Bioengineering a Safe and Efficient Vector Technology for Stem Cell Transfection

描述（由申请人提供）：为了设计干细胞作为癌症治疗的基因传递载体，他们在体外用转基因（非整合）转染，以短暂表达感兴趣的治疗。整合载体（如慢病毒）具有潜在的致癌性，不适合用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会随着时间的推移而发生变化/突变（通常在8次传代之后），从而为处理提供了有限的机会窗口。除了效率之外，转染载体还需要对干细胞无致癌作用，因为它们有可能将正常干细胞转化为致癌细胞并导致肿瘤形成。因此，在干细胞工程中使用的载体具有很高的安全性。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，并且还没有全面的研究密切关注载体的遗传毒性、致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种重要工具，但它并没有得到很好的应用

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.