Lentiviral gene tranfer to porcine airway epithelia

慢病毒基因转移至猪气道上皮细胞

• 批准号: 7741445
• 负责人: PAUL B MCCRAY
• 金额: $ 28.86万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7741445
• 关键词: Alleles; Animal Model; Animals; Apical; Baculoviruses; Binding; Bronchoscopy; Cells; Cloning; Code; Complementary DNA; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Defect; Disease; Disease Progression; Electrolytes; Engineering; Epithelium; Exocrine pancreatic insufficiency; Family; Family suidae; Feline Immunodeficiency Virus; Gene Delivery; Gene Expression; Gene Transfer; Genes; Goals; Health; Human; Ileus; Immune response; Infection; Inflammation; Instruction; Kinetics; Knock-in Mouse; Lentivirus Vector; Libraries; Life; Liquid substance; Liver; Lung; Lung diseases; Measures; Meconium; Modeling; Morbidity - disease rate; Mutagenesis; Nasal Epithelium; Nose; Organ; Outcome Measure; Phenotype; Pneumonia; Point Mutation; Property; Proteins; Pulmonary Cystic Fibrosis; Relative (related person); Reporter; Resolution; Screening procedure; Serial Passage; Somatic Cell; Specificity; Subfamily lentivirinae; Surface; System; Testing; Tracheal Epithelium; Tropism; Vesicular stomatitis Indiana virus; Viral; Virus; X-Ray Computed Tomography; airway epithelium; base; cell type; cellular transduction; clinical application; deltaF508-CFTR protein; directed evolution; gene therapy; improved; in vivo; mortality; mutant; novel; positional cloning; prevent; progenitor; programs; receptor binding; respiratory; stem; transduction efficiency; vector; virus envelope

Cystic fibrosis lung disease might be corrected by delivering a normal copy of the CFTR cDNA to sufficient epithelia to restore electrolyte and liquid transport. Towards this goal, our Project has developed lentiviral vectors based on feline immunodeficiency virus (FIV) because of their ability to integrate and persist. We also identified a number of virus envelopes ("pseudotypes") that confer apical entry on polarized epithelia. The testing of the utility of gene transfer for CF has been hindered by lack of an animal model that recapitulates the human phenotype. Recently, this PPG developed a pig CF model. Animals heterozygous for a CFTR null or AF508 knock in allele were generated. Remarkably, live born CFTR'' and CFTR^^''^^ piglets share many features of human babies with CF including pancreatic insufficiency, meconium ileus, liver involvement, and respiratory abnormalities. We will use this animal model to evaluate the efficacy of lentiviral gene transfer in modifying CF pulmonary disease. There are 3 aims. Aim 1. Which lentiviral pseudotype achieves the best apical targeting and persistent gene transfer? We propose a thorough comparison of several candidate envelopes in pig and human cells to select an optimal vector pseudotype. We hypothesize that one or more pseudotyped lentivirus will target surface airway epithelia that express CFTR and possess progenitor capacity. Aim 2. Can we increase the gene transfer efficiency of the GP64 envelope by directed evolution on airway epithelia? We identified the baculovirus GP64 envelope as having very favorable properties for in vivo airway transduction. We hypothesize that we can further improve lentiviral gene transfer efficiency and specificity by directed evolution of GP64. We will use a reverse genetics system to engineer a replication competent GP64 pseudotyped VSV and perform serial passage on primary pig and human airway epithelia using wild type GP64 to evolve an ainway adapted envelope. In a complementary approach, the GP64 receptor binding domain will be randomly modified by PCR mutagenesis, and this library of novel lentivirus mutants screened on ainA/ay epithelia. These same directed evolution approaches can be applied to other candidates that emerge from Aim 1. Aim 3. Will lentiviral gene delivery correct the CFTR defect and prevent or slow lung disease progression? We will perform gene transfer to the pig ainA/ays using our optimized vectors to identify the pseudotypes with efficient gene transfer and persistence. We hypothesize that FIV gene transfer of CFTR to the ainA/ays of cFTR'^^¿^''^''^¿^ pigs will Drevent or significantly slow lung disease progression. Outcome measures will include in vivo bioelectrics, ligh resolution CT scans, screening for infection and inflammation by bronchoscopy, and other measures of lung health. RELEVANCE (See instructions);

囊性纤维化肺疾病可以通过将CFTR cDNA的正常拷贝递送至 足够的上皮细胞来恢复电解质和液体运输。为了实现这一目标，我们的项目已经开发 基于猫免疫缺陷病毒（FIV）的慢病毒载体，因为它们能够整合和 坚持。我们还鉴定了一些病毒包膜（“假型”）， 极化上皮基因转移对CF的效用的测试由于缺乏一种有效的方法而受到阻碍。 再现人类表型的动物模型。最近，PPG开发了猪CF模型。 产生CFTR无效或AF 508敲入等位基因杂合的动物。值得注意的是， CFTR“和CFTR仔猪与患有CF的人类婴儿有许多共同特征，包括胰腺 功能不全、胎粪性肠梗阻、肝脏受累和呼吸异常。我们会用这种动物 模型，以评估慢病毒基因转移在改善CF肺病中的功效。有3 目标。目标1。哪种慢病毒假型实现了最佳的顶端靶向和持久基因 转移？我们提出了一个彻底的比较几个候选信封在猪和人类细胞， 选择一个最佳向量伪类型。我们假设一个或多个假型慢病毒将靶向 表达CFTR并具有祖细胞能力的表面气道上皮细胞。目标二。才能增加 GP 64包膜定向进化对气道上皮细胞的基因转移效率？我们 鉴定了杆状病毒GP 64包膜对于体内气道具有非常有利的性质， 转导我们假设我们可以进一步提高慢病毒基因转移效率， 通过GP 64的定向进化的特异性。我们会用反向遗传学系统设计一个复制品 胜任GP 64假型VSV并在原代猪和人气道上进行连续传代 使用野生型GP 64来进化通道适应性包膜。在一种补充方法中， GP 64受体结合结构域将通过PCR诱变随机修饰，并且该新的GP 64受体结合结构域文库将通过PCR诱变随机修饰。 在AinA/ay上皮上筛选慢病毒突变体。这些定向进化方法可以 适用于目标1中出现的其他候选人。目标3.慢病毒基因传递会纠正 CFTR缺陷和预防或减缓肺部疾病进展？我们将对猪进行基因转移 我们使用我们优化的载体来鉴定具有有效基因转移的假型， 坚持不懈我们假设，将CFTR的FIV基因转移到cFTR猪的血管中， 避免或显著减缓肺部疾病进展。结果测量将包括体内生物电， 高分辨率CT扫描，通过支气管镜检查筛查感染和炎症，以及其他措施 肺部健康 相关性（参见说明）;