Molecular Evolution of Chondroitinase ABCI for SCI

描述（由申请人提供）：脊髓损伤（SCI）是一种破坏性事件，仅在美国每年就影响约11，000人。虽然大多数受害者都能活下来，但患者往往会终生瘫痪。60%的伤害发生在16至30岁的人群中。每位患者的终身医疗费用平均约为150万美元，加上失去的收入潜力，使SCI成为个人和国家层面的主要负担。SCI导致永久性损伤的部分原因是神经元不能通过损伤后形成的胶质瘢痕再生。这种神经胶质瘢痕含有细胞外基质分子，包括硫酸软骨素蛋白聚糖（CSPG）。软骨素酶ABCI（cABCI）是催化硫酸软骨素碳水化合物链（如在SPG上发现的那些）降解的细菌酶。最近在SCI动物模型中的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。虽然在动物模型中观察到的益处是显著的，并且可能转化为人类状况，但软骨素酶疗法受到cABCI在体温下的相对热不稳定性的挑战。短的酶半衰期需要频繁给药，限制了动物试验的规模，也使人类治疗具有挑战性。在体温下具有增加的稳定性的cABCI酶将极大地促进研究，并通过允许使用留置泵和持续释放制剂而不是重复鞘内注射来使人类治疗更安全、更容易和更具有成本效益。该补助金的第一阶段旨在提高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。鉴于在这一初步工作中取得的成功，第二阶段将推进主要候选人进入动物疗效和临床前开发。cABCI的热稳定性将通过VerdiZyme的定向基因组装（DGA）技术得到改善。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。在该提议中，从这些成功的DE实验中的几个中提取的技术将用于构建具有增加的热稳定性的cABCI酶的变体，其通过（i）基于知识的合理蛋白质重新设计，（ii）随机诱变，和（iii）通过重组分离有利的和有害的突变。将对变体进行筛选以增强热稳定性，并重新组合以使用DGA进行优化。主要候选人将被净化和定性。该建议的目的是1）产生多达100，000个软骨素酶突变体的文库2）筛选文库以增强软骨素酶的热稳定性，3）重新分类鉴定的菌株以分离有利的组合和4）纯化和表征软骨素酶产物候选物。

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.