Gene Addition
Obstacles Success Stories Other Vectors
Viral Vectors

Gene addition inserts a functioning copy of a misfunctioning or nonfunctional native gene. Viral based gene addition involves the "domestication" of viral genomes as vectors. This was the first method of gene addition successfully used to treat a patient in the U.S. (Thompson, 1990). Retroviruses were the first type of virus used, since the normal life cycle of this class of virus is well adapted for delivering genes into cells.

A retroviral genome contains 3 genes important in the infection cycle. The first of the retroviral genes, gag, encodes structural proteins. The second, pol, encodes various enzymes, including reverse transcriptase. Lastly, env encodes the envelope glycoprotein. Long Terminal Repeat (LTR) regulatory sequences, found at the insertion points of the genome, are essential for integration of the virus into the host genome. (See Figure 4)
Figure 4. Formation of a Retroviral RNA Vector
In order to create a gene addition vector from a retroviral genome, the three genes involved in the infection cycle (gag, pol, and env) must be removed, and a therapeutic gene must be added between the LTR regulatory sequences. This allows for the insertion activity of the genome to remain intact, but ensures that the infective action is eliminated.
Adapted from Walters & Palmer, 1997
Once a recombinant Viral RNA genome is created, the next step is the integration of this nucleic acid strand into the patients genome (See Figure 5)
Figure 5. Schematic of Viral Vector Integration
Click on Image for Larger Version and Explanation --Adapted from Walters & Palmer, 1997

Obstacles to Gene Addition. The most significant obstacle that currently faces the application of gene addition therapy is the inability to specify the insertion point of the recombinant genes.

Although no disease conditions have yet occurred within treatment subjects, it is imagined by some that such a random "stone throwing" (shotgun cloning) approach to gene insertion could put patients at risk for cancer-causing mutations.

In addition to such hypothesized risks, the current procedure of random insertion of therapeutic genes is, at best, an inefficient method of administering gene therapy.

Gene Addition Therapy Success Stories

On September 14, 1990, the first officially sanctioned human somatic-cell gene therapy treatment was applied, using ex-vivo gene addition techniques. A four year old girl, suffering from the rare genetic disorder, adenosine deaminase deficiency (ADA) was, so far as is currently known, successfully treated using these techniques. (Thompson, 1990)

As of 1998 there were at least 100 NIH Recombinant DNA Advisory Committee protocols that involve gene addition techniques. These studies examine possible treatment of diseases such as rheumatoid arthritis, peripheral artery disease, cystic fibrosis and cancer. (Thompson, 1990)

Other Vectors and Gene Delivery Methods

  • Microinjection -- microscopic "cell surgery" in which DNA is physically injected into cells using micropipettes
  • Calcium Phosphate Coprecipitation -- Although poorly understood, Calcium Phosphate causes cells to take up extracellular DNA.
  • Electroporation -- Cells exposed to rapid pulses of high voltage create resealing holes in the membrane, through which DNA is taken up from solution.
  • Liposomes -- Phospholipid sacs, containing DNA, which spontaneously fuse with cell membranes, spilling their contents into the cytosol of the cell
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