{"doi":"10.1101/2022.07.12.499831","title":"Genetically Corrected <i>RAG2</i> -SCID Human Hematopoietic Stem Cells Restore V(D)J-Recombinase and Rescue Lymphoid Deficiency","abstract":"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 αβ and γδ 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 CD56 bright 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 POINTS Human hematopoietic stem cells can be corrected to restore endogenous RAG2 gene expression while preserving durable engraftment potential. Gene-corrected RAG2 locus restores V(D)J recombination in RAG2 -SCID patient stem cells, promoting T and B-cells’ receptor formation.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":300266,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9496,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":281848,"name":"Cameron L. Gardner","orcid":"0000-0002-7884-4794","position":1,"is_corresponding":false},{"id":492812,"name":"Yusuke Nakauchi","orcid":"0000-0002-6943-3811","position":2,"is_corresponding":false},{"id":281849,"name":"Tomoki Kawai","orcid":"0000-0003-0375-9752","position":3,"is_corresponding":false},{"id":104168,"name":"Ottavia M. Delmonte","orcid":"0000-0002-4772-0799","position":4,"is_corresponding":false},{"id":391316,"name":"Boaz Palterer","orcid":"0000-0003-2499-8250","position":5,"is_corresponding":false},{"id":281845,"name":"Marita Bosticardo","orcid":"0000-0002-1771-4523","position":6,"is_corresponding":false},{"id":281846,"name":"Francesca Pala","orcid":"0000-0003-4537-9061","position":7,"is_corresponding":false},{"id":990722,"name":"Sébastien Viel","orcid":"0000-0002-5085-443X","position":8,"is_corresponding":false},{"id":238416,"name":"Harry L. Malech","orcid":"0000-0001-5874-5775","position":9,"is_corresponding":false},{"id":704891,"name":"Hana Y. Ghanim","orcid":"0000-0002-4102-2367","position":10,"is_corresponding":false},{"id":642880,"name":"Nicole M. Bode","orcid":"0000-0001-7209-303X","position":11,"is_corresponding":false},{"id":18370,"name":"Gavin Kurgan","orcid":"0000-0002-4047-1427","position":12,"is_corresponding":false},{"id":356489,"name":"Christopher A. Vakulskas","orcid":"0000-0001-8510-4332","position":13,"is_corresponding":false},{"id":274203,"name":"Adam Sheikali","orcid":"0000-0002-0083-4297","position":14,"is_corresponding":false},{"id":991312,"name":"Sherah T. Menezes","orcid":null,"position":15,"is_corresponding":false},{"id":991313,"name":"Jade Chrobok","orcid":null,"position":16,"is_corresponding":false},{"id":991314,"name":"Elaine M. Hernández González","orcid":null,"position":17,"is_corresponding":false},{"id":492814,"name":"Ravindra Majeti","orcid":"0000-0002-5814-0984","position":18,"is_corresponding":false},{"id":231247,"name":"Luigi D. Notarangelo","orcid":"0000-0002-8335-0262","position":19,"is_corresponding":false},{"id":251111,"name":"Matthew H. Porteus","orcid":"0000-0002-3850-4648","position":20,"is_corresponding":false},{"id":274206,"name":"Mara Pavel-Dinu","orcid":"0000-0003-2719-2498","position":0,"is_corresponding":true}],"reference_count":51,"raw_metadata":null,"created_at":"2026-07-19T00:31:53.559757Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}