Pharmacologic evaluation of fVIII expression in retroviral mediated gene therapy for hemophilia A Open Access
Dooriss, Kerry Titus (2010)
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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