Pharmacological analysis of liver-directed AAV-fVIII gene therapy and anti-fVIII inhibitor incidence in a preclinical model of hemophilia A Open Access

Abstract

Hemophilia A is an X-linked, monogenic bleeding disorder caused by mutations in the F8 gene

leading to defective or deficient production of coagulation factor VIII (fVIII). Hemophilia A is

estimated to occur in 1 of 4,000 males globally and approximately 1 in 5,000 males in the United

States. 10 in 100,000 of these males have the severe phenotype of this disease, bringing with it

severe morbidity and mortality risks and major impacts to quality of life. Those patients that

develop anti-fVIII neutralizing antibodies (inhibitors) to their fVIII replacement protein

therapeutic incur similar risks as those of untreated patients along with the increased cost of nonfVIII

alternative therapeutics and immune tolerance induction strategies, which are not always

successful. While the immune response to exogenous fVIII replacement therapy remains poorly

understood, even less clarity exists regarding the immune response to fVIII after adeno-associated

viral (AAV) vector gene therapy delivery encoding functional fVIII (AAV-fVIII) for endogenous

expression by transduced cells. Most preclinical programs do not include formal immunogenicity

testing and no previously untreated patients (PUPs) have received AAV-fVIII.

The goal of the current project is to define the mechanisms and parameters that govern the antifVIII

inhibitor response following AAV-fVIII gene therapy and help guide the development of

safe and effective AAV gene therapies. Animal models of disease remain our premiere resource

for preclinical candidate evaluation prior to clinical testing, and this study utilizes an established

murine model of hemophilia A to evaluate the efficacy and transgene product immunogenicity of

four AAV-fVIII vectors over a range of clinically relevant doses. The results provide a

pharmacokinetic model of fVIII protein product expression kinetics and anti-fVIII inhibitor

outcomes providing reliable predictions of immunogenicity risk and efficacy associated with

vector potency and dose. This study provides a platform for future investigations of mechanisms

of immune tolerance to fVIII following gene therapy, for testing strategies to promote tolerance,

and for further establishment of frameworks that are appropriate for development of genetic

medicines in order to more properly assess their safety and efficacy. Greater understanding of these

areas is critical for the potential of AAV-fVIII gene therapy to reach the entire hemophilia A

patient population and for progress in the field of gene therapy in general.

Table of Contents

Abstract.…………………………………………………….……………………………….........................……4

Acknowledgements ................................................................................................................6

Table of Contents.......……………………….………………………………………....................…………....7

List of Figures and tables.......……………………….…………………………...................………………...9

List of Abbreviations.......……………………….………………………………......................………………12

Chapter 1.......……………………….………………………………………………………..........................…16

1.1 Coagulation Factor VIII (fVIII)......…………………………………………………….......................…17

1.1.1 FVIII: the protein.….………………………………………………………………………...................…17

1.1.2 FVIII in hemostasis and hemophilia A………………..……………………………………..........…….19

1.2 Treating hemophilia A……………………………………….………………………………..............……..23

1.2.1 Advances in fVIII protein replacement therapeutics………………..……………………………......23

1.2.2 Non-fVIII alternatives for hemophilia A treatment…………………..……………………......……..26

1.2.3 Preclinical models for hemophilia A drug development……………..………………………......…..28

1.3 The Immune Response to Exogenous fVIII…………………………….………………………............…30

1.3.1 The anti-fVIII inhibitor response……………………………………….……………............…………..30

1.3.2 The T and B cell response……………………………………………….………………...............……….33

1.3.3 Protein drug factors contributing to the anti-fVIII immune response……..………………….…...38

1.4 Immune Tolerance to fVIII………………………………………………….…………………...............…..40

1.4.1 Antigen-specific immune tolerance……………………………………….……………………..............41

1.4.2 FVIII immune tolerance induction (ITI)…………………………………………………............……...46

1.5 FVIII Gene Therapy…………………………………………………………………………...................…….48

1.5.1 The development of gene therapy for hemophilia A………………………………………..…........…48

1.5.2 AAV-fVIII product design and its effects on efficacy and immunogenicity……………....…..……52

1.5.3 Concerns and challenges facing AAV-fVIII gene therapy…………………………........………..…...60

1.6 Overarching Thesis Hypotheses and Objectives………………………………............…………….……65

Chapter 2 …………………………………………………………………………………............................….….66

2.1 Abstract…………………………………………………………………………………....................…………..67

2.2 Introduction……………………………………………………………………………...................…………...68

2.3 Materials and Methods………………………………………………………………….................……….….71

2.4 Results………………………………………………………………………………………….....................……75

2.4.1 The comparative anti-fVIII IgG response to bioengineered fVIII proteins...............................75

2.4.2 The characterization of four AAV2/8-fVIII vectors................................................................81

2.4.3 A potential conditioning strategy for transplantation of LV-fVIII transduced HSPCs...............90

2.5 Discussion………………………………………………………………………………………..................…...96

2.6 Acknowledgments………………………………………………………………………….…….................….99

2.7 Supplemental Information………………………………………………………………….….................….100

Chapter 3 ………………………………………………………………………………….…................................104

3.1 Abstract……………………………………………………………………………………….....................……105

3.2 Introduction……………………………………………………………………………………....................….105

3.3 Materials and Methods……………………………………………………………………..................……...111

3.4 Results…………………………………………………………………………………….....................………..119

3.5 Discussion………………………………………………………………………………....................…….……162

3.6 Acknowledgments……………………………………………………………………..................…….….…..168

3.7 Supplemental Information…………………………………………………………………..................…….169

Chapter 4 ………………………………………………………………………………...............................….….176

4.1 Discussion of Results…………………………………………………………………….................…...….…177

4.2 Translational and Clinical Impact…………………………………………………………….....................185

4.3 Limitations and Future Directions………………………………………………………….................…...189

4.4 Conclusion References ………………………………………………………………………...................…..191

References ………………………………………………………………………………..............................….….193

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School	Laney Graduate School
Department	Clinical Research
Degree	M.S.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Health Sciences, Pharmacology Health Sciences, Immunology
Keyword	Immune response Gene therapy Therapeutic protein
Committee Chair / Thesis Advisor	Christopher Doering, Emory University
Committee Members	Henry Blumberg, Emory University Renhao Li, Emory University Sean Stowell, Emory University Trent Spencer, Emory University

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