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Targeted Transgene Insertion into th...

Chang, Felix Tay.

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Targeted Transgene Insertion into the AAVS1 Locus via Zinc Finger Nuclease Technology for Cancer Immunotherapy.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Targeted Transgene Insertion into the AAVS1 Locus via Zinc Finger Nuclease Technology for Cancer Immunotherapy./
作者:	Chang, Felix Tay.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2018,
面頁冊數:	217 p.
附註:	Source: Dissertations Abstracts International, Volume: 80-09, Section: C.
Contained By:	Dissertations Abstracts International80-09C.
標題:	Medicine. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13850184
ISBN:	9781083485533

Targeted Transgene Insertion into the AAVS1 Locus via Zinc Finger Nuclease Technology for Cancer Immunotherapy.
Chang, Felix Tay.

Targeted Transgene Insertion into the AAVS1 Locus via Zinc Finger Nuclease Technology for Cancer Immunotherapy. - Ann Arbor : ProQuest Dissertations & Theses, 2018 - 217 p.

Source: Dissertations Abstracts International, Volume: 80-09, Section: C.

Thesis (Ph.D.)--National University of Singapore (Singapore), 2018.

This item must not be sold to any third party vendors.

Cancer remains to be one of the leading causes of death. However, the use of traditional cancer treatments such as chemotherapy, radiotherapy and surgery are limited by their side effects such as the weakening of the immune system, destruction of healthy cells and cancer relapses. In recent years, cancer immunotherapy has been considered to be the next frontier for the treatment of cancers. Our immune system consists of effector cells that are able to recognise and kill cancer cells. Therefore, cancer immunotherapy works on the principle of boosting this defence system to target and destroy tumours. Cancer immunotherapy has also benefitted from the advances in the rapidly evolving field of genetic modification by utilizing gene delivery techniques to boost the tumour killing abilities of immune effector cells. A lot of attention has been given lately to the development of site-specific gene delivery systems, one of which being zinc finger nucleases (ZFNs). They are artificial endonucleases consisting of a zinc finger DNA-binding domain and a FokI endonuclease, which has the ability to recognize, bind and cleave a specific genomic site. By introducing a DNA template, transgenes can be efficiently delivered into the predefined genomic site. Several sites in the human genome, such as the adeno-associated virus integration site 1 (AAVS1) locus, have been identified as "safe harbour" sites for transgene insertions that permits persistent expression of the transgene. In this study, we developed a baculoviral vector-mediated zinc finger nuclease (BV-ZFN) technology, which consists of two baculoviral transduction systems: one for the delivery of zinc finger nuclease (ZFN) and another for the DNA donor template. Using this system, we achieved a high targeting efficiency of 93.8% and observed stability in expression of the inserted transgene in the AAVS1 locus of human induced pluripotent stem cells (hiPSCs). Following which, human fibroblasts was efficiently reprogrammed into a state of pluripotency by integrating the OKSM (Oct3/4, Klf4, Sox2, and c-myc) transcription factor genes into the AAVS1 locus via the BV-ZFN technology. Through this technology, hiPSC colonies were obtained at a high efficiency of 12% with all characterized hiPSC clones carrying the transgenic cassette only at the ZFN-specified AAVS1 locus. Besides the efficient targeting efficiency, flexibility in transgene exchange through baculovirus transduced, Cre recombinase-mediated cassette exchange (BV-Cre-RMCE) was demonstrated with the addition of heterospecific loxP sequences in the donor cassette. Using the same reprogramming technique, hepatocellular carcinoma (HCC) cell line, HepG2, was also successfully reprogrammed to generate cancer stem cell (CSC)-like cells that exhibited classical in vitro and in vivo CSC characteristics. Lysates generated from CSC-like cells could successfully generate cytotoxic T-Lymphocytes (CTL) that targeted HCC CSCs, showing the potential of these CSC-like cells as an infinite source of CSC-associated antigens for the dendritic cell (DC)-based vaccine against HCC CSC.

ISBN: 9781083485533Subjects--Topical Terms:

641104
Medicine.

Targeted Transgene Insertion into the AAVS1 Locus via Zinc Finger Nuclease Technology for Cancer Immunotherapy.
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