Dr. Leone is the Principal Investigator (PI) on a human gene therapy protocol for Canavan Disease (CD) patients at UMDNJ - SOM, and a co-PI of the neuroimaging portion of the same clinical protocol at the Children Hospital of Philadelphia, and the PI of the N.I.H./ Recombinant DNA Advisory Committee protocol. Dr. Leone is also the FDA Sponsor-Investigator of the AAV-ASPA (IND#9119) and is responsible for protocols and Standard Operating Procedures (SOP) on the production of the clinical grade sector and for the training of Clinical Research Investigators and Clinical Scientists. She is currently the principal investigator of the first NINDS-sponsored clinical gene therapy Phase I study for Gene Therapy of Canavan Disease using AAV vectors. In addition to conducting a clinical research study on CD, Dr. Leone's laboratory conducts basic research projects that directly study CD, a disorder that has been the focus of her research for nearly a decade.

Dr. Leone's laboratory is studying and characterizing the Canavan rat model and the "tremor rat" model. These studies run in parallel with the characterization of oligodendrocytes, cells responsible for white matter development in the human brain. Furthermore Dr. Leone's laboratory is also studying the effect of common and novel CD genetic mutations and the development and characterizing of altered genes and pathways in CD. Dr. Leone is also conducting a research project designed to generate pre-clinical data for a cell-based therapy for CD and other white matter disorders. The successful completion of these studies would provide invaluable safety and efficacy data that would be required for the submission of a FDA protocol for a phase I study. Furthermore this data would be available for cross-reference to other clinical applications, and would be of significant relevance to future stem cell applications in Canavan Disease.

In Dr. Popko and Dr. Traka studies, an authentic mouse model of Canavan Disease (CD) has been identified and characterized. These mice have an ASPA gene mutation; similar to those that occur in humans. Moreover, the mutant animals display many of the phenotypic characteristics of CD, including clinical and histological abnormalities. Thus, these animals represent an extremely important tool to better understanding the mechanism of disease progression. In addition, these mice should serve as an ideal model to test therapeutic strategies for CD. The two have donated these mice to the mouse distributor, The Jackson Laboratory, making that the model easily available to any researcher interested in this disorder.

One note of encouragement from their analysis of the CD mutant mice is that throughout the course of the disease there appears to be an endogenous effort to repair and remyelinate the nervous system. Once therapeutic strategies are developed, which will counteract the effect of the ASPA mutation, it would seem likely that the nervous system will have the potential to repair itself even in individuals with advanced myelin abnormalities.

The ASPA mutation that we have characterized arose in a highly inbred strain of mice. Dr. Popko and Dr. Traka noticed that when they crossed these mice to other inbred strains of mice that disease severity was altered, indicating that the effects of the ASPA mutation are “modified” by other genes. More specifically, they have identified a recessive genetic effect that increases the severity of CD in their mice. Using a similar approach, which allowed the doctors to identify the original ASPA mutation, they have been mapping this CD modifier locus and have narrowed its location to a relatively small region of the mouse genome. The identification of this gene, and the elucidation of the mechanism by which it modifies disease severity, should provide insight into CD disease pathogenesis. The presence of such modifier loci helps explain the variability of disease severity seen in human patients with CD.

Following his postdoctoral work in chemistry at the University of Chicago and Mt. Sinai, New York, Dr. Ledeen began his studies in neurobiology as a junior faculty member at Albert Einstein College of Medicine, New York. He became Professor of Biochemistry in Neurology in 1976, with research interests in (a) the biochemistry of myelin and (b) the functional role of gangliosides in the nervous system. These remained the themes of his research when he transferred to New Jersey Medical School in 1991. Since beginning his research, Dr. Leddem has had continuous research funding from the NIH, grants from the National Multiple Sclerosis Society and various other foundations. These studies have resulted in approximately 150 publications; most of them refereed contributions to neurobiological journals.

Current research efforts of Dr. Ledeen's group in the area of Canavan Disease (CD) are focused on determining the function and cellular locus of aspartoacylase (ASPA), the deficient mutated enzyme in CD. His studies are providing conclusive evidence that this enzyme in normal brain has bimodal loci in the cytoplasm of oligodendrocytes and the myelin sheath. This will be useful information in designing strategies to replace the missing enzyme in patients with CD. His studies suggests that ASPA has an important role in the production and maintenance of the myelin sheath. Biochemical analysis of myelin from the tremor rat, an animal with mutated (inactive) ASPA, has revealed specific depletion of certain myelin lipids. These studies, when completed, are expected to explain the reduced level of myelin in CD children and the inability of the remaining myelin to function normally within the central nervous system. They should also provide a chemical yardstick for quantifying recovery in CD children following efforts to replace the defective ASPA enzyme and deficient myelin components.

The long-term goals of this research program are to understand the functional roles of NAA and NAAG in the nervous system, with an immediate focus on understanding the pathogenesis of Canavan Disease (CD) and developing safe and effective treatment strategies.

Three primary goals are being pursued at the present time in their laboratory. First, a multidisciplinary effort to pursue preclinical translational studies aimed at developing acetate supplementation as a therapeutic strategy for CD. Simultaneously, studies will focus on understanding the pathogenic mechanisms of CD that are unrelated to acetate deficiency.

Second, efforts to characterize the molecular mechanisms of NAA biosynthesis with the goal of cloning the gene or genes involved are progressing rapidly. This approach will address the question of excessive NAA as a potential factor in CD pathogenesis. Furthermore, this approach is expected to help the program to ascertain the role of NAA in neuronal energy metabolism and helps explain the decreases in NAA detected by magnetic resonance spectroscopy in numerous brain diseases.

Finally, a goal to understand the mechanisms of NAAG biosynthesis from NAA. NAAG is a critical neuroprotective peptide found in the brain, which is related to NAA. A cell culture system based on SH-SY5Y human neuroblastoma cells is providing valuable leads in determining the mechanisms of NAAG biosynthesis.

The objective of the NYU Canavan Disease Research Program is to understand the pathophysiology of Canavan Disease-the chain of events causing brain damage. The group proposes that glutamic acid toxicity contributes to the brain injury via NAAG, one of the products, which accumulates along with NAA in Canavan Disease. Since NAAG is believed to contribute to the pool of brain glutamic acid, its accumulation in Canavan Disease could create an excess of this amino acid. Under normal circumstances, glutamic acid serves as an excitatory neurotransmitter in the central nervous system but in excess it is known to cause neurodegeneration. In similar fashion, this mechanism of tissue injury could also contribute to the brain pathology in Canavan Disease.

The research group has teamed up with a laboratory in Italy that uses capillary zone electrophoresis to study NAAG levels in blood and urine of Canavan patients with different degrees of clinical involvement. They plan to refine their test for aspartoacylase, the enzyme deficient in Canavan Disease, by using NAAG (rather than NAA) as a substrate for the reaction so that they can measure more exactly the amount of residual aspartoacylase present in Canavan patients. Further, they are setting up an antibody-based analyze for residual aspartoacylase protein in cell lines from patients with Canavan Disease, by the so-called Western blot method. This will provide them with another indication of the severity of different types of mutations in the aspartoacylase gene.

The researchers continue to examine sequence data for the aspartoacylase gene in three Canavan Disease cases for which they have not yet been able to find one of their two disease alleles. This information is important for genotype-phenotype correlation and for genetic counseling and prenatal diagnosis in these families. Three publications describing our mutation analyses of the aspartoacylase gene are already in print.

In summary, the groups work aims to improve our knowledge of the causes of brain pathology in Canavan Disease and thereby open up new opportunities to intervene for the benefit of patients.