Long-term serological, virological and histological responses to RNA inhibition by ARC-520 in Chinese chronic hepatitis B patients on entecavir treatment.

OBJECTIVE: We examined the serological, virological (in serum and liver) and histological profiles in chronic hepatitis B virus (HBV) patients during and after completion of multiple dose (MD) ARC-520. DESIGN: The present phase 1b study was a multidose, open-label extension cohort of patients that had received single dose ARC-520 in our previous study. Eight patients received 4-9 4 weekly doses of MD ARC-520 and entecavir. Liver biopsies were performed in six patients. Intrahepatic and serum HBV DNA, HBV RNA and viral antigens were measured. RESULTS: All patients had 28.9-30.4 months of follow-up after the last MD. All three hepatitis B e antigen (HBeAg)-positive patients had profound reductions in hepatitis B surface antigen (HBsAg), HBeAg, hepatitis B core-related antigen and HBV RNA with two undergoing HBeAg seroconversion. One further achieved HBsAg seroconversion (anti-HBs level of 25.1 IU/L) and the remaining two had HBsAg reductions of -1.7 and -3.5 log IU/mL >30 months after MD. Among the five HBeAg-negative patients, four had modest HBsAg reduction >29 months after completion of MD and one achieved HBsAg seroconversion (anti-HBs level of 152.5 IU/L) and was negative for liver HBsAg staining. Entecavir was successfully stopped in this patient 12 months after HBsAg seroconversion. Temporally related alanine aminotransferase elevations preceded by HBsAg reductions were observed in three patients suggesting immune activation. HBcAg staining was negative in all six biopsied patients. Two patients with <10% HBsAg positive staining of hepatocytes had correspondingly low serum HBsAg levels of 1.5 and 11.5 IU/mL. CONCLUSIONS: MD ARC-520 therapy achieved sustained and profound reductions of viral antigens and HBV RNA. HBsAg seroclearance was achievable. TRIAL REGISTRATION NUMBER: NCT02065336.

RNA Interference Therapy for Chronic Hepatitis B Predicts the Importance of Addressing Viral Integration When Developing Novel Cure Strategies.

Chronic hepatitis B infection remains a globally important cause of morbidity and mortality and has recently undergone a renaissance in therapeutic interest with increased pre-clinical and clinical testing of new drug classes. One of the first new classes in the clinic was RNA interference agents, which have the potential to impact the entire viral life cycle by reducing all virus-produced mRNA. Early clinical testing with the first of these agents in the clinic, ARC-520, demonstrated that rapid and deep reductions in viral proteins, RNA and DNA could be produced with this approach, but also the surprising insight that HBsAg production from incomplete HBV DNA integrated into the host genome appears to play a heretofore unappreciated and important role in maintaining circulating HBsAg, thought to play a fundamental role in preventing host clearance of the virus. Thus, accounting for viral DNA integration in novel HBV treatment approaches may prove to be essential to achieving successful finite therapies of this difficult to treat chronic infection.

Development of an RNAi therapeutic for alpha-1-antitrypsin liver disease.

The autosomal codominant genetic disorder alpha-1 antitrypsin (AAT) deficiency (AATD) causes pulmonary and liver disease. Individuals homozygous for the mutant Z allele accumulate polymers of Z-AAT protein in hepatocytes, where AAT is primarily produced. This accumulation causes endoplasmic reticulum (ER) stress, oxidative stress, damage to mitochondria, and inflammation, leading to fibrosis, cirrhosis, and hepatocellular carcinoma. The magnitude of AAT reduction and duration of response from first-generation intravenously administered RNA interference (RNAi) therapeutic ARC-AAT and then with next-generation subcutaneously administered ARO-AAT were assessed by measuring AAT protein in serum of the PiZ transgenic mouse model and human volunteers. The impact of Z-AAT reduction by RNAi on liver disease phenotypes was evaluated in PiZ mice by measuring polymeric Z-AAT in the liver; expression of genes associated with fibrosis, autophagy, apoptosis, and redox regulation; inflammation; Z-AAT globule parameters; and tumor formation. Ultrastructure of the ER, mitochondria, and autophagosomes in hepatocytes was evaluated by electron microscopy. In mice, sustained RNAi treatment reduced hepatic Z-AAT polymer, restored ER and mitochondrial health, normalized expression of disease-associated genes, reduced inflammation, and prevented tumor formation. RNAi therapy holds promise for the treatment of patients with AATD-associated liver disease. ARO-AAT is currently in phase II/III clinical trials.

RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.

Chronic hepatitis B virus (HBV) infection is a major health concern worldwide, frequently leading to liver cirrhosis, liver failure, and hepatocellular carcinoma. Evidence suggests that high viral antigen load may play a role in chronicity. Production of viral proteins is thought to depend on transcription of viral covalently closed circular DNA (cccDNA). In a human clinical trial with an RNA interference (RNAi)-based therapeutic targeting HBV transcripts, ARC-520, HBV S antigen (HBsAg) was strongly reduced in treatment-naïve patients positive for HBV e antigen (HBeAg) but was reduced significantly less in patients who were HBeAg-negative or had received long-term therapy with nucleos(t)ide viral replication inhibitors (NUCs). HBeAg positivity is associated with greater disease risk that may be moderately reduced upon HBeAg loss. The molecular basis for this unexpected differential response was investigated in chimpanzees chronically infected with HBV. Several lines of evidence demonstrated that HBsAg was expressed not only from the episomal cccDNA minichromosome but also from transcripts arising from HBV DNA integrated into the host genome, which was the dominant source in HBeAg-negative chimpanzees. Many of the integrants detected in chimpanzees lacked target sites for the small interfering RNAs in ARC-520, explaining the reduced response in HBeAg-negative chimpanzees and, by extension, in HBeAg-negative patients. Our results uncover a heretofore underrecognized source of HBsAg that may represent a strategy adopted by HBV to maintain chronicity in the presence of host immunosurveillance. These results could alter trial design and endpoint expectations of new therapies for chronic HBV.

Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses.

The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites. Compared to PCR- or ELISA-based methods, this assay enables separate quantitation of non-phosphorylated full-length guide strands from 5΄-phosphorylated forms that may associate with RNA-induced silencing complexes (RISC). Biodistribution studies in mice indicated that ARC-520 guide strands predominantly accumulated in liver. 5΄-phosphorylation of guide strands was observed within 5 min after ARC-520 injection, and was detected for at least 4 weeks corresponding to the duration of HBV mRNA silencing. Guide strands detected in RISC by AGO2 immuno-isolation represented 16% of total 5΄-phosphorylated guide strands in liver, correlating with a 2.7 log10 reduction of HBsAg. The PNA method enables pharmacokinetic analysis of RNAi triggers, elucidates potential metabolic processing events and defines pharmacokinetic-pharmacodynamic relationships.

Synthetic RNAi triggers and their use in chronic hepatitis B therapies with curative intent.

Current therapies for chronic hepatitis B virus infection (CHB) - nucleos(t)ide analogue reverse transcriptase inhibitors and interferons - result in low rates of functional cure defined as sustained off-therapy seroclearance of hepatitis B surface antigen (HBsAg). One likely reason is the inability of these therapies to consistently and substantially reduce the levels of viral antigen production. Accumulated evidence suggests that high serum levels of HBsAg result in exhaustion of the host immune system, rendering it unable to mount the effective antiviral response required for HBsAg clearance. New mechanistic approaches are required to produce high rates of HBsAg seroclearance in order to greatly reduce off-treatment disease progression. Already shown to be a clinically viable means of reducing gene expression in a number of other diseases, therapies based on RNA interference (RNAi) can directly target hepatitis B virus transcripts with high specificity, profoundly reducing the production of viral proteins. The fact that the viral RNA transcripts contain overlapping sequences means that a single RNAi trigger can result in the degradation of all viral transcripts, including all messenger RNAs and pregenomic RNA. Advances in the design of RNAi triggers have increased resistance to degradation and reduced nonspecific innate immune stimulation. Additionally, new methods to effectively deliver the trigger to liver hepatocytes, and specifically to the cytoplasmic compartment, have resulted in increased efficacy and tolerability. An RNAi-based drug currently in clinical trials is ARC-520, a dynamic polyconjugate in which the RNAi trigger is conjugated to cholesterol, which is coinjected with a hepatocyte-targeted, membrane-active peptide. Phase 2a clinical trial results indicate that ARC-520 was well tolerated and resulted in significant, dose-dependent reduction in HBsAg for up to 57days in CHB patients. RNAi-based therapies may play an important role in future therapeutic regimes aimed at improving HBsAg seroclearance and eliminating the need for lifelong therapy. This paper forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B."

Targeted in vivo delivery of siRNA and an endosome-releasing agent to hepatocytes.

The discoveries of RNA interference (RNAi) and short interfering RNAs (siRNAs) have provided the opportunity to treat diseases in a fundamentally new way: by co-opting a natural process to inhibit gene expression at the mRNA level. Given that siRNAs must interact with the cells' natural RNAi machinery in order to exert their silencing effect, one of the most fundamental requirements for their use is efficient delivery to the desired cell type and, specifically, into the cytoplasm of those cells. Numerous research efforts involving the testing of a large number of delivery approaches using various carrier molecules and inventing several distinct formulation technologies during the past decade illustrate the difficulty and complexity of this task. We have developed synthetic polymer formulations for in vivo siRNA delivery named Dynamic PolyConjugates™ (DPCs) that are designed to mimic the features viruses possess for efficient delivery of their nucleic acids. These include small size, long half-life in circulation, capability of displaying distinct host cell tropism, efficient receptor binding and cell entry, disassembly in the endosome and subsequent release of the nucleic acid cargo to the cytoplasm. Here we present an example of this delivery platform composed of a hepatocyte-targeted endosome-releasing agent and a cholesterol-conjugated siRNA (chol-siRNA). This delivery platform forms the basis of ARC-520, an siRNA-based therapeutic for the treatment of chronic hepatitis B virus (HBV) infection. In this chapter, we provide a general overview of the steps in developing ARC-520 and detailed protocols for two critical stages of the discovery process: (1) verifying targeted in vivo delivery to hepatocytes and (2) evaluating in vivo drug efficacy using a mouse model of chronic HBV infection.

Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

Current status of pharmaceutical and genetic therapeutic approaches to treat DMD.

Duchenne muscular dystrophy (DMD) is a genetic disease affecting about one in every 3,500 boys. This X-linked pathology is due to the absence of dystrophin in muscle fibers. This lack of dystrophin leads to the progressive muscle degeneration that is often responsible for the death of the DMD patients during the third decade of their life. There are currently no curative treatments for this disease but different therapeutic approaches are being studied. Gene therapy consists of introducing a transgene coding for full-length or a truncated version of dystrophin complementary DNA (cDNA) in muscles, whereas pharmaceutical therapy includes the use of chemical/biochemical substances to restore dystrophin expression or alleviate the DMD phenotype. Over the past years, many potential drugs were explored. This led to several clinical trials for gentamicin and ataluren (PTC124) allowing stop codon read-through. An alternative approach is to induce the expression of an internally deleted, partially functional dystrophin protein through exon skipping. The vectors and the methods used in gene therapy have been continually improving in order to obtain greater encapsidation capacity and better transduction efficiency. The most promising experimental approaches using pharmaceutical and gene therapies are reviewed in this article.

Dose response in rodents and nonhuman primates after hydrodynamic limb vein delivery of naked plasmid DNA.

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle. Expression from the CMV promoter was highest. For mice, rats, and rhesus monkeys, the linear range for pDNA dose response could be defined by the mass of pDNA relative to the mass of target muscle. Correlation between pDNA dose and expression was linear between a threshold dose of 75 µg/g and maximal expression at approximately 400 µg/g. One HLV injection into rats of a dose of CMV-LacZ yielding maximal expression resulted in an average transfection of 28% of all hind leg muscle and 40% of the gastrocnemius and soleus. Despite an immune reaction to the reporter gene in monkeys, a single injection transfected an average of 10% of all myofibers in the targeted muscle of the arms and legs and an average of 15% of myofibers in the gastrocnemius and soleus.

Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes. It was batch dependent and correlated with the quantity of bacterial genomic DNA (gDNA) that copurified with the pDNA. To determine whether such an effect was due to bacterial DNA or simply to fragmented DNA, mice were treated by HLV injection with sheared bacterial or murine gDNA. As little as 20 µg of the large fragments of bacterial gDNA caused muscle damage that morphologically resembled damage caused by the toxic pDNA preparations, whereas murine gDNA caused no damage even at a 10-fold higher dose. Toxicity from the bacterial gDNA was not due to endotoxin and was eliminated by DNase digestion. We conclude that pDNA itself does not cause muscle damage and that purification methods for the preparation of therapeutic pDNA should be optimized for removal of bacterial gDNA.

Myofiber Damage Evaluation by Evans Blue Dye Injection.

Evans blue dye (EBD) can be used in live mice to study muscle pathology or injury, including exercise-induced muscle damage. EBD is excluded from intact cell membranes but leaks into cells, including muscle fibers, when the cell membrane is ruptured. EBD can be visualized by its autofluorescence under a fluorescence microscope. EBD-stained myofibers can be quantified from microscope images of muscle cross-sections. These myofibers are often in clusters that lend themselves to morphometric analysis. When the damaged myofibers are interspersed among intact myofibers, however, a more suitable approach is to count individual myofibers in the field of view. A much faster approach to measure EBD in muscles from different strains of mice or between treatment groups is to extract the EBD from muscle samples and quantitate it using a spectrophotometric microplate reader. The advantages and disadvantages of using each of these approaches are discussed here. Curr. Protoc. Mouse Biol. 1:463-488 © 2011 by John Wiley & Sons, Inc.

Evaluation of hydrodynamic limb vein injections in nonhuman primates.

The administration route is emerging as a critical aspect of nonviral and viral vector delivery to muscle, so as to enable gene therapy for disorders such as muscular dystrophy. Although direct intramuscular routes were used initially, intravascular routes are garnering interest because of their ability to target multiple muscles at once and to increase the efficiency of delivery and expression. For the delivery of naked plasmid DNA, our group has developed a hydrodynamic, limb vein procedure that entails placing a tourniquet over the proximal part of the target limb to block all blood flow and injecting the gene vector rapidly in a large volume so as to enable the gene vector to be extravasated and to access the myofibers. The present study was conducted in part to optimize the procedure in preparation for a human clinical study. Various injection parameters such as the effect of papaverine preinjection, tourniquet inflation pressure and duration, and rate of injection were evaluated in rats and nonhuman primates. In addition, the safety of the procedure was further established by determining the effect of the procedure on the neuromuscular and vascular systems. The results from these studies provide additional evidence that the procedure is well tolerated and they provide a foundation on which to formulate the procedure for a human clinical study.

Use of Evans blue dye to compare limb muscles in exercised young and old mdx mice.

Evans blue dye (EBD) is used to mark damaged and permeable muscle fibers in mouse models of muscular dystrophy and as an endpoint in therapeutic trials. We counted EBD-positive muscle fibers and extracted EBD from muscles sampled throughout the hindlimbs in young adult and old mdx mice to determine if the natural variability in morphology would allow measurement of a functional improvement in one limb compared to the contralateral limb. Following one bout of rotarod or treadmill exercise that greatly increased serum creatine kinase levels, the number of EBD(+) muscle fibers in 12-19-month-old mdx mice increased 3-fold, EBD in the muscles increased, and, importantly, contralateral pairs of muscles contained similar amounts of EBD. In contrast, the intra- and interlimb amounts of EBD in 2-7-month-old mdx mice were much too variable. A therapeutic effect can more readily be measured in old mdx mice. These results will be useful in the design of therapy protocols using the mdx mouse.

Functional efficacy of dystrophin expression from plasmids delivered to mdx mice by hydrodynamic limb vein injection.

In these studies we delivered by hydrodynamic limb vein (HLV) injection plasmid DNA (pDNA) expressing the full-length mouse dystrophin gene to skeletal muscles throughout the hind limbs of the mdx mouse model for Duchenne muscular dystrophy (DMD). We evaluated the levels and stability of dystrophin expression and measured the resulting muscle protection, using Evans blue dye (EBD) to mark the damaged myofibers. Plasmid delivery was as efficient in the dystrophic mice as in wild-type mice and equally efficient in young adult and old mice, as long as the dose of pDNA was adjusted for the target muscle weight. The HLV gene delivery procedure was tolerated well by the dystrophic mice and repeat injections could be performed over an extended period of time. Multiple gene deliveries additively increased the amount of dystrophin protein and also increased the percentages of dystrophin-expressing myofibers. Plasmids expressing dystrophin from a cytomegalovirus (CMV) promoter construct containing the HMG1 intron provided stable dystrophin expression for the life of the mouse and provided significant benefit to the limbs. EBD staining showed that dystrophin gene delivery preserved myofibers in the CMV-HMGi-mDys-injected leg by 2.5- to 5-fold in large groups of muscles and by 2.5-fold throughout the injected legs, compared with the contralateral control legs injected with a nonexpressing plasmid. A similar degree of protection was measured in young adult mice evaluated soon after the last gene delivery and in aged mice injected over an extended period of time. This degree of protection resulted from 18 to 20% of the normal level of dystrophin protein, with 11-16% dystrophin-expressing myofibers. These studies show promise for the use of HLV injections to deliver therapeutic doses of full-length dystrophin-expressing plasmids for long-lasting protection of skeletal muscles in patients with DMD.

Sustained liver-specific transgene expression from the albumin promoter in mice following hydrodynamic plasmid DNA delivery.

BACKGROUND: To properly study gene expression in vivo, often long-term expression is desired. Previous studies using plasmid DNA (pDNA) vectors have typically resulted in short-term expression. Here, we evaluated combinations of the albumin promoter with different enhancers and untranslated regions for liver-specific expression in mice. METHODS: A series of pDNA secreted alkaline phosphatase (SEAP) reporter gene expression vectors was constructed using the albumin promoter and various other expression cassette elements. Each was evaluated for level and duration of SEAP expression in mice following hydrodynamic tail vein delivery. RESULTS: Sustained liver expression was obtained from vectors combining the albumin promoter with an albumin 3' untranslated region (3'UTR). The level of expression was increased by inclusion of enhancers and a 5' intron. The optimal expression vector consisted of the albumin promoter combined with an alpha-fetoprotein MERII enhancer, 5' intron from the factor IX gene, and the 3'UTR from the albumin gene including intron 14. With this vector, SEAP reporter gene expression levels remained high for 1 year, at levels comparable to those obtained from the cytomegalovirus (CMV) promoter on day 1. Expression of human apolipoprotein E3 (hApoE) in ApoE knockout mice provided a dose-dependent correction of their hypercholesterolemia. CONCLUSIONS: Liver-specific sustained transgene expression can be obtained at very high levels from optimized pDNA vectors, without the use of integration systems. Such vectors will further facilitate biological studies of genes in vivo and may find application in gene therapy.

Long-term RNA interference from optimized siRNA expression constructs in adult mice.

DNA constructs for small interfering RNA (siRNA) expression in mammalian cells have the potential for longer-term target gene knockdown than synthetic siRNAs. We compared in adult mice the efficacy and longevity of target gene knockdown from siRNA expression cassettes contained in plasmids, PCR-generated linear constructs or PCR constructs containing "dumbbell" ends using the hydrodynamic delivery method. Plasmid siRNA expression constructs were more effective than PCR constructs for target gene knockdown. The efficacy of the PCR constructs was improved by addition of short extensions beyond the transcription termination signal and greatly improved by addition of dumbbell ends. Constructs containing the H1 promoter were significantly less effective in mice than those containing the U6 promoter, whereas both promoters functioned equally well in cultured cells. Target gene knockdown perdured for at least 20 weeks in mice after delivery of either PCR or plasmid siRNA expression cassettes. These results will help guide RNAi vector design.