The most common disease-causing mutation of factor XIII deficiency is corrected by CRISPR/CAS9 gene editing system.
Journal
Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis
ISSN: 1473-5733
Titre abrégé: Blood Coagul Fibrinolysis
Pays: England
ID NLM: 9102551
Informations de publication
Date de publication:
01 Apr 2022
01 Apr 2022
Historique:
pubmed:
1
3
2022
medline:
7
6
2022
entrez:
28
2
2022
Statut:
ppublish
Résumé
Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.
Identifiants
pubmed: 35221320
doi: 10.1097/MBC.0000000000001126
pii: 00001721-202204000-00003
doi:
Substances chimiques
Factor XIII
9013-56-3
CRISPR-Associated Protein 9
EC 3.1.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
153-158Informations de copyright
Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.
Références
Dorgalaleh A, Rashidpanah J. Blood coagulation factor XIII and factor XIII deficiency. Blood Rev 2016; 30:461–475.
Kohler H, Ichinose A, Seitz R, Ariens R, Muszbek L XIIIF. Diagnosis and classification of factor XIII deficiencies. J Thromb Haemost 2011; 9:1404–1406.
Dorgalaleh A, Tabibian S, Shams M, Majid G, Naderi M, Casini A, et al., editors. A unique factor XIII mutation in southeastern Iran with an unexpectedly high prevalence: Khash factor XIII. Semin Thromb Hemost 2019: 45:043–049.
Muszbek L, Katona É, editors. Diagnosis and management of congenital and acquired FXIII deficiencies. Seminars in thrombosis and hemostasis. Thieme Medical Publishers; 2016; 42:429–439.
Nakamura T, Morishige S, Ozawa H, Kuboyama K, Yamasaki Y, Oya S, et al. Successful correction of factor V deficiency of patient-derived iPSCs by CRISPR/Cas9-mediated gene editing. Haemophilia 2020; 26:826–833.
Guan Y, Ma Y, Li Q, Sun Z, Ma L, Wu L, et al. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med 2016; 8:477–488.
Huai C, Jia C, Sun R, Xu P, Min T, Wang Q, et al. CRISPR/Cas9- mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice. Hum Genet 2017; 136:875–883.
Zheng Q, Cai X, Tan MH, Schaffert S, Arnold CP, Gong X, et al. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. Biotechniques 2014; 57:115–124.
Flynn R, Grundmann A, Renz P, Hänseler W, James WS, Cowley SA, Moore MD. CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells. Exp Hematol 2015; 43:838–848.e3.
Noroozi-Aghideh A, Kashanikhatib Z, Naderi M, Dorgalaleh A, Azad M, Alizadeh Sh. Shabanf Expression and methylation status of vascular endothelial growth factor and thrombospondin-1 genes in congenital factor XIII-deficient patients with intracranial hemorrhage. Blood Coagul Fibrinolysis 2021; 32:317–322.
Varshney GK, Pei W, LaFave MC, Idol J, Xu L, Gallardo V, et al. High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome Res 2015; 25:1030–1042.
Vidigal JA, Ventura A. Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries. Nat Commun 2015; 6:8083.
Nathwani AC, Davidoff AM, Tuddenham EGD. Gene therapy for hemophilia. Hematol Oncol Clin North Am 2017; 31:853–868.
Nathwani AC, Reiss U, Tuddenham E, Chowdary P, Mclontosh J, Riddel A, et al. Adeno-associated mediated gene transfer for hemophilia B: 8 year follow up and impact of removing ‘empty viral particles’ on safety and efficacy of gene transfer. Blood 2018; 132: (Suppl 1): 491.
Nathwani AC. Gene therapy for hemophilia. Hematology Am Soc Hematol Educ Prog 2019; 6:1–8.
Peyvandi F, Palla R, Menegatti M, Siboni SM, Halimeh S, Faeser B, et al. Coagulation factor activity and clinical bleeding severity in rare bleeding disorders: results from the European Network of Rare Bleeding Disorders. J Thromb Haemost 2012; 10:615–621.
Stephens CJ, Lauron EJ, Kashentseva E, Lu ZH, Yokoyama WM, Curiel DT. Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9. J Control Release 2019; 298:128–141.
Park C-Y, Kim DH, Son JS, Sung JJ, Lee J, Bae S, et al. Functional correction of large factor VIII gene chromosomal inversions in hemophilia A patient-derived iPSCs using CRISPR-Cas9. Cell Stem Cell 2015; 17:213–220.
Dorgalaleh A, Naderi M, Shamsizadeh M. Morbidity and mortality in a large number of Iranian patients with severe congenital factor XIII deficiency. Ann Hematol 2015; 95:451–455.