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MIDGE Transfection Vector

Viral Vectors

A virus contains genetic information but cannot reproduce itself. To replicate, it must invade another cell and use parts of that cell's reproductive machinery.The elaborated systems for gene transfer and gene expression used by viruses constitute powerful tools for the construction of transfection vectors. Thus animal viruses have been used for gene transfer to mammalian cells.

In general, a virus is per se immunogenic, so are viral vectors.

Retroviral vectors

Retroviruses are RNA viruses which replicate through a DNA intermediate. The viral DNA integrates into the host genome. The viral genes required for replication are removed from the retrovirus and replaced by the therapeutic gene. Only regulatory elements remain of the original virus genome. Retroviral vectors are most frequently based upon the Moloney murine leukaemia virus (Mo-MuLV). The AIDS virus (HIV) is also a retrovirus.

Retroviral vectors enter the target cells, transcribe their RNA into DNA and integrate stably into the host genome, sometimes with very high efficiency.

A disadvantage of the murine retroviral vectors is their size limitations for the transgene. Retroviral integration and expression of viral genes requires that the target cells should be dividing. This limits gene therapy to mitotic cells. Another issue is the potential for producing recombinant viruses that can replicate, thus posing a severe threat of the patients safety. Furthermore, MuLV carry proto-oncogenes, which when mutated can induce oncogenesis (reviewed in Gray).

Lastly, using retrovirus is quite expensive, largely because of the need to test for contaminating virus, bacteria, and fungus. Particularly, the virus’ envelope makes purification difficult and costly.

Adenovirus

Adenoviruses are the second most popular choice of viral gene delivery vectors. There are over forty serotype strains of adenovirus; however serotype 2 or 5 are predominantly used as vectors.

Adenoviral vectors are very efficient at transducing target cells and delivering its genetic cargo to the nucleus (Shenk, 1996). Viral replication occurs without integration into the host genome, leading to transient expression of the transferred gene (Verma&Somia). Adenovirus can be used for a wide range of cells, dividing and non-dividing.

The main disadvantage of the adenovirus is its immunogenicity, provoking the host immune system to attack the virus and render the therapy ineffective. Furthermore, adenovirus has been shown to cause damage in the brain and other organs. Adenovirus was used in the gene therapy study which led to the death of a teenager in September 1999.

Adeno-associated viral vector

Adeno-associated virus is a member of the parvovirus family that requires adeno or herpes virus for replication.

The adeno-associated virus is a single-stranded DNA virus, which combines the advantages of retroviral and adenoviral vectors. It has little immunogenicity and can infect a wide range of cell types, including both dividing and non-dividing cells.

In the absence of a helper virus, AAV integrates into the host genome on the short arme of chromosome 19 (Rivadeneira, 1998).

The main problems is that AAV vectors can only transport rather small genes, 5 kb is the upper limit (Smith, 1995). And, as with all viral vectors, there exists the danger of insertional mutagenesis and the generation of replication competent virus.

indextop I next: Non-viral vectors

 

 


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