Investigation on therapeutic nucleic acids delivery system using fusion peptide-based self-assembled complex for gene therapy = 융합 펩타이드 기반 자기조립 복합체를 이용한 유전자 치료용 핵산 전달 시스템 연구

다국어 초록 (Multilingual Abstract)

Gene therapy is a transformative approach that introduces therapeutic nucleic acids, including DNA and RNA, to address genetic anomalies responsible for intractable diseases at their molecular foundation. The FDA’s recent endorsement of short interfering RNA treatments, the ascendance of messenger RNA vaccines amidst the COVID-19 pandemic, and the advancements in CRISPR/Cas9 gene editing technology are notable not just for earning Nobel distinctions but also for their immense prospective market value. Such potential is fueling intensive research efforts in these domains.
For successful gene therapy strategies, such as gene silencing or gene editing, functional nucleic acids must navigate various intracellular obstacles to reach their target cells effectively. Entry into the cell necessitates crossing the cell membrane, followed by their release from endosomes/lysosomes into the cytosol. DNA, distinct from RNA, requires further transport into the nucleus. Nucleic acids, given their size and the negative charge stemming from their phosphate backbone and phosphodiester bonds, inherently display hydrophilic properties. These characteristic challenges their ability to interact with the cell’s lipid bilayer membrane. For RNA-based approaches, there’s an added emphasis on the need for safety-enhancing technologies. In light of these challenges, there’s a pressing need to develop technologies that ensure the efficient and secure intracellular delivery of these functional nucleic acids.
In this research, while examining the elements of the gene delivery system, the focus was placed on two innovative approaches for gene silencing and gene mutation. These approaches employed fusion methodologies with peptides characterized as non-viral vectors and utilized system strategies rooted in self-assembled complexes.
In the first study, a novel small RNA (sRNA)/fusion peptide complex was devised, incorporating a fusion peptide that merges two distinct cell-penetrating peptides (CPPs) for the efficient and safe intracellular delivery of sRNA, leveraging the RNA interference mechanism to initiate gene silencing. Each of these peptides effectively traverses the cell membrane through unique mechanisms. While prior research predominantly utilized a skin permeating and cell entering (SPACE) peptide centered on its covalent association with siRNA, standalone cationic peptides encountered limitations in nucleic acid complex formation and delivery efficacy. Addressing these challenges, our team devised a fusion peptide that combines the SPACE peptide with a cationic oligoarginine sequence. This innovation facilitated the development of self-assembled complexes, grounded in electrostatic interactions with sRNA.
As the result, the sRNA/fusion peptide complex demonstrated superior efficiency in complex formation compared to standalone cationic peptides. This enhanced performance can be attributed to the utilization of multiple non-covalent interactions, encompassing electrostatic, hydrophobic, and hydrogen bonds. In a serum environment, the complex not only bolstered the stability of sRNA but also outperformed both standalone peptide complexes and commercial liposomes in terms of sRNA delivery to various cell lines, including HeLa, HDFn, and RAW 264.7. Specifically, in HeLa and RAW 264.7 cells, the complex managed to suppress the target gene’s mRNA expression by 61.3% and 66.2%, respectively, showing results on par with Lipofectamine. Investigations into the cellular uptake pathway of the complex revealed lipid-mediated endocytosis as the predominant mechanism. Furthermore, in vivo tests substantiated the complex’s increased stability post-local injection, alongside notable downregulation of the target gene expression.
In the second study, our objective was to efficiently deliver large plasmid DNA to facilitate the expression of the CRISPR genome editing tool. To this end, a peptide-assisted lipoplex (PAL) was established as a novel complex using a lipoplex with a fusion peptide that integrates a nuclear localization signal (NLS) and CPP. However, commercial liposomes faced challenges in efficiently encapsulating larger plasmids. Their inability to neutralize the pronounced negative charge of DNA culminated in compromised stability and reduced efficiency in nuclear entry of these complexes. It is also noteworthy that while electroporation is a prevalent method for delivering large plasmids, it frequently induces cellular damage, underscoring the pressing need for more benign alternatives.
This PAL system notably enhanced the ability to neutralize the negative charge of the plasmid, resulting in a more stable complex formation. When benchmarked against electroporation, PAL achieved a 2-fold and 1.6-fold increase in delivery efficiency for 9,283 bp and 29,350 bp plasmids, respectively. Furthermore, it elevated cell viability and activity by an impressive 29-fold. Across diverse cell lines, PAL outperformed conventional lipoplexes, delivering plasmids with 2- to 4.4-fold greater efficiency. In an in vivo mouse study, the expression of cells with the PAL-introduced plasmid was maintained for up to 3 days. Impressively, when PAL encapsulated a Cas9 plasmid targeting overactive cancer genes, including the CRISPR RNA, it achieved a mutation efficiency of up to 44.1% in the targeted genes. When used synergistically with chemotherapy, this approach hindered tumor growth in a mouse tumor model. In essence, our work pioneers a fusion peptide-centric complex, offering a promising avenue in gene therapy delivery that balances both efficacy and safety.
In summary, our research successfully pioneered two distinct systems: (1) an sRNA/fusion peptide complex for gene silencing, and (2) a fusion peptide-assisted lipid complex tailored for the delivery of large plasmids. Merging high efficiency with safety, these systems have demonstrated exceptional capabilities in gene silencing and gene editing through the effective delivery of small RNA and large plasmids, respectively. These achievements underscore the promising potential of advanced gene delivery platforms, setting the stage for potential treatments of a wide range of genetic disorders in the coming years.

국문 초록 (Abstract)

유전자 치료는 치료적 효능을 가진 DNA 또는 RNA와 같은 핵산을 외부에서 주입하여 난치병의 원인이 되는 유전적 결함을 분자 수준에서 근본적으로 치료할 수 있는 방법이다. 최근 FDA 승인을 획득한 짧은 간섭 RNA 치료제, COVID-19 팬데믹으로 인해 널리 알려진 mRNA 백신, 그리고 CRISPR/Cas9 유전자 편집 기술은 모두 노벨상을 수상했을 뿐 아니라 미래의 거대한 유전자 치료제 시장의 잠재력을 가지고 있어 이에 대한 연구가 활발하게 진행되고 있다.
유전자 침묵이나 유전자 편집과 같은 유전자 치료 전략을 실행하는 기능적 핵산은 목표 세포에 전달되어 효과를 나타내기 위해 여러 세포 내 장벽을 통과해야 한다. 세포 내로의 진입을 위해서는 세포막을 통과하고, 세포 내에서는 엔도좀/리소좀에서 세포기질로 방출되어야 하며, DNA의 경우 RNA와는 다르게 핵 내로까지 전달되어야 한다. 그러나 핵산은 대형 분자로, 뉴클레오티드의 인산 골격과 인산다이에스터 결합 사이의 인산기로 인해 강한 음전하를 가진다. 이러한 특성은 핵산이 친수성을 갖게 하며 인지질 이중층 구조를 가진 세포막과의 상호작용을 방해한다. 또한 RNA의 경우 세포 내에서 분해에 취약하므로 안정성을 향상시킬 수 있는 기술이 필요하다. 이러한 도전을 극복하기 위해 기능적 핵산의 효율적이며 안전한 세포 내 전달을 지원하는 기술 개발을 필요로 하게 되었다.
본 연구에서는 이러한 유전자 전달 시스템의 구성 요소를 고려하여, 비바이러스성 벡터로 분류되는 펩타이드의 융합 전략과 자기조립 복합체를 이용한 제형 전략을 활용하여 유전자 침묵 및 유전자 편집을 위한 두 가지의 새로운 시스템을 중점적으로 연구하였다.
첫 번째 연구에서는 RNA 간섭 메커니즘을 활용하여 유전자 침묵을 유도하는 짧은 RNA (siRNA, shRNA)의 효율적이고 안전한 세포 내 전달을 목표로, 고유한 메커니즘을 통해 세포막을 효과적으로 투과할 수 있는 두 종류의 세포 투과성 펩타이드를 융합한 혁신적인 융합 펩타이드를 개발하였다. 기존 연구에서 단일 SPACE (skin permeating and cell entering) 펩타이드는 짧은 간섭 RNA와의 공유 결합을 중심으로 활용되었으며, 단독으로 사용된 양이온성 펩타이드는 핵산과의 복합체 형성 및 전달 효율에 한계가 있었다. 이에 따라 본 연구에서는 SPACE 펩타이드와 양이온성 올리고아르기닌 서열을 융합한 새로운 융합 펩타이드를 설계하여, 짧은 RNA와의 전기적 상호작용을 기반으로 한 자기조립 복합체를 생성하였다.
실험 결과, 짧은 RNA/융합 펩타이드 복합체는 전기적 상호작용 및 소수성 상호작용, 수소 결합 등의 비공유 결합을 활용하여 기존의 단독 양이온성 펩타이드보다 향상된 복합체 형성 효율을 보였다. 복합체는 혈청 환경에서 짧은 RNA의 안정성을 증가시키고 HeLa, HDFn, 그리고 RAW 264.7 등의 다양한 세포주에서 단독 펩타이드 복합체나 상용 리포좀보다 짧은 RNA의 전달 효율을 향상시켰다. HeLa와 RAW 264.7 세포에서 복합체는 표적 유전자의 mRNA 발현을 각각 61.3%와 66.2% 감소시켜, 리포펙타민과 유사한 효과를 나타냈다. 세포 내에서의 복합체 이입 메커니즘은 지질 중재 세포 내 흡수로 확인되었고, in vivo 실험에서는 국소적으로 주입된 복합체의 향상된 안정성 및 표적 유전자의 발현 저하를 확인하였다.
두 번째 연구에서는 CRISPR/Cas9 유전자 가위의 효율적인 발현을 통한 유전자 편집을 가능하게 하는 대형 플라스미드 DNA의 전달을 목표로, 핵 위치 신호 (peptide nuclear localization signal; NLS)와 세포 투과성 펩타이드를 융합한 융합 펩타이드와 리포플렉스를 조합한 복합체를 개발하였다. 상용 리포좀은 대형 플라스미드의 패키징에 한계를 보이며, DNA의 강한 음전하를 충분히 중화하지 못해 복합체의 안정성과 핵 내 투과 효율이 저하되었다. 또한 대형 플라스미드 전달에 주로 사용되는 전기천공법은 세포 손상을 유발하기 때문에 이에 대한 대체 전략의 필요성이 대두되었다.
따라서 본 연구에서는 SV40 NLS와 양이온성 올리고아르기닌 서열을 융합하여 리포플렉스에 조합한 펩타이드 보조 리포플렉스 (Peptide-assisted lipoplex; PAL)를 개발하였다. PAL은 플라스미드의 음전하 중화 능력을 향상시켰으며, 복합체 형성과 안정성 또한 강화하였다. 전기천공법에 비해 PAL은 9,283 bp 및 29,350 bp의 대형 플라스미드의 세포 전달 효율을 각각 2배 및 1.6배 증가시켰으며, 세포 생존율과 활성도를 29배 향상시켰다. 다양한 세포주에서 PAL은 기존 리포플렉스 대비 2배에서 4.4배 향상된 대형 플라스미드 전달 효율을 나타내었다. In vivo 마우스 모델에서는 3일 동안 대형 플라스미드 발현 세포를 유지시켰다. 특히, 과발현되는 암 관련 유전자를 표적으로 하는 CRISPR RNA가 삽입된 Cas9 플라스미드를 포함한 PAL의 전달은 최대 44.1%의 표적 유전자 변이 효율을 보였으며, 화학요법과 결합한 효과로 in vivo 마우스 종양 모델에서 종양 성장을 억제하였다. 실험 결과를 통해, 본 연구의 융합 펩타이드 기반 자기조립 복합체는 유전자 치료용 핵산 전달 시스템으로서 효율성과 안전성을 겸비한 새로운 접근법을 제시하였다.
종합적으로 본 연구를 통해 제시된 (1) 짧은 RNA/융합 펩타이드 나노복합체 기반 유전자 침묵 시스템 및 (2) 융합 펩타이드 보조 지질복합체를 활용한 대형 플라스미드 전달 시스템이 성공적으로 개발되었다. 이 두 시스템은 고효율과 안전성을 결합하여 짧은 RNA 및 대형 플라스미드의 전달을 통해 각각 유전자 침묵과 유전자 편집에 탁월한 성능을 보였다. 이러한 결과는 첨단 유전자 전달 플랫폼으로의 응용 가능성을 크게 높여주어 미래에 다양한 유전적 질환 치료에 적용할 수 있을 것으로 기대되었다.

목차 (Table of Contents)

• Abstract · i
• Table of Contents · iv
• List of Tables · x
• List of Figures · xi
• Chapter 1. Research background · 1
• 1.1. Gene therapy · 1
• 1.1.1. RNA interference · 9
• 1.1.1.1. Short interfering RNA · 11
• 1.1.1.2. Short hairpin RNA · 14
• 1.1.2. CRISPR/Cas9 · 14
• 1.1.2.1. Gene editing tools · 14
• 1.1.2.2. CRISPR/Cas9 · 16
• 1.2. Gene delivery systems · 22
• 1.2.1. Gene delivery · 22
• 1.2.2. Gene delivery systems · 25
• 1.2.2.1. Viral vectors · 27
• 1.2.2.2. Non-viral vectors · 30
• 1.2.2.2.1. Physical methods · 32
• 1.2.2.2.2. Chemical methods · 33
• 1.3. Functional peptides in gene delivery · 36
• 1.3.1. Cell-penetrating peptides · 37
• 1.3.1.1. Mechanisms of cell-penetrating peptides · 39
• 1.3.1.1.1. Direct translocation · 41
• 1.3.1.1.2. Endocytosis · 42
• 1.3.1.1.3. Other mechanisms · 46
• 1.3.1.2. Types of cell-penetrating peptides · 46
• 1.3.1.2.1. Classification of cell-penetrating peptides based on physicochemical properties · 46
• 1.3.1.2.2. Classification of cell-penetrating peptides based on origins · 49
• 1.3.2. Nuclear localization signals · 50
• 1.3.2.1. Types of nuclear localization signals · 52
• 1.3.2.2. Mechanisms of nuclear localization signals · 54
• 1.3.3. Other peptides · 58
• 1.3.4. Fusion peptide strategy · 59
• 1.4. Self-assembled complex in gene delivery · 60
• 1.4.1. Peptide-mediated gene delivery systems · 60
• 1.4.2. Mechanisms of self-assembled complex in gene delivery · 61
• 1.4.3. Influential factors in self-assembled complex · 62
• 1.4.4. Properties and potentials of self-assembled complex · 63
• 1.5. Research objectives · 64
• Chapter 2. Development of an efficient and safe small RNA delivery system using fusion
• peptide-mediated nanocomplex for gene silencing
• 2.1. Introduction · 67
• 2.2. Experimental procedures · 72
• 2.2.1. Materials · 72
• 2.2.2. Preparation of small RNA/fusion peptide nanocomplexes · 75
• 2.2.3. Characterization of small RNA/fusion peptide nanocomplexes · 75
• 2.2.3.1. Gel retardation assay · 75
• 2.2.3.2. Size and zeta-potential measurement · 76
• 2.2.3.3. Scanning electron microscope imaging · 76
• 2.2.3.4. RiboGreen assay · 77
• 2.2.3.5. Stability test in serum · 77
• 2.2.3.6. Release profiles of small RNA · 78
• 2.2.4. Intracellular delivery of nanocomplexes · 78
• 2.2.4.1. Cellular uptake efficiency using flow cytometry · 78
• 2.2.4.2. Fluorescence microscope imaging · 79
• 2.2.4.3. Confocal microscope imaging · 80
• 2.2.5. Gene knockdown analysis · 81
• 2.2.5.1. Quantitative reverse transcription polymerase chain reaction · 81
• 2.2.5.2. In vivo fluorescence imaging · 83
• 2.2.6. Endocytosis pathway study · 84
• 2.2.7. Biocompatibility test of fusion peptide · 84
• 2.2.7.1. Cytotoxicity assay · 84
• 2.2.7.2. Cell viability assay · 85
• 2.2.8. In vivo fluorescence monitoring and imaging · 85
• 2.2.9. Histological analysis · 85
• 2.2.10. Statistical analysis · 87
• 2.3. Results and discussion · 87
• 2.3.1. Characterization of small RNA/fusion peptide nanocomplex · 87
• 2.3.1.1. Confirmation of small RNA/fusion peptide nanocomplex formation · 87
• 2.3.1.2. Stability of small RNA and nanocomplex · 96
• 2.3.2. Evaluation of cellular uptake efficiency of nanocomplex · 103
• 2.3.2.1. Evaluation of small RNA delivery efficiency of nanocomplex in HeLa, HaCaT, and HDFn · 103
• 2.3.2.2. Evaluation of small RNA delivery efficiency of nanocomplex in macrophages · 106
• 2.3.3. Gene silencing effect of nanocomplex · 111
• 2.3.3.1. GAPDH knockdown in HeLa and HaCaT cells · 111
• 2.3.3.2. IL-23p19 knockdown in macrophages · 113
• 2.3.3.3. In vivo mCherry silencing effect · 113
• 2.3.4. In vivo small RNA retention effect · 116
• 2.3.5. Safety assessment of fusion peptide and nanocomplex · 118
• 2.3.5.1. Biocompatibility of fusion peptide in primary cells · 118
• 2.3.5.2. Biocompatibility of fusion peptide in macrophages · 118
• 2.3.5.3. Biocompatibility of nanocomplex in vivo · 120
• 2.3.6. Mechanism study of nanocomplex · 120
• 2.4. Conclusions · 124
• Chapter 3. Development of an efficient and safe large plasmid DNA delivery system
• using peptide-assisted lipoplex for gene editing
• 3.1. Introduction · 125
• 3.2. Experimental procedures · 129
• 3.2.1. Materials · 129
• 3.2.2. Complexation of peptide-assisted lipoplex · 130
• 3.2.3. Characterization of peptide-assisted lipoplex · 131
• 3.2.3.1. Gel retardation assay · 131
• 3.2.3.2. Scanning electron microscopy imaging · 131
• 3.2.3.3. Size and zeta-potential measurement · 132
• 3.2.3.4. Stability test of plasmid DNA within peptide-assisted lipoplex · 132
• 3.2.3.5. Stability test of peptide-assisted lipoplex by size distribution analysis · 132
• 3.2.4. Transfection of peptide-assisted lipoplex · 133
• 3.2.4.1. Fluorescence cell counting · 133
• 3.2.4.2. Electroporation · 134
• 3.2.4.3. Cell viability assay · 135
• 3.2.4.4. Fluorescence measurement · 136
• 3.2.4.5. In vivo fluorescence imaging · 136
• 3.2.5. Mechanism study of peptide-assisted lipoplex · 137
• 3.2.5.1. Treatment of endocytosis inhibitors · 137
• 3.2.5.2. Lysotracker staining and fluorescence imaging · 138
• 3.2.5.3. Cell cycle-arrested cells transfection · 138
• 3.2.6. pCas9-crRNA transfection · 139
• 3.2.6.1. Cloning of crRNAs into pCas9 · 139
• 3.2.6.2. pCas9-crmCherry transfection · 141
• 3.2.6.3. Tracking of indels by decomposition analysis · 141
• 3.2.6.4. Cell viability assay · 142
• 3.2.6.5. Live/Dead cell imaging · 144
• 3.2.6.6. In vivo intratumoral delivery · 144
• 3.2.7. Histological analysis · 145
• 3.2.8. Statistical analysis · 145
• 3.3. Results and discussion · 146
• 3.3.1. Characterization of peptide-assisted lipoplex · 146
• 3.3.1.1. Confirmation of peptide-assisted lipoplex formation · 146
• 3.3.1.2. Stability of plasmid DNA and peptide-assisted lipoplex · 148
• 3.3.2. Evaluation of transfection efficiency and cellular activity of peptide-assisted lipoplex · 152
• 3.3.2.1. N:P ratio optimization of SV40-R11 peptide in PAL-pCas9 · 152
• 3.3.2.2. Cell recovery rate and viability of PAL-pCas9 · 157
• 3.3.2.3. Evaluation of transfection efficiency of PAL-pMasterE · 159
• 3.3.2.4. Cell recovery rate and viability of PAL-pMasterE · 161
• 3.3.2.5. Evaluation of transfection efficiency of peptide-assisted lipoplex in various cell lines · 164
• 3.3.2.6. In vivo fluorescence analysis in mice injected with Cas9-mCherry expressing cells · 166
• 3.3.3. Mechanism study of peptide-assisted lipoplex · 169
• 3.3.3.1. Endocytosis mechanism of peptide-assisted lipoplex · 169
• 3.3.3.2. Endosomal escape and nuclear localization of peptide-assisted lipoplex · 172
• 3.3.3.3. Evaluation of transfection efficiency on cell cycle-arrested cells · 173
• 3.3.4. Gene editing efficiency of peptide-assisted lipoplex · 174
• 3.3.4.1. Construction of pCas9-crRNA · 174
• 3.3.4.2. Evaluation of mCherry gene editing efficiency using fluorescence-positive cell counting · 174
• 3.3.4.3. Evaluation of PLK1 and BCL2 editing efficiency using TIDE analysis · 177
• 3.3.5. Therapeutic application of peptide-assisted lipoplex for cancer · 184
• 3.3.5.1. Effect of PAL-crRNA on cancer cell viability · 184
• 3.3.5.2. Synergistic effects of PAL-crRNA and chemical drugs on cancer cell viability · 187
• 3.3.5.3. Therapeutic effects of PAL-crRNA/Dox and safety of peptide-assisted lipoplex in vivo · 191
• 3.4. Conclusions · 193
• Chapter 4. Overall conclusions · 195
• 4.1. Conclusions · 195
• 4.2. Future directions · 197
• References · 200
• 국문초록 · 228
• Acknowledgements · 232