CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer's Disease.
CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer's Disease.
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DOI：
10.1016/j.omtn.2018.03.007
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发表时间：
2018-06-01
期刊：
影响因子：
--
通讯作者：
Ingelsson M
中科院分区：
文献类型：
--
作者：
György B;Lööv C;Zaborowski MP;Takeda S;Kleinstiver BP;Commins C;Kastanenka K;Mu D;Volak A;Giedraitis V;Lannfelt L;Maguire CA;Joung JK;Hyman BT;Breakefield XO;Ingelsson M
淀粉样蛋白前体蛋白 (APP) 基因中的 APPswe（瑞典语）突变会导致显性遗传性阿尔茨海默病 (AD)，这是由于 β 分泌酶对淀粉样蛋白 -β (Aβ) 前体蛋白的裂解增加所致。这会导致 Aβ 水平异常高，不仅在大脑中，而且在突变携带者的外周组织中也是如此。在这里，我们使用 CRISPR 选择性破坏了人类突变体 APPSW 等位基因。通过应用来自化脓性链球菌的 CRISPR/Cas9，我们生成了 APPSW 或 APPWT 的等位基因特异性删除。通过 ELISA 测量，目标患者来源的成纤维细胞的条件培养基显示分泌的 Aβ 减少了约 60%。接下来，APPSW 特异性指导 RNA (gRNA) 和 Cas9 的编码序列被包装到单独的腺相关病毒 (AAV) 载体中。在从 APPSW 转基因小鼠胚胎 (Tg2576) 分离的原代神经元中以及将这些载体共注射到成年小鼠的海马体中后，都实现了位点特异性插入缺失形成。综上所述，我们在此提供了概念验证数据，表明 CRISPR/Cas9 可以在体外和体内选择性破坏 APPSW 等位基因，从而减少致病性 Aβ。因此，该系统可能有潜力被开发为针对由 APPswe 和其他与 Aβ 增加相关的点突变引起的 AD 的基因治疗工具。
The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo—and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.
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