Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL

The tRNA 3′-processing endoribonuclease (tRNase Z or 3′ tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3′ trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression

Oral Pre-Exposure Prophylaxis by Anti-Retrovirals Raltegravir and Maraviroc Protects against HIV-1 Vaginal Transmission in a Humanized Mouse Model

Sexual HIV-1 transmission by vaginal route is the most predominant mode of viral transmission, resulting in millions of new infections every year. In the absence of an effective vaccine, there is an urgent need to develop other alternative methods of pre-exposure prophylaxis (PrEP). Many novel drugs that are currently approved for clinical use also show great potential to prevent viral sexual transmission when administered systemically. A small animal model that permits rapid preclinical evaluation of potential candidates for their systemic PrEP efficacy will greatly enhance progress in this area of investigation. We have previously shown that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and displays CD4 T cell loss typical to that seen in the human. Thus far systemic PrEP studies have been primarily limited to RT inhibitors exemplified by tenofovir and emtricitabine. In these proof-of-concept studies we evaluated two new classes of clinically approved drugs with different modes of action namely, an integrase inhibitor raltegravir and a CCR5 inhibitor maraviroc as potential systemically administered chemo-prophylactics. Our results showed that oral administration of either of these drugs fully protects against vaginal HIV-1 challenge in the RAG-hu mouse model. Based on these results both these drugs show great promise for further development as orally administered PrEPs

RNA and DNA viral loads in mice administered with maraviroc.

<p>RAG-hu mice were challenged by vaginal route after oral administration of raltegravir as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015257#s2" target="_blank">Methods</a>. Blood was collected weekly. Viral RNA was extracted from the plasma fraction and DNA was extracted from the cellular fraction. Viral RNA and DNA loads were determined by Q-RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015257#s2" target="_blank">methods</a>. A. RNA viral loads B. DNA viral loads.</p

Oral administration of raltegravir or maraviroc protects humanized mice against vaginal HIV-1 challenge.

<p>RAG-hu mice were challenged by vaginal route after oral administration of raltegravir or maraviroc as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015257#s2" target="_blank">Methods</a>. Blood was collected weekly from infected mice and the status of HIV-1 infection was determined by Q-RT-PCR. The viral challenge experiments were performed at same time for both of the drugs and the same set of control non-treated infected mice were used for comparison. Kaplan-Meier plots of time course of appearance of viremia in drug treated versus non-treated virus challenged mice. A. Raltegravir treated B. Maraviroc treated.</p

Summary of human cell engraftment levels in humanized (RAG-hu) mice^*.

<p>*Peripheral blood was collected from human CD34 cell reconstituted mice at 10–12 weeks post engraftment. White blood cell fraction was stained with human CD45 FITC conjugated antibody and analyzed by FACS to confirm human cell engraftment prior to drug treatments and vaginal HIV challenges.</p

CD4 T cell decline in non-treated vaginally challenged mice in contrast to mice protected with raltegravir and maraviroc treatment.

<p>Levels of CD4 T cells were monitored on a weekly basis by FACS to determine their decline in treated versus non-treated mice. Baseline values for each of the mice were established prior to infection as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015257#s2" target="_blank">Methods</a>. A. Raltegravir treated, B. Maraviroc treated.</p