Leveraging biodiversity and utilizing genetic engineering to expand the structure and function of silk fibroin biopolymers for biomedical applications

利用生物多样性和基因工程扩展丝素蛋白生物聚合物的结构和功能，用于生物医学应用

• 批准号: 10680505
• 负责人: Whitney L Stoppel
• 金额: $ 36.16万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680505
• 关键词: Address; Agriculture; Amino Acid Sequence; Award; Bacteria; Binding; Biochemical; Biodiversity; Biological; Biophysics; Biopolymers; Bombyx; Bombyx mori; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Embryo; Fiber; Fibroins; Future; Genetic Engineering; Goals; Growth Factor; Healthcare; Human; Humidity; Immune response; Implant; In Vitro; Injections; Insecta; Interruption; Investigation; Length; Lepidoptera; Life Cycle Stages; Mammalian Cell; Medical Device; Medicine; Modification; Moths; Muscle rehabilitation; Natural regeneration; Outcome; Peptides; Pharmacologic Substance; Phase; Phenotype; Polymers; Population; Process; Production; Proteins; Public Health; Pupa; Research; Research Personnel; Silk; Site; Solvents; Stimulus; Structure; Temperature; Tissue Engineering; Traction; Transcriptional Regulation; Translations; United States Food and Drug Administration; Work; aqueous; design; implantation; improved; in vivo; manufacture; mechanotransduction; pathogen; protein aminoacid sequence; scale up; success; surgery material; tissue reconstruction; tool

Project Summary Materials for applications in healthcare and medicine usually come from two main groups (a) synthetic polymers specifically designed to achieve a certain goal or (b) naturally derived biopolymers that are leveraged in their native or slightly modified state for a specific goal. The advantage of being a synthetic chemist is that technically, if you can synthesize it, the possibilities are infinite, but the downfall is that often solvents or portions of the polymer cause cytocompability issues or concerns when it comes to translation and implantation in a human. Alternatively, unmodified natural biopolymers, or proteins, have an easier path toward Food and Drug Administration's approval, but lack the customizability afforded in synthesis or chemical modification. Genetic engineering via production of small peptides in bacteria has improved the availability of customizable short peptides, but proteins on the order of hundreds of kilodaltons cannot be produced this way. This is the case for the silk fibroin biopolymers isolated from caterpillars in the Lepidoptera order, where the heavy chain of silk fibroin is known to be over 300 kilodaltons in length. Genetic engineering, using tools such as CRISPR or PiggyBac, provides an avenue for theoretically modifying the sequence of silk proteins, which has been attempted with limited success in the domesticated silkworm, Bombyx mori. However, silk is collected from the cocoon of the B. mori pupae, meaning that the life cycle of this silkworm is interrupted, making it difficult to maintain these modified populations or assess phenotypes in a high-throughput manner. To address this, silk fibroin will be isolated from an entirely different silk-producing species: Plodia interpunctella, or the Indianmeal moth. Under specific conditions, this agricultural pest produces sheets of silk prior to entering the cocooning phase. These easily collectable sheets of silk fibers can then be cleaned, degummed, and regenerated to an aqueous biopolymer solution. Moreover, unlike B. mori, P. interpunctella silk collection does not interrupt the life cycle of the silkworm/moth and these silkworms are easier to stably genetically modify though embryo injections compared to B. mori. In this Maximizing Investigators' Research Award, genetic engineering will be leveraged to modify the silk fibroin protein sequence at the organismal level, adding in new peptide sequences such as mammalian cell binding motifs or sites for human growth factor sequestration. Scale-up of the process will be achieved via transcriptional regulation of silk fibroin as a function of external stimuli such as humidity or pathogens. Together, these two strategies for enhancing the bio-functionality of the silk fibroin protein and the scale-up required for advanced manufacturing of medical devices or materials will be explored. The outcomes of this work include full biophysical, biochemical, and in vivo characterization of these materials through analysis of systemic and local immune responses in vivo, complete characterization of the new biopolymer structures, and investigation of mechanotransduction in these materials in vitro. Future work aims to leverage this new class of biopolymers for specific applications in pharmaceutical delivery, tissue engineering, and muscle rehabilitation.

项目摘要 医疗保健和医学应用的材料通常来自两个主要组（a）合成聚合物 专门为实现某个目标而设计的，或（b）自然得出的生物聚合物 特定目标的本地或略微修改状态。成为合成化学家的优点是从技术上讲， 如果您可以合成它，那么可能性是无限的，但是倒下的是溶剂或部分的部分 当涉及到人类的翻译和植入时，聚合物会引起细胞相应性问题或关注点。 或者，未修饰的天然生物聚合物或蛋白质具有更轻松的食物和药物途径 政府的批准，但缺乏合成或化学修改中提供的可定制性。遗传 通过细菌中的小肽生产工程提高了可自定义短的可用性 肽，但不能以这种方式产生数百公斤的蛋白质。就是这样 从鳞翅目命令中从毛毛虫中分离出的丝正肌蛋白生物聚合物，丝绸的重链 众所周知，纤维蛋白的长度超过300千达的。基因工程，使用CRISPR或等工具 Piggybac，为理论上修改丝蛋白的顺序提供了一种途径， 在驯养的蚕（Bombyx Mori）中取得了有限的成功。但是，丝绸是从 B. mori pupae的茧，这意味着这种蚕的生命周期被中断，使得很难 维持这些改良的人群或以高通量方式评估表型。为了解决这个问题，丝绸 纤维素将与完全不同的丝绸生产物种隔离：Plodia interpunctella或Indianmeal 蛾。在特定条件下，这种农业害虫在进入茧之前会产生丝绸床单 阶段。然后可以清洁，脱胶并再生到一个易于收集的丝绸纤维 水性生物聚合物溶液。此外，与B. Mori不同 蚕/蛾和这些蚕的循环更容易在胚胎注射时稳定地遗传修饰 与B.莫里相比。在最大化研究人员的研究奖中，基因工程将被利用 修改丝正肌蛋白蛋白在生物水平上的序列，以新的肽序列（例如 哺乳动物细胞结合基序或人类生长因子隔离的位点。该过程的扩展将是 通过对丝纤维蛋白的转录调节与外部刺激的函数（例如湿度或 病原体。加在一起，这两种策略是增强丝纤维蛋白蛋白的生物功能和 将探索先进制造医疗设备或材料所需的扩展。结果 这项工作包括通过分析对这些材料的完整生物物理，生化和体内表征 在体内的系统性和局部免疫反应，新生物聚合物结构的完全表征， 并研究了这些材料在体外的机械转导。未来的工作旨在利用这一新课程 用于药物分娩，组织工程和肌肉康复中的特定应用的生物聚合物。