药用高分子材料-siRNA药物课件

蛋白质表达
转录mRNA
化学修饰的siRNA
Figure 2. Chemical modifications of siRNAs. Shown are structures of sugar, backbone and base modifications and of the cholesterol conjugate.
基于纳米载体的 siRNA给药系统
脂质体类siRNA纳米载体
脂质体类siRNA纳米载体
Stable Nucleic Acid Lipid Particle (SNALP)
Mean particle size: 140±12 nm Mean encapsulation efficiency: 93%±3% Nucleic acid/lipid ratio: 42±4g/mol
水溶性阳离子高分子纳米载体:
NUCLEIC ACIDS RESEARCH 32 (19): Art. No. e149 2004
Cancer Research 2005, 65, 8984-8992
Ligand
PEG
Degradable linkage
siRNA
JACS, 127, 1624-1625
Байду номын сангаас
Removing the organic solvent
Second emulsion
More water
polymer
siRNA
Cationic lipid
PEG-PLA/Cationic lipid/siRNA nanoparticles
First emulsion
Cationic Lipid Assisted Nanoparticles: CLAN
Natue Chemical Biology, 2 (2006), 711-719.
Nature reviews drug discovery, 6 (2007), 443-453
Nature，2004，432：173-178
化学修饰的siRNA
Challenge 1: 稳定 Challenge 2: 长循环 Challenge 3: 靶向 Challenge 4: 渗透 Challenge 5: 细胞摄取 Challenge 6: 制剂化
高分子类 siRNA给药系统
Steric Coat (Inert)
Active Core
Exposed Targeting Ligands
Cleavable Link (Shedding)
Ligands
Steric polymer
Carrier
Cleavable linkage
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siRNA Payload
siRNA-SNALP with a longer half-life in plasma and liver reduced serum HBV DNA titers in mouse model following intravenous injection
Nature Biotechnology 23, 1002 - 1007 (2005)
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Sun TM, et al., ACS Nano, 2011, 5, 1483-1494
Days post-xenografting
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44
Tumor Volume (mm3)
100
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PaclitaxelMicelleplexNCsiRNA
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siRNA
亲水嵌段
疏水嵌段
带正电荷嵌段
阳离子胶束/siRNA复合纳米颗粒:
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FAM-siRNA
Rho-PTX
Rho-PTXMicelleplexFAM-siRNA
FAM-siRNA
PBS
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Rho-PTXMicelleplexNCsiRNA + MicelleplexFAM-siRNA
siRNA包载效率低下，仅为21%！ 1 g siRNA目前的价格为200万！
基于聚乙二醇-聚乳酸的siRNA纳米给药体系
Organic phase (polymer/lipid)
aqueous phase (siRNA)
Such nanoparticles, which is based FDA approved PEG-PLA, exhibit high encapsulation efficiency of siRNA (about 96%).
姚老师作业：聚乳酸的本体聚合工艺，相应机理和反应方程
第六章 高分子纳米药物-siRNA药物
病变组织的基因活性
正常表达
过度表达
低表达
小分子 抑制剂?
蛋白疗法?
抑制剂: RNA干扰疗法, 反义RNA, …
下调
上调
增强表达: 质粒DNA Viral Vectors
Emulate Drug?
抗体: Bind protein, but only extracellular epitopes Low throughput, high cost 反义RNA/核酶: Stop gene expression (production), most proteins Chemical stabilization essential Barriers: sequence selection, potency, toxicities, … RNA干扰: Stop gene expression (production), most proteins Good activity without chemical stabilization Rapidly replacing antisense: really better?
靶向修饰的水溶性高分子-siRNA键合物:
JACS, 126 (42), 13612 -13613
Loading siRNA post nanoparticle formation PEG: prolonged blood circulation Biocompatible Degradable, potentially intracellular siRNA release Co-delivery of chemotherapeutic drug for synergistic effect
Clinical
Lead Discovery & Optimization
Preclinical
药物递送是关键挑战
siRNA药物?
siRNA药物: 革命性突破
难以被细胞摄取：大分子，阴离子
脱靶效应：非特异性沉默， 引起免疫刺激等
难以富集在肿瘤部位：体内不稳定， 半衰期短
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Potential of siRNA-Based Therapy
RNA干扰疗法的潜力
siRNA药物临床引用的关键
表达siRNA的质粒DNA的传递 病毒（Nature Biotechnology, 2002, 20: 1006-10） 非病毒类载体 化学合成的siRNA的传递 非病毒类载体：脂质体和高分子 物理手段: electroporation, high pressure/high volume injection
特异性基因下调手段
RNAi 作用机制
Nature，2004，430：161
Log [Oligo] (M)
Normalized Gene Expression Level
Antisense siRNA
Controls
From H. Winkler, IBC Conference “RNA Interference”, San Diego, CA, Feb 2003
基于CLAN技术设计的siRNA给药系统
基于CLAN技术设计的靶向siRNA给药系统
Nature Biotechnol. 23, 709–717 (2005)
抗体类 siRNA给药系统
M Sioud1, Gene Therapy, 2006, 13, 194–195
思考题
RNAi原理和siRNA siRNA给药载体有几种，分别是什么，并简单描述。 siRNA和质粒DNA的差别，它们的给药方式有何区别以及一致的地方。 siRNA和质粒DNA针对疾病治疗的原理有什么差异。
siRNA
dsRNA
siRNA
RNAse
shRNA
In Vitro
In Vivo
In Vivo
miRNA
Adapted From Dykxhoorn et al., Nature Reviews 4, 457, 2003
具有RNAi机制的药物
Target ID
siRNA Target Validation Growing
Potential of siRNA-Based Therapy
RNA干扰疗法的潜力
siRNA的传递：体内RNAi技术应用的关键
内涵体逃避
靶向 结合 吞噬
核转运
DNA释放 到细胞质
形成RISC
内涵体逃避
靶向 结合 吞噬
siRNA释放 到细胞质
siRNA的传递
DNA的传递
组织转运
DAN和siRNA传递的比较
PaclitaxelMicelleplexsiPlk1
PaclitaxelMicelleplexNCsiRNA +MicelleplexsiPlk1
MicelleplexsiPlk1
PBS
Blank Micelles
MicelleplexNCsiRNA
Sun TM, et al., ACS Nano, 2011, 5, 1483-1494
β-actin
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Plk1 mRNA (% of Control)
MicelleplexNCsiRNA
PBS
Blank micelles
MicelleplexsiPlk1
PaxlitaxelMicelleplex
PaxlitaxelMicelleplexsiPlk1
PaxlitaxelMicelleplexNCsiRNA +MicelleplexsiPlk1
Plk1
Kim A.Woodrow, W. Mark Saltzman et al., Nature Materials, 2009, 8, 526 – 533 Christopher J. Cheng, W. Mark Saltzman, Biomaterials, 2011, 32, 6194–6203