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

成人克拉伯病发病机制及其基因治疗基本途径的研究

• 批准号: 08670714
• 负责人: FURUYA Hirokazu
• 金额: $ 1.41万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670714/
• 关键词: Krabbe disease; galactosylceramidase(GALC); lysosmal enzyme; psychosine; sphingolipid; gene therapy; gene replacement therapy; リソソーム酵素; galactosylceramidase; 分子生物学; アミノ酸置換; 遺伝子性疾患

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.

我们检测了5例日本成人型球形细胞脑白质营养不良（克拉伯病）（AO-GLD）患者的半乳糖神经酰胺酶（GALC）cDNA和基因。我们发现了三个错义突变（I66 M，G270 D，L 618 S）和一个外显子6跳跃（535- 573 de 1）。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。在这些经验中，显示（1）AO-GLD突变，包括在此发现的那些，位于GALC酶的N（I66 M，G270 D，535- 573 de 1）或C（L 618 S）末端。而报道的婴儿型（IF-GLD）突变位于中央结构域。（2）虽然GALC（80 kDa）与50和30 kDa亚基一致，但前者存在于细胞外，后者存在于细胞内，两者均具有酶活性。然而，这些亚基在独立表达后的混合物显示没有GALC活性。（3）根据抑制试验，GALC被认为是在加工成两个亚基后被摄取到溶酶体囊泡中的。（4）在AO-GLD中发现的一些突变显示GALC加工受到抑制。我们还准备利用逆转录病毒和腺相关病毒载体构建基因治疗的表达载体。

项目成果

Kukita Y et al.: "Characterization of GALC gene in three Japanes patients with adult-onset Krabbe disease." Genetic Testing. (in press).

Kukita Y 等人：“三名日本成人克拉伯病患者的 GALC 基因特征。”

Furuya H et al.: "Adult onset globoid cell leukodystrophy(Krabbe disease):Analysis of galactosylceramidase cDNA from four Japanese patients." Hum Genet. 100. 450-456 (1997)

Furuya H 等人：“成人发病的球状细胞脑白质营养不良（克拉伯病）：四名日本患者的半乳糖神经酰胺酶 cDNA 分析。”

Satoh JI et al.: "Adult-onset Krabbe disease with homozygous T1853C mutation in thegalactocerebrosidase gene exhibits unique MRI findings of the pure corticospinal tract demyelination" Neurology. 49. 1392-1399 (1997)

Satoh JI 等人：“半乳糖脑苷酶基因纯合 T1853C 突变的成人发病克拉伯病表现出纯皮质脊髓束脱髓鞘的独特 MRI 发现”。

Yoji KUKITA et al.,: "Characterization of galactosylceramidase (GALC) gene in three Japanese patients with adult-onset Krabbe disease" Human Mutation. (in press).

Yoji KUKITA 等人：“三名日本成年发病克拉伯病患者的半乳糖神经酰胺酶 (GALC) 基因特征”人类突变。