National Research Council of Italy: Project application

Title: Novel experimental therapies to treat blindness in Wolfram syndrome
Applicant: Dr. Vania Broccoli

San Raffaele Hospital/CNR-Institute of Neuroscience, Milan Italy

Eye gene therapy for restoring WFS1 gene function in Wolfram mice

AAV-based gene therapy is based on intra-vitreal injections of AAV vector expressing a therapeutic gene. This strategy is already in clinical use for treating Leber hereditary optic neuropathy (LHON) and clinical trials performed in multiple centres have reaches beneficial effects without any troublesome side-effects. Strong advantages of this approach areå the ease of the surgical intervention, the efficacy of the AAV infection and the durable expression of the therapeutic genes for many years if not forever. Thus, we are developing an AAV-based replacement gene therapy to express a functional copy of the Wfs1 in retinal tissue to eb tested in Wolfram mice. However, our recent results indicate that Wolframin is significantly more expressed in glial cells where it controls MCT1 protein levels and its inactivation leads to RGC death through a non-cell autonomous mechanism of energy deprivation. These results clearly implicate that restoring Wfs1 gene expression only in RGCs might not be sufficient to protect from progressive visual loss. Our previous results indicate that both astrocytes in the retina and oligodendrocytes in the optic nerve play a crucial role in supplying energetic molecules to RGCs and this function is affected when Wolframin is inactivate in these cells. Thus, the question is in which cells the reintroduction of Wfs1 will have the best therapeutic effects in promoting RGC survival and functions. To answer to this question and to establish the most efficient strategy of gene therapy, we have been generating 4 different AAV vectors where the WFS1 gene is under the control of different promoters that combined with a specific delivery route will allow the expression of the therapeutic WFS1 gene copy in either only RGCs, optic nerve oligodendrocytes, retinal astrocytes, both glial cell types or all these type together. We will produce in our lab the AAV viral particles for intra-vitreal injections in the Wolfram mice. Six groups of mice will be prepared (4 animals for each group) to be treated each group with a different AAV vector at 2 months of age. Then, treated mice will be subjected to the visual acuity test (Optodrum machine) every 2 months. After 10 months from the treatment, the eyes will be isolated and analyzed for retinal morphology, RGC numbers and optic nerve anatomy by electron microscopy. This analysis will define in which cell type is most relevant to express the WFS1 functional gene copy to obtain the best protection of visual functions in Wolfram mice.

Although we expect that the AAV supporting the expression of WFS1 in both RGC and glial cells will provide the most beneficial effects, it is possible that WFS1 expression only in retinal astrocytes will trigger significant improvements. In that case, this last option might be preferable given that it might be easier to transduce glial cells respect to RGCs in the retina with AAV particles. This study will determine the best AAV gene therapy method considering both the ease of the administration route and its beneficial effects on visual functions. These results will pave the way to the clinical exploitation of this approach in Wolfram patients for establishing the first neuroprotective approach to arrest RGC loss in the disease.

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