© 2018 ABIVAX

FIGURE 4.miR-124 upregulation by ABX464 originates from splicing of a long non-coding RNA at the miR.124.1 locus. 4a. There are three miR-124 genes, miR-124-1, miR-124-2, and miR-124-3, located on chromosomes 8 and 20 in the human genome. 4b. We employed a targeted-RNA capture and sequencing strategy to determine which gene is induced by ABX464. In both infected and noninfected cells, treatment with ABX464 leads to upregulation of miR-124 from the miR.124.1 locus, whereas a control locus miR.429 is not affected. 4c. Locus miR.124.1 contains a long non-coding RNA (lncRNA599-205) whose splicing is stimulated by ABX464. 4d. Counts of the reads at splice junctions (J1, J2, J3, and J4), exon-exon (J5 and J6) and the miR-124 75 bp region (miR-124) quantified by RNA CaptureSeq in PBMCs treated with ABX464. 4e. Quantification of the expression of miR-124 following transfection of a lncRNA599-205 plasmid in HeLa cells.

Background

ABX464 is a first-in-class small molecule currently in clinical development with at least three biological effects when given to HIV-infected patients:

1. An antiviral effect;

2. A reduction of total HIV DNA in PBMCs; and

3. An increased expression of the anti-inflammatory microRNA, miR-124 in rectal tissue.1

The antiviral effect has been previously shown to be mediated through enhanced splicing of viral mRNA, while the virus needs unspliced in order to be able to replicate.2

Objective

The objective of this study was to further elucidate the mechanism of action of ABX464 in order to better understand the means of reduction of HIV DNA in PBMCs, and the anti-inflammatory response in clinical trials.

Methods

■ PBMCs from healthy donors were infected (or not) with Ada-M and YU2 HIV strains and treated with ABX464.

■ HIV RNA and miR-124 sequences were captured on a NimbleGen SeqCap® EZ Developer Library (Roche/NimbleGen).

■ EZ Oligo pool was made against Ada-M R5 and YU2 strains and the three loci encoding miR-124.

■ Total RNA was extracted from PBMC samples using TRIzol® reagent (Invitrogen).

■ Libraries for sequencing were prepared and sequenced using the Illumina protocol.

■ Affymetrix array V2 was used to unravel microRNA regulated by ABX464.

1a. 1c.

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1d. Contigs Assembly (Raw Counts)

ABX464, by binding to the CBC 80/20 complex, enhances pre-mRNA splicing, resulting in the generation of novel HIV-derived RNA species and in increased expression of the anti-inflammatory miR-124

Conclusions

These findings substantiate a mechanism of action for ABX464 that starts with CBC 80/20 binding and enhancement of mRNA splicing which leads to the following:

■ Viral load reduction and elimination of HIV from the latent reservoir through immune response to putative new peptides generated by viral RNA splicing

■ A negligible effect of ABX464 on cellular splicing

■ ABX464 upregulates the anti-inflammatory miR-124

■ Upregulation of miR-124 by ABX464 is caused by splicing of long non-coding RNA (lncRNA599-205)

References

1. Steens JM, et al. Antimicrob Agents Chemother. 2017 Jul 27;61(7) 13;

2. Campos N, et al. Retrovirology. 2015 Apr 9;12:30.

Acknowledgements

This work was supported by the collaborative laboratory ABIVAX, OSEO-ISI CaReNA grant. We would like to thank Yannick Marie and Laurent Journot from the sequencing facilities plateforms ICM Hôpital de la Salpêtrère Paris and IGF, Montpellier, respectively. The authors wish to thank Pierre de la Grange from Genosplice for generating and analyzing the microRNA data, David Piquemal from ACOBIOM for miR-124 quantification from HIV patients and Salah Chetibi from Roche for the design and synthesis of SeqCap® EZ.

Audrey Vautrin 2,3, Laurent Manchon 2,3, Aude Garcel 2, Noëlie Campos 2, Abdelhamid Mahdi Laaref 1, Didier Scherrer 2, Hartmut Ehrlich 2, and Jamal Tazi 1*1IGMM, CNRS, University Montpellier, Montpellier, France; 2ABIVAX, Montpellier, France; 3These authors contributed equally to this work; *Corresponding author

5, rue de la Baume 75008 Paris, FranceE-mail: info@abivax.com Tel.: +33(0)1 53 83 08 41www.abivax.com

FIGURE 1.Interaction of ABX464 with CBC complex and a change in the conformation of the complex (Figure 1a and 1b) leads to enhanced splicing of HIV RNA and generation of a new viral RNA (Figure 1c-1d). 1a. Ribbon representation of the CBC complex (CBC20 in brown and CBC80 in purple) with the position of the cap RNA (in blue/red) on CBC20 and the putative peptide of CBC20 that interacts with ABX464 (yellow). 1b. Images of CryoEM negative staining of CBC and CBC with ABX464 showing changes in the conformation of the complex. 1c. Organization of HIV-1 genome and different mRNA splicing products. The 5’ ss (D1–D4) and 3’ ss (A1–A7) are indicated. ORFs of coding exons of each mRNA product are indicated with a different colour code alluding to the corresponding encoded proteins of the HIV genome. The noncoding exons are boxed in grey. 1d. While in all samples treated by ABX464 most of assembled contigs (90%) correspond to spliced RNA, or small RNA containing gag-pol sequences that can be released introns, in untreated samples the spliced RNAs represent a minor fraction (less than 23%) and the majority corresponds to full length unspliced viral RNA (more than 78%).

Results

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Not Infected Infected

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4d. Mapped Reads (Raw Counts) at Exon-Intron and Exon-Exon Junction

4e. Transfection of lncRNA599-205 and Expression of miR-124

4c.Chr. 8

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<miR-124-1 miRNA

Poster LBPEA009Presented at the 22nd International AIDS ConferenceAmsterdam, Netherlands 23-27 July, 2018

FIGURE 3.ABX464 upregulates a single microRNA, the anti-inflammatory miR-124. 3a. Microarray analysis of small RNA from 6 donors’ PBMCs infected or noninfected treated or nontreated with ABX464. Volcano plots show that infection leads to this large variation in small non-coding RNAs (right panel), whereas ABX464 induced a reproducible upregulation of a single microRNA miR-124 in infected and noninfected cells (middle and left panels respectively). 3b. Quantification of miR-124 in the different conditions using TaqMan™ technology. 3c. Using purified CD4, CD8 and macrophages, we show that this upregulation concerns lymphoid cells but not monocyte derived macrophage where the expression of miR-124 is undetectable. 3d. None of the antiretrovirals maraviroc, efavirenz, darunavir, or AZT was able to modulate the expression of miR-124, whereas a molecule that has the same properties as ABX464, ABX530 does upregulate miR-124. 3e. Rectal biopsies from HIV-infected patients receiving antiretroviral therapy (ART) and treated with ABX464 (N=9) were used to monitor the expression of miR-124. miR-124 was downregulated in HIV patients treated with ART compared to its expression in colon biopsies from healthy donors (N=10). Treatment with ABX464 normalized expression of miR-124 to the level of healthy donors. Stopping the treatment for 28 days lead to return of miR-124 expression to the levels before treatment.

FIGURE 2.ABX464 has no global effect on cellular splicing. 2a. The effect of ABX464 on infected (I) and noninfected (NI) cells was tested by high-throughput RNAseq approach. Sixteen libraries were constructed with 4 conditions noninfected (DMSO-NI), noninfected treated with ABX464 (464-NI), infected (DMSO-I) or infected treated with ABX464 (464-I) corresponding to 4 donors. Approximately 38 million reads (more than 50% of the total raw reads) were aligned to exon of the human genome sequence in each of the samples. 2b. Multidimensional Scaling analysis (MDS) was used to interpret major trends in the data. The MDS of our gene expression data showed without any outliers that the different donors separated well and distributed for the DMSO and ABX464 treatments (infected or not), which suggests that the molecule does not induce a major difference in gene expression. 2c. Alternative splicing events of cellular genes were classified into five major groups: Alternative 5’ splice site (A5SS), Alternative 3’ splice site (A3SS), Skipped exon (SE), Mutually exclusive exons (MXE) and Retained intron (RI). No switch like events were detected in infected or noninfected T CD4 samples compared to ABX464 treated samples. The exact numbers of common and differential splicing events between infected and noninfected T CD4 cells treated with ABX464 is very low (less than 10 events) while depletion of NCBP1 in stem cells by only 50% (Nuclear Cap Binding protein subunit 1) gives rise to large variations with 385 alternative exon, 59 A 5’ SS, 60 A 3’ SS and 656 IR. By comparing exon coverage reads on a common highly expressed gene (B2M) between ABX464 and DMSO conditions on the 4 donors, we confirmed that ABX464 did not increase splicing events on particular gene. 2d. Volcano plot of DMSO-I vs DMSO-NI, DMSO-I vs 464-I and DMSO-NI vs 464-NI. The gene expression variation generated by ABX464 treatment was very low in infected (6 downregulated) and noninfected (6 downregulated) samples.

464-NI vs DMSO-NI 464-I vs DMSO-I3a. DMSO-I vs DMSO-NI

3c. miR-124 Modulation by ABX464

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