Successful Compensation for Dystrophin Deficiency by a Helper-dependent Adenovirus Expressing Full-length Utrophin

Abstract

Helper-dependent adenovirus vector (AdV)-mediated full-length dystrophin expression leads to significant mitigation of the dystrophic phenotype of the mdx mouse. However, dystrophin, as a neoantigen, elicits antibody formation. As an alternative approach, we evaluated gene transfer of full-length murine utrophin, a functional homologue of dystrophin that is normally present only at the neuromuscular junction. A single injection in the tibialis anterior (TA) muscle of the helper-dependent adenovirus vector encoding utrophin provided very good transduction, with 58% of fibers demonstrating sarcolemmal utrophin expression in the neonates, and 35% utrophin-positive (Utr(+)) fibers in adults. The presence of utrophin prevented extensive necrosis in the neonates, halted further necrosis in the adults, and led to restoration of sarcolemmal expression of dystrophin-associated proteins up to 1 year after injection. Marked physiological improvement was observed in both neonates and adults. Neither increased humoral responses nor cellular immune responses were evident. However, there was a time-related decline of the initial high utrophin expression. Although viral DNA persisted in animals that were injected in the neonatal stage, viral DNA levels decreased in muscles of adult mice. These results demonstrate that although utrophin gene transfer leads to amelioration of the dystrophic phenotype, the effects are not sustained upon loss of utrophin expression.

Progress and prospects: gene therapy for genetic diseases with helper-dependent adenoviral vectors

“…For example, intravenous injection of HDAd expressing the canine coagulation factor IX in hemophilia B dogs resulted in sustained phenotypic improvement of the bleeding diathesis for the duration of the experiment of over 1 year after vector administration. 3 These, as well as other recent studies, [4][5][6][7][8][9][10][11][12][13] provide compelling evidence for using HDAd to treat genetic disorders.…”

“…To overcome this problem, several strategies have been successfully utilized including co-delivery of immunomodulatory molecules such as CTL4Aig, 10,50 and use of an alternative transgene such as utrophin, a functional homolog of dystrophin. 51 HDAds have also been explored for in utero gene therapy of DMD. The immaturity of the immune system of the fetus coupled with the survival advantage of dystrophin-expressing HDAd-mediated gene therapy N Brunetti-Pierri and P Ng muscle fibers over dystrophin-deficient muscle fibers may offer a significant advantage for this promising therapeutic strategy.…”

Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin

“…Except for some spatial and/or temporal differences in the expression pattern and a minor difference in the rod-domain length, utrophin seems sufficient to correct virtually all the cellular defects caused by dystrophin deficiency (Tinsley et al, 1998). Encouraging results from gene therapy, protein therapy and pharmacological interventions have further substantiated the therapeutic promise of utrophin (Cerletti et al, 2003;Deol et al, 2007;Khurana and Davies, 2003;Miura and Jasmin, 2006;Odom et al, 2008;Sonnemann et al, 2009).…”

Targeting Artificial Transcription Factors to the Utrophin A Promoter

“…Some sections were immunostained with both utrophin and EGFP antibody. Additional sections were also stained using antibody against ␤-dystroglycan (NCL-43DAG, Novocastra), and ␣-sarcoglycan (NCL-50DAG, Novocastra), as described previously (9,10,32). Muscle sections were also counterstained with hematoxylin-eosin to allow determination of the number of central nuclei.…”

“…Muscle sections were also counterstained with hematoxylin-eosin to allow determination of the number of central nuclei. The number of utrophin-positive myofibers on the entire muscle cross-section was counted as previously described (10,32) and expressed as the percentage of the entire muscle fiber number.…”