Cardiac-specific gene expression facilitated by an enhanced myosin light chain promoter.
BACKGROUND
Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression.


METHODS
To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc) reporter gene (Ad.4 x MLC250.Luc). Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 x MLC.Luc or control vectors. For in vivo testing, Ad.4 x MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera.


RESULTS
In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells) was 20.4 versus 0.9 (Ad.4 x MLC250.Luc vs. Ad.CMV). In vivo: Ad.4 x MLC250.Luc significantly reduced luc activity in liver (38.4-fold), lung (16.1-fold), and kidney (21.8-fold) versus Ad.CMV (p =.01); whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 x MLC.Luc) versus 1:1.4 (Ad.CMV.Luc).


CONCLUSIONS
This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.
Klinikum Grosshadern.
Wolfgang
W
Boecker
Oliver Y
OY
Bernecker
Joseph C
JC
Wu
Xinsheng
X
Zhu
Tomohiro
T
Sawa
Luanda
L
Grazette
Anthony
A
Rosenzweig
Federica
F
del Monte
Ulrich
U
Schmidt
Roger J
RJ
Hajjar
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007091:Image Processing, Computer-Assisted; D018374:LLC-PK1 Cells; D008156:Luciferases / Q000378:metabolism; D008297:Male; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018994:Myosin Light Chains / Q000235:genetics*; D009928:Organ Specificity / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine; D013997:Time Factors; D019076:Transgenes
D018994:Myosin Light Chains; D008156:Luciferases