Bioengineering a Safe and Efficient Vector Technology for Stem Cell Transfection

描述(申请人提供)：为了设计干细胞作为癌症治疗的基因载体，它们在体外被转基因(非整合)导入，以瞬时表达感兴趣的治疗方法。整合载体(如慢病毒)是潜在的致癌基因，不适合于基于干细胞的癌症治疗。用于干细胞转基因的载体需要高效，因为快速生产无限量未分化干细胞的方法尚未完善。此外，细胞培养中的干细胞随着时间的推移而变化/突变(通常在八代之后)，因此提供了一个有限的加工机会窗口。除了效率外，转基因载体还需要对干细胞是非致癌的，因为它们可能会将正常干细胞转化为癌症起始细胞，并导致肿瘤形成。因此，干细胞工程中使用的载体预计会有很高的安全性。不幸的是，为了证明安全性，许多非整合载体只是简单地评估了它们对干细胞代谢活动的影响，还没有全面的研究来密切关注载体对遗传毒性、癌基因上调和其他有害影响的潜力。而代谢活性(增殖)试验是评价毒性的一个重要工具，但它不是 把整个故事都讲出来。需要更深入的毒性分析来评估真正的毒性，特别是当其意图是使用干细胞作为治疗癌症的手段时。因此，它是 这是确保工程干细胞在转基因过程中不会致癌所必需的。这项拟议的研究旨在解决目前存在的两个重大缺陷：1)非病毒载体在干细胞转染中的低效率，以及2)与干细胞工程中使用非病毒基因递送系统相关的毒性数据(如细胞存活率、膜完整性、微核形成、致瘤性和癌基因上调)的不足。本研究的目的是开发一种非遗传毒性/非致癌的载体，该载体可以高效地(80%)转染干细胞，同时保持干细胞的活性和肿瘤趋向性。目前，还没有非整合病毒或非病毒载体可用于 在保持低毒的同时，高效地(50%)转化干细胞。为了实现这一目标，将设计两种类型的重组载体：靶向和非靶向。靶向载体配备了与血管内皮生长因子受体(VEGFR)结合的多肽，以便于细胞进入。这种受体在干细胞的膜上高度表达，是细胞进入的安全途径。非靶向载体配备了有效的非阳离子细胞穿透肽，这些肽能够通过 细胞壁。所提出的重组融合载体的结构新颖，以前从未设计过。这些载体被设计成包含有效的干细胞转染所需的所有主要基序。同时，由于其非阳离子特性和生物降解性，所提出的载体可以保持低毒。

DESCRIPTION (provided by applicant): To engineer stem cells as gene delivery vehicles for cancer therapy, they are transfected ex-vivo with transgenes (non-integrating) to transiently express the therapeutics of interest. Integrating vectors (e.g. lentiviral) are potentially oncogenc and not suitable for stem cell based cancer therapy. The vector that is used for stem cell transfection needs to be highly efficient because the methods to rapidly produce unlimited quantities of undifferentiated stem cells have not yet perfected. Moreover, stem cells in cell culture change/mutate over time (usually after eight passages), thereby providing a limited window of opportunity for processing. In addition to efficiency, transfection vectors need to be non-oncogenic to stem cells because they could potentially transform normal stem cells into cancer initiating cells and result in tumor formation. Therefore, high levels of safety are expecte from vectors that are used in stem cell engineering. Unfortunately, for demonstration of safety many non-integrating vectors have been simply evaluated for their impact on metabolic activity of stem cells and there has been no comprehensive study that has closely looked at vectors' potential for genotoxicity, upgregulation of oncogenes and other detrimental effects. While metabolic activity (proliferation) assay is one important tool to evaluate toxicity but it does not tell the whole story. More in depth toxicity analysis is required to evaluate the true toxicity especially when the intension is to use stem cells as a means for treating cancer. Therefore, it is essential to ensure that the engineered stem cells don't become tumorigenic during the transfection process. The proposed research intends to address two significant deficiencies that currently exist: 1) low efficiency of non-viral vectors in stem cell transfection, and 2) insufficint toxicity data (e.g., cell viability, membrane integrity, micronuclei formation, tumorigenicity and upregulation of oncogenes) related to the use of non-viral gene delivery systems in stem cell engineering. The objective of this research is to develop a non-genotoxic/non-oncogenic vector that could transfect stem cells with high efficiency (>80%) while preserving their viability and tumor tropism. Currently, there is no non- integrating viral or non-viral vector available that can transfect stem cells with high efficiency (>50%) while maintaining low toxicity. To achieve the objective, two types of recombinant vectors will be engineered: targeted and non-targeted. The targeted vectors are equipped with peptides that bind to Vascular Endothelial Growth Factor Receptor (VEGFR) for cellular entry. This receptor is highly expressed on the membrane of stem cells and a safe route for cellular entry. The non-targeted vectors are equipped with efficient non-cationic cell penetrating peptides which are able to enter the stem cells through the cell wall. The structures of proposed recombinant fusion vectors are novel and never been designed before. These vectors are designed to contain all the major motifs necessary for efficient stem cell transfection. Concurrently, the proposed vectors can maintain low toxicity because of their non-cationic characteristics and biodegradability.