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E - Disease Models and Clinical Applications -> Hematologic and Immunologic Diseases

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

Type: Oral Abstract Session

Presentation Details
Session Title: Gene Therapy for Immunologic Diseases
Location: Room 202
Start Time: 5/17/2022 17:00
End Time: 5/17/2022 17:15

Introduction: CD3δ severe combined immunodeficiency (SCID) is a devastating inborn error of immunity caused by a homozygous mutation in the CD3D gene. The predominant disease-causing substitution, CD3D C202T, is present in a Mennonite population, and results in the failure to produce CD3δ protein, an essential, invariable chain of the CD3/T-cell receptor (TCR) complex. Patients living with CD3δ SCID present with a complete absence of mature T cells and are severely susceptible to lethal infections, often leading to infant mortality. Methods and Results:
We compared two gene editing approaches: (1) CRISPR/Cas9 homology-directed repair (HDR) with a single-stranded oligodeoxynucleotide (ssODN) homologous donor and (2) adenine base editing (ABE)-correction, to precisely revert the CD3D C202T mutation and rescue CD3 expression and T-cell signaling. A CD3 Knockout (KO) Jurkat T-cell disease model, containing the C202T mutation, was developed to evaluate the two editing strategies. We rationally designed and investigated novel editing reagents, including three adenine base editor variants, to target the C202T mutation in the CD3 KO Jurkat T-cell model. Our most efficient editing reagents demonstrated up to 88%, 85%, 83%, and 55% correction of the CD3D C202T mutation by NRTH-ABEmax, NRTH-ABE8e, NG-ABE8e, and CRISPR/Cas9 RNP + ssODN-mediated editing, respectively. Editing efficiency correlated with functional rescue of CD3 protein complex surface expression, with results demonstrating up to 85%, 79%, 78%, and 59% of CD3 complex restoration in manipulated CD3 KO Jurkat T cells by NRTH-ABEmax, NRTH-ABE8e, NG-ABE8e, and RNP + ssODN treatment, respectively. Stimulation of edited-CD3 KO Jurkat T cells with anti-CD3 and anti-CD28 produced wildtype-level rescue of calcium flux in NRTH-ABEmax, NRTH-ABE8e, and NG-ABE8e experimental arms, while RNP + ssODN treatment restored calcium flux to 58% of wildtype levels. To validate our best-performing base editing strategy in healthy donor CD34+ hematopoietic stem and progenitor cells (HSPCs), we introduced the CD3D C202T disease target by lentiviral integration followed by treatment with mRNA base editor. We observed up to 75% precise and stable therapeutic base editing of the C202T mutation 15 days post-editing, suggesting similarly high levels of correction will be obtained in CD3δ SCID patient HSPCs.We are actively assessing functional restoration of T-cell development from base-edited CD3δ SCID patient HSPCs in-vitro in the artificial thymic organoid (ATO) system, which can support differentiation of CD3δ SCID HSPCs to mature T cells, if the C202T mutation is corrected.
Conclusions: Our studies demonstrate a novel base editing strategy for CD3δ SCID, capable of restoring 88% of CD3 protein expression and functionally correcting CD3/TCR signaling to wildtype levels in a CD3 KO Jurkat T-cell disease model. Additionally, our approach displays high rates of precise CD3D C202T editing in clinically relevant CD34+ HSPCs. Altogether, these results indicate a potentially curative, novel base editing gene therapy in HSPCs for CD3δ SCID to be translated to the clinic.

Grace E. McAuley1, Gloria Yiu2, Gregory A. Newby3, Sung Hae L. Kang4, Amber J. Garibay4, Jeffrey A. Butler4, Valentina S. Christian4, Sorel Fitz-Gibbon5, Ryan L. Wong6, Kelcee A. Everette3, Zulema Romero1, Nicola Wright7, David R. Liu3, Gay M. Crooks4,8, Donald B. Kohn1,6,8

1Department of Microbiology, Immunology & Molecular Genetics, UCLA, Los Angeles, CA,2Department of Rheumatology, UCLA, Los Angeles, CA,3Broad Institute of Harvard and MIT, Cambridge, MA,4Department of Pathology & Laboratory Medicine, UCLA, Los Angeles, CA,5Department of Molecular, Cell, & Developmental Biology, UCLA, Los Angeles, CA,6Department of Molecular & Medical Pharmacology, UCLA, Los Angeles, CA,7Department of Pediatrics, University of Calgary, Calgary, AB, Canada,8Division of Pediatric Hematology-Oncology, UCLA, Los Angeles, CA
 G.E. McAuley: None.

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