BY JAMIE TALAN
An international team of scientists from Italy, France, and the United States have delivered gene-packed viral vectors to the liver and successfully engineered the tailored genes to make the missing or deficient protein that causes Pompe disease, a rare and potentially lethal glycogen storage disease caused by the lack of the enzyme acid-alpha glucosidase (GAA). So far, they have shown in mice and a handful of non-human primates that the protein is secreted into plasma, where it circulates and is taken up by vulnerable tissues in muscle, brain, and the spinal cord.
The findings, published in the November 29 issue of Science Translational Medicine, offer proof of concept that adeno-associated vector-mediated liver delivery can be used successfully to make and secrete an engineered version of the GAA enzyme. The scientists are now conducting more studies aimed at initiating a clinical trial in patients.
Pompe disease is a severe neuromuscular disorder caused by mutations in the gene that encodes for the GAA enzyme. Normally, the enzyme does its job in the lysosomes – the cellular housekeeping system. These molecular scavengers ingest glycogen, a sugar, and the enzyme converts glycogen to glucose, which is used to fuel muscles. Patients with Pompe can’t metabolize glycogen. Without the enzyme, glycogen accumulates in tissues, primarily in the heart and other muscles such as the diaphragm.
Fatal forms of the disease manifest in infancy and other forms can trigger symptoms in adolescence or adulthood. All the mutations (about 300 have been identified) are linked back to an enzyme deficiency.
A drug to replace the GAA enzyme replacement therapy — lysosomal glycogen-specific enzyme alglucosidase alfa (Lumizyme) — was approved by the Food and Drug Administration in 2006 but the treatment has several limitations; among them, it requires twice monthly infusions, it’s unable to deliver enzyme to the brain and spinal cord, and it’s immunogenic.
With these concerns in mind, Federico Mingozzi, PhD, Giuseppe Ronzitti, PhD, scientists at Genethon, Inserm, and Pierre and Marie Curie University in Paris, and their colleagues set about to design a gene therapy that could work in the liver to produce the missing enzyme.
Dr. Ronzitti explained that they have developed liver-targeted vectors for other diseases and believe that the approach to use the liver as a manufacturing site for the gene product could overcome some of the limitations of enzyme replacement therapy.
STUDY METHODS, FINDINGS
To conduct the research, the team developed transgenes that encode for GAA and could be efficiently expressed and secreted by liver cells. They then used an adeno-associated virus serotype 8 (AAV8) vector to target the liver and establish a steady supply of the GAA enzyme. They introduced a low vector and high vector dose of gene therapy in GAA knockout mice — an animal model of Pompe disease — and performed a series of tests to see whether it prevented the accumulation of glycogen in muscle and in the central nervous system. The knockout animals generally take six to ten months before severe symptoms develop. They delivered the therapy intravenously at the first sign of muscle weakness, around four months.
Dr. Ronzitti said that the AAV8 vectors, because of their high affinity for the liver, targeted hepatocytes and started manufacturing the missing protein. The enzyme was then secreted into the circulatory system and delivered to skeletal muscle and other organs. And unlike enzyme replacement therapy, the needed protein also made it into the brain and spinal cord.
The scientists reported that the enzyme reduced abnormal levels of glycogen, almost 100 percent in skeletal muscle and 50 percent in the central nervous system. The mice that received the gene therapy had no evidence of cardiac hypertrophy, muscle weakness, and respiratory problems compared to the knockout mice who did not receive the active treatment.
Pompe symptoms were averted and the animals lived longer. Normally, the knockout mice start dying at seven months. The mice that received gene therapy were still living at 14 months. The animals also did not mount an antibody response to the treatment.
The researchers also performed tests comparing the secretable transgene to a native form of GAA that was also delivered through viral vectors and found much higher levels of the enzyme in muscle. The enzyme levels peaked by the third month of treatment and remained stable for the rest of the study. Dr. Ronzitti said the treatment preserves muscle structure and normalizes autophagy, cell damage, and death.
The scientists also showed that delivering these transgenes to the liver induces a strong peripheral immune tolerance towards the transgene. This prevented an immune reaction against the foreign protein. Dr. Ronzitti said that this is critically important. One of the real worries with enzyme replacement therapy, he said, is that some patients develop antibody to the enzyme and this abnormal immune response makes it too risky to keep administering the therapy.
They also delivered the gene therapy to monkeys as a proof of concept as they move forward in designing a clinical trial in patients with Pompe disease. Again, there was strong evidence that the liver was secreting the enzyme. There was higher GAA activity in plasma and in peripheral tissues.
“These findings suggest that we may be able to develop a similar strategy for patients,” said Dr. Mingozzi. He said that they are conducting more studies to compare the gene therapy to enzyme replacement therapy.
The study was funded mostly by grants by Genethon, a nonprofit research institute focused on developing gene therapies for rare genetic diseases, and the French Multiple Dystrophy Association. Several of the authors, including Dr. Mingozzi and Dr. Ronzitti hold patents related to AAV-mediated liver gene transfer and/or Pompe disease.
“The key [to the success here] is that the scientists are targeting the liver,” said Juan M. Pascual, MD, PhD, director of the Complex and Undiagnosed Brain Diseases Program at The University of Texas Southwestern Medical Center at Dallas and professor of neurology and neurotherapeutics, physiology, and pediatrics. “Having the gene in the liver is enough to make the enzyme and have it circulate into muscle, brain and spinal cord. It is a very complete study. We think it is very possible that it will work in patients.”
“I think it is an encouraging step,” said Jeffery M. Vance, MD, PhD, director of the Center for Genomic Education & Outreach in the John P. Hussman Institute for Human Genomics and professor of neurology in the Dr. John T. Macdonald Foundation department of human genetics at the University of Miami Health System.
Dr. Vance said that there are several advantages of this experimental approach compared with enzyme replacement therapy. “They were able to get a relatively longstanding induction of the enzyme through the hepatic system with a much lower immune response,” he explained. “And it looks like it is getting into the central nervous system. It has the potential to be a one-time treatment.”
“This is good preclinical data,” said Priya Kishnani, MD, C.L. and Su Chen professor of pediatrics and medical director of the Alice and YT Chen Pediatric Genetics and Genomics Center and division chief of medical genetics at Duke University Medical Center. “There have been similar gene therapy approaches for Pompe disease and other conditions where the transgene was optimized for hepatic expression and the liver secretes the therapeutic protein. In this case, it is GAA, the enzyme missing in Pompe disease. What is important data in this paper is the ability to get enzyme into the central nervous system."
“Using a liver-specific promoter, as done by the authors on this paper has the advantage that it can induce immune tolerance and builds on previous reports that have shown the same," Dr. Kishnani said. "There is more of a risk for an immune response when genes are delivered directly to muscle. One of the biggest challenges with enzyme replacement therapy has been the development of antibodies against the therapeutic enzyme.”
The commentators disclosed no competing conflicts of interest.
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Puzzo F, Colella P, Biferi MG, et al. Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid a-glucosidase. Science Transl Med 2017; Epub 2017 Nov 29.