Periodic Reporting for period 4 - CRISS (CRISPR Gene Correction for Severe Combined Immunodeficiency Caused by Mutations in Recombination-activating gene 1 and 2 (RAG1 and RAG2))
Berichtszeitraum: 2022-04-01 bis 2023-09-30
SCIDs represent a group of severe primary immunodeficiency disorders primarily caused by genetic mutations that disrupt the development of T-cells. SCIDs can also lead to abnormalities in the function and quantities of B-cells and natural killer cells. Without treatment, SCID is fatal within the first year of life. Currently, the primary method for definitively treating SCID patients involves allogeneic bone marrow transplantation. However, this treatment approach presents substantial challenges, including the difficulty of finding a compatible donor and the potential for immunological complications like graft-versus-host disease (GVHD). Gene therapy using viral vectors containing a corrective transgene is being developed for some of these disorders. However, for other SCID disorders, such as those caused by genetic mutations in RAG1 and RAG2, the transgene needs to be expressed in a precise, developmental, and lineage-specific manner to achieve functional gene correction and to avoid the risks of cellular transformation. The emergence of genome-editing techniques, especially the use of targeted nucleases like CRISPR-Cas9, has ushered in a new era in gene therapy research, offering a promising solution for blood and immune system disorders. The overall objective of the CRISS project was to develop robust and safe CRISPR-genome editing in hematopoietic stem and progenitor cells (HSPCs) to treat RAG-SCIDs and complete pre-clinical efficacy and safety studies to show the approach has a clear path towards future clinical trials. This study led to the development of CRISPR-Cas9/rAAV6 gene-correction approach for disease and correction modeling of SCID in human HSPCs. We showed a first-of-its-kind functional gene correction of RAG2-SCID patient-derived HSPCs and established an innovative HDR-mediated gene-correction strategy that enables the preservation of endogenous regulatory elements. Moreover, we established an efficient analytical pipeline that characterizes and quantifies off-target CRISPR events which is being used for pre-clinical and clinical trials. Taken together, this research will broadly impact the field of genome editing since it addresses the current issues holding potential gene therapies back not only for RAG2-SCID but for many monogenic disorders of the blood and immune system.
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.
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.
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. Our innovation hinges on a crucial insight: to efficiently trigger CRISPR-Cas9 HDR-mediated genome editing for precise coding sequence replacement, it's essential to separate the distal homology arm from the cleavage site and align it with the sequence immediately downstream of the segment needing replacement. In this process, elongating the distal homology arm length in the donor is crucial. Our approach preserves endogenous regulatory elements and intronic sequences, faithfully reproducing natural gene expression levels and reducing the risks of unregulated gene expression. This groundbreaking technique, which involves replacing entire coding sequences or exons while retaining critical regulatory elements, brings hope to RAG2-SCID patients and holds promise for treating other genetic disorders.