Department of Periodontics, University of Alabama at Birmingham School of Dentistry, Birmingham, Alabama, United States of America.
PLoS One. 2011;6(10):e24281. doi: 10.1371/journal.pone.0024281. Epub 2011 Oct 7.
To explore gene therapy strategies for amelogenesis imperfecta (AI), a human ameloblast-like cell population was established from third molars of an AI-affected patient. These cells were characterized by expression of cytokeratin 14, major enamel proteins and alkaline phosphatase staining. Suboptimal transduction of the ameloblast-like cells by an adenovirus type 5 (Ad5) vector was consistent with lower levels of the coxsackie-and-adenovirus receptor (CAR) on those cells relative to CAR-positive A549 cells. To overcome CAR -deficiency, we evaluated capsid-modified Ad5 vectors with various genetic capsid modifications including "pK7" and/or "RGD" motif-containing short peptides incorporated in the capsid protein fiber as well as fiber chimera with the Ad serotype 3 (Ad3) fiber "knob" domain. All fiber modifications provided an augmented transduction of AI-ameloblasts, revealed following vector dose normalization in A549 cells with a superior effect (up to 404-fold) of pK7/RGD double modification. This robust infectivity enhancement occurred through vector binding to both α(v)β3/α(v)β5 integrins and heparan sulfate proteoglycans (HSPGs) highly expressed by AI-ameloblasts as revealed by gene transfer blocking experiments. This work thus not only pioneers establishment of human AI ameloblast-like cell population as a model for in vitro studies but also reveals an optimal infectivity-enhancement strategy for a potential Ad5 vector-mediated gene therapy for AI.
为了探索成釉不全（AI）的基因治疗策略，我们从 AI 患者的第三磨牙中建立了一个人成釉细胞样细胞群体。这些细胞通过角蛋白 14、主要釉蛋白和碱性磷酸酶染色来表达。腺病毒 5 型（Ad5）载体对成釉细胞样细胞的转导效果不佳，与 CAR 阳性的 A549 细胞相比，这些细胞上的柯萨奇-腺病毒受体（CAR）水平较低。为了克服 CAR 缺陷，我们评估了带有各种遗传衣壳修饰的衣壳修饰型 Ad5 载体，包括在衣壳蛋白纤维中掺入“pK7”和/或“RGD”基序的短肽，以及带有 Ad 血清型 3（Ad3）纤维“knob”结构域的纤维嵌合体。所有纤维修饰都增强了 AI-成釉细胞的转导，在 A549 细胞中通过载体剂量归一化后显示出更高的效果（高达 404 倍），pK7/RGD 双修饰的效果最好。这种强大的感染性增强是通过载体与高度表达于 AI-成釉细胞的α(v)β3/α(v)β5 整联蛋白和硫酸乙酰肝素蛋白聚糖（HSPGs）结合而发生的，这一点通过基因转移阻断实验得到了证实。因此，这项工作不仅开创了人 AI 成釉细胞样细胞群体作为体外研究模型的建立，还揭示了一种优化的感染性增强策略，为潜在的 Ad5 载体介导的 AI 基因治疗提供了可能性。
