Background: ABX464 is a first-in-class, clinical stage small molecule with at least three biological effects when given to HIV patients: 1) an antiviral effect, 2) a reduction in HIV-DNA in PBMCs, and 3) an anti-inflammatory effect through increased expression of miR-124 in rectal tissue. In earlier studies, the antiviral effect was shown to be mediated through enhanced splicing of viral mRNA, while there was only a negligible effect on pre-mRNA splicing of cellular genes.
Methods: Using customized library probes targeting HIV sequences, cDNAs were prepared from infected PBMCs (+/- ABX464) of 6 donors. Given that the target of ABX464, the CBC complex, is also involved in the biogenesis of small non coding RNA including microRNAs, we also performed a microarray analysis of these RNAs from 6 healthy donors.
Results: In all samples treated with ABX464, 90% of assembled contigs corresponded to spliced RNA.In contrast, in untreated samples, >90% of the viral RNA is full length and unspliced. After filtering and analysis of splicing events in treated samples, 3 novel species of spliced RNA were identified. The capacity of these novel RNAs to generate immunogenic peptides is currently being tested.
While infection leads to large variations in the expression of small non coding RNAs, ABX464 induced a reproducible upregulation of a single microRNA, i.e. miR124, in both infected and non infected cells. This upregulation was only observed with locus miR124.1, which happens to have different long non coding transcripts that could be responsible for the biogenesis of miR124. ABX464 enhanced the splicing of a single long non coding RNA in this locus.
Conclusions: Our findings substantiate a mechanism of action of ABX464 that starts with CBC 80/20 binding, leading to a conformational change of this regulatory protein and enhanced splicing of a viral as well as a non coding RNA. HIV viral load reduction and, via upregulation of miR124, the generation of a therapeutic anti-inflammatory response are direct consequences of this mechanism. We are currently studying whether the observed reduction of HIV reservoir cells could be caused by the initiation of an immune response, e.g. through novel peptides/proteins generated by the modulation of splicing.

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