Mending RAG2: gene editing for treatment of RAG2 deficiency

In this issue of Blood Advances, Pavel-Dinu et al1 demonstrated successful immune reconstitution in a mouse model of severe combined immunodeficiency (SCID) using RAG2-deficient human hematopoietic stem cells that were genetically corrected with a CRISPR/Cas9 system. By insertion of a codonoptimized transgene within the native RAG2 locus, their approach provides a potential universal gene therapy that could be used with most RAG mutations. Autosomal recessive mutations in RAG1 and RAG2 genes are the second most common cause of SCID in the United States, resulting in low to absent T and B cells and normal numbers of natural killer (NK) cells.2 Hypomorphic RAG mutations lead to a broad range of immune deficiency disorders, which include Omenn syndrome, combined immunodeficiency, and autoimmunity with granulomatous disease.3 Although newborn screening has enabled detection of SCID in many countries, unscreened patients may not present until onset of infections or autoimmune disease, which may be delayed in onset.4 Hematopoietic stem cell transplantation is potentially curative, and although the use of a matched related donor can achieve acceptable immune reconstitution without conditioning in some patients, this is not an option for most patients. Reduced intensity or myeloablative conditioning is typically required for good immune reconstitution when using haploidentical or unrelated marrow donors for patients with RAG SCID but involves risk of chemotherapy-related toxicity.5 Ex vivo gene therapy allows for the use of a patient's autologous hematopoietic stem cells, minimizing exposure to chemotherapy and negating risk of acute and chronic graft-versus-host disease. Gene addition have been used in many previous clinical trials via integrating viral vectors, with evidence of efficacy and improved safety with modern lentiviral vectors.6 However, lentiviral gene addition is not a viable option for many highly regulated genes. Previous investigators have demonstrated immune reconstitution of RAG1 deficiency via lentiviral gene addition in a murine model, with only subtle defects in the T-cell repertoire after gene therapy.7 Although a study of lentiviral-based gene therapy for RAG1 deficiency is ongoing, gene insertion using CRISPR/Cas9 represents an attractive option to provide the corrected RAG gene under the endogenous promoter. In this article, Pavel-Dinu et al used CRISPR/Cas9 editing for targeted insertion of a codon-optimized RAG2 transgene (coRAG2) within the RAG2 coding region. They used an adeno-associated virus 6 (AAV6) vector for coRAG2 delivery to this region via homologous recombination in RAG2-null cells. They showed high specificity for the RAG2 gene with no increase in off-targeting vs mock-edited cells. Gene-modified hematopoietic stem cell progenitors (HSPCs) showed normal development of multiNext, frozen peripheral blood HSPCs from a patient with RAG2 deficiency with compound heterozygous mutations (RAG2 null HSPCs) were treated with CRISPR/Cas9-AAV6. A higher multiplicity of infection of AAV6 (5000) resulted in viral toxicity and reduced viability of gene-corrected HSPCs. This was ameliorated by use of a p53 inhibitor (i53) to inhibit nonhomologous end joining, which allowed a