CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications

Area of Science:

• Immunology
• Molecular Biology
• Gene Therapy

Background:

• RAG2-SCID is a primary immunodeficiency stemming from mutations in the Recombination-activating gene 2 (RAG2).
• Current treatment involves allogeneic hematopoietic stem cell transplantation (HSCT), which has limitations.
• Ex-vivo gene editing of patient hematopoietic stem and progenitor cells (HSPCs) presents a potential alternative therapy.

Purpose of the Study:

• To develop an innovative RAG2 gene correction strategy for RAG2-SCID.
• To preserve endogenous gene regulation and locus architecture by replacing the entire coding sequence.
• To demonstrate the efficacy of this strategy in restoring T cell development and repertoire diversity.

Main Methods:

• Utilized CRISPR-Cas9/rAAV6 gene editing for ex-vivo manipulation of HSPCs.
• Implemented a strategy to replace the endogenous RAG2 coding sequence (CDS) with a corrective transgene.
• Assessed T cell development (CD3+TCRαβ+, CD3+TCRγδ+) and T cell receptor (TRB, TRG) repertoire diversity in an in-vitro platform.

Main Results:

• Successful expression of the corrective RAG2 transgene was achieved.
• The gene-corrected cells developed into functional CD3+TCRαβ+ and CD3+TCRγδ+ T cells.
• Highly diverse TRB and TRG repertoires were established, indicating restored lymphocyte maturation.

Conclusions:

• This proof-of-concept study demonstrates a viable gene therapy approach for RAG2-SCID.
• The strategy of replacing the RAG2 CDS holds promise for safer gene therapies targeting tightly regulated genes.
• This method could offer a safer alternative to HSCT for RAG2-SCID patients.