Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice.

Yao-Ming Shih,Mi-Hua Tao,Karl-Dimiter Bissig,Yu-Chen Enya Chen,Hui-Hsien Chou,Ping-Yi Wu,Tzu-Hui Wu,Chun-Chi Chen,Cheng-Pu Sun

doi:10.1038/srep15259

Abstract

Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients.

Highlights

Patients chronically infected with hepatitis B virus (HBV) typically produce high viral DNA titers[1,2] and viral protein levels[3,4], both of which are associated with a high risk of developing several severe liver complications, including cirrhosis and hepatocellular carcinoma
In the 5 week samples collected from the GL2 small hairpin RNA (shRNA)-treated mice, no mutations were seen in the P28 target region and the ratio of wild-type to T472C in the S1 target region was the same as the input ratio. These results show that the HBV variants that dominated in the week 5 sample of the associated viral vector serotype 8 (AAV8)/P28-treated mouse were generated under the selection pressure of P28 shRNA
We demonstrated that a HBV variant, T472C HBV, identified in the pre-existing quasispecies pool of a chronic HBV patient, was able to escape from S1 treatment and become the dominant population in vivo (Figs 1 and 4)

Summary

Introduction

Patients chronically infected with hepatitis B virus (HBV) typically produce high viral DNA titers[1,2] and viral protein levels[3,4], both of which are associated with a high risk of developing several severe liver complications, including cirrhosis and hepatocellular carcinoma. We identified a highly potent anti-HBV small hairpin RNA (shRNA), designated as S1, and demonstrated that a single treatment of HBV transgenic mice with S1 shRNA delivered by adeno-associated viral vector serotype 8 (AAV8) reduced the serum HBV titer by up to 1,000- to 10,000-fold, but, more importantly, substantially decreased serum and hepatic levels of the HBV proteins[12]. One major problem for antiviral RNAi therapy is the selection of escape mutants with a point mutation or deletion within the target sequence that could abolish the RNAi activity This is a particular concern for RNA viruses whose replication polymerases lack proof-reading activity and are prone to mutant generation. Selection of RNAi-resistant variants could be a potential problem in the treatment of chronic HBV patients due to the high error rate of HBV polymerase and the presence of many genetically distinct viral populations in these patients. We used human liver chimeric mice as a HBV infection model to evaluate the therapeutic efficacy, and monitor the dynamics of HBV variants during treatment with a single shRNA or a combination of different shRNAs

Methods

Results

Conclusion