Specific targeting of a neuronal subset using a novel chimeric vector pseudotype

描述（由申请人提供）：本提案将研究狂犬病病毒包膜糖蛋白（RvG）可用作制备新型嵌合包膜糖蛋白（称为RvGN 2）的基础的假设，通过插入来自神经生长因子（NGF）的环2代替RvG的“毒素样”环。环2赋予NGF结合TrkA受体的能力，TrkA受体是主要在感觉神经元上表达的受体，包括从背根投射到脊髓的板层I和II中的那些。RvGN 2嵌合体仍将保持逆行转运的能力，通过允许微创递送策略的潜力，使载体对临床应用更具吸引力。该项目更具体的目标是测试RvG可以作为工程重定向的功能可行基础的假设。这将通过免疫细胞化学进行评估，以检测转染细胞表面上的RvGN 2表达。下一步将是检验RvGN 2报告病毒将在体外优先感染表达TrkA的细胞而不是允许野生型狂犬病病毒感染的细胞的假设。TrkA受体的瞬时转染也将用于“拯救”抗性细胞系。最后一个目的将检验以下假设：当通过注射到脊髓中将RvGN 2报告病毒体内施用给大鼠时，将维持体外靶向特征。该目的的第二部分是显示通过肌内或皮内注射的外周递送将保持这种靶向特征。然后将免疫组织化学与抗体一起用于组织切片，以检测指示不同神经元子集的不同标记物。将该染色模式与报告病毒的转导模式进行比较，以评估哪些神经元亚群被转导。目前，病毒基因治疗载体不能区分神经元的亚群，这使得它们在治疗许多疾病方面不切实际。选择性靶向神经元亚群的能力将为治疗从慢性疼痛到帮助脊髓再生的多种疾病奠定基础。而且，逆行转运的能力将允许这些载体的侵入性更小的递送，而不是直接注射到脊髓中。

DESCRIPTION (provided by applicant): This proposal will investigate the hypothesis that the rabies virus envelope glycoprotein (RvG) can be used as a base for making a novel chimeric envelope glycoprotein, to be called RvGN2, by inserting Loop 2 from Nerve Growth Factor (NGF) in place of the "toxin-like" loop of RvG. Loop 2 confers the ability of NGF to bind the TrkA receptor, which is a receptor that is primarily expressed on sensory neurons including those projecting from the dorsal root into lamina I & II of the spinal cord. The RvGN2 chimera will still maintain the ability to be retrogradely transported, making the vector more attractive for clinical applications by allowing the potential for a less invasive delivery strategy. The more specific aims of this project will be to test the hypothesis that the RvG can act as a functionally viable basis for engineered retargeting. This will be assessed by immunocytochemistry to detect RvGN2 expression on the surface of transfected cells. The next step will be to test the hypothesis that RvGN2 reporter viruses will preferentially infect cells in vitro that express TrkA over cells that are permissive to wild- type Rabies virus infection. Transient transfection of the TrkA receptor will also be used to "rescue" resistant cell lines. The last aim will test the hypothesis that the in vitro targeting profile will be maintained when RvGN2 reporter virus is administered to rats in vivo, by injection into the spinal cord. The second part of this aim is to show that peripheral delivery by intramuscular or intradermal injection will preserve this targeting profile. Immunohistochemistry will then be used on tissue sections with antibodies to detect different markers indicative of separate subsets of neurons. This staining pattern will be compared to the transduction pattern of the reporter viruses to assess which neuronal subpopulations were transduced. At this time viral gene therapy vectors cannot differentiate among subsets of neurons, making them impractical to treat many disorders. The capability to selectively target subsets of neurons will allow the basis for treatment of a multitude of disorders, from chronic pain to aiding in spinal cord regeneration. And, the ability to be retrogradely transported will allow for less invasive delivery of these vectors as opposed to direct injections into the spinal cord.