CRISPR-Cas9 RAG2 Correction via Coding Sequence Replacement to Preserve Endogenous Gene Regulation and Locus Structure

Abstract

RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient’s own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show a first-of-its-kind RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) to preserve the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene led to successful development into CD3+TCRαβ+ and CD3+TCRγδ+ T cells and promoted the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. We believe that a CDS replacement technique to correct tightly regulated genes, like RAG2, while maintaining critical regulatory elements and conserving the locus structure could bring safer gene therapy techniques closer to the clinic.

Homology-Directed-Repair-Based Genome Editing in HSPCs for the Treatment of Inborn Errors of Immunity and Blood Disorders

“…After the ssDNA sheds its viral protein coat and enters the nucleus, it undergoes second-strand synthesis to dsDNA, with dsDNA serving as a predominant template for HDR-mediated gene targeting [ 69 , 85 , 86 ] ( Figure 2 D). AAV donor DNA sequences have been shown to induce high-quality HDR in CD34 + HSPCs with minimum homology arm lengths of ~300–400 bp, although, in certain cases, extending the homology arms to as long as 1.6–2.2 kb has shown marked improvement in HDR efficiency in HSPCs and several other primary cells [ 20 , 25 , 87 , 88 ]. The ease of use and early success in using AAV for transgene delivery in gene-therapy applications has boosted its popularity as a therapeutic vector.…”

“…The ease of use and early success in using AAV for transgene delivery in gene-therapy applications has boosted its popularity as a therapeutic vector. Pre-clinical studies using AAV vectors to develop potential treatments for SCD and β-thalassemia [ 20 ], SCID-X1 [ 30 , 89 ], WAS [ 32 ], IPEX [ 31 ], CGD [ 90 , 91 , 92 ], Hemophilia [ 93 ], XLA (X-Linked Agammaglobulinemia) [ 94 ], XMEN (X-linked Immunodeficiency with Magnesium Defect, Epstein–Barr Virus (EBV) Infection, and Neoplasia) [ 95 ], and most recently RAG2 -SCID [ 29 , 87 , 96 , 97 ] have been conducted in CD34 + HSPCs, showing highly efficient frequencies of HDR. Interestingly, most AAV donor structures have the homology arms flanking the immediate genomic sequences 5′ and 3′ to the Cas9-induced DSB, leading to the insertion of the transgene into the break site.…”

“…Interestingly, most AAV donor structures have the homology arms flanking the immediate genomic sequences 5′ and 3′ to the Cas9-induced DSB, leading to the insertion of the transgene into the break site. However, recent works have shown that distancing the 3’ homology arm from the DSB site allows for the replacement of up to several kilobases of genomic DNA with the transgene [ 20 , 87 ] ( Figure 3 ). Allen et al utilized this replacement strategy to replace the entire endogenous RAG2 coding sequence, allowing for transgenes to maintain critical endogenous regulatory elements while preserving the integrity and 3D architecture of the genomic locus as much as possible by limiting the introduction of kilobases of new DNA [ 87 ].…”

“…However, recent works have shown that distancing the 3’ homology arm from the DSB site allows for the replacement of up to several kilobases of genomic DNA with the transgene [ 20 , 87 ] ( Figure 3 ). Allen et al utilized this replacement strategy to replace the entire endogenous RAG2 coding sequence, allowing for transgenes to maintain critical endogenous regulatory elements while preserving the integrity and 3D architecture of the genomic locus as much as possible by limiting the introduction of kilobases of new DNA [ 87 ]. This replacement strategy is particularly useful for correcting tightly controlled genes, such as RAG2 , that are regulated by spatiotemporal elements in the surrounding locus [ 98 ].…”

“…Additionally, Shin et al demonstrated that HDR could be increased through controlled cycling and quiescence of the edited cells [ 106 , 137 ]. Lastly, when HDR efficiencies are low, extending the homology arms of the donor DNA to as long as 1.6–2.2 kb has shown a marked improvement in HDR frequencies via the use of AAV in HSPCs and several other primary cells [ 20 , 25 , 87 , 88 ].…”