Adenovirus

Recombinant, replication deficient adenoviral vectors (AdV) represents one of the most sophisticated expression systems available today. This exciting technology is made widely accessible thanks to the detailed protocols and comprehensive components provided with the Adeno-Quest™ and AdEasy™ kits for recombinant Ad construction.

• Adeno-Quest™ system, based on recombinant AdV production in 293 cells.


• AdEasy™ system, based on recombination in bacteria.

Advantages of using a recombinant adenovirus

1. Broad host range and low pathogenicity in humans.

AdV systems have been used extensively to express human as well as non-human proteins. Adenoviruses can infect a broad range of mammalian cells and therefore permit the expression of recombinant proteins in most mammalian cell lines and tissues.

Adenoviruses can infect virtually all cell types with the exception of some lymphoid cells, which are more resistant to AdV infection. AdV is the best system to study gene expression in primary non-replicative cells. This allows for a direct comparison of results obtained with transformed cell lines and primary cells. On the other hand, retroviruses can only infect replicative cells. Transfection of purified DNA necessitates the maintenance of cells in culture and cannot be done in non-replicative cells.

3. Replicates efficiently to high titers.

AdV system allows production of 10¹⁰ to 10¹¹ VP/mL which can be concentrated up to 10¹³ VP/mL. This feature makes it the best-suited vector system for gene therapy applications.

To provide additional cloning space, the E1 and E3 early regions of AdV have been deleted. Additionally, AdV can normally encapsidate a viral DNA molecule slightly bigger than the normal DNA (105%). These combined features allow for the insertion of an expression cassette containing a gene or multiple genes of up to 7.5kb into one recombinant AdV.

This is the first expression system that can be easily designed to express multiple genes in the same cell line or tissue. The simplest way to proceed is to insert two genes in a double expression cassette of the Ad transfer vectors. Alternatively, using different recombinant viruses each expressing a different protein, a co-infection of the desired cell lines can be performed. Determining the MOI ratio of the different recombinant viruses will provide the proper relative co-expression of the recombinant proteins.

6. No insertional mutagenesis; remains epichromosomal.

Retroviruses integrate randomly into the host chromosome and can inactivate genes or activate oncogenes. AdV remains epichromosomal in all known cells except eggs and therefore does not interfere with other host genes. The integration of only one copy of virus in zona-free eggs is a better system to produce transgenic animals with specific characteristics.

293 cells can be adapted to grow in suspension. This adaptation permits a production scale-up. There are numerous examples of 1 to 20 liter bioreactor batches of recombinant proteins expressed in suspension cultures of 293 cells.

The AdV vector system uses a human virus as a vector and human cells as a host. It therefore provides the ideal environment for proper folding and exact post-translational modifications of human proteins. Most human proteins are expressed at high levels and are fully functional.

Potential Applications for Recombinant Adenovirus

Adenoviral vectors (AdV) have been successfully used to express a wide variety of viral and cellular genes in mammalian cells (1,2,3). Proteins produced with the adenovirus expression system may be used for biochemical characterization (4,5), the development of commercial applications, including production of subunit vaccine (6), and the development of new diagnostic kits. Adenoviral vectors (AdV) can also be used to transfect cells for protein expression and in vivo characterization studies as well as gene therapy related experiments.

Transfection

The isolation and characterization of a specific gene is frequently followed by studies of its function, localization, transport and/or molecular interaction in mammalian cells. The need for transfection of recombinant DNA molecules into mammalian cells has precipitated the development of many different transfection methods, including calcium phosphate co-precipitation, electroporation, cationic lipid based transfection and most recently the gene gun. Even though these methods are effective for transfecting many common cell lines, many cell types cannot be routinely or efficiently transfected by any of these methods. Their limitations are even greater when it comes to transfection of primary cell cultures, which are, in most cases, extremely difficult to transfect by current methods. This constitutes a great disadvantage, since in many cases primary cultures offer major advantages over immortalized cell lines for biochemical and immunochemical analysis of specific cell functions.

The AdV has great potential for use as a routine transfection method, since Ad efficiently infects many different cell lines. The virus enters the cell but does not replicate because the essential E1 early genes are absent. This abortive infection may be seen as a transfection system for introducing a functional gene into cells.

The advantages of using Ad to introduce genes into cells include:

1. It is quick, simple, and does not require any special equipment.



2. Contrary to many other methods, it is well tolerated, in fact post-infection viability is almost 100%. Therefore, Ad has the ability to transfer genetic material into cells with negligible apparent toxic effects (7).



3. Most cell types can be infected.



4. Gene expression can be analyzed as early as a few hours post infection.



5. More than one protein can be expressed simultaneously.

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Overexpression in Human Cells

For historical, practical as well as economical considerations, Eschericihia coli expression systems have been the first choice for the expression of heterologous proteins. Despite the versatility and efficacy of its expression vectors, there are several limitations to the E. coli system for the expression of different categories of heterologous proteins. This is especially relevant for those proteins that undergo complex post-translational modifications as observed with many viral and mammalian proteins. These limitations include inappropriate or lack of post-translational modifications, incorrect folding, proteolytic degradation, inefficient secretion, and amino acid misincorporation (8). Because of these limitations, efforts have been directed towards the development of more sophisticated expression systems including other prokaryotes, lower eukaryotes such as yeast, and higher eukaryotes such as mammalian and insect cells. Although it is clear that there is no one universally accepted expression system recombinant adenovirus offers several advantages:

1. Expression in human cells gives human proteins with identical post-translational modifications to native proteins (Figure 1).



2. A series of adenoviral transfer vectors allow for the production of high levels of heterologous proteins (Figure 2).



3. The recombinant protein represents the most abundant polypeptide (20-30%) of total cellular protein.



4. The expression system can be scaled up with 293 suspension cultures of several liters yielding up to 90 mg/L under optimal conditions (9).

Gene Therapy

Gene therapy can be defined as the transfer of genetic material into the cells of an individual resulting in a therapeutic benefit to this individual. Instead of altering the disease phenotype through the use of agents which interact with the gene products (proteins) or which are themselves gene products, gene therapy allows the modification of specific genes. This results in the permanent treatment of the disease, hopefully with few or no side effects in the process. The scientific discoveries which led to the demonstration that viruses are able to transfer genetic material to infected cells is the basic principle behind gene therapy.(10,11) Targeted tissues for gene therapy include, among others, the lungs (cystic fibrosis, emphysema), the muscles (muscular dystrophy), the liver (Wilson's disease), the kidneys (nephrotic syndrome), the spleen (Gaucher's disease) and the nervous system (Parkinson, Alzheimer, etc.). The initial goal of gene therapy was the treatment of genetic disorders, but many forms of cancer and other chronic and infectious diseases such as AIDS are also targeted for treatment by gene therapy. Among the viral vectors, AdV is one of the most promising candidates as a gene delivery vehicle for human gene therapy.