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

The development of highly active anti-retroviral therapy (HAART) has considerably improved life expectancy of HIV-1 positive patients by transforming this infection, which once was lethal, into a manageable chronic illness. Although a significant suppression of viral replication under undetectable level is guaranteed following a constant therapeutic adherence, this therapy fails to completely eliminate the infection due to the persistence of HIV-1 into reservoirs, which represent therefore the main obstacle to a definite cure. Furthermore, a lifelong adherence to treatment is associated with drug toxicities and persistent immune dysfunction, which can lead to discontinuation of therapy and the onset of drug resistance. These hurdles, together with the high economic costs of providing HAART to more than 35 million people, which are currently affected by HIV-1, contribute to render HIV-1/AIDS pandemic one of the most important global health challenge. In this scenario, the search for a curative strategy is necessary. Recent successes in inherited immune deficiencies treatment and cancer immunotherapy have raised interest in gene and cell modification to treat HIV-1 infection with the final aim of inducing permanent resistance to HIV-1. In particular, anti HIV-1 gene therapy (GT) protocols, based on engineering of autologous T cells or their progenitors, such as the CD34+ hematopoietic stem cells (HSCs), appear a promising approach to repopulate the immune system after a single therapeutic intervention. Long-term HIV-1 remission in the “patient of Berlin”, who received an heterologous stem cell transplant for acquired immunodeficiency syndrome-related lymphoma from a CCR5 homozygous null HLA-matched donor (CCR5 -/-), even after discontinuation of conventional therapy, has energized the field. However, due to the limited chance of finding matching Δ32 CCR5 donors, recapitulating this clinical success on a large scale appears to be difficult. Moreover, autologous regimens are potentially less toxic, as they may not require full bone marrow ablation or subsequent immune suppression for engraftment. In this setting, the goal would be to disable the CCR5 gene in enough target cells to confer benefit and transplanted back into the patient. Different GT strategies to artificially disrupt the CCR5 gene or transcript have proved to be successful in primary T cells, HSCs, as well as in humanized mice, and are recently been tested in clinical trials. Inhibitors of entry or of early step of viral replication prior the virus integration are expected to lead an advantage selection of gene modified cells, thus preventing the establishment of chronic HIV-1 infection and limit the continued replenishment of viral reservoirs. However, a major goal of gene therapy is to target simultaneously multiple viral sites and endogenous host factors, interfering with different steps of viral cycle, hoping to reduce the onset of virus variants. Among antiviral agents, small interfering RNAs (siRNAs) are less immunogenic than protein-based ones and represent a powerful tool to silence gene expression post-transcriptionally in a sequence specific manner. In order to obtain a stable and long expression, necessary for a chronic disease, multiple anti-HIV-1 siRNAs can be accommodated into self-inactivating (SIN) lentiviral vectors, which are currently preferred for their ability to efficiently transduce target cells, and then because they confer a potentially safer integration site profile, compared to gammaretroviral vectors. For these reasons, different combinatorial platforms based on SIN lentiviral vectors were previously developed to express multiple siRNAs against highly conserved regions of cellular and viral genes, including the cellular co-receptor CCR5, the vif and tat/rev viral factors, involved in different phases of HIV-1 replication and pathogenesis. These siRNAs were placed under the control of different human Polymerase III promoters (such as U6, 7SK and H1) either as independent transcriptional units or as extended short hairpin RNA (e-shRNA), able to express the three siRNA under the control of a single promoter. The most potent antiviral activity in transduced human primary CD4+ T lymphocytes was conferred by two effective anti-HIV-1 combinatorial vectors (i.e. pLL3.7 U6shCCR5-7SKshvif-H1lhtat/rev and pLL3.7 H1e-shRNA). However, HIV-1 can use an alternative coreceptor (i.e. CXCR4) for entering into target cells, that is less favored as target of siRNA or novel gene-editing technologies, because its disruption can compromise fundamental physiological functions, especially the maturation of HSCs. At the same time, CXCR4 tropic viruses are relevant in the pathogenesis of AIDS. Thus, to improve the efficacy of this approach, the two selected vectors were optimized by the insertion of a small membrane anchored C-peptide (maC46) fusion inhibitor, which has been shown to protect against a broad range of HIV-1 isolates and it has been tested in a phase 1 clinical trial. When expressed on target cell surface, the maC46 peptide, which derives from the C-terminal heptad repeat (HR2) of the HIV-1 gp41 envelope glycoprotein, blocks the membrane fusion by interacting with the N-terminal coiled coil domain of the gp41 intermediate structure and preventing the six-helix bundle formation. In these constructs, the expression of the peptide, either fused in frame with the enhanced green fluorescence protein (eGFP) or alone, is driven by the human Elongation Factor 1 promoter (EF1), a cellular-derived enhancer/promoter, which has been shown to confer high level of transgene expression in HSCs and a more safety profile, since decreased cross-activation of nearby promoters. Additionally, the new developed lentiviral vectors carries an optimized version of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE*), which enhances the vector titer and lacks oncogenic properties. Furthermore, control vectors either lacking the maC46 encoding sequence or characterized by a scrambled sequence in place of the siRNA encoding ones were generated. Once transfected into different cell lines, the developed vectors led to the expression of the maC46 peptide, that correctly localized at the plasma membrane, as shown with immunofluorescence cell staining. Furthermore, recombinant lentiviral particles (RLVPs) were produced and titrated either using Reverse Transcriptase (RT) Assay and Fluorescence-activated cell sorting (FACS). The efficacy of the maC46 peptide fusion inhibitor, in combination with the silencing activity of the expressed siRNAs, was evaluated in challenge experiments, in which transduced T lymphoblastoid Jurkat cells were infected with HIV-1 HXBc2 Vpr+/Vpu+/Nef+ R4-tropic molecular clone at different multeplicity of infection (M.O.I.). In parallel, in collaboration with the Baum’s group at the Department of Experimental Hematology of the Hannover Medical School, the mutagenic potential of these vectors was assessed by means of in vitro immortalization assay. The results obtained so far clearly show that the optimized vectors, combining for the first time a potent fusion inhibitor with three short hairpin (sh)RNAs, appear to be extremely promising as anti-HIV-1 approach. Importantly, these vectors showed a strongly reduced insertional transformation potential compared to a positive gammaretroviral control vector. This strategy could be now extended to primary cells and in particular to HSCs, the ultimate target of this gene therapy approach, with the final aim of accomplishing a high level of protection from HIV-1 infection over sustained lengths of time, without cell toxicity, and loss of stemness, proliferation capability and differentiation potential for possible future clinical applications.