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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要全球有超过 3 亿人受到遗传健康状况的影响超过 4,400 种遗传病。已发现的疾病几乎全部被认为是罕见的，这限制了研究的数量基因疗法因其独特的优势而成为治疗遗传病的一种有吸引力的方法。多功能性和广泛的适用性，例如 CRISPR-Cas9、碱基编辑和 prime 编辑已经彻底改变了基因治疗研究和生命科学的其他领域，然而，很少有基因编辑治疗方法已进入市场，临床转化仍面临重要挑战。需要安全有效的基因治疗递送工具及其创建平台。在这个项目中，我们将设计和测试一类新型可编程、非病毒基因治疗载体和货物 – 受病毒启发的 DNA 折纸 (VIDO) 载体和修复模板 – 以及 Essemblix GT，一种纳米工程与其他基因治疗递送工具相比，VIDO 产品是为其生产量身定制的平台。模块化且易于修改以适应不同的疾病，而且它们结构明确，几乎没有什么问题。据我们所知，这将有助于促进监管批准和临床转化。第一个研究使用 DNA 折纸封装和递送基因编辑剂的项目。在目标 1 中，我们将证明通过折叠和压缩提高 CRISPR-Cas9 敲入效率采用 DNA 折纸方法的同源定向修复 (HDR) 模板将是 VIDO 折叠报告模板。通过电穿孔递送至 HEK293T 和 Jurkat 人类细胞时与非结构化对照进行比较两个不同基因组插入位点的细胞系将通过共聚焦显微镜确定。荧光团标记的模板和敲入效率将通过流式细胞术评估。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 载体可以封装并共同传递 CRISPR-Cas9 编辑剂和 VIDO 模板，很容易被细胞吸收并诱导敲入效率可与通过病毒样颗粒 (VLP) 递送相同药物相媲美。内体逃逸和基因表达将通过共聚焦显微镜和流式细胞术进行追踪。目的是通过 Illumina 测序确认正确的基因组整合。成功完成这些目标将建立 VIDO 载体和模板作为新的可编程基因与现有替代品相比，治疗产品具有关键优势，使其易于快速设计和使用。通过创建此类 VIDO 产品，Essemblix GT 纳米工程平台可以改变基因治疗研究迈向基因治疗工程范例，从而使研究人员能够为罕见病提供更多治疗方法疾病更快地传播给更多患者。