Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency

• Published in: Science translational medicine 2024-02, Vol.16 (733), p.eadh8162-eadh8162
• Main Authors: Castiello, Maria Carmina, Brandas, Chiara, Ferrari, Samuele, Porcellini, Simona, Sacchetti, Nicolò, Canarutto, Daniele, Draghici, Elena, Merelli, Ivan, Barcella, Matteo, Pelosi, Gabriele, Vavassori, Valentina, Varesi, Angelica, Jacob, Aurelien, Scala, Serena, Basso Ricci, Luca, Paulis, Marianna, Strina, Dario, Di Verniere, Martina, Sergi Sergi, Lucia, Serafini, Marta, Holland, Steven M, Bergerson, Jenna R E, De Ravin, Suk See, Malech, Harry L, Pala, Francesca, Bosticardo, Marita, Brombin, Chiara, Cugnata, Federica, Calzoni, Enrica, Crooks, Gay M, Notarangelo, Luigi D, Genovese, Pietro, Naldini, Luigi, Villa, Anna
• Format: Article
• Language: English
• Subjects: Animals; Cell differentiation; Exons; Gene Editing - methods; Genetic engineering; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hematopoietic Stem Cells - metabolism; Hemopoiesis; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Homology; Humans; Immune system; Immunodeficiency; Mice; Mutation; Progenitor cells; RAG1 protein; Toxicity; Transplantation
• ISSN: 1946-6234; 1946-6242; 1946-3242
• DOI: 10.1126/scitranslmed.adh8162

Recombination activating genes ( ) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human in HSPCs from patients with hypomorphic mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.