Targeting HIV-1 gene expression: towards a new approach for an antiretroviral therapy

• Applicant: Professor Dr. Heiner Schaal
• Subject Area: Virology
• Term: from 2016 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 314480083

During HIV 1 replication the viral (+)RNA genome is reverse transcribed, converted into double stranded DNA and integrated into the host cell genome. Transcription of the provirus generates a polycistronic pre-mRNA that leaves the cell nucleus either in a spliced or unspliced form. A significant fraction of the spliced mRNAs also contains introns. Since the virus critically depends on a balanced expression of these spliced and unspliced mRNAs, any disturbance of this ratio dramatically affects HIV 1 infectivity and pathogenesis. Most recently, we could show that two conserved splicing regulatory elements within the HIV-1 genome, both the GI3-2 and the ESEtat element play a major role in the generation of viral mRNA species such as vif, vpr and tat mRNAs. Furthermore, mutations of these elements inhibited viral replication. With regard to the development of an alternative antiretroviral therapy, which targets HIV-1 gene expression, we masked these splicing regulatory elements with antisense oligonucleotides also resulting in a block of HIV-1 particle production. As oligonucleotides we used locked nucleic acids (LNAs), modified nucleotides which are constrained in the ideal conformation for Watson-Crick binding due to an extra methylene bridge. Besides many advantages over other antisense oligonucleotides LNAs can be delivered into cells without transfection reagents (gymnosis) which makes these oligonucleotides attractive for therapeutic applications.We propose that LNAs provide a promising tool to develop a novel antiretroviral therapy targeting HIV-1 gene expression. Our preliminary results showed that gymnotically delivered LNAs, even more than transfected LNAs, display a very strong influence on the viral replication. Surprisingly, LNAs delivered unassisted, localize within the cytoplasm and here seem to induce degradation of viral mRNAs containing their target sequence. If the LNAs at a later time point also accumulate within the cell nucleus, and here disturbing viral alternative splicing by masking binding sites for splicing regulatory proteins, has to be analyzed. Therefore, we wish to analyze the cellular localization and distribution of gymnotically delivered LNAs in a time-course experiment and unravel the underlying LNA-mediated RNA degradation mechanisms (on-target effect). In addition, we want to investigate possible off-target effects including stimulation of innate immune responses. Furthermore, we aim at identifying additional LNA targets within the HIV-1 genome to develop an LNA cocktail as an alternative antiretroviral therapy.

• DFG Programme: Research Grants