Viral Vectors

Different types of viruses, including retroviruses, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, and others, have been engineered as gene transfer vectors. A virus-mediated gene delivery system uses the cell receptor recognition system of the virus for binding to a specific cell type, which is followed by the internalization of the vector DNA and its transport to the cell nucleus. Some viruses have mechanisms for integration of the vector DNA into a chromosome site, and with others the input DNA is maintained as an extrachromosomal element. [50]

 Because of their ability to sneak into cells, viruses appear to be efficient delivery vehicles for replacing mutant genes. But first three major obstacles stand in the way:

1. Scientists must find a way to block the ability of the virus to replicate its own genome.

2. They must stop the production of viral messenger RNA that codes for the proteins that help the virus escape into the cell.

3. They must insert the therapeutic gene in such a way that the formation of the capsid will be normal, allowing the virus to get into the cell.

• RNA Viruses:

            • Retroviruses:

The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of the hosts is in the form of DNA. When a retrovirus infects a host cell, it introduces its RNA together with some enzymes into the cell. The RNA molecule must produce a DNA copy from its RNA molecule before it can be considered part of the genetic material of the host cell. Retroviruses are a class of virus that can create double-stranded DNA copies of their RNA genomes. Because the genetic material is RNA rather than DNA, retroviruses produce an enzyme known as reverse transcriptase. Because they make this enzyme, they can transform their RNA into DNA, which can be permanently integrated into the DNA of the host cells. Scientists were the first to use these vectors, which are easily cloned and work best in actively dividing cells. Critical retroviral genes are removed so that the virus cannot reproduce after it delivers its genetic cargo. However, because cells in the body do not divide often, retroviruses are used primarily ex vivo (i.e., outside the body). The process works in the following way:

• DNA Viruses:

            • Adenoviruses:

This class of virus has double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. They efficiently enter most cells and can infect stationary cells. Advantages of working with this class of virus include high levels of replication and expression, ease of handling, and their capacity to infect many types of human cells, including nondividing ones. A disadvantage of working with adenoviruses is that the immune system responds, and expels the foreign material from the body. Researchers are trying to move large portions of unessential DNA, hoping the body will not reject the virus and its payload. The virus that causes the common cold is an adenovirus. Adenovirus type 2 (AD-2) has been used in trials targeting T lymphocytes (the cells of the immune system) and a number of tumor cells. A form of AD-2 has been therapeutically injected directly into the liver for treatment. The problem here is tricky. Too little will cause insufficient gene expression; too much can infect other cells. Also, the expression of the therapeutic gene tends to decline after a week or so.

             • Adeno-associated viruses (AAVs):

These viruses are small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19. They cause no known diseases in humans and have long-term expression. They have the ability to target nondividing cells located in muscle and in the brain, liver, and lungs, and can insert their genome into the genome of the recipient. They also appear to evade the assault of the immune system. Because of their staying power, they hold promise for the treatment of such chronic diseases as hemophilia. A 2006 study by scientists at the University of Florida evaluated a method of delivering three subtypes of adeno-associated virus, which are not known to trigger an immune response reaction. They tested the ability of AAV-1,AAV-8, and AAV 9 to insert genes into skeletal and heart muscle. Tests revealed that AAV-9 was taken into the heart in amounts 200 times the level at which AAV was taken in.[18]

            • Lentiviruses:

Lentiviruses (LVs) are derived from a special group of viruses, of which HIV is a member. HIV has adapted itself to enter human cells in an effective manner. For this reason HIV has been difficult to eradicate, but for the same reason, the virus may develop into be a very efficient vector. Researchers must engineer a way to make virus vectors less dangerous. For example, removing just six genes from HIV makes it less virulent. LVs have been evaluated in clinical human trials. A Phase I trial for the treatment of patients with HIV/AIDS was successfully completed at the University of Pennsylvania, which showed excellent safety profiles.

        LVs have vast potential as drug discovery tools, including their possible use in target validation and in generation of engineered cell lines and transgenic animals. However, here perception becomes part of the problem. Who wants a debilitated HIV virus injected into his or her body?[24]

             • Herpesviruses:

            • Poxviruses: