Molecular Evolution of Chondroitinase ABCI for SCI

SCI 软骨素酶 ABCI 的分子进化

• 批准号: 6932827
• 负责人: ANTHONY O CAGGIANO
• 金额: $ 18.44万
• 依托单位: ACORDA THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2006-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6932827
• 关键词: biotechnology; chondroitin sulfates; directed evolution; drug screening /evaluation; enzyme activity; enzyme therapy; genetic library; hyaluronidase; mass spectrometry; molecular genetics; nervous system disorder chemotherapy; nucleic acid sequence; peptide chemical synthesis; polymerase chain reaction; protein engineering; protein purification; proteoglycan; spinal cord injury; synthetic enzyme; thermostability

DESCRIPTION (provided by applicant): Spinal cord injury (SCI) is a devastating event affecting approximately 11,000 individuals each year in the US alone. Although most victims survive, the individual is often paralyzed for the rest of his or her life. Sixty percent of injuries occur in individuals between the ages of 16 and 30. Life-time medical costs average about $1.5 million dollars per patient and combined with lost earning potential make SCI a major burden both on an individual and national level. SCI results in permanent injury in part because neurons fail to regenerate through the glial scar that is formed after injury. This glial scar contains extracellular matrix molecules including chondroitin sulfate proteoglycans (CSPGs). Chondroitinase ABCI [cABCI) is a bacterial enzyme that catalyzes the degradation of chondroitin sulfate carbohydrate chains such as those found on SPGs. Exciting recent work in animal models of SCI has shown that treatment with cABCI dramatically improves both motor and autonomic functional outcomes after injury. Although the benefits observed in animal models are significant and will likely translate to the human condition, chondroitinase therapies are challenged by the relative thermal instability of cABCI at body temperatures. The short enzyme halflife necessitates frequent dosing, limiting the size of animal trials and making human therapy challenging as well. A cABCI enzyme with increased stability at body temperatures will greatly facilitate research and make human therapies safer, easier and more cost effective by allowing use of indwelling pumps and sustained release formulations rather than repeated intrathecal injections. Phase 1 of this grant is designed to improve the thermal stability of cABCI to make it a more useful agent and potential human therapeutic. Given success during this initial work, phase 2 will advance the lead candidate into animal efficacy and preclinical development. Thermal stability of cABCI will be improved through VerdiZyme's Directed Gene Assembly (DGA) technology. This directed evolution (DE) approach has been used previously to increase the thermal stability of numerous enzymes. In this proposal, techniques drawn from several of these successful DE experiments will be used to construct variants of the cABCI enzyme with increased thermal stability through (i) knawledge-based-rational protein redesign, (ft) random mutagenesis, and (Hi) separation of advantageous and detrimental mutations through-recombination. Variants will be screened for enhanced thermal stability and reassorted for optimization with DGA. Lead candidates will be purified-and characterized. The aims of this proposal are 1) Produce a library of up to 100,000 chondroitinase mutants 2) Screen library for enhanced chondroitinase .thermal stability, 3) Re-assort identified aEeles to isolate advantageous combinations and 4) Purify and characterize chondroitinase product candidates.

描述(申请人提供)：脊髓损伤(SCI)是一种毁灭性的事件，仅在美国每年就有大约11,000人受到影响。尽管大多数受害者幸存下来，但个人往往会在其余生中瘫痪。60%的伤害发生在16岁至30岁之间的个人身上。每个患者的终身医疗成本平均约为150万美元，再加上收入潜力的丧失，使脊髓损伤成为个人和国家层面的主要负担。脊髓损伤会导致永久性损伤，部分原因是神经元不能通过损伤后形成的胶质疤痕再生。这种胶质瘢痕含有细胞外基质分子，包括硫酸软骨素蛋白多糖(CSPGs)。软骨素酶ABCI[cABCI]是一种细菌酶，催化硫酸软骨素碳水化合物链的降解，如SPGS上发现的那些。最近在脊髓损伤动物模型中令人兴奋的研究表明，cABCI治疗显著改善了损伤后的运动和自主神经功能结果。尽管在动物模型中观察到的益处是显著的，并可能转化为人类的情况，但软骨素酶疗法受到cABCI在体温下相对热不稳定的挑战。酶的半衰期很短，需要频繁给药，限制了动物试验的规模，也使人类治疗具有挑战性。一种在体温下具有更高稳定性的cABCI酶将极大地促进研究，并通过允许使用留置泵和缓释制剂而不是重复鞘内注射，使人类治疗更安全、更容易和更具成本效益。这笔赠款的第一阶段旨在提高cABCI的热稳定性，使其成为更有用的制剂和潜在的人类治疗药物。鉴于这项初步工作的成功，第二阶段将推动主要候选药物进入动物疗效和临床前开发。通过VerdiZyme的定向基因组装(DGA)技术，cABCI的热稳定性将得到改善。这种定向进化(DE)方法以前曾被用来增加许多酶的热稳定性。在这项提案中，从几个成功的DE实验中提取的技术将被用于构建cABCI酶的变体，通过(I)基于知识的合理蛋白质重新设计，(Ft)随机突变，以及(Hi)通过重组分离有利和有害的突变，从而提高热稳定性。将对变种进行筛选，以增强热稳定性，并通过DGA进行重新组合以进行优化。将对候选铅进行提纯-并对其进行表征。该建议的目的是：1)建立高达100,000个软骨素酶突变体文库；2)筛选增强热稳定性的软骨素酶文库；3)对已鉴定的基因进行重新分类，以分离出有利的组合；4)纯化和鉴定软骨素酶候选产物。