Research Highlights Netherland's
Scientists Produce Human Molecular Genetics, January 1998 "Generalized glycogen storage and cardiomegaly in a knockout mouse model of Pompe disease" by A. J. J. Reuser/A. T. van der Ploeg, et al Enzyme Replacement Therapy (ERT).......... Therapy for Glycogen Storage Disease
Type II by AGNES BIJVOET, Ph.D. Agnes Bijvoet received her Ph.D., in June 1999, from Erasmus University Rotterdam, the Netherlands. Dr. Bijvoet worked in conjunction with Arnold J.J. Reuser, Ph.D., Erasmus University Rotterdam, and Ans T. van der Ploeg, M.D., Ph.D., Sophia Children’s Hospital Rotterdam, to develop a mouse model for Pompe’s disease. This animal model was used to test enzyme produced in transgenic rabbits. Following are excerpts from her thesis entitled, "Therapy for Glycogen Storage Disease Type II--Acid alpha-glucosidase production in milk and enzyme replacement therapy in a mouse model." "The aims of the experimental work described in this thesis were to investigate the pathogenesis of glycogen storage disease type II and to test the feasibility of enzyme replacement therapy using recombinant human acid alpha-glucosidase produced in the milk of transgenic mammals. A knockout mouse model of the disease was made and used as an experimental tool in these studies." The methodology is described and "documents the acid alpha-glucosidase deficiency in the homozygous knockout mice and illustrates the lysosomal glycogen storage in liver, heart and skeletal muscle." The thesis also documents the generation of transgenic mice (to produce) recombinant human acid alpha-glucosidase in their milk. "The aim of these studies was to investigate the feasibility of large-scale production of therapeutic lysosomal protein in farm animals.....The recombinant acid alpha-glucosidase was tested for its in vitro and in vivo effect." .....The experiments described..... demonstrate the degradation of lysosomal glycogen in heart, skeletal and smooth muscle and the reversal of tissue pathology." "Based on these promising results, the genomic construct was used for the industrial production of recombinant human acid alpha-glucosidase in the milk of transgenic rabbits. The potential therapeutic effect of this latter enzyme was demonstrated by treating GSD II knockout mice over a six months period." "The work reported in this thesis demonstrates the feasibility of recombinant human acid alpha- glucosidase production in the milk of transgenic rabbits and the potential value of this enzyme for treatment of patients with GSD II. It has laid a solid basis for the start of the phase II clinical trial of enzyme replacement therapy, which has begun at Sophia Children’s Hospital Rotterdam. "The work described in this thesis has contributed to the understanding of glycogen storage disease type II and will help to elucidate the molecular and pathological processes in patients with this disease. Furthermore, it has stimulated and steered the development of enzyme replacement therapy for this and other lysosomal diseases by the presentation of a new method for the large-scale production of lysosomal enzymes. The potential efficacy of enzyme replacement therapy for GSD II has been demonstrated in a mouse model of this disease. This animal model has additional value for dose finding, for fundamental studies on enzyme replacement therapy, and for testing of alternative therapeutic approaches in the future." The studies described in this thesis were performed in the Netherlands at the department of Medical Biotechnology of the Leiden University and the department of Clinical Genetics of the Erasmus University Rotterdam. The research was supported financially by the "Prinses Beatrix Fonds", the Sophia Foundation for Medical Research, the Association for Glycogen Storage Disease (UK), the Acid Maltase Deficiency Association (USA), and the Foundation of Clinical Genetics, Rotterdam. Publication of the thesis was financially supported by Genzyme BV and Pharming BV. Gene Therapy.......... Human Gene Therapy 9:1609-1616
(July 20, 1998) Adenovirus-Mediated
Transfer of Human Acid Maltase Gene S. Tsujino 1), N.
Kinoshita 1), T. Tashiro 1), K. Ikeda 1), ABSTRACT Acid maltase deficiency (AMD) causes a lysosomal glycogenosis inherited as an autosomal recessive trait. The infantile type of AMD (Pompe disease) leads to early death due to severe dysfunction of cardiac and respiratory muscles and no effective therapy is available. Replication-defective adenovirus vectors offer a promising tool for in vivo gene delivery and gene therapy. We constructed a recombinant adenovirus containing the human acid maltase (AM) cDNA downstream of the CAG promoter, composed of modified chicken beta-actin promoter and CMV IE enhancer (AxCANAM). Japanese quail with AMD was used for this study as an animal model for human AMD. When cultured fibroblasts from AMD quail were infected with AxCANAM, AM activity in the cells increased in proportion to the multiplicity of infection (MOI). When AxCANAM (4.5 x 10(8) PFU) was injected into unilateral superficial pectoral muscle of AMD quail, PAS staining showed that glycogenosomes disappeared and stainablility of acid phosphatase was reduced in the injected area as compared with the contralateral muscle of the same birds. Biochemically, AM activity increased and glycogen content decreased in the injected muscle. Western blot analysis showed that AMD quail muscle injected with AxCANAM expressed human AM protein processed to active forms. These results suggest that the human AM cDNA transferred by an adenovirus vector was sufficiently expressed, leading to a marked reduction of the glycogen accumulation in the skeletal muscle of AMD quail. Gene Therapy.......... Modified Adenovirus for GSD-II The following article by Laura Spinney appears in BioMednews Reports (6- 25-99,#57) Glycogen storage disease type II causes enlargement of certain muscles, particularly the heart and tongue, due to their inability to break down glycogen. A team of American scientists is developing a method of delivering the enzyme required for that breakdown to all the body's muscles with a single intravenous injection of adenovirus. They presented their findings at the Second Annual Meeting of the American Society of Gene Therapy in Washington, D.C., held June 9-13, 1999. Glycogen storage disease type II (GSD-II) is an autosomal recessive disorder in which the enzyme acid alpha-glucosidase (GAA), which normally clears glycogen from the muscles, is missing or reduced. The disease occurs in neonatal, juvenile, and adult forms with high mortality, and babies born with it usually die by the age of 18 months. In the lethal neonatal form, GSD-II first shows itself as a general weakness and susceptibility to colds and pneumonia. But the hallmark is a massively enlarged heart, and occasionally tongue, caused by glycogen buildup. There is currently no cure. GSD-II, along with Duchenne muscular dystrophy, is a perfect example of the kind of disease that would be amenable to virus-mediated gene therapy. Any potential treatment would have to reach all the muscles of the body, including the notoriously inaccessible cardiac muscle, to be effective. That means either multiple injections of the enzyme into different muscle types, or else some kind of systemic delivery. At Duke University Medical Center in Durham, North Carolina, Dr. Andrea Amalfitano and his colleagues have been testing both strategies. Clinical trials for the direct enzyme therapy got underway last week. But the approach that may hold out most promise in the long run involves systemic delivery of GAA via a modified adenovirus vector that Dr. Amalfitano's team has been studying in a mouse model of GSD-II. The idea is simple, and requires the organs of the affected individual to do most of the work. An adenovirus that has been rendered harmless by the removal of two genes (called E1 and E2b) that it needs to replicate, but that is still capable of infecting cells and transforming their DNA, is loaded with the gene that encodes for GAA and then injected into a vein. Once in the bloodstream, the virus infects cells, migrating naturally to the liver where the transgene is expressed and the enzyme product is secreted back into the blood. Thereafter it travels to all the muscles in the body. "We transform the liver into an enzyme factory," says Amalfitano. And because the adenovirus is so infectious, a single injection is capable of transducing all the cells of a particular organ, in this case the liver. Within twelve days of the injection, the researchers measured GAA activity in the livers of the treated mice and found that it had increased 100-fold. Levels of a precursor of the enzyme circulating in the blood had also risen. The muscles of the leg showed a 10-fold increase in GAA expression while in the diaphragm the increase was 100-fold. Even the heart muscle showed quadrupled GAA activity. But most importantly staining for glycogen revealed that it had been reduced to a normal, healthy level. "There's nothing out there that can deliver genetic information as efficiently as adenovirus," says Amalfitano. But he says that although the findings are promising, there is a lot more work to be done. For instance, his team found some evidence of an immune response to either the vector, the transgene, or both that could herald possible problems to explore the treatment's long term consequences, including the virus's ability to survive in vivo, the duration of the GAA enzyme's activity once it enters the muscle, and the speed with which glycogen builds up again after the end of the treatment. Gene Therapy.......... Duke Duke Scientists
Reverse a August 2, 1999 Using a modified virus to deliver a therapeutic gene, scientists at Duke University Medical Center have shown that, in mice, they can reverse the damage caused by an inherited muscle-wasting disease with a single injected dose. The study findings, which appear in the Aug. 3 issue of the Proceedings of the National Academy of Science (see abstract below), show for the first time that it appears possible to deliver a therapeutic gene product throughout all of the muscles of the body to reverse muscle wasting, a result that has implications for treating dozens of forms of muscular dystrophy. The researchers note, however, that to date they have only demonstrated a short-term reversal of symptoms in laboratory mice, and further experiments are needed to determine if the approach could become practical for use in people. The study is part of a large, collaborative effort at Duke to find an effective treatment for Pompe disease, a rare inherited disorder in which the body cannot process glycogen.... People born with Pompe disease have a defect in the enzyme...acid alpha-glucosidase (GAA), which normally processes glycogen and converts it to sugar. Several forms of Pompe’s affects more than 5,000 people in the US.........Duke pediatric geneticist Y.T. Chen has been simultaneously pursuing....... replacing the missing GAA enzyme and replacing the faulty gene. The first method uses cells grown in the laboratory that secrete a special form of GAA that, when injected intravenously, is easily taken up by muscle cells and processed...... Chen and his colleagues have developed a way to make the enzyme in large quantities and licensed that technology to Synpac (N.C.) Inc., a drug development company..... (part of) Synpac Pharmaceuticals Ltd. of Cambois, England. The company is funding an on-going clinical trial to test the enzyme therapy in up to 3 infants at Duke...... Chen collaborated with Dr. Andrea Amalfitano, a pediatric geneticist...and the two designed an experimental system to deliver the genetic information.....using a modified adenovirus....Amalfitano has developed a form of the virus that appears to be able to evade detection by the immune system.....The modified virus tends to normally infect liver cells since the liver filters all blood within the body..... (Human Gene Therapy, Feb. 1999). "The liver normally makes and secretes a large number of enzymes and we used that to our advantage in designing our gene delivery system," said Amalfitano .......When the researchers injected the virus containing the specially designed genetic information into a mouse that develops Pompe disease, the virus went to the liver, which then began making and secreting the special enzyme into the blood stream... The idea worked-the mice that received the modified GAA gene in their livers subsequently had reduced accumulation of glycogen in muscles throughout the body. "The heart and diaphragm muscles appeared to be especially responsive to the treatment," said Amalfitano. "This is significant because failure of the heart or respiratory muscles are the primary cause of death in many people with Pompe disease.....This is the first example of the simultaneous correction of multiple muscle groups after a single, simple, intravenous administration of a gene therapy vector," Amalfitano said,. "a hurdle that has always made the potential of gene therapy to treat muscle disease very difficult to envision." A.J. McVie-Wylie, H. Hu, and T.L. Dawson of Duke and N. Raben, P. Plotz of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH also contributed to the work. Gene Therapy.......... Applied Biological Sciences Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid alpha glucosidase............. National Academy of Sciences Vol. 96, Issue 16, 8861-8866, 08/03/99 http://www.pnas.org/cgi/content/abstract/96/16/8861 Abstract Enzyme Replacement Therapy (ERT),
Gene Therapy New York University Medical
Center
There are five main areas of research ongoing for glycogen storage disease type II or Acid Maltase Deficiency:
Research ParticipationTitle of Research Study: Project Director: Frank Martiniuk, Ph.D., is undertaking a study to establishing cell lines in order to determine the mutations present at the acid maltase gene. He is asking for donations of blood and urine from patients of AMD. If parents of patients want to donate samples, that would be helpful, also. The purpose of the study is to understand the genetic reason for Acid Maltase Deficiency. The potential benefits to you or
to others: If you are interested in volunteering for this study, contact AMDA for consent forms and more information. Diet, Exercise,
& Ephedrine Study
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Office of Communications
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Media contact: John Cramer (410) 955-1534
E-mail: jcramer@welchlink.welch.jhu.edu
October 29, 1996
SCIENTISTS MAKE
PROGRESS IN
GENE THERAPY FOR HEART DISEASE
Johns Hopkin's University scientists have successfully used a virus to supply a missing gene and its enzyme product to muscle cells in animals and humans for an extended period. The achievement could have implications in the treatment of an inherited fatal heart disease in children called Pompe's disease.
The two Hopkins-led studies are the first to demonstrate long-term production of the normal enzyme without toxicity and therefore the possibility of single treatment by this method. Results will be presented at 9:30 a.m. and 10 a.m., Nov. 11 at the American Heart Association's 69th annual Scientific Session in New Orleans.
Pompe's disease, which causes cardiomyopathy (an enlargement and weakening of heart muscle) in infants, is caused by an inherited metabolic disorder. Heart muscle dysfunction results from a missing or defective enzyme (proteins that stimulate chemical reactions in living tissue) caused by a gene mutation.
Hopkins scientists isolated the defective gene in Pompe's disease and, with Avigen, Inc., researchers, used a harmless virus as a vehicle to carry the normal gene's DNA into mice and into human muscle cells in laboratory dishes. The cells came from children who had died of Pompe's disease.
Once inserted into the abnormal cells, the healthy gene material replaced the missing enzyme and restored normal enzyme function over a prolonged period.
"In principle, we know a single gene disorder could be treated by replacing a defective gene," says Paul D. Kessler, M.D., senior author and an assistant professor of medicine at Hopkins. "But the problem has always been a lack of good vectors for getting the normal gene into cells long term."
The Hopkins group used an adeno-associated virus to deliver the gene for the enzyme acid alpha-glucosidase. Healthy DNA was injected into muscles in the mice and began producing the enzyme two weeks later and continued to produce it for at least three months. Further animal studies are planned.
"These results demonstrate that adeno-associated viral vectors can effectively transfer genes into muscles in animals and lead to sustained expression of a therapeutic protein," says Barry J. Byrne, M.D., Ph.D., lead author and an assistant professor of pediatric cardiology at Hopkins.
Gene therapy involves treating diseases by delivering genes into cells to restore normal cells actions or to stop abnormal cell actions. The genes are delivered by vectors, harmless viruses that enter the target cell, delivering the gene with them.
The studies were supported by the Peter Belfer Laboratories for Myocardial Research at Hopkins, the American Heart Association's Delaware branch, the March of Dimes Birth Defects Foundation and the W.W. Smith Foundation.
© AMDA, Acid Maltase Deficiency Association, Inc. 1997
