262. siRNA Delivery to the Central Nervous System Mediated by Shape-Controlled Nanoparticles

NEUROLOGICAL DISEASES residual ATP7A-mediated copper transport. Thus, morbidity and mortality remain high (75-80%) for this illness even when diagnosis and medical intervention is optimal. To address this dismal natural history, we are developing a path forward to viral gene therapy for Menkes disease. We previously illustrated that brain (lateral ventricle)-directed rAAV5-ATP7A + brain (lateral ventricle)-directed copper chloride could prevent early lethality (13d) in a naturally occurring animal model of this illness, the mottled-brindled mouse. However, subtle abnormalities in growth and neuromotor function persisted in long-term surviving mice. We now report far superior outcomes in this model, using rAAVrh10-ATP7A delivered to the cerebrospinal fluid in combination with a series of several peripheral subcutaneous injections of copper histidine. Copper histidine is an investigational new drug (IND#34,166, Orphan Product Designation #12-3663) that we employ in a current clinical trial for Menkes disease. The rAAVrh10 mouse rescue proved dose-dependent; low-dose (1.6e9 vg total) treatment extended life span only slightly (median survival 18d, n=5), whereas both intermediate (5e9 vg total) and high (1.6e10 vg total) dose treatment markedly enhanced survival (55% combined long-term survival, n=11). As of this writing, current ages of three high-dose survivors are 106d and 85d (2 mice) and, for the intermediate dose, 48d and 42d (2 mice). Importantly, in contrast to our previous rescue with rAAV5 (30% long-term survival), rAAVrh10 treated animals have shown no deficits in serial rotarod and wire hang tests to date compared to normal littermate controls. In addition, whereas rAAV5-ATP7A transduction appeared limited to choroid plexus epithelia, western blot analysis of whole brain from animals receiving high or intermediate rAAVrh10-ATP7A indicated robust global brain transgene expression. We conclude that CSF-directed rAAVrh10-ATP7A gene addition in combination with peripheral subcutaneous copper injections represents a highly promising approach to classic severe Menkes disease unresponsive to copper treatment alone. Together with effective newborn screening for this condition (also in development), viral gene therapy may help to transform the natural history of this difficult disease in the near future.

thus achieving selective and efficient modulation of these proteins. We then evaluated the effect of different shapes of nanoparticles on its delivery efficiency to the CNS in awake and freely moving non-transgenic mice via intraventricular infusion of up to 64 μg of siRNA over a 7-day period. Using confocal microscopic analysis, we confirmed that nanoparticles gained access to the cytoplasm of cells near the infusion site. Importantly, whereas we observed only modest knockdown of BACE1 levels by worm-like and spherical nanoparticles, in the forebrain, a more robust knockdown can be achieved by rod-shaped nanoparticles (Fig. 2). Interestingly, we also observed BACE1 knockdown in the hippocampi and the spinal cords of mice infused with nanoparticles. Our data suggests that rod-shaped nanoparticles serve as optimal vehicles for the delivery of siRNAs to the CNS. A continued exploration of the effect of nanoparticle shape on siRNA delivery will allow for the creation of a translatable platform towards developing a viable therapeutic approach.

262. siRNA Delivery to the Central Nervous System Mediated by Shape-Controlled Nanoparticles

Rishab Shyam,1 Jason Lee,1 Yong Ren,1 Philip Wong,1 Hai-Quan Mao.1 1 Johns Hopkins University, Baltimore, MD. Identification of parameters that mediate efficient delivery of siRNA via nanoparticles to the central nervous system (CNS) have important therapeutic implications for diseases such as Alzheimer’s Disease, a progressive degenerative brain disorder characterized pathologically by the accumulation of amyloid-β (Aβ) species and neurofibrillary tangles. β-secretase (BACE1), a key enzyme required for the generation of Aβ from the amyloid precursor protein (APP), is a well-validated therapeutic target amenable to the RNA interference therapeutic strategy. As a proof-of-principle, we demonstrate here the utility of shape-controlled nanoparticles for the delivery of siRNA to knockdown BACE1 and APP in in vitro and in vivo models as a potential therapeutic strategy to attenuate Aβ amyloidosis. We prepared siRNA micellar nanoparticles using linear polyethylenimine (LPEI) grafted with varying degrees of polyethylene glycol (PEG) as a condensing agent, and showed that the shape of siRNA-loaded nanoparticles can be controlled to form worm-like, rod-like, or spherical morphologies (Fig. 1). These particles were made at an N/P ratio of 5, to mitigate cytotoxic effects observed at higher N/P ratios, and stabilized with disulfide crosslinks. They did not exhibit aggregation, and were stable in water and under physiological conditions. We first showed that LPEI17k encapsulating siRNA was able to reduce the protein levels of BACE1 and APP, in an N2a cell culture model, by 63.3 ± 25.4% and 75.6 ± 10.2%, respectively, S100

263. Evaluation of Intrathecal rAAV Vectors in Canine Mucopolysaccharidosis VII

Brittany Gurda,1 Ping Wang,1 Peter Bell,2 Jessica Bagel,1 Tracey Sikora,1 Patricia O’Donnell,1 Yanqing Zhu,2 Hongwei Yu,2 Therese Ruane,1 Roberto Calcedo,2 Margret Casal,1 Charles Vite,1 Katherine Ponder,3 James Wilson,2 Mark Haskins.1 1 Pathobiology & Clinical Studies, University of Pennsyvania, Philadelphia, PA; 2Medicine, University of Pennsylvania, Philadelphia, PA; 3Internal Medicine, Washington University, St. Louis, MO. There are many diseases that involve the central nervous system (CNS) that could potentially be treated with gene therapy. In mice, some AAV vector serotypes have been able to enter the brain after Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy