There are over 40 lysosomal storage disorders, and most of these diseases affect adversely the central nervous system (CNS). The mainstay of treatment of the lysosomal storage disorders is enzyme replacement therapy (ERT). However, ERT is not effective for the brain, because the enzymes do not cross the brain capillary wall, which forms the blood-brain barrier (BBB) in vivo. Without treatment of the CNS, the young patients are destined to progressive neurodegeneration and death, even with ERT. The limiting factor in the future treatment of these diseases is the transport of the enzyme across the BBB. Bypass of the BBB with direct injection into the brain is not effective, because only a small part of the brain is treated with a trans-cranial delivery system, and nearly all cells of the brain are affected in lysosomal storage disorders. Conversely, virtually all cells of the brain can be treated with a trans-vascular delivery system that enables the enzyme to cross the BBB following intravenous administration. A new approach to the BBB delivery of large molecules such as enzymes is the molecular Trojan horse technology. A bi-functional fusion protein is produced with genetic engineering, wherein the missing recombinant enzyme is fused to a BBB molecular Trojan horse. The latter is a genetically engineered protein that is able to cross the human BBB by receptor-mediated transcytosis on endogenous BBB peptide transport systems. Pre-clinical studies show that a large enzyme with a molecular weight >100,000 Daltons, can be delivered to brain via transport across the BBB, following attachment to a BBB receptor-specific Trojan horse. The present work will produce a novel fusion gene encoding a lysosomal enzyme and a genetically engineered molecular Trojan horse, which will allow the production of the corresponding fusion protein, AGT-180. The fusion gene will be incorporated in a eukaryotic expression vector followed by transfection of COS cells. The bi-functionality of the secreted fusion protein will be demonstrated by ELISA, BBB receptor binding assays, and enzyme activity assays. These phase I studies will enable future phase II work and the development of a permanently transfected CHO cell line producing the recombinant AGT-180 fusion protein. Fusion proteins comprised of BBB targeting antibodies and recombinant enzymes could be therapeutic in the treatment of the brain in human lysosomal storage disorders. ? ?
