Construction and analysis of compact muscle-specific promoters for AAV vectors

Abstract

Adeno-associated viral (AAV) vectors have been broadly used for gene transfer in vivo for various applications. However, AAV precludes the use of most of the original large-sized tissue-specific promoters for expression of transgenes. Efforts are made to develop highly compact, active and yet tissue-specific promoters for use in AAV vectors. In this study, we further abbreviated the muscle creatine kinase (MCK) promoter by ligating a double or triple tandem of MCK enhancer (206-bp) to its 87-bp basal promoter, generating the dMCK (509-bp) and tMCK (720-bp) promoters. The dMCK promoter is shorter but stronger than some previously developed MCK-based promoters such as the enh358MCK (584-bp) and CK6 (589-bp) in vitro in C2C12 myotubes and in vivo in skeletal muscles. The tMCK promoter is the strongest that we tested here, more active than the promiscuous cytomegalovirus (CMV) promoter. Furthermore, both the dMCK and tMCK promoters are essentially inactive in nonmuscle cell lines as well as in the mouse liver (4200-fold weaker than the CMV promoter).The dMCK promoter was further tested in a few lines of transgenic mice. Expression of LacZ or minidystrophin gene was detected in skeletal muscles throughout the body, but was weak in the diaphragm, and undetectable in the heart and other tissues. Similar to other miniature MCK promoters, the dMCK promoter also shows preference for fast-twitch myofibers. As a result, we further examined a short, synthetic muscle promoter C5-12 (312-bp). It is active in both skeletal and cardiac muscles but lacks apparent preference on myofiber types. Combination of a MCK enhancer to promoter C5-12 has increased its strength in muscle by two-to threefold. The above-mentioned compact muscle-specific promoters are well suited for AAV vectors in muscle-directed gene therapy studies.

Systemic human minidystrophin gene transfer improves functions and life span of dystrophin and dystrophin/utrophin‐deficient mice

“…4,9,10 rAAV1 vector stocks were produced and purified using a previously published protocol. 11 The vector titers were approximately 5 Â 10 12 viral genome (v.g.)…”

AAV-based shRNA silencing of NF-κB ameliorates muscle pathologies in mdx mice

“…On the basis of high efficiency of AAV9 vectors that we observed in murine 25 and large DMD 20 models, the use of an AAV9 vector in this study ensures a high efficiency of shRNA delivery in mdx muscle by i.m. injection.…”

“…25 More importantly, the AAV9-mediated delivery of the mini-dystrophin gene in the small and large DMD models did not stimulate a host immune issue. 20,25 We constructed an AAV vector expressing either a p65-shRNA or a control shRNA (ct-shRNA) from an U6 promoter and the ZsGreen reporter from the human cytomegalovirus (CMV) promoter.…”

“…25 More importantly, the AAV9-mediated delivery of the mini-dystrophin gene in the small and large DMD models did not stimulate a host immune issue. 20,25 We constructed an AAV vector expressing either a p65-shRNA or a control shRNA (ct-shRNA) from an U6 promoter and the ZsGreen reporter from the human cytomegalovirus (CMV) promoter. Initially, to determine the efficacy of AAV9 vector-mediated shRNA gene transfer, the extent of ZsGreen reporter gene expression in treated gastrocneminus (GAS) muscles was examined following injection of 3.5 Â 10 11 viral particles of AAV9_U6_p65-shRNA_CMV_ZsGreen.…”

“…Moreover, anti-inflammatory approaches combined with gene replacement can also minimize the administration dose of viral vectors for gene replacement, and also diminish the potential immune response in the host through an improvement in the inflamed microenvironment. Furthermore, the carrying capacity of an AAV vector allows the p65-shRNA silencing cassette (o350 bp) and minidystrophin (D3849) expressing cassette package (o4.5 kb) 17,25 into a single-stranded AAV vector, which provides a possibility to combine gene replacement and anti-inflammatory therapy in a one-step treatment. In the future, a synergistic therapeutic effect could be achieved by combining dystrophin gene replacement with p65-shRNA-based anti-inflammation in a single AAV vector for DMD treatment, especially by muscle-specific expression of mini-dystrophin and gene silencing of NF-kB/p65 in damaged muscle cells and immune cells.…”