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The Adenovirus System – Introduction

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Video Summary

Viral vectors are tools commonly used to express exogenous genetic materials in vitro or in vivo. The virulence aspect of the recombinant viral vectors is usually altered so that they are safe to use by molecular biologists. Among them, the most commonly used viral vectors to date are retrovirus, lentivirus, adenovirus and adeno-associated virus (AAV). This section will focus on the recombinant adenovirus expression system.



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Adenovirus Structure and Biology

Adenovirus (Ad) was first isolated from adenoid tissue in 1953 and to date at least 57 different human serotypes have been identified. The most commonly studied adenovirus serotypes are Type 2 and 5, both of which are the basis of the recombinant expression system in research today. Adenovirus is a non-enveloped virus with a linear, double-stranded DNA genome of 30-40 kb. Its nucleocapsid consists of 252 proteins in the form of 3 major types: fiber, penton and hexon based proteins (Figure 1).

Adenovirus Structure


Figure 1 – Adenovirus Structure

Hexon based proteins are the structural basis of Adenovirus and compose most of the viral capsid. Both the fiber and penton based proteins are for receptor binding and internalization of the adenovirus into host cells (1). Almost all serotypes adenoviruses use the coxsakie- and adenovirus- receptor (CAR) as the primary receptor on the host cell. After initial attachment via CAR, the Arg-Gly-Asp -motif in the penton based protein binds to cell surface integrin receptors such as avb3 and avb5 which act as a secondary or internalization receptor. This step induces the virus uptake by the host cell via endocytosis. Once inside the host cell, the capsid of virus is dismantled and the core protein-coated viral genome is transported into the nucleus. It is inside the nucleus where the viral genes are expressed via the host cell’s replication machineries (Figure 2). Viral replication and assembly occurs in the nucleus of infected cells and mature viruses leave the host cell via disintegration. In contrast to other viruses, adenovirus DNA is not integrated into the host genome and remains in an episomal state (2). Episomes are non-integrated extrachromosomal closed circular DNA molecule that may be replicated in the nucleus (3).

Adenovirus Transduction


Figure 2 – Transduction of Adenovirus

The first gene to be expressed once the adenovirus enters a host cell is the immediate early E1A gene. Proteins encoded by E1A gene are essential for adenovirus replication and their presence also trigger the expression of the delayed early genes in E1B, E2, E3 and E4 transcription units. The expression of adenoviral E1B, E2, E3 and E4 genes will alter a group of cellular genes in the host cell to facilitate adenovirus replication (4). The organization of adenovirus genome is shown in (Figure 3A). ITR is the abbreviation for Inverted Terminal Repeats sequence at each end of the genome which contains all the cis-acting elements necessary for replication and packaging. Understanding the Adenovirus replication cycle has enabled researchers to manipulate the vector to develop replication-defective (RD Ad Vector) versions for use in research.

Schematic of the genome of adenovirus type 5 (Ad5) and Ad5-based vetors


Figure 3 – Schematic of the genome of adenovirus type 5 (Ad5) and Ad5-based vetors

Our recombinant adenovirus vectors are replication-incompetent (-E1/-E3) human Adenovirus Type 5 (Ad5).
Variations of Modern Day Recombinant Adenoviral Vectors

Replication-defective adenovirus vectors (RD Ad vector)

Recombinant Ad expression vectors exploit the high nuclear transfer efficiency and the low pathogenicity of the virus to deliver genes to the host cell. Most adenovirus vectors used in research are derived from Ad serotype 5 (Ad5). To date, human adenovirus vectors are involved in more than 400 gene therapy trials. Most of them are intended for cancer, while some are for vaccines in which the vector expresses a foreign antigenic protein or for gene therapy in which the vector expresses a functional protein to correct a genetic defect (1). One common theme between the adenovirus vectors in clinical trials is that they are all modified viruses, (i.e. first- and second-generation, or helper-dependent), that differ from the wild-type replication competent Ad5.

First- and second-generation of recombinant adenovirus vectors (Figure 3B)

Usually a recombinant virus vector can be produced by inserting or substituting gene of interest into three regions of the viral genome: a region in E1, E3 and a short region between E4 and end of the genome.

For the first generation vectors, E1 gene region is replaced by the transgene or therapeutic gene to be delivered to the target cell with a high activity promoter such as cytomegalovirus immediate early (CMV) promoter which drives expression of transgenes (1). Additional deletion in E3 gene is also feasible which make the total transgene capacity of these vectors up to 8kb. Because E1 gene is essential for replication, lack of E1 in the transgene expression makes the recombinant virus incapable of reproduction under normal conditions. This strategy provides a safer alternative to using the wild-type Ad5 in clinical settings. In order to propagate the recombinant virus that lacks E1 transgene expression, a complementing producer cell line such as HEK293 or PER.C6 is required. Both cell lines stably express the viral E1A and E1B proteins and thereby provide the essential E1 proteins in trans for adenovirus replication.

Despite of the E1 and E3 deletion, evidences from animal experiments and human clinical studies demonstrated that viral genes are still expressed at low levels in cells transduced with first-generation vectors which can cause direct toxicity and immune response in vivo. Moreover, these immune responses also result in the clearance of vector-transduced cells by T cell response and consequently, these vectors only allow short term expressions of the transgenes. Thus, the second-generation vectors were constructed featured by additional deletions of E2 and/or E4 function which have improved transgene persistence and decreased inflammatory response in some studies. These E4- and E2-deleted vectors must be grown on cell lines that complement the E1, E4, and E2 deletions. However, these vectors still exhibit leaky expression of viral genes which did not thoroughly solve the immunogenicity problems. The advantage though, is that the second generation recombinant vectors can accommodate bigger transgene sizes.

The construction of the vector genome usually involves the direct cloning recombination or transposition techniques in E. coli cells. The vector genome is then processed (i.e restriction digest) and released from the plasmid or cosmid backbone, and transfected into the complimenting producer cell lines for virus packaging (5). Alternatively, Cre recombinase/loxP site-specific recombination system can also be used for efficient production of recombinant adenovirus vectors (6).

Helper-dependent adenovirus vectors (HD Ad vector) (Figure 3C)

Helper-dependent adenovirus vectors, or ‘gutless’ adenovirus vectors are deleted of all viral coding genes but retained the cis-acting sequences such as ITRs as well as the packaging sequence that is required for the genome to replicate and be packaged. Up to 36kb of non-viral DNA can be integrated into helper-dependent vector so that larger cDNAs, longer tissue-specific or regulatable promoters, several expression cassettes or even small genomic loci can be transferred into the host cells. Deletion of the whole viral gene from these vectors greatly reduces their toxicity and immunogenicity in vivo and enables the long-term transgene expression. In one study, the α1-antitrypsin gene transferred to liver cells has been expressed for more than 1 year in mice (7).

The production of helper-dependent adenovirus vectors is usually based on recombinase-mediated excision of the packaging signal in the helper virus, which is flanked by loxP recognition sites (8). The recombinase expression is harbored by the producer cell line. During the production stage, the E1- and recombinase- expressing producer cells are transfected with recombinant HD-Ad vector DNA carrying the transgene and co-infected with the helper virus. Inside the producing cells, the packaging signal in the helper-virus is excised by recombinase, making it unable to be packaged into viral particles, but provide regulatory and structural proteins required for replication and packaging of the HD-Ad vector DNA. While helper-dependent advenovirus vectors can accommodate greater transgene sizes, the downside is that an extra step is required for viral purification. Cesium chloride (CsCl) gradient centrifugation is often required to remove the transgene adenoviral particles from the remaining contaminating helper-virus particles with un-excised packaging signals (Figure 4) (1).

Production of helper-dependent adenovirus vectors (HD-Ad vectors)


Figure 4 – Production of helper-dependent adenovirus vectors (HD-Ad vectors)

Replication-competent (RC) adenovirus vectors (RC Ad vector)

While most replication-incompetent adenovirus vectors are used in research, it is not uncommon to have replication-competent adenovirus for clinical trials. In fact, conditionally replication-competent adenoviruses (no transgene) are often exploited for their cytotoxicity and are used exclusively on targeting tumor cells (9, 10). RC-Ad vectors can lyse the cancer cells as the end results of their life cycle, with their progeny virus particles infecting and destroying neighboring tumor cells. Cancer cells are more permissive to adenovirus replication than quiescent or non-cancerous cells, majorly because the gene expression pattern of cancer cells greatly facilitates adenovirus replication (11, 12). For example, one type of conditionally replicative competent (CRC-) adenovirus vector features a deletion in the gene encoding protein named E1B-55K (Figure 3D). This protein is essential for adenovirus replication in cells. However, many cancer cell lines can offer the same function of this protein even in the absence of this protein. Thus vectors can still replicate in cancer cells but not in normal cells and finally lyse only the tumor cells (13, 14). One of the E1B-55K-deleted vectors, Oncorine, was approved by Chinese SFDA for treating nasopharyngeal carcinoma in combination with chemotherapy (15).

We offer a comprehensive library of first-generation replication-defective adenovirus vectors containing full-length ORFs for human, mouse and rate genes driven by a strong CMV promote with a choice of His-Tag, HA-Tag or GFP Reporter.
Amplification of Adenovirus in Laboratory Setting

The general procedure of adenovirus amplification is outlined in the following paragraph. Initially, producer cells are transfected with recombinant RD-Ad vector carrying the transgene. The vector viruses are then replicated and packaged in the producer cells. After several rounds of infection using the producer cells with vector lysate for amplification, transgene vector particles are purified and then used to infect the targeted cells (Figure 5). You can find a detailed amplification protocol here.

Can’t find your gene? We can generate a custom adenovirus for you! Check out our Custom Adenovirus Service page for more details.
Advantages and Disadvantages of Recombinant Adenoviral Vectors

Many different viruses are being developed as gene transfer vectors, but the most advanced ones are adenovirus, retrovirus (including lentivirus) and adeno-associated virus (AAV) (16). One of the major advantages of adenoviral vectors is that they provide the most efficient gene transfer among other viral vector systems for a wide variety of cell types. Especially, they can transfer genes into both proliferating and quiescent cells (17). Moreover, they don’t integrate into the host cell genome so that there are low disturbances in genes or cellular processes within the body (18). From a production point of view, adenovirus vectors have a very high vector yield (> 1012 pfu/ml).

Require high titer adenovirus for your experiments? We offer adenovirus amplification and purification service up to 1012 pfu/ml.

Due to their replication-deficiency and separation from the host genome, adenovirus vectors mediated gene expression is transient, ranging from two weeks to a few months. Therefore, they are not suitable for long term correction of chronic disorders but proper for therapeutics that needs high and transient expression (13). Table 1 below compares some key features among some common recombinant viral vectors.


Table 1 – Comparison between Lentivirus, Adenovirus and AAV viral vectors

Features Lentivirus Adenovirus AAV
Efficiency ••• ••••† •••
Cell Type Most Dividing/Non-Dividing Cells Dividing Cells Most Dividing/Non-Dividing Cells and High Transduction Rate Towards Primary Cells All Cell Types
Integrating Yes No 90% Not, 10% May Integrate
Immune Response ••• ••••• ••

†Up to 36 kb for helper-dependent adenovirus vectors

We can help you find the most suitable Lentivirus, Adenovirus and AAV for your research project any time! Contact us at [email protected].
Clinical use of Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy

Gene therapy utilizes directed gene transfer to treat diseases. It is aimed to revolutionize the practice of medicine by treating the causes of disease rather than the symptoms. The fundamental obstacle in gene therapy is to develop efficient and adequate gene transfer vehicles such as delivery vectors with high efficacy and specificity. In the past decade, we have witnessed significant improvements in recombinant vector technology which manifested great clinical benefits in gene therapy (19). Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Therefore adenoviral vector-based clinical trials have increased remarkably. Below we outline some examples of adenovirus technology in gene therapy.

Tuberculosis

An RD Ad5-based AdHu5Ag85A vaccine, developed at McMaster University, has shown promising effects against tuberculosis (20). This vector expresses an immune-dominant antigen of Mycobacterium tuberculosis and aims to boost the host’s T cell responses to the pathogen. In a phase I study with 26 volunteers previously immunized with BCG and patients naive to BCG, injection of the virus vectors boosted T cell responses in the previously BCG vaccinated group. Moreover, pre-existing immunity to Ad5 did not affect the safety or efficacy of the vaccine.

Cancer Treatment

RD Ad-based vectors have also been used to treat cancer. For example, Advexin and Gendicine are RD Ad5 vectors expressing p53. Nearly half of the cancers lack functional p53, and almost all cancers have a dysfunctional p53 tumor suppressor pathway. Thus the forced expression of p53 by cancer cells with Advexin and Gendicine will cause cell cycle arrest or apoptosis. In a phase III clinical trial for advanced recurrent HNSCC (head and neck squamous cell carcinoma), Advexin was well tolerated and demonstrated anti-tumor activity (21). Similarly, Gendicine was approved in 2003 by the State Food and Drug Administration (SFDA) in China for intratumoral treatment of HNSCC in combination with chemotherapy (22). There are many other RD Ad vectors expressing different transgenes being examined or currently under investigation in clinical trials such as those expressing functional OTC, CFTR, VEGF, GM-CSF, human IL-2, human IL-12, human IFNα, etc.



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References
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