Eric Schon

Most genetic diseases are caused when mutations in a gene render the protein it encodes nonfunctional. With this in mind, researchers in neuromuscular diseases and other areas of medicine are exploring "gene therapy" -- attempts to treat these diseases at their source by providing cells with healthy copies of the damaged genes.

MDA grantee Eric Schon believes that the same gene therapy techniques being developed for the muscular dystrophies and other diseases should be applicable to mitochondrial diseases caused by mutations in the chromosomal DNA. This type of DNA resides in a cell's nucleus and it's what's almost always referred to when scientists talk about genetic diseases.

But the body has another type of DNA, one that resides inside the cells' mitochondria (mtDNA), and defects in this DNA can also lead to mitochondrial disorders.

"When you start talking about mtDNA, there's a whole other order of complexity to it [gene therapy] for a number of reasons," says Schon, who has been working on gene therapy to fix a mitochondrial gene called ATP synthase subunit 6. Defects in this gene lead to maternally inherited Leigh's syndrome or MILS.

Schon explains that when the defective gene is in the mitochondria, delivering a functional gene to the cell is only half the battle. Although the new DNA can get into the cell, it can't get into the mitochondria, because mtDNA doesn't use the same genetic code as chromosomal DNA.

"So you couldn't just stick a new gene in the nucleus and expect it to work," Schon says. "It wouldn't make the proper protein."

(If you've ever tried to coax a PC computer to read a Macintosh file you'll understand the problem. Although the PC may eventually be persuaded to open the file, it interprets the Macintosh computer code incorrectly and displays gibberish on the screen.)

To get around this problem, Schon and the members of his laboratory have painstakingly "translated" the mtDNA code of the ATP synthase 6 gene into a code that the rest of the cell can understand. Presumably it doesn't matter if the mitochondrial gene can't get into the mitochondria because the cell nucleus should be able to make the proper protein.

Schon also added a bit of DNA sequence to the gene to give the resulting protein a special routing tag instructing it to go into the mitochondria.

Now the modified mtDNA gene can be read outside of the mitochondria and the resulting protein is automatically taken up by the mitochondria.

"The good news," Schon says, "is that we don't think that you have to import a lot of the protein into the mitochondria to correct function. So, that's not such a tall order then.

"Now we're trying to see if the protein goes into the right part of the ATP synthase complex," Schon says. If that works, he'll test the procedure in human cells from a person with MILS.

Schon's laboratory is also trying to find ways to fix problems with the mitochondrial transfer RNAs (tRNAs). The tRNAs aren't proteins, but are molecules needed to manufacture proteins from the genes. Many mtDNA diseases, including MELAS and MERFF, can result from defects in one of the 22 mitochondrial tRNAs.

Schon has met with some initial success in his search for a way to get healthy tRNAs into the mitochondria.

Gene therapy is one way to go, but researchers are also studying a different strategy for dealing with defective mtDNA. This strategy takes advantage of the fact that the cells of almost all people with mtDNA mutations are heteroplasmic -- that is, each cell has a mixture of normal and mutant mtDNA.

This quality of having the good mixed with the bad may be useful because the mitochondria in individual cells are constantly dividing. If something could be done to selectively block the replication of the mutated mtDNA, then the cells might gradually be able to replace the defective mtDNA with normal mtDNA.

"The trick is, how are you going to get the bad ones specifically?" asks Schon.

A group of researchers, led by R.N. Lightowlers of the University of Newcastle upon Tyne in the United Kingdom, is attempting to do just this.

The researchers are using little molecules called peptide nucleic acids that are like homing missiles: They're designed to seek out and bind to specific sequences of mutated mtDNA. The scientists hope these molecules will prevent the mutated mtDNA from being copied so that the only mtDNA being produced is normal.

This type of therapy would require very specific tailor-made molecules for each person's mutation.

Schon's laboratory is also trying to find a way to increase the amount of good vs. bad mtDNA in the muscles of people with mitochondrial diseases. They're currently experimenting with a toxin called oligomycin.

"Oligomycin is poisonous -- it will kill you," Schon says.

"But we have found that if we add tiny amounts of oligomycin to cells that are heteroplasmic for the mtDNA mutation that causes MILS, over a period of one week we could shift the heteroplasmy in the cells in a good direction toward more normal mtDNA and less mutant. Now we're working on analogues of oligomycin that are less toxic. We've made a little list that includes, among other things, the AIDS drug AZT."

Another approach being evaluated by Schon's laboratory is the way good and bad mtDNA are mixed within muscle cells. Some muscle fibers have sharp dividing lines between areas that contain normal mtDNA and areas that contain mutant mtDNA, Schon explains. His idea is to try to spread out the normal mtDNA more evenly in the muscle fibers so that most fibers have some functional mitochondria.

This strategy may work because often only a small percentage of normal mitochondria is needed to get a significant improvement in cell function.

Schon also speculates that mixing the two types of mtDNA may shut down production of the mutant kind. His laboratory is currently testing a compound that may promote mixing at the cellular boundaries between normal and mutant mtDNA.

Another strategy along the same lines that doesn't involve any fancy designer molecules and has already shown some initial benefit to one patient is being tried by Eric Shoubridge of Montreal Neurological Institute in Quebec. Shoubridge and his colleagues have managed to shift the ratio of mutant to normal mtDNA for the better in the arm muscles of one man with PEO through a specific type of exercise.

During exercise, some damage is normally done to the muscle. It's repaired when immature muscle cells called satellite cells divide and fuse with the damaged muscle to help it regenerate.

In the Montreal experiment, the subject had normal mtDNA everywhere in his body, except in his mature skeletal muscle cells (giving him a "pure myopathy"). Because the satellite cells were full of normal mtDNA, the researchers wondered whether exercise-induced muscle damage that stimulated satellite cell fusion might be a way to add more normal mtDNA to the mature muscle cells.

When they tried this strategy, the researchers found that "concentric" exercises, which involve shortening muscle contractions, increased the proportion of normal mtDNA in the arm muscles of the patient from 12 percent to 33.4 percent.

Despite this improvement in the muscle's genetic makeup, the man didn't experience any measurable increase in strength in the exercised arm. Shoubridge and colleagues suspect the exercise program didn't last long enough to yield any noticeable increases in strength.

This type of therapy would only apply to those people who have normal satellite cell DNA and mutated muscle cell mtDNA. The technique wouldn't correct problems that occur in organs other than muscle.

"When I got into this business, there was no way that I could see my way clear to any rational approach to treating any mitochondrial diseases, and it looked almost hopeless to me," Schon says. "Nobody even thought about therapies back then.

"But we've come a long way pretty fast, and I don't feel that way anymore. Now we do a lot of thinking about therapies for mitochondrial diseases. And a lot of that has come because of all the MDA-funded research."

Recently Schon collaborated with Ian Wilmut of the Roslin Institute in Scotland, the creator of Dolly the famous cloned sheep, to answer the important question: "Where do Dolly's mitochondria come from?"

It turns out that Dolly may be a clone, but her mitochondria come from the donor cell. These results were published in the September issue of Nature Genetics.

This finding may be of interest to women with mtDNA diseases who want to have children.

It may prove possible to put the nucleus of one of a woman's egg cells into a donor egg cell from which the nucleus has been removed. If the situation in humans is similar to that in sheep, the resulting child may have nuclear genes from both natural parents, but derive its mitochondrial genes from the healthy egg cell donor.

This technique has already been performed successfully by Jacque Cohen of the Institute for Reproductive Medicine and Science at St. Barnabas Medical Center in New Jersey, although the woman didn't have a verified mitochondrial myopathy.