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theses

Stem cell mediated gene delivery has steadily gained momentum in the past decade as a new strategy to improve the safety and efficacy of current cancer gene therapy methods. Recent evidence indicates that systemically administered mesenchymal stem cells can migrate and deliver therapeutic genes to the tumor site. In order to engineer stem cells as gene delivery vehicles for cancer therapy, the cells are first transfected ex-vivo with transgenes to transiently express the therapeutic of interest. While viruses are effective vectors for delivering exogenous genes to cells, concerns related to insertional mutagenesis, lack of tropism, immunogenicity and high production costs necessitate the development of non-viral methods. Non-viral gene delivery vectors hold great promise for stem cell gene therapy due to the safety concerns with viral vectors. However, the application of non-viral vectors is hindered by their low transfection efficiency and toxicity. Vectors used for stem cell transfection must be non-genotoxic, non-immunogenic and highly efficient, in order to circumvent the potential transformation of normal stem cells into cancer-initiating cells. Herein, in order to tackle these challenges, we strived to develop non-viral vectors with efficient gene delivery and low toxicity to hard-to-transfect mesenchymal stem cells.

This doctoral dissertation will focus on the design and application of efficient non-viral vectors, for the genetic modification of stem cells without any negative somatic or genetic impact. The first part of the dissertation describes the characterization of mesenchymal stem cells and neural stem cells. These stem cells were screened for over-expressed cell surface receptors by systematically developed protocol, which laid the foundation for the development of vectors that recognize the port of entry to the stem cells. The next part of the dissertation describes the design and production of vectors in bacterial system. A number of parameters were compared, including the choice of expression hosts, metal affinity columns and expression conditions, in order to identify the most effective means to obtain highly pure vectors. The final portion of the dissertation describes characterization, efficiency and toxicity studies of the developed vectors in mesenchymal stem cells. All vectors were evaluated for their transfection efficiency, impact on metabolic activity, cell membrane integrity and micronuclei formation (chromosomal aberrations). The results of this study showed that the bioengineered vector utilizing receptors for cellular entry could transfect mesenchymal stem cells with high efficiency without inducing genotoxicity and negative impact on gene function. The genetically engineered vector in this study proved that it can be safely and efficiently used to genetically modify stem cells with potential applications in cancer gene therapies.

ETD_10635

Ph.D.

Includes bibliographical references

doi:10.7282/t3-8yk9-vt93

ETD doctoral

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