Studies of the adenovirus 5 L1 gene aimed at developing L1 gene deficiencies for use in gene therapy vectors
Gene therapy is a novel approach to the treatment of human disease that is in its very early\ud stages of development. Its purpose is to add to and/or alter the pattern of gene expression in\ud cells so as to achieve a therapeutic benefit and is being developed for application to such\ud diverse conditions as simple inherited diseases, cancer, AIDS, and cardiovascular disease.\ud Poor uptake of DNA by cells in vivo is a significant technical barrier for gene therapy.\ud Viruses have evolved to carry their nucleic acid contents into cells very efficiently and are\ud thus considered as potential vectors for gene therapy, provided their pathogenicity and other\ud adverse features can be overcome.\ud \ud Adenovirus is a virus type, which is a prime candidate for development as a vector for\ud efficient transfer of genes into human tissues. In recent years, there have been many studies\ud in order to develop replication deficient adenoviral vectors as gene therapy vectors. Current\ud disadvantages of these vectors include their limited capacity to accommodate exogenous\ud DNA and elucidation of a host immune response against these vectors. Currently they have\ud a capacity for about 9 kilobasepairs of DNA, which is not sufficient for some therapeutic\ud purposes.\ud \ud The work described in this thesis aimed to provide a way to produce highly deleted\ud adenoviral vectors containing deletions in their L1 52/55-kD, L1 52/55-kD and IVa2, or L1\ud 52/55-kD and IX coding regions. These genes encode proteins, which contribute to virus\ud particles, and their assembly and some of which have been reported to increase expression\ud of the viral late (structural protein) genes. Together with the deletions available in currently\ud used vectors, these new deletions would create vectors with an increased capacity to\ud accommodate exogenous DNA. Furthermore, these deletions were expected to make the\ud vectors more replication-deficient and less immunogenic by decreasing the expression of\ud residual viral genes.\ud \ud Initially, cell lines expressing adenovirus L1 52/55-kD, L1 52/55-kD and IVa2, or L1 52/55-kD\ud and IX proteins were constructed for use as complementing cell lines. These were\ud designed to supply in trans the relevant proteins that would be missing during attempts to\ud construct recombinant viruses with deletion mutations in these gene(s). 293-L1 cells were\ud proven in their ability to complement missing L1 52/55-kD protein function using existing\ud adenovirus L1 52/55-kD point mutants ts369 and H5pmSOO1. However, attempts to isolate\ud an L1 52/55-kD coding region deleted recombinant adenovirus using this cell line were\ud unsuccessful, possibly due to the nature of sequences missing in the deleted L1 52/55-kD\ud coding region which might have currently undefined cis acting regulatory functions.\ud \ud L1 52/55-kD protein is known to form a complex with IVa2 and the latter protein has been\ud reported to activate the major late promoter. The effects of L1 52/55-kD protein on the\ud adenovirus major late promoter transactivation were therefore investigated by transient\ud expression experiments carried out by transfection of COS cells with a major late promoter-dependant reporter gene (CAT) and expression vectors for L1 52/55-kD and/or IVa2. These\ud experiments did not reveal any role for the L1 52/55-kD protein in the activation of\ud adenovirus major late promoter.
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