Pharmacologic evaluation of fVIII expression in retroviral mediated gene therapy for hemophilia A Público

Dooriss, Kerry Titus (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/2801pg42m?locale=es
Published

Abstract

Hemophilia A is an excellent candidate for gene therapy because it can be cured by the transfer of a single gene into any cell with access to the bloodstream. Unfortunately, the success of gene therapy has been hampered by low and transient transgene expression, the risk of insertional mutagenesis, and the frequent induction of an immune response. We hypothesized that the development of a safer viral vector incorporating a high-expressing fVIII transgene for the transduction and transplantation of hematopoietic stem cells can overcome the barriers of low and transient expression with a decreased risk of insertional mutagenesis. With respect to the development of a high-expressing transgene, a number of human, porcine, and human/porcine chimeric transgenes were developed for the purpose of determining which transgene exhibits the highest fVIII expression following lentiviral-mediated hematopoietic stem cell transduction and transplantation in a mouse model of hemophilia A. We
discovered that porcine fVIII and one chimeric fVIII transgene express at significantly higher levels than any of the human constructs. We also
demonstrated sustained fVIII expression in hemophilic mice following lentiviral-mediated transduction and transplantation of gene-modified hematopoietic stem cells. To evaluate the ability of a lentivirus to mediate lineage-specific fVIII expression in gene-modified bone marrow cells, self-inactivating lentiviral vectors were developed to express porcine fVIII under the control of the β-globin promoter and locus control region. We demonstrated high-level fVIII expression following lentiviral mediated gene-transfer into a myelogenous leukemic cell line, as well as from gene-modified hematopoietic cells in transplanted mice. Although we confirmed this lentiviral-mediated gene transfer platform is capable of GFP and fVIII expression in mice, hematopoietic stem cell transduction efficiency was limiting. Transduction efficiency was lower with the porcine fVIII-containing lentivirus compared to the GFP-containing lentivirus. Together, these studies demonstrate that a porcine or human/porcine chimeric transgene is able to mediate high level fVIII expression, that erythroid-specific fVIII expression is possible, and that hematopoietic stem cell transduction is the limiting factor in the future success of this erythroid-specific lentiviral-mediated gene transfer system.

Table of Contents

TABLE OF CONTENTS

Chapter 1: General Introduction

1.1 Hemostasis and the blood coagulation cascade (2)
1.2 Genetic and clinical implications of hemophilia A (4)
1.3 Gene therapy as a cure for hemophilia A (6)
1.4 Successes and setbacks in gene therapy (9)
1.5 Improving the safety of gene therapy 2(2)
1.6 Hematopoietic stem cell-mediated gene therapy (24)

Chapter 2: Comparison of factor VIII transgenes bioengineered for
improved expression in gene therapy of hemophilia A

2.1 Abstract (29)
2.2 Introduction (30)
2.3 Materials and Methods (34)
2.4 Results (49)
-Expression of bioengineered fVIII constructs in heterologous mammalian cells
-Comparison of fVIII expression using targeted single transgene integration
-High level fVIII expression from a human/porcine chimeric fVIII transgene
-Characterization of fVIII production rates
-High-level expression of pfVIII using lentiviral gene transfer
-In vivo comparison of lentiviral-mediated hfVIII and pfVIII expression
2.5 Discussion (84)


Chapter 3: Erythroid-restricted lentiviral gene therapy of hemophilia A
incorporating high expression porcine factor VIII

3.1 Abstract (95)
3.2 Introduction (96)
3.3 Materials and Methods (100)
3.4 Results (108)
-Comparison of strength and specificity of two erythroid promoters
-High level pfVIII expression in hemophilia A mice controlled by the

beta-globin promoter
-Optimization of HSC purification and transplant from murine bone marrow
-Gene-modification of HSCs using Sca-1+/CD150+ bone marrow cells
-Induction of immune tolerance by transient erythroid fVIII expression
3.5 Discussion (142)

Chapter 4: Characterization of lentiviral transduction efficiencies

4.1 Abstract (152)
4.2 Introduction (153)
4.3 Materials and Methods (156)
4.4 Results (160)
-5-FU pretreatment for HSC enrichment
-Comparison of SIV and HIV-based viruses for erythroid promoter-
driven pfVIII expression
-Comparison of lentiviral production protocols
-Characterization of HIV-GFP and HIV-pfVIII viral titers
-Determination of transgene-specific or cell-specific differences in lentiviral transduction
4.5 Discussion (186)

Chapter 5: Conclusions and future directions

5.1 Conclusions (192)
-Development of recombinant fVIII transgenes
-Gene transfer platforms
-Gene therapy transgene promoters
-Hematopoietic stem cell transduction
-Lentiviral quantification
-Variable viral transduction efficiencies

5.2 Future directions (205)

References (218)

Copyright authorizations (238)

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