Development and characterization of a triple combination gene therapy vector inhibiting HIV-1 multiplication

BACKGROUND: RNA-based approaches are promising for long-term gene therapy against HIV-1. They can target virtually any step of the viral replication cycle. It is also possible to combine anti-HIV-1 transgenes targeting different facets of HIV replication to compensate for limitations of any individual construct, maximizing efficacy and decreasing chances of escape mutations. We have previously developed two strategies to inhibit HIV-1 multiplication. One was a short hairpin RNA targeting the host factor cyclophilin A implicated in HIV-1 replication. Additionally, an antisense derivative of U7 small nuclear RNA was designed to induce the skipping of the HIV-1 Tat and Rev internal exons. RESULTS: In the present study, we have established an additional tRNAval promoter-driven shRNA against the coding sequence of viral infectivity factor. When human T-cell lines or primary CD4+ T cells are transduced with a triple lentiviral vector encoding these three therapeutic RNAs, HIV-1 multiplication is very efficiently suppressed. Moreover, all three therapeutic RNAs exhibit antiviral effects at early stages of the viral replication cycle (i.e. prior to viral cDNA integration or gene expression). CONCLUSIONS: These findings make this triple lentiviral vector an attractive candidate for a gene therapy against HIV/AIDS.

Identification of potential HIV restriction factors by combining evolutionary genomic signatures with functional analyses

Jérémie Perreaud, Daniel Sauter, Amalio Telenti, Danielle Thompson

Background: Known antiretroviral restriction factors are encoded by genes that are under positive selection pressure, induced during HIV-1 infection, up-regulated by interferons, and/or interact with viral proteins. To identify potential novel restriction factors, we performed genome-wide scans for human genes sharing molecular and evolutionary signatures of known restriction factors and tested the anti-HIV-1 activity of the most promising candidates. Results: Our analyses identified 30 human genes that share characteristics of known restriction factors. Functional analyses of 27 of these candidates showed that over-expression of a strikingly high proportion of them significantly inhibited HIV-1 without causing cytotoxic effects. Five factors (APOL1, APOL6, CD164, TNFRSF10A, TNFRSF10D) suppressed infectious HIV-1 production in transfected 293T cells by >90% and six additional candidates (FCGR3A, CD3E, OAS1, GBP5, SPN, IFI16) achieved this when the virus was lacking intact accessory vpr, vpu and nef genes. Unexpectedly, over-expression of two factors (IL1A, SP110) significantly increased infectious HIV-1 production. Mechanistic studies suggest that the newly identified potential restriction factors act at different steps of the viral replication cycle, including proviral transcription and production of viral proteins. Finally, we confirmed that mRNA expression of most of these candidate restriction factors in primary CD4+ T cells is significantly increased by type I interferons. Conclusions: A limited number of human genes share multiple characteristics of genes encoding for known restriction factors. Most of them display anti-retroviral activity in transient transfection assays and are expressed in primary CD4+ T cells.

Biomed Central Ltd2015

Tissue- and sex-specific small RNAomes reveal sex differences in response to the environment

Alexandra Bezler, Raffaella Conconi, Pierre Gönczy, Laurent Keller, Frédéric Schütz

RNA interference (RNAi) related pathways are essential for germline development and fertility in metazoa and can contribute to inter- and trans-generational inheritance. In the nematode Caenorhabditis elegans, environmental double-stranded RNA provided by feeding can lead to heritable changes in phenotype and gene expression. Notably, transmission efficiency differs between the male and female germline, yet the underlying mechanisms remain elusive. Here we use high-throughput sequencing of dissected gonads to quantify sex-specific endogenous piRNAs, miRNAs and siRNAs in the C. elegans germline and the somatic gonad. We identify genes with exceptionally high levels of secondary 22G RNAs that are associated with low mRNA expression, a signature compatible with silencing. We further demonstrate that contrary to the hermaphrodite germline, the male germline, but not male soma, is resistant to environmental RNAi triggers provided by feeding, in line with previous work. This sex-difference in silencing efficacy is associated with lower levels of gonadal RNAi amplification products. Moreover, this tissue- and sex-specific RNAi resistance is regulated by the germline, since mutant males with a feminized germline are RNAi sensitive. This study provides important sex- and tissue-specific expression data of miRNA, piRNA and siRNA as well as mechanistic insights into sex-differences of gene regulation in response to environmental cues. Author summary Environmental factors such as heat or pathogens influence living organisms, and in some cases these experiences can be passed on to the next generation. One such environmental factor is double stranded RNA that can be taken up by some animals and induce changes in gene expression via small RNA mediated silencing. Here, we use the roundworm Caenorhabditis elegans to show that the female and male germline react differently to the presence of such an environmental factor that may affect transmission to following generations. Sequencing of gonadal small RNA suggests that the underlying sex-differences affect downstream processing but not uptake. Furthermore, we describe the naturally occurring small RNA populations of female and male gonads and use genetic mutants to assign small RNAs to the two tissues of the gonad, namely the somatic gonad or the germline. These sex-and tissue-specific small RNA expression data provide an important resource for future research. In addition, this work reveals mechanistic insights into sex-differences in response to environmental cues.

PUBLIC LIBRARY SCIENCE2019

Tissue- and sex-specific small RNAomes reveal sex differences in response to the environment

Alexandra Bezler, Raffaella Conconi, Pierre Gönczy, Laurent Keller, Frédéric Schütz

PUBLIC LIBRARY SCIENCE2019