摘要
背景:越来越多的阿尔茨海默症患者亟需更有效的疗法。尽管抗阿尔茨海默症免疫疗法取得了可观的成功,但它依然面临脑内和细胞内输送的挑战。这个实验提出了脑内注射PEG-PAsp(DET)/mRNA聚合物后原地产生抗淀粉样β(Aβ)抗体这一新型免疫治疗途径,它比腺病毒编码的高全身抗体剂量和给药更安全。 方法:我们使用mRNA编码三种不同的Aβ特异性单链抗体可变区基因片段(scFv),被动免疫治疗有分泌信号。单链抗体可变区基因片段包括一个6x组氨酸标记的免疫监控。IL2(IL2ss)的分泌信号被添加为允许细胞外老年斑的结合。单链抗体可变区基因片段的Aβ亲和性由表面等离子共振测量。为了允许细胞内运输,scFv与我们智能的共聚体聚乙二醇聚[ N’N-(2-氨基乙基)- 氨乙基]天冬酰胺 [PEG-PAsp (DET)]形成了复合物。我们通过Western blot和ELISA评估scFv细胞内表达,通过硫黄素T法检测它的分解淀粉样蛋白聚集体的能力。此外,体内表达和治疗效果在淀粉样变性小鼠模型中分别由抗6x组氨酸标记-ELISA和抗-Aβ ELISA测定。 结果:选定的抗β淀粉样特异性单链抗体可变区基因片段对Aβ表现出亲和力,能在体内分解Aβ纤维 。然而在癌细胞中DNA和mRNA转染均能引起scFv表达,只有在原发性神经元中mRNA引起可观察的scFv。此外,由原发性神经元分泌的3,4-折叠scFv造成IL2ss使用的增加超过了mRNA复合物分泌信号的缺乏。在体内,3到11倍的颅内抗体水平测定mRNA与DNA形成体内抗体水平和更高的含il2ss mRNA在非获得分泌表达。与il2ss血型βmRNA形成颅内注射导致40%个β减少急性变性模型;没有降低检测单链mRNA和DNA形成的控制。然而,没有在更具挑战性的阿尔茨海默病转基因模型检测到Aβ的减少 。 结论:我们的研究结果不仅为阿尔茨海默氏病的治疗,也为神经系统疾病的免疫治疗介绍了一种协调的方法。我们的平台的有效性所需的抗βscFv颅内递送mRNA没有DNA,与il2ss分泌序列有利于在淀粉样变性模型β参与mRNA的表达,对神经细胞的高效转染智能共聚物络合,达到检测mRNA的表达在大脑中的淀粉样变性模型急性48h。淀粉样蛋白的减少只能通过这三个因素(mRNA编码单链抗体,聪明的共聚物,il2ss)集成到一个单一的配方。
关键词: 阿尔茨海默症,信使RNA,被动免疫疗法,单链抗体可变区基因片段
Current Alzheimer Research
Title:Improved Brain Expression of Anti-Amyloid β scFv by Complexation of mRNA Including a Secretion Sequence with PEG-based Block Catiomer
Volume: 14 Issue: 3
Author(s): Federico Perche, Satoshi Uchida, Hiroki Akiba, Chin-Yu Lin, Masaru Ikegami, Anjaneyulu Dirisala, Toshihiro Nakashima and Keiji Itaka, Kohei Tsumoto, Kazunori Kataoka.
Affiliation:
关键词: 阿尔茨海默症,信使RNA,被动免疫疗法,单链抗体可变区基因片段
摘要: Background: The ever-increasing number of people living with Alzheimer’s disease urges to develop more effective therapies. Despite considerable success, anti-Alzheimer immunotherapy still faces the challenge of intracerebral and intracellular delivery. This work introduces in situ production of anti-amyloid beta (Aβ) antibody after intracerebral injection of PEG-PAsp(DET)/mRNA polyplexes as a novel immunotherapy approach and a safer alternative compared to high systemic antibodies doses or administration of adenovirus encoding anti- Aβ antibodies.
Methods: We used mRNA encoding three different Aβ-specific scFV with a secretion signal for passive immunotherapy. scFv contained a 6xHis-tag for immuno-detection. The secretion signal from IL2 (IL2ss) was added to allow extracellular engagement of senile plaques. Aβ affinity of scFv was measured by surface plasmon resonance. To allow intracellular delivery, scFv were administered as polyplexes formed with our smart copolymer polyethylene glycol-poly[N’-[N-(2-aminoethyl)-2-aminoethyl] aspartamide] [PEG-PAsp (DET)]. We evaluated scFv expression in cellulo by Western blot and ELISA, their ability to disaggregate amyloid aggregates by thioflavine T assay. Moreover, in vivo expression and therapeutic activity were evaluated in a murine amyloidosis model, by anti-6xHis-tag ELISA and anti- Aβ ELISA, respectively. Results: The selected anti-amyloid beta scFv showed affinity towards Aβ and disaggregated Aβ fibers in vitro. Whereas both DNA and mRNA transfection led to scFV expression in cancer cells, only mRNA led to detectable scFv expression in primary neurons. In addition, the use of IL2ss increased by 3.4-fold scFv secretion by primary neurons over mRNA polyplexes devoid of secretion signal. In vivo, a 3 to 11- fold of intracranial scFv levels was measured for mRNA compared to DNA polyplexes and higher in vivo scFv levels were obtained with mRNA containing IL2ss over non-secreted mRNA. Intracranial injection of anti-Aβ mRNA polyplexes with IL2ss resulted in 40 % Aβ decrease in an acute amyloidosis model; with no decrease detected with control scFv mRNA nor DNA polyplexes. However, no Aβ decrease was detected in a more challenging transgenic model of Alzheimer’s disease. Conclusion: Our results introduce a concerted approach not only for Alzheimer’s disease treatment but also for immunotherapy against neurological diseases. The effectivity of our platform required the intracranial delivery of anti-Aβ scFv as mRNA not DNA, as mRNA with an IL2ss secretion sequence to favor engagement of Aβ in the amyloidosis model, complexation with a smart copolymer for efficient transfection of primary neurons and to achieve detectable mRNA expression in the brain during 48h. Amyloid burden decrease in an acute amyloidosis model was only achieved when these three factors (mRNA coding scFv, smart copolymer, IL2ss) were integrated into a single formulation.Export Options
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Cite this article as:
Federico Perche, Satoshi Uchida, Hiroki Akiba, Chin-Yu Lin, Masaru Ikegami, Anjaneyulu Dirisala, Toshihiro Nakashima and Keiji Itaka, Kohei Tsumoto, Kazunori Kataoka. , Improved Brain Expression of Anti-Amyloid β scFv by Complexation of mRNA Including a Secretion Sequence with PEG-based Block Catiomer, Current Alzheimer Research 2017; 14 (3) . https://dx.doi.org/10.2174/1567205013666161108110031
DOI https://dx.doi.org/10.2174/1567205013666161108110031 |
Print ISSN 1567-2050 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5828 |
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