factor, Thrombopoietin, Fms-like tyrosine kinase 3 ligand (all 100ng/mL) and UM171 for two days prior to editing. Base editing is performed by electroporation (MaxCyte Inc.) of cells with ABE8e SpRY (CELLSCRIPT LLC.) and synthetic guide (Synthego) RNAs. For in vitro killing activity, T cells were co-cultured with EBV-B cells at a ratio of 1:1 for 48 hours and analyzed for apoptosis (Apoptracker Green+ 7AAD-), necrosis (Apoptracker Green+ 7AAD+) and degranulation (CD8+ CD107a+). HSPCs were transplanted into mice two days after EP and harvested 17-20 weeks after transplant.

Results:Base editing of XMEN T cells: Flow cytometric analysis 2 days post BE show restored expression of biomarker NKG2D in XMEN CD8+ T cells (75.5%) compared to HD (93.2%) and naïve XMEN (2.39%). CD8+ cytotoxic activity against EBV-B cells was restored in BE XMEN patient cells; as indicated by increased apoptosis (BE: 6.78%, naïve: 4.82%, HD: 8.17%), necrosis (BE: 9.35%, naïve: 4.54%, HD: 7.19%), and degranulation (BE: 22.80%, naïve: 15.50%, HD: 21.40%), compared to naïve MAGT1 CD8+ T cells.In an in vivo tumor model, BE XMEN T cells injected into mice with induced luminescent EBV-B cell tumors lysed tumors and reduced average bioluminescence flux (photons/sec) (7 weeks) (8.23 × 10⁷ ± 1.06 × 10⁸) compared to naïve T cells (3.39 × 10⁹ ± 2.22 × 10⁹ and HD T cells (1.45 × 10⁹ ± 2.22 × 10⁹).

Base editing of HSPCs: Immunodeficient NSGS pups transplanted with BE XMEN HSPCs engrafted and differentiated normally into lymphocytes with a normalized CD4:CD8 ratio in spleen (BE: 0.79 ± 0.14, naïve: 3.45 ± 1.1, HD: 1.3 ± 0.40) and peripheral blood (BE: 2.16 ± 0.84, naïve: 5.56 ± 2.32, HD: 1.79 ± 0.70) (Fig 1a). Importantly, restoration of biomarker NKG2D expression selectively on CD8+ T cells in spleen (BE: 80.5% ± 8.61%, naïve: 3.63% ± 1.45%, HD: 68.9% ± 10.32%) and peripheral blood (BE: 86.1% ± 2.88%, naïve: 2.25% ± 1.45%, HD: 62.6% ± 12.44%) (Fig 1b) confirmed correction of MAGT1 function in the glycosylation of critical NKG2D protein. High throughput targeted sequencing of on-target locus shows minimal bystander edits and additional off-target analysis is ongoing.

Conclusion: Our data show highly efficient and specific base editing repair of MAGT1 mutation in XMEN T lymphocytes and CD34+ HSPCs. BE XMEN T cells increased cytotoxic activity in vitro and killing of tumor cells in mice. In vivo studies with mice transplanted with BE CD34+ HSPCs also confirm long term engraftment and differentiation into CD8+ T cells with restored NKG2D necessary for activation and cytolytic activity of CD8+ T and NK cells. The highly efficient and specific correction with base editing holds great promise for gene therapy for XMEN disease.

Meis:CELLSCRIPT LLC: Current Employment. Dahl:CELLSCRIPT: Current Employment, Current equity holder in private company. Malech:Prime Medicine: Research Funding; CSL Behring: Research Funding.