Introduction to Adenovirus Technology
The year 1953 saw the isolation of the Adenovirus which was soon recognized as an invaluable tool for investigating mammalian molecular biology. Several of the distinguished features of Adenovirus have made it the preferred vehicle for gene transfer and transgene expression in mammalian cells. The following presents a small overview of the biology of Adenovirus.
Adenoviruses are associated with a number of disorders (eg. common cold), most of which are mild. The pathology is primarily from inflammation and loss of infected epithelial cells. Viruses of subgroup C (serotype 2, 5) cause various respiration infections in confined groups (elderly, military recruits and children).
Adenovirus is a non-enveloped 80-110 nm diameter virus presenting an icosahedral symmetry. Human Adenoviruses contain a linear, double stranded DNA genome, with a terminal protein (TP) attached covalently to the 5' termini. The DNA, which has a length of approximately 36,000 bp, is wrapped in a histone-like protein and has inverted terminal repeats (ITRs) of 50-200 bp, which act as origins of replication.
The hexon, penton base, and knobbed fiber are the most important capsid proteins with regards to gene delivery. Hexon is the major protein forming the 20 triangular faces of the viral capsid. The 240 hexon capsomers in the capsid are trimers, each interacting with six other trimers. The 12 vertices are formed by the penton capsomere, a complex of five copies of the penton base, and three copies of fiber. Each penton capsomere interacts with five hexon capsomeres, one from each of the five faces that converge at the vertex. The knobbed fiber protrudes from the fiber base.
Adsorption and entry into the cell
The adsorption of the virus to target cell receptors involves high-affinity binding via the knob portion of the fibre. The prime receptor for human Adenovirus serotype 5 is identical to that for coxsackie B virus and has been named the Coxsackie/Adenovirus receptor (CAR). After the attachment step, interaction between the penton base and av integrins on the cell surface leads to internalisation of the virus through endocytosis. Once inside the cell, the virus escapes the endosome with help of the penton base, and translocates to the nuclear pore complex, where the viral DNA is released into the nucleus and transcription begins. Transcription, replication and viral packaging take place in the nucleus of the infected cell.
Binding and internalization of Adenovirus
A complex series of splicing accompanies transcription, and genes are transcribed from both strands. Adenovirus transcription is a two-phase event, early and late, occurring before and after viral DNA replication, respectively. The early transcribed regions are E1, E2, E3 and E4. The E1 gene products can be further subdivided into E1A and E1B. E1 gene products are involved in the replication of the virus. The E2 region is subdivided into E2A and E2B. These proteins provide the machinery for viral DNA replication and the ensuing transcription of late genes. Most of the E3 proteins are involved in modulating the immune response of infected cells, a function not essential for viral growth in vitro. The gene products encoded by the E4 region (called ORFs 1-6/7) are involved in the metabolism of virus messenger RNA and provide functions that promote virus DNA replication and shut-off of host protein synthesis. Furthermore, E4 products prevent viral DNA concatenation.
Adenovirus has been adapted so that it can be used as both gene delivery and gene therapy tools. Recombinant Adenovirus (rAd) generated with the technologies offered by Qbiogene have their E1 and E3 regions deleted. The E1 deletion prevents the recombinant Adenovirus from replicating and therefore no cell lysis occurs. Once packaged into a complementing cell line, i.e. a cell line that provides the E1 products in trans (e.g. QBI-HEK 293A cells), viral replication is made possible. The E3 region, not essential for viral growth, is also deleted. These two deletions allow the introduction of the transgene of interest into the virus. In addition to these two deletions, Qbiogene offers custom services with protease (PS) deleted recombinant Adenovirus. The main components of the PS deleted system are also available under a license agreement. For more info on PS deleted Adenovirus, please refer to the MERLIN® Custom Services section of this catalog.
Gene delivery, gene therapy and protein over-expression with recombinant Adenovirus
Gene delivery consists in introducing DNA and RNA into cells, tissues, or organisms, in order to study regulation and function of genes and proteins. The biggest hurdle that gene delivery technologies have to overcome is the cell membrane, which is impermeable to negatively charged macromolecules such as DNA and RNA. Numerous different gene delivery methods using either chemical, physical or biological pathways have been used in recent years and are constantly being improved upon. The advantage of recombinant Adenovirus lies in its potential to bind and efficiently enter mammalian cells through its naturally occurring receptor (CAR) (see figure 2).
Recombinant Adenovirus is largely used in gene therapy which aims at treating both genetic (e.g. cancer, haemophilia) and infectious diseases (e.g. AIDS) by introducing new genetic material into selected cells.
Recombinant Adenovirus can also be used in vaccination by expressing a gene product that triggers an immune response.
Recombinant Adenovirus technology is also used to overexpress proteins of interest and to subsequently study their functions. In contrast to prokaryotic or insect-based systems, the use of human cells permits the complex post-translational protein modifications required to ensure the proper folding and post-translational modifications of the protein. In addition, the CMV5 promoter, being substantially stronger than the standard CMV promoter, allows high expression of the protein of interest.
Advantages of using Recombinant Adenovirus
There are many advantages in using an Adenovirus to introduce genetic material into host cells.
- Represents a homologous system for human genes: adenoviral vectors use a human virus as vector and human cells as host. Therefore, human proteins have identical post-translational modifications as native proteins
- Has the ability to infect most mammalian cell types (both replicative and non-replicative)
- Allows propagation in suspension thus permitting production of large volumes
- May be grown at high titer (1010 VP/mL, which can be concentrated up to 1013 VP/mL)
- Accommodates reasonably large transgenes (up to 7.5 kb)
- Supports simultaneous expression of multiple genes
- Allows a high expression of the recombinant protein (up to 35% of total cellular protein)
- Is well tolerated, with post-infection viability of the host cells being 100%
- Remains epichromosomal, i.e. does not integrate into the host chromosome, inactivating genes or activating oncogenes
- Can be generated without any special equipment
- Can be used in combination with an inducible expression system (Q-mate™) to express potentially toxic proteins
Coronal Plane Section of Rat Striatums Injected with Ad5.CMV-GFP. Courtesy of Dr. Michael Y. Chen, NIH
Rat Spinal Cord Injected with Ad5.CMV-LacZ. Courtesy of Dr. Michael J. Iadarola, NIH
All these advantages, and the extensive knowledge of viral genetics, have made recombinant Adenovirus the vector of choice for functional genomics research, protein-over-expression, pre-clinical studies and clinical trials.
See also our new Adenovirus website: www.adenovirus.com
Qbiogene Adenoviral Vector Systems
Four different Adenoviral expression systems are offered for the production of recombinant adenovirus. Two of these systems are based on homologous recombination in E. coli, one uses homologous recombination in human QBI-HEK 293A cells, and our most recent system takes advantage of site-specific transposition in E.coli.
The major differences between the four systems lie in the mode of recombination and the transfer vectors. With each of the kits, several transfer vectors are available. Some transfer vectors come with a promoter and a poly A site, others benefit from a user-supplied expression cassette. Several of our transfer vectors are available with an MCS (multiple cloning site), whereas others have a single cloning site. In addition, the strength of the promoter, and therefore the level of expression of the protein, is an important consideration for the researcher in the choice of the kit to use.
The plasmids, that carry part of the Ad5 genome and are used in the recombination step, are all of the first generation, i.e. ΔE1/E3. The gene of interest, or the expression cassette, is inserted into the deleted E1 region.
In addition, a new second generation Adenovirus deleted for the essential protease (PS) gene has been developed by Qbiogene. In the absence of E1, the PS deleted recombinant Adenovirus is completely replication deficient, whereas in the presence of E1, a single round of replication occurs, allowing a greatly enhanced transgene expression without viral shedding which is of particular interest in vaccination, in situ therapy for tumors, protein production, and Adeno-Associated Virus (AAV) production. Furthermore, the ectopic expression of the PS gene permits the positive selection of recombinant Adenovirus (rAD) with 100% efficiency allowing the construction of Ad-based libraries (AdLibTM) having numerous applications in functional genomics studies. The generation of a rAd using the PS deleted viral backbone, as well as the generation of Ad-based libraries, are available as Custom Adenovirus Service.
Each kit is available with the principal components and controls for the generation of 5 recombinant viruses and includes a comprehensive applications manual.