A nanoengineering platform for programmable gene editing therapies against rare diseases

用于针对罕见疾病的可编程基因编辑疗法的纳米工程平台

• 批准号: 10699037
• 负责人: Steven L Armentrout
• 金额: $ 32.5万
• 依托单位: PARABON NANOLABS, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10699037
• 关键词: Affect; Algorithms; Biological Sciences; Bone Marrow; CRISPR/Cas technology; Capital; Carrying Capacities; Cell Line; Cells; Charge; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Computer-Aided Design; Confocal Microscopy; DNA; Disease; Dose; Effectiveness; Electroporation; Elements; Encapsulated; Endosomes; Endothelial Cells; Engineering; FDA approved; Face; Flow Cytometry; Gene Delivery; Gene Expression; Gene Transfer; Genes; Genetic; Genetic Diseases; Genome; Genomics; Goals; Health; Heritability; Human Cell Line; Immune response; Inflammatory; Insertional Mutagenesis; Jurkat Cells; Knock-in; Label; Machine Learning; Marketing; Medical; Medical Technology; Methods; Molecular; Nanostructures; Nanotechnology; Nuclear; Nucleic Acids; Outcome; Patients; Peptides; Persons; Phase; Polymers; Production; Rare Diseases; Reporter; Reporting; Research; Research Personnel; Safety; Services; Shapes; Site; Small Business Innovation Research Grant; Software Design; Specificity; Structure; Surface; System; Testing; Therapeutic Studies; Transgenes; Vendor; Viral Vector; Virus; Virus-like particle; base editing; capsule; clinical translation; commercial application; commercialization; delivery vehicle; design; falls; fluorophore; gene therapy; genome editing; human stem cells; improved; in vivo; lipid nanoparticle; manufacturing cost; nanocarrier; nanoengineering; nanofabrication; non-viral gene delivery; non-viral gene therapy; novel; operation; particle; prime editing; rare genetic disorder; repaired; scale up; therapeutic genome editing; vector

PROJECT SUMMARY More than 300 million people worldwide are affected by a genetic health condition. Over 4,400 genetic diseases have been identified; nearly all of which are considered rare, which limits the amount of research each receives. Gene therapy is an attractive approach for treatment of genetic disease because of its versatility and broad applicability. Genome editing systems such as CRISPR-Cas9, base editing and prime editing have revolutionized gene therapy research and other fields of life science, however, few gene editing treatments have reached the market and clinical translation still faces important challenges. Among them is the need for safe and effective gene therapy delivery vehicles and platforms for their creation. In this project, we will design and test a new class of programmable, non-viral gene therapy carriers and cargo – virus-inspired DNA origami (VIDO) vectors and repair templates – and Essemblix GT, a nanoengineering platform tailored for their production. In contrast to other gene therapy delivery vehicles, VIDO products are modular and easily modified for different diseases. Moreover, they are structurally well-defined with little intermolecular variability, facilitating regulatory approval and clinical translation. To our knowledge, this will be the first project to investigate the use of DNA origami for encapsulation and delivery of gene editing agents. In Aim 1, we will demonstrate that CRISPR-Cas9 knock-in efficiency is improved by folding and compacting homology-directed repair (HDR) templates with DNA origami methods. VIDO-folded reporter templates will be compared against unstructured controls when delivered via electroporation to HEK293T and Jurkat human cell lines at two different genome insertion sites. Nuclear entry will be determined by confocal microscopy of fluorophore-labeled template and knock-in efficiency will be assessed by flow cytometry. In Aim 2, using the same cell lines and genomic targets, we will demonstrate VIDO vectors can encapsulate and co-deliver CRISPR-Cas9 editing agents and VIDO templates, are readily taken up by cells and induce knock-in efficiency that is competitive with delivery of the same agents via virus-like particles (VLP). Endosomal escape and gene expression will be tracked via confocal microscopy and flow cytometry. In both aims, correct genomic integration will be confirmed via Illumina sequencing. Successful completion of these aims will establish VIDO vectors and templates as new, programmable gene therapy products with key advantages over existing alternatives. By making it practical to rapidly design and create such VIDO products, the Essemblix GT nanoengineering platform could shift gene therapy research toward a paradigm of gene therapy engineering, thus enabling researchers to deliver more treatments for rare diseases to more patients more quickly.

项目摘要 全世界有3亿多人受到遗传健康状况的影响。超过4,400个基因 疾病已经被确定;几乎所有这些都被认为是罕见的，这限制了研究的数量 每个人都收到。基因治疗是治疗遗传性疾病的一种有吸引力的方法，因为其 通用性和广泛的适用性。基因组编辑系统，如CRISPR-Cas9，碱基编辑和引物 基因编辑已经彻底改变了基因治疗研究和其他生命科学领域，然而，很少有基因编辑 治疗方法已进入市场，临床翻译仍面临重大挑战。其中之一是 需要安全和有效的基因治疗递送载体和平台来创建它们。 在这个项目中，我们将设计和测试一类新的可编程的，非病毒的基因治疗载体和货物 - 病毒启发的DNA折纸（VIDO）载体和修复模板-以及纳米工程 为他们的生产量身定制的平台。与其他基因治疗载体相比，VIDO产品 模块化且易于修改以适应不同疾病。此外，它们在结构上定义明确， 分子间变异性，促进监管批准和临床转化。据我们所知，这将是 第一个研究DNA折纸用于基因编辑剂的封装和递送的项目。 在目标1中，我们将证明CRISPR-Cas9敲入效率通过折叠和压缩而提高。 同源定向修复（HDR）模板与DNA折纸方法。VIDO折叠报告模板将 与通过电穿孔递送至HEK 293 T和Jurkat人细胞时的非结构化对照相比 两个不同的基因组插入位点。将通过共聚焦显微镜测定细胞核进入。 通过流式细胞术评估荧光团标记的模板和敲入效率。 在目标2中，使用相同的细胞系和基因组靶标，我们将证明VIDO载体可以包封 并且共递送CRISPR-Cas9编辑剂和VIDO模板，容易被细胞摄取并诱导细胞凋亡。 与通过病毒样颗粒（VLP）递送相同试剂竞争的敲入效率。 将通过共聚焦显微镜和流式细胞术跟踪内体逃逸和基因表达。无论是 因此，将通过Illumina测序确认正确的基因组整合。 这些目标的成功完成将建立VIDO载体和模板作为新的可编程基因 与现有替代品相比具有关键优势的治疗产品。通过使快速设计和 创建这样的VIDO产品，Eschemblix GT纳米工程平台可以改变基因治疗研究 走向基因治疗工程的范式，从而使研究人员能够提供更多的治疗罕见的 让更多的病人更快地得到治疗。