초음파 활성 나노 입자를 이용한 탈모 치료를 위한 Cas9-RNP 전달 시스템
Cas9-RNP delivery system for hair loss therapy by an ultrasound active particels
- 주제(키워드) ultrasound , microbubble , nanoliposome , CRISPR/Cas9 , androgenic alopecia
- 발행기관 아주대학교
- 지도교수 윤태종
- 발행년도 2017
- 학위수여년월 2017. 8
- 학위명 석사
- 학과 및 전공 일반대학원 약학
- 실제URI http://www.dcollection.net/handler/ajou/000000025906
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Protein-based therapy has conventional limitations because of its low efficiency and the difficulty of targeted delivery. To overcome this, we designed that lecithin-based liposome (NL) containing a Cas9/sgRNA ribonucleoprotein (Cas9-RNP) complex were linked to a microbubble (MB). Importantly, the bursting of the MB under acoustic pressure released the NL and enhanced their intracellular delivery owing to a sonoporation effect. We demonstrated that the Cas9-RNP within the NL used for SRD5A2 gene editing improved cell proliferation by blocking conversion of testosterone from dihydrotestosterone (DHT) which induces cell death. Interestingly, multiple treatments of the microbubble-nanoliposome (MN) exhibited higher efficacy than a single treatment. In summary, these results demonstrated that the ultrasound active MN system incorporating Cas9-RNP for hair loss therapy enhanced gene editing efficiency in human dermal papilla cells by sonoporation. Also, we expect that this MN system reduce side effects by provide opportunities to local delivery of Cas9-RNP to dermal papilla cells in hair follicles.
more목차
1. Introduction 1
2. Materials and Methods 3
2.1 Materials 3
2.2 Design and cloning of sgRNA 3
2.3 In vitro transcription of sgRNA 4
2.4 Expression and purification of Cas9 protein 5
2.5 In vitro cleavage of genomic DNA 6
2.6 Preparation of Cas9-RNP encapsulated nanoliposome 6
2.7 Preparation of microbubble-nanoliposome (MN) 6
2.8 Characterization of MN 7
2.9 Cell culture and transfection 7
2.10 Intracellular delivery of MN 7
2.11 CLSM analysis of MN into cells 7
2.12 Cell viability 8
2.13 Quantitative RT-PCR analysis 8
2.14 Western blot analysis 8
2.15 Human SRD5A2 ELISA 9
2.16 Caspase-3 assay 9
2.17 Statistical analysis 9
3. Results 10
3.1 Screening sgRNA for SRD5A2 gene targeting 10
3.2 Preparation of Cas9 protein and complexation with sgRNA 10
3.3 Synthesis of NL and encapsulation of the Cas9-RNP using PEI 10
3.4 Preparation of MBs and complexation with NL(Cas9-RNP(+)) 11
3.5 MN echogenicity 11
3.6 Effect of testosterone in DPCs 11
3.7 Delivery of MN particles to DPCs 11
3.8 Gene editing in DPCs by MN 12
3.9 Inhibition of testosterone-induced cell death after MN delivery to DPCs 12
3.10 Effect of SRD5A2 gene editing on caspase-3 activity in cell death 12
4. Conclusion and discussion 14
References 30
국문요약 33
