A Modified Neprilysin for Gene Therapy

Increasing the clearance of amyloid 13 peptides (Af3) is receiving considerable attention as a method of treatment for Alzheimer's disease (AD). Immunological approaches, A13 binding compounds, and A13 degrading peptidases are all being investigated for this purpose. Our laboratory has focused on the use of the peptidase neprilysin (INEP) as a way to increase Af3 clearance. Studies from this and other laboratories indicate that this can be accomplished by expressing NEP in either the brain or the periphery (Mar et al, 2003, Marr et a!, 2004, Leissring et al., 2003a, Eckman and Eckman, 2005, Liu et a!., 2007). A limitation in the use of NEP for treating Alzheimer's disease is the lack of specificity of the enzyme. NEP cleaves a number of physiological peptides including the enkephalins, substance P, bradykinin, etc. In order to overcome this lack of specificity we propose to develop an NEP variant more specific for A13 that can be used as a therapeutic treatment for AD. The following specific aims are proposed to accomplish this goal: Specific Aim 1: To use semi-rational mutagenesis of known substrate binding residues to select a modified NEP that more efficiently and selectively binds Aft Specific Aim 2: To use semi-rational mutagenesis of known active site residues of NEP to select a variant that more rapidly degrades A13.