Evolutionary approaches to coagulation factor VIII biopharmaceutical engineering Public
Zakas, Philip Michael (2016)
Published
Abstract
Deficiencies of coagulation factor VIII (FVIII) result in the bleeding disorder hemophilia A. Current treatments are limited to protein replacement through intravenous infusions of recombinant or plasma-derived FVIII. Despite adequate management of the disease in several countries, FVIII replacement therapy remains unavailable to 75% of the global population. Gene therapy through adeno-associated or lentiviral vector delivery offers the potential for a long-term treatment or cure; however, in vivo biosynthesis of FVIII has not achieved therapeutic levels at clinically tolerable viral doses. This biosynthetic limitation is the largest obstacle in the development of improved protein therapeutics and the establishment of gene therapy protocols. Characterization of orthologous FVIII molecules from mammalian species has led to translational discoveries regarding FVIII biosynthesis and biochemistry. Mouse FVIII demonstrates a six-fold increase in stability following thrombin activation. Canine FVIII demonstrates a two-fold increase in coagulant activity per molecule. Porcine FVIII demonstrates 10-100 fold enhanced biosynthesis compared to human FVIII in heterologous expression systems. Incorporation of porcine domains into human FVIII resulted in a hybrid molecule that retains high biosynthesis, demonstrating the ability to bioengineer a FVIII molecule with enhanced therapeutic properties. To expand this ortholog-based bioengineering approach, we characterize a novel FVIII ortholog derived from sheep for unique biochemical characteristics. Traditional bioengineering efforts for FVIII through rational design or directed-evolution are not feasible. Structural data regarding FVIII is limited. Directed-evolution approaches require large quantities of recombinant protein and are likely to result in an inactive molecule; 1437 unique missense mutations within the 2332 FVIII residues have been documented in hemophilia A patients. A novel approach to bioengineering is critical for the development of improved FVIII therapies. In pursuit of this, we investigate the molecular evolution of extant and predicted FVIII sequences through ancestral sequence reconstruction and establish this methodology as a platform for bioengineering. We constructed and characterized predicted ancestral sequences to the most studied extant FVIII molecules and found incremental changes in amino acid sequence that result in significant changes in biochemical properties. Using this platform, we engineered novel FVIII molecules with enhanced biochemical properties through minimal amino acid substitutions.
Table of Contents
Introduction. 1
Chapter I Engineered Hematopoietic Stem Cells as Therapeutics for Hemophilia A (Published). 29
Chapter II: Development and Characterization of Recombinant Ovine
Coagulation Factor VIII (Published)
Abstract. 53
Introduction. 55
Materials and Methods. 57
Results. 65
Discussion. 78
Chapter III: Expanding the Ortholog Approach for Hemophilia
Treatment Complicated by Factor VIII Inhibitors (Published)
Summary. 84
Introduction. 86
Materials and Methods. 89
Results. 93
Discussion. 110
Chapter IV: Bioengineering Coagulation Factor VIII through Ancestral
Sequence Reconstruction
Materials and Methods. 114
Abstract/ Introduction/ Results. 120
Discussion. 145
References. 155
About this Dissertation
| School | |
|---|---|
| Department | |
| Subfield / Discipline | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Mot-clé | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Evolutionary approaches to coagulation factor VIII biopharmaceutical engineering () | 2018-08-28 15:14:07 -0400 |
|
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
Dissertation special pages.docx () | 2018-08-28 15:18:01 -0400 |
|