Single and Double AAV Somatic Gene Amelioration of Mouse Models of LAMA2 Related Dystrophy

Rutgers Biomedical And Health Sciences, Newark NJ

Investigators

Abstract

ABSTRACT: Laminin-211 (Lm211) is an ~800 kDa glycoprotein of neuromuscular basement membranes (BMs) required for BM assembly, structure, and functions. Missense and truncating mutations of the LAMA2 gene cause a congenital muscular dystrophy and neuropathy (LAMA2-Related Dystrophy, MDC1A). The clinical spectrum ranges from mild (ambulatory) to severe (non-ambulatory), depending on the mutation, with corresponding mouse models covering the disease spectrum. The goal of this application is to develop BM structure-altering therapies in two mouse models. Our strategy employs non-replicating adeno-associated virus (AAV) delivery of genes coding for small laminin-binding linker proteins that restore missing Lm211 functions of defective or compensating laminin. The approach is built upon our experience studying BM assembly and structure-function relationships with engineering of novel interactive proteins. Aim I. The dy2J/dy2J mouse, a model for ambulatory dystrophy, bears a mutation within the 2LN domain that prevents polymerization. We found the disease phenotype and pathology are substantially ameliorated by AAV9 delivery of a gene coding for a laminin-binding protein (LNNdG2'). A. To understand dose-benefit relationships, we will compare mice treated at different doses with evaluation of linker and laminin DNA/protein levels, ambulation, strength, histology and seek to better understand molecular mechanisms responding to enablement of laminin polymerization, e.g. effects on apoptosis, myofiber proliferation, and inflammation. B. We will determine the degree of amelioration achievable at later AAV delivery ages. C. We plan to modify the WPRE enhancing sequence and poly A tail to increase expression. Aim II. Most LAMA2-RD patients have little or no Lm2 expression. They remain non-ambulatory with profound muscle weakness, joint-contractures, and life-threatening respiratory impairment accompanied by neuropathy and seizures. The Lama2-/- (dy3K/dy3K) mouse serves as a model. Here the Lm4 subunit is expressed in compensation, mostly as Lm411, and overall trimeric laminin expression is reduced. Preliminary data reveal that muscle and peripheral neuropathy are ameliorated by simultaneous AAV9 expression of two small genes, one coding for LNNdG2' to enable polymerization and the other coding for miniagrin (mag) to link the Lm411 coiled-coil domain to the alpha-dystroglycan receptor. We plan to: A Extend the preliminary analyses to include determination of survival, ambulation, grip-strength, linker/endogenous DNA and protein levels, histology, BM and receptor expression, apoptosis, and regeneration, distinguishing polymerization and receptor anchorage contributions; B Design and evaluate single laminin-binding proteins carrying both needed activities, first in vitro and then in dy3K mice, and C Evaluate mag and dual-function single linker constructs in a myotropic AAV to reduce liver expression/toxicity and dose. Amyelination will be prevented by low-dose AAV9-CBh-LNNdG2'.