Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection

Abstract

The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to miR-122 leads to long-lasting suppression of HCV viremia, with no evidence of viral resistance or side effects in the treated animals. Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.

Fig. 1. Silencing of miR-122 by SPC3649 in chimpanzees with chronic hepatitis C virus infection

(A) Analysis of HCV RNA levels in HCV-infected chimpanzees during the study. The HCV titers are shown as genomic equivalents (GE) for the high-dose animals (4×0513 blue triangles, 4×0514 magenta diamonds) and low-dose animals (4×0267 orange squares, 4×0358 red dots) in serum (GE/mL, solid lines) and liver (GE/µg liver RNA, dashed lines). The placebo and active treatment periods are indicated below. (B) Northern blot analysis of RNA from chimpanzee liver biopsies using LNA-modified probes detecting free and sequestered miR-122 (upper panel) and SPC3649 (lower panel). The first two lanes are positive controls for free miR-122 and preformed miR-122:SPC3649 heteroduplexes, respectively. (C) Detection of sequence variants in the miR-122 seed sites (boxed) by deep sequencing of the HCV 5’ NCR from the high dose animals at four time points: baseline, end of treatment, viral rebound and end of the follow-up period. (D) The two miR-122 seed sites (boxed) in the HCV 5’ NCR are conserved in all HCV genotypes and subtypes (see SOM for details).

Fig. 2. Functional antagonism of miR-122 by SPC3649 in HCV-infected chimpanzees

(A) Assessment of liver transcriptome changes after SPC3649 treatment in each animal by microarray expression profiling of liver biopsies. The liver mRNA 3’ UTRs were analysed for the presence of different types of canonical miR-122 seed match sites. The cumulative fraction plots show the distribution of log₂ fold changes for each seed match type and a Kolmogorov-Smirnov test was used to compare the three miR-122 seed match types to mRNAs with no seed sites in the 3’ UTR. (B) Expression profiles of interferon-regulated genes (green lines correspond to IRGs with decreased expression from baseline to end of treatment, while red lines indicate IRGs showing an increase) and serum HCV RNA levels (black dashed line) in HCV-infected chimpanzees during the study. (C) Serum IP-10 levels (color coding and plot symbols as in Fig. 1A) and the serum HCV titer (dashed black line) in HCV-infected chimpanzees during the study. (D) Serum cholesterol levels (color coding and plot symbols as in Fig. 1A) and serum HCV RNA levels (dashed black line) in HCV-infected chimpanzees during the study.

Fig. 3. Treatment of HCV-infected chimpanzees with SPC3649 was well tolerated

(A) Plasma trough levels of SPC3649, (B) alanine aminotransferase (ALT) levels and (C) Creatinine levels in HCV-infected chimpanzees during the study. (D–F) Photomicrographs of hematoxylin and eosin stained sections from biopsies of (D) a normal chimpanzee liver, (E) the animal 4×0513 at week −4, (F) week 19, and (G) week 25, respectively.