Lentivirus Transfection Protocol

American and European scientists used lentiviruses in a ground-breaking clinical trial aimed at curing adrenoleukodystrophy (ALD), a fatal brain disease in young boys that is caused by a mutated ALD gene. The lentivirus delivered a correct version of the ALD gene to two young patients and in 2009, the scientists announced that they had stopped progression of the disease.

Lentiviruses

• Lentiviruses are retroviruses, since their genetic information is contained in RNA rather than DNA. They infect human cells and a special enzyme converts their RNA into DNA, which integrates into the cellular DNA. The cells replicate these genes, which make more viruses that infect other cells.

Modifications

• Scientists modified the lentiviruses. Instead of delivering the virus genes to a cell, the modified lentivirus delivers a therapeutic gene such as ALD. They placed the genes for the viral structural proteins in plasmids, which are small pieces of DNA. A separate plasmid contains the ALD gene.

Transfection

• Scientists produce the modified lentiviruses by transfection. They mix the plasmids with salts or lipids that allow the plasmids into cells where they are replicated. The cells produce the viral structural proteins that self-assemble into virus particles, each containing a piece of RNA with the ALD gene. These modified particles exit the cells into the growth medium, where they are collected for use in gene therapy trials.

Uses

• The viruses are a popular tool for modifying cells for research and industrial uses, in addition to gene therapy. For example, cells can be made that express insulin for pharmaceutical production or Green Fluorescent Protein for tracking cells in migration studies.