Diseases of the nervous system are an important and increasing challenge to America's health care system. Treatment of these diseases from Alzheimer's disease to Zn toxicity require novel and non- traditional approaches because of the relative inability of small and large therapeutic molecules to penetrate the brain capillary endothelium, which makes up the blood-brain barrier (BBB). The long- term goal of this proposed research is to create a novel gene delivery system for targeting brain neuropathies and neurological disorders such as Glut-1 deficiency syndrome (GLUT-1 DS). The integral membrane protein Glut-1 is the predominant glucose transporter at the BBB and is expressed at high levels in brain endothelial cells. Mutations in the human GLUT-1 gene cause GLUT-1 DS and leads to defective glucose transport resulting in hypoglycorrhachia with associated developmental encephalopathy. GLUT-1 DS is inherited in an autosomal-dominant pattern and a varied spectrum of heterozygous mutations in the GLUT-1 gene can result in haploinsufficiency. To date, there is no effective and enduring therapy for this orphan disease. The goal of the experiments outlined in this proposal is to populate the brain vasculature with endothelial cells engineered to express the GLUT-1 gene. The proposal entails a stem cell-based therapeutic approach to engineer brain endothelial cells to express genomic copies of GLUT-1 using a novel, non-viral based mammalian artificial chromosome expression system (ACE) amenable to a broad-range of potential gene therapy applications. The eventual goal of this project is to ameliorate the GLUT-1 haploinsufficiency utilizing engineered endothelial progenitor cells (EPCs) containing ACE chromosomes expressing GLUT-1. A unique pre-clinical mouse model of GLUT-1 haploinsufficiency will be utilized in these studies. To accomplish the goals of this project we will complete the following three specific aims: 1) Rescue brain glucose transport in GLUT-1 deficient mice by incorporating wild type endothelial cells into the brain vasculature, 2) Generate ACE-platform engineered endothelial progenitor cells carrying variable numbers of a human GLUT1 PAC clones, 3) Rescue brain glucose transport in GLUT-1 deficient mice by incorporating ACE engineered EPCs into the brain vasculature. The proposed work is innovative because it capitalizes on a novel means of engineering adult-derived stem cells utilizing a mammalian artificial chromosome-based vector system. In addition, this project combines the expertise and promotes interactions across a number of disciplines including stem cell engineering, artificial chromosome production and blood-brain barrier biology. Public Health Relevance Statement A methodology designed to therapeutically alter brain blood vessel physiology would have profound clinical significance in the treatment of brain vasculature genetic disorders and in the regulation of drug entry into the brain. This proposal outlines experiments designed to create such a novel therapy by incorporating genetically engineered blood vessel progenitor cells into the brain vasculature under hypoxic conditions. ? ? ? ? ? ?
