Choroid plexus-directed gene therapy for lysosomal storage diseases. The Section's U01 project in collaboration with John Wolfe, VMD, PhD (Penn) continues studies focused toward a first-in-human clinical trial of AAV gene therapy for the central nervous system in alpha-mannosidosis (AMD), a prototypical lysosomal storage disease (LSD) for which no suitable therapies currently exist. Our novel research strategy of targeting the choroid plexuses (CP) to treat the brain disease has narrowed the AAV therapeutic candidates to two specific serotypes (AAV1 and AAV6) based on testing a total of five in animal models. The optimal serotype and dose, once finalized, will undergo preclinical toxicological evaluation in concert with FDA guidance and be proposed as the IND for the human clinical trial, also included in the current application. An ongoing natural history study of AMD patients will continue to completion (40% enrollment to date). Several distinctive cerebrospinal fluid (CSF) biochemical and proteomic biomarkers identified from our preliminary data represent solid candidates for tracking the effects of CSF-directed viral gene therapy. We have also made substantial progress on identifying distinctive magnetic resonance spectroscopy (MRS) biomarkers in human AMD subjects that may be used to assess responses to therapy. AAV vectors injected into CSF target the CP, highly vascularized structures that project into the brain ventricles. The polarized CP epithelia (CPE) produce CSF, regulate the biochemical milieu of the brain, and secrete numerous proteins. We hypothesized that LSDs could benefit from a CP-targeted approach by secreting vector-encoded normal lysosomal enzyme (LE) into the CSF, which extends throughout the ventricular system to the subarachnoid space, from which molecules ultimately reach the entire brain. Secreted normal lysosomal enzymes (LE) are taken up and utilized metabolically by diseased neural cells. AAV-mediated gene therapy provides a long-term if not permanent source of normal LE since the vector is sustained as an episomal transgene in cells, and CPE cells have an extremely slow rate of turnover. Furthermore, we have found that CSF injection of AAV1 also delivers the normal gene to brain cells in the parenchyma, generating additional sources of therapeutic LE production. Despite the exciting progress attained in the first period of this award, additional factors need to be addressed in the continuation studies in order to develop a human clinical trial that has the greatest likelihood of being beneficial to patients. These include: dose optimization to achieve complete histological resolution in the whole brain; evaluation of the accuracy of MRS changes relative to histological correction; and determination as to whether the vector dose scales proportionally from cat to monkey brains (5-6 times larger) in order to develop an estimated dose required in humans. The aims of this U01 continuation proposal are to complete our pre-clinical studies, conduct additional studies requested by the FDA, obtain an IND, and begin a phase I clinical trial. The potential impact in the field of LSDs is high since, if the proposed aims are achieved, the largest current barriers to health for patients with AMD and other LSDs affecting the brain would be circumvented.