DESCRIPTION (provided by applicant): The proposed studies test the efficacy of an in vivo, therapeutic, silencing strategy for dysmyelinating leukodystrophies that result from production of abnormal proteolipid protein (PLP). Our approach resolves the practical problem of blood brain barrier access, which has been a major obstacle to silencing therapies for CNS diseases. The PLP1 gene codes for both PLP, the major intrinsic CNS myelin protein, as well as the less abundant DM20 protein. The gene is almost 100% conserved across mammalian species, and PLP1 mutations in humans result in a spectrum of relatively rare, X-linked disorders ranging in severity from extreme forms of Pelizaeus-Merzbacher Disease (PMD), a developmental disorder in which the CNS can be essentially devoid of myelin, to milder forms of an allelic condition, Spastic Paraplegia 2 (SPG2). In 60% of cases, PMD is due to gene duplications that result in overproduction of PLP/DM20. The most severe forms of PMD are associated with missense mutations in introns or exons of the PLP1 gene. Interestingly, complete gene deletion results in a milder neurologic impairment. The biological basis of PMD/SPG2 dysmyelination is still not completely understood. PMD patients experience early nystagmus, dystonia, and fail to meet developmental motor milestones. In severe cases, this progresses to hypotonia, difficulty with breathing and feeding, and death. There is no palliative therapy or cure. We reversed certain PLP-related cell defects in culture studies using gene silencing. Based on that work, we pioneered an in vivo PLP-silencing strategy using normal mice. A poly-arginated form of a rabies virus glycoprotein recognition epitope peptide was used to bind and deliver PLP-siRNA to the CNS after peripheral vascular injection. This non-infectious and non-pathogenic strategy enables abundant CNS entry and delivery to specific cells, accompanied by dramatic reductions in normal PLP in both brain and spinal cord. Proposed studies use bona fide murine models of two major mutations causing human PMD to test the efficacy of siRNA on clinically relevant outcomes. Aim 1 studies optimize siRNA treatment parameters in transgenic mice that over-express normal PLP, assessing effects on longevity, and using behavioral (rotarod, grip strength) and histological/biochemical outcome measures. In Aim 2, similar studies are conducted using siRNAs appropriate for jimpy mice, which have a naturally occurring point mutation that codes for a mutant PLP. These proof of concept studies are comprehensive in that they test a similar strategy in well-characterized models of two major forms of PMD. Based on proof of efficacy, we intend to conduct studies that develop PLP-siRNA therapeutics for patients with PMD/SPG2. Further, the strategy of silencing developed here should be applicable to other diseases involving dominant, negative mutations.

The CNS proteolipid protein (PLP) constitutes about half the total protein in mature myelin membranes. Mutations in the PLP1 gene are associated with a spectrum of rare, X-linked CNS disorders in humans, a subset of which (Pelizaeus-Merzbacher Disease~ PMD) are extremely disabling and invariably fatal. The most severe PMD cases are caused by dominant, negative mutations, either a duplication of the normal PLP gene, or point mutations. Based on solid preliminary data, we test the hypothesis that PLP siRNAs delivered to the CNS can increase longevity and decrease neuropathology in two animal models of PMD. siRNAs will be delivered peripherally, using a novel approach that permits penetration of the blood-brain-barrier.