AEROSOL DELIVERY OF CATIONIC LIPID & DNA COMPLEX INTO NON HUMAN PRIMATE LUNGS

阳离子脂质的气溶胶输送

• 批准号: 6277815
• 负责人: GEOFF AKITA
• 金额: $ 7.56万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6277815
• 关键词: Mammalia; Primates; biological products; biotechnology; gene therapy; model design /development; respiratory system

Direct gene transfer via cationic lipid/plasmid DNA (pDNA) complexes offers an attractive therapeutic approach to cystic fibrosis. The objective of this study was to examine the safety and efficacy of aerosol delivery of up to 12 doses of lipid/pDNA into the lungs of nonhuman primates. The time interval between doses was r 7 days. Aerosol exposures prior to the last exposure were to a mid-level dose of lipid/pDNA with a plasmid carrying the gene for the cystic fibrosis transmembrance conductance regulator (CFTR). The last aerosol exposure was to the same dose of lipid/pDNA with a plasmid carrying the gene for chloramphenicol acetyltransferase (CAT). Rhesus monkeys (Macaca mulatta) were trained by the New England Regional Primate Research Center personnel to sit in a standard primate restraint chair and to wear a fitted face mask through which aerosolized lipid/pDNA was delivered for up to 3 hours. Prior to aerosol administrations thoracic radiographs, serum chemistries, CBCs and antibody to lipid and or DNA was determined. During aerosol administrations the animal's heart rate, blood oxygen saturation and clinical condition was monitored. At predetermined intervals during aerosol treatments animals were anesthetized and thoracic radiographs, nasal brushings, bronchoscope guided bronchial brushes were obtained. Serum chemistries and CBCs were also performed. Bronchial and nasal brushes were analyzed for pDNA specific DNA and mRNA by fluorescent in situ hybridization (FISH) and RT-PCR, respectively. Two days following the last lipid/pDNA exposure thoracic radiographs, a serum chemistry, CBC and serum for detection of any antibodies to lipid or pDNA were obtained. The animal was euthanized and underwent a complete necropsy. The lungs were evaluated for transgene expression, and pDNA specific mRNA and DNA. The lungs, livers, kidneys, spleens, hearts and any other organs displaying abnormalities on gross examination were examined histologically. The first animal received 12 aerosol administrations of lipid/pDNA with one week between each dose. The second animal received 6 aerosol administrations of lipid/pDNA with an interval of s 2 months between doses. Throughout aerosol exposures and just prior to euthanasia thoracic radiographs, serum chemistries and CBCs were within normal limits. Analysis of transgene expression indicated that a longer interval between exposures resulted in higher levels of transgene expression. Vector DNA was detected in nasal and bronchial brushes. Transgene expression was detected in lung but not on brushes, suggesting that the lack of transgene expression on brushes was a sampling artifact. In the first animal there was minimal inflammation in the lung and all other organs

通过阳离子脂质/质粒DNA（pDNA）的直接基因转移 复合物为囊性提供了吸引人的治疗方法 纤维化。 这项研究的目的是检查安全性和 将多达12剂脂质/pDNA的气溶胶递送到该气溶胶的功效 非人类灵长类动物的肺。 剂量之间的时间间隔为r 7 天。 在上次接触之前的气溶胶暴露是 脂质/pDNA的中级剂量，带有基因的质粒 囊性纤维化透射电导调节剂（CFTR）。 最后 气溶胶暴露于具有质粒的同一剂量的脂质/pDNA 携带氯霉素乙酰转移酶（CAT）的基因。 恒河 猴子（Macaca Mulatta）接受了新英格兰地区的培训 灵长类动物研究中心的人员坐在标准灵长类 约束椅子并戴上合适的口罩 雾化的脂质/pDNA最多可输送3小时。 之前 气雾剂施用胸腔X光片，血清化学，CBCS 确定脂质和或DNA的抗体。 在气雾剂期间 管理动物的心率，血氧饱和度和 监测临床状况。 在预定的间隔 气溶胶处理动物被麻醉和胸部X光片， 获得了鼻刷，支气管镜指导的支气管刷。 还进行了血清化学和CBC。 支气管和鼻腔 通过荧光在 原位杂交（FISH）和RT-PCR。 两天 遵循最后的脂质/pDNA暴露胸部X光片，血清 化学，CBC和血清，用于检测任何脂质抗体或 获得pDNA。 该动物被安乐死并接受了 完整的尸检。 评估肺的转基因表达， 和pDNA特异性mRNA和DNA。 肺，肝脏，肾脏，脾脏， 心脏和其他任何有异常情况的器官 检查检查。 第一只动物收到 12个脂质/pDNA的气溶胶管理 剂量。 第二只动物接收了6个气溶胶管理 脂质/pDNA的间隔为2个月的剂量之间。 自始至终 气溶胶暴露，就在安乐死胸腔X光片之前， 血清化学和CBC在正常范围内。 分析 转基因表达表明在 暴露导致更高水平的转基因表达。 向量 在鼻和支气管刷中检测到DNA。 转基因表达 在肺中检测到，但没有在刷子上检测到 刷子上的转基因表达是一种采样伪影。 在第一个 动物肺和所有其他器官的炎症很少