Structured DNA Nanoparticles Therapeutic mRNA and CRISPR/Cas9 Delivery

项目摘要 治疗性基因编辑CRISPR核糖核蛋白（RNP）复合物向细胞的靶向递送提供了主要的 然而，实现这一目标需要一种工具来保护， 将CRISPR RNP引导至其靶细胞和组织。目前，还没有车辆可以纳入 CRISP RNP用于体内细胞递送。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RNP的多功能载体，利用RNP和靶向递送载体之间的碱基配对， DNA-NP以促进RNP掺入和受控释放。DNA-NP将被设计成具有化学计量比。 控制它们内部携带的RNP有效载荷的数量，并在外部用抗体进行功能化 以及控制细胞靶向和细胞内运输的脂质结合部分。我们会尽量减少巨噬细胞 通过聚乙二醇（PEG）表面功能化， 和其他生物相容性钝化技术，使用纳米颗粒生理和 免疫相互作用CRISPR RNP从DNA-NP的结合和释放将在体外测定，并且 DNA-RNP碱基配对将确保稳定的结合和货物从媒介物的容易释放。基因组编辑将 通过下一代测序和靶向PCR进行测定，这将确保酶促 货物的活动在交付期间保持完整。将CRISPR RNP靶向培养的细胞亚群 将评价表达细胞特异性表面受体的细胞，特别关注靶基因 编辑感兴趣的细胞。我们的RNP交付平台的通用性提供了一个独特的， 通过靶向治疗一系列致命的和目前无法治疗的遗传疾病和癌症的方法 CRISPR的传递。

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.