Non-genotoxic anti-CD117 conditioning for hematopoietic stem cell transplantation and gene therapy applications Open Access

Russell, Athena (Summer 2021)

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Nearly 1.5 million hematopoietic stem cell transplants have been performed world-wide since this life-changing and lifesaving procedure was first performed nearly 65 years ago. This powerful therapy can be used in the treatment or cure of a broad range of malignant and non-malignant hematologic diseases. However, despite its robust therapeutic potential, hematopoietic stem cell transplantation is underutilized in many non-malignant disease settings for which it can be curative. A critical component of the procedure is the pre-transplantation conditioning or preparative regimen. The conditioning regimen serves multiple functions: (a) ablation of endogenous bone marrow resident hematopoietic stem cells to make space for transplanted cells to engraft and restore hematopoiesis; (b) immune suppression, in the setting of allogeneic transplantation, to prevent graft rejection; (c) elimination of disease in the case of hematologic malignancy. Agents that are conventionally used for hematopoietic stem cell transplantation conditioning are DNA-damaging, genotoxic agents such as ionizing radiation or chemotherapeutic alkylating drugs. Though effective, these agents cause cytotoxicity not specific to the target cells, simultaneously inducing damage to non-target healthy cells and tissues. As a result, genotoxic conditioning agents are associated with many short- and long-term adverse effects and significant regimen-related toxicity and treatment-related mortality. These risks are more acceptable in the setting of active malignancy, however for patients with non-malignant disease for which alternative therapies exist, the risk-benefit ratio of enduring genotoxic conditioning is often deemed too great. Therefore, improving the safety profile of the hematopoietic stem cell transplantation conditioning regimen is a major unmet need in the field. Developing safer and less toxic conditioning agents would not only expand the patient population in whom traditional hematopoietic stem cell transplantation can be safely performed, but also broaden its applicability to providing safe, curative ex vivo lentiviral modified autologous hematopoietic stem cell gene therapy options for monogenic diseases.

The work presented herein represents a preliminary step towards realizing this objective. We evaluated the use of a non-genotoxic immunotoxin targeting the stem cell factor receptor CD117 and its utility in enabling pre-transplant conditioning in murine models of ex vivo lentiviral hematopoietic stem cell gene therapy for hemophilia A and allogeneic hematopoietic stem cell transplantation for ataxia telangiectasia. We hypothesized that a non-genotoxic immunotherapy could replace genotoxic radiation and chemotherapy in both allogeneic and autologous gene therapy hematopoietic stem cell transplantation settings. CD117 immunotoxin selectively depletes hematopoietic stem cells in bone marrow and allows donor cell engraftment and multilineage chimerism following transplantation, correcting or ameliorating disease phenotypes while sparing healthy tissues and minimizing adverse effects. This dissertation presents proof-of-concept data in support of efforts towards clinical translation of similar non-genotoxic antibody-based regimens for human application.

Table of Contents

Chapter 1 1

1.1 Hematopoietic Stem Cell Transplantation 2

1.1.1 History of Bone Marrow Transplantation 2

1.1.2 Current Platforms of Hematopoietic Stem Cell Transplantation 3

1.1.3 Conditioning for Hematopoietic Stem Cell Transplantation 13

1.1.4 Next generation Non-genotoxic Antibody- and Immunotoxin-based Conditioning Agents 16

1.1.5 Hematopoietic Stem Cell Gene Therapy 22

1.2 Hemophilia A 29

1.2.1 Hemophilia A Background 29

1.2.2 Factor VIII and Its Role in Blood Coagulation 30

1.2.3 Current and Developing Therapies for Hemophilia A 32

1.2.4 Gene Therapy for Hemophilia A 36

1.3 Ataxia Telangiectasia 40

1.3.1 Ataxia Telangiectasia Background 40

1.3.2 ATM Kinase: Central Role in DNA Double-strand Break Repair and Other Diverse Functions 42

1.3.3 Current and Developing Therapies for Ataxia Telangiectasia 47

1.3.4 Allogeneic Hematopoietic Stem Cell Transplantation for Ataxia Telangiectasia 51

1.4 Hypothesis 54

Chapter 2 56

2.1 Abstract 57

2.2 Introduction 57

2.3 Materials and Methods 60

2.4 Results 66

2.5 Discussion 88

2.6 Acknowledgements 96

2.7 Supplemental Information 98

Chapter 3 112

3.1 Abstract 113

3.2 Introduction 114

3.3 Materials and Methods 117

3.4 Results 121

3.5 Discussion 136

3.6 Acknowledgements 139

3.7 Supplemental Information 141

Chapter 4 146

4.1 Summary of Results 147

4.2 Implications of Findings 150

4.3 Limitations and Future Directions 152

4.4 Conclusions 157

References 158

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