Examination of the Immunological Aspects of Gene Therapy for the Treatment of Hemophilia A Open Access

Lytle, Allison (Fall 2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/6m311p28w?locale=en
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Abstract

 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.   

Table of Contents

Abstract

Acknowledgements

Table of Contents

List of Figures and Tables

List of Abbreviations

Chapter 1 – Introduction to Gene Therapy.................................................................... 1

1.1 History of Clinical Gene Therapy...................................................................... 2

A.  Developing Ex Vivo Gene Transfer Methods............................................... 4

B.   Current Integrating Vectors.......................................................................... 7

C.   Developing In Vivo Gene Transfer Methods.............................................. 12

D.  Current Non-Integrating Vectors............................................................... 14

1.2 Hemophilia A................................................................................................... 18

A.  Physiology of Coagulation......................................................................... 20

B.   Coagulation Factor VIII............................................................................. 25

1.3 Clinical Gene Therapy for Hemophilia A......................................................... 27

1.4 Immunogenicity of FVIII................................................................................. 29

1.5 Hypothesis....................................................................................................... 30

Chapter 2 – Bioengineered Coagulation Factor VIII Enables Long-Term Correction of   Murine Hemophilia A Following Liver-Directed Adeno-Associate Viral Vector Delivery......................................................................... 32

2.1 Abstract............................................................................................................ 33

2.2 Introduction...................................................................................................... 33

2.3 Materials and Methods..................................................................................... 35

2.4 Results............................................................................................................. 39

2.5 Discussion....................................................................................................... 52

2.6 Acknowledgements.......................................................................................... 61

2.7 Supplemental Information................................................................................ 62

Chapter 3 – Immunogenicity is a Barrier to Liver-Directed AAV Gene Therapy of Hemophilia A   69

3.1 Abstract............................................................................................................ 70

3.2 Introduction...................................................................................................... 70

3.3 Materials and Methods..................................................................................... 72

3.4 Results............................................................................................................. 75

3.5 Discussion....................................................................................................... 91

3.6 Acknowledgements.......................................................................................... 98

3.7 Supplemental Information.............................................................................. 100

Chapter 4 – Developing an Immunotoxin Based Preparative Regimen for HSCT Gene Therapy for the Treatment of Hemophilia A     106

4.1 Abstract.......................................................................................................... 107

4.2 Introduction.................................................................................................... 107

4.3 Materials and Methods................................................................................... 112

4.4 Results........................................................................................................... 115

4.5 Discussion..................................................................................................... 130

4.6 Acknowledgements........................................................................................ 133

Chapter 5 – Developing Alternative Conditioning Regimens Using Lamprey-Antibody Based Immunotoxins      134

5.1 Abstract.......................................................................................................... 135

5.2 Introduction.................................................................................................... 135

5.3 Material an Methods...................................................................................... 136

5.4 Results........................................................................................................... 139

5.5 Discussion..................................................................................................... 145

5.6 Acknowledgements........................................................................................ 146

Chapter 6 – General Discussion................................................................................... 147

6.1 Summary of Results....................................................................................... 148

6.2 Implications of Findings................................................................................ 150

6.3 Limitations and Future Directions.................................................................. 153

6.4 Conclusions................................................................................................... 157

References....................................................................................................................... 160

List of Figures and Tables

Figure 1.1 Schematic of gamma-retroviral vectors using either an LTR or an internal promoter/self-inactivating design to drive transgene expression........................................................................................................................... 9

Figure 1.2 Different serotypes demonstrate variable tissue-tropism in mice............. 16

Figure 1.3 The extrinsic and intrinsic pathways converge into the common pathway of the coagulation cascade    23

Figure 1.4 Model of loss of vector potency with increasing transgene length............ 28

Figure 2.1: Viral Vector Design...................................................................................... 41

Figure 2.2: Molecular assembly of rAAV-HCR-ET3 vector particles........................ 44

Figure 2.3: Dose finding of rAAV-HCR-ET3 in a murine model of hemophilia A... 46

Figure 2.4: Formation of FVIII inhibitors following rAAV-HCR-ET3 administration           49

Figure 2.5: In vivo viral genome copy number.............................................................. 51

Figure 2.6: Phenotypic correction of the bleeding diathesis......................................... 53

Supplemental Table 2.1: Biotinylated probes used for detection of AAV-HCR-ET3 viral genomes  62

Supplemental Table 2.2: Primers used for regional transgene analysis..................... 63

Supplemental Table 2.3: Hydrodynamic injection of FVIII encoding AAV expression plasmids      64

Supplemental Figure 2.1: Standard curves for quantitative PCR analysis............... 65

Supplemental Figure 2.2: Sequence alignment of porcine-substituted domains of ET3 and HSQ     66

Supplemental Figure 2.3: ET3 C2 domain sequence RNA is present in liver of treated mice 68

Figure 3.1 Immune tolerance to FVIII through LV HSCT gene therapy.................. 77

Figure 3.2: Dose dependent tolerance achieved through AAV-FVIII........................ 80

Figure 3.3: Efficacy of AAV ET3 in the context of pre-existing immunity to hFVIII 83

Figure 3.4: Efficacy of AAV-ET3 in the context of pre-existing immunity to ET3... 85

Figure 3.5: Rescue HSCT of AAV-ET3 treated mice with anti-ET3 inhibitory antibodies     88

Figure 3.6: Quantification of transgene and mRNA copies per cell in the livers of AAV treated mice           90

Figure 3.7: Efficacy ofAAV-FVIII in the setting of low-titer anti-FVIII immunity. 92

Supplemental Figure 3.1: AAV-HLP-ET3 expression cassette sequence................. 100

Supplemental Figure 3.2: Mean percent of initial titers in mice with AAV-ET3 pre-existing immunity to hFVIII  104

Supplemental Figure 3.3 Mean percent of initial titers in mice with AAV-ET3 with pre-existing immunity to ET3 105

Figure 4.1 Hematopoiesis of stem and progenitor cells.............................................. 109

Figure 4.2: Conjugation of Sap-255 with CD45 antibody......................................... 117

Figure 4.3: CD45-Sap255 mediated depletion of stem and progenitor cells in hemophilia A mice     119

Figure 4.4: CD45-Sap255 mediated depletion of bone marrow mononuclear cells. 122

Figure 4.5: HSCT gene therapy for hemophilia A using a CD45-Sap255 regimen. 123

Figure 4.6: CD45-Sap255 conditioned mice maintain CD48- depletion 16 weeks post injection         126

Figure 4.7: Engraftment of experimental cohort after 2nd transplantation of FVIII gene-modified cells       127

Figure 4.8: Clearance of anti-CD45 clone 104 in naïve hemophilia A...................... 129

Figure 5.1: Flow cytometric analysis of yeast surface display................................... 141

Figure 5.2: Transient transfection of VLR sequences in HEK 293 cells................... 142

Figure 5.3: SDS-Page analysis of elution fractions from the purification of VLR-fusion proteins     143

Figure 5.4: Flow cytometric analysis of VLR-fusion protein FITC conjugates to Lin- Sca+ cKit+ stem cells within the bone marrow compartment ... 144

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