Recipient Organization
UNIV OF PENNSYLVANIA
(N/A)
PHILADELPHIA,PA 19104
Performing Department
School Of Veterinary Medicine
Non Technical Summary
The explanation of molecular mechanisms underlying the development of Best disease, and consequent striving for a therapeutic cure requires reliable laboratory models for investigation and testing of potential therapies. Induction of Vmd2 malfunction in the mouse has been done in order to create models that resemble the human phenotype. However, satisfactory models are lacking (Marmorstein, ARVO 2006). It has previously been shown that spontaneous mutations in canine genes expressed in the retina and/or RPE not only often resemble the human disease phenotype, but also provide excellent models to develop different therapeutic strategies. While the dog eye lacks the foveo-macular region typical of primates, the similarities in clinical and pathological features of cmr and Best disease are striking. Thus, the dog model will serve as a unique resource to understand the molecular mechanisms of disease and develop these potential therapies. We have described the first spontaneous animal model for human Best disease for which breeding colonies are currently established. Screening of breeds exhibiting compatible disease may further increase the number of mutations identified in the dog, and provide a number of different mutations; a situation similar to that observed in human disease. The ascertainment of phenotype to genotype correlations will shed light on the underlying molecular change, and allow follow up analysis in primary RPE cell culture. Functional studies related to these disorders will allow to identify key molecules for therapeutic intervention and establish appropriate carrier systems for retinal therapies.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Establish breeding colony. The cmr1 mutation identified in the Great Pyrenees and English mastiff breeds is already present in our breeding colony in heterozygous state. Carriers are currently bred to express this mutation homozygously. Additional cmr models will be included in the future as they become available. Clinical and histological evaluation. In collaboration with Drs. Acland, Aguirre, Beltran, and Jacobson, affected and carrier animals will undergo complete ophthalmologic assessment to establish a more detailed clinical description of cmr than has been available so far. Colony produced animals can be studied longitudinally, and will be available for more sensitive examinations such as electrooculogram (EOG), which is one of the few consistent means of diagnosis for Best disease, and optical coherence tomography (OCT). Additional molecular studies will allow to completely charaterize the disease. Assay spatial distribution of VMD2 expression in the eye. It has recently been suggested that the localized expression of the Best disease phenotype to the human macula might be a result of varying protein expression throughout the mature human retina (Ed Stone and associates, personal communication). While the clinical disease in the dog presents multifocal lesions, the individual lesions closely resemble the human phenotype. It is likely that the pattern in both species is a direct result of changes in the relative VMD2 expression level. A detailed comparative spatial map of VMD2 expression of the canine retina will be established to correlate the disease phenotype to the relative protein expression. Screen for additional mutation. Based on the phenotypical similarities, the VMD2 gene will be screened for either shared or additional mutations responsible for the disease. New mutations will be integrated into the breeding strategy to increase the number of mutation models. Expression studies to identify disease pathway. Functional studies are extremely difficult or impossible in intact tissues. Expression studies will be initiainitiated in primary dog RPE cell lines to produce a model system for gene expression and functional studies. These studies will provide the foundation to functional analysis of the bestrophin channel with additional methods such as patch clamping. Furthermore, the expression of the besbestrophin protein is critical for the identification of interacting molecules. Develop a carrier system for gene therapy. Based on the out outcomes of the planned experiments, a carrier system will be established to provide reliable bestrophin expression specifically targeted to the basal membrane of the RPE. The multifocal development of the disorder in the dog allows administration of the therapeutic agent to on or a few lesions of an affected dog eye, providing an immediate control system in the same eye. This will be the first step in the development of gene therapy specific to the cmr/BMD disorder.
Project Methods
Breeding colony. A breeding colony has been established at the RDS. Based on well established protocols, carrier and affected animals for the VMD2 mutations will be bred and held available for proposed studies. Clinical exam. Our laboratory has several veterinary ophthalmologicsts that have expertise in evaluating changes of the retina due to pathological processes of the retina or RPE (Drs. Acland, Aguirre, Beltran, and Komaromy). Furthermore, established collaboration with Dr. Jacobson at Scheie Eye Institute will allow for ERG, EOG, and OCT analysis. Pathology and Immunochemistry. Eyes will be fixed in 4% paraformaldehyde and embedded in OCT to provide tissue for cryosectioning. 10μm sections are transferred onto microscope slides for morphology and IMH staining. Pathology will be evaluated on HE, PAS, Sudan black, and other stains; protein detection is obtained through cross reaction with rabbit or mouse anti human polyclonal VMD2 antibody and secondary fluorescent labeled goat anti rabbit antibody. Spatial expression of VMD2. Retinas will be sampled for 4mm punches for the central and peripheral region. Midperipheral punches will be collected of the inferior, superior, nasal, and temporal areas. Expression of VMD2 will be determined on the RNA level (RT-PCR) and protein level (Western blot) in comparison to RPE65. Exon screening. Blood samples will be collected from animals affected with disease phenotypically similar to cmr for DNA extraction. PCR primers used for the exon screening are available for a whole gene screen. PCR products will be purified and sequenced. Sequence comparison is used to identify common or new sequence changes, and evaluate their potential consequence on gene function. Owners of animals with mutations in the VMD2 gene will be contacted to gain access to breeding material in order to introduce novel mutations into our breeding stock. Cell culture and gene expression. Primary RPE cell culture will be grown and harvested as described elsewhere and tested with the same antibody used for IMH for VMD2 expression. Expression will then be optimized by using selective substrates, modified cultures, and/or fetal cell lines to ensure sufficient VMD2 production. The VMD2 gene will be modified through mutagenesis and expression and distribution of the mutated gene will be compared to the normal allele. Our laboratory has extensive experience in tissue culture of dog and cat RPE. Functional analysis and interaction of bestrophin. Expression of the VMD2 gene in its natural and mutated form provides the foundation to determine function and interactive proteins. Functional analysis of the ion channel properties of the VMD2 gene will be determined in collaboration with Dr. Claire Mitchell (Department of Physiology, Univ. of Pennsylvania) using patch clamping. Protein interaction of bestrophin will be evaluated in a yeast-two hybrid system in collaboration with Dr. Ed Stone's group. Develop carrier system for gene therapy. Based on the results of above experiments, a suitable carrier system will be established to spspecifically restore VMD2 expression in affected cells following methods previously developed in our laboratory.