T cell correction pipeline for Inborn Errors of Immunity
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants for autologous cell therapies for Inborn Errors of Immunity (IEI). The present IEI correction strategies mainly focus on the knock-in of therapeutic cDNAs, or knockout of the disease-causing gene when feasible. These strategies address many single-gene defects but may disrupt gene expression and require significant optimization for each newly discovered IEI-causing gene, highlighting the need for complementary platforms that can precisely correct diverse pathogenic variants. Here, we present a safe and efficient T cell single nucleotide variant (SNV) correction pipeline based on homology-directed repair (HDR), suitable for diverse monogenic mutations. By using founder mutations of Deficiency of ADA2 (DADA2), Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and Cartilage Hair Hypoplasia (CHH) as IEI models, we show that our pipeline can achieve up to 80% bi-allelic editing, with resultant functional correction of the disease phenotype in patient T cells. We do not find detectable pre-malignant off-target effects or karyotypic, transcriptomic or proteomic aberrations upon profiling patient T cells with GUIDE-seq, single cell RNA sequencing, PacBio based long-read whole genome sequencing, and high-throughput proteomics. This study demonstrates that HDR-based SNV editing is a safe and effective option for IEI T cell correction and that it could be developed to an autologous T cell therapy, as the presented protocol is scalable for a GMP-compatible workflow. This study is a step towards the development of gene correction platform that targets a broad number of monogenic mutations.
Authors 28
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Oslo University Hospital · University of Oslo
Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Department of Pediatrics, Oslo University Hospital, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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Affiliation as printed
Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
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Affiliation as printed
Computational Biology Unit, Department of Informatics, University of Bergen, Norway
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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University of Helsinki · HiLIFE – Elämäntieteiden Instituutti
Affiliation as printed
Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland
Systems Biology/Pathology Research Group, University of Helsinki, Helsinki, Finland
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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Oslo University Hospital · University of Oslo
Affiliation as printed
Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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University of Helsinki · HiLIFE – Elämäntieteiden Instituutti
Affiliation as printed
Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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Oulu University Hospital · University of Oulu
Affiliation as printed
ERN-RITA Core Center Member, RITAFIN Consortium, Infectious Diseases Clinic, Oulu University Hospital, Oulu, Finland
Research Unit of Internal Medicine and Biomedicine, University of Oulu, Oulu, Finland
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Oslo University Hospital · University of Oslo · Institute for Molecular Medicine Finland · HiLIFE – Elämäntieteiden Instituutti
Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Department of Medical Genetics, Oslo University Hospital, Oslo, Norway
Institute for Molecular Medicine Finland, HiLIFE, Helsinki, Finland
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Affiliation as printed
Department of Obstetrics, Division of Obstetrics and Gynecology, Oslo University Hospital, Oslo, Norway
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Camilla Schalin‐Jäntti Aachen
University of Helsinki · Helsinki University Hospital · Endo-ERN
Affiliation as printed
Endocrinology, Abdominal Center, Helsinki University Hospital, Helsinki, Finland
University of Helsinki, ENDO-ERN (European Reference Network on Rare Endocrine Conditions), Helsinki, Finland
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Oslo University Hospital · University of Oslo
Affiliation as printed
Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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Affiliation as printed
Folkhälsan Institute of Genetics, and Stem Cells and Metabolism Research Program, University of Helsinki, Finland
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University of Helsinki · HiLIFE – Elämäntieteiden Instituutti
Affiliation as printed
Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland
Systems Biology/Pathology Research Group, University of Helsinki, Helsinki, Finland
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University of Oslo · University of Bergen
Affiliation as printed
Computational Biology Unit, Department of Informatics, University of Bergen, Norway
Department of Biosciences, University of Oslo, Oslo, Norway
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Oulu University Hospital · University of Oulu
Affiliation as printed
ERN-RITA Core Center Member, RITAFIN Consortium, Infectious Diseases Clinic, Oulu University Hospital, Oulu, Finland
Research Unit of Internal Medicine and Biomedicine, University of Oulu, Oulu, Finland
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Affiliation as printed
Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden
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University of Helsinki · Helsinki University Hospital
Affiliation as printed
Inflammation Center, Department of Infectious Disease, Helsinki University Hospital and University of Helsinki, Helsinki, Finland
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Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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Emma Maria Haapaniemi corresponding
Oslo University Hospital · University of Oslo
Affiliation as printed
Centre for Molecular Medicine Norway, University of Oslo, Oslo, Norway
Department of Pediatrics, Oslo University Hospital, Oslo, Norway
Precision Immunotherapy Alliance, University of Oslo, Oslo, Norway
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