Genetically corrected <i>RAG2</i> -SCID human hematopoietic stem cells restore V(D)J-recombinase and rescue lymphoid deficiency
Mara Pavel-Dinu, Cameron L. Gardner, Yusuke Nakauchi, Tomoki Kawai, Ottavia M. Delmonte, Boaz Palterer, Marita Bosticardo, Francesca Pala, Sébastien Viel, Harry L. Malech, Hana Y. Ghanim, Nicole M. Bode, Gavin Kurgan, Angela M. Detweiler, Christopher A. Vakulskas, Norma Neff, Adam Sheikali, Sherah T. Menezes, Jade Chrobok, Elaine M. Hernández González, Ravindra Majeti, Luigi D. Notarangelo, Matthew H. Porteus
Abstract
ABSTRACT: Recombination-activating genes (RAG1 and RAG2) are critical for lymphoid cell development and function by initiating the variable (V), diversity (D), and joining (J) (V(D)J)-recombination process to generate polyclonal lymphocytes with broad antigen specificity. The clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCIDs), causing life-threatening infections and death early in life without hematopoietic cell transplantation (HCT). Despite improvements, haploidentical HCT without myeloablative conditioning carries a high risk of graft failure and incomplete immune reconstitution. The RAG complex is only expressed during the G0-G1 phase of the cell cycle in the early stages of T- and B-cell development, underscoring that a direct gene correction might capture the precise temporal expression of the endogenous gene. Here, we report a feasibility study using the CRISPR/Cas9-based "universal gene-correction" approach for the RAG2 locus in human hematopoietic stem/progenitor cells (HSPCs) from healthy donors and RAG2-SCID patient. V(D)J-recombinase activity was restored after gene correction of RAG2-SCID-derived HSPCs, resulting in the development of T-cell receptor (TCR) αβ and γδ CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of CDR3 length and preserved usage of the distal TRAV genes. We confirmed the in vivo rescue of B-cell development with normal immunoglobulin M surface expression and a significant decrease in CD56bright natural killer cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit RAG2-deficient patients.