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Title: Long-term genetic modification of the ocular outflow tract and the retinal pigment epithelium with feline immunodeficiency viral vectors
Author: Loewen, Nils Axel
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2008
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The central goal to engineer feline immunodeficiency viral (FIV) vectors into a more modular platform for long-term ocular gene therapy was accomplished collaboratively by separating envelope, packaging and transfer function, the latter of which I optimized. FIV mediated gene transfer to the aqueous humor outflow tract and to the retina was established and long-term vector function and biocompatibility were confirmed. Transduction of the outflow tract was highly efficient and resulted in genetic modification of nearly the entire trabecular meshwork. For this purpose, I developed methods for administration and sensitive monitoring of FIV vectors in the anterior chamber and a novel subretinal injection technique. Bicistronic FIV vectors generated high levels of two different transgenes eGFP and β-galactosidase, which allowed live in vivo tracking and sensitive yet specific cell labeling in tissue specimen, respectively. An integrase mutant control vector that differed in only one amino acid resulted in no significant transduction while preserving all other biochemical properties. I developed a novel protocol for scaled-up production FIV vectors using cell factories and large-volume concentration that proved useful in the animal studies of this thesis. Especially the results achieved in the larger animal model, the domestic cat, validated the protocol for large-scale production. Transient transfection of 10 times lessDNA into 293T cells within high surface area cell factories and high volume, fixed-angle ultracentrifugation resulted in high titer vectors.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral