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.
