Bioengineering a Safe and Efficient Vector Technology for Stem Cell Transfection

描述（由申请人提供）：为了将干细胞工程化作为癌症治疗的基因递送载体，用转基因（非整合）离体转染干细胞以瞬时表达感兴趣的治疗剂。整合载体（例如慢病毒）是潜在致癌的，并且不适合于基于干细胞的癌症治疗。用于干细胞转染的载体需要高效，因为快速产生无限量未分化干细胞的方法尚未完善。此外，细胞培养物中的干细胞随时间变化/突变（通常在八次传代后），从而提供有限的加工机会窗口。除了效率之外，转染载体需要对干细胞是非致癌的，因为它们可能将正常干细胞转化为癌症起始细胞并导致肿瘤形成。因此，期望用于干细胞工程的载体具有高水平的安全性。不幸的是，为了证明安全性，许多非整合载体仅评估了它们对干细胞代谢活性的影响，并且还没有全面的研究仔细研究载体的遗传毒性、致癌基因的上调和其他有害作用的潜力。虽然代谢活性（增殖）测定是评价毒性的一个重要工具，但它并不 把事情的来龙去脉说出来需要更深入的毒性分析来评估真正的毒性，特别是当意图是使用干细胞作为治疗癌症的手段时。因此有 这对于确保工程干细胞在转染过程中不会致瘤至关重要。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）毒性数据不足（例如，细胞活力、膜完整性、微核形成、致瘤性和癌基因的上调）与在干细胞工程中使用非病毒基因递送系统有关。本研究的目的是开发一种无遗传毒性/非致癌性的载体，该载体可以以高效率（>80%）转染干细胞，同时保留其活力和肿瘤嗜性。目前，没有可用的非整合病毒或非病毒载体可以 以高效率（>50%）捕获干细胞，同时保持低毒性。为了实现该目标，将工程化两种类型的重组载体：靶向的和非靶向的。靶向载体配备有与血管内皮生长因子受体（VEGFR）结合以进入细胞的肽。这种受体在干细胞膜上高度表达，是细胞进入的安全途径。非靶向载体配备有有效的非阳离子细胞穿透肽，所述肽能够通过微通道进入干细胞。 细胞壁所提出的重组融合载体的结构是新颖的，并且以前从未设计过。这些载体被设计成含有有效干细胞转染所需的所有主要基序。同时，由于它们的非阳离子特性和生物降解性，所提出的载体可以保持低毒性。

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.