Adenoviruses as Viral Vectors

History
The discovery and initial description of Adenoviruses (Ad) took place in the early 1950s. They were first isolated from human adenoid tissue cultures (Rowe et al., 1953). Since then several different serotypes of human, avian, reptilian, amphibian and other mammalian adenoviruses have been isolated and characterized giving birth to the Adenoviridae family of over 50 members (Davison et al., 2003; Shenk et al, 2001).

Classification
In order to provide a systematic organization the International Committee on Taxonomy of Viruses (ICTV), first founded in the late 1960s, established the following taxonomic classification for adenoviruses. It uses the familiar systematic taxonomy scheme of Order, Family, Subfamily, Genus and Species (no Kingdoms, Phyla, or Class of viruses have been described within this scheme): (Dimmock AJ. Introduction to Modern Virology. 2007)

Structure
The general architecture of most viruses consist in a molecule containing their vital information, the genome, made of DNA or RNA; a proteic coverage called capsid that provides protection, stability and interaction elements which are needed during the cell-infection process. The virus also carries a few enzymes required for early events into the host cell. For some viruses an extra lipidic envelope is also required. (Dimmock AJ. Introduction to Modern Virology. 2007)

The genome of Adenoviruses is a linear, double-stranded DNA with a terminal protein (TP) attached to one of its ends and a molecular weight of 30-40 Kb. All of these viruses have a characteristic morphology with an icosahedral capsid consisting of three major proteins called Hexon (or protein II), penton base (or protein III) and a knobbed fiber (or protein IV) along with a number of other minor proteins, VI, VIII, IX, IIIa and Iva2. They have an average diameter of 50-100 nm.

Adenovirus in Human Pathology
Most of the human serotypes are responsible for a wide variety of organ- specific affections, specially respiratory, gastrointestinal and ocular acute infections mainly among children under the age of 5 (Carballal G., Oubiña J. Virología Medica. 3° ed. Buenos Aires: El Ateneo 1998; Lopez E. Manual práctico de infectologia pediatrica. 2° ed. Actualizada. 1999)

It is known that most of the world population will eventually have contact with some type of adenovirus during their life.

Main Events in Adenovirus Infection
The Adenovirus can infect a wide variety of cells. The infection cycle can be differentiated into two separate phases:(Russell WC, 2000)

The first or early phase involves the attachment of viral particles to the cell surface, the entry into the host cell, the passage of the virus genome to the cell nucleus and the selective transcription and translation of the early genes. These events can take up to 6-8 h to complete.


 * The attachment of virus to target cell (adsorption) requires high-affinity receptors that bind viruses via the knob portion of the capsid´s fiber. The major receptor for human adenoviruses (with the exception of Human adenovirus B) is the Coxsackie/Adenovirus Receptor (CAR), a 46 kDa plasma membrane protein belonging to the immunoglobulin superfamily. However, other proteins have been recently involved in the virus-cell interaction process such as integrins, MHC I and sialoglycoprotein receptors.


 * The entry of the virus proceeds via clathrin-mediated endocytosis and requires the presence of cellular integrins αvβ3 and αvβ5 for adenovirus internalization. Once the virus enters the cell the capsid is disrupted by proteolysis and transported towards the nuclear membrane. The DNA genome gets through the nuclear pore and into the nucleus where the primary transcription events are initiated. Most of the viral genes codify for proteins that modulate cell metabolism and cell division, assist in the replication of viral DNA, help avoid host defenses, release the mature virus from the cell, etc.

The second or late phase leads to the assembly in the cell nucleus of the structural viral proteins (encapsidation) and the maturation of infectious virus within 4-6 h, yielding viruses which are prepared to exit the host cell and infect new ones. The permeabilization of the nuclear membrane facilitates the egress of the virus into the cytoplasm and is followed by the lysis of the plasma membrane and release of viral particles.

Adenoviruses as Gene Vectors
Because of the ability of adenovirus for infecting a great variety of mitotic and post-mitotic cells (meaning those which no longer will divide), even highly differentiated tissues such as skeletal muscle, lung, heart and brain, they are one of the best candidates for the delivery and expression of therapeutic DNA sequences within the context of gene therapy. Other characteristics that benefit the use of adenoviruses for this innovative and ambitious technique are:
 * Delivery of therapeutic DNA directly into the nucleus
 * Standard capacity for incorporating foreign DNA (2-6 Kb)
 * Relative ease of preparation and purification
 * No risk of insertional mutations and oncogenesis

However, the use of wild-type adenoviruses for human therapy involves the risk of high morbidity and mortality. Therefore these viruses are genetically manipulated and modified in order to eliminate the pathogenic viral genes and incorporate a foreign gene of interest obtaining finally a recombinant adenoviral vector (RAd).

This type of vectors can be utilized for gene delivery into cancerous cells for tumor suppression, gene delivery to tissues to enhance the expression of defective genes or supplement the cells with a particular therapeutic or non therapeutic DNA sequence. (Russell W. 2000)

There are several techniques for constructing this recombinant adenoviral vectors. One of the first and most popular is the one described by Dr. Frank Graham and known as the “two plasmid method” (now available in commercial kits). In this system a circular DNA (plasmid) that carries the modified viral genome is combined with another plasmid containing the gene of interest (shuttle) and resulting in a new circular DNA carrying most of the information. This procedure is called cotransfection and takes place into a specific cell line called HEK 293 (Bett AJ, et al. 1994)

Finally, the mature viral particles exit the HEK 293 cells by lysis and are ready to infect other cells of the same culture. Afterwards, the generated Rad is amplified, purified and characterized using in-vitro and in-vivo models.