In contrast to conventional medications, therapeutic viral vectors are given as a single dose and their levels cannot be adjusted to meet the therapeutic needs of an individual patient. Thus, one cannot overestimate the importance of animal model-based preclinical studies as a means to characterize the efficacy and safety of gene therapy regimens. Most preclinical trials are based on a rodent model comprising a large number of animals of the same strain, and a large animal model involving a smaller number of animals. Importantly, the effects of the genetic background of mouse strains employed in preclinical studies on the safety and efficacy of viral vectors has not been characterized. Similarly the effects of the patient's geneti background on the outcome of gene therapy regimens cannot be evaluated. Consequently, several gene therapy clinical trials have resulted in major adverse effects, which could not be observed in relevant earlier preclinical studies. The overall goal of the proposed studies is to characterize the effects of the host genetic variation on the efficacy and safety of lentiviral vector-based gene replacement therapy. Our experimental approach is premised on the Collaborative Cross (CC) mouse strains, which were derived by a funnel breeding of 8 genetically diverse, inbred founder strains. The genomes of these mouse strains are well defined, and will be utilized to study the overall effects of the host genetic background on the overall efficacy and safety of lentiviral vectors, as well as to identify putative genetic loci affecting these processes, test these variants' effects on the safety and efficiency of transducing human cells, and to evaluate the approach of employing an in vitro strain (potentially patient)-specific mouse embryo fibroblast (MEF's)-based system as a means to predict the safety and efficacy of hepatic gene delivery.
In Aim 1, we will focus on characterizing the effects of the hos genetic background on the ability of lentiviral vectors to deliver and maintain long-term hepatic transgene expression, as well as lentiviral vector pattern of integration. F2 intercrosses of CC strains with contrasting phenotypes will be employed to identify genetic loci and the putative candidate genes involved in these processes. The optimal mouse strains for lentiviral vector-based hepatic gene delivery (demonstrating highest long-term transgene expression) will be identified.
In Aim 2, we will employ MEF's from the above CC strains to study the effects of the host genetic background on the early steps of, as well as overall lentiviral vector transduction. Gene loci involved in these processes will be identified and their effects on safety and efficacy of vector transduction of human cells will be determined. The ability of the MEF-based model to predict in vivo transduction efficiency will be determined, as will this prediction's efficacy acros genetically diverse strains.
In Aim 3, we will characterize the effects of the host genetic background on: the ability of lentiviral vectors to maintain therapeutic levels of factor IX (FIX) n vivo, the immune responses to FIX and vector- transduced hepatocytes, and on the development of thrombotic pathologies in the presence of high levels FIX.
Viral vector-based gene replacement therapy has proven efficacious in human clinical trials. It is expected that in the near future, viral vectors will bethe therapy of choice for an increasing number of human genetic and acquired diseases. Currently animal model-based preclinical studies are the sole means to characterize the efficacy and safety of gene therapy regimens. However, the effects of the genetic backgrounds of mouse strains employed in these studies on the safety and efficacy of viral vectors has not been characterized. Similarly the effects of the patient's genetic background on the outcome of gene therapy regimens cannot be evaluated. Indeed, several gene therapy clinical trials have resulted in major adverse effects (including death), which could not be anticipated by the findings obtained in relevant preclinical studies. The overall goal of the proposed studies is to characterize the effects of the host genetic background on the efficacy and safety of lentiviral vector-based gene replacement therapy. Our experimental approach is premised on 40 Collaborative Cross (CC) mouse strains, which were derived by multiple crossing of 12 known founder strains. The genetic backgrounds of these mouse strains are well defined and will be utilized to identify putative genetic loci affecting overall lentiviral vector efficacy and safety n vivo. In addition, we will employ cell lines derived from the aforementioned CC mouse strains to identify putative gene loci involved in the early steps of lentiviral vector transduction, and we wll evaluate the ability of in vitro cell line-based studies to predict the outcome of lentiviral vecto-based gene delivery in vivo. Finally, we will use the CC mouse strains to determine the effects of the host genetic background on lentiviral vector-based gene replacement therapy. Specifically, we will test the ability of lentiviral vectors to deliver and maintain therapeutic leels of factor IX in the above mouse strains, and will characterize the emergence of immune responses to vector transduced hepatocytes and the development of thrombotic adverse effects following expression of vector delivered factor IX.
