Publications
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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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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/.
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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.
Full text in Research Archive
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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
Show summary
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
Show summary
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
(2022).
Precise editing for ADA2 deficiency.
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CRISPR/Cas9 technology has been shown to be able to correct harmful mutations in variety of human cells and enable numerous clinical trials to be started already. Here, we took advantage of convenience and elegance of this editing system and introduced model SNPs into ADA2 mutation specific region in human CD34+ cord blood haematopoietic stem and progenitor cells (HSPCs). First, we screened 7 sgRNAs together with ssODN repair templates spanning C→T (R169Q) ADA2 mutation in HSPCs. We validated the editing outcomes by Next generation amplicon sequencing, Digital-Droplet PCR (ddPCR) and ICE analysis. To ensure the edited cells with the best performing sgRNA were fully functional, we utilized standard NSG mouse model NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (Charles Rivers) for xenotransplantation study including optimized irradiation protocol. Finally, we did not observe suppression of engraftment levels and were able to detect HDR events in subpopulations of HSPCs and differentiated immune cells after 12 weeks post intravenous injection. Nevertheless we saw donor to donor variation in editing and engraftment potential. Additionally, we tested chemically modified ssODNs and DNA repair interfering factors GSE56, Ad5 E4 Orf6&7 and i53 and secured HDR improvement in HSPCs in vitro. Taken together, we presented workflow and evidence for future application of precise and tailored approach for editing and engrafting of ADA2-SCID patients derived CD34+ cells.
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Kopcil, Pavel
(2026).
CRISPR/Cas9-based Strategies for ADA2 Inborn Error of Immunity.
Universitetet i Oslo.
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We show that gene editing can correct a rare immune disorder called DADA2. Using CRISPR, a “molecular scissor,” we fixed a single-letter error in the ADA2 gene with high precision (up to 80%) and tested adding a healthy copy of the gene as a broader solution. Edited blood stem cells stayed healthy, made balanced blood cell types, and worked in mice.
DADA2 often starts in childhood and can cause serious illness. Current treatment can be a stem cell transplant from a donor, but donors are scarce and the procedure can fail. Our work points to a patient-tailored therapy that uses a person’s own stem cells, and a near-universal option that could help many patients regardless of their exact mutation.
The tools are powerful and flexible, and they could change how we treat rare genetic diseases. But cutting DNA can introduce risks, such as unwanted changes. Therefore, we tested safety in several ways and found encouraging results, yet more work is needed to ensure long-term safety before clinical use.
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Published
Nov. 3, 2020 3:03 PM
- Last modified
Mar. 14, 2025 10:41 PM
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