Research for pathogenesis of adult onset Krabbe disease and basic approach for its gene therapy

我们检测了5例日本成人起病的球状细胞脑白质营养不良(Krabbe病)患者半乳糖基神经酰胺酶(GALC)基因的表达。我们发现了三个错义突变(I66M、G270D、L618S)和一个外显子6跳过(-573de1)。我们构建了突变的GALC cDNA，并在COS-1细胞中瞬时表达，在CHO细胞中稳定表达。这些研究表明：(1)AO-GLD突变，包括这里发现的突变，位于GALC酶的N(I66M，G270D，-573de1)或C(L618S)末端。而已报道的婴儿形式的突变(IF-GLD)位于中心区。这种突变位点的差异可能影响GLD临床特征的表型。(2)尽管GALC(80 KDa)与50 kDa和30 kDa亚基一致，但前者存在于细胞外，后者存在于细胞内，两者都具有酶活性。(3)根据抑制实验，GALC被认为是在加工成两个亚单位后摄取到溶酶体囊泡中的；(4)在一些在AO-GLD中发现的突变中显示了对GALC加工的抑制。我们还准备利用逆转录病毒和腺相关病毒载体构建用于基因治疗的表达载体。

We examined galactosylceramidase (GALC) cDNA and gene in five Japanese patients with adult onset globoid cell 1eukodystrophy (Krabbe disease) (AO-GLD). We identified three missense mutations (I66M,G270D,L618S) and one exon 6 skipping (535-573de1). We constructed mutated GALC cDNAs and expressed them in COS-1 cells transiently and in CHO cell stably. In these experiences, it is shown that (1) AO-GLD mutations, including those found here, are located in the N (I66M,G270D,535-573de1) or C (L618S) terminus of the GALC enzyme. Whereas the reported mutations in the infantile form (IF-GLD) are in the central domain. This difference in mutation sites may affect the phenotype of the clinical features of GLD,(2) Although GALC (80kDa) is consistent with 50 and 30 kDa subunits, the former exits in extracellular, the latters in intrace11ular, both of which have enzymatic activity. However, mixture of these subunits after independent expression show no GALC activity, (3) According to the inhibition assay, GALC is considered to uptake into the lysosomal vesicle following with processing into two subunits in it and (4) In some mutation found in AO-GLD show inhibition of GALC processing. We also preparing to construct expression vectors for gene therapy using retroviral and adeno associated viral vectors.