The 1950’s and 1960’s gave rise to the era of recombinant DNA and the notion that “good DNA” could be transferred into individuals with heritable diseases as a curative therapy. Decades were spent developing methods to produce high titer virus and modifying recombinant vectors to improve infectivity. Finally, in 1990, the Food and Drug Administration approved their first clinical gene therapy trial for the treatment of adenosine deaminase severe combined immunodeficiency (ADA-SCID). Gene therapy was on the rise and the entire country had high hopes that this kind of therapy would revolutionize modern medicine and provide a cure for over 1500 diseases recently shown to be genetically determined. Unfortunately, several clinical trials resulted in severe and in some cases fatal adverse events, sending the field back into the laboratory to focus on improving the efficacy and safety of viral mediated gene transfer.
Since these events, the field of gene therapy has made significant progress improving the efficacy and safety of both ex vivo and in vivo gene transfer methods. Clinical trials have been initiated covering over 8 different indications with at least 100 trials beginning in 2016 alone. While a significant amount of effort has been dedicated to vector development and safety, very few preclinical studies have examined the immunological implications of gene transfer. Hemophilia A (HA), an X-linked heritable bleeding disorder caused by a deficiency in coagulation factor VIII (FVIII), offers a unique opportunity to compare the immunomodulatory capabilities of both ex vivo and in vivo methods of gene transfer in the context of the same disease.
Both methods of gene transfer have been described to facilitate a tolerogenic immune state in which transgene specific immunoregulatory cells are generated to provide surveillance and prevent immune responses. However, neither of these mechanisms have been studied in the context of FVIII gene transfer, a coagulation factor known to be immunogenic and cause serious complications in 20-30% of patients with severe HA. Current clinical gene therapy trials only include patients that have previously received protein replacement therapy and are considered tolerized to recombinant FVIII protein. However, we must consider what the immunological implications of gene therapy would be in previously untreated patients, or patients with suboptimal gene therapy outcomes that would require some level of exogenous FVIII infusions. This dissertation will focus on the immunological aspects of FVIII gene transfer for two different gene therapy paradigms demonstrated to be curative in preclinical models of HA. We hypothesize that in vivo gene transfer methods targeting hematopoietic stem cells result in transplantation tolerance of FVIII gene-modified cells, and is a more durable mechanism of tolerance induction compared to in vivo methods targeting hepatocytes. Furthermore, this dissertation will discuss the development of novel pharmacological agents used to improve the safety of hematopoietic stem cell transplantation gene therapy.
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About this Dissertation
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