Reverse transfection studies with synthetic RNAs demonstrated that each shRNA derived from PF-05095808 delivers an independent antiviral effect against the HCV Con1b replicon. Administration of PF-05095808 AAV-8 to standard HCV replicon and infectious virus systems also revealed dose-dependent antiviral activity, consistent with a sequence-specific effect. Replicon sequences were described on the basis of data from molecular clones. The poor efficiency of AAV-8 vectors in cultured cells continually presented a major challenge to the in vitro characterization of PF-05095808.
There have been numerous reports that RNAi-based agents such as PF-05095808 deliver antiviral activity in HCV replicon systems (14
). Our studies were intended to go beyond this proof of principle and to identify methodologies that would enable the linking of in vitro
activity with the potential to predict in vivo
efficacy. A key step in understanding the potential efficacy of a novel therapeutic is the accurate determination of potency in in vitro
activity assays. Our results unequivocally demonstrated that observed potency in vitro
was impacted by the transduction efficiency of the vector delivery system, to the extent of a 4-log range in EC50
s between the viral variants. There was some concordance in EC50
s (~30 vg/cell) against the Con1b replicon when the most efficient AdV and LV vectors for delivery of PF-05095808 were used although it is important that reported virus titers were determined by different methodologies and therefore may not be absolutely comparable. For viral vectors with a good correspondence between in vitro
and in vivo
transduction efficiencies, one approach to understanding efficacy may be direct measurement of virus genomes within transduced cells. To properly understand the efficacy of PF-05095808, an assay which measured the quantity of RNAi effector molecules required for antiviral activity was required.
A universal qPCR assay for direct quantification of the shRNA was therefore developed. This assay was suitable for both in vitro
and in vivo
derived sample analysis and so provides a method for linking activity in tissue culture systems with exposure and efficacy in the clinical setting. Using this assay, we were able to reproducibly determine the level of shRNA expression associated with HCV replicon inhibition. The number of shRNAs associated with an approximate EC50
effect across viral vector systems proved remarkably consistent, within a 0.5-log range; this despite a 3-log difference in the virus inoculum needed to deliver antiviral activity. We included the measurement of miR-103 RNA levels as an endogenous control in all experiments to give an independent measure of any nonspecific effects on host cell miRNA levels. In studies with the AdV system at high doses, we did detect indicators of toxicity in both miR-103 RNA and WST-1 assays, consistent with previous literature observations (33
). However, our data support the idea that, using AAV-based delivery systems, it is possible to achieve antiviral activity against the HCV replicon by an RNAi-driven mechanism, in the absence of overt cytotoxicity or nonspecific effects on host cell miRNA levels.
We sought to determine the number of shRNA molecules needed for antiviral activity in the replicon system, with a view to building a framework for understanding the required doses for antiviral activity in the clinic. The limiting in vitro
transduction efficiency of PF-05095808 in the replicon cell line meant that we were not able to confidently determine shRNA levels associated with a 90 or 99% effective concentration (EC90
, respectively) effect. The shRNA levels associated with an EC50
effect were in the order of 500 to 2,000 copies/cell under these experimental conditions. Replicon copy numbers are reported to be in the range of 1,000 copies/cell (2
). Our data therefore suggest that, at least in this in vitro
system, effector RNAi molecule levels do not need to be in great stoichiometric excess over target levels. The work presented in the manuscript uses qPCR methodologies that capture all processed shRNAs, regardless of termini, and thus the quantities reported here are slightly higher than those recorded in parallel PF-05095808 studies (Suhy et al., submitted). There are many challenges, however, in trying to understand the translation between in vitro
and in vivo
efficacy for agents of this type. Processing of the viral vector prodrug to the active RNAi molecules may vary between cell lines. HCV replication kinetics in the infected patient are poorly understood, and the numbers of genome copies per cell are reported to be significantly lower than in the artificially optimized HCV 1b replicon (23
). Given these and other uncertainties, the true potential of this agent to treat chronic HCV infection may fully be assessed only through clinical trials.
To better understand the mode of action of this agent, we screened for resistance emergence following serial passage with short RNAs. Resistance emergence to individual siRNAs and shRNAs was anticipated and has been previously reported (3
). Indeed, exposure to siRNA22 and shRNA22 resulted in sequence and sensitivity changes at the target site in the Con1b replicon. Clonal sequencing revealed a greater diversity of changes associated with shRNA treatment than with siRNA treatment (10 versus 4 sites). Literature reports (1
) and our own experimental data (unpublished observations) support the finding that Dicer processing of double-stranded RNA results in families of shRNAs with different termini being generated. The diversity of changes associated with shRNA treatment may be reflective of selective pressure from a number of related siRNA molecules.
The predominant target site sequence changes of C9198G and C9206A were associated with a reduction in sensitivity to shRNA22 challenge. The reduction was augmented when the changes were presented as double mutations. The C9206A change lies at the heart of the predicted cleavage domain mediated by the RNA-induced silencing complex (RISC) and as such is consistent with current ideas of the preferred site of RNA cleavage, i.e., nucleotide 9/10 from the 5′ terminus (8
). In contrast, the C9198G change is distal to the predicted cleavage site, where it is thought that changes will more likely be tolerated. Sequencing data of 50 further clones revealed nucleotide changes spread over the target domain and brings supporting evidence that changes across the breadth of an shRNA target site can impact cleavage. The nucleotide changes arising from prolonged shRNA22 exposure are consistent with sequence-specific selective pressure and therefore are likely to derive from an RNAi-directed mechanism. Further support for an authentic siRNA mode of action underlying the antiviral activity of PF-05095808 was obtained by 5′ rapid amplification of cDNA ends (RACE) characterization of replicon RNA cleavage products (unpublished observations).
Importantly, individual treatments of shRNA6 or shRNA19 remained fully active and effective against all shRNA22 replicon variants. Not only does this further support a sequence-specific mechanism of action consistent with RNAi activity, it also suggests that activity of PF-05095808 in a clinical setting might be entirely abrogated only in cases where HCV genomes have disruptive, mismatched sequences at all three targeted sites. Experiments testing the activity of PF-05095808 on the shRNA22 variants were not performed because of the difficulty of teasing apart the relative contributions of each of the expressed hairpins toward abrogating replicon activity. Far more sensitive cleavage assays would need to be developed for that type of analysis and is well beyond the scope of the studies presented here. Thus, understanding the barrier to resistance of this novel agent will be very difficult in the context of the current HCV model systems. Short hairpin RNA6 will likely provide the most rigorous barrier to the emergence of resistant strains because conformational restraints require that the primary sequence targeted by shRNA6 is exquisitely conserved. Indeed, to our knowledge there are no reports of resistant variants emerging in the HCV 5′ UTR. There is emerging evidence that with careful selection of direct-acting antiviral agents (DAAs), HCV inhibitors which in isolation suffer from resistance emergence have the potential in dual-therapy regimes to clear virus (25
). An HCV therapy which delivers a high barrier to resistance in a single agent would be a big step forward for the field.
The spectrum of activity of specific sequences expressed from PF-05095808 was interrogated using an RNA cleavage reporter assay. This enabled an exclusive focus on the impact of individual sequence changes on RNA cleavage, without the inevitable disturbance of replication capacity of the HCV replicon. Although the clinical isolate data set predicts the relative cleavage efficiencies of different RNA sequences, it does not provide any measure of absolute efficacy. The observation that target sequences were cleaved more efficiently in short RNA domains is likely explained by reduced target accessibility in the extended HCV sequences. This further reinforces the idea that data obtained from such reporter assays must be analyzed in the appropriate context of the known complexity of the secondary structure for the full-length HCV genome.
Across the clinical isolate panel, small but measureable decreases in RNA cleavage efficiency were associated with changes in target sequences, with the exception of sample 18. The result for sample 18 may reflect sequence changes not identified by population sequencing (detection limit of 25%). The majority of single nucleotide changes had no detectable effect on observed cleavage activity (for shRNA19, 7/8 sequence changes; for shRNA22, 2/3 sequence changes). All clinical isolates showed some sensitivity to shRNA cleavage. To provide a measure of the effectiveness of PF-05095808 as an entire system, the three shRNA data sets derived from each individual plasma sample were analyzed to determine the number of target sites inhibited at ≥60%; Con1b control cleavage efficiency was 60 to 80%. For 17 of the 18 plasma HCV RNA samples, ≥60% inhibition of activity was observed with at least two of the shRNAs. The data generated using this novel RNA reporter assay provide supporting evidence that PF-05095808 will be efficacious against commonly circulating clinical isolates of HCV. Additionally the consistent and reproducible data generated support the use of this assay format to characterize any potential resistance emergence following PF-05095808 administration in clinical trials.
HCV therapy is a rapidly evolving field. New inhibitors bring the promise of improved sustained viral response rates but on top of the punishing regime that consists of interferon and ribavirin combination therapy. Not all patients can tolerate or will be suitable for the current SOC-based regime; indeed, there may soon be clearer stratification of those suitable for SOC therapy on the basis of IL28B and associated single nucleotide polymorphisms (12
). PF-05095808 represents the prototype of a new approach to HCV therapy, a single-dose treatment that can be administered alone or in combination with other anti-HCV agents. These studies demonstrated that PF-05095808 delivers sequence-specific antiviral activity in the absence of overt cytotoxicity. These studies have also shown the potential of PF-05095808 to target commonly circulating strains of HCV. Existing HCV model systems were used to probe efficacy and resistance emergence; however, clinical trials will be needed to truly understand the therapeutic potential of this compound. For many HCV-infected patients, particularly those living in regions with less developed health care systems, novel therapeutics such as PF-05095808 may provide the best option for combating this insidious infection.