In vitro and In vivo Model Systems for Hemophilia A Gene Therapy

Abstract

Hemophilia A is a hereditary disorder caused by various mutations in factor VIII gene resulting in either a severe deficit or total lack of the corresponding activity. Recent success in gene therapy of a related disease, hemophilia B, gives new hope that similar success can be achieved for hemophilia A as well. To develop a gene therapy strategy for the latter, a variety of model systems are needed to evaluate molecular engineering of the factor VIII gene, vector delivery efficacy and safety-related issues. Typically, a tissue culture cell line is the most convenient way to get a preliminary glimpse of the potential of a vector delivery strategy. It is then followed by extensive testing in hemophilia A mouse and dog models. Newly developed hemophilia A sheep may provide yet another tool for evaluation of factor VIII gene delivery vectors. Hemophilia models based on other species may also be developed since hemophiliac animals have been identified or generated in rat, pig, cattle and horse. Although a genetic nonhuman primate hemophilia A model has yet to be developed, the non-genetic hemophilia A model can also be used for special purposes when specific questions need to be addressed that cannot not be answered in other model systems. Hemophilia A is caused by a functional deficiency in the factor VIII gene. This X-linked, recessive bleeding disorder affects approximately 1 in 5000 males [1–3]. Clinically, it is characterized by frequent and spontaneous joint hemorrhages, easy bruising and prolonged bleeding time. The coagulation activity of FVIII dictates severity of the clinical symptoms. Approximately 50% of all cases are classified as severe with less than 1% of normal levels of factor VIII detected [4]. This deficiency may lead to spontaneous joint hemorrhages or life-threatening bleeding. In contrast, patients with 5–30% of normal factor VIII activity exhibit mild clinical manifestations.

Successful Phenotype Improvement following Gene Therapy for Severe Hemophilia A in Privately Owned Dogs

“…Hemophilia is a relatively common disease among privately owned dogs, with an estimated 60 new cases diagnosed each year in the US through the Comparative Coagulation Laboratory at Cornell University (personal communication, Marjory Brooks, 2015). Although the prevalence of HA in dogs in unknown, most of these cases are HA, as in humans, suggesting that an endogenous mechanism of mutation in the F8 gene is relatively common among mammals, as naturally occurring HA has also been reported in rats [ 25 ], sheep [ 26 ], cats, and horses (review in [ 27 ]).…”

Coagulant activity of recombinant human factor VII produced by lentiviral human F7 gene transfer in immortalized hepatocyte-like cell line

“…Human mesenchymal stem cells (hMSCs) have been recently established as a new tool for delivering secreted proteins because they are easy to isolate, expand, and manipulate with genetic modification [10]. Several studies showed that hMSCs derived from adipose tissue, bone marrow or umbilical cord blood are capable of differentiating into hepatocyte-like cells in specialized in vitro culture conditions [11–13].…”