The Objective of Gene Therapy
The idea behind most gene therapy is to either correct a genetic disease by adding a new functional gene to cells with a defective version the gene, or to add a gene that somehow alters the target cells to provide a treatment or cure for a disease. For example, a new gene could be introduced to fix a congenital genetic defect, or to label some diseased cells, like cancer cells, so the immune system will attack them or make them more susceptible to a subsequent treatment. The most efficient way to introduce a new gene into a cell's DNA is to use organisms naturally designed for it. Viruses have evolved to enter cells, splice a copy of their DNA into the genome, then replicate it and make more viruses. For gene therapy, a patient's cells are infected with a modified virus that cannot replicate but carries the functional gene. Cells can be isolated from the patient, infected, the put back into the patient (called an ex vivo protocol), or the virus can directly injected in the patient to infect the target cells (called an in vivo protocol). With either approach, the idea is that certain affected cells, after infection, continue on as they normally would but with a working version of the gene.
Vectors to Carry Genes into Cells
In animal models, researchers use several types of viral vectors, as well as other non-viral approaches to get DNA into cells. However, many of these are not suitable for human gene therapy due to safety concerns. For human gene therapy, DNA is introduced into cells mostly using highly modified DNA from natural viruses, although other chemical approaches can sometimes be used too. Obviously, vectors made from viruses need to be extensively altered before they can safely be used for gene therapy. For example, the components that enable a virus to replicate itself and affect the function of other genes in cells, need to be removed. Only very well understood viruses can be modified appropriately, and then, the vectors derived from these modifications must be extensively tested and evaluated. DNA vectors made from viruses only contain enough of the viral DNA to enable them to enter a cell and set up a gene. The parts that enable a virus to control the cell make copies of itself or coat itself in proteins so it can infect other cells are deleted. Most human gene therapy studies use DNA vectors derived from one of three virus types: retrovirus, adenoviruses, and adeno-associated viruses.
Adenovirus Vectors for Temporary Fixes
Vectors made from adenoviruses do not become part of a cell's core DNA. As with chemical methods to introduce DNA into cells, genes introduced to cells using adenovirus vectors do not integrate into the chromosomes and become part of the genome. The DNA from an adenovirus is put in the same compartment of the cell as the genomic DNA (i.e., the nucleus) and the cell decodes the gene just like other genes in the genome. However, the gene stays on a extra piece of adenovirus DNA and is eventually lost after the cells divide several times. For temporary changes, for example, making specific cancer cells more noticeable to the immune system so it will respond and kill them, adenovirus vectors offer a safer option than ones like retroviruses that insert a gene directly into genomic DNA. For this reason, the adenovirus vector can be used for protocols where the viral vector is injected directly into the patient. However, a permanent cure to a genetic disease requires a different sort of vector and different approach.
True Genome Changes with DNA Integration
Unlike adenovirus vectors, DNA vectors made from retroviruses insert a new gene right into the genome. It becomes part of the core cellular DNA in the chromosomes and is copied and maintained in daughter cells when the cell divides. The cell and its descendants are permanently altered. For this reason, when these genome-integrating vectors are employed, an ex vivo protocol used, which means cells are extracted from a patient, infected in a cell culture plate in the lab, then put back in the patient later. Since a retroviral vector places a gene virtually anywhere in the genome, one safety risk with retroviral vectors involves disruption of native cellular genes at the insertion site. This sort of disruption led to the development of leukemia in some patients in early trials. However, making a permanent genetic change to cure a congenital disease requires this type of vector. A lot of work has been done to reduce the risk of retroviral vectors, much of it using vectors made from lentiviruses, a specific type of retrovirus.
Vectors made from adeno-associated viruses (AAV) can also be used to permanently introduce DNA to a cell's genome. AAV almost always inserts at a specific spot on chromosome 19 so it minimizes the risk of disrupting other genes. AAV vectors, though, are somewhat difficult to produce and manipulate and they can only be used to insert small genes. However, AAV vectors have been shown to be very effective to treat Leber congenital amaurosis a congenital blindness caused by a defective retinal pigment gene (RPE65).