Introduction
Gene therapy is used to describe any medical precedure that uses genetic material to try to correct a disease. This ranges from simply inserting plain DNA or RNA into a cell to attempt to temporarily express the gene, to sophisticated packages of genes and promoters in a complex viral vector, in an effort to completely replace a defective gene. gene therapy has great promise for curing many genetic disorders, and could also be used to reduce the incidence of inherited cancer or coronary heart disease, but cannot currently be used because of the difficulties in getting the body to express foreign DNA, many of which are not fully understood, and the stringent clinical trials that any gene therapy product would have to undergo.
DNA
The DNAor RNA used in gene therapy is composed of at least two components, a correct, therapeutic gene, and a promoter, a sequence that tells the cell to express the therapeutic gene. Ideally the promoter should be specific to the area of the body affected by the disease, but at the moment generic promoters are used. Other sequences to increase gene expression, integration or specificity can be included, but this is limited by the size of the vector.
vectors
In order to insert material into a complex
organism like a
human, a
vector has to be used, which will perform a number of functions:
1. The
vector must be capable of avoiding the
immune system, which would otherwise destroy the foreign
DNA/
RNA as it entered the body.
2. The
vector must be capable of entering
cells and passing through
membranes. The main reason naked
DNA/
RNA cannot be used is that
DNA/
RNA has a negatively charged
sugar-
phosphate backbone, which is repelled by
phospolipid membranes which are also negatively charged.
3. The
vector must allow the
DNA/
RNA to interact with transcription/translation mechanisms inside the
cell, but must also resist digestion by nuclease enzymes inside the
cell.
Five main classes of
vector have been used, four of them viral, one a completely artificial, non-viral
vector. These are:
Adenoviruses:
Adenoviruses are viruses that normally cause respiratory infections in
humans. This makes them especially good for treatment of
cystic fibrosis, one of the main targets of
gene therapy research. WIth the
genes that can stimulate an
immune response removed,
adenoviruses can deliver a therapeutic
gene into a
cell, however, the
gene expression is low and temporary as the
gene is no integrated into the
genome.
Adeno-Associated Virus (AAV)
AAV is a good candidate for a
vector as it naturally inserts itself into an apparently unused patch of the
human chromosome 19, one of the best types of integration that can occur, as not only is there the possibility of permenant
gene expression, but the integration is very unlikely to do any damage to the existing
genome. Unfortunately this specificity is lost as some of the viral
genes are removed to insert the
gene. Only very small
genes can be used as the original AAV
genome is very small.
Herpes Simplex Virus
The latent
Herpes Simplex Virus is nonpathogenic and is particularly attracted to
nerve cells making it important for targeting
nerves, however issues about its safety and difficulties in making sure the virus remains latent, and does not start killing
cells make it an unlikely candidate for any immediate
gene therapy products.
Retroviruses
Retroviruses are
viruses that can stably integrate into a
genome without causing any serious
immune response, however, they are limited by the inability to infect nondividing
cells and the difficulties in purification. A more promising subclass of
retroviruses, called lentiviruses, are being investigated, that have the abliity to infect nondividing
cells.
All these viruses are usually manufactured by using packaging
cell lines, where a
plasmid containing the
DNA for both the therapeutic
gene and the viral
DNA required is assembled as a
plasmid, and inserted into a
bacteria (almost always
E.coli )to produce the
virus.
Nonviral vectors
A number of nonviral
gene therapy
vectors have been created, most using cationic
lipid complexes. These are groups of positively charged
lipids that bind to the negatively charged
sugar-
phosphate backbone of the
DNA. These complexes have a net positive charge, so they are attracted to and pass through negatively charged
cell membranes. They are not capable of integrating the
DNA into the
genome, although the idea of using them to introduce an entire artificial
chromosome to a
cell.
The Future
Gene therapy treatments for both cystic fibrosis and heamophilia, using the CFTR and clotting factor IX genes repectively, are now being heavily researched by several organisations, and it seems likely that a commercial product will be available withing the next 5-10 years, depending on the clinical trials required. The possibilities for gene therapy are huge once the technology becomes reliable (gene therapy is still not a precise science). Many genetic disorders will be permenantly eliminated by gene therapy in utero before they even appear, while more advanced human genetic engineering will become possible.
sources:
http://www.tulane.edu/~dmsander/WWW/335/peel/peel1.html
http://www.endocrinology.org/SFE/training/mew/mew2_ray.htm