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 用于医疗保健和医药的材料通常来自两大类：(A)合成聚合物 专门设计以实现特定目标或(B)天然衍生的生物聚合物，这些聚合物在其 特定目标的原生状态或稍作修改的状态。作为一名合成化学家的优势在于，从技术上讲， 如果你能合成它，可能性是无限的，但缺点是通常是溶剂或部分 当涉及到人类体内的翻译和植入时，聚合物会引起细胞相容性问题或担忧。 或者，未经修饰的天然生物聚合物或蛋白质更容易走向食品和药物 政府批准，但缺乏在合成或化学修饰中提供的定制化。遗传 通过在细菌中生产小肽的工程技术提高了可定制短肽的可用性 多肽，但数百千道尔顿量级的蛋白质不能用这种方法生产。情况就是这样。 从鳞翅目毛虫中分离出的丝素生物聚合物，其中丝素的重链 众所周知，丝素蛋白的长度超过300千道尔顿。基因工程，使用CRISPR或 IggyBac，为从理论上修改丝蛋白序列提供了一条途径，该序列已经被 在驯化的家蚕上进行了尝试，但收效甚微。然而，丝绸是从 桑蚕蛹的茧，这意味着这种家蚕的生活史被打断，使其很难 保持这些修改的种群或以高通量的方式评估表型。为了解决这个问题，丝绸 丝素蛋白将从一种完全不同的丝素生产物种中分离出来：间点突起丝菌，或印度粉 飞蛾。在特定的条件下，这种农业害虫在进入结茧之前会产生一片片丝绸。 相位。然后，这些容易收集的丝纤维薄片可以被清洁、脱胶并再生为 含水生物聚合物溶液。此外，与B.Mori不同的是，点状P.interplatea丝绸收集不会中断生命 家蚕/蛾的周期和这些家蚕更容易通过胚胎注射稳定地进行遗传修改 与B.Mori相比。在这个最大化研究人员奖中，基因工程将被用来 在生物体水平上修改丝素蛋白序列，添加新的肽序列，如 人生长因子的哺乳动物细胞结合基序或结合部位。该流程的扩展将是 通过丝素蛋白的转录调节来实现的，作为外部刺激的函数，如湿度或 病原体。总之，这两种增强丝素蛋白生物功能性的策略和 将探索先进的医疗器械或材料制造所需的扩大规模。结果是 这项工作包括通过分析对这些材料进行全面的生物物理、生化和活体表征 体内系统和局部免疫反应的研究，新生物聚合物结构的完整表征， 并对这些材料的体外机械转导进行了研究。未来的工作旨在利用这一新类 生物聚合物在药物输送、组织工程和肌肉康复中的特定应用。