Genetic engineering of a lysosomal enzyme fusion protein for targeted delivery across the human blood-brain barrier
Genetic engineering of a lysosomal enzyme fusion protein for targeted delivery across the human blood-brain barrier
复制标题
DOI：
10.1002/bit.21602
复制
发表时间：
2008-02-01
影响因子：
3.8
通讯作者：
Pardridge, William M.
中科院分区：
文献类型：
--
作者：
Boado, Ruben J.;Zhang, Yun;Pardridge, William M.
I型粘多糖样变性，Hurler综合征，是一种影响大脑的溶酶体储存障碍。缺失的酶α-L-艾杜糖醛酸酶（IDUA）不能穿过血脑屏障（BBB）。为了使酶能够通过BBB转运，将人IDUA与人胰岛素受体（HIR）嵌合单克隆抗体（MAb）重链的羧基末端融合。HIRMAb在内源性胰岛素受体上穿过BBB，并作为分子特洛伊木马将IDUA运送到脑中。COS细胞的转染导致培养基和细胞内空间中IDUA酶活性的高水平。如用蛋白质印迹和针对人IDUA或人IgG的抗体测量的，融合重链的大小相对于亲本HIRMAb的重链的大小增加约80 kDa。亲和纯化的HIRMAb-IDUA融合蛋白的IDUA酶比活性为363 +/-37 U/μ g蛋白，其与重组IDUA的比活性相当。通过用HIRMAb-IDUA融合蛋白处理，Hurler成纤维细胞中糖基氨基聚糖的积累减少70%。共聚焦显微镜显示融合蛋白靶向溶酶体。HIRMAb-IDUA融合蛋白以高亲和力与HIR结合，并且在静脉内施用后迅速转运到成年恒河猴的脑中。HIRMAb-IDUA融合蛋白是治疗Hurler综合征的一种新疗法，它已被专门设计为穿过人类BBB。
Mucopolysaccharidosis Type I, Hurler's Syndrome, is a lysosomal storage disorder that affects the brain. The missing enzyme, alpha-L-iduronidase (IDUA), does not cross the blood-brain barrier (BBB). To enable BBB transport of the enzyme, human IDUA was fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb crosses the BBB on the endogenous insulin receptor, and acts as a molecular Trojan horse to ferry into brain the IDUA. Transfection of COS cells resulted in high levels of IDUA enzyme activity both in the medium and in the intracellular space. The size of the fusion heavy chain, as measured with Western blotting and antibodies to either human IDUA or human IgG, was increased about 80 kDa, relative to the size of the heavy chain of the parent HIRMAb. The IDUA enzyme specific activity of the affinity purified HIRMAb-IDUA fusion protein was 363 +/- 37 U/mu g protein, which is comparable to specific activity of recombinant IDUA. The accumulation of glycosoaminoglycans in Hurler fibroblasts was decreased 70% by treatment with the HIRMAb-IDUA fusion protein. Confocal microscopy showed targeting of the fusion protein to the lysosome. The HIRMAb-IDUA fusion protein bound with high affinity to the HIR, and was rapidly transported into the brain of the adult Rhesus monkey following intravenous administration. The HIRMAb-IDUA fusion protein is a new treatment for Hurler's syndrome, which has been specifically engineered to cross the human BBB.