Bioengineering Viral Transgenes for the Treatment of the Hemophilias Open Access

Brown, Harrison Carroll (2016)

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Since its inception in the mid-1960s, gene therapy has remained an attractive yet elusive strategy for the treatment of inherited genetic disease. The first successful introduction of therapeutic DNA was into a human was performed in 1990, where a gene encoding adenosine deaminase was introduced in cells from an adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID) patient. Since then, over 2,300 clinical trials have been conducted worldwide, and over $7 billion were invested in commercial gene therapy in 2015 alone. Despite these efforts, only one clinical gene therapy product has been approved in a regulated market to date. Over the last 15 years, there has been substantial scientific and financial investment in producing a clinically viable gene therapy for the hemophilias, A and B. At the time of writing, there are 8 active gene therapy clinical trials for hemophilia, with 4 actively recruiting. These trials propose to use adeno-associated virus to deliver therapeutic transgenes to the liver of patients with hemophilia. However, a second modality of using lentivirus to deliver the transgene to hematopoietic cells is also under pre-clinical investigation. Experience with previous adeno-associated viral trials for hemophilia B have been hampered by low-levels of protein expression at clinically safe viral doses. This problem has been anticipated in ongoing trials, and substantial effort has been made to increase protein expression while maintaining a safe vector dose. The work presented herein furthers these efforts, both by expanding on previously utilized methods of increasing protein production as well as introducing novel strategies tailored toward gene therapy. We hypothesized that there are two primary factors limiting efficient protein production from these gene therapy systems. First, that the oversized nature of the transgene utilized in the context of AAV-mediated gene therapy limits the amount of functional vector DNA that is delivered to a patient's cells, and that this could be overcome by engineering smaller, more efficient regulatory control regions. Second, that the intracellular biosynthesis of the protein itself limits protein production, and that this could be overcome by both engineering the protein itself or by modifying the coding sequence of the vector DNA for optimal expression within the particular cellular milieu in which it is expressed. Through a combination of these techniques, we have developed AAV vectors for both hemophilia A and B that pre-clinical in vivo studies predict are of sufficient potency to delivery curative levels of protein expression at clinical safe viral vector doses, achieving a long-sought milestone in the history of viral gene therapy for the hemophilias.

Table of Contents

Chapter 1 - Introduction. 1

1.1 History of gene therapy. 2

1.2 Types of gene therapy vectors. 4

A. Integrating vectors. 5

B. Non-integrating vectors. 7

1.3 AAV as a gene therapy vector. 9

1.4 History of the Hemophilias. 13

1.5 Coagulation Physiology. 15

1.6 Challenges in Modern Hemophilia Treatment. 17

1.7 Landscape of Clinical AAV for the Hemophilias. 20

1.8 Strategies for Improving AAV Vectors for the Hemophilias. 24

A. Capsid Discover and Engineering. 25

B. Protein Bioengineering. 25

C. Size Reduction. 26

D. Codon Optimization. 27

1.9 Concluding Remarks. 27

1.10 Thesis Hypothesis. 29

Chapter 2 - Bioengineered Coagulation Factor VIII Enables Long-Term Correction of Murine Hemophilia A Following Liver-Directed Adeno-Associate Viral Vector Delivery. 30

2.1 Abstract. 31

2.2 Introduction. 31

2.3 Materials and Methods. 33

2.4 Results. 38

2.5 Discussion. 49

2.6 Acknowledgements. 59

2.7 Supplemental Information. 60

Chapter 3 - Liver-directed bioengineering of AAV-FVIII transgenes. 67

3.1 Abstract. 68

3.2 Introduction. 68

3.3 Materials and Methods. 70

3.4 Results. 73

3.5 Discussion. 89

3.6 Acknowledgements. 94

3.7 Supplemental Information. 95

Chapter 4 - General Discussion. 96

4.1 Collective Results. 97

4.2 Implications of Findings. 99

4.3 Limitations and Future Directions. 101

4.4 Conclusions. 104

References. 106

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