Novel mini-dystrophin gene dual adeno-associated virus vectors restore neuronal nitric oxide synthase expression at the sarcolemma

Abstract

Six- to 8-kb mini-dystrophin genes are promising candidates for Duchenne muscular dystrophy (DMD) gene therapy. Several dual adeno-associated virus (AAV) mini-dystrophin vectors have been tested in dystrophin-deficient mice. Despite the encouraging preclinical results, none of the existing dual AAV vectors can restore sarcolemmal neuronal nitric oxide synthase (nNOS) expression. Localization of nNOS to the sarcolemma may greatly improve the therapeutic outcome in DMD (Lai, Y., Thomas, G.D., Yue, Y., et al. [2009]. J. Clin. Invest. 119, 624-635). In this study, we developed a series of dual AAV expression vectors to express a synthetic minigene that carries the nNOS localization domain. To help validate dual vector reconstitution, we also included a FLAG tag and a GFP reporter at different ends of the minigene. These dual AAV vectors were packaged in Y445F tyrosine mutant AAV-6 and tested in dystrophin-null mdx4cv mice by direct muscle injection. All dual vectors expressed GFP/FLAG-tagged mini-dystrophin and restored sarcolemmal nNOS. However, the reconstitution efficiency was significantly different among different sets. The dual vector set YZ27/YZ22 yielded the highest transduction efficiency (∼90%). Further development of this set dual vector may lead to more effective DMD gene therapy.

FIG. 1.

Schematic outline of the mini-dystrophin gene structure and dual adeno-associated virus (AAV) constructs. (A) The ΔR2–15/ΔR18–19 mini-dystrophin gene contains the N-terminal, cysteine-rich (CR), and C-terminal domains and an abbreviated middle rod domain. R16/17 is the neuronal nitric oxide synthase (nNOS) anchoring domain. The dotted line marks the location of the deletion. The structure of the deleted regions is presented above the minigene. (B) The cartoon presentation of the dual vector sets used to express the ΔR2–15/ΔR18–19 minigene. The FLAG tag and GFP gene were fused in-frame with the minigene in dual vectors. The viral genome is bracketed by AAV inverted terminal repeats (depicted as the hairpin-like structure). CMV, the cytomegalovirus promoter; pA, the polyadenylation signal.

FIG. 2.

Comparative evaluation of four different sets of the mini-dystrophin dual AAV vectors in mdx4cv mice. (A) Representative Western blot results. Open arrowhead, full-length dystrophin; filled arrowhead, ΔR2–15/ΔR18–19 mini-dystrophin. (B) Representative fluorescence photomicrographs revealing different levels of GFP expression in mdx4cv anterior tibialis muscles infected by different sets of dual AAV minigene vectors. Scale bar applies to all images. (C) Quantification of GFP-positive cells in muscle cross-sections. *Significantly different from other groups. β-actin was used as the loading control.

FIG. 3.

Dual AAV vectors restored sarcolemmal nNOS expression. Left and middle panels, representative serial muscle cross-section photomicrographs from dual AAV vector set IV–infected mdx4cv muscle. Top left panel, GFP fluorescence image; top middle panel, indirect immunofluorescence staining with a human dystrophin-specific antibody; bottom left panel, immunofluorescence staining with a polyclonal nNOS antibody; bottom middle, a histochemistry image of NOS activity staining. Right panels, representative NOS activity staining in normal BL6 and dystrophin-null mdx4cv muscles. Scale bar applies to all images. Asterisk marks the same myofiber in serial sections. Black asterisk, a myofiber transduced by the dual AAV vector; gray asterisk, a myofiber that was not transduced by AAV.