You may find it hard to imagine that something as simple as a single base change—specifically, the appearance of a cytosine instead of a thymine somewhere along the 3-trillion-base-long human genome—could lead to a condition as profound as blindness. Yet, in certain cases of congenital blindness known as Leber congenital amaurosis, this is exactly what happens. On first glance, it seems tantalizingly simple to fix disorders that are caused by mutations in a single gene: Simply insert the correct form of the mutant gene into the cells that need the functioning gene, then stand back and watch the disease disappear. However, the field of gene therapy, in which scientists work to treat disorders at the level of the sequence of mutated genes, is much more complicated than it first appears.
How to Get Therapeutic DNA Inside Cells
![Gene targeting with recombinant adenoviral vectors](javascript:dispOrigImg("/scitable/content/22302/10[1].1038_nrmicro1266-f2_full.gif", "Gene targeting with recombinant adenoviral vectors", "true", "Figure 1", "A nonfunctional mutant of a reporter gene (W in the figure) is introduced or is present in a cell (a). In the recombinant targeting vector (b), the viral genes are replaced by a sequence that is homologous to the chromosomal locus targeted for modification. Usually, the modification is introduced between stretches of homology called 5' and 3' homology arms. Z designates an inactivating mutation in the viral repair template. This recombinant adeno-associated virus (rAAV) is used to transduce the cells. Recombination with the chromosomal target (c) can result in repair of the defect and recovery of a healthy cell (d). The frequency of gene targeting is determined as the fraction of infected cells that expresses a functional reporter. Stable integration is confirmed by antibiotic selection and Southern analysis. (In the figure, crosses [X] mark regions of homology between the chromosomal and viral DNA, and ITR indicates an inverted terminal repeat.)", "true", "All rights reserved.", '500', '450', 'http://www.nature.com');)
Figure 1One major obstacle facing effective gene therapy involves finding the best way to deliver therapeutic genes into target cells within the body. Scientists have tested a number of methods, some of which utilize liposomes or cell surface receptors. But the most common method of DNA delivery employs something that is perfectly evolved to enter cells: the virus.
Viruses are obligate intracellular parasites, designed through the course of evolution to infect cells, often with great specificity to particular cell types. Viruses efficiently transfect their own DNA into a host cell, then use the host's cellular machinery to replicate their DNA and synthesize certain viral proteins, thereby producing more viral particles. When it comes to gene therapy, scientists can capitalize on this process by removing the disease-causing portions of the viral genome and adding a foreign therapeutic gene. The engineered genome is then repackaged into the viral protein coat and allowed to infect the proper cell target. Now, instead of producing viral toxins and causing disease, the cell infected by this virus is able to produce the protein or enzyme it lacks.
One of the more common ways of introducing the wild-type version of a gene into cells with a defective copy is by way of an adenovirus. It is possible to use recombinant DNA technology to introduce a wild-type gene into an adenoviral vector. Once the adenovirus has infected the target cells, homologous recombination can occur, resulting in replacement of the native, mutant copy of the gene with the wild-type version from the vector (Figure 1).
