Bioengineering a Safe and Efficient Vector Technology for Stem Cell Transfection

生物工程安全高效的干细胞转染载体技术

• 批准号: 8701678
• 负责人: Arash Hatefi
• 金额: $ 23.25万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8701678
• 关键词: Address; Adenovirus Vector; Adenoviruses; Binding; Biological Assay; Biomedical Engineering; Bystander Effect; Cell Culture Techniques; Cell Death; Cell Survival; Cell Therapy; Cell Wall; Cell membrane; Cells; Cessation of life; Characteristics; Charge; Data; Disease; Electroporation; Engineering; Ensure; Enzymes; Ganciclovir; Gene Delivery; Genes; Genetic; Genetic Vectors; Genome; Goals; Image; Infection; Laboratories; Life; Lipids; Malignant - descriptor; Malignant Neoplasms; Mediating; Membrane; Metabolic; Methods; Mutate; Non-Viral Vector; Nude Mice; Oncogenes; Oncogenic; Patients; Peptides; Polymers; Process; Prodrugs; Protocols documentation; Recombinants; Research; Route; Safety; Scientist; Solid Neoplasm; Stem cells; Structure; Surface; Technology; Therapeutic; Thymidine Kinase; Time; Toxic effect; Transfection; Transgenes; Tropism; Tumor Stem Cells; Tumorigenicity; Undifferentiated; Up-Regulation; Ursidae Family; Vascular Endothelial Growth Factor Receptor; Viral; Viral Vector; base; cancer cell; cancer therapy; cellular engineering; design; effective therapy; gene delivery system; gene therapy; genotoxicity; improved; interest; meetings; micronucleus; nanoparticle; nanosized; non-oncogenic; non-viral gene delivery; novel; overexpression; particle; plasmid DNA; public health relevance; receptor; stem cell technology; therapeutic gene; tool; trafficking; transduction efficiency; transgene expression; tumor; tumor xenograft; tumorigenesis; tumorigenic; uptake; vector

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.

描述（由申请人提供）：为了将干细胞改造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药物。整合载体（例如慢病毒）具有潜在的致癌性，不适合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会随着时间的推移（通常在八次传代后）发生变化/突变，从而提供有限的处理机会。除了效率之外，转染载体还需要对干细胞非致癌性，因为它们有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，干细胞工程中使用的载体预计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，并且没有全面的研究来仔细研究载体的遗传毒性、癌基因上调和其他有害影响的潜力。虽然代谢活性（增殖）测定是评估毒性的重要工具之一，但它并不 讲述整个故事。需要更深入的毒性分析来评估真正的毒性，特别是当目的是使用干细胞作为治疗癌症的手段时。因此，它是 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效率低下，2）毒性数据不足（例如细胞活力、膜完整性、微核形成、致瘤性和癌基因上调） ）与干细胞工程中非病毒基因传递系统的使用有关。本研究的目的是开发一种非基因毒性/非致癌载体，能够高效（>80%）转染干细胞，同时保留其活力和肿瘤趋向性。目前，还没有可用的非整合病毒或非病毒载体可以 高效率（>50%）转染干细胞，同时保持低毒性。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。靶向载体配备有与血管内皮生长因子受体（VEGFR）结合以进入细胞的肽。该受体在干细胞膜上高度表达，是细胞进入的安全途径。非靶向载体配备有高效的非阳离子细胞穿透肽，能够通过 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被设计过。这些载体被设计为包含有效干细胞转染所需的所有主要基序。同时，所提出的载体由于其非阳离子特性和生物降解性而可以保持低毒性。