Anti HIV-1 gene therapy approach combining multiple siRNA with the membrane anchored fusion inhibitor C peptide maC46

Background HIV-1 infection is normally characterized by sustained viral replication and a progressive loss of CD4+ T cells, leading to AIDS. Combined antiretroviral therapy (cART) suppresses viral replication and drastically reduces mortality. However, cART alone is not able to eradicate the virus, which persists in reservoirs, and it is still associated with considerable comorbidities and the emergence of drug-resistant escape mutants. A cure for HIV-1 infection is needed to bypass the limitations of the current therapy and restore health. Advances in the fields of gene-targeting strategies, based on intracellular immunization of autologous T cells or their progenitors, i.e. the CD34+ hematopoietic stem cells (HSC), appear encouraging to repopolate the immune system. Materials and Methods Self-inactivating (SIN) lentiviral vectors were generated in order to express multiple HIV-1 inhibitors. Recombinant particles were produced in HEK 293T cells and titrated by flow cytometry and Reverse Transcriptase (RT) activity assay. To address the antiviral activity of the combinatorial vectors, CD4+ T lymphoblastoid Jurkat cells were transduced with lentiviral particles, then underwent to MTT cell viability assay and infected with the HIV-1 HXBc2 Vpr+/Vpu+/Nef+ strain (MOI=0.1 TCID50/cell). In order to test the mutagenic potential of this SIN lentiviral platform, the in vitro immortlization (IVIM) assay was employed. Results The generated vectors were characterized by different combinations of siRNA targeting the CCR5 cellular gene as well as vif and tat/rev viral transcripts, along with or without an HIV membrane-anchored fusion inhibitor (maC46) , which has been shown to efficiently block the entry of both CXCR-4 and CCR5-using viruses. Specifically, the siRNA were either expressed as single transcriptional unit under the control of different human RNA polymerase III promoters (U6, 7SK, H1), or simultaneously, as an extended shRNA. The maC46, which derives from the C-terminal heptad repeat (HR2) domain of gp41, was cloned under the transcriptional control of the human elongation factor 1 alpha (EF1 alpha) promoter. Finally, an optimized version of the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE*), able to reduce the risk of oncogenicity, was introduced in all vectors. The EF1 alpha promoter led to a high level of maC46 expression, with a correct localization of the peptide at the plasma membrane. The developed lentiviral particles efficiently transduce Jurkat cells, without cytotoxic effect, and potently block HIV-1 replication up to 4 weeks post-infection. Furthermore, no impact on the viability or the proliferation of transduced murine cells with these vectors was observed in the IVIM assay. Conclusions The new generation vectors, combining a potent fusion inhibitor with 3 shRNAs, appear to be extremely promising as anti-HIV-1 vectors. Once challenge experiments in different cell lines will be completed, the best performing vectors will be assayed in primary CD4+ cells and CD34+ HSCs in order to further validate this approach for clinical application in selected HIV-1+ patients.