Respiratory Syncytial Virus Based Vectors for CFTR

许多病毒载体已经被开发出来，最终目标是高效地将替换基因传递到人体器官，而不会对目标细胞造成损害。然而，人类呼吸道上皮的管腔细胞通常对大多数载体都是折射的。呼吸道病毒如呼吸道合胞病毒(RSV)是独一无二的，因为它们专门针对这些细胞。典型的感染会在几天后导致细胞死亡，但现在可以通过改变RSV的方式使其不再具有细胞毒性。 因此，RSV可能是一种有用的载体，可以将转基因(如CFTR通道)输送到囊性纤维化患者的呼吸道上皮细胞。我们现在知道RSV特异性地感染由PPG细胞培养核心培养的人高分化呼吸道上皮(HAE)培养物中的纤毛细胞，并在一周内对这些细胞具有细胞毒性。在这项提议中，将进一步开发一些用于呼吸道特异性基因传递的试剂。具体地说，RSV的减毒疫苗候选毒株将进行测试，以确定其在HAE细胞中没有细胞毒性。除了减毒的RSV毒株外，最近利用RSV‘复制子’所做的工作缺乏病毒糖蛋白基因，因此无法传播，现在可以用于进一步的媒介开发。为了从这些复制子中产生具有感染性的载体，病毒糖蛋白将以类似于以下诱导方式从整合的基因拷贝中提供 逆转录病毒包装系统。非复制型RSV还提供了传递受控(剂量依赖)的CFTR转基因的机会，这可能足以纠正诸如囊性纤维化之类的遗传缺陷。能够在产生细胞中产生最高数量的载体和在目标HAE细胞中产生最低细胞毒性的RSV载体将被设计成携带CFTR转基因。这种PPG的独特之处在于，能够在囊性纤维化患者的HAE培养中测试产生的RSV-CFTR载体，用于修复生物电缺陷、恢复呼吸道表面液体深度和恢复粘液纤毛清除。

Numerous viral vectors have been developed with the ultimate goal of efficiently delivering replacement genes to human organs, without causing damage to the targeted cells. However, the luminal cells of the human airway epithelium are generally refractive to most vectors. Respiratory viruses like respiratory syncytial virus (RSV) are unique in that they specifically target these cells. Typical infection causes cell death after several days however it is now possible to alter RSV in such a way that it may no longer be cytotoxic. As a result, RSV could be a useful vector for delivering a transgene, such as the CFTR channel, to the respiratory epithelial cells of cystic fibrosis patients. We now know that RSV specifically infects the ciliated cells in primary cultures of human well-differentiated airway epithelial (HAE) cultures developed by the PPG cell culture core, and is cytotoxic on these cells over the course of a week. In this proposal, a number of reagents will be further developed for airway specific gene delivery. Specifically, attenuated vaccine candidate strains of RSV will be tested for lack of cytotoxicity in HAE cells. In addition to attenuated RSV strains, recent work with RSV 'replicons' that lack the viral glycoprotein genes and therefore are unable to spread are now available for further vector development. To generate infectious vector from these replicons, the viral glycoproteins will be provided from integrated gene copies in an inducible manner similar to retroviral packaging systems. Non-replicating RSV also provide the opportunity to deliver controlled (dose dependent) amounts of the CFTR transgene which may be enough to correct a genetic defect such as cystic fibrosis. RSV vectors capable of producing the highest amounts of vector in producer cells and the lowest cytotoxicity in target HAE cells will be engineered to carry CFTR transgene. Unique to this PPG is the ability to test resulting RSV-CFTR vectors in HAE cultures derived from cystic fibrosis patients for repair of bioelectric defects, restoration of airway surface liquid depth, and the restoration of mucociliary clearance.