Novel hemophilia B mouse models exhibiting a range of mutations in the Factor IX gene

Abstract

Animal models have been critical to the development of novel therapeutics in hemophilia. A deficiency of current murine models of hemophilia B is that they are all due to gene deletions, a type of mutation that is relatively rare in the human hemophilia population. We generated mice with a range of mutations in the Factor IX (F.IX) gene; these more faithfully reflect the types of mutations that cause disease in the human population. Transgenic mice expressing either wild-type human F.IX (hF.IX), or F.IX variants with premature translation termination codons, or missense mutations, under the control of the murine transthyretin promoter, were generated and crossed with mice carrying a large deletion of the murine F.IX gene. Gene copy number, F.IX transcript levels in the liver, intrahepatocyte protein expression, and circulating levels of F.IX protein in the mice were determined and compared with data generated by transient transfection assays using the same F.IX variants. Mice were injected with a viral vector expressing hF.IX and displayed a range of immune responses to the transgene product, depending on the underlying mutation. These new mouse models faithfully mimic the mutations causing human disease, and will prove useful for testing novel therapies for hemophilia.

Transgene expression levels and kinetics determine risk of humoral immune response modeled in factor IX knockout and missense mutant mice

“…gene therapy of this missense R333Q-hFIX mouse model does not elicit inhibitors across a range of vector doses that stimulate inhibitor development in the FIXKO mouse. Recently, Sabatino et al 38 have reported a transgenic approach to the creation of mice carrying a variety of additional hFIX mutations (between 1 and 68 FIX gene copies/cell, expressed from a non-native promoter). This group also examined responses to lowdose AAV2 i.m.…”

“…Therefore, a CRM+ hemophilia B mouse developed by this group (MS-2 with Anti-factor IX antibodies after AAV1 and AAV2 FIX T-P Zhang et al 15 copies of the missense FIX gene) displayed tolerance of hFIX gene therapy. 38 The study we wished to perform of relative role of transgenic FIX expression kinetics upon inhibitor risk could not be studied in their model, however, because of the interference of background CRM+ protein with circulating transgene measurements.…”

“…40 Although our goal was to create a clinically relevant model of hemophilia B to study hFIX therapies, a mouse expressing wild-type hFIX would provide an interesting control for these experiments. Sabatino et al 38 have reported that a transgenic mouse expressing wild-type hFIX did not make any Ig directed against FIX after AAV2-hFIX i.m. It is not known whether very high expression in the wildtype hFIX-expressing mouse would lead to non-inhibitory IgG anti-hFIX formation, analogous to what we see after our highest dose of AAV1 in the R333Q-hFIX mouse.…”

“…administration of AAV1 serotype expressing FIX does not, per se, trigger inhibitor antibody formation, as has been suggested. 38 Beginning with our highest dose of 1 Â 10 11 vg/animal, we treated separate cohorts of mice with decreasing doses of AAV1 so as to reproduce the low levels of FIX expression achieved by AAV2-hFIX. The development of inhibitor antibody in the CRMÀ mouse was not influenced by factors related directly to the AAV capsid, but was instead determined by the amount, and perhaps the kinetics, of expression of the transgene FIX.…”

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

“…Furthermore, AAV-hF.IX gene transfer to muscle of animals transgenic for hF.IX expressed in the liver (the normal site of F.IX synthesis) or the skin does not break tolerance. 28,29 However, accumulation and binding of muscle-derived F.IX to the extracellular matrix surrounding muscle fibers may contribute to undesired antigen uptake and processing by APCs as described for cell surface-associated ovalbumin, leading to an immune response in animals not tolerant to the F.IX antigen. 14,30 Role of antigen levels in muscle gene transfer While neutralizing antibody formation to hF.IX occurred over a wide range of vector doses in the context of gene transfer to skeletal muscle, this response was partially overcome within 2 months in the high-dose group.…”