Validation of a cell-based assay for BoNT/A detection against the mouse bioassay

描述(申请人提供)：肉毒杆菌神经毒素(BONT)是人类已知的毒性最大的物质，中毒后会导致呼吸衰竭。尽管BoNTs具有致命性，但它具有临床健康和美容应用，目前已获FDA批准用于治疗颈部肌张力障碍、眼睑痉挛、颅神经VII障碍、眉间纹(皱纹)和宇宙。BONT通过使引起肌肉收缩的神经元沉默来缓解肌肉紧张。对于许多疾病，基于BONT的治疗提供了显著和持久的疼痛减轻。BONT精致的神经元特异性和较长的作用时间使其成为治疗常规治疗失败的神经和肌肉疾病的首选药物。目前，基于BONT的治疗的质量控制和量化是通过小鼠生物测定实现的，该方法包括将样本注射到小鼠体内，并计算注射后一天或多天死亡的小鼠的数量。以BONT为基础的药物的每个制造商都使用独特的小鼠生物检测方案；结果是非标准化的活动单位。这种不一致导致了对制造商对制造商活动和等效剂量的混淆，从而将患者置于危险之中。此外，小鼠实验低通量、费时费力，而且会牺牲大量动物。这项第一阶段提案的总体目标是验证BioSentinel独特的基于细胞的检测BONT/A的方法与小鼠生物检测方法的对比。由于BONT具有三种活性--细胞受体结合和摄取、囊泡转位和靶向切割--因此，只有基于细胞的检测方法才能以与小鼠生物检测相同的保真度有效地测量BONT活性。BioSentinel与Metabiologics合作，建议优化BioSentinel基于细胞的BONT/A检测方法，生成BONT/A参考标准，确定该分析是否提供了比小鼠生物测定更高的精密度，并使用热应激的BONT/A样本，确定基于细胞的分析是否以与小鼠生物测定相同的保真度准确测量BONT/A样本的活性。BioSentinel经过验证的基于细胞的分析将使研究人员和制造商能够对BONT/A制剂进行量化，对BONT/A抑制剂进行高通量筛选，并使用无动物、成本效益高的系统开发基于BONT的新疗法。本试验也将满足SBIR征集PA-09-179《用于评估A型肉毒神经毒素效力的体外试验的发展》的要求。 公共卫生相关性：肉毒杆菌神经毒素是一种极其致命的细菌毒素，也被广泛用于化妆品和药物应用，缓解肌肉收缩和疼痛。目前，基于肉毒杆菌神经毒素的治疗是使用小鼠生物测定进行量化的，其中动物死亡是测定的终点。BioSentinel建议验证其无动物、基于细胞的肉毒杆菌神经毒素检测方法，以取代以肉毒杆菌神经毒素为基础的产品的质量控制和量化的小鼠生物检测方法。

DESCRIPTION (provided by applicant): Botulinum neurotoxins (BoNT) are the most toxic substances known to humans, causing respiratory failure upon intoxication. Despite their lethality, BoNTs have clinical health and cosmetic applications and are currently FDA-approved for treating cervical dystonia, blepharospasm, cranial nerve VII disorders, glabellar lines (wrinkles), and cosmoses. BoNT provides relief of muscle tension by silencing neurons that cause muscle contraction. For many disorders, BoNT-based treatments provide significant and long-lasting reductions in pain. BoNT's exquisite specificity for neurons and long time of action make it a lead candidate for the treatment of neurological and muscle disorders where conventional treatments have failed. Quality control and quantification of BoNT-based therapies are currently achieved using the mouse bioassay which involves injecting samples into mice and counting the number of mice that die one or more days post-injection. Each manufacturer of a BoNT-based pharmaceutical uses a unique protocol for the mouse bioassay; the result is non-standardized units of activity. This non-uniformity has led to confusion about manufacturer-to-manufacturer activity and equivalent dosing, thus putting patients at risk. In addition, the mouse assay is low-throughput, time- and labor-intensive, and sacrifices large numbers of animals. The overall objective of this Phase I proposal is to validate BioSentinel's unique cell-based assay for BoNT/A detection against the mouse bioassay. Because BoNT has three activities-cell receptor binding and uptake, vesicle translocation, and target cleavage-only a cell-based assay can effectively measure BoNT activity with the same fidelity as the mouse bioassay. BioSentinel, in collaboration with Metabiologics, proposes to optimize BioSentinel's cell-based assay for BoNT/A detection, generate a BoNT/A reference standard, determine whether the assay offers improved precision compared to the mouse bioassay, and, using thermally stressed BoNT/A samples, determine whether the cell-based assay accurately measures the activity of BoNT/A samples with the same fidelity as the mouse bioassay. BioSentinel's validated cell-based assay will enable researchers and manufacturers to quantitate BoNT/A preparations, perform high-throughput screens for BoNT/A inhibitors, and develop new BoNT-based therapies using an animal-free, cost-effective system. This assay will also satisfy the requirements of SBIR solicitation PA-09-179, "Development of In-Vitro Assays to Assess the Potency of Botulinum Neurotoxin Type A." PUBLIC HEALTH RELEVANCE: Botulinum neurotoxins are extremely lethal bacterial toxins that are also widely used for cosmetic and pharmaceutical applications providing relief of muscle contraction and pain. Currently, botulinum neurotoxin-based therapies are quantified using the mouse bioassay where animal death is the assay endpoint. BioSentinel proposes to validate its animal-free, cell-based assay for detecting botulinum neurotoxin against the mouse bioassay with the goal of replacing the mouse bioassay for quality control and quantification of botulinum neurotoxin-based products.