Molecular Evolution of Chondroitinase ABCI for SCI

描述（由申请人提供）：脊髓损伤（SCI）是一种毁灭性的事件，仅在美国每年就影响大约11,000人。虽然大多数受害者都能活下来，但他们往往会瘫痪一生。60%的受伤发生在16到30岁之间。每位患者一生的医疗费用平均约为150万美元，再加上失去的收入潜力，使脊髓损伤成为个人和国家层面的主要负担。脊髓损伤导致永久性损伤，部分原因是神经元无法通过损伤后形成的神经胶质瘢痕再生。胶质瘢痕含有细胞外基质分子，包括硫酸软骨素蛋白聚糖（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，如在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治疗可显著改善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，并且很可能转化为人类的情况，但由于cABCI在体温下的相对热不稳定性，软骨素酶治疗受到挑战。由于酶的半衰期短，需要频繁给药，这限制了动物试验的规模，也使人体治疗具有挑战性。一种在体温下稳定性更高的cABCI酶将极大地促进研究，并使人类治疗更安全、更容易和更具成本效益，因为它允许使用留置泵和缓释制剂，而不是重复的鞘内注射。该资助的第一阶段旨在改善cABCI的热稳定性，使其成为更有用的药物和潜在的人类治疗药物。鉴于这一初步工作的成功，第二阶段将推进主要候选药物的动物疗效和临床前开发。通过VerdiZyme的定向基因组装（direct Gene Assembly， DGA）技术，cABCI的热稳定性将得到改善。这种定向进化（DE）方法以前已被用于增加许多酶的热稳定性。在本提案中，从这些成功的DE实验中提取的技术将用于构建具有更高热稳定性的cABCI酶变体，通过(i)基于知识的合理蛋白质重新设计，（ft）随机诱变，以及（Hi）通过重组分离有利和有害突变。变体将筛选增强热稳定性和重新组合优化与DGA。将对主要候选人进行纯化和鉴定。本研究的目标是：1)建立一个包含100,000个骨素酶突变体的文库；2)筛选增强型骨素酶文库。3)重新排序鉴定的aEeles，以分离有利的组合；4)纯化和表征软骨素酶候选产品。

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.