Gramlich Paul A, Westbroek Wendy, Feldman Ricardo A, Awad Ola, Mello Nicholas, Remington Mary P, Sun Ying, Zhang Wujuan, Sidransky Ellen, Betenbaugh Michael J, Fishman Paul S
Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA; Research Service, Veterans Affairs Maryland Health Care Service, Baltimore, MD, USA.
Section on Molecular Neurogenetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
J Biotechnol. 2016 Mar 10;221:1-12. doi: 10.1016/j.jbiotec.2016.01.015. Epub 2016 Jan 18.
Although recombinant glucocerebrosidase (GCase) is the standard therapy for the inherited lysosomal storage disease Gaucher's disease (GD), enzyme replacement is not effective when the central nervous system is affected. We created a series of recombinant genes/proteins where GCase was linked to different membrane binding peptides including the Tat peptide, the rabies glycoprotein derived peptide (RDP), the binding domain from tetanus toxin (TTC), and a tetanus like peptide (Tet1). The majority of these proteins were well-expressed in a mammalian producer cell line (HEK 293F). Purified recombinant Tat-GCase and RDP-GCase showed similar GCase protein delivery to a neuronal cell line that genetically lacks the functional enzyme, and greater delivery than control GCase, Cerezyme (Genzyme). This initial result was unexpected based on observations of superior protein delivery to neurons with RDP as a vector. A recombinant protein where a fragment of the flexible hinge region from IgA (IgAh) was introduced between RDP and GCase showed substantially enhanced GCase neuronal delivery (2.5 times over Tat-GCase), suggesting that the original construct resulted in interference with the capacity of RDP to bind neuronal membranes. Extended treatment of these knockout neuronal cells with either Tat-GCase or RDP-IgAh-GCase resulted in an >90% reduction in the lipid substrate glucosylsphingosine, approaching normal levels. Further in vivo studies of RDP-IgAh-GCase as well as Tat-GCase are warranted to assess their potential as treatments for neuronopathic forms of GD. These peptide vectors are especially attractive as they have the potential to carry a protein across the blood-brain barrier, avoiding invasive direct brain delivery.
尽管重组葡糖脑苷脂酶（GCase）是遗传性溶酶体贮积病戈谢病（GD）的标准治疗方法，但当中枢神经系统受到影响时，酶替代疗法并不有效。我们构建了一系列重组基因/蛋白质，其中GCase与不同的膜结合肽相连，包括Tat肽、狂犬病糖蛋白衍生肽（RDP）、破伤风毒素的结合域（TTC）和一种类破伤风肽（Tet1）。这些蛋白质中的大多数在哺乳动物生产细胞系（HEK 293F）中表达良好。纯化后的重组Tat-GCase和RDP-GCase向基因上缺乏功能性酶的神经元细胞系递送GCase蛋白的情况相似，且递送量比对照GCase（Cerezyme，健赞公司生产）更高。基于以RDP作为载体向神经元递送蛋白质效果更佳的观察结果，这一初步结果出人意料。在RDP和GCase之间引入来自IgA的柔性铰链区片段（IgAh）的重组蛋白，其向神经元递送GCase的能力显著增强（比Tat-GCase高2.5倍），这表明原始构建体导致RDP与神经元膜结合的能力受到干扰。用Tat-GCase或RDP-IgAh-GCase对这些基因敲除神经元细胞进行延长处理，可使脂质底物葡糖神经酰胺减少>90%，接近正常水平。有必要对RDP-IgAh-GCase以及Tat-GCase进行进一步的体内研究，以评估它们作为神经元型GD治疗方法的潜力。这些肽载体特别有吸引力，因为它们有可能携带蛋白质穿过血脑屏障，避免侵入性的直接脑内给药。
