Dramatic results from combo therapy surprises Krabbé-disease researchers

The Washington University School of Medicine in St. Louis researchers who made the discovery set out with low hopes for the combination therapy because on its own, each treatment was only modestly effective for the sick mice.

The mice are called Twitcher mice because the damage that happens in their nervous systems results in twitching. In people, the same genetic defect causes Krabbé disease, or globoid-cell leukodystrophy, an inherited disorder that destroys both the brain and the nerves of the body.

“We had everything we needed to test the combination treatment, so we said, 'Let's just give it a try,'” says the study's senior author, Mark Sands, Ph.D., associate professor of medicine and of genetics. “Alone, these therapies resulted in a small survival benefit, but with the combination we were seeing mice with dramatically extended life spans. Ninety-five percent of the Twitcher mice that had the combination treatment were still alive after all the other Twitcher mice had died.”

The researchers gave newborn Twitcher mice injections of a gene therapy vector into the brain and infused them with bone marrow from healthy mice. Mice receiving the dual therapy lived more than twice as long (greater than 100 days), on average, as untreated mice or those given a single therapy and had improved motor skills. The research is reported in the January issue of Molecular Therapy.

Although not considered a cure at this stage, the combination therapy offers hope for treatment of diseases like Krabbé, which are termed lysosomal storage diseases. These disorders include Niemann-Pick and Tay-Sachs diseases and affect 1 in 5,000 children born each year.

“This may become the paradigm for the treatment of lysosomal storage diseases that have a profound effect on the central nervous system,” Sands says.

About 45 lysosomal storage diseases are known, each caused by a genetic defect that knocks out an enzyme needed for normal function of lysosomes, cellular organelles that digest proteins, fats, carbohydrates and nucleic acids. When a lysosomal enzyme is missing, the material it normally breaks down builds up to harmful levels inside cells. The enzyme missing in Twitcher mice and Krabbé sufferers is responsible for getting rid of a particular kind of fat common in nerve cells.

“Lysosomal storage diseases are some of the most devastating diseases around,” Sands says. “Children appear normal at birth, but slowly deteriorate. Between 5 and 10 years old they are often deaf, blind, mentally retarded and confined to wheelchairs, and the disease just marches on. The infantile form of Krabbé disease is usually fatal by age 2.”

Sands and his colleagues previously showed they could increase enzyme levels in the brains of Twitcher mice to as much as 25 times normal levels by injecting directly into the brain viral vectors that held the gene for the missing enzyme.

This procedure is a form of gene therapy, a technique for correcting defective genes responsible for disease development. A similar injection procedure is currently being used in a Phase I clinical trial at Cornell University as a treatment for children with late infantile Batten disease, another lysosomal storage disease. Unfortunately, gene therapy alone didn't fully correct the disease in Twitcher mice in Sands' early experiments, and it only slightly increased their lifespan. On the other hand, decades of prior research with Twitcher mice had shown that under extreme treatment conditions bone marrow transplantation could extend the lives of these mice to an average of about 80 days (untreated Twitcher mice live to approximately 38 days).

“Lysosomal enzymes are located inside the cell, and at first glance, there's no reason to think that transplanting normal bone marrow would do anything at all,” Sands says. “But it turns out that lysosomal enzymes can escape from cells and get incorporated into other cells. When bone marrow is given, the marrow cells circulate to every organ and tissue in the body, sharing their lysosomal enzymes.”

However, this mechanism is relatively inefficient in the brain. Transplanted bone marrow cells have a hard time getting across the blood-brain barrier, and not much enzyme was delivered to brain cells by this treatment. So why was the treatment working for the Twitcher mice?

“We went back to the drawing board and asked what are each of these approaches doing?” Sands says.

The researchers could see that gene therapy was getting functional genes to brain cells, but they began to suspect that bone marrow transplantation was working in an entirely different way — it was reducing inflammation in the brain by a mechanism that hasn't yet been clearly defined.

“We hypothesized that if we could supply high levels of enzyme to the brain with gene therapy and at the same time decrease inflammation in the brain with bone marrow transplantation, we might have an effect,” Sands says. “However, we never imagined that the two approaches would synergize to the degree we saw.”

Inflammation in the brain in Krabbé disorder is an important factor in neural damage. Sands believes reducing inflammation allowed the gene therapy to be much more effective in the brain, even in areas far from the injection site that received low doses of the gene.

“Nothing we used here hasn't already been used for other disorders,” Sands says. “We are going to work on optimizing the therapy in the lab, and I think the combination approach could potentially be in the clinic in a few years.”