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Genetic engineering RGD-containing siRNA protein carrier

含有RGD的基因工程siRNA蛋白载体

基本信息

批准号：
10019543
负责人：
Hong Yan Liu
金额：
$ 13.2万
依托单位：
DOTQUANT, LLC
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-18 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10019543
关键词：
3-Dimensional Address Affinity Antibodies Aptamer Technology Arginine Aspartic Acid BT 474 Binding Biodistribution Blood Circulation Blood Vessels Buffers Carrier Proteins Cells Charge Chimera organism Clinical Cloning Complex Consumption Diagnosis Disease Double-Stranded RNA Double-Stranded RNA Binding Domain Endosomes Endothelial Cells Engineering Evaluation FDA approved Gene Silencing Genetic Engineering Glycine Goals Granulocyte Colony-Stimulating Factor Histidine Human Image Individual Integrin alphaVbeta3 KDR gene Label Life Ligand Binding Ligands Malignant Neoplasms Micelles Modeling Molecular Conformation Mus Neoplasms in Vascular Tissue Non-Viral Vector Pegfilgrastim Peptides Pharmaceutical Preparations Polymers Process Production Protein Binding Domain Proteins RGD (sequence)RNA RNA Interference Rest Safety Serum Small Interfering RNA Specificity Therapeutic Time Toxic effect Tumor Cell Line Variant Viral Vector Work Xenograft Model angiogenesis aptamer base body system cancer therapy cell type clinical application cost cyanine dye 5 cytotoxicity density image guided imaging agent immunogenicity in vivo innovative technologies nanoparticle neoplastic cell neovasculature novel therapeutics siRNA delivery targeted delivery tumor vector

项目摘要

siRNA has shown the potential in treating undruggable diseases such as cancer. Despite its potential, therapeutic application of siRNA has been greatly impeded by the lack of safe and effective delivery vectors. Viral vectors have safety concern and have been precluded for clinical application. Current most non-viral vectors including lipoplexes, polymers, inorganic nanoparticles, micelles, and cell penetrating peptide are positively charged and suffer from low serum stability, off-target effect, cytotoxicity and batch-to-batch variation. Additionally, inorganic nanoparticles and polymers have serious safety concerns due to non-degradable and poor clearance. Therefore, current carriers are unsuitable for systemic delivery of siRNA. In previous study, we have developed a non-cationic protein based aptamer-siRNA chimera carrier by adding 18 Histidine (His) peptide on human origin dsRNA binding domain (dsRBD) protein. DsRBD-18his does not rely on high positive charges to interact with RNA molecules; instead, it binds dsRNA via specific 3-D conformation. Histidine molecules have pKa value about 6, at neutral pH, they are uncharged, and charged in acidic condition such as endosome. 18His can confer sufficient buffering capacity to drive cargo endosomal escape. However, current siRNA vectors including dsRBD-18his lacking cell type specificity, and targeting molecules such as antibody, aptamer, or ligand have to be added into each cargo. That is a time consuming and costly process. RGD (Arg- Gly-Asp) peptide has been well characterized as a binding ligand of an integrin αvβ3, which is highly expressed on tumor neo-vasculature as well as some tumor cells, but not present in resting endothelial cells and normal organ systems. RGD peptide is a well validated tumor targeting molecule, and has been used for guiding imaging agents and drugs for tumor diagnosis and therapy in clinical setting. In this project, we will genetic engineer three tumor and tumor blood vessel targeted vectors by fusing RGD peptide into N-, C-, or both N-and C termini of dsRBD- 18His protein. New vectors will have three functions including siRNA binding, endosomal escape, and tumor targeting. New vectors will be uncharged, low toxicity, biodegradable, cell type specific, and ease of mass production. The specific aims of this proposal are 1) cloning, expression, and characterization of RGD vectors, and 2) evaluation of tumor and tumor neo-vasculature targeting capabilities in cancer xenograft models. New vectors with load-to-go capability will simplify siRNA delivery in vivo, and any siRNAs can be loaded and targeting delivered to tumors or tumor blood vessels.

siRNA在治疗癌症等不治之症方面显示出了潜力。尽管它