Generation of Mouse Model of Hemophilia A by Introducing Novel Mutations, Using CRISPR/Nickase Gene Targeting System

Document Type : Short Communication

Authors

1 Department of Animal Biotechnology, Institute Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

2 Department of Molecular Medicine, Institute of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

3 Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

Abstract

Developing mouse models of hemophilia A has been shown to facilitate in vivo studies to explore the probable
mechanism(s) underlying the disease and to examine the efficiency of the relevant potential therapeutics. This study
aimed to knockout (KO) the coagulation factor viii (fviii) gene in NMRI mice, using CRISPR/Cas9 (D10A/nickase) system,
to generate a mouse model of hemophilia A. Two single guide RNAs (sgRNAs), designed from two distinct regions on
NMRI mouse FVIII (mFVIII) exon 3, were designed and inserted in the pX335 vector, expressing both sgRNAs and
nickase. The recombinant construct was delivered into mouse zygotes and implanted into the pseudopregnant female
mice’s uterus. Mutant mice were identified by genotyping, genomic sequencing, and mFVIII activity assessment. Two
separate lines of hemophilia A were obtained through interbreeding the offspring of the female mice receiving potential
CRISPR-Cas9-edited zygotes. Genomic DNA analysis revealed disruptions of the mfviii gene reading frame through
a 22-bp deletion and a 23-bp insertion in two separate founder mice. The founder mice showed all the clinical signs
of hemophilia A including; excessive bleeding after injuries, and spontaneous bleeding in joints and other organs.
Coagulation test data showed that mFVIII coagulation activity was significantly diminished in the mFVIII knockout
(FVIIIKO) mice compared to normal mice. The CRISPR/nickase system was successfully applied to generate mouse
lines with the knockout fviii gene. The two novel FVIIIKO mice demonstrated all clinical symptoms of hemophilia A, which
could be successfully inherited. Therefore, both of the developed FVIIIKO mouse lines are eligible for being considered
as proper mouse models of hemophilia A for in vivo therapeutic studies.

Keywords

Main Subjects

References

  1. De Brasi CD, Slavutsky IR, Larripa IB. Genética molecular de la hemofilia A [Molecular genetics of hemophilia A]. Medicina (B Aires). 1996; 56(5 Pt 1): 509-517.
  2. Cafuir LA, Kempton CL. Current and emerging factor VIII replacement products for hemophilia A. Ther Adv Hematol. 2017; 8(10): 303-313.
  3. Kaufman RJ, Dorner AJ, Fass DN. von Willebrand factor elevates plasma factor VIII without induction of factor VIII messenger RNA in the liver. Blood. 1999; 93(1): 193-197.
  4. Batty P, Lillicrap D. Hemophilia gene therapy: approaching the first licensed product. Hemasphere. 2021; 5(3): e540.
  5. Luo L, Zheng Q, Chen Z, Huang M, Fu L, Hu J, et al. Hemophilia a patients with inhibitors: Mechanistic insights and novel therapeutic implications. Front Immunol. 2022; 13: 1019275.
  6. Booth CJ, Brooks MB, Rockwell S, Murphy JW, Rinder HM, Zelterman D, et al. WAG-F8(m1Ycb) rats harboring a factor VIII gene mutation provide a new animal model for hemophilia A. J Thromb Haemost. 2010; 8(11): 2472-2477.
  7. Nielsen LN, Wiinberg B, Häger M, Holmberg HL, Hansen JJ, Roepstorff K, et al. A novel F8 -/- rat as a translational model of human hemophilia A. J Thromb Haemost. 2014; 12(8): 1274-1282.
  8. Kashiwakura Y, Mimuro J, Onishi A, Iwamoto M, Madoiwa S, Fuchimoto D, et al. Porcine model of hemophilia A. PLoS One. 2012; 7(11): e49450.
  9. Notley C, Killoran A, Cameron C, Wynd K, Hough C, Lillicrap D. The canine factor VIII 3’-untranslated region and a concatemeric hepatocyte nuclear factor 1 regulatory element enhance factor VIII transgene expression in vivo. Hum Gene Ther. 2002; 13(13): 1583- 1593.
  10. Porada CD, Sanada C, Long CR, Wood JA, Desai J, Frederick N, et al. Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A. J Thromb Haemost. 2010; 8(2): 276-285.
  11. Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian HH Jr. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet. 1995; 10(1): 119-121.
  12. Healy PJ, Sewell CA, Exner T, Morton AG, Adams BS. Haemophilia in hereford cattle: factor VIII deficiency. Aust Vet J. 1984; 61(4): 132-133.
  13. Turecek PL, Gritsch H, Richter G, Auer W, Pichler L, Schwarz HP. Assessment of bleeding for the evaluation of therapeutic preparations in small animal models of antibody-induced hemophilia and von Willebrand disease. Thromb Haemost. 1997; 77(3): 591-599.
  14. Øvlisen K, Kristensen AT, Tranholm M. In vivo models of haemophilia- status on current knowledge of clinical phenotypes and therapeutic interventions. Haemophilia. 2008; 14(2): 248-259.
  15. Bi L, Sarkar R, Naas T, Lawler AM, Pain J, Shumaker SL, et al. Further characterization of factor VIII-deficient mice created by gene targeting: RNA and protein studies. Blood. 1996; 88(9): 3446-3450.
  16. Sarkar R, Gao GP, Chirmule N, Tazelaar J, Kazazian HH Jr. Partial correction of murine hemophilia A with neo-antigenic murine factor VIII. Hum Gene Ther. 2000; 11(6): 881-894.
  17. Sabatino DE, Nichols TC, Merricks E, Bellinger DA, Herzog RW, Monahan PE. Animal models of hemophilia. Prog Mol Biol Transl Sci. 2012; 105: 151-209.
  18. Austin CP, Battey JF, Bradley A, Bucan M, Capecchi M, Collins FS, et al. The knockout mouse project. Nat Genet. 2004; 36(9): 921-924.
  19. Gama Sosa MA, De Gasperi R, Elder GA. Animal transgenesis: an overview. Brain Struct Funct. 2010; 214(2-3): 91-109.
  20. Dow LE. Modeling disease in vivo with CRISPR/Cas9. Trends Mol Med. 2015; 21(10): 609-621.
  21. Ge XA, Hunter CP. Efficient homologous recombination in mice using long single stranded DNA and CRISPR Cas9 nickase. G3 (Bethesda). 2019; 9(1): 281-286.
  22. Chao BN, Baldwin WH, Healey JF, Parker ET, Shafer-Weaver K, Cox C, et al. Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene. J Thromb Haemost. 2016; 14(2): 346-355.
  23. Yen CT, Fan MN, Yang YL, Chou SC, Yu IS, Lin SW. Current animal models of hemophilia: the state of the art. Thromb J. 2016; 14 Suppl 1: 22.
  24. Chao BN, Baldwin WH, Healey JF, Parker ET, Shafer-Weaver K, Cox C, et al. Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene. J Thromb Haemost. 2016; 14(2): 346-355.
  25. Hall B, Limaye A, Kulkarni AB. Overview: generation of gene knockout mice. Curr Protoc Cell Biol. 2009; Chapter 19: Unit 19.12 19.12.1-17.
  26. Santacroce R, Acquila M, Belvini D, Castaldo G, Garagiola I, Giacomelli SH, et al. Identification of 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A. J Hum Genet. 2008; 53(3): 275-284.
  27. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013; 154(6): 1380- 1389.
Cell Journal (Yakhteh)
Volume 25, Issue 9 - Serial Number 116
September 2023
Pages 655-659

Files

Share

How to cite

Statistics