Optimization of a Lentiviral Vector for use in an HSC Gene Therapy Protocol for Hemophilia A Open Access

Johnston, Jennifer Marie (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/g732d948n?locale=en


Gene therapy for hemophilia A has been hindered by limited expression of factor VIII (fVIII). In order to overcome the expression barrier, several high-expression fVIII transgene sequences were compared and identified enhanced expression of a B domain deleted porcine fVIII sequence. As a result, a high-expression fVIII transgene was engineered and used to genetically modify HSCs and effectively treat hemophilia A mice. Prior to clinical application, the components of the self-inactivating lentiviral vector system needed to be optimized in regards to viral production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: 1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), 2) HIV versus SIV viral vector systems, and 3) various internal promoters. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone devoid of a WPRE. In addition, two obstacles faced by the field of gene therapy were confronted. Firstly, lentiviral vectors containing fVIII transgenes consistently have lower titers compared to similar vectors containing other transgenes. An alternative approach to lentiviral production was attempted in BHK-M cells. However, the transfection efficiency was too low to produce substantial amounts of virus. The second obstacle addressed is that of the inefficiency of lentiviral vectors to transduce HSCs. Limited transduction efficiency has been observed clinically with the use of lentiviral vectors and will need to be enhanced in scenarios in which a growth advantage is not expected as is the case for hemophilia A. Therefore, a high-throughput screen was performed to identify compounds that could enhance the transduction of HSCs by lentiviral vectors. A library of 1280 pharmacologically active compounds was screened using K562 cells in the presence of a SIN lentiviral vector encoding a GFP transgene. Among the positive hits were known enhancers of transduction, as well as phorbol 12-myristate 13-acetate (PMA). Transduction was enhanced in K562, EU1 and most notably CD34+ cells following treatment with PMA prior to transduction. The results from this report were used to help develop a lentiviral vector that is currently being considered by the FDA for a clinical trial to treat hemophilia A.

Table of Contents


Page #

Chapter 1: Introduction to Gene Therapeutic Approaches for Hemophilia A

1.1-- Hemostasis and Blood Coagulation 2

1.2-- Hemophilia A: A Deficiency of Factor VIII 13

1.3-- Pharmacological Principles of Gene Therapy 21

1.4-- Historical Perspective of Gene Therapy 31

1.5-- Preclinical Gene Therapeutic Applications to cure hemophilia A 34

1.6-- Limitations of Gene Therapy 51

Chapter 2: Pharmacological Evaluation of Lentiviral Vector Optimized for fVIII Expression

2.1-- Introduction 59

2.2-- Materials and Methods 62

2.3-- Results 71

2.31-- Assessment of the WPRE sequence 71

2.32-- Comparison of three lentiviral vectors 80

2.33-- Analysis of three internal promoters 90

2.34-- Hematopoietic stem cell gene therapy for hemophilia A 92

utilizing the optimized lentiviral vector

2.4-- Discussion 96

Chapter 3: Assessment of Alternative Approaches to Lentiviral Production and Transduction

3.1-- Introduction 102

3.2-- Materials and Methods 106

3.3-- Results 108

3.31-- Toxicity of VSVG in BHK-M cells 108

3.32-- Effect of alternatively pseudotyping an HIV-based lentiviral 115


3.33-- Knocking down p21 118

3.34-- Utilizing spinoculation as a means for enhancing sca-1+ 122


3.4-- Discussion 133

Chapter 4: Pharmacologic Enhancement of Lentiviral Transduction

4.1-- Introduction 138

4.2-- Materials and Methods 140

4.3-- Results 146

4.31-- High-throughput screen identifies lead compounds 146

4.32-- High-throughput screen identifies false positives 151

4.33-- High-throughput screen identifies previously confirmed 158


4.34-- Enhancement of the Positive Hit, PMA is cell dependent 162

4.35-- Mechanism for PMA effect on K562 cells 179

4.4-- Discussion

Chapter 5: Conclusions and Future Perspectives

5.1-- Conclusions 192

5.2-- Future Perspectives 204

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