Structured DNA Nanoparticles Therapeutic mRNA and CRISPR/Cas9 Delivery

项目总结 靶向将治疗性基因编辑CRISPR核糖核蛋白(RNP)复合体输送到细胞可提供主要 然而，下一代疗法的机会，实现这一目标需要一个工具来保护和 将CRISPR RNP定向到它们的目标细胞和组织。目前，还没有车辆可以将 用于体内细胞递送的脆性RNPs。在这里，我们建议使用结构化DNA纳米颗粒(DNA-NPs)作为 用于靶向递送RNPs的多功能载体，利用RNP和RNP之间的碱基配对 DNA-NP促进RNP的掺入和控制释放。DNA-NPs将按照化学计量比进行设计 对其内部携带的RNP有效载荷数量进行控制，并在外部使用抗体进行功能化 和脂结合部分，以控制细胞靶向和细胞内转运。我们将最大限度地减少巨噬细胞 聚乙二醇(PEG)表面功能化，吞噬和增强血液相容性 和其他生物兼容钝化技术，使用标准的纳米颗粒生理和 免疫相互作用。将在体外检测DNA-NPs中CRISPR RNPs的结合和释放，以及 DNA-RNP碱基配对将确保稳定结合和方便地从车辆上释放货物。基因组编辑将 通过下一代测序和靶向PCR进行检测，这将确保酶 货物在交付过程中的活动保持不变。以CRISPR RNPs为靶点的培养亚群 将对表达细胞表面受体的细胞进行评估，并特别注意靶向基因。 在感兴趣的单元格中编辑。我们的RNP交付平台的通用性提供了一种独特且具有变革性的 靶向治疗一系列致命和目前无法治疗的遗传病和癌症的方法 CRISPR的交付。

PROJECT SUMMARY Targeted delivery of therapeutic gene editing CRISPR ribonucleoprotein (RNP) complexes to cells offers major opportunities for next-generation therapies, however, realization of this goal requires a vehicle to protect and direct CRISPR RNPs to their targeted cells and tissues. Currently, there are no vehicles that can incorporate CRISP RNPs for cellular delivery in vivo. Here, we propose to use structured DNA nanoparticles (DNA-NPs) as versatile carriers for the targeted delivery of RNPs, taking advantage of base-pairing between the RNP and the DNA-NP to facilitate RNP incorporation and controlled release. DNA-NPs will be designed with stoichiometric control over the number of RNP payloads they carry internally, and functionalized externally with antibodies and lipid binding moieties to control cellular targeting and intracellular trafficking. We will minimize macrophage engulfment and enhance blood compatibility through surface functionalization with polyethylene glycol (PEG) and other biocompatible passivation techniques, using standard assays for nanoparticle physiological and immunological interactions. Binding and release of CRISPR RNPs from DNA-NPs will be assayed in vitro, and DNA-RNP base pairing will ensure stable binding and facile release of cargo from vehicle. Genome editing will be assayed through both next generation sequencing and targeted PCR, which will esure that the enzymatic activity of the cargo remains intact during delivery. Targeting CRISPR RNPs to sub-populations of cultured cells expressing cell specific surface receptors will be evaluated, with particular attention paid targeted gene editing in cells of interest. The generality of our RNP delivery platform offers a unique and transformative approach to treating a range of deadly and currently untreatable genetic diseases and cancers by targeted delivery of CRISPR.