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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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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Chlubnova, Marketa; Christophersen, Asbjørn Otto; Sandve, Geir Kjetil Ferkingstad; Lundin, Knut Erik Aslaksen; Jahnsen, Jørgen & Dahal-Koirala, Shiva
[Show all 7 contributors for this article]
(2023).
Identification of gluten T cell epitopes driving celiac disease.
Science Advances.
9(4),
p. 1–8.
doi:
10.1126/sciadv.ade5800.
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CD4+ T cells specific for cereal gluten proteins are key players in celiac disease (CeD) pathogenesis. While several CeD-relevant gluten T cell epitopes have been identified, epitopes recognized by a substantial proportion of gluten-reactive T cells remain unknown. The identification of such CeD-driving gluten epitopes is important for the food industry and in clinical settings. Here, we have combined the knowledge of a distinct phenotype of gluten-reactive T cells and key features of known gluten epitopes for the discovery of unknown epitopes. We tested 42 wheat gluten–reactive T cell clones, isolated on the basis of their distinct phenotype and with no reactivity to known epitopes, against a panel of synthetic peptides bioinformatically identified from a wheat gluten protein database. We were able to assign reactivity to 10 T cell clones and identified a 9-nucleotide oligomer core region of five previously uncharacterized gliadin/glutenin epitopes. This work represents an advance in the effort to identify CeD-driving gluten epitopes.
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Christophersen, Asbjørn Otto; Dahal-Koirala, Shiva; Chlubnova, Marketa; Jahnsen, Jørgen; Lundin, Knut & Sollid, Ludvig Magne
(2022).
Phenotype-Based Isolation of Antigen-Specific CD4+ T Cells in Autoimmunity: A Study of Celiac Disease.
Advanced Science.
9(10),
p. 1–10.
doi:
10.1002/advs.202104766.
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The pathogenic immune response in celiac disease (CeD) is orchestrated by phenotypically distinct CD4+ T cells that recognize gluten epitopes in the context of disease-associated HLA-DQ allotypes. Cells with the same distinct phenotype, but with elusive specificities, are increased across multiple autoimmune conditions. Here, whether sorting of T cells based on their distinct phenotype (Tphe cells) yields gluten-reactive cells in CeD is tested. The method′s efficiency is benchmarked by parallel isolation of gluten-reactive T cells (Ttet cells), using HLA-DQ:gluten peptide tetramers. From gut biopsies of 12 untreated HLA-DQ2.5+ CeD patients, Ttet+/Tphe+, Ttet−/Tphe+, and Ttet−/Tphe− cells are sorted for single-cell T-cell receptor (TCR)-sequencing (n = 8) and T-cell clone (TCC)-generation (n = 5). The generated TCCs are TCR sequenced and tested for their reactivity against deamidated gluten. Gluten-reactivity is observed in 91.2% of Ttet+/Tphe+ TCCs, 65.3% of Ttet−/Tphe+ TCCs and 0% of Ttet−/Tphe− TCCs. TCR sequencing reveals clonal expansion and sequence sharing across patients, features reflecting antigen-driven responses. The feasibility to isolate antigen-specific CD4+ T cells by the sole use of phenotypic markers in CeD outlines a potential avenue for characterizing disease-driving CD4+ T cells in autoimmune conditions.
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Dahal-Koirala, Shiva; Balaban, Gabriel; Neumann, Ralf Stefan; Scheffer, Lonneke; Lundin, Knut & Greiff, Victor
[Show all 9 contributors for this article]
(2022).
TCRpower: quantifying the detection power of T-cell receptor sequencing with a novel computational pipeline calibrated by spike-in sequences.
Briefings in Bioinformatics.
ISSN 1467-5463.
23(2),
p. 1–14.
doi:
10.1093/bib/bbab566.
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T-cell receptor (TCR) sequencing has enabled the development of innovative diagnostic tests for cancers, autoimmune diseases and other applications. However, the rarity of many T-cell clonotypes presents a detection challenge, which may lead to misdiagnosis if diagnostically relevant TCRs remain undetected. To address this issue, we developed TCRpower, a novel computational pipeline for quantifying the statistical detection power of TCR sequencing methods. TCRpower calculates the probability of detecting a TCR sequence as a function of several key parameters: in-vivo TCR frequency, T-cell sample count, read sequencing depth and read cutoff. To calibrate TCRpower, we selected unique TCRs of 45 T-cell clones (TCCs) as spike-in TCRs. We sequenced the spike-in TCRs from TCCs, together with TCRs from peripheral blood, using a 5′ RACE protocol. The 45 spike-in TCRs covered a wide range of sample frequencies, ranging from 5 per 100 to 1 per 1 million. The resulting spike-in TCR read counts and ground truth frequencies allowed us to calibrate TCRpower. In our TCR sequencing data, we observed a consistent linear relationship between sample and sequencing read frequencies. We were also able to reliably detect spike-in TCRs with frequencies as low as one per million. By implementing an optimized read cutoff, we eliminated most of the falsely detected sequences in our data (TCR α-chain 99.0% and TCR β-chain 92.4%), thereby improving diagnostic specificity. TCRpower is publicly available and can be used to optimize future TCR sequencing experiments, and thereby enable reliable detection of disease-relevant TCRs for diagnostic applications.
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Ciacchi, Laura; Farenc, Carine; Dahal-Koirala, Shiva; Petersen, Jan; Sollid, Ludvig Magne & Reid, Hugh H.
[Show all 7 contributors for this article]
(2022).
Structural basis of T cell receptor specificity and cross-reactivity of two HLA-DQ2.5-restricted gluten epitopes in celiac disease.
Journal of Biological Chemistry.
ISSN 0021-9258.
298:101619(3),
p. 1–11.
doi:
10.1016/j.jbc.2022.101619.
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Celiac disease is a T cell-mediated chronic inflammatory condition often characterized by human leukocyte antigen (HLA)-DQ2.5 molecules presenting gluten epitopes derived from wheat, barley, and rye. Although some T cells exhibit cross-reactivity toward distinct gluten epitopes, the structural basis underpinning such cross-reactivity is unclear. Here, we investigated the T-cell receptor specificity and cross-reactivity of two immunodominant wheat gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). We show by surface plasmon resonance that a T-cell receptor alpha variable (TRAV) 4+-T-cell receptor beta variable (TRBV) 29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1 with similar affinity, whereas a TRAV4- (TRAV9-2+) TCR recognized HLA-DQ2.5-glia-ω1 only. We further determined the crystal structures of the TRAV4+-TRBV29-1+ TCR bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1, as well as the structure of an epitope-specific TRAV9-2+-TRBV7-3+ TCR-HLA-DQ2.5-glia-ω1 complex. We found that position 7 (p7) of the DQ2.5-glia-α1a and DQ2.5-glia-ω1 epitopes made very limited contacts with the TRAV4+ TCR, thereby explaining the TCR cross-reactivity across these two epitopes. In contrast, within the TRAV9-2+ TCR-HLA-DQ2.5-glia-ω1 ternary complex, the p7-Gln was situated in an electrostatic pocket formed by the hypervariable CDR3β loop of the TCR and Arg70β from HLA-DQ2.5, a polar network which would not be supported by the p7-Leu residue of DQ2.5-glia-α1a. In conclusion, we provide additional insights into the molecular determinants of TCR specificity and cross-reactivity to two closely-related epitopes in celiac disease.
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Zhou, Chunyan; Østerbye, Thomas; Bach, Emil; Dahal-Koirala, Shiva; Høydahl, Lene Støkken & Steinsbø, Øyvind
[Show all 11 contributors for this article]
(2022).
Focused B cell response to recurring gluten motif with implications for epitope spreading in celiac disease.
Cell reports.
41:111541(4),
p. 1–16.
doi:
10.1016/j.celrep.2022.111541.
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Antibodies to deamidated gluten peptides are accurate diagnostic markers of celiac disease. However, binding of patient antibodies to all possible gluten epitopes has not previously been investigated. Here, we assess serum antibody specificity across the gluten proteome by use of high-density peptide arrays. We confirm the importance of deamidation for antibody binding, and we show that the response is remarkably focused on the known epitope QPEQPFP (where E results from deamidation of Q). In addition, we describe an epitope in native (non-deamidated) gluten, QQPEQII (where E is gene encoded), which is associated with both B cell and T cell reactivity. Antibodies to this native epitope are cross-reactive with the major deamidated epitope due to recognition of the shared PEQ motif. Since cross-reactive B cells can present peptides to different gluten-specific T cells, we propose that such B cells play a role in epitope spreading by engaging T cells with multiple specificities.
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Qiao, Shuo-Wang; Dahal-Koirala, Shiva; Eggesbø, Linn Margrethe; Lundin, Knut E. A. & Sollid, Ludvig Magne
(2021).
Frequency of Gluten-Reactive T Cells in Active Celiac Lesions Estimated by Direct Cell Cloning.
Frontiers in Immunology.
12:646163,
p. 1–8.
doi:
10.3389/fimmu.2021.646163.
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Chronic inflammation of the small intestine in celiac disease is driven by activation of CD4+ T cells that recognize gluten peptides presented by disease-associated HLA-DQ molecules. We have performed direct cell cloning of duodenal biopsies from five untreated and one refractory celiac disease patients, and three non-celiac disease control subjects in order to assess, in an unbiased fashion, the frequency of gluten-reactive T cells in the disease-affected tissue as well as the antigen fine specificity of the responding T cells. From the biopsies of active disease lesions of five patients, 19 T-cell clones were found to be gluten-reactive out of total 1,379 clones tested. This gave an average of 1.4% (range 0.7% - 1.9%) of gluten-reactive T cells in lamina propria of active celiac lesions. Interestingly, also the patient with refractory celiac disease had gluten-reactive T cell clones in the lamina propria (5/273; 1.8%). In comparison, we found no gluten-reactive T cells in any of the total 984 T-cell clones screened from biopsies from three disease control donors. Around two thirds of the gluten-reactive clones were reactive to a panel of peptides representing known gluten T-cell epitopes, of which two thirds were reactive to the immunodominant DQ2.5-glia-α1/DQ2.5-glia-α2 and DQ2.5-glia-ω1/DQ2.5-glia-ω2 epitopes. This study shows that gluten-reactive T cells in the inflamed duodenal tissue are prevalent in the active disease lesion, and that many of these T cells are reactive to T-cell epitopes that are not yet characterized. Knowledge of the prevalence and epitope specificity of gluten-specific T cells is a prerequisite for therapeutic efforts that target disease-specific T cells in celiac disease.
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Risnes, Louise Fremgaard; Eggesbø, Linn Margrethe; Zühlke, Stephanie; Dahal-Koirala, Shiva; Neumann, Ralf Stefan & Lundin, Knut Erik Aslaksen
[Show all 8 contributors for this article]
(2021).
Circulating CD103+ γδ and CD8+ T cells are clonally shared with tissue-resident intraepithelial lymphocytes in celiac disease.
Mucosal Immunology.
ISSN 1933-0219.
14(4),
p. 842–851.
doi:
10.1038/s41385-021-00385-8.
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Gut intraepithelial γδ and CD8+ αβ T lymphocytes have been connected to celiac disease (CeD) pathogenesis. Based on the previous observation that activated (CD38+), gut-homing (CD103+) γδ and CD8+ αβ T cells increase in blood upon oral gluten challenge, we wanted to shed light on the pathogenic involvement of these T cells by examining the clonal relationship between cells of blood and gut during gluten exposure. Of 20 gluten-challenged CeD patients, 8 and 10 had increase in (CD38+CD103+) γδ and CD8+ αβ T cells, respectively, while 16 had increase in gluten-specific CD4+ T cells. We obtained γδ and αβ TCR sequences of >2500 single cells from blood and gut of 5 patients, before and during challenge. We observed extensive sharing between blood and gut γδ and CD8+ αβ T-cell clonotypes even prior to gluten challenge. In subjects with challenge-induced surge of γδ and/or CD8+ αβ T cells, as larger populations of cells analyzed, we observed more expanded clonotypes and clonal sharing, yet no discernible TCR similarities between expanded and/or shared clonotypes. Thus, CD4+ T cells appear to drive expansion of clonally diverse γδ or CD8+ αβ T-cell clonotypes that may not be specific for the gluten antigen.
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Dahal-Koirala, Shiva; Risnes, Louise Fremgaard; Neumann, Ralf Stefan; Christophersen, Asbjørn; Lundin, Knut E. A. & Sandve, Geir Kjetil
[Show all 8 contributors for this article]
(2021).
Comprehensive Analysis of CDR3 Sequences in Gluten-Specific T-Cell Receptors Reveals a Dominant R-Motif and Several New Minor Motifs.
Frontiers in Immunology.
12:639672,
p. 1–13.
doi:
10.3389/fimmu.2021.639672.
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Gluten-specific CD4+ T cells are drivers of celiac disease (CeD). Previous studies of gluten-specific T-cell receptor (TCR) repertoires have found public TCRs shared across multiple individuals, biased usage of particular V-genes and conserved CDR3 motifs. The CDR3 motifs within the gluten-specific TCR repertoire, however, have not been systematically investigated. In the current study, we analyzed the largest TCR database of gluten-specific CD4+ T cells studied so far consisting of TCRs of 3122 clonotypes from 63 CeD patients. We established a TCR database from CD4+ T cells isolated with a mix of HLA-DQ2.5:gluten tetramers representing four immunodominant gluten epitopes. In an unbiased fashion we searched by hierarchical clustering for common CDR3 motifs among 2764 clonotypes. We identified multiple CDR3α, CDR3β, and paired CDR3α:CDR3β motif candidates. Among these, a previously known conserved CDR3β R-motif used by TRAV26-1/TRBV7-2 TCRs specific for the DQ2.5-glia-α2 epitope was the most prominent motif. Furthermore, we identified the epitope specificity of altogether 16 new CDR3α:CDR3β motifs by comparing with TCR sequences of 231 T-cell clones with known specificity and TCR sequences of cells sorted with single HLA-DQ2.5:gluten tetramers. We identified 325 public TCRα and TCRβ sequences of which 145, 102 and 78 belonged to TCRα, TCRβ and paired TCRαβ sequences, respectively. While the number of public sequences was depended on the number of clonotypes in each patient, we found that the proportion of public clonotypes from the gluten-specific TCR repertoire of given CeD patients appeared to be stable (median 37%). Taken together, we here demonstrate that the TCR repertoire of CD4+ T cells specific to immunodominant gluten epitopes in CeD is diverse, yet there is clearly biased V-gene usage, presence of public TCRs and existence of conserved motifs of which R-motif is the most prominent.
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Ting, Yi Tian; Dahal-Koirala, Shiva; Kim, Hui Shi Keshia; Qiao, Shuo Wang; Neumann, Ralf Stefan & Lundin, Knut Erik Aslaksen
[Show all 10 contributors for this article]
(2020).
A molecular basis for the T cell response in HLA-DQ2.2 mediated celiac disease.
Proceedings of the National Academy of Sciences of the United States of America (PNAS).
ISSN 0027-8424.
117(6),
p. 3063–3073.
doi:
10.1073/pnas.1914308117.
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The highly homologous human leukocyte antigen (HLA)-DQ2 molecules, HLA-DQ2.5 and HLA-DQ2.2, are implicated in the pathogenesis of celiac disease (CeD) by presenting gluten peptides to CD4 + T cells. However, while HLA-DQ2.5 is strongly associated with disease, HLA-DQ2.2 is not, and the molecular basis underpinning this differential disease association is unresolved. We here provide structural evidence for how the single polymorphic residue (HLA-DQ2.5-Tyr22α and HLA-DQ2.2-Phe22α) accounts for HLA-DQ2.2 additionally requiring gluten epitopes possessing a serine at the P3 position of the peptide. In marked contrast to the biased T cell receptor (TCR) usage associated with HLA-DQ2.5–mediated CeD, we demonstrate with extensive single-cell sequencing that a diverse TCR repertoire enables recognition of the immunodominant HLA-DQ2.2-glut-L1 epitope. The crystal structure of two CeD patient-derived TCR in complex with HLA-DQ2.2 and DQ2.2-glut-L1 (PFSEQEQPV) revealed a docking strategy, and associated interatomic contacts, which was notably distinct from the structures of the TCR:HLA-DQ2.5:gliadin epitope complexes. Accordingly, while the molecular surfaces of the antigen-binding clefts of HLA-DQ2.5 and HLA-DQ2.2 are very similar, differences in the nature of the peptides presented translates to differences in responding T cell repertoires and the nature of engagement of the respective antigen-presenting molecules, which ultimately is associated with differing disease penetrance.
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Dahal-Koirala, Shiva; Neumann, Ralf Stefan; Jahnsen, Jørgen; Lundin, Knut Erik Aslaksen & Sollid, Ludvig Magne
(2019).
On the immune response to barley in celiac disease: Biased and public T-cell receptor usage to a barley unique and immunodominant gluten epitope.
European Journal of Immunology.
ISSN 0014-2980.
p. 1–14.
doi:
10.1002/eji.201948253.
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Celiac disease (CeD) is driven by CD4+ T‐cell responses to dietary gluten proteins of wheat, barley, and rye when deamidated gluten epitopes are presented by certain disease‐associated HLA‐DQ allotypes. About 90% of the CeD patients express HLA‐DQ2.5. In such patients, five gluten epitopes dominate the anti‐gluten T‐cell response; two epitopes unique to wheat, two epitopes present in wheat, barley, and rye and one epitope unique to barley. Despite presence of barley in commonly consumed food and beverages and hence being a prominent source of gluten, knowledge about T‐cell responses elicited by barley in CeD is scarce. Therefore, in this study, we explored T‐cell response toward the barley unique epitope DQ2.5‐hor‐3 (PIPEQPQPY) by undertaking HLA‐DQ:gluten peptide tetramer staining, single‐cell T‐cell receptor (TCR) αβ sequencing, T‐cell cloning, and T‐cell proliferation studies. We demonstrate that majority of the CeD patients generate T‐cell response to DQ2.5‐hor‐3, and this response is characterized by clonal expansion, preferential TCR V‐gene usage and public TCR features thus echoing findings previously made for wheat gluten epitopes. The knowledge that biased and public TCRs underpin the T‐cell response to all the immunodominant gluten epitopes in CeD suggests that such T cells are promising diagnostic and therapeutic targets.
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Zühlke, Stephanie; Risnes, Louise Fremgaard; Dahal-Koirala, Shiva; Christophersen, Asbjørn; Sollid, Ludvig Magne & Lundin, Knut Erik Aslaksen
(2019).
CD38 expression on gluten-specific T cells is a robust marker of gluten re-exposure in coeliac disease.
United European Gastroenterology Journal (UEG Journal).
ISSN 2050-6406.
7(10),
p. 1337–1344.
doi:
10.1177/2050640619874183.
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Christophersen, Asbjørn; Risnes, Louise Fremgaard; Dahal-Koirala, Shiva & Sollid, Ludvig Magne
(2019).
Therapeutic and diagnostic implications of T cell scarring in celiac disease and beyond.
Trends in Molecular Medicine.
ISSN 1471-4914.
25(10),
p. 836–852.
doi:
10.1016/j.molmed.2019.05.009.
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Few therapeutic and diagnostic tools specifically aim at T cells in autoimmune disorders, but are T cells a narrow target in these diseases? Lessons may be learned from celiac disease (CeD), one of the few autoimmune disorders where the T cell driving antigens are known, i.e. dietary gluten proteins. T cell clonotypes specific to gluten are expanded, persist for decades and express a distinct phenotype in CeD patients. Cells with this phenotype are increased also in other autoimmune conditions. Accordingly, disease-specific CD4+ T cells form an immunological scar in CeD and probably other autoimmune disorders. We discuss approaches how such T cells may be targeted for better treatment and diagnosis via their antigen specificity or via their expression of characteristic phenotypic markers.
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Christophersen, Asbjørn; Lund, Eivind Gard; Snir, Omri; Solà, Elsa; Kanduri, Srinivasa Kalyana Chakravarthi & Dahal-Koirala, Shiva
[Show all 15 contributors for this article]
(2019).
Distinct phenotype of CD4+ T cells driving celiac disease identified in multiple autoimmune conditions.
Nature Medicine.
ISSN 1078-8956.
25(5),
p. 734–737.
doi:
10.1038/s41591-019-0403-9.
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Combining HLA-DQ–gluten tetramers with mass cytometry and RNA sequencing analysis, we find that gluten-specific CD4+ T cells in the blood and intestines of patients with celiac disease display a surprisingly rare phenotype. Cells with this phenotype are also elevated in patients with systemic sclerosis and systemic lupus erythematosus, suggesting a way to characterize CD4+ T cells specific for disease-driving antigens in multiple autoimmune conditions.
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Dahal-Koirala, Shiva; Ciacchi, Laura; Petersen, Jan; Risnes, Louise Fremgaard; Neumann, Ralf Stefan & Christophersen, Asbjørn
[Show all 11 contributors for this article]
(2018).
Discriminative T-cell receptor recognition of highly homologous HLA-DQ2-bound gluten epitopes.
Journal of Biological Chemistry.
ISSN 0021-9258.
294(3),
p. 941–952.
doi:
10.1074/jbc.RA118.005736.
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Celiac disease (CeD) provides an opportunity to study the specificity underlying human T-cell responses to an array of similar epitopes presented by the same human leukocyte antigen II (HLA-II) molecule. Here, we investigated T-cell responses to the two immunodominant and highly homologous HLA-DQ2.5–restricted gluten epitopes, DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF). Using HLA-DQ2.5–DQ2.5-glia-α1a and HLA-DQ2.5–DQ2.5-glia-ω1 tetramers and single-cell αβ T-cell receptor (TCR) sequencing, we observed that despite similarity in biased variable-gene usage in the TCR repertoire responding to these nearly identical peptide–HLA-II complexes, most of the T cells are specific for either of the two epitopes. To understand the molecular basis of this exquisite fine specificity, we undertook Ala substitution assays revealing that the p7 residue (Leu/Gln) is critical for specific epitope recognition by both DQ2.5-glia-α1a– and DQ2.5-glia-ω1–reactive T-cell clones. We determined high-resolution binary crystal structures of HLA-DQ2.5 bound to DQ2.5-glia-α1a (2.0 Å) and DQ2.5-glia-ω1 (2.6 Å). These structures disclosed that differences around the p7 residue subtly alter the neighboring substructure and electrostatic properties of the HLA-DQ2.5–peptide complex, providing the fine specificity underlying the responses against these two highly homologous gluten epitopes. This study underscores the ability of TCRs to recognize subtle differences in the peptide–HLA-II landscape in a human disease setting.
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Risnes, Louise Fremgaard; Christophersen, Asbjørn; Dahal-Koirala, Shiva; Neumann, Ralf Stefan; Sandve, Geir Kjetil & Sarna, Vikas Kumar
[Show all 9 contributors for this article]
(2018).
Disease-driving CD4+ T cell clonotypes persist for decades in celiac disease.
Journal of Clinical Investigation.
ISSN 0021-9738.
128(6),
p. 2642–2650.
doi:
10.1172/JCI98819.
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Little is known about the repertoire dynamics and persistence of pathogenic T cells in HLA-associated disorders. In celiac disease, a disorder with a strong association with certain HLA-DQ allotypes, presumed pathogenic T cells can be visualized and isolated with HLA-DQ:gluten tetramers, thereby enabling further characterization. Single and bulk populations of HLA-DQ:gluten tetramer–sorted CD4+ T cells were analyzed by high-throughput DNA sequencing of rearranged TCR-α and -β genes. Blood and gut biopsy samples from 21 celiac disease patients, taken at various stages of disease and in intervals of weeks to decades apart, were examined. Persistence of the same clonotypes was seen in both compartments over decades, with up to 53% overlap between samples obtained 16 to 28 years apart. Further, we observed that the recall response following oral gluten challenge was dominated by preexisting CD4+ T cell clonotypes. Public features were frequent among gluten-specific T cells, as 10% of TCR-α, TCR-β, or paired TCR-αβ amino acid sequences of total 1813 TCRs generated from 17 patients were observed in 2 or more patients. In established celiac disease, the T cell clonotypes that recognize gluten are persistent for decades, making up fixed repertoires that prevalently exhibit public features. These T cells represent an attractive therapeutic target.
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Sarna, Vikas Kumar; Skodje, Gry Irene; Reims, Henrik Mikael; Risnes, Louise Fremgaard; Dahal-Koirala, Shiva & Sollid, Ludvig Magne
[Show all 7 contributors for this article]
(2017).
HLA-DQ:gluten tetramer test in blood gives better detection of coeliac patients than biopsy after 14-day gluten challenge.
Gut.
ISSN 0017-5749.
67(9),
p. 1606–1613.
doi:
10.1136/gutjnl-2017-314461.
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Objective: Initiation of a gluten-free diet without proper diagnostic work-up of coeliac disease is a frequent and demanding problem. Recent diagnostic guidelines suggest a gluten challenge of at least 14 days followed by duodenal biopsy in such patients. The rate of false-negative outcome of this approach remains unclear. We studied responses to 14-day gluten challenge in subjects with treated coeliac disease. Design: We challenged 20 subjects with biopsy-verified coeliac disease, all in confirmed mucosal remission, for 14 days with 5.7 grams per oral gluten daily. Duodenal biopsies were collected. Blood was analysed by multiplex assay for cytokine detection, and by flow cytometry using HLA-DQ:gluten tetramers. Results: Nineteen participants completed the challenge. Villous blunting appeared at end of challenge in 5 of 19 subjects. Villous height to crypt depth ratio reduced with at least 0.4 concomitantly with an increase in intraepithelial lymphocyte count of at least 50% in 9 of 19 subjects. Interleukin-8 plasma concentration increased by more than 100% after 4 hours in 7 of 19 subjects. Frequency of blood CD4+ effector-memory gut-homing HLA-DQ:gluten tetramer-binding T cells increased by more than 100% on day 6 in 12 of 15 evaluated participants. Conclusion: A 14-day gluten challenge was not enough to establish significant mucosal architectural changes in majority of patients with coeliac disease (sensitivity ≈25%–50%). Increase in CD4+ effector-memory gut-homing HLA-DQ:gluten tetramer-binding T cells in blood 6 days after gluten challenge is a more sensitive and less invasive biomarker that should be validated in a larger study.
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Gunnarsen, Kristin Støen; Høydahl, Lene Støkken; Risnes, Louise Fremgaard; Dahal-Koirala, Shiva; Neumann, Ralf Stefan & Bergseng, Elin
[Show all 15 contributors for this article]
(2017).
A TCRα framework–centered codon shapes a biased T cell repertoire through direct MHC and CDR3β interactions.
JCI Insight.
2(17).
doi:
10.1172/jci.insight.95193.
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Selection of biased T cell receptor (TCR) repertoires across individuals is seen in both infectious diseases and autoimmunity, but the underlying molecular basis leading to these shared repertoires remains unclear. Celiac disease (CD) occurs primarily in HLA-DQ2.5+ individuals and is characterized by a CD4+ T cell response against gluten epitopes dominated by DQ2.5-glia-α1a and DQ2.5-glia-α2. The DQ2.5-glia-α2 response recruits a highly biased TCR repertoire composed of TRAV26-1 paired with TRBV7-2 harboring a semipublic CDR3β loop. We aimed to unravel the molecular basis for this signature. By variable gene segment exchange, directed mutagenesis, and cellular T cell activation studies, we found that TRBV7-3 can substitute for TRBV7-2, as both can contain the canonical CDR3β loop. Furthermore, we identified a pivotal germline-encoded MHC recognition motif centered on framework residue Y40 in TRAV26-1 engaging both DQB1*02 and the canonical CDR3β. This allowed prediction of expanded DQ2.5-glia-α2–reactive TCR repertoires, which were confirmed by single-cell sorting and TCR sequencing from CD patient samples. Our data refine our understanding of how HLA-dependent biased TCR repertoires are selected in the periphery due to germline-encoded residues. This research was originally published in JCI Insight. © 2018 American Society for Clinical Investigation
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Dahal-Koirala, Shiva; Risnes, Louise Fremgaard; Christophersen, Asbjørn; Sarna, Vikas Kumar; Lundin, Knut Erik Aslaksen & Sollid, Ludvig Magne
[Show all 7 contributors for this article]
(2016).
TCR sequencing of single cells reactive to DQ2.5-glia-α2 and DQ2.5-glia-ω2 reveals clonal expansion and epitope-specific V-gene usage.
Mucosal Immunology.
ISSN 1933-0219.
9(3),
p. 587–596.
doi:
10.1038/mi.2015.147.
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CD4+ T cells recognizing dietary gluten epitopes in the context of disease-associated human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 molecules are the key players in celiac disease pathogenesis. Here, we conducted a large-scale single-cell paired T-cell receptor (TCR) sequencing study to characterize the TCR repertoire for two homologous immunodominant gluten epitopes, DQ2.5-glia-α2 and DQ2.5-glia-ω2, in blood of celiac disease patients after oral gluten challenge. Despite sequence similarity of the epitopes, the TCR repertoires are unique but shared several overall features. We demonstrate that clonally expanded T cells dominate the T-cell responses to both epitopes. Moreover, we find V-gene bias of TRAV26, TRAV4, and TRBV7 in DQ2.5-glia-α2 reactive TCRs, while DQ2.5-glia-ω2 TCRs displayed significant bias toward TRAV4 and TRBV4. The knowledge that antigen-specific TCR repertoire in chronic inflammatory diseases tends to be dominated by a few expanded clones that use the same TCR V-gene segments across patients is important information for HLA-associated diseases where the antigen is unknown.
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Fykerud, Tone Aase; Knudsen, Lars Mørland; Totland, Max; Sørensen, Vigdis; Dahal-Koirala, Shiva & Lothe, Ragnhild A
[Show all 8 contributors for this article]
(2016).
Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding.
Cell Cycle.
ISSN 1538-4101.
15(21),
p. 2943–2957.
doi:
10.1080/15384101.2016.1231280.
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In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed “mitotic nanotubes,” were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.
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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/.
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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
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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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).
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Published
Nov. 8, 2021 2:01 PM
- Last modified
Mar. 20, 2025 5:24 AM
