Advancing CNS drug delivery via epigenetic modulation

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Abstract Lysosomal storage disorders (LSDs) are a group of inherited diseases characterized by dysfunctions in lysosomes, with cumulative frequency of 1 in 7000 live births. Over 2/3 of LSD patients present an involvement of the central nerve system (CNS) with a broad spectrum of severity (nLSD), which makes LSDs the most common cause of pediatric neuronopathic diseases. Allogeneic hematopoietic stem cell transplantation (HSCT) or enzyme replacement therapy (ERT) are main treatment options for LSDs. However, they are largely unsuccessful in reversing neurological complications due to the poor penetration of the enzymes into the CNS, a major obstacle in treating nLSD. The impact of the proposed study is driven by the unmet medical need for efficient treatment of inherited nLSDs AND the major limitation of enzyme-delivery into the CNS. The cation-independent mannose-6-phosphate receptor (M6PR) plays a critical role in lysosomal enzyme trafficking and intercellular transfer for the majority of lysosomal enzymes, which is essential for metabolic cross- correction in treating LSDs. Developmental decline of M6PR on blood-brain-barrier (BBB) during early postnatal period in mouse and human is attributable to the lack of CNS enzyme delivery into adult brain. Using a dual luciferase reporter system with site-mutagenesis, we have recently identified microRNA-143 (miR143) as an epigenetic modulator to reduce M6PR protein levels on brain microvessels (BrMV). Using a mouse model of Hurler syndrome (severe mucopolysaccharidosis type I, MPS I), which is caused by the deficiency of α-L- iduronidase (IDUA), we demonstrated functional rescue of M6PR-mediated IDUA transfer in the brain of double- knockout (MPS/miR-143KO) mice with long-term CNS therapeutic benefits, as well as in human vascular endothelial cells by sequestration of miR-143 with miR-143-sponge sequences. The data provide strong scientific premise for the development of a novel approach that would selectively “open” BBB to systemic enzymes provided by any current treatment options or future enzyme/gene/cell therapies for synergistic CNS benefits in many nLSDs. In this proposal, we aim to develop an adeno-associated viral vector (AAV)-based translatable platform to “restore” M6PR pathway on mature BBB for advanced delivery of therapeutic enzymes into the CNS with 3 aims, including developing optimal artificial miR143 inhibitor (143in) and expression cassette(s) for robust and targeted reduction of miR143 on brain endothelia cells (Aim 1), in vivo examination of “on-target” and “off-target” expression and effects in mice with AAV/143in delivery (Aim 2), as well as preclinical evaluation of BrMV-targeted AAV/143in in correcting CNS abnormalities in MPS I mice by enzyme therapy derived from genetically modified erythroid/megakaryocytic lineages (Aim 3). The studies will provide a proof- of concept for a new in vivo miRNA-inhibitor mediated, brain-targeted approach that could be applicable for many other nLSDs involving M6PR pathway AND neurological diseases benefiting from advanced CNS delivery of therapeutics via adapting M6PR-mediated transport pathway by modification with M6P residues or IGF2-tag.