A nanoengineering platform for programmable gene editing therapies against rare diseases

项目总结 全球有超过3亿人受到基因健康问题的影响。超过4,400个基因 疾病已经被发现；几乎所有的疾病都被认为是罕见的，这限制了研究的数量 每个人都收到了。基因治疗是治疗遗传病的一种有吸引力的方法，因为它具有 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PRIME 编辑使基因治疗研究和其他生命科学领域发生了革命性的变化，然而，很少有基因编辑 治疗已经推向市场，临床翻译仍然面临着重要的挑战。其中包括 需要安全和有效的基因治疗提供工具和平台来创造。 在这个项目中，我们将设计和测试一种新型的可编程的、非病毒的基因治疗载体和货物 -病毒激发的DNA折纸(VIDO)载体和修复模板-以及纳米工程EsSemblix GT 为他们的生产量身定做的平台。与其他基因治疗载体相比，Vido产品 模块化，易于针对不同的疾病进行修改。此外，它们在结构上定义良好，几乎没有 分子间的可变性，便于监管批准和临床翻译。据我们所知，这将是 第一个项目是研究DNA折纸在基因编辑剂的封装和传递中的应用。 在目标1中，我们将证明CRISPR-Cas9的插入效率通过折叠和紧凑来提高 使用DNA折纸方法的同源定向修复(HDR)模板。视频折叠的记者模板将是 通过电穿孔传递给HEK293T和Jurkat人细胞时与非结构对照的比较 在两个不同的基因组插入点处。核进入将通过共聚焦显微镜确定 荧光标记的模板和敲入效率将通过流式细胞术进行评估。 在目标2中，使用相同的细胞系和基因组靶点，我们将演示Vido载体可以包裹 和共同交付的CRISPR-Cas9编辑剂和VIDO模板，很容易被细胞吸收和诱导 与通过病毒样颗粒(VLP)输送相同试剂相比，敲击效率具有竞争力。 内体逃逸和基因表达将通过共聚焦显微镜和流式细胞仪进行跟踪。在这两个地方 目的，正确的基因组整合将通过Illumina测序得到确认。 这些目标的成功完成将使Vido载体和模板成为新的可编程基因 与现有替代品相比具有关键优势的治疗产品。通过使快速设计和实现 创造这样的Vido产品，EsSemblix GT纳米工程平台可能会改变基因治疗的研究 走向基因治疗工程的范例，从而使研究人员能够为罕见的疾病提供更多的治疗方法 疾病更快地传给更多的患者。

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.