P.328In vivo comparison of different AAV-microutrophin constructs

Abstract

Over-expression of the dystrophin-related gene utrophin is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. AAV-mediated delivery of utrophin to the mdx/utrn double-knockout mice showed improvements in dystrophic phenotype (Odom et al). The aim of this study was to create the microutrophin-coding AAV vectors for effective gene delivery to muscles, evaluate microutrophin gene expression from prepared constructions and assess the functional response after intravenous (iv) administration of microutrophin under constitutive (CMV) and muscle-specific (SPc5-12) promoters in comparison with microdystrophin gene delivery to adult mdx mice. Using bioinformatic approaches we developed constructs, encoding human microutrophin, and performed codon-optimization for effective expression in muscle tissues. Obtained codon-optimized human microutrophin sequence was cloned in AAV vector under muscle-specific SPc5-12 promoter. Proteins expression was confirmed using IHC and WB analyses. To assess muscle function in mice we used hanging wire test and force deficit measurement after lengthening contraction of hindlimb muscle. Results: Five weeks after iv administration in adult mice three types of microutrophin were detected in skeletal and heart muscles. The maximum hanging time was 3-fold increased after iv administration of human codon-optimized microutrophin and 2,5-fold increased after administration of murine microutrophin. Increase in hanging time of mice injected with microutrophin under SPc512 promoter was not significant. Differences in force deficit after lengthening contraction and creatine kinase level were not detected. Iv administration of microdystrophin under CMV promoter led to more significant increase in hanging time and reduction in CK level in with group injected with microutrophin, but because of lower immune toxicity of microutrophin, delivery of this gene is still promising.