700. Targeted Delivery of a Recombinant Protein to Neurons and Astrocytes by Transport and Uptake Via the Low Density Lipoprotein (LDL) Receptor for Treatment of Neurodegenerative Disorders

Abstract

Inherited lysosomal storage disorders occur in approximately 1 in 8000 births worldwide resulting from deficient activity of a key enzyme involved in catalysis of glucosaminoglycans resulting in symptoms ranging from skeletal deformities to progressive neuronal degeneration. Enzyme replacement therapy has been used to treat the peripheral symptoms; however, no treatment effectively treats the neurological disorders associated with the diseases. Gaucher's disease is caused by a deficiency of the lysosomal enzyme glucocerebrosidase (GC). To test the potential of gene therapy for this disorder, the GC gene was fused at the C-terminus with the LDL receptor-binding domain of Apolipoprotein B or Apolipoprotein E, thus targeting the protein for uptake via the LDL receptor and transport to the lysosome. These constructs were tested in vitro for their ability to express and secrete enzymatically active GC enzyme. They were then inserted into the lentivirus vector and delivered intra-peritonealy to 4–6 week old Gaucher heterozygote knockout mice. Plasma samples taken from these mice showed increasing GC activity throughout the 14 days of the experiment. At 14 days post-delivery, liver, lung, spleen and whole brain was removed and homogenized to form lysates, which were then tested for GC enzyme activity. GC assays of the liver, lung and spleen showed increased activity in all mice that were injected with the lentivirus expressing the GC protein; however, only those mice that had received the virus expressing the GC – ApoE or ApoB fusion proteins showed elevated enzyme activity in the brain. Immunofluorescence staining of the liver of injected animals showed the recombinant enzyme was expressed from sinusoidal cells. Coronal sections of the CNS of those mice that had received the GC fusion construct showed co-staining of the recombinant enzyme with Calretinin, GFAP, and lectin indicating the uptake of the protein via neurons, astrocytes and endothelial cells. In addition, co-staining with LAMP1 indicated delivery of the enzyme to the lysosome. These results suggest not only a possible treatment for the neurological aspects of Gaucher's disease but also a general method for protein delivery to neurons and astrocytes of the CNS via hepatic gene delivery.