1. Volume 2, Issue 1, Pages 16-25 (July 2000)
Analysis of Muscle Creatine Kinase Regulatory Elements in Recombinant Adenoviral Vectors 
Michael A. Hauser, Ann Robinson, Dennis Hartigan-O'Connor, DeeAnn Williams-Gregory, Jean N. Buskin, Steve Apone, Christopher J. Kirk, Stephen Hardy, Stephen D. Hauschka, Jeffrey S. Chamberlain 
Molecular Therapy 
Volume 2, Issue 1, Pages (July 2000)
DOI: /mthe
Copyright © 2000 American Society for Gene Therapy Terms and Conditions

2. FIG. 1
Muscle creatine kinase promoter and enhancer. (a) The sequence of a 3355-bp genomic fragment of the murine MCK transcriptional regulatory region extending from –3348 to +7 relative to the transcriptional start site has been deposited in the GenBank database (Accession No. AF188002), and the corresponding restriction map is shown. (b) The sequence of the 206-bp MCK upstream enhancer is shown, with protein binding sites underlined (27, 51, 56). The sequence alterations corresponding to the 2R and S5 modifications are indicated above the wild-type sequence. The NcoI site indicated marks the upstream boundary of the MEF2 deletion in construct CK5.
Molecular Therapy 2000 2, 16-25DOI: ( /mthe )
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3. FIG. 2
Transcriptional regulatory cassettes based on the muscle creatine kinase promoter and enhancer. CK3 contains the full 3355-bp region extending from –3348 to +7 relative to the transcriptional start site. CK2 extends from –1256 to +7. CK5 contains part of the 2RS5 enhancer, extending from nucleotides –1256 to –1091, thereby deleting the enhancer MEF2 site, and a promoter extending from –944 to +7. Previous deletion studies have indicated no control elements within the –1050 to –945 MCK promoter region that was deleted (30, 33). CK6 contains the full 2RS5 enhancer sequence in Fig. 1 and a promoter extending from –358 to +7. CK4 contains the full 2RS5 enhancer and a promoter extending from –80 to +7. The CMV promoter used in plasmid constructs extends from –525 to +1 relative to its transcriptional start site. All constructs include the 150-bp minx intron, a nuclear targeted lacZ transgene, and the SV40 polyadenylation signal. This schematic diagram at the bottom shows an expression cassette inserted into a recombinant adenoviral vector so that transcription proceeds away from the viral left ITR. MCK regulatory elements in this orientation direct muscle-specific expression, while those in the opposite orientation allow leaky transcription in nonmuscle cells.
Molecular Therapy 2000 2, 16-25DOI: ( /mthe )
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4. FIG. 3
Tissue specificity of expression depends on cassette orientation. Sections of muscle from a 6-week-old mdx mouse, stained with X-gal 5 days after injection with recombinant adenoviral vectors containing (a) CK5lacZ and (b, c) CK6lacZ expression cassettes. When the marker gene is oriented so that it is transcribed away from the left adenoviral ITR, expression of lacZ is limited to the myonuclei (a–c, large panels). When the expression cassette is oriented in the opposite direction, rare positively staining nonmuscle nuclei are observed (inset in a).
Molecular Therapy 2000 2, 16-25DOI: ( /mthe )
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5. FIG. 4
MCK- and CMV-driven expression in muscle. (a) Muscles injected with the MCK enhancer containing virus AdCK6lacZ exhibit strong staining of muscle nuclei, but no staining of cells in the mononuclear infiltrate characteristic of dystrophic mdx mice. In contrast, muscles injected with the CMV enhancer containing virus H5.010CBlacZ exhibit strong staining of both muscle (b) and nonmuscle (c) nuclei. Muscles of 6-week-old mdx mice were stained with X-gal 5 days after injection of adenoviral vectors.
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6. FIG. 5
MCK- and CMV-driven expression in liver. Six-week-old mdx mice were injected in the tail vein with 2 × 109 pfu of the CMV enhancer containing virus H5.010CblacZ (a) and sacrificed 5 days later. X-gal staining of liver cryosections shows high levels of lacZ expression. Livers of animals injected with the same amount of the MCK enhancer containing viruses AdCK5lacZ (b) and AdCK6lacZ (c) exhibit no detectable lacZ expression.
Molecular Therapy 2000 2, 16-25DOI: ( /mthe )
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7. FIG. 6
Minimal expression from CK promoters in human dendritic cells and extended β-galactosidase expression in vivo. (A) Dendritic cell expression: 2.5 × 108 particles of each virus were added to 2.5 × 105 human dendritic cells in 24-well plates. Cells were washed the following day and cell extracts were prepared 72 h after the start of infection. AdCK5lacZ and AdCK6lacZ produced about 1000 times less β-galactosidase than AdCMVlacZ, a control virus containing the CMV promoter. The amount of β-galactosidase produced by MCK viruses was not statistically greater than that produced by a virus containing no transgene (CK5, n = 4; CK6, n = 8). (B) Longevity of expression: For each time point, four tibialis anterior muscles were injected with 1.58 × 1011 particles of AdCK6lacZ. Muscles were harvested, assayed individually for β-galactosidase activity, and normalized to the activity observed after 3 days. The asterisk indicates a statistically significant decline in total β-gal activity by 4 months after injection, compared to the activity observed at 3 days.
Molecular Therapy 2000 2, 16-25DOI: ( /mthe )
Copyright © 2000 American Society for Gene Therapy Terms and Conditions