Publications
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Labun, Kornel; Rio, Oline Eriksen; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia; Valen, Eivind & Haapaniemi, Emma Maria
(2026).
SNIPSNP: precision design of CRISPR/Cas9 knock-in reagents for variant correction and disease modeling.
Nucleic Acids Research (NAR).
ISSN 0305-1048.
54(W1),
p. W145–W153.
doi:
10.1093/nar/gkag409.
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We present SNIPSNP (crisprtools.org/snipsnp), a comprehensive bioinformatics pipeline for designing experiments for CRISPR-induced homology-directed repair (HDR). The tool addresses the critical challenge of Cas9 re-cleavage by simplifying the selection of “blocking” silent variants that are effective at inhibiting RNP binding upon donor-templated editing. SNIPSNP handles complex edits, including indels, and uses multi-objective optimization to balance editing efficiency with biological safety. From user-defined wild-type and desired HDR alleles, the pipeline identifies candidate guides, annotating them with integrated efficiency scores and genome-wide off-target assessments. Uniquely, SNIPSNP evaluates guide binding against the post-edit genome to determine whether the therapeutic variant alone disrupts repeated Cas9 recognition. When necessary, it introduces synonymous blocking variants, prioritizing PAM and seed regions to minimize re-cleavage probability and editing of the wild-type (WT) allele when editing heterozygous variants. All candidate modifications undergo safety profiling and prioritization of known benign variants from dbSNP. We experimentally validated SNIPSNP and benchmarked it on pathogenic inborn error of immunity variants in primary patient T-cells. Across loci, SNIPSNP-designed templates outperform standard “correction-only” strategies, demonstrating enhanced precision editing, and reduced re-cleavage, establishing SNIPSNP as a robust platform for genome editing and disease modeling.
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Labun, Kornel; Rio, Oline Eriksen; Tjeldnes, Håkon; Swirski, Michał; Komisarczuk, Anna Zofia & Haapaniemi, Emma Maria
[Show all 7 contributors for this article]
(2025).
CHOPOFF: symbolic alignments enable fast and sensitive CRISPR off-target detection.
bioRxiv - the preprint server for biology.
doi:
10.1101/2025.01.06.603201.
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ABSTRACT CRISPR/Cas systems offer powerful tools for genome editing, but their therapeutic application is hampered by the risk of unintended off-target mutations. Many molecular methods have been established to detect off-target editing, however, their sensitivity depends on first identifying potential sites using in silico methods. However, these in silico prediction methods are challenged by a trade-off between speed and sensitivity, and can fail to comprehensively detect all edited off-target sites. Here, we demonstrate that ignoring bulges has led to missing editing at off-target sites in previous studies and that continuing this practice can lead to inflated claims of fidelity. As a solution, we introduce the concept of symbolic alignments to efficiently identify off-targets without sacrificing sensitivity. We further present specialized data structures that enable rapid, alignment-free probabilistic ranking of guide RNAs based on their predicted off-target burden. Implemented in the tool CHOPOFF, these innovations accommodate mismatches, bulges (insertions/deletions), and incorporate genomic sequence variants for personalized off-target assessment. Benchmarking demonstrates that CHOPOFF significantly outperforms state-of-the-art tools in both prediction accuracy and computational speed. Availability CHOPOFF command line available at https://github.com/JokingHero/CHOPOFF.jl CHOPOFF web server available at https://crisprtools.org/chopoff
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Kopcil, Pavel; Ervik, Carolina W.; Reint, Ganna; Mamia, Katariina; Szymanska, Monika & Dahal-Koirala, Shiva
[Show all 17 contributors for this article]
(2025).
Editing of ADA2 Point Mutation in Human Hematopoietic Stem Cells.
bioRxiv (preprint article).
doi:
10.1101/2025.08.14.670303.
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Abstract Background The homozygous ADA2 : c.506G>A (p.Arg169Gln; p.R169Q) variant accounts for majority of Deficiency in Adenosine Deaminase 2 (DADA2). This monogenic disorder may be amenable to ex vivo gene therapy by correcting the pathogenic mutation in CD34+ hematopoietic stem and progenitor cells (HSPCs). Objective To apply CRISPR-Cas9 and homology-directed repair (HDR) as a surrogate strategy to model correction of the pathogenic ADA2 c.506G>A variant in healthy cord blood HSPCs. Methods HSPCs were electroporated with optimised CRISPR-Cas9 editing reagents, and editing outcomes, including HDR and on-target deletions, were quantified by ddPCR. Cell functionality was assessed through colony-forming unit (CFU) assays and by xenotransplantation into NOD SCID Gamma (NSG) mice. Two HDR enhancement strategies were tested: (1) genetic inhibitors of p53 and non-homologous end joining (NHEJ) pathways, and (2) pharmacological NHEJ inhibition. Results Small-molecule NHEJ inhibitors increased HDR efficiency approximately two-fold (from ∼40 % to ∼80 %). Edited HSPCs retained normal CFU capacity and successfully engrafted in NSG mice. However, up to 8 % of edited cells exhibited on-target chromosome loss, though this declined over time. Up to 40 % of T cells and fibroblasts demonstrated similar losses under NHEJ inhibitors treatment. In contrast, genetically encoded inhibitors did not improve HDR. Conclusion The ADA2 p. c.506G>A variant can be effectively edited employing surrogate strategy in HSPCs without impairing functionality. Although pharmacological inhibition of NHEJ enhances HDR efficiency, it also increases the risk of on-target chromosome aberrations, highlighting the need for careful consideration of the associated risks and benefits in therapeutic gene editing. Key messages 1) The ADA2 p.R169Q variant can be efficiently corrected via HDR, and the edited CD34+ HSPCs retain their engraftment capability in NSG mice. 2) Pharmacological inhibition of NHEJ using small-molecule inhibitors increases HDR efficiency but is associated with significant on-target deletions and chromosomal arm loss, particularly in differentiated cell types, and in a donor-dependent manner. Capsule summary The ADA2 p.R169Q variant is a viable target for precision gene editing in hematopoietic stem cells. Although inhibition of NHEJ improves HDR efficiency, it concomitantly increases the risk of large on-target deletions, particularly in differentiated cells.
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Mamia, Katariina Aino Inkeri; Kolbeinsdottir, Solrun; Labun, Kornel; Li, Zhuokun; Komisarczuk, Anna Zofia & Keskitalo, Salla
[Show all 32 contributors for this article]
(2025).
Precision T cell correction platform for Inborn Errors of Immunity.
Molecular Therapy.
ISSN 1525-0016.
33(11),
p. 5624–5643.
doi:
10.1016/j.ymthe.2025.08.018.
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CRISPR/Cas9 gene editing is a promising tool to correct pathogenic variants for autologous cell therapies targeting Inborn errors of immunity (IEI). Current strategies, such as gene knockout or cDNA knock-in, address many single-gene defects but can disrupt gene expression, highlighting the need for precise correction platforms. While transplanting corrected autologous hematopoietic stem cells is a curative approach, it is unsuitable for patients with advanced disease, inflammation or acute infections. As correcting T cells is an alternative therapeutic strategy for lymphoid IEIs, we present an efficient T cell single nucleotide variant (SNV) correction platform based on homology-directed repair (HDR). By using STAT1 gain-of-function (STAT1-GOF), Cartilage Hair Hypoplasia (CHH), Deficiency of ADA2 (DADA2), and Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) as IEI models, we demonstrate that our platform achieves up to 80% correction, with resultant functional correction of the disease phenotype in the selected models. Furthermore, we performed safety profiling using GUIDE-seq, single-cell RNA sequencing, long-read genome sequencing, and proteomics analysis and detected no genomic, transcriptomic, or proteomic aberrations. This study establishes HDR-based SNV editing as a portable method for developing clinical autologous T cell therapies and represents a promising step toward a broad-spectrum gene correction platform for treating diverse monogenic immune disorders.
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Kaustio, Meri; Szymanska, Monika; Li, Weiwei; Braathen, Ragnhild Selvig; Campbell, Tessa M. & Haugen, Frida Loe
[Show all 23 contributors for this article]
(2025).
Heterozygous loss of MAP4K1 causes immune dysregulation by amplifying T-cell responses.
Journal of Allergy and Clinical Immunology.
ISSN 0091-6749.
156(4),
p. 1024–1037.
doi:
10.1016/j.jaci.2025.07.010.
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Background
MAP4K1 encodes hematopoietic progenitor kinase 1 (HPK1), a serine/threonine kinase that negatively regulates T-cell receptor signaling via phosphorylation of the adaptor proteins SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kDa) and Gads. While common MAP4K1 variants have been implicated in polygenic immune-mediated diseases, the impact of rare germline variants on human immunity remains undefined.
Objective
We investigated the immunologic and functional consequences of HPK1 deficiency in individuals with suspected inborn errors of immunity.
Methods
We performed genomic linkage analysis and exome sequencing to identify disease-associated variants in patients with inborn errors of immunity. Immunophenotyping, RNA sequencing, and functional assays were conducted on patient-derived lymphocytes, complemented by CRISPR-Cas9–mediated MAP4K1 disruption and correction in primary T cells.
Results
Heterozygous MAP4K1 loss-of-function variants were identified in two kindreds presenting with diverse immune dysregulatory symptoms, including recurrent fevers, inflammatory arthritis, Epstein-Barr virus–related complications, and nephritis. These variants led to reduced HPK1 protein levels and SLP-76Ser376 phosphorylation. While lymphocyte development was largely preserved, T cells from HPK1-deficient individuals displayed hyperresponsiveness to T-cell receptor stimulation, characterized by elevated secretion of proinflammatory cytokines, particularly IFN-γ and TNF. CRISPR-Cas9–mediated knockout recapitulated, and variant correction partially reversed this phenotype. Transcriptomic profiling of stimulated CD4+ T cells further revealed upregulation of immune signaling pathways—including NF-κB, JAK/STAT, and AP-1—as well as increased expression of multiple T-cell cytokines, consistent with enhanced T-cell receptor signaling and T-cell responses in HPK1-deficient individuals.
Conclusion
HPK1 deficiency, caused by heterozygous loss of MAP4K1, is a novel monogenic cause of immune dysregulation. Increased T-cell activation and proinflammatory cytokine production are implicated in disease pathogenesis
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Kaustio, Meri; Nayebzadeh, Naemeh; Hinttala, Reetta; Tapiainen, Terhi; Åström, Pirjo & Mamia, Katariina Aino Inkeri
[Show all 37 contributors for this article]
(2021).
Loss of DIAPH1 causes SCBMS, combined immunodeficiency, and mitochondrial dysfunction.
Journal of Allergy and Clinical Immunology.
ISSN 0091-6749.
148(2),
p. 599–611.
doi:
10.1016/j.jaci.2020.12.656.
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Background Homozygous loss of DIAPH1 results in seizures, cortical blindness, and microcephaly syndrome (SCBMS). We studied 5 Finnish and 2 Omani patients with loss of DIAPH1 presenting with SCBMS, mitochondrial dysfunction, and immunodeficiency. Objective We sought to further characterize phenotypes and disease mechanisms associated with loss of DIAPH1. Methods Exome sequencing, genotyping and haplotype analysis, B- and T-cell phenotyping, in vitro lymphocyte stimulation assays, analyses of mitochondrial function, immunofluorescence staining for cytoskeletal proteins and mitochondria, and CRISPR-Cas9 DIAPH1 knockout in heathy donor PBMCs were used. Results Genetic analyses found all Finnish patients homozygous for a rare DIAPH1 splice-variant (NM_005219:c.684+1G>A) enriched in the Finnish population, and Omani patients homozygous for a previously described pathogenic DIAPH1 frameshift-variant (NM_005219:c.2769delT;p.F923fs). In addition to microcephaly, epilepsy, and cortical blindness characteristic to SCBMS, the patients presented with infection susceptibility due to defective lymphocyte maturation and 3 patients developed B-cell lymphoma. Patients’ immunophenotype was characterized by poor lymphocyte activation and proliferation, defective B-cell maturation, and lack of naive T cells. CRISPR-Cas9 knockout of DIAPH1 in PBMCs from healthy donors replicated the T-cell activation defect. Patient-derived peripheral blood T cells exhibited impaired adhesion and inefficient microtubule-organizing center repositioning to the immunologic synapse. The clinical symptoms and laboratory tests also suggested mitochondrial dysfunction. Experiments with immortalized, patient-derived fibroblasts indicated that DIAPH1 affects the amount of complex IV of the mitochondrial respiratory chain. Conclusions Our data demonstrate that individuals with SCBMS can have combined immune deficiency and implicate defective cytoskeletal organization and mitochondrial dysfunction in SCBMS pathogenesis.
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Reint, Ganna; Li, Zhuokun; Labun, Kornel; Keskitalo, Salla; Soppa, Inkeri & Mamia, Katariina Aino Inkeri
[Show all 19 contributors for this article]
(2021).
Rapid genome editing by CRISPR-Cas9-POLD3 fusion.
eLIFE.
11.
doi:
10.7554/eLife.75415.
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Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.
View all works in NVA
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Rio, Oline Eriksen; Labun, Kornel; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia; Valen, Eivind & Haapaniemi, Emma Maria
(2026).
SNIPSNP: precision design of CRISPR reagents.
Full text in Research Archive
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Ervik, Carolina Wieczorek; Kopcil, Pavel; Szymanska, Monika; Kristiansen, Oddrun; Mamia, Katariina Aino Inkeri & Dahal-Koirala, Shiva
[Show all 11 contributors for this article]
(2025).
ENHANCING CRISPR GENE CORRECTION IN HEMATOPOIETIC STEM/PROGENITOR CELLS.
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CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of IDT HDR Enhancer v2, and two PKc inhibitors (KU0060684 and AZD7648) on enhancing HDR in CD34+ cells. We measured the colony forming unit (CFU) output of edited cells was compared: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR as a method to quantify edited cells, we found that PKc inhibitors increased HDR up to 80% in CD34+ cells. Our results suggest that PKc inhibitors is a promising HDR modulator for gene correction in HSPCs. Viability and proliferation were affected in IDT and KU0060684, but in AZD7648 function as well as mock and control. In conclusion, our preliminary results indicates that AZD7648 could lead to the development of more effective gene therapies for monogenic blood diseases in the future. Furthermore, we are optimizing the in vitro culturing of the long-term repopulating hematopoietic stem cells to keep stemness and potentially expanding this population. While initial in vivo testing has been performed, we will also create a novel human xenograft model for modelling diseases e.g. monogenetic disease and leukemia that are irradiation free.
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Ervik, Carolina Wieczorek; Kopcil, Pavel; Szymanska, Monika; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva & Komisarczuk, Anna Zofia
[Show all 10 contributors for this article]
(2025).
Enhancing CRISPR gene correction in hematopoetic stem/progenitor cells.
doi:
https:/iuis2025.org/.
Full text in Research Archive
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CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of IDT HDR Enhancer v2, and two PKc inhibitors (KU0060684 and AZD7648) on enhancing HDR in CD34+ cells. We measured the colony forming unit (CFU) output of edited cells was compared: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR as a method to quantify edited cells, we found that PKc inhibitors increased HDR up to 80% in CD34+ cells. Our results suggest that PKc inhibitors is a promising HDR modulator for gene correction in HSPCs. Viability and proliferation were affected in IDT and KU0060684, but in AZD7648 function as well as mock and control. In conclusion, our preliminary results indicates that AZD7648 could lead to the development of more effective gene therapies for monogenic blood diseases in the future. Furthermore, we are optimizing the in vitro culturing of the long-term repopulating hematopoietic stem cells to keep stemness and potentially expanding this population. While initial in vivo testing has been performed, we will also create a novel human xenograft model for modelling diseases e.g. monogenetic disease and leukemia that are irradiation free.
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Haugen, Frida Loe; Mamia, Katariina Aino Inkeri; Rio, Oline Eriksen; Sollano, Anniken Solveig Matheson; Li, Zhuokun & Labun, Kornel
[Show all 20 contributors for this article]
(2025).
CRISPR-Cas9 pipeline for custom editing in autologous T cells.
doi:
https:/event.fourwaves.com/crisprmed25/abstracts/b5911e8e-de8e-4ee4-b8e2-9f08cc2f31d8.
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CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants in Inborn Errors of Immunity (IEIs). Current IEI correction strategies rely on cDNA knock-in or gene knockout, which can disrupt gene expression and need extensive optimization. To address this, we developed a safe, efficient CRISPR/Cas9-based T cell editing pipeline using HDR for SNV correction.
We observe a rapid and selective expansion of CD3+ T cells from patient peripheral blood following an 8-day cytokine stimulation. To enhance HDR outcomes, we explored multiple strategies and developed an optimized T cell pipeline capable of achieving up to 80% HDR. We found no pre-malignant off-target effects or karyotypic, transcriptomic, or proteomic abnormalities in patient T cells analysed with GUIDE-seq, scRNA-seq, long-read WGS, and proteomics. We observed functional correction of the disease phenotype in patient T cells demonstrated on transcriptomic level by single cell RNA sequencing, on proteomic level by Mass Spectrometry and in cell culture-based assays.
So far, we have implemented the pipeline and corrected 12 unique SNV mutations causing 8 diverse IEIs. This platform is designed to streamline CRISPR tool discovery/optimization and can be further developed into a GMP-compatible, scaled-up process for salvage therapy in T cell-related IEIs.
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Kopcil, Pavel; Ervik, Carolina Wieczorek; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Szymanska, Monika & Komisarczuk, Anna Zofia
[Show all 8 contributors for this article]
(2024).
Enhancing CRISPR Gene Correction In Hematopoietic Stem/Progenitor Cells.
Full text in Research Archive
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CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of Compound X, a small molecule inhibitor selected from a chemical compounds screen in T cells, on enhancing HDR in CD34+ cells. We also compared two methods for measuring the colony forming unit (CFU) output of edited cells: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR (ddPCR) as a method to quantify edited cells, we found that Compound X increased HDR up to 80% in CD34+ cells, but reduced CFU output by 2-3 folds. Our results suggest that Compound X is a promising HDR modulator for gene correction in HSPCs, and could lead to the development of more effective gene therapies for monogenic blood diseases in the near future; however, its side effects need to be further investigated. We are currently performing in vivo experiments with Compound X and exploring novel editing strategies, while also working to align our protocols with current good manufacturing practice (cGMP) standards. Furthermore, we aim to assess and optimize the efficacy of Compound X, as well as similar compounds, in long-term repopulating hematopoietic stem cells (LT-HSCs), given their significant clinical importance and the crucial need to enhance editing efficiency in these cells.
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Kopcil, Pavel; Ervik, Carolina Wieczorek; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Szymanska, Monika & Komisarczuk, Anna Zofia
[Show all 8 contributors for this article]
(2024).
Enhancing CRISPR Gene Correction in Hematopoietic Stem/Progenitor Cells.
Full text in Research Archive
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CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of Compound X, a small molecule inhibitor selected from a chemical compounds screen in T cells, on enhancing HDR in CD34+ cells. We also compared two methods for measuring the colony forming unit (CFU) output of edited cells: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR (ddPCR) as a method to quantify edited cells, we found that Compound X increased HDR up to 80% in CD34+ cells, but reduced CFU output by 2-3 folds. Our results suggest that Compound X is a promising HDR modulator for gene correction in HSPCs, and could lead to the development of more effective gene therapies for monogenic blood diseases in the near future; however, its side effects need to be further investigated. We are currently performing in vivo experiments with Compound X and exploring novel editing strategies, while also working to align our protocols with current good manufacturing practice (cGMP) standards. Furthermore, we aim to assess and optimize the efficacy of Compound X, as well as similar compounds, in long-term repopulating hematopoietic stem cells (LT-HSCs), given their significant clinical importance and the crucial need to enhance editing efficiency in these cells.
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Ervik, Carolina Wieczorek; Kopcil, Pavel; Mamia, Katariina Aino Inkeri; Dahal-Koirala, Shiva; Komisarczuk, Anna Zofia & Bak, Rasmus O.
[Show all 7 contributors for this article]
(2024).
Enhancing CRISPR Gene Correction In Hematopoietic Stem/Progenitor Cells.
Full text in Research Archive
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CRISPR/Cas9 is a powerful tool for precise gene correction of monogenic blood diseases, such as deficiency of adenosine deaminase 2 (DADA2), a rare disorder that causes recurrent inflammation and vascular damage. However, the efficiency of homology-directed repair (HDR) in CD34+ hematopoietic stem/progenitor cells (HSPCs) is low and limits the therapeutic potential of this approach. In this study, we used a Finnish founder mutation (R169Q) in ADA2 as a model locus for gene correction. We introduced four silent single nucleotide polymorphisms (SNPs) by electroporation using RNPs (ribonucleoproteins) and single-stranded oligodeoxynucleotide (ssODN) repair template in wild-type CD34+ cells isolated from umbilical cord blood. We evaluated the effect of Compound X, a small molecule inhibitor selected from a chemical compounds screen in T cells, on enhancing HDR in CD34+ cells. We also compared two methods for measuring the colony forming unit (CFU) output of edited cells: a solid assay based on morphology and pigment of cells, and a liquid assay based on cell surface markers (CD14, CD15, CD235), enabling a higher-throughput setup. Using droplet digital PCR (ddPCR) as a method to quantify edited cells, we found that Compound X increased HDR up to 80% in CD34+ cells, but reduced CFU output by 2-3 folds. Our results suggest that Compound X is a promising HDR modulator for gene correction in HSPCs, and could lead to the development of more effective gene therapies for monogenic blood diseases in the near future; however, its side effects need to be further investigated. We are currently performing in vivo experiments with Compound X and exploring novel editing strategies, while also working to align our protocols with current good manufacturing practice (cGMP) standards. Furthermore, we aim to assess and optimize the efficacy of Compound X, as well as similar compounds, in long-term repopulating hematopoietic stem cells (LT-HSCs), given their significant clinical importance and the crucial need to enhance editing efficiency in these cells.
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Rio, Oline Eriksen; Komisarczuk, Anna Zofia; Labun, Kornel & Haapaniemi, Emma Maria
(2023).
CRISPR-Cas9 corrected T cells for personalized
primary immunodeficiency disease (PIDD) therapy.
Full text in Research Archive
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Rio, Oline Eriksen; Komisarczuk, Anna Zofia; Labun, Kornel & Haapaniemi, Emma Maria
(2023).
CRISPR-Cas9 corrected T cells for personalized primary immunodeficiency disease (PIDD) therapy.
Full text in Research Archive
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Rio, Oline Eriksen; Haapaniemi, Emma Maria; Komisarczuk, Anna Zofia & Labun, Kornel
(2023).
CRISPR-Cas9 corrected T cells for personalized
primary immunodeficiency disease (PIDD) therapy.
Full text in Research Archive
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Naseri, Nafasgol; Dahal-Koirala, Shiva; Haapaniemi, Emma Maria & Falnes, Pål
(2026).
Investigation of on-target Chromosomal deletions and loss during HDR-enhanced CRISPR-Cas9 correction of a CARMIL2 point mutation in patient-derived T cells.
University of Oslo (UiO).
Full text in Research Archive
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Borg, Nora Helene Hammersten; Komisarczuk, Anna Zofia; Falnes, Pål & Haapaniemi, Emma Maria
(2026).
Development of a Multimodal Screening Platform for Comparative Evaluation of Prime Editing Efficiency. Establishing an experimental framework through reporter cell line generation, prime editing vector construction, and detection assay development.
Universitetet i Oslo.
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--- Abstract will be published: 2027-05-19
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Mamia, Katariina Aino Inkeri & Haapaniemi, Emma Maria
(2025).
Precision T cell correction platform for Inborn Errors of Immunity.
Universitetet i Oslo.
Full text in Research Archive
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In this study, Katariina Mamia and colleagues developed an efficient and safe gene editing platform that can correct genetic “typos” in human T cells. This platform can correct single-gene mistakes in T cells from patients with rare diseases affecting the immune system, also known as Inborn Errors of Immunity (IEI).
Currently, some IEIs can be treated with stem cell transplants from healthy donors, but this approach carries serious risks and is not an option for everyone. To address this unmet need, Katariina and colleagues used CRISPR/Cas9 gene editing technology to modify the patients’ own T cells instead. In this approach, T cells are isolated from blood samples, corrected in a laboratory – and one day, transplanted back into the patients.
The platform achieved up to 80% correction efficiency and was shown to improve T cell functions in the corrected T cells. Extensive safety testing detected no harmful effects or unwanted genetic changes within the methods used.
This study presents a first-generation T cell correction platform that can repair various single-gene defects with one workflow. This study presents a first-generation T cell correction platform that can repair various single-gene defects with one workflow. It represents an early step towards clinical translation of gene therapies for IEI patients who currently have limited treatment options.
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Komisarczuk, Anna Zofia; Ganeshan, Janaarthan; Haapaniemi, Emma Maria & Winge, Per
(2024).
epegRNA Screening Platform for Primary Immunodeficiency Mutations in HEK293 cells and Hematopoietic Stem and Progenitory Cells (HSPCs).
Norges teknisk-naturvitenskapelige universitet.
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Mangel på adenosindeaminase (DADA2) er en primær immunsvikt forårsaket av autosomale recessive mutasjoner i adenosindeaminasegen (CECR1, ADA2). Korrekte behandlingsmetoder er først og fremst avhengig av sykdomsfenotypen, og understreker behovet for målrettet genkorreksjon. Prime editing (PE) er et allsidig genomredigeringsverktøy som er i stand til å introdusere innsetting, sletting, alle 12 typer nukleotidendringer og kombinasjoner derav. PE kan potensielt korrigere opptil 90 % av genetiske lidelser, inkludert primære immunsvikt. Evaluering av redigeringseffektiviteten til prime-redigeringsguidRNA (pegRNA) krever imidlertid betydelig innsats.
I denne studien beskriver vi en optimalisert prime-redigeringsscreeningsplattform for å vurdere redigeringseffektiviteten til grunnleggende (pegRNA) og konstruert pegRNA (epegRNA) rettet mot pR169Q-mutasjon i ADA2 (ENSG00000093072). Vi evaluerte sekvensriktigheten til epegRNA- og pegRNA-plasmidbiblioteket ved Sanger-sekvensering og genererte lentivirusbiblioteker. Vi evaluerte også den smittsomme titeren ved bruk av digital dråpe-PCR (ddPCR), genererte cellebiblioteker for stabil ekspresjon av epegRNA og pegRNA, og optimaliserte PCR-forholdene for dyp amplikonsekvensering.
Vi demonstrerte en ddPCR-basert tilnærming for å kvantifisere lentivirus-kopinummer med høy sensitivitet og lavt standardavvik. Det dynamiske området for ddPCR var mellom 300 og 30 000 målkopier per reaksjon, som ble brukt til estimering av kopiantallvariasjon, beregning av infeksiøs titer og infeksjonsmangfold for epegRNA- og pegRNA-cellebibliotek-preparat. Vi observerte at PCR-tilstanden brukt med epegRNA-plasmid ikke var forenlig med gDNA oppnådd fra cellebiblioteket når biblioteket ble klargjort for amplikonsekvensering.
Oppsummert la denne studien grunnlaget for pålitelig og effektiv screening av epegRNA og pegRNA. Vi fremhevet viktigheten av å optimalisere PCR-forhold for amplikonsekvensering. Implementering av denne optimaliserte plattformen lover å effektivisere prosessen med å teste pegRNA-er for monogene mutasjoner.
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Sundell, Tuva; Carlsen, Harald & Haapaniemi, Emma Maria
(2023).
The impact of ADA2 downregulation on T-cell functioning of CRISPR/Cas9 edited PBMCs.
Norges miljø- og biovitenskapelige universitet (NMBU).
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Adenosine Deaminase 2 Deficiency (DADA2) is a rare autosomal genetic syndrome characterized by auto-inflammatory symptoms, where the development of T lymphocytes and B lymphocytes is malfunctioning. The deficiency is caused by the loss of function mutations in the CECR1 gene, which encodes the enzyme adenosine deaminase 2. Adenosine Deaminase 2 (ADA2) is involved in the metabolism of adenosine and has a role in regulating the immune response, and in addition it has been linked to the regulation of T-cell activation and differentiation. Recent studies suggest that downregulation of ADA2 can lead to altered T-cell activation and proliferation. The pathogenesis of the disease is still not completely understood. The aim of this study was to study the effect of knock out of ADA2 and knock in of ADA2 R169Q mutation on the activity and expression of the ADA2 gene. and study the impact of ADA2 downregulation on T-cells functioning. The editing on T-cells was done to understand more of the mechanism of DADA2. Electroporation was used as delivery method for CRISPR/Cas9 in PBMCs (Peripheral Blood Mononuclear Cells) to knock out the ADA2 gene and knock in a mutation of ADA2 (R169Q), a mutation linked to the development of ADA2 deficiency. Further the cells were analyzed to evaluate ADA2 enzyme activity and gene expression, and the effect of ADA2 knockout and R169Q mutation on T-cells were studied using flow cytometry in T-cell activation and proliferation downstream assays. A decrease in ADA2 activity and gene expression were observed for the ADA2 KO and KI of R169Q samples compared to the non-edited samples. However, for the study of T-cell functioning, some problems occurred for long cultivation of the edited T-cells, which was further discussed and compared with cells undergoing a shorter cultivation period. Thus, future experiments are needed with a wider set of samples from multiple donors with enhanced editing efficiency. It is crucial to optimize the experimental procedures for utilizing edited cells to gain deeper insights into the effects of ADA2 downregulation on T-cell functionality. Taking these factors into consideration will contribute to a more comprehensive understanding of the impact of ADA2 downregulation on the functioning of T-cells.
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Published
Jan. 8, 2019 2:18 PM
- Last modified
Mar. 18, 2025 11:23 PM
