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유전자 치료를 위한 유전자 전달체와 표적 특이적 치료유전자 발현 시스템의 개발

Title
유전자 치료를 위한 유전자 전달체와 표적 특이적 치료유전자 발현 시스템의 개발
Author
김현아
Advisor(s)
이민형
Issue Date
2012-02
Publisher
한양대학교
Degree
Doctor
Abstract
Gene therapy is a promising strategy for treating genetic andacquired diseases, such as cancers, ischemic diseases, immune disorders and diabetes.Gene carriers and therapeutic genes are two important prerequisites for successful gene therapy. However, some problems such as cytotoxicity, immunogenicity and oncogenesis have limited the application of gene therapy. To overcome these drawbacks, non-toxic and efficient gene carriers and site-specific gene regulatory systems should be developed. In this study, gene carriers based on polymer and peptide were developed and evaluated in the lung disease model. In addition, site-specific therapeutic genes were developed and evaluated in glioblastoma and islets. Dexamethasone was conjugated to low molecular weight polyethylenimine (2 kDa, PEI2k). Dexamethasone conjugated PEI2k (PEI-Dexa) was evaluated as a gene carrier to lung epithelial cells in vitro. Transfection assay showed that PEI-Dexa had higher transfection efficiency than PEI2k and lipofectamine. Furthermore, PEI-Dexa had lower toxicity than PEI25k and lipofectamine. To evaluate in vivo gene delivery efficiency, the PEI-Dexa/p??-Luc complexes were administrated into mouse lung by intratracheal injection. The results showed that PEI-Dexa was more efficient than PEI25k and PEI2k in mice lung in terms of delivery efficiency. These results suggest that PEI-Dexa is a useful carrier for lung cells. Furthermore, PEI-Dexa had anti-inflammatory effect, due to its dexamethasone part in vitro and in vivo, suggesting that PEI-Dexa may be more useful in gene delivery to inflammatory diseases. For active targeting to lung epithelial cells, lung epithelial binding peptide (LEBP) was directly linked to high mobility group box-1 A box (HMGB-1A). LEBP-HMGB-1A was synthesized and purified by recombinant DNA technology. HMGB-1A is a cationic peptide, which is originated from nuclear protein. Unlike PEI-Dexa, PEI2k was not directly linked to LEBP-HMGB-1A. Instead, PEI2k and LEBP-HMGB-1A mixture was used to form complexes with plasmid DNA (pDNA).Therefore, PEI2k and HMGB-1A bound to pDNA forming complex with LEBP decoration on the surface of the complex. In vitro transfection assay showed that LEBP increased transfection efficiency specifically to lung epithelial cells. In addition, due to the low cationic charge of HMGB-1A, the toxicity of HMGB-1A/PEI2k/pDNA complex had less toxicity than PLL control. Hypoxia is an important characteristic of various diseases. Ischemic diseases and solid tumors have hypoxic regions in their tissues. In the process of transplantation, cells or tissues are subjected to hypoxia. Therefore, hypoxia is an excellent target for disease specific gene therapy. For tumor-hypoxia specific gene therapy, I constructed luciferase and herpes simplex virus thymidine kinase (HSV-TK) pDNAs with hypoxia and glioma specific promoters. Among the constructed pDNAs, the combination of nestin intron-2 (NI2) and the erythropoietin (Epo) enhancer (pEpo-NI2-SV-TK) showed the highest specificity in terms of hypoxia and glioma specific gene expression in C6 glioblastoma cells. Hypoxia specific gene expression is also useful to protect islets from hypoxic insult in the process of transplantation. For hypoxia specific gene expression, the RTP801 promoter was inserted into the vascular endothelial growth factor (VEGF) expression plasmid, constructing in pRTP-VEGF. In vitro transfection to rat islets showed that hypoxia specific expression of VEGF. In conclusion, PEI-Dexa and LEBP-HMGB-1A may be useful for gene delivery to lung cells with low toxicity. Hypoxia specific gene expression systems such as pEpo-NI2-SV-TK and pRTP801-VEGF will be useful for targeting gene therapy without side-effects.
URI
http://dcollection.hanyang.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000062674https://repository.hanyang.ac.kr/handle/20.500.11754/137408
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIOENGINEERING(생명공학과) > Theses (Ph.D.)
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