Genetically Corrected RAG2-SCID Human Hematopoietic Stem CellsRestore V(D)J-Recombinase and Rescue Lymphoid Deficiency

Pavel-Dinu, M.; Gardner, C. L.; Nakauchi, Y.; Kawai, T.; Delmonte, O. M.; Palterer, B.; Bosticardo, M.; Pala, F.; Viel, S.; Malech, H. L.; Ghanim, H. Y.; Bode, N. N.; Kurgan, G. L.; Vakulskas, C. A.; Sheikali, A.; Menezes, S. T.; Chrobok, J.; Gonzalez, E. M. H.; Majeti, R.; Notarangelo, L. D.; Porteus, M. H.

doi:10.1101/2022.07.12.499831

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bioRxiv · 10.1101/2022.07.12.499831

Genetically Corrected RAG2-SCID Human Hematopoietic Stem CellsRestore V(D)J-Recombinase and Rescue Lymphoid Deficiency

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Abstract

Recombination-activating genes (RAG1 and RAG2) are critical in lymphoid cell development and function for initiating the V(D)J-recombination process to generate polyclonal lymphocytes with broad antigen-specificity. Clinical manifestations of defective RAG1/2 genes range from immune dysregulation to severe combined immunodeficiencies (SCID), causing life-threatening infections and death early in life in the absence of hematopoietic cell transplantation (HCT). 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 phases of the cell cycle at the early stages of T and B cell development, underscoring that a direct gene correction would capture the precise temporal expression of the endogenous gene, is a promising therapeutic approach for RAG1/2-deficiencies. 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) in healthy donors and one RAG2-SCID patient. V(D)J recombinase activity was restored following gene correction of RAG2-SCID-derived HSPCs, resulting in the development of TCR {beta} and {gamma}{delta} CD3+ cells and single-positive CD4+ and CD8+ lymphocytes. TCR repertoire analysis indicated a normal distribution of the CDR3 length and preserved usage of distal TRAV genes. We confirmed in vivo rescue of B-cell development, with normal IgM surface expression and a significant decrease in CD56bright NK cells. Together, we provide specificity, toxicity, and efficacy data supporting the development of a gene-correction therapy to benefit all RAG2-deficient patients. KEY POINTSO_LIHuman hematopoietic stem cells can be corrected to restore endogenous RAG2 gene expression while preserving durable engraftment potential. C_LIO_LIGene-corrected RAG2 locus restores V(D)J recombination in RAG2-SCID patient stem cells, promoting T and B-cells receptor formation. C_LI

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Pavel-Dinu, M., Gardner, C. L., Nakauchi, Y., Kawai, T., Delmonte, O. M., Palterer, B., Bosticardo, M., Pala, F., Viel, S., Malech, H. L., Ghanim, H. Y., Bode, N. N., Kurgan, G. L., Vakulskas, C. A., Sheikali, A., Menezes, S. T., Chrobok, J., Gonzalez, E. M. H., Majeti, R., Notarangelo, L. D., Porteus, M. H.. 2022-07-13. Genetically Corrected RAG2-SCID Human Hematopoietic Stem CellsRestore V(D)J-Recombinase and Rescue Lymphoid Deficiency. https://doi.org/10.1101/2022.07.12.499831

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