The goal of this Phase I proposal is to develop critical reagents that will support the production of high vector titers for transfusion medicine ie. hematopoietic gene therapy for treatment of blood disorders as well as other applications. Attempts at concentrating these vectors has generally resulted in a significant loss of infectious activity. One possibility is that the virus titer maybe dependent upon the composition of the medium in which the viral producing cells (VPC) are grown, therefore Dr. Brown proposes to study different media compositions in an effort to improve the vector titer produced by the VPC. A second issue for the medium design is the safety and infusibility of the medium into human patients. The ability to produce the vector supernatant in reagent medium would minimize the risk of immunizing the patient against foreign serum proteins in the media that might produce toxicities that could limit the usefulness of the therapy. This would allow the direct injection of high titer virus and reduce the treatment time required for injected autologous VPC to produce the vectors. As a model system the PA3l7/LNL6 packaging cell line that contains the LNL6 vector will be employed. The LNL6 vector carries the bacterial gene for neomycin resistance. Vector titer will be assessed using G418 resistant colony forming units by the G418 selection of infected NIH-3T3 fibroblasts. The regent medium will be developed with the eventual goal of producing a medium that is desi ned for direct infusibility into human patients.
