The Generation and Characterisation of Novel Mouse Models of Haemophilia A

Abstract

Haemophilia A (HA) is a hereditary, X-linked recessive disease caused by mutations in the F8 gene, with the absence or deficiency in the coagulation factor, Factor VIII (FVIII), resulting in a bleeding diathesis. The phenotype of HA varies extensively among HA patients with respect to bleeding frequency, anti-FVIII inhibitor response and haemophilic arthropathy development. The underlying cause of this patient heterogeneity remains elusive. Thus, F8-/- animal models are required.

In this thesis, I generated a new C57BL/6J strain mouse model of HA, F8em1Pfal (F8em1), targeting exon 1 of the mouse F8 gene using CRISPR/Cas9 technology. F8em1 mice were compared to C57BL/6J-F8tm1Kaz (F8tm1) which has a mutation in F8 exon 16 for validation and phenotypic comparison. Both male hemi- and female homozygous C57BL/6J-F8em1 had a prolonged aPTT and undetectable FVIII:C activity relative to WT mice and comparable to F8tm1 mice. In addition, C57BL/6J-F8em1 mice showed a prolonged bleeding time in a tail snip assay when compared to WT C57BL/6J mice which was identical to the F8tm1 model. Moreover, severe overt bleeding was identified in male hemizygous C57BL/6J-F8em1 mice but not in female F8em1 homozygotes. In comparison, frequency and severity of bleeds in male F8tm1 hemizygotes was significantly lower. Histological examination of C57BL/6J-F8em1 hind legs with overt bleeds identified pathology indicative of haemophilic arthropathy.

C57BL/6J-F8em1 mice were backcrossed onto three different inbred mouse strain backgrounds- A/J, BALB/c and DBA/1J -for >10 generations. When backcrossed to three other inbred mouse strains, all F8em1 hemizygous males experienced prolonged time to clot formation in an aPTT assay and undetectable chromogenic FVIII:C activity relative to their respective WT counterparts. Bleeding time post tail snip and incidence of overt bleeding episodes considerably differed among the F8em1 males on different inbred strains.

Finally, when anti-FVIII IgG response was assessed; male hemizygous F8em1 mice had a significantly higher total anti-FVIII IgG titre relative to naïve controls as well as male WT, F8tm1 hemizygotes and female F8em1 homozygotes following FVIII immunisation. Taken together, this study has generated four novel mouse models to experimentally explore the underlying mechanisms of the heterogenous disease phenotype seen in patients with HA.