Gene Therapy Strategies for RAG1 Deficiency: Challenges and Breakthroughs.

Mutations in the recombination activating genes (RAG) cause various forms of immune deficiency. Hematopoietic stem cell transplant (HSCT) is the only cure for patients with severe manifestations of RAG deficiency; however, outcomes are suboptimal with mismatched donors. Gene therapy aims to correct autologous hematopoietic stem and progenitor cells (HSPC) and is emerging as an alternative to allogeneic HSCT. Gene therapy based on viral gene addition exploits viral vectors to add a correct copy of a mutated gene into the genome of HSPCs. Only recently, after a prolonged phase of development, viral gene addition has been approved for clinical testing in RAG1-SCID patients. In the meantime, a new technology, CRISPR/Cas9, has made its debut to compete with viral gene addition. Gene editing based on CRISPR/Cas9 allows to perform targeted genomic integrations of a correct copy of a mutated gene, circumventing the risk of virus-mediated insertional mutagenesis. In this review, we present the biology of the RAG genes, the challenges faced during the development of viral gene addition for RAG1-SCID, and the current status of gene therapy for RAG1 deficiency. In particular, we highlight the latest advances and challenges in CRISPR/Cas9 gene editing and their potential for the future of gene therapy.

Engineering spacer specificity of the Cre/loxP system.

Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing Cre discrimination between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking. To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the plasticity of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations were used to investigate the possible mechanisms behind the specificity switch. Our findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications.