Development of nanoparticle-based artificial transcription factors for improve myogenesis efficiency of human iPSC = 인간 유도만능줄기세포의 근육분화 효율 향상을 위한 나노입자 기반 인공 전사인자 개발

다국어 초록 (Multilingual Abstract)

Transcription factors are multidomain proteins that serve as key regulators of gene expression by enabling precise transcriptional control. In this study, we expanded the gold nanoparticle–based artificial transcription factor platform, NanoScript, by developing a NanoScript-TFO (NS-T) system that employs a triplex-forming oligonucleotide (TFO) as the DNA-binding domain. By incorporating TFOs, this platform minimizes competition with endogenous transcription factors and enables a novel transcriptional regulation strategy targeting non-coding regulatory DNA regions. NS-T exhibited high stability under physiological conditions, showed no detectable cytotoxicity, and achieved efficient nuclear localization. By modulating the orientation of the gold nanoparticle and the genomic position of the target site, we identified that transcriptional activation was most effective when NS-T targeted non-coding DNA with the gold nanoparticle positioned downstream. Application of NS-T, designed with optimized TFO sequences, to human iPSC-derived satellite-like cells resulted in upregulation of a broad range of myogenic genes, including MYOD and MYOG, and led to accelerated myotube formation. Collectively, the NS-T platform presented in this study offers a safe, non-viral, and rationally designable approach for gene regulation, highlighting its broad technological potential for applications in skeletal muscle regeneration as well as diverse fields of regenerative medicine and transcriptional modulation research.

국문 초록 (Abstract)

전사인자는 다중 도메인 구조를 갖는 단백질로서 유전자의 발현을 정밀하게 조절하는
핵심 인자이다. 본 연구에서는 금 나노입자 기반의 인공 전사인자 플랫폼인
NanoScript 를 확장하여, 삼중체 형성 올리고뉴클레오타이드(TFO)를 DNA 결합
도메인으로 활용한 NanoScript-TFO (NS-T)를 개발하였다. TFO 를 도입함으로써
자연 전사인자와의 경쟁을 최소화하며, 비부호화 (non-coding) 조절 영역을 표적하는
새로운 전사 조절 접근법을 제안하였다.
NS-T 는 생리적 조건에서도 안정적으로 유지되었으며, 세포 독성이 없고 핵 내 전달이
효율적으로 이루어졌다. 또한 gold nanoparticle 의 방향성과 표적 위치를 조절한 결과,
non-coding DNA 이며 gold 의 위치가 downstream 방향을 타깃할 때 전사 활성화가
가장 효과적임을 확인하였다. 최적의 TFO 서열을 기반으로 NS-T 를 human iPSC
유래 satellite-like cell 에 적용한 결과, MYOD 와 MYOG 를 비롯한 광범위한 근육
관련 유전자 발현이 증가하였으며, myotube 형성이 가속화되는 효과가 관찰되었다.
본 연구에서 제시한 NS-T 플랫폼은 비바이러스성 방식으로 유전자를 조절할 수 있는
안전성과 설계의 용이성을 갖추고 있어, 향후 근육 재생을 포함한 다양한 재생의학 및
유전자 조절 연구에 폭넓게 활용될 수 있는 기술적 잠재력을 지닌다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials & Methods 3
• 2.1 Synthesis of Peptides 3
• 2.2 Synthesis of Triplex Forming Oligonulceotides (TFO) 3
• 2.3 Synthesis of CTB 3
• 2.4 Synthesis of NanoScript-TFO 3
• 2.5 Dynamic light scattering 4
• 2.6 Zeta potential 5
• 2.7 Transmission Electron Microscopy 5
• 2.8 iPS cells culture and differentiation into myotube 5
• 2.9 Intracellular and nucleus localization of NanoScripts imaging 6
• 2.10 Cell viability test 6
• 2.11 RT-qPCR analysis 7
• 2.12 Immunocytochemisty 7
• 3. Results and Discussion 11
• 3.1 Synthesis and Characterization of Nanoscript-TFO 11
• 3.2 Positional effect of gold nanoparticles on target gene transcription 21
• 3.3 Identification of effective TFOs activating MyoD and MyoG transcription 27
• 3.4 Acceleration of myogenic differentiation in iPSCs using NS-T 29
• 3.4.1 Differentiation of iPSCs into satellite-like cells 31
• 3.4.2 NS-T accelerate myogenic gene expression in satellite-like cells 35
• 3.4.3 NS-T accelerate myogenic differentiation toward myotube formation 39
• 4. Conclusion 43
• 5. Reference 45
• 6. Abstract in Korean 48