6.1 Gene addition:

In present scenario gene addition is one of the most practical approaches to gene therapy. It involves the insertion of an active copy of a defective innate gene. In the year 1990, viral vector based gene addition was successfully done to treat a patient named Thompson in the U.S. In the viral vector based gene addition Retroviruses were used at first because they were supposed and found to be most fit among all viruses for their adaptive nature for delivering genes into cells. This therapy is a promising one because it has been proven to be a substitute for gene replacement and is having a very high efficiency for curing any disease. Even though gene addition therapy is quite satisfactory but it has some benefits and drawbacks over the gene replacement therapy. Its advantages are that the cells that do not ordinarily express a specific gene can be manipulated to express that gene. For example, blood clotting factors could be synthesized in any somatic cell, making somatic tissues other than the liver convenient objectives for clotting factor gene transfer. Alternately, completely new genes might be used to cure the disease. For example, the transmission of human immunodeficiency virus (HIV) could be restricted by transfer of genes that encode ribozymes competent of degrading HIV RNA. A disadvantage of gene addition therapy is the disordered insertion of genes into the genome. Thus, the inserted genes may be erroneously expressed or may cause inappropriate expression of genes immediate to the insertion site.

The outcome of gene addition relies on the effective insertion of therapeutic   genes   at the suitable target spot within the host cell, avoiding any cell damage,   mutations or any kind of adverse immune response . Zinc finger nucleases (ZFNs) have become essential reagents for engineering the genomes of many plants and animals including drosophila melanogaster . Specially constructed laboratory, ZFNs suggests a common method to deliver a site-specific double strand breaks (DSB) to the genome, and stimulate a local homologous recombination by considerable amount of magnitude. The ZFN-encoding plasmid-based approach has the efficacy to avoid all the problems linked with the viral delivery of therapeutic genes. ZFNs drive exceedingly effective genome editing by generating a site-specific DNA DSB at a fixed site in the genome. Consequent restoration of this DSB break through the homology-directed repair (HDR) leads to targeted gene addition.

In some wet lab experiments addition of a break at a specific target site causes gene addition using a homologous donor template on using CCR5-specific ZFNs as a model system. Efficacy for a single stranded break to direct repair pathway choice may prove beneficial for some therapeutic applications such as the targeted correction of human disease-causing mutations.