Design of Tissue-specific Regulatory Cassettes for High-level rAAV-mediated Expression in Skeletal and Cardiac Muscle

Abstract

Systemic delivery of recombinant adeno-associated virus (rAAV) 6 vectors mediates efficient transduction of the entire striated musculature, making this an attractive strategy for muscle gene therapy. However, owing to widespread transduction of non-muscle tissues, optimization of this method would benefit from the use of muscle-specific promoters. Most such promoters either lack high-level expression in certain muscle types or are too large for inclusion in rAAV vectors encoding microdystrophin. Here, we describe novel regulatory cassettes based on enhancer/promoter regions of murine muscle creatine kinase (CK) and alpha-myosin heavy-chain genes. The strongest cassette, MHCK7 (770 bp), directs high-level expression comparable to cytomegalovirus and Rous sarcoma virus promoters in fast and slow skeletal and cardiac muscle, and low expression in the liver, lung, and spleen following systemic rAAV6 delivery in mice. Compared with CK6, our previous best cassette, MHCK7 activity is approximately 400-, approximately 50-, and approximately 10-fold higher in cardiac, diaphragm, and soleus muscles, respectively. MHCK7 also directs strong microdystrophin expression in mdx muscles. While further study of immune responses to MHCK7-regulated microdystrophin expression is needed, this cassette is not active in dendritic cell lines. MHCK7 is thus a highly improved regulatory cassette for experimental studies of rAAV-mediated transduction of striated muscle.

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

“…8,9,13 A recent report by Hauschka's group, however, has described a number of improved MCK-based promoters such as the MHCK7 that are not only active in the heart and diaphragm muscles, but also show reduced myofibertype preference. 26,27 Our own effort here also identified a hybrid muscle-specific synthetic promoter, in which a modified MCK enhancer was fused with an original synthetic muscle promoter (C5-12). 33 This 520 bp hybrid promoter (E-Syn) is 2-3 stronger than the original C5-12 promoter.…”

“…19,[20][21][22][23] However, most comprehensive work has been done by Hauschka's laboratory on the modification and optimization of MCK-based muscle-specific promoters. 9,[24][25][26][27] We have made our own efforts to further reduce the size and retain/improve the strength and specificity of the truncated MCK promoters, as the minidystrophin genes we tested for gene therapy have the coding sequences around 4.0 kb, whereas the AAV vectors could only afford less than 4.7 kb of foreign gene cassette. This has left little room to accommodate the promoter and polyA sequences in addition to the minidystrophin gene.…”

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

“…Most muscle‐specific promoters such as that of the muscle creatine kinase (MCK) gene are very large (6.5 kb), and thus incompatible with AAVs having a 4.5 kb capacity (Wu et al ., 2010). Efforts have been made to shorten the MCK promoter, giving rise to the dMTK (509 bp) or tMCK (720 bp) promoter that show skeletal muscle‐specific expression (Yue et al ., 2015) and the MHCK7 (770 bp) promoter that drives cardiac and skeletal muscle expression (Salva et al ., 2007). …”

Promoter analysis of bovine cardiomyopathy-associated protein 1 gene

“…Based on the putative transcription factor binding site (Wu et al, 2013), we chose the 1155 bp (+20/-1135) upstream of the CMYA1 gene 5'-flanking region to analyze the core promoter. Some muscle-specific gene promoters can regulate gene expression in myocytes but not in fibroblasts (Salva et al, 2007). In the present study, pEGFP-1-CMYA1-P1 and pEGFP-1-CMYA1-P7 fragments were transfected into C2C12 cells, bovine myoblast cells, and bovine fibroblasts cells.…”