During this grant period, our research team has made significant strides in advancing curative therapies for RAG-SCIDs through genome editing. Initially, our focus was on enhancing on-target genome editing at the RAG loci in HSPCs to advance CRISPR gene correction for RAG-SCIDs. We documented our findings in a paper published in Molecular Therapy- Methods & Clinical Development (Shapiro et al., 2020, doi: 10.1016/j.omtm.2020.04.027) providing a comprehensive workflow for this optimization process. Collaborating with IDT, a global leader in DNA synthesis and sequencing products, we demonstrated that chemically synthesized gRNAs, delivered as RNP complexes into HSPCs, achieved high on-target activity. This work has influenced CRISPR-based studies worldwide, contributing to the development of efficient CRISPR-Cas9 protocols.
Next, addressing the challenge of limited SCID patient-derived samples, we innovated a platform utilizing CRISPR-Cas9, rAAV6 donors, and an in vitro T-cell differentiation system. This platform enables the modeling of both SCID and the therapeutic outcomes of gene-editing therapies for SCID, employing HSPCs derived from healthy donors. Our work, published in "Molecular Therapy–Nucleic Acids" (Iancu et al., 2022, doi.org/10.1016/j.omtn.2022.12.006) introduces a proof-of-concept CRISPR-Cas9/rAAV6 gene-correction approach in cells from healthy donors, and the successful generation of T cells from corrected RAG2-SCID patient-derived HSPCs. Furthermore, our recent publication in "Nature Communications" (Allen et al., 2023, doi:10.1038/s41467-023-42036-5) introduces an innovative gene editing technique designed to replace the entire RAG2 coding sequence while preserving endogenous gene regulation and locus structure. This versatile replacement strategy can be employed to replace complete coding sequences or exons in genes susceptible to disease-causing mutations. These two studies present a pivotal proof-of-concept gene therapy strategies with broad applicability to various blood and immune system disorders.
Importantly, we established an efficient analytical pipeline that characterizes and quantifies off-target events which is being used for pre-clinical and clinical trials. Detecting and understanding undesirable genome-editing outcomes, like harmful off-target editing and genotoxic effects, is crucial for safe CRISPR technology use. Our pipeline addresses this issue by applying the cell-based GUIDE-seq methodology for the identification of potential off-target sites, followed by quantification of their off-target activity using rhAmpSeq technology (Shapiro et al., 2020), coupled with the CRISPECTOR analysis tool. As detailed in our publication in “Nature Communications” (Amit et al., 2021, doi: 10.1038/s41467-021-22417-4) CRISPECTOR is a software tool developed and implemented, to overcome issues of low signal-to-noise ratio as well as false negative/positive outcomes that other editing tools suffer from. CRISPECTOR accurately and reliably quantifies CRISPR-Cas adverse off-target effects, including translocation events, that can occur during genome editing experiments. Concerns about DNA damage response were also addressed through innovative imaging flow cytometry and γH2AX staining, detailed in “The CRISPR Journal” (Allen et al., 2022, doi: 10.1089/crispr.2021.0128). Leveraging deep learning for image analysis, our platform accurately quantifies DDR levels in CD34+ HSPCs after CRISPR-Cas9 and/or rAAV6 treatment, ensuring safety and minimizing genotoxicity.
In summary, our multidimensional approach to CRISPR-Cas9 genome editing for RAG1/2 genes represents a leap forward in the treatment of SCID and offers a versatile platform for addressing a spectrum of genetic disorders. This transformative work brings us closer to a future where targeted gene therapies provide hope and healing for those affected by severe genetic conditions.
