Currently, there is no viral vector available that allows the stable integration and expression of genes in postmitotic cells. As a lentivirus, HIV-1 is able to transport its preintegration complex through the nuclear pores and to integrate into nondividing cells like macrophages or dendritic cells. One of the goals of this study is to develop safe defective HIV-1 particles that could function as a gene delivery and expression vector for postmitotic cells. We have earlier developed a defective HIV-1 helper virus construct, HDPack1. This construct was now used to generate HIV-1 particles that contained either the ecotropic or amphotropic Env proteins of Moloney murine leukemia virus. The genomic RNA of these particles consisted of a mini-HIV-1 RNA encoding a neomycin resistance gene. These pseudotype HIV-1 particles were able to infect other species such as mouse cells and confer neomycin resistance to these cells. These results demonstrated for the first time that all functions and structural elements necessary for the generation of the defective HIV-1 vector were properly provided by our helper virus DNA construct. This construct will now be used for the generation of an expression vector for postmitotic cells. Several attempts to isolate a stable cell line that constitutively sheds defective HIV-1 particles were not successful. A single cell clone was isolated but it produced low levels of virus particles for only five weeks. It has recently been described that the Vpr protein of HIV-1 which is encoded by HDPack1 resulted in very high virus titers, suggesting that HDPack1 is not toxic but that a clonal expansion to a cell line may not be possible with Vpr present. Since transient transfections of HDPack1 in 293 cells have yielded up to 108 HIV-1 like particles per ml, we will try to use this transient system to generate sufficient amounts of the viral expression vector.
