Study on cytoplasmic internalization of cytotransmab-biomolecule conjugates

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dc.description학위논문(석사)--아주대학교 일반대학원 :분자과학기술학과,2019. 2-
dc.description.tableofcontents1. Introduction 1 1.1 Cell penetrating antibody, cytotransmab 1 1.2 Antisense technology 3 1.2.1 Antisense oligonucleotides 3 1.2.2 RNA interference 5 1.3 Delivery of oligonucleotides 7 1.3.1 Nanoparticles 8 1.3.2 Ligand conjugates 8 1.4 Aim of study 10 2. Materials and Methods 12 2.1 Cell line 12 2.2 THIOMAB production 12 2.3 Antibody production 12 2.4 Reduction & re-oxidation 12 2.5. Conjugation of antibody and oligonucleotides 13 2.6. Flow cytometry 13 2.7. Split GFP assay 14 2.8. Confocal microscopy 14 3. Results and discussion 16 3.1 Cytotransmab inCT99-siRNA conjugation and mRNA knock down test. 16 3.2 Determination of cysteine introducing site for site-specific conjugation 20 3.3. Conjugation of inCT99 (N424C)-GFP11 and oligonucleotides 25 3.4. Binding of inCT99 (N424C)-GFP11-oligonucleotide conjugates 30 3.5. Cytoplasmic internalization of inCT99 (N424C)-GFP11-oligonucleotide 32 4. Conclusion 35 5. References 37 6. Abstract in Korean 42-
dc.publisherThe Graduate School, Ajou University-
dc.rights아주대학교 논문은 저작권에 의해 보호받습니다.-
dc.titleStudy on cytoplasmic internalization of cytotransmab-biomolecule conjugates-
dc.title.alternativeBo Seok Jeong-
dc.contributor.affiliation아주대학교 일반대학원-
dc.contributor.alternativeNameBo Seok Jeong-
dc.contributor.department일반대학원 분자과학기술학과- 2-
dc.description.alternativeAbstractAntisense therapy using oligonucleotides has attracted many attentions because of its high efficiency of manipulating the expression of disease-causing genes. To endow the therapeutic molecules with an ability to target specific cells, oligonucleotides have been conjugated to targeting molecules such as ligands and antibodies. In particular, the antibody has been a focus of research because of its pharmacokinetic and physicochemical features. However, these conjugate molecules still have a limitation of low efficiency to locate in the cytoplasm or nucleus where the oligonucleotide works. In this study, I investigated the capability of cytotransmab, which can internalize via endocytosis and locate in the cytoplasm by escaping the endosome, to deliver oligonucleotides. The thiol group of unpaired Cys residues were used for conjugation with oligonucleotides modified with a linker having the maleimide group. Several positions of IgG were evaluated for the reduction and re-oxidation steps to generate unmodified thiol groups in the introduced Cys residues, and position N425 in the heavy chain was chosen for substitution with Cys. Single- or double-stranded oligonucleotides with different length were conjugated to cytotransmab, and binding affinity and cytoplasmic localization of these conjugates were analyzed using flow cytometry and split GFP complimentary assay, respectively. The reduction, re-oxidation and oligonucleotides were not affect the affinity of inCT99 (N424C)-GFP11 against integrin αvβ5. In addition, it was shown that conjugates can translocate into cytosol, and the length of oligonucleotide is a factor which can affect the escaping from endosome of cytotransmab.-
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Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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