Metachromatic Leukodystrophy

Bethanys Hope Leukodystrophy Research Laboratory

Bethanys Hope Leukodystrophy Research Laboratory is directed by Dr. Tony Rupar. Dr. Rupar is the Director of the Biochemical Genetics Laboratory of the London Health Sciences Centre and the Medical Genetics Program of Southwestern Ontario, Canada. Dr. Rupar is also chairman of the division of Clinical Biochemistry Department of Biochemistry. and the program director of the Molecular Genetics Program, Childrens Health Research Institute at University of Western Ontario, London, Ontario, Canada.

Current full time research staff in Bethanys Hope Leukodystrophy Research Laboratory includes Dr. Jiahui Li and Cathy Regan, MSc. Several students including most recently Geoffrey Siu, Tanya Guilland and Peter Polischuk have contributed to the research. Dr. John Barranger, scientific adviser to Bethany’ Hope Leukodystrophy Research Laboratory has had a distinguished carrier, both in academia and business, dedicated to the understanding and treatment of lysosomal storage diseases.

Laboratory Mission

Bethanys Hope Leukodystrophy Research Laboratory was established by Bethanys Hope Foundation to conduct research to find a treatment and cure for metachromatic leukodystrophy. The laboratory has focused on studying the disease process in the mouse model of MLD and developing viral – derived gene transfer vectors to treat MLD.

The laboratory also provides DNA mutation identification services for patients and families who otherwise do not have access to this information. The mutation identification service is provided in co-operation with the Biochemical Genetics Laboratory, a licensed diagnostic laboratory (www.qmpls.org/ ) in the Province of Ontario, Canada.

Highlights of Research in Bethanys Hope Leukodystrophy Research Laboratory

The Mouse Model of MLD

The MLD mouse developed in Dr. Gieselman’s laboratory has a subtle disease without the devastating leukodystrophy that is present in the human disease. Detailed histological studies of the cerebellum in the MLD mouse brain demonstrated a small increase in the number of apoptotic (dying) cells when compared to age matched control mice.

The reason for the relatively small difference between the MLD and control mice is because the MLD mice accumulate only slightly more sulfatide in their brain than control mice. We have recently developed an HPLC method for quantifying sulfatide in tissues including urine sediment. This data indicates that the MLD mouse accumulates significant amounts of sulfatide in its kidneys but not its brain.

However, when sulfatide is added to primary cultures of murine astrocytes in vitro it causes significant retraction of cell processes and cell death. This indicates that in MLD, sulfatide and not another metabolite is responsible for the damage to the nervous system.

Control

Treated with sulfatide

Gene transfer vectors for MLD

A focus of research in Bethanys Hope Leukodystrophy Research Laboratory has been to develop gene transfer vectors that are capable of delivering a correct ASA gene when the vectors are directly introduced into the brain of the MLD mouse.

Gene transfer vectors are developed from naturally occurring viruses. Viruses have the abilities to attach to cells and deliver their DNA into target cells.

Some of these vectors, based on the foamy virus, have been developed in collaboration with Dr. Myra McClure. The foamy virus is a retrovirus and integrates its DNA into the target cell’s DNA. The foamy virus has not been associated with causing any disease in humans. This makes them attractive as the basis of therapeutic gene transfer vectors.

A major focus of the laboratory has also been the construction of lentivirus derived gene transfer vectors. These vectors are also able to integrate the ASA gene into the target cells DNA.

Gene transfer vectors created from both the foamy virus and the lentivirus are able to correct MLD fibroblasts by expressing ASA.

ASA produced in human fibroblasts can be taken up fibroblasts from MLD patients and primary neural cultures from ASA KO mice. The ASA is enzymatically active

ASA assay using BODIPY sulfatide and TLC analysis. Lanes 2-4 are fibroblast homogenates assayed with BODIPY sulfatide. Lane 1 BODIPY sulfatide, lane 2 human MLD fibroblasts after HFV ASA transfection, lane 3 control fibroblasts, lane 4 MLD fibroblasts.

Lanes 5 and 6 are ASA assay of primary ASA KO neural cell cultures with BODIPY sulfatide. Lane 5 after HFV ASA transfection, lane 6 ASA KO.

Foamy virus derived gene transfer vectors remain present for more than six months when delivered to the MLD mouse brain. There is no sign of inflammation or other adverse effects.