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Date of Award

1995

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

Professor Frank L. Graham

Abstract

In recent years adenovirus (Ad) vectors have been used for the expression of foreign genes in mammalian cells, have been studied as vaccine vectors and more recently as gene transfer vectors for gene therapy. A number of properties make the Ad system a good candidate for each of these applications not the least of which is the extensive understanding of their structure and biology that has been gained through their use as a model system for studying all aspects of gene expression and DNA replication. The construction of recombinant Ad vectors involves the insertion of foreign DNA sequences into the Ad genome usually in place of compensating deletions made in early region 1 (E1) or E3. Deletions of up to 2.9 kb have been made in E1, which is not required for viral replication in complementing 293 cells, allowing the construction of conditional helper independent vectors with a capacity of 4.7-4.9 kb. A variety of deletions have been made in E3, a region which is nonessential for viral replication in any normally permissive cells. The most commonly used deletion of 1.9 kb allows the construction of nonconditional helper independent vectors with a capacity of 3.7-3.9 kb.

Even with our extensive knowledge of Ads a more detailed understanding of vectorology is required before recombinants can be confidently used in humans for vaccine and gene therapy purposes. For example it was generally assumed that adenovirus vectors are relatively stable and can package DNA up to 105% of the wt genome length. To more thoroughly address this issue the genetic stability of Ad5 vectors with E3 substitutions representing net insertions of up to 8.3% of the AdS genome was analyzed after serial passages in 293 cells. This investigation revealed that vector stability correlated with net genome size and vectors with the largest genomes rearrange extremely rapidly. This observation prompted the development of a new vector system (the BHG system) with larger deletions in E1 (3.2 kb) and E3 (2.7 or 3.2 kb) that can accommodate larger inserts. This system is the most versatile yet developed and allows the insertion of genes into E1 or E3 or both and mutations or deletions can be readily introduced elsewhere in the viral genome. The system provides a capacity of 8.3 kb.

Using the information gained in vector construction, and the new vectors systems that were developed, vectors were constructed that expressed various simian immunodeficiency virus (SIV) genes. There should be little doubt that the development of a safe, effective vaccine for human immunodeficiency virus (HIV) will provide the most secure and cost effective means of controlling HIV and AIDS. The infection of macaque monkeys with SIV provides one of the most relevant animal models for studying AIDS and for developing vaccines (Desrosiers and Ringler, 1989; Fultz, 1993). Information gained through the development and characterization of Ad/SIV vectors will have important implications in terms of developing a potential Ad/HIV vaccine.

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