Generation of hPSCs Using Reprogramming Proteins-Encapsulated Nanoparticles

描述（由申请人提供）：干细胞有可能通过实现再生医学来彻底改变医疗实践。该提案旨在通过利用一类新的基于纳米颗粒（NP）的载体来开发高效的人诱导多能干细胞（hiPSC）的产生。这些NP能够递送重编程转录因子（TF），即OCT 4、SOX 2、KLF 4和c-MYC。目前的重编程系统引起了安全性问题，因为它们利用四种关键TF的病毒表达，导致具有多种病毒整合的hiPSC。最近已经通过将细胞穿透肽融合的重编程TF引入人体细胞中来证明用于产生hiPSC的更安全的策略，从而避免遗传操作的任何潜在危险。尽管如此，TF的有效递送仍然是产生hiPSC的关键障碍。加州大学洛杉矶分校的三个研究小组，专业知识涵盖合成化学，纳米颗粒，微流体（Tseng和Lu）和干细胞生物学（Pyle）合作并取得了初步成果，包括以下基本概念验证研究：（i）用于递送TF的超分子纳米颗粒（SNP）的自组装生产，（ii）（ii）单蛋白纳米胶囊技术，（iii）用于大规模筛选的数字微反应器，（iv）用于单个hiPSC的定量表型分析的微流控图像细胞术（MIC）技术，以及（v）在生成hiPSC方面的丰富经验。拟议的研究将利用多学科团队实现以下两个具体目标：1）我们将合成各种聚合物构建块，交联剂，功能配体，以及含有四种重编程TF的纳米胶囊，用于自组装基于SNP的递送载体。2)我们将产生重编程TF封装的SNP的组合文库（即，OCT 4/SOX 2/ KLF4/c-MYC？通过对四种含TF的纳米胶囊、聚合物结构单元、交联剂和功能性配体进行比例混合，制备了一种新的单核苷酸多态性（SNP）。将使用具有OCT 4-EGFP报告基因的人ESC衍生的成纤维细胞作为靶细胞。随后，将采用MIC技术通过测量SNP处理的细胞中的多能标记物和集落形成来量化重编程性能。如果效率太低，我们建议共同递送凋亡抑制剂（例如，siRNA_p53）与使用siRNA/TF的四种重编程TF？基于SNP的载体我们预期凋亡抑制剂和重编程TF的组合可以显著提高重编程的效率。 公共卫生相关性：这项研究计划的长期目标是探索使用超分子纳米颗粒（SNP）作为一种新的人工载体类别，用于同时递送四种重编程转录因子（即，OCT 4、S 0X 2、KLF 4和c-MYC）导入人体细胞中以诱导重编程为诱导多能细胞（iPSC）状态。

DESCRIPTION (provided by applicant): Stem cells have the potential to revolutionize medical practice by enabling regenerative medicine. This proposal aims to develop highly efficient generation of human induced pluripotent stem cells (hiPSCs) by utilizing a new class of nanoparticle (NP)-based vectors. These NPs are capable of delivering reprogramming transcription factors (TFs), namely OCT4, SOX2, KLF4 and c-MYC. Current reprogramming systems raise safety concerns because they utilize viral expression of the four key TFs, resulting in hiPSCs with multiple viral integrations. Safer strategies for generating hiPSCs have recently been demonstrated by introducing cell- penetrating peptide-fused reprogramming TFs into human somatic cells, averting any potential dangers of genetic manipulation. Still, effective delivery of TFs remains a key obstacle in creating hiPSCs. Three research groups at UCLA with expertise covering synthetic chemistry, nanoparticles, microfluidics (Tseng and Lu) and stem cell biology (Pyle) have collaborated and accomplished preliminary results consisting of the following fundamental proof-of-concept studies: (i) self-assembly production of supramolecular nanoparticles (SNPs) for delivery of TFs, (ii) (ii) a single protein nano-capsule technology, (iii) digital microreactors for large-scale screening, (iv) microfluidic image cytometry (MIC) technology for quantitative phenotyping of single hiPSCs, and (v) extensive experience in generating hiPSCs. The proposed research will leverage the multidisciplinary team to implement the following two specific aims: 1) We will synthesize a variety of polymer building blocks, cross linkers, functional ligands, as well as, nano- capsules containing the four reprogramming TFs for self-assembly of SNP-based delivery vectors. 2) We will generate a combinatorial library of reprogramming TF-encapsulated SNPs (i.e., OCT4/SOX2/ KLF4/c-MYC?SNPs) by performing ratiometric mixing of the four TF-containing nano-capsules, polymer building blocks, cross linkers and functional ligands. Human ESC-derived fibroblast cells with an OCT4-EGFP reporter will be employed as target cells. Subsequently, the MIC technology will be employed to quantify reprogramming performance by measuring pluripotent markers and colony formation in the SNPs-treated cells. If the efficiency is too low, we propose to co-deliver apoptotic inhibitors (e.g., siRNA_p53) with the four reprogramming TFs using siRNA/TFs?SNPs-based vectors. We anticipate that combination of apoptotic inhibitors and reprogramming TFs could dramatically increase the efficiency of reprogramming. PUBLIC HEALTH RELEVANCE: The long-term objective of this research proposal is to explore the use of supramolecular nanoparticles (SNPs) as a new category of artificial vector for simultaneous delivery of the four reprogramming transcription factors (i.e., OCT4, SOX2, KLF4 and c-MYC) into human somatic cells to induce reprogramming to the induced pluripotent cell (iPSC) state.