Functional Substitution by TAT-Utrophin in Dystrophin-Deficient Mice

Background: The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or Delta R4-21 micro'' utrophin (mu Utr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice. Methods and Findings: Recombinant TAT-Utr and TAT-mu Utr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-mu Utr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290 +/- 920 U versus 5,950 +/- 61,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54% +/- 65% versus 37% +/- 64% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72% +/- 65% versus 40% +/- 68% drop; PBS versus TAT), and increased specific force production (9.7 +/- 1.1 N/cm(2) versus 12.8 +/- 60.9 N/cm(2); PBS versus TAT). Conclusions: These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin.