In Vitro Studies

The methods outlined above have been used to make a large number of first-generation E1", E3~ adenoviral vectors. Among the most widely used are those that express readily monitored reporter genes such as p-galactosidase, luciferase, chloramphenicol acetyl transferase, and green fluorescent protein (34,35). As a control, viruses with the same promoter-driving expression of no transgene (AdNull) are used.

Using the reporter gene Ad vectors, there are many studies examining the ease of gene transfer to different primary cells and cell lines. Some primary epithelial cells are easily infected by wild-type adenovirus type 5 and, as expected, are easily transfected by adenoviral vectors. By contrast, macrophages (36) and lymphocytes (37,38) are more difficult to infect and only very high multiplicities of infection in concentrated cell suspensions are effective. The discovery that CAR is the ade-noviral receptor partially accounts for the relative ease of infection. There are several studies in which the overexpression of CAR was shown to be sufficient to make an otherwise refractory cell line susceptible to gene transfer by Ad (36,39). But integrins and postinternalization factors must also affect the efficiency of gene transfer.

A large number of cancer cell lines have been shown to be susceptible to gene transfer, including cells derived from hepatoma (40,41), glioblastoma (42), myeloma (43,44), melanoma (45), prostate cancer (46), and ovarian cancer (47,48). In contrast, lymphoma cell lines are resistant to infection (44). Studies in which cells are infected in vitro are instructive in indicating what cell types and therefore diseases might be candidates for adenoviral gene therapy. It is difficult to evaluate if studies with reporter genes show that therapeutic levels of proteins are achievable in any cell type due to the use of Ad vectors with different promoters, reporter genes, multiplicities of infection, and times of exposure.

In cells infected in vitro with E1 deleted, replication-deficient adenovirus vectors, a low level of transcription of early and late genes (49,50), and a small amount of DNA synthesis (51) can be detected. The reason for this is not entirely understood but is hypothesized to result from E1-like activities in the target cell, which support expression of Ad genes. In dividing cells there would also be a high level of E2F, which would support adenoviral transcription. However, measurements of viral load in cultures suggest that this does not translate into the production of infectious viral particles in the absence of contaminating wild-type adenovirus. Although cells may continue to divide after infection, the absolute level of vector does not increase or decrease even if the amount of vector per cell decreases to the point when a small minority of cells are infected. Based on this evidence, the Ad genome is likely to remain episomal and is not integrated into the cellular genome,

Figure 4 Production of the genome of a replication-deficient adenovirus vector by homologous recombination in E. coli. (A) A new cDNA is cloned into the kanamycin-resistant plasmid pShuttleCMV between the CMV promoter and the polyadenylation (poly A) site of SV40. Analogous plasmids are available that allow other promoters to be used (31). The expression cassette lies between 2 portions of the Ad genome, nt 1-480 and 3542-5780. (B) Homologous recombination between Pmel-linearized pShuttleCMV containing the gene of interest and supercoiled pEasy1 results in a kanamycin-resistant plasmid with a full-length adenovirus genome bordered by Pacl sites. Plasmid pEasy-1 is used to make E1-E3-E4+ vectors or pEasy-2 to make E1-E3-E4-vectors. The recombinant prepared in large amounts for transfection into E1 complementing cell lines, where it will give rise to the desired vector.

Figure 4 Production of the genome of a replication-deficient adenovirus vector by homologous recombination in E. coli. (A) A new cDNA is cloned into the kanamycin-resistant plasmid pShuttleCMV between the CMV promoter and the polyadenylation (poly A) site of SV40. Analogous plasmids are available that allow other promoters to be used (31). The expression cassette lies between 2 portions of the Ad genome, nt 1-480 and 3542-5780. (B) Homologous recombination between Pmel-linearized pShuttleCMV containing the gene of interest and supercoiled pEasy1 results in a kanamycin-resistant plasmid with a full-length adenovirus genome bordered by Pacl sites. Plasmid pEasy-1 is used to make E1-E3-E4+ vectors or pEasy-2 to make E1-E3-E4-vectors. The recombinant prepared in large amounts for transfection into E1 complementing cell lines, where it will give rise to the desired vector.

although this is difficult to prove, as it is hard to detect integrated DNA at a very low frequency.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment