Gene transfer and expression of humanα-galactosidase from mouse muscle in vitro andin vivo
Gene transfer and expression of humanα-galactosidase from mouse muscle in vitro andin vivo
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小鼠肌肉中人α-半乳糖苷酶的体内外基因转移和表达
DOI：
10.1038/sj.gt.3300410
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发表时间：
1997
期刊：
影响因子：
5.1
通讯作者：
K. Macdermot
中科院分区：
文献类型：
--
作者：
FJ Novo;D. Górecki;G. Goldspink;K. Macdermot
溶酶体胆积症可通过酶替代治疗。通过直接注射表达载体对肌肉进行遗传修饰可能代表了一种提供缺陷酶的替代方法，如果可以在肌肉中实现足够和持久的表达水平。我们利用C2 C12小鼠肌源性细胞系研究了肌肉特异性调控元件组合对人溶酶体酶α-半乳糖苷酶（α-gal）表达的影响。在分化的肌管中，含有肌球蛋白轻链1/3增强子与人巨细胞病毒启动子组合的构建体导致比相同增强子与兔β-肌球蛋白重链启动子组合或仅含有CMV启动子的构建体更高的表达。在细胞提取物和上清液中均可检测到酶活性增加。此外，缺乏α-gal的人成纤维细胞能够从转染成肌细胞的条件培养基中摄取酶。在存在甘露糖-6-磷酸的情况下未发生这种情况，这表明摄取是通过甘露糖-6-磷酸受体进行的。据我们所知，这是第一份报告，其中正确加工形式的人α-gal表达和分泌分化的肌肉细胞。将质粒表达载体直接注射到小鼠胫骨前肌中，在注射后7天显示α-gal水平显著增加。
Lysosomal storage disorders are amenable to treatment by enzyme replacement. Genetic modification of muscle via direct injection of expression vectors might represent an alternative method of providing the defective enzymes, if adequate and long-lasting expression levels can be achieved in muscle. We have used the C2C12 mouse myogenic cell line to study the effect of combination of muscle-specific regulatory elements on the expression of the human lysosomal enzyme α-galactosidase (α-gal). In differentiated myotubes, a construct containing the myosin light chain 1/3 enhancer in combination with the human cytomegalovirus promoter resulted in higher expression than constructs combining the same enhancer with the rabbit β-myosin heavy chain promoter, or containing the CMV promoter only. Increased enzymatic activity was detectable both in cell extracts and in supernatants. Furthermore, human fibroblasts deficient in α-gal were able to take up the enzyme from medium conditioned by transfected myoblasts. This did not occur in the presence of mannose-6-phosphate which indicates that the uptake was via mannose-6-phosphate receptors. To our knowledge, this is the first report in which a correctly processed form of human α-gal was expressed and secreted from differentiated muscle cells. Direct injection of a plasmid expression vector into mouse tibialis anterior muscle showed significantly increased levels of α-gal 7 days after injection.
转染的兔肌球蛋白重链β基因启动子的肌肉特异性调节。
DOI：
--
发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Cribbs,LL;Shimizu,N;Yockey,CE;Levin,JE;Jakovcic,S;Zak,R;Umeda,PK
通讯作者：
Umeda,PK