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 用于医疗保健和医学应用的材料通常来自两大类：（a）合成聚合物 或（B）天然衍生的生物聚合物，其在其用途中被利用， 原始状态或为特定目标而稍微修改的状态。作为一名合成化学家的优势在于， 如果你能合成它，可能性是无限的，但失败的是，往往溶剂或部分的 当涉及到在人体内的翻译和植入时，聚合物引起细胞相容性问题或担忧。 另外，未经修饰的天然生物聚合物或蛋白质更容易进入食品和药物领域。 管理部门的批准，但缺乏合成或化学修饰提供的定制性。遗传 通过在细菌中产生小肽的工程化提高了可定制的短肽的可用性， 肽，但数百千道尔顿量级的蛋白质不能以这种方式产生。就是这种情况 从鳞翅目毛虫中分离的丝素蛋白生物聚合物，其中丝的重链 已知丝心蛋白的长度超过300千道尔顿。基因工程，使用CRISPR或 PiggyBac为理论上修饰丝蛋白序列提供了一种途径，该途径已被 在家蚕中尝试了有限的成功。然而，丝绸是从 B的茧这意味着这种蚕的生命周期被打断， 维持这些修饰的群体或以高通量方式评估表型。为了解决这个问题，丝绸 丝心蛋白将从一种完全不同的产丝物种中分离出来：Plodia interpunctella或Indianmeal 蛾在特定的条件下，这种农业害虫在进入茧层之前会产生一层丝 相位这些容易收集的丝纤维片然后可以被清洁、脱胶和再生为 生物聚合物水溶液。而且，不像B。桑、间斑家蚕丝采集不中断生活 蚕/蛾的周期，这些蚕更容易通过胚胎注射进行稳定的遗传修饰 与B相比。桑。在这个最大限度地提高研究人员的研究奖，基因工程将被利用， 在生物体水平上修饰丝素蛋白质序列，加入新的肽序列， 哺乳动物细胞结合基序或用于人生长因子螯合的位点。该进程的规模扩大将是 通过丝纤蛋白的转录调节作为外部刺激如湿度或 病原体总之，这两种用于增强丝素蛋白的生物功能性的策略和用于增强丝素蛋白的生物功能性的策略是有效的。 将探讨医疗设备或材料的先进制造所需的规模扩大。成果 这项工作包括通过分析这些材料的完整生物物理、生物化学和体内特性 体内全身和局部免疫反应，新生物聚合物结构的完整表征， 并在体外研究了这些材料的力学传导。未来的工作旨在利用这个新类 用于药物输送、组织工程和肌肉康复的特定应用的生物聚合物。