Publikasjoner
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Kopcil, Pavel; Ervik, Carolina W.; Reint, Ganna; Mamia, Katariina; Szymanska, Monika & Dahal-Koirala, Shiva
[Vis alle 17 forfattere av denne artikkelen]
(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
[Vis alle 32 forfattere av denne artikkelen]
(2025).
Precision T cell correction platform for Inborn Errors of Immunity.
Molecular Therapy.
ISSN 1525-0016.
33(11),
s. 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
[Vis alle 23 forfattere av denne artikkelen]
(2025).
Heterozygous loss of MAP4K1 causes immune dysregulation by amplifying T-cell responses.
Journal of Allergy and Clinical Immunology.
ISSN 0091-6749.
156(4),
s. 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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Reint, Ganna; Li, Zhuokun; Labun, Kornel; Keskitalo, Salla; Soppa, Inkeri & Mamia, Katariina Aino Inkeri
[Vis alle 19 forfattere av denne artikkelen]
(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.
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Poulsen, Lars La Cour; Edelmann, Reidunn Jetne; Krüger, Stig; Diéguez-Hurtado, Rodrigo; Shah, Akshay & Stav-Noraas, Tor Espen
[Vis alle 22 forfattere av denne artikkelen]
(2018).
Inhibition of endothelial NOTCH1 signaling attenuates inflammation by reducing cytokine-mediated histone acetylation at inflammatory enhancers.
Arteriosclerosis, Thrombosis and Vascular Biology.
ISSN 1079-5642.
38(4),
s. 854–869.
doi:
10.1161/ATVBAHA.117.310388.
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Objective: Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context. Approach and Results: Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1β stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB–directed inflammatory enhancers. Conclusions: Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype.
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Szymanska, Monika; Fosdahl, Anne Marthe; Nikolaysen, Filip; Pedersen, Mikkel W; Grandal, Michael M & Stang, Espen
[Vis alle 7 forfattere av denne artikkelen]
(2016).
A combination of two antibodies recognizing non-overlapping epitopes of HER2 induces kinase activity-dependent internalization of HER2.
Journal of Cellular and Molecular Medicine.
ISSN 1582-1838.
20(10),
s. 1999–2010.
doi:
10.1111/jcmm.12899.
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Sak, Malgorzata Magdalena; Szymanska, Monika; Bertelsen, Vibeke; Hasmann, Max; Madshus, Inger Helene & Stang, Espen
(2013).
Pertuzumab counteracts the inhibitory effect of ErbB2 on degradation of ErbB3.
Carcinogenesis.
ISSN 0143-3334.
34(9),
s. 2031–2038.
doi:
10.1093/carcin/bgt173.
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Ervik, Carolina Wieczorek; Kopcil, Pavel; Szymanska, Monika; Kristiansen, Oddrun; Mamia, Katariina Aino Inkeri & Dahal-Koirala, Shiva
[Vis alle 11 forfattere av denne artikkelen]
(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
[Vis alle 10 forfattere av denne artikkelen]
(2025).
Enhancing CRISPR gene correction in hematopoetic stem/progenitor cells.
doi:
https:/iuis2025.org/.
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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
[Vis alle 20 forfattere av denne artikkelen]
(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
[Vis alle 8 forfattere av denne artikkelen]
(2024).
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 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
[Vis alle 8 forfattere av denne artikkelen]
(2024).
Enhancing CRISPR Gene Correction in Hematopoietic Stem/Progenitor Cells.
Fulltekst i vitenarkiv
Vis sammendrag
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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Saarela, Janna Saija; Szymanska, Monika; Bøhn, Siv Kjølsrud & Lurås, Sanne Iversen
(2024).
Deciphering ADA2 Deficiency: Creating an ADA2 deficiency model using CRISPR/Cas genome editing.
Norges miljø- og biovitenskapelige universitet (NMBU).
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Mangel på adenosindeaminase 2 (DADA2) er en sjelden autoinflammatorisk lidelse forårsaket av bialleliske mutasjoner i adenosindeaminase 2 (ADA2) genet. Pasienter med DADA2 viser et bredt spekter av kliniske manifestasjoner, og tilstanden kan være livstruende. Sykdomsmekanismene til DADA2 er i stor grad ukjente og for å øke kunnskapen i dette feltet, hadde denne studien som mål å lage DADA2-cellemodeller og studere effekten av nedslått ADA2 genuttrykk på to sentrale immune celle mekanismer: proliferasjon av monocytter og makrofagpolarisering. For å gen redigere ADA2 ble guide RNA (gRNA) og CRISPR-assosiert protein 9 (Cas9) introdusert inn i THP-1 celler ved bruk av elektroporering. De gen redigerte cellene ble videre sortert ved bruk av fluorescensaktivert cellesortering (FACS), hvor til slutt fire av de utvalgte klonene viste en homozygot delesjon i ADA2-genet som er forventet å forårsake en leseramme forskyvning i budbringer-RNA (mRNA). En proliferasjon analyse som ble utført på ADA2-redigerte, ikke-sorterte celler ved bruk av Incucyte S3 viste at de redigerte cellene hadde en signifikant lavere proliferasjon sammenlignet med kontroll celler. Effekten av DADA2 på makrofagpolarisering ble testet på sorterte ADA2-redigerte celler og evaluert med spesifikke markører ved hjelp av kvantitativ PCR (qPCR). Cellene ble først differensiert til M0 og deretter polarisert til M1 og M2a makrofager. Resultatene indikerte at DADA2-cellemodeller viste en favorisert polarisering mot M1 makrofager. Eksperimentelle begrensninger oppsto, og ytterligere testing må gjøres for å trekke pålitelige konklusjoner.
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Publisert
20. sep. 2019 08:41
- Sist endret
17. mars 2025 13:17
