Transfer of the α5(IV) collagen chain gene to smooth muscle restores in vivo expression of the α6(IV) collagen chain in a canine model of Alport syndrome
Transfer of the α5(IV) collagen chain gene to smooth muscle restores in vivo expression of the α6(IV) collagen chain in a canine model of Alport syndrome
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DOI：
10.1016/s0002-9440(10)63883-7
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发表时间：
2003-03-01
影响因子：
6
通讯作者：
Thorner, PS
中科院分区：
文献类型：
--
作者：
Harvey, SJ;Zheng, KQ;Thorner, PS
x连锁Alport综合征是一种由编码α 5（IV）胶原蛋白链的COL4A5基因突变引起的进行性肾脏疾病。作为Alport综合征基因治疗的第一步，我们报道了重组alpha51（IV）胶原蛋白在体外和体内的表达。通过转染能合成α 1（IV）和α 2(IV)，但不能合成α 3（IV）到α 6（IV）胶原链的HEK293细胞，克隆并体外表达了全长cdna编码犬α 5（IV）胶原链。通过Northern blotting，表达了一个5.2 kb的alpha5(IV) mRNA转录物，免疫细胞化学检测了重组蛋白。该链以190 kd的单体形式分泌到培养基中；未检出三螺旋菌种。转染后的细胞合成了含有alpha1（IV）和alpha2（IV）链的细胞外基质，但重组的alpha5（IV）链未掺入。这些发现与α 5（IV）链需要一个或多个α (IV)、α 4（IV）或α 6（IV）链进行三螺旋组装的概念是一致的。体内研究是在患有x连锁阿尔波特综合征的狗身上进行的。将含有alpha5（IV）转基因的腺病毒载体注射到缺乏alpha5（IV）和alpha6（IV）链的膀胱平滑肌中。注射后5周，注射部位的平滑肌细胞均表达alpha5（IV）和alpha6（IV）链，呈基底膜分布。因此，重组α 5（IV）链能够恢复第二α (IV)链的表达，而第二α (IV)链需要α 5（IV）链的存在才能并入胶原三聚体中。该载体将为进一步探索阿尔波特综合征的基因治疗提供有用的工具。
X-linked Alport syndrome is a progressive renal disease caused by mutations in the COL4A5 gene, which encodes the alpha5(IV) collagen chain. As an initial step toward gene therapy for Alport syndrome, we report on the expression of recombinant alpha51(IV) collagen in vitro and in vivo. A full-length cDNA-encoding canine alpha5(IV) collagen was cloned and expressed in vitro by transfection of HEK293 cells that synthesize the alpha1(IV) and alpha2(IV), but not the alpha3(IV) to alpha6(IV) collagen chains. By Northern blotting, an alpha5(IV) mRNA transcript of 5.2 kb was expressed and the recombinant protein was detected by immunocytochemistry. The chain was secreted into the medium as a 190-kd monomer; no triple helical species were detected. Transfected cells synthesized an extracellular matrix containing the alpha1(IV) and alpha2(IV) chains but the recombinant alpha5(IV) chain was not incorporated. These findings are consistent with the concept that the alpha5(IV) chain requires one or more of the alpha(IV), alpha4(IV), or alpha6(IV) chains for triple helical assembly. In vivo studies were performed in dogs with X-linked Alport syndrome. An adenoviral vector containing the alpha5(IV) transgene was injected into bladder smooth muscle that lacks both the alpha5(IV) and alpha6(IV) chains in these animals. At 5 weeks after injection, there was expression of both the alpha5(IV) and alpha6(IV) chains by smooth muscle cells at the injection site in a basement membrane distribution. Thus, this recombinant alpha5(IV) chain is capable of restoring expression of a second alpha(IV) chain that requires the presence of the alpha5(IV) chain for incorporation into collagen trimers. This vector will serve as a useful tool to further explore gene therapy for Alport syndrome.