The main purpose of this project is to develop an antiviral strategy directed against HIV-1 which would delay the onset of AIDS. Unlike the use of antiviral drugs, our strategy is a biological approach which employs novel defective interfering HIV-1 particles (HIV-1 Dls) targeted to infect HIV-1-expressing cells. Replication and gene expression of these Dl particles are totally dependent on the regulatory and structural proteins provided by HIV-1. We anticipate that progeny Dl particles will not be pathogenic. They will be able to spread the interfering genes specifically to other HIV-1 infected cells or to cells HIV-1 would normally infect. Unlike naturally occurring defective interfering particles of other virus groups, these Dl particles will be dominant and they will uniquely use HIV-1 as their host. The anticipated spread of the interfering genes within the monocyte/macrophage population throughout the body will be slow and it will occur during the asymptomatic phase of the HIV-1 infection. Important is that the particles should also target cells in the central nervous system. It is our hope that the Dl particles will slowly limit or down-regulate the HIV-1 load, and thereby delay or possibly even prevent the onset of AIDS. We have developed six generations of these HIV-1 Dl genomes. Coexpression of each of these constructs with HIV-1 resulted in a dramatic inhibition of HIV-1 replication in HeLa T4 cells. Interference was caused by the HIV-1 Tat dependent expression of a chimeric CD4/env receptor protein encoded by the HIV-Dl genome. Expression of the receptor caused a downregulation of the HIV-1 Env protein on the cell surface. The HIV-1 Dls did not inhibit HIV-1 gene expression, but they caused virus progeny to be less infectious. Novel catalytic RNAs (multitarget-ribozymes) were developed which were designed to target and to specifically cleave at ten different, highly conserved sites in the envelope region of the HIV-1 RNA. These multitarget-ribozymes showed unique properties as compared to single cleavage ribozymes. They were highly functional even when they were part of a large transcript. Based on sequence analyses they should be active against most if not all HIV-1 variants presently sequenced. From the fourth HIV-1 Dl generation on, these multitarget-ribozymes were part of the HIV-1 Dl genomes. We have shown that these HIV-1 Dls interfere with the replication of HIV-1 by two different mechanisms: 1. the complex formation between the HIV-1 Env with the chimeric receptor and 2. the specific in vivo cleavage of HIV env mRNA by the multitarget-ribozyme. In fact, the genomic RNA of the HIV-1 Dl itself functioned as a multitarget-ribozyme. To generate the HIV-1 Dls in the absence of HIV-1, a new HIV-1 packaging DNA was developed. Cotransfection with the HIV-1 Dl DNAs resulted in the efficient assembly of HIV-1 Dls. The packaging efficiencies of the HIV-1 Dl genomic RNAs No. 4 through No. 6 are currently under investigation. Cocultivation of HIV-1 Dl expressing cells with cells persistently infected with HIV-1 has currently been started. This will allow a first evaluation of the effectiveness of our antiviral strategy in tissue culture. It is our hope that the HIV-1 Dls will perform safely as outlined in our strategy and that they can be used in a biological antiviral therapy in the future.
