Research Updates September-October 2007

Diseased featured in this article include: Duchenne muscular dystrophy, Friedreich's ataxia and myotonic musuclar dystrophy.

ID of utrophin brake raises DMD treatment hopes

MDA-supported researchers at the University of Pennsylvania and the University of Ottawa report finding a molecule that keeps the muscle protein known as utrophin confined to one small area of a muscle fiber and thereby limits its potential as a therapy for Duchenne muscular dystrophy (DMD). Releasing this brake by blocking this molecule could become a strategy for treating the disease.

MDA grantee Tejvir Khurana at the University of Pennsylvania and colleagues, including MDA-supported Bernard Jasmin at the University of Ottawa, say a compound called ERF (ETS-2 repressor factor) helps regulate utrophin production in mature muscles.

The utrophin protein is very similar in structure to the dystrophin protein, which is missing in boys with DMD. However, its instructions are in a gene on chromosome 6, in contrast to those for dystrophin, which are on the X chromosome and are defective in DMD-affected animals and people. The utrophin gene isn’t affected in DMD.

During fetal development and up until shortly after birth, utrophin is produced (expressed) all around muscle fibers. But as the fibers mature, most of the utrophin is replaced by dystrophin, except in the small areas where the nerve and muscle fibers meet, called the neuromuscular junctions.

Figuring out a strategy for returning utrophin expression to the whole muscle fiber has been a goal of MDA research ever since experiments in mice in the 1990s showed that utrophin can, at least in part, compensate for dystrophin deficiency.

Increasing utrophin’s presence might in some situations be easier than replacing or repairing dystrophin genes. And, utrophin would be highly unlikely to cause an unwanted immune response, since boys with DMD already make some utrophin.

The investigators, who published their findings online May 16 in Molecular Biology of the Cell, say ERF apparently interacts with a molecular switch (promoter) to repress the activation of utrophin instructions. When they reduced ERF levels, utrophin promoter activity and utrophin expression outside the neuromuscular junctions increased.

“Together, these studies suggest ‘repressing repressors’ as a potential strategy for achieving utrophin upregulation (increase) in DMD,” the researchers say.

Gene transfer using herpes virus transporter helps mice with Friedreich's Ataxia

Scientists in Madrid have successfully treated mice with Friedreich's ataxia (FA) by transferring genes for the frataxin protein into their nervous systems, using a viral transporter.

FA is a progressive disease that causes weakness and incoordination. It results from mutations in the gene for frataxin, a protein needed for energy production in nerve cells.

When Filip Lim and colleagues at Autonoma University of Madrid injected human frataxin genes, encased in modified herpes simplex viruses, into the brainstems of frataxin-deficient mice, they found they had restored the animals’ ability to stay on a rotating rod.

Their functional recovery was “surprisingly complete,” the researchers say in their report, published in the June issue of Molecular Therapy.

They note that humans who are carriers of FA but have no symptoms of the disease only have about 40 percent of the normal level of frataxin, indicating that high-level frataxin restoration probably isn’t necessary.

Delivery of genes to the nervous system is perhaps the most challenging form of gene therapy, but the modified herpes simplex virus appears promising as a vehicle.

The investigators note that it naturally targets nerve cells when it causes an infection; that it’s large enough to carry a lot of therapeutic DNA; and that it doesn’t integrate into any of a cell’s chromosomes, which makes it safer than viruses that do.

“All therapeutic strategies aimed at alleviating neurodegeneration in FA and many other neurological conditions have failed so far, emphasizing the need to explore other options, such as gene therapy,” the authors write.

They also say new mice, developed by Mark Pook at Brunel University in the United Kingdom, and colleagues, will provide better tools for future studies, because they have genetic mutations exactly like those that cause human FA and have a disease that more closely mimics the human one.

Delivering genes via bacteria may become an option

The possibility of using modified bacteria, instead of modified viruses, as vehicles to carry therapeutic genes into cells, is moving closer to reality, say researchers at Purdue University in West Lafayette, Ind., who described the process online June 10 in Nature Nanotechnology.

Bacteria are much larger than viruses and can carry considerably more DNA, as well as other compounds.

Demir Akin and colleagues attached various genes, including those for luciferase (a protein that lights up and is easy to detect), to the surface of a bacterium known as Listeria monocytogenes.

Although bacteria are generally engulfed by cells and destroyed in acidic compartments, this bacterium escapes from these compartments because it has a special toxin that destroys their walls.

In the process, any cargo becomes separated from the bacterial transporter, which is later destroyed by the cell. The cargo DNA travels to the cell nucleus, the home of cellular chromosomes and the place where the first steps toward protein synthesis from genes occur.

When the investigators injected gene-carrying Listeria bacteria into the abdominal cavity in mice, they found the transported genes penetrated the nuclei of several types of cells, including some in the central nervous system. The DNA cargo was used for protein manufacturing, as evidenced by the lighting up of the cells that took up the luciferase genes and other tests for the other cargo.

The researchers say they’ll now concentrate on developing a nontoxic strain of Listeria (the one they used was fairly toxic); targeting delivery to specific cells; and increasing the bacterium’s carrying capacity to include larger molecules.

Z-Shaped construct has potential for blocking hazardous genes

Scientists at the Karolinska Institute in Stockholm, Sweden, have developed a new way to block harmful genes, adding to an expanding array of gene-silencing strategies that have potential for treating diseases in which something toxic, rather than something missing, is the underlying problem. Most dominantly inherited conditions, in which a genetic flaw from only one parent is needed to cause symptoms, are in this category.

Rongbin Ge and colleagues, who published their results in the June issue of the FASEB (Federation of American Societies for Experimental Biology) Journal, have created a Z-shaped construct that sticks to DNA and thereby specifically silences genes in cells grown in laboratory containers. Other silencing strategies are aimed at blocking RNA, the genetic “message” derived from DNA instructions (genes). The cell uses the RNA message to create protein molecules.

The Swedish scientists have dubbed their compound Zorro locked nucleic acid (LNA) and say it sticks to each of the two strands that make up a typical stretch of DNA, as the DNA begins to “unzip” just before RNA synthesis.

RNA silencing will probably be an effective treatment for some conditions, in which only the protein product of the gene is toxic. But for disorders in which the RNA itself appears to be toxic, blocking DNA instructions is likely to be a better option. Types 1 and 2 myotonic dystrophy (MMD) are in this category.

“It’s still very early days when it comes to this technology,” said study co-author C.I. Edvard Smith, “but we are working hard to find out more about its potential. At least in the test tube and following microinjection into cells, we believe that the Zorro LNA construct has strand-invading properties. However, we still have a long way to go before we know about the clinical efficacy of this potential drug.”