Summary excerpt: RNA interference (RNAi) as a novel therapeutic strategy for the treatment of chronic hepatitis B

There are approximately 290 million chronic hepatitis B virus infections worldwide, and existing nucleoside (acid) analogues and interferon drugs cannot meet clinical needs due to long-term or even lifelong use or tolerance issues, as well as the rare ability to achieve current functional cure goals. Therefore, there is an urgent need to develop new mechanism of action drugs.

Nowadays, there are many new mechanism drugs for the treatment of chronic hepatitis B under clinical development, including virus entry inhibitor, polymerase inhibitor, RNA silencing therapy, capsid assembly regulator, viral protein transport inhibitor, farnesol X receptor agonist (FXR), therapeutic vaccine, gene editing technology, etc.

Among them, RNA silencing (RNAi) therapy is one of the earlier developed therapies and has shown promising clinical results. RNAi involves using homologous nucleotide chains to target transcribed HBV mRNA, thereby inhibiting downstream viral protein production. RNAi can suppress HBsAg to low levels, which is an important alternative result for predicting HBsAg serum clearance. By reducing HBsAg through RNAi, T cell function may be reversed and host immune response may be reconstructed.

Man Fang Yuen team of the School of Medicine of the University of Hong Kong recently published a review on RNAi as a new treatment for chronic hepatitis B B, in which researchers reviewed the mechanism and evidence of RNAi for chronic hepatitis B. Lue Xiaoxue has selected some of the content from this review to share with everyone. Below are the details of the content

RNA interference (RNAi)

Mechanism of RNAi action

RNA interference (RNAi) refers to the highly conserved and highly specific degradation of homologous mRNA induced by double stranded RNA (dsRNA) during evolution. In 1998, Fire et al. confirmed that the phenomenon of justice RNA inhibiting homologous gene expression discovered by Guo et al. was caused by contamination of trace amounts of non coding double stranded RNA (dsRNA) in RNA prepared by in vitro transcription. This non coding double stranded RNA was named small interfering RNA (siRNA) and this phenomenon was named RNAi.

SiRNA contains both a sense chain and an antisense chain, with the antisense chain complementary to the target mRNA. SiRNA enters the cytoplasm through endocytosis and interacts with Dicer (RNase III endonuclease), Argonaute (RNase), and RNA binding cofactor to form RISC (RNA Induced Silencing Complex) complex (RLC), which further forms RISC that can bind to target mRNA with complementary sequences to siRNA guide chains. After binding to target mRNA, RISC induces gene silencing through various mechanisms, which may vary from organism to organism.

RNAi as a therapy for viral infectious diseases

In 2018, the US FDA approved Patisiran, an siRNA therapy targeting genetic transthyretin amyloidosis, and since then, the field of siRNA therapy has rapidly expanded. We are currently researching its applications in various diseases, including viral infections, genetic diseases, heart failure, chronic kidney disease, and malignant tumors.

Currently, siRNA is being studied for the treatment of chronic viral infections that cannot be eliminated, such as chronic hepatitis B and human immunodeficiency virus (HIV) infections, as well as some viral infections for which there are no effective treatment methods, such as respiratory syncytial virus, poliovirus, and Ebola virus.

A key consideration in developing siRNA antiviral drugs is appropriate sequence selection. The selected RNA sequence should have high specificity for conserved sequences in the target virus genome in order to exert pan genotypic antiviral effects. Specific siRNA sequences can also reduce off target effects on the host genome, thereby avoiding adverse drug toxicity. SiRNA is generally about 21-23bp long, with two nucleotides protruding from the 3 'end, as longer sequences increase the risk of off target effects.

Structural optimization is crucial for siRNA antiviral drugs. Due to the naturally occurring nucleases, unmodified siRNA rapidly decomposes in human serum. In addition, due to the presence of phosphate backbone and anionic charge, unmodified siRNA is hydrophilic and cannot diffuse through negatively charged cell membranes. Then, siRNA has immunostimulatory effects and can induce unwanted non-specific interferon responses through double stranded RNA dependent protein kinases and Toll like receptors.

The chemical modification of siRNA phosphate backbone can solve the three challenges of siRNA instability, cell entry, and accidental immune activation. By replacing the 2 '- OH group with 2' - O-methyl or 2 '- F-nucleotide on the phosphate backbone, siRNA can be protected from the influence of serum nucleases, reducing off target effects, greatly reducing unwanted immune stimulation reactions, and increasing efficacy by 500 times.

At present, both viral and non viral vectors have been studied for carrying siRNA into target cells. Non viral vectors include polymers, aptamers, peptides, liposomes, antibodies, and lipid nanoparticles. Adenovirus vectors are commonly used viral vectors because they are non-toxic, easy to produce, and have sufficient experience in vaccine applications. In addition, the choice of delivery system also depends on the required siRNA target. For example, N-acetylglucosamine (GalNAc) is an ideal choice for liver targeted siRNA because GalNAc binds to the asialoglycoprotein receptor (ASGPR), which is abundant and specific in liver cells.

Design RNA interference therapy for hepatitis B virus

The characteristic of HBV is its four open reading frames (ORFs), which encode the hepatitis B pre core/core, polymerase, surface, and X protein, respectively. The four ORFs have overlapping sequences and share the same polyadenylation signal at the 3 'end of the core protein coding region. This allows a single siRNA to interfere with all four HBV transcripts, thereby reducing the production of all downstream viral proteins and pre genomic RNA. Virus replication can also be reduced by siRNA, which may indirectly decrease the cccDNA storage library. Therefore, siRNA can directly or indirectly interfere with multiple steps of the virus lifecycle. The following figure illustrates the mechanism of RNAi against HBV.

When designing siRNA targeting HBV, viral mutations are a concern that needs to be considered, as single base mismatches may lead to the loss of siRNA effectiveness. According to reports, mutant strains rapidly appear in RNA viruses (including HIV and HCV) after siRNA treatment. However, there are currently no reports of siRNA induced HBV mutations, but siRNA can exert selective pressure on pre-existing resistant HBV quasispecies. It is crucial to use targeted and conserved regions or multiple siRNA triggers in siRNA therapy to minimize the potential impact of HBV mutations.

SiRNA can also restore the host's immune response to HBV. One of the main factors contributing to the sustained chronic existence of HBV is host immune suppression after long-term exposure to high levels of immunosuppressive viral antigens, especially HBsAg. CHB is associated with impaired host immune pathways, including Toll like receptor signaling, immune checkpoint signaling, and increased activity of immunosuppressive T regulatory cells. These effects ultimately lead to a reduction and dysfunction of HBV specific T cell clones, resulting in anti HBV deficiencies in T cell quantity and function. Through its effective role in reducing HBV viral antigens, siRNA can indirectly rebuild the immune system.

Anti sense oligonucleotides, another form of gene silencing

Firstly, siRNA is a double stranded RNA with an ideal length of 21 nucleotides plus two 3 '- terminal protruding nucleotides. In contrast, ASO is a single stranded DNA with a length of 15 to 25 nucleotides. ASO is designed as a Gapmer structure, with a central unmodified complementary DNA sequence flanked by modified RNA like fragments. The Gapmer structure enhances the affinity of ASO for its target sequence and increases its resistance to nuclease degradation.

Secondly, unmodified siRNA requires a vector to enter cells, while unbound ASO can be absorbed into liver cells through receptor-mediated pathways. After entering the cell, siRNA accumulates in the endosome and is administered less frequently, while ASO accumulates in the cytoplasm and requires more frequent administration. Although ASO does not require carrier modification, GalNAc modification in ASO can enhance liver cell uptake and reduce systemic exposure, similar to the effect of GalNAc modification in siRNA.

Thirdly, siRNA needs to form RISC in order to bind to target mRNA, as the removal of the sense strand is essential in the exposure of complementary nucleotides on the antisense strand, while ASO can bind to target mRNA alone.

Fourthly, siRNA and ASO have different gene silencing mechanisms. As mentioned above, siRNA mediates its effect through RISC. In contrast, ASO most commonly mediates its function by recruiting RNase-H (endonuclease family) in the cytoplasm and nucleus to cleave target RNA. ASO can also inhibit RNA translation by suppressing 5 'cap formation or blocking ribosomal subunit attachment.

Preclinical and clinical evidence of chronic hepatitis B

In this review, researchers described the research data of investigational drugs currently in preclinical and clinical trial stages one by one. RNAi drugs include ARC-520, ARB-1467, AB-729, RG-6346 (DCR HBVS), VIR-2218 (BRII-835), JNJ-3989 (ARO-HBV) ALG-125918、ALG-125755， Anti sense oligonucleotide (ASO) drugs include RO7062931, GSK3389404, and Bepirovesen (GSK3228836, GSK836) 、ALG-020572， And the combination therapy of VIR-2218, JNJ-6379, ALG-12755 (siRNA)+ALG-020572 (ASO), ALG-125903 (siRNA)+ALG-020579 (ASO)+ALG-010133. Since the data related to the above drugs were almost published in the history of the liver time WeChat official account, we will not repeat it here. Friends who want to view the drugs can enter the name of the relevant drugs in the history for viewing.

Overview of current evidence and conclusions

Existing clinical trials have consistently confirmed that siRNA is generally safe, with most adverse events being mild injection reactions or flu like symptoms. A sudden increase in ALT can occur during siRNA therapy, but it is usually short-lived and associated with a decrease in HBsAg, indicating immune reconstitution and elimination of infected liver cells.

SiRNA has been shown to effectively reduce HBsAg. In the new generation siRNA (excluding the old generation ARC-520 and ARB-1467), an average reduction of 2-2.5 log in HBsAg can be achieved, with over 90% of patients in the high-dose treatment group achieving HBsAg reduction of over 1 log IU/ml, and 50-97% of patients having HBsAg suppressed to below 100 IU/ml. The reduction of HBsAg in siRNA after treatment is sustainable, and the incidence of HBsAg serological clearance has been recorded.

However, it is still unclear whether these effective HBsAg lowering effects can translate into persistent HBsAg serological clearance. In addition, larger scale studies comparing HBeAg positive and negative patients are needed to determine whether drug effects can be extended to different subgroups of CHB. The long-term data from trials involving different CHB populations are highly anticipated.

The current standard administration regimen for siRNA is multiple doses, as multiple doses are more effective in reducing HBsAg and have a longer lasting effect than single dose administration. The current evidence supports that siRNA can be administered once a month, and AB-729 even shows effective dosing effects when administered once every 8 or 12 weeks.

The total duration of siRNA administration (total course of treatment) is still unclear, as different treatment durations (ranging from two months to one year) were used in the current trial. The determination of the ideal duration of treatment may depend on the persistence of HBsAg suppression after treatment, and longer follow-up trials are required to monitor HBsAg serological clearance. These issues are of great significance for the future development of RNAi therapy.

Similar to siRNA, ASO is generally safe and well tolerated. Although siRNA can be administered once a month, ASO requires more frequent administration (once a week or every two weeks), reflecting the inherent pharmacokinetic differences between siRNA and ASO.

Before deciding whether it is necessary to combine RNAi with other novel antiviral drugs to achieve functional cure in all patients, it is necessary to determine the likelihood of subsequent HBsAg serological clearance in patients who achieve low HBsAg levels (e.g.<100 IU/mL) through RNAi. If the HBsAg serum clearance rate is significantly high within a short period of time after stopping RNAi, it may not be necessary to combine RNAi with other new drugs of different categories. However, for patients who still have high HBsAg levels, combination therapy may be necessary.

With the established efficacy and safety of NA, future RNAi treatment plans may use NA as a cornerstone. Adding a third anti HBV drug to RNAi and NA may result in a synergistic antiviral effect.

Due to the potential role of RNAi in immune reconstitution, the addition of immune modulators may further enhance the host's antiviral immune system. In fact, combining VIR-2218 with pegylated interferon alpha-2a can produce greater HBsAg inhibition than using either drug alone. The current data indicates that combining siRNA with interferon can produce a synergistic effect.

However, other combinations of siRNA with immunomodulators (including siRNA+therapeutic vaccines and siRNA+Toll like receptor agonists) are still undergoing clinical trial research. Research is also underway on the combination therapy of siRNA with novel virus targeting agents.

It is worth noting that preclinical experiments have demonstrated the synergistic effect of combining siRNA and ASO, two RNA silencing agents that share the same target (mRNA), although the potential mechanism and clinical efficacy of this combination have not been determined.

On the other hand, the combination of siRNA and capsid assembly modulators did not produce a synergistic effect in the ongoing experiments. This highlights the necessity of carefully selecting drugs in combination with siRNA, and a series of combination trials involving drugs with different mechanisms are still ongoing.

In summary, RNAi is a safe technique that can induce effective and sustainable HBsAg inhibition. The research on RNAi is rapidly developing, and early data is very promising. Multiple trials are currently underway, and with further development, RNAi may become a novel therapeutic strategy that changes the paradigm of CHB treatment.