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Development of Non-Viral Gene Delivery System Using RNA Interference and CRISPR Interference for the Treatment of Inflammatory Diseases

Title
Development of Non-Viral Gene Delivery System Using RNA Interference and CRISPR Interference for the Treatment of Inflammatory Diseases
Author
Jee Young Chung
Advisor(s)
김용희
Issue Date
2018-02
Publisher
한양대학교
Degree
Doctor
Abstract
Targeted gene regulation on a genome-wide scale level is a great strategy for interrogating, perturbing, and engineering cellular systems. Diverse mechanism regarding targeted gene delivery has made it possible to perform precise genome editing. Genetic reprogramming of cells requires predictable methods for expression or repression of target sets of genes. RNA interference has the potential for knockdown of targeted genes but faces tremendous barriers before accumulating to target cytoplasm. The clustered regularly interspaced short palindromic repeats (CRISPR) system provides a platform for targeted genomic regulation. Cas proteins direct the degradation of complementary sequences present within the plasmid DNA together with single guide RNAs to target and cleave DNA in a sequence-specific manner, however the efficient delivery tool and off-target effects remains a hurdle to overcome. An efficient systemic carrier system for both passive and active targeting using RNAi and CRISPRi mechanism was developed for inflammatory disorders including cancer, obesity and obesity induced diabetes and rheumatoid arthritis. RNA interference (RNAi) provides a very effective tool for developing anti-tumor therapies
however, its application to date has remained limited by a lack of effective small interfering RNA (siRNA) delivery systems in vivo. In the first study, a polymeric gene carrier system based on PEGylation of a cationic cysteine-ended 9-mer arginine oligopeptide (CR9C) was designed for effective systemic siRNA delivery. PEG500-CR9C complexed with siRNA targeting vascular endothelial growth factor (VEGF) provided improved blood circulation, enhanced protection from serum proteases, reduced uptake in the liver and kidneys, enhanced tumor targeting and down-regulated intratumoral VEGF level, which comprehensively resulted in improved anti-tumor efficacy without significant toxicity in vivo. In the second study, dual expression short hairpin RNA (shRNA) was designed to target metallothionein and vascular endothelial growth factor. Repeated injections of cisplatin induces multi-drug resistance by interaction with cysteine-rich protein, metallothionein. sh(MT/VEGF) and PEG500-CR9C complex demonstrated efficient transfection to silence MT and VEGF over-expression in cisplatin resistant cancer cell line. Co-administration of shMT/rPOA oligo-peptoplex and cisplatin in vivo tumor model showed noticeable tumor-suppressing effect by inducing reversal of cisplatin resistance following effective intracellular delivery of shMT. This study provides a potential remedy of inhibiting platinum drug resistance by the RNA interference method. CRISPR interference (CRISPRi) mechanism based on catalytically dead Cas9 and single guide RNA was combined with targeted non-viral gene delivery system to treat obesity and obesity induced type 2 diabetes. Obesity is an increasing pathophysiological problem in developed societies and currently available commercial anti-obesity drugs have shown limited efficacy. Anti-obesity drug development is focusing on targeting adipose tissues that store excess fat. In the third study, adipocyte fusion peptide targeting vascular marker of adipose tissues, prohibitin was developed by conjugation of adipocyte targeting sequence (CKGGRAKDC) to 9-arginine (ATS-9R). dCas9 and single RNA against fatty acid binding protein-4 (FABP4) complexed with ATS-9R caused FABP4 knock-down in pre and mature adipocytes. The system was further applied to high fat diet induced mouse model and the treatment using CRISPRi system reduces inflammation in adipose tissues, ameliorate obesity and restore hepatic steatosis. This RNA-guided DNA recognition platform provides a safe therapeutic approach to regress and treat obesity and obesity induced metabolic syndromes. CRISPRi system was also applied for treatment of rheumatoid arthritis. Vasculature structure plays an important feature of RA in the destructive regions, as there is an increased number and density of synovial blood vessels that are required to supply the expansion of synovial tissue leading to development of the invasive nature of RA synovium. In the final study, a peptide targeting the inflamed synovial joints in rheumatoid arthritis (NQR-9R) was designed to deliver dCas9 and sgRNA to knock-down tumor necrosis factor-alpha converting enzyme (TACE). dCas9/sgRNA complexed with NQR-9R caused TACE knock-down in primary endothelial cells and fibroblast like synoviocytes and showed therapeutic effects in CIA induced RA mouse model. In this dissertation, non-viral gene delivery system revealed effective delivery of therapeutic gene and siRNA for cancer, obesity induced metabolic syndrome and rheumatoid arthritis. Despite future challenges regarding immunogenicity and stability of the non-viral gene delivery system, the proposed data will promote a platform of a simple approach for selectively perturbing gene expression on a genome-wide scale.
URI
http://www.dcollection.net/handler/hanyang/000000106198http://repository.hanyang.ac.kr/handle/20.500.11754/68166
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIOENGINEERING(생명공학과) > Theses (Ph.D.)
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