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Development of poly(mannitol-co-PEI) gene transporter modified with rabies virus glycoprotein for brain targeted delivery of therapeutic siRNA
광견병 바이러스 당단백질로 수식된 뇌 표적 치료용 폴리만니톨계 siRNA 전달체의 개발

DC Field Value Language
dc.contributor.advisor최윤재-
dc.contributor.author박태은-
dc.date.accessioned2017-07-13T08:22:10Z-
dc.date.available2017-07-13T08:22:10Z-
dc.date.issued2015-02-
dc.identifier.other000000025409-
dc.identifier.urihttps://hdl.handle.net/10371/119483-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2015. 2. 최윤재.-
dc.description.abstractAlzheimers disease (AD) is the worlds most common dementing illness, but no defensive treatments are available currently. A key reason is that AD drug development has been focused on symptomatic management without addressing basic cause of the disease. Hence, genetic intervention to suppress AD causative genes could represent an alternative to standard pharmacological approach. With the advent in therapeutic approach of RNA interference (RNAi), down-regulation of AD problematic genes including BACE1 has been extensively studied using viral vectors. Although viral vectors offered potential advantages for AD RNAi therapeutics, their clinical applications are limited by safety issue associated with viral-mediated carcinogenesis and immunogenicity, which led to the finding of nanotechnology-based non-viral vectors.
It is generally accepted that none of non-viral vectors is comparable to viral-vectors for delivery of genetic materials into host cells because non-viral vectors themselves are not equipped with modules for overcoming extracellular and intracellular barriers. Once delivered in body, therapeutic genes encounter extracellular barriers such as serum degradation, immune clearance, non-specific cell binding, and poor penetration of blood-brain barrier until they reach to target cells. After therapeutic genes are arrived in target cells, intracellular barriers inhibit RNAi activity which mainly includes cytotoxicity, poor endosomal escape and lysosomal degradation. The aim of the study is development of non-viral vector for AD RNAi therapeutics equipped with modules to conquer the biological barriers, which is divided into three parts
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dc.description.abstracti) study 1: development of non-viral vector to overcome intracellular barriers, ii) study 2: surface functionalization of the non-viral vector to overcome extracellular barriers, and iii) study 3: verification of AD RNAi therapeutic potential of the developed non-viral vector in vivo.
Study 1 mainly focused on an approach of controlling cellular uptake mechanism and consequent intracellular route of complexes to overcome the intracellular barriers. Since it became clear that uptake mechanism by which complexes are internalized determines their intracellular fates, caveolar endocytosis has emerged as an important endocytic target because it provides avoidance of lysosomal degradation. Caveolae vesicles are generally immobilized at the plasma membrane by the actin cytoskeleton, and internalized by certain stimulation via ligand-receptor interaction or osmotic stress. With a view to generating osmotically active gene carrier which facilitates caveolar endocytosis, degradable poly(mannitol-co-PEI) gene transporter (PMT) was generated by crosslinking low molecular weight PEI providing proton sponge effect and mannitol diacrylate as an osmolyte linker. PMT/DNA showed lower cytotoxicity compared to PEI/DNA complexes due to easily degradable ester groups and partially negatively charged mannitol backbone which can shield the remaining cationic charges of PEI. The proton sponge effect of PEI backbone partially contributed to gene delivery of PMT. More importantly, selective stimulation of caveolar endocytosis by mannitol backbone provided enhanced transfection efficiency via successful avoidance of lysosomal degradation of cargo. The action mechanism of PMT/DNA complexes on stimulation of caveolae-mediated endocytosis was associated with the activation of Src-kinase by mannitol part of PMT.
In study 2, PMT was functionalized to overcome the extracellular barriers. Delivery of therapeutic siRNA to the brain is one of the biggest challenges for successful brain gene therapy because blood-brain barrier (BBB) permits selective entry of only few substances into the brain. In this study, Trojan horse strategy was employed for ferrying PMT/siRNA complexes across the BBB. For this, PMT was conjugated with BBB-permeable rabies virus glycoprotein (RVG) via poly(ethylene glycol) (PEG) linker generating R-PEG-PMT. The PEG linker was expected to provide advantageous spatial arrangement of RVG peptide and stealth property on gene carrier. To determine the BBB penetration of complexes, in vitro BBB culture constructed by co-culture of bEnd.3 cells (mouse brain capillary endothelioma) and B23 cells (rat astrocytoma) on transwell system was utilized. The RVG ligand led to BBB penetration of PMT/siRNA complexes through receptor-mediated transcytosis via nicotinic acetylcholine receptors expressed on BBB. In mechanistic study, it was determined that improved BBB penetration of R-PEG-PMT/siRNA was achieved by stimulated receptor-mediated caveolar transcytosis. The enhanced accumulation in brain and biodistribution property of R-PEG-PMT/siRNA complexes was also demonstrated when systemically administrated.
In study 3, RNAi therapeutic potential of R-PEG-PMT/siBACE1 complexes was examined in vivo using normal C57/BL6 mice. Intravenous administration of R-PEG-PMT/siBACE1 for three times within 2 weeks has shown brain-targeted suppression of BACE1 without significant systemic toxicity. Furthermore, decreased BACE1 led to inhibition of beta-amyloid poduction, a marker of AD pathogenesis in hippocampus and cortex parts.
The overall results inferred that R-PEG-PMT is a promising tool for AD RNAi therapeutics. In aspect of overcoming intracellular barriers, i) degradable ester linkage of PMT reduced the cytotoxicity ii) PEI backbone of PMT has shown proton sponge effect, and iii) mannitol backbone provided the avoidance of lysosomal degradation via stimulation of caveolar endocytosis. In aspect of overcoming extracellular barriers, i) RVG offered neuronal cell targeting specificity, ii) stealth effect of PEG prevented the immune clearance in vivo, and iii) RVG directed the complexes across the BBB via receptor-mediated transcytosis and PMT facilitated trafficking of caveolae vesicle during transcytosis. The brain-targeted RNAi therapeutic effect of R-PEG-PMT/siBACE1 was demonstrated by suppression of BACE1 expression and beta-amyloid production. Consequently, R-PEG-PMT is a powerful gene carrier which can conquer the biological barriers itself, and possess the potential to be widely applicable in neurodegenerative diseases as a safe and efficient brain-targeted gene carrier.
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dc.description.tableofcontentsSummary Contents
List of Tables and Figures
List of Abbreviations
Introduction
Review of Literature
1. Overview of gene therapy for neurodegenerative disease
1) Historical evolution of gene therapy
2) Progress and prospect of gene therapy for neurodegenerative disease
2. Gene delivery systems
1) Viral vectors
2) Non-viral vectors
3. Extracellular barriers and coping strategies for brain gene delivery 30
1) BBB
2) Reticuloendothelial system (RES) recognition
4. Intracellular barriers and coping strategies for brain gene delivery
1) Protection and release of genes in cytosol
2) Intracellular trafficking in endocytosis pathways
5. Alzheimers disease: pathogenesis and neuropathology
Chapter 1. Selective stimulation of caveolae-mediated endocytosis by an osmotically active poly(mannitol-co-PEI) gene transporter
1. Introduction
2. Materials and methods
3. Results and discussion
1) Synthesis and characterization of a PMT
2) Physiochemical characterization of PMT/DNA complexes
3) In vitro cytotoxicity of PMT/DNA complexes
4) In vitro transfection efficiency of PMT/DNA complexes with or without serum
5) Transfection efficiency of PMT/DNA complexes
6) Mechanism of gene delivery of PMT
4. Conclusion
Chapter 2. Enhanced BBB permeability of poly(mannitol-co-PEI) gene transporter modified with rabies virus glycoprotein via selective stimulation of caveolr endocytosis
1. Introduction
2. Materials and methods
3. Results and discussion
1) Synthesis and physiochemical characteristics of R-PEG-PMT
2) In vitro cytotoxicity of R-PEG-PMT/siRNA complexes
3) In vitro targeting specificity of R-PEG-PMT/siRNA complexes
4) In vitro BACE1 silencing effects of R-PEG-PMT/siRNA complexes
5) In vitro BBB permeability of R-PEG-PMT/siRNA complexes
6) Mechanistic study of in vitro BBB penetration of R-PEG-PMT/siRNA complexes
7) Optical imaging of distribution of R-PEG-PMT/siRNA complexes
4. Conclusion
Chapter 3. RNA interference therapeutic potential of poly(manntiol-co-PEI) modified with rabies virus glycoprotein in Alzheimers disease
1. Introduction
2. Materials and methods
3. Results and discussion
1) Targeting specificity of R-PEG-PMT/siRNA
2) Dose-optimization of R-PEG-PMT/siRNA complexes
3) BACE1 knock-down efficiency of R-PEG-PMT/siRNA complexes
4. Conclusion
Overall Conclusion and Future Prospects
Literature Cited
Summary in Korean
Appendix
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dc.formatapplication/pdf-
dc.format.extent14606218 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectblood-brain barrier-
dc.subjectRNA interference-
dc.subjectpolyethylenimine-
dc.subjectmannitol-
dc.subjectAlzheimer’s disease-
dc.subjectBACE1-
dc.subjectcaveolar endocytosis-
dc.subject.ddc630-
dc.titleDevelopment of poly(mannitol-co-PEI) gene transporter modified with rabies virus glycoprotein for brain targeted delivery of therapeutic siRNA-
dc.title.alternative광견병 바이러스 당단백질로 수식된 뇌 표적 치료용 폴리만니톨계 siRNA 전달체의 개발-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pagesxviii, 214-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2015-02-
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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