Volume 15, Issue 2, Pages 320-329 (February 2007) Design of Tissue-specific Regulatory Cassettes for High-level rAAV-mediated Expression in Skeletal and Cardiac Muscle  Maja Z Salva, Charis L Himeda, Phillip WL Tai, Eiko Nishiuchi, Paul Gregorevic, James M Allen, Eric E Finn, Quynh G Nguyen, Michael J Blankinship, Leonard Meuse, Jeffrey S Chamberlain, Stephen D Hauschka  Molecular Therapy  Volume 15, Issue 2, Pages 320-329 (February 2007) DOI: 10.1038/sj.mt.6300027 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Regulatory cassettes for striated muscle-specific expression. (a) Enhancer and proximal promoter regulatory regions of murine muscle CK1 cassette and (b) the α-MHC enhancer, showing the identified transcription factor binding sites within each region. CK7 modifications, a 63-bp deletion between the enhancer right E-box and MEF2 sites, mutation to an initiator consensus sequence at the transcription start site, and the inclusion of MCK 5′-UTR sequence to +50, are indicated by gray shading. (c) Regulatory cassettes based on the MCK and α-MHC regions. The enhancer in the CK6 cassette (*) has the left E-box replaced with the right E-box and contains a 5-bp insertion 3′ of the right E-box. The CK7 modifications (gray shading) are shown in a. (d) Schematic of elements that comprise expression constructs for testing regulatory cassettes in the context of rAAV vectors: regulatory cassette, human placental AP cDNA, and the simian virus 40 polyA signal, flanked with AAV2 inverted terminal repeats. Molecular Therapy 2007 15, 320-329DOI: (10.1038/sj.mt.6300027) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Cassette activity in vitro. (a) Proliferating MM14 mouse muscle cells were co-transfected with a test plasmid expressing AP under control of the indicated cassette and a reference plasmid expressing β-galactosidase under control of the −1256/+7 region of the MCK promoter, and switched to differentiation media 4 h later. (b) HEK 293 cells, a human fibroblast cell line, (c) L cells, a mouse fibroblast cell line, and (d) JAWSII mouse immature dendritic cells were transfected with the same test plasmids as MM14 cells and a reference plasmid expressing β-galactosidase under control of the CMV promoter. Cell lysates were harvested 48 h (MM14, HEK 293, and L cells) or 24 h (JAWSII cells) after transfection and AP and β-galactosidase activity were measured by luminometric assays. Activity is shown as a percentage of CMV activity. Error bars represent SDs, n=6 (MM14 myocytes) or 3 (non-muscle cells). Molecular Therapy 2007 15, 320-329DOI: (10.1038/sj.mt.6300027) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Cassette activity in cardiac muscle. A total of 6 × 1011 vector genomes of rAAV6 vectors were administered by tail vein injection and hearts were harvested 4 weeks later for assaying. Each heart was subdivided into three portions, one for tissue lysate preparation, one for DNA extraction, and one for histology. (a) AP activity of cardiac tissue lysates is shown after subtracting activity of uninjected tissue lysates, normalized to total protein and number of vector genome copies. Error bars represent SDs, n=4. (b) AP activity in sections of cardiac muscle stained with BCIP/NBT substrate for 1 h. Representative images obtained using a 10 × objective are shown. Bar=200 μm. Molecular Therapy 2007 15, 320-329DOI: (10.1038/sj.mt.6300027) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Quantitative analysis of AP expression following systemic delivery of rAAV6 vectors. A total of 6 × 1011 vector genomes of rAAV6 vectors encoding AP driven by the indicated cassettes (key shown in (a) is used throughout the entire figure) were administered by tail vein injection and tissues were harvested 4 weeks later. (a) Viral genome distribution in muscles and non-muscle tissues. Total DNA was extracted from each tissue and the relative number of vector genomes was determined by quantitative PCR using plasmid DNA as a standard curve. Data are expressed as vector genomes per nanogram of total DNA. Error bars represent SDs, n=4. (b) Cassette activity in skeletal muscles. Muscles from one side were used for tissue lysate preparation and genome extraction, whereas muscles from the contralateral side were used for histology. AP activities of tissue lysates of soleus muscle, diaphragm, quadriceps muscle, and gastrocnemius muscle are shown after subtracting the activity from uninjected tissue lysates, normalized to total protein and number of vector genome copies. Error bars represent SDs, n=4. (c) Cassette activity in non-muscle tissues. AP activities in tissue lysates of liver, spleen, lung, intestine, and testes are shown after subtracting the activities of tissue lysates from uninjected mice. All values are normalized to total protein and number of vector genome copies. Error bars represent SDs, n=4. Activities in kidney, aorta, and brain lysates were at background level for all constructs (data not shown). Background level activity in (b and c) is indicated by an asterisk. Molecular Therapy 2007 15, 320-329DOI: (10.1038/sj.mt.6300027) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 AP activity in cross sections of (a) diaphragm, (b) soleus muscle, (c) quadriceps muscle, and (d) gastrocnemius muscle stained with BCIP/NBT substrate for 1 h. Representative images obtained using a 10 × objective lens are shown. Bar=200 μm. Molecular Therapy 2007 15, 320-329DOI: (10.1038/sj.mt.6300027) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 MyHC isotype expression and AP activity in individual soleus muscle fibers. Left panels show immunofluorescent staining for MyHC types I (blue) and IIa (red) in sections from mice injected with (a) CK1, (b) CK7, and (c) MHCK7. Right panels show adjacent sections stained for AP activity with BCIP/NBT substrate for 25 min. All images were obtained using a 20 × objective lens. Bar=100 μm. Matching fibers in adjacent sections are denoted with the following: type I (−), type IIb (o), type IIb, or IId/x non-blue/red fibers (*). Molecular Therapy 2007 15, 320-329DOI: (10.1038/sj.mt.6300027) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 MHCK7-mediated expression of microdystrophin in mdx mice following systemic delivery of rAAV6 vectors. A total of 5 × 1011 vector genomes of rAAV6 vectors encoding MHCK7-driven microdystrophin were administered by tail vein injection to 5- to 6-week-old mdx mice and tissues were harvested 10 weeks later. Microdystrophin expression in frozen sections was detected by immunofluorescent staining using a primary antibody against the N-terminal of dystrophin and an Alexa 488-labeled secondary antibody. Sections from quadriceps, cardiac muscle, and diaphragm from wild-type, untreated, and treated mdx mice were photographed using a 20 × objective lens. Bar=50 μm. Molecular Therapy 2007 15, 320-329DOI: (10.1038/sj.mt.6300027) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions