Chen Chen, Przedpelski Amanda, Tepp William H, Pellett Sabine, Johnson Eric A, Barbieri Joseph T
Medical College of Wisconsin, Microbiology and Molecular Genetics, Milwaukee, Wisconsin, USA.
The University of Wisconsin-Madison, Bacteriology, Madison, Wisconsin, USA.
mBio. 2015 Aug 11;6(4):e00734. doi: 10.1128/mBio.00734-15.
Cholera toxin (CT) and the related heat-labile enterotoxins (LT) of Escherichia coli have been implicated as adjuvants in human therapies, but reactivity upon intranasal delivery dampened efforts to develop other clinical applications. However, each CT family member variant has unique biological properties that may warrant development as therapeutic platforms. In the current study, a nontoxic variant of the heat-labile enterotoxin IIa (LTIIa) was engineered to deliver heterologous, functional proteins into the cytosol of neurons. As proof of principle, the LTIIa variant delivered two cargos into neurons. LTIIa delivered β-lactamase efficiently into cells containing complex gangliosides, such as GD1b, as host receptors. LTIIa delivery of β-lactamase was sensitive to brefeldin A, an inhibitor that collapses the Golgi compartment into the endoplasmic reticulum, but not sensitive to treatment with botulinum neurotoxin D (BoNT/D), an inhibitor of synaptic vesicle cycling. LTIIa delivered a single-chain, anti-BoNT/A camelid antibody that inhibited SNAP25 cleavage during post-BoNT/A exposure of neurons. Delivery of functional, heterologous protein cargos into neurons demonstrates the potential of LTII variants as platforms to deliver therapies to inactivate toxins and microbial infections and to reverse the pathology of human neurodegenerative diseases.
This study engineered a protein platform to deliver functional, heterologous proteins into neurons. The protein platform developed was a variant of the heat-labile enterotoxin IIa (LTIIa) which lacked the catalytic domain, yielding a nontoxic protein. As proof of principle, LTIIa variants delivered two functional proteins into neurons, β-lactamase and a camelid antibody. These studies show the utility of LTIIa variants to deliver therapies into neurons, which could be extended to inactivate toxins and microbial infections and potentially to reverse the progression of neurological diseases, such as Alzheimer's disease and Parkinson's disease.
霍乱毒素（CT）及大肠杆菌相关的不耐热肠毒素（LT）已被用作人类治疗中的佐剂，但经鼻给药后的反应性阻碍了其他临床应用的开发。然而，每个CT家族成员变体都具有独特的生物学特性，可能值得开发为治疗平台。在本研究中，对不耐热肠毒素IIa（LTIIa）的无毒变体进行了改造，以将异源功能性蛋白递送至神经元的细胞质中。作为原理验证，LTIIa变体将两种货物递送至神经元中。LTIIa能有效地将β-内酰胺酶递送至含有复杂神经节苷脂（如GD1b）作为宿主受体的细胞中。LTIIa介导的β-内酰胺酶递送对布雷菲德菌素A敏感，布雷菲德菌素A是一种能使高尔基体区室塌陷到内质网中的抑制剂，但对肉毒杆菌神经毒素D（BoNT/D，一种突触小泡循环抑制剂）处理不敏感。LTIIa递送了一种单链抗BoNT/A骆驼科抗体，该抗体在神经元暴露于BoNT/A后抑制SNAP25的裂解。将功能性异源蛋白货物递送至神经元证明了LTII变体作为递送疗法以灭活毒素和微生物感染以及逆转人类神经退行性疾病病理的平台的潜力。
本研究构建了一个蛋白质平台，用于将功能性异源蛋白递送至神经元。所开发的蛋白质平台是不耐热肠毒素IIa（LTIIa）的变体，其缺乏催化结构域，产生无毒蛋白。作为原理验证，LTIIa变体将两种功能性蛋白β-内酰胺酶和骆驼科抗体递送至神经元中。这些研究表明LTIIa变体在向神经元递送疗法方面的效用，这可扩展至灭活毒素和微生物感染，并有可能逆转神经疾病（如阿尔茨海默病和帕金森病）的进展。
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