Rational Design of Viral-Mimetic Lipid Gene Vectors

病毒模拟脂质基因载体的合理设计

基本信息

批准号：
8501446
负责人：
FRANCIS C. SZOKA
金额：
$ 30.92万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-22 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8501446
关键词：
Animals Betaine Biophysics Blood Circulation Carnitine Cells Cellular biology Charge Chemical Structure Chimeric Proteins Complex Confocal Microscopy Cytoplasm DNA Disease Dose Drug Formulations Encapsulated Endosomes Engineering Enzymes Esters Exhibits Fluorescence Gene Transfer Genes Head Hepatocyte Hepatotoxicity Hereditary Disease Hydrophobicity Infection Inflammatory Injection of therapeutic agent Intravenous Lead Ligands Link Lipid Binding Lipids Liposomes Liquid substance Liver Location Luciferases Measures Mediating Membrane Membrane Fusion Molecular Mus Nucleic Acids Organ Particulate Pathway interactions Peptides Phase Process Property Reaction Research Serum Surrogate Markers Synthetic Genes Tail Techniques Testing Toxic effect Transfection Viral Viral Vector Virus base carboxylate cytokine cytotoxicity design gene therapy immune clearance improved in vivo interfacial macromolecule mimetics neoplastic novel particle phase change plasmid DNA preference public health relevance success trafficking tumor tumor progression vector

项目摘要

DESCRIPTION (provided by applicant): The objective of this mechanism-based research is to devise a safe and efficient, synthetic lipid- based gene carrier for use in parenteral gene therapy in animals. We postulate that molecular strategies to deliver nucleic acids by viral membrane fusion proteins can be mimicked using less complex lipids. We will design and synthesize lipids that mediate a triggered-membrane fusion and assemble them with plasmid DNA to create a sub-100 nm, nanolipid particle (NLP) that mediates high transfection with low toxicity in animals. The research plan contains four specific aims. Specific aim 1: To design, synthesize and characterize novel lipids that alter their charge, interfacial hydrophobicity and phase preference at low pH. The design explores the effect of systematic alterations in the chemical structure of the head group, linker region and hydrophobic portion of the lipids on liposome collapse at low pH. Specific aim 2: To formulate lipids synthesized in aim 1 so that they undergo a low pH triggered lamellar to a hexagonal phase change and to characterize the formulations to confirm their phase preference. Specific aim 3: To measure the transfection activity and cytotoxicity of NLP formulations encapsulating luciferase encoding plasmid DNA as a function of the amount of lipid that binds to the cell. To measure the rate, location and extent of cytoplasmic contents delivery using fluorescence confocal microscopy and correlate this with the pH of the compartment when delivery is first observed. To test the hypothesis that incorporation of an appropriate targeting ligand or membrane destabilizing peptide can further increase gene transfer. Specific aim 4: To characterize the extent and duration of gene transfer in liver hepatocytes or in a flank tumor in mice as a function of NLP composition after a low volume intravenous dose of an optimized targeted NLP. Completion of these specific aims will result in a 'lipid engineered' synthetic gene carrier with an in vivo gene transfer efficiency approaching that of current viral vectors. The resulting NLP will be a non- inflammatory vector in circulation and mediate rapid cytoplasmic transfer of nucleic acids after internalization into cells. Success in this research will enable safe and effective gene therapy for a variety of currently untreatable genetic disorders and neoplastic diseases.

描述（由申请人提供）：基于机制的研究的目的是设计一种安全有效的基于合成脂质的基因载体，用于在动物中用于肠胃外基因治疗。我们假设可以使用较不复杂的脂质模仿通过病毒膜融合蛋白输送核酸的分子策略。我们将设计和合成脂质，介导触发的膜融合并用质粒DNA组装它们，从而产生亚100 nm的纳米脂质颗粒（NLP），该纳米脂质颗粒（NLP）介导了高毒性，而动物中的毒性低。研究计划包含四个具体目标。特定目标1：设计，合成和表征新的脂质，这些脂质改变了它们的电荷，界面疏水性和低pH值的相位偏好。该设计探讨了头部组，接头区域和脂质的疏水部分对脂质体塌陷的系统变化的影响。特定目标2：为AIM 1合成的脂质制定脂质，以便它们经历低pH值触发了层状片段变化，并表征制剂以确认其相位偏好。特定目的3：测量NLP配方的转染活性和细胞毒性，封装了荧光素酶编码质粒DNA，这是与细胞结合的脂质量的函数。测量使用荧光共聚焦显微镜递送的细胞质含量的速率和程度，并在首次观察到递送时将其与隔室的pH相关。为了测试以下假设：掺入适当的靶向配体或膜破坏肽可以进一步增加基因转移。特定目标4：表征肝肝细胞中基因转移的程度和持续时间或小鼠侧面肿瘤中的函数NLP组成的函数，在低体积静脉内静脉内剂量优化的靶向NLP之后。这些特定目标的完成将导致具有体内基因转移效率接近当前病毒载体的“脂质工程”合成基因载体。所得的NLP将是循环中的非炎性载体，并在内部化到细胞后介导核酸的快速细胞质转移。这项研究的成功将使目前各种目前不可治疗的遗传疾病和肿瘤疾病的安全有效的基因治疗能够。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection.

DOI：
10.1016/j.jconrel.2008.12.016
发表时间：
2009-04-17
期刊：
JOURNAL OF CONTROLLED RELEASE
影响因子：
10.8
作者：
Cohen, Richard N.;van der Aa, Marieke A. E. M.;Macaraeg, Nichole;Lee, Ai Ping;Szoka, Francis C., Jr.
通讯作者：
Szoka, Francis C., Jr.

Nucleic acid delivery: the missing pieces of the puzzle?