Chronic hepatitis B (CHB), caused by persistent hepatitis B virus (HBV) infection, remains a significant global public health challenge (World Health Organization, 2016). In April 2024, the World Health Organization released the “Global Hepatitis Report 2024” (World Health Organization, 2024). Global epidemiological data from 2022 indicate that approximately 254 million individuals were living with HBV infection worldwide. Over time, chronic HBV infection contributes to severe hepatic complications, including liver fibrosis (Zhang Q. et al., 2022; He et al., 2023), cirrhosis, and hepatocellular carcinoma (HCC) (Ringelhan et al., 2024). HBV is a non-cytopathic hepatotropic DNA virus (Tong et al., 2017; Wang et al., 2022). HBV infection can lead to diverse clinical outcomes, ranging from acute self-limiting infection to chronic persistence. The host’s age at the time of HBV exposure plays a critical role in determining disease progression (Wu and Chang, 2015). Immunocompetent adults typically develop acute, self-limited infections and eventually clear the virus (Trépo et al., 2014). In contrast, 90% of people exposed to HBV in the perinatal period develop chronic infection, and approximately 25%–30% of patients infected with HBV in infancy develop chronic infection (Hou et al., 2017). Beyond its clinical implications, chronic HBV infection imposes substantial economic burdens and psychological distress on affected individuals and their families. The current clinical management of CHB primarily involves two classes of antiviral agents: nucleoside/nucleotide analogues (NAs) (Fung et al., 2011; Hsu et al., 2023; Yang et al., 2023) and interferon-alpha (IFN-α) (Jiang et al., 2023; Zhao et al., 2024). While both drug classes can effectively suppress viral replication and mitigate liver disease progression, neither achieves complete eradication of HBV (Lee and Banini, 2019). NAs are associated with high relapse rates upon discontinuation, and prolonged use may increase the risk of drug resistance (Yang et al., 2023). IFN-α exhibits a limited immune response rate in patients and is contraindicated in cases of decompensated cirrhosis (Fung et al., 2022). Currently, the inability to effectively eliminate cccDNA and integrated HBV-DNA precludes complete or virological cure for CHB. Consequently, current clinical goals focus primarily on achieving a “functional cure” (Phillips et al., 2022; Zheng et al., 2022), defined as sustained hepatitis B surface antigen (HBsAg) loss with undetectable serum HBV-DNA (Wong et al., 2022), with or without hepatitis B surface antibody (HBsAb) seroconversion. However, residual cccDNA remains in hepatocytes, highlighting the need for novel therapeutic strategies. In response, emerging CHB therapeutics are being actively developed, targeting either direct antiviral mechanisms (inhibiting various stages of the HBV life cycle) or indirect approaches (modulating host immune responses) (Asselah et al., 2019; Xia and Liang, 2019; Maepa et al., 2021; Tang et al., 2022; Li J. et al., 2024). Among these, small nucleic acid drugs have garnered significant attention as promising candidates in pharmaceutical research.

Small nucleic acid drugs, generally refer to nucleotide sequences of no more than 30 base pairs (Chan et al., 2006), acting upstream of protein synthesis at the post-transcription and pre-translation stages (Kanasty et al., 2013; Soriano et al., 2017; Halloy et al., 2022). In contrast to conventional small-molecule drugs and monoclonal antibodies (Takakusa et al., 2023), small nucleic acid drugs are designed to target the viral post-transcriptional messenger RNA (mRNA) and pregenomic RNA (pgRNA) through the design of specific nucleotide sequences (Hui et al., 2022), This mechanism triggers gene silencing, which, in turn, effectively inhibits the production of HBV antigens and viral replication (Sun et al., 2024). As a result, it can prevent the continuous deterioration of liver damage. Unlike traditional therapeutics constrained by protein druggability requirements, small nucleic acid drugs target mRNA based on the principle of base complementary pairing, they circumvent the limitations imposed by protein druggability, thereby expanding the range of potential therapeutic targets. Furthermore, their high specificity and precision in target recognition contribute to accelerated drug development cycles. In addition, small nucleic acid drugs demonstrate significantly extended pharmacological activity compared to traditional small-molecule drugs. Through rational chemical modifications, these engineered oligonucleotides achieve enhanced metabolic stability, prolonging their plasma half-life and enabling sustained therapeutic effects with reduced dosing frequency. Although small nucleic acid drugs hold considerable promise for the treatment of hepatitis B, their translation into clinical applications remains challenging. Unmodified small nucleic acids suffer from poor stability and potential off-target effects. Current research efforts are therefore focused on addressing these limitations through advanced chemical modifications and novel delivery strategies.

In this review, we provide a comprehensive overview of the current state and future trends in the development of small nucleic acid drugs for the treatment of CHB. First, we delve into the distinctive structure of the HBV genome and explore the potential of designing small nucleic acid drugs based on this unique structure. We then introduce the main classifications and mechanisms of small nucleic acid drugs, including single-stranded antisense oligonucleotides (ASOs) and double-stranded RNAs (dsRNAs) such as small interfering RNAs (siRNAs) and microRNAs (miRNAs). Moreover, we elaborate on the pathways through which small nucleic acid drugs can achieve a functional cure. These pathways primarily involve the inhibition of viral replication and their influence on immune regulation. However, small nucleic acid drugs are highly susceptible to degradation in vivo, which poses numerous challenges during their development. One of the key issues is how to effectively enrich these drugs in target tissues and enable them to act on target genes. In addition to relying on appropriate chemical modifications, the precise delivery of small nucleic acid drugs using efficient delivery systems represents an important research avenue. Subsequently, we present the current research on small nucleic acid drugs for CHB treatment, covering both preclinical studies and clinical studies. We also discuss the combination therapy of small nucleic acid drugs. Finally, we have explored the future development trend of small nucleic acid drugs.

HBV is a small enveloped DNA virus (Dandri, 2020), consisting of only 3200 nucleotides (nt) (Tsukuda and Watashi, 2020). The viral genome exists as a partially double-stranded relaxed circular form (rcDNA) in mature virions. Upon host cell entry, rcDNA is transported to the nucleus, where it is converted into covalently cccDNA (Nassal, 2015; Wang et al., 2016; Wei and Ploss, 2021). The episomal form persists in infected cells and serves as the transcriptional template for viral replication. Every nucleotide in HBV DNA has coding capacity, and the whole genome exhibits high informational density (Tu et al., 2017). The HBV genome comprises four overlapping open reading frames (ORFs), which are the S region, C region, P region, and X region (Michel and Tiollais, 1987) (Figure 1). Since transcription is initiated by four distinct promoters at different positions in the HBV genome, it can generate four types of polyadenylated RNAs (3.5 kb preC/C mRNA, 2.4 kb PreS1 mRNA, 2.1 kb PreS2/S mRNA, and 0.7 kb X mRNA). These transcripts are exported to the cytoplasm for protein translation and DNA replication (Tong and Revill, 2016). The 0.7 kb mRNA encodes the regulatory X protein (HBxAg), while the 2.1 kb and 2.4 kb mRNAs can encode the S, M, and L surface proteins (HBsAg), The 3.5 kb mRNA consists of two species: the longer variant, termed precore RNA, encodes the precore protein (HBeAg), whereas the shorter variant, known as pgRNA, serves as a template for reverse transcription and encodes both the core protein (HBcAg) and the P protein (HBV DNA polymerase). Despite these transcripts are initiated from different positions, they terminate at the same polyadenylation site (Weinberg and Arbuthnot, 2010). If the site is utilized as a target for small nucleic acid drug design, it is theoretically possible to simultaneously target multiple viral mRNAs, induce their degradation, and inhibit viral protein production. Moreover, if pgRNA can be targeted, it is possible to silence the HBV gene, thereby inhibiting viral protein expression and suppressing replication. However, it is worth noting that HBV gene fragments can integrate into the host genome during viral replication. This integration enables the persistence of HBsAg in the serum of patients undergoing long-term antiviral therapy, primarily derived from integrated HBV DNA fragments. Therefore, small nucleic acid drugs should also target these integrated fragments, as they retain both the complete HBsAg coding sequence and a truncated HBxAg coding region. In theory, such targeting could reduce the expression of these viral proteins (Budzinska et al., 2018; Zhang et al., 2021).

Currently, small nucleic acid drugs used for the treatment of CHB can be categorized into RNA interference (RNAi) and ASOs according to their mechanism (Figure 2).

RNAi is a unique gene regulatory mechanism mediated by dsRNAs, which induces post-transcriptional gene silencing (PTGS) and is widely observed in mammals (Tabara et al., 1998; Setten et al., 2019). This process can prevent protein synthesis by triggering the efficient and specific degradation of homologous mRNA molecules (López-Fraga et al., 2009). RNAi is highly conserved in evolution, and it plays a crucial role in gene regulation and serves as a defense mechanism against viral RNA and transposon invasion (Agrawal et al., 2003). The RNAi pathway primarily involves two classes of small RNA molecules: siRNAs and miRNAs, which exhibit biogenesis and mechanistic differences. During siRNA biogenesis, dsRNA is imported into the cytoplasm, where it associates with the RNase III enzyme Dicer and the transactivating response RNA-binding protein (TRBP). Subsequently, Dicer processes the extended dsRNA into short interfering RNA fragments (Tiemann and Rossi, 2009). The siRNA duplex consists of a guide strand (antisense strand) and a passenger strand (sense strand). The short siRNA interacts with the RNA-induced silencing complex (RISC), which is composed of several proteins, such as Argonaute 2(AGO 2, the catalytic engine of RISC) (Rand et al., 2005; Marimani et al., 2013). After binding, the passenger strand is degraded, while the guide strand directs RISC to complementary target mRNA. AGO 2 then cleaves the mRNA, leading to its degradation (Carthew and Sontheimer, 2009; Purewal and Doshi, 2024). In contrast, miRNA is generated in the nucleus, where RNA polymerase II transcribes primary miRNA (pri-miRNA). Following transcription, the nuclear microprocessor complex, consisting of the RNase III enzyme Drosha and its cofactor DGCR8 (Lee et al., 2003; Han et al., 2006; Ranganathan and Sivasankar, 2014; Medley et al., 2021), processes pri-miRNA into a hairpin-structured precursor miRNA (pre-miRNA). Pre-miRNA is then exported to the cytoplasm via Exportin5, where Dicer further trims it by removing its terminal loop into a ∼22nt miRNA duplex. Similar to siRNA, the miRNA is loaded into RISC, retaining only the guide strand. However, unlike siRNA, which requires complete Watson-Crick base pairing for target recognition, miRNA binds to mRNA through partial base pairing (Carthew and Sontheimer, 2009). It mainly achieves gene silencing by inhibiting translation rather than mRNA degradation (Bobbin and Rossi, 2016).

ASOs are single-stranded DNA, RNA, or chimeric oligonucleotides composed of a dozen to dozens of bases. Through Watson-Crick base pairing, ASOs bind to complementary mRNA sequences, thereby modulating their function (Crooke et al., 2018). ASOs exert gene regulatory effects via multiple mechanisms, primarily including target mRNA degradation, translational inhibition, and splicing modulation (Halloy et al., 2022; Hui et al., 2022; Chen et al., 2024; Miao et al., 2024). Target mRNA degradation occurs through both Ribonuclease H1 (RNase H1)-dependent and RNase H1-independent pathways. In the RNase H1-dependent mechanism, ASOs bind to target mRNAs and form the ASO-mRNA complexes (Hörberg et al., 2024), which are recognized and degraded by endogenous RNase H1. Notably, RHase H1 is distributed in both the cytoplasm and the nucleus, which allows ASOs to target not only mRNAs in the cytoplasm but also pre-mRNAs in the nucleus (Liang et al., 2017). RNase H1-independent degradation may involve alternative pathways, such as Ago2-mediated mRNA degradation, which plays an important role in RNAi regulation of mRNA degradation. Translational inhibition by ASOs primarily relies on steric hindrance. Steric-hindrance ASOs demonstrate high binding affinity to target mRNAs, and while they cannot induce mRNA degradation, they effectively interfere with RNA processing and translational functions. This interference is achieved through sequence-specific masking, which obstructs critical mRNA modification processes including splicing, polyadenylation, and ribosomal engagement. For instance, ASOs targeting the 5′cap region prevent translation initiation factors (e.g., eIF-4a) and other regulatory proteins from binding to the mRNA, thereby inhibiting translation. Similarly, ASOs complementary to upstream translation initiation codons can obstruct the binding of mRNA to protein complexes (such as ribosomal subunits), further inhibiting protein synthesis (Roberts et al., 2020). In addition, ASOs can function as splice-switching oligonucleotides (SSOs) by binding to pre-mRNA and interfering with the binding of the spliceosome to the splicing site. This modulation can lead to exon skipping or inclusion, ultimately altering gene expression (Disterer et al., 2014). Based on the aforementioned mechanism, ASOs effectively inhibit HBV replication by targeting viral RNA for degradation and suppressing gene expression. Billioud et al. (Billioud et al., 2016) demonstrated through cellular and animal experiments that ASOs directed against the highly conserved X region of HBV effectively suppress HBV RNA levels in hepatocytes. Additionally, these ASOs reduced HBsAg, HBeAg, and HBV DNA levels in cell culture supernatants and animal sera.

Although siRNAs, miRNAs, and ASOs all utilize base complementation for PTGS, they exhibit several key differences. First, their origins are different. siRNAs are typically derived from exogenous dsRNAs, whereas miRNAs are encoded by endogenous genes (Mainini and Eccles, 2020). In contrast, ASOs are synthesized artificially in vitro. Second, their structural features vary. siRNAs are short dsRNAs with 2 nt overhangs at the 3′end, generally 21–25 nt in length (Herbert, 2003). miRNAs, processed from hairpin-shaped pre-miRNAs, are single-stranded RNAs (ssRNAs) of approximately 19–25 nt (Kim, 2005; Liu et al., 2022). ASOs, typically 15–25 nt in length, are single-stranded oligonucleotides, often designed as gapmers (Seth et al., 2020), a structure comprising a DNA fragment in the middle and RNA wings on both sides (Aguti et al., 2024). The gapmer design not only enhances ASO binding affinity to target mRNA but also reduces its degradation by nucleases. Third, their sites of action differ. siRNAs function in the cytoplasm, whereas miRNAs undergo nuclear processing before entering the cytoplasm. ASOs can play a role in both the nucleus and the cytoplasm. Fourth, their cellular uptake mechanisms are different. Both siRNAs and miRNAs require carrier-mediated delivery (Torres-Vanegas et al., 2021; Vaillant, 2022), whereas ASOs can enter hepatocytes in carrier-bound or free form. Binding with the carrier can increase the targeting ability to hepatocytes, reduce systemic exposure, and lower toxic side effects. Finally, ASOs generally exhibit a shorter half-life than siRNAs and miRNAs due to their mechanism of directly binding to and blocking the translation of target mRNAs. In contrast, siRNAs and miRNAs can be recycled in vivo, and the half-life can be up to several months (Revia et al., 2019).

Current challenges in achieving a functional cure for hepatitis B include the persistence of cccDNA reservoir within hepatocyte nuclei that evade complete eradication, as well as the integrated HBV DNA fragments in the host genome that sustain residual viral replication. Furthermore, chronic HBV infection induces immune tolerance and functional impairment of the host immune system, resulting in impaired host antiviral immune responses (Gane, 2020). Therefore, a functional cure necessitates dual strategies: suppression of viral replication and restoration of the host immune system. Small nucleic acid drugs exhibit unique advantages in addressing both aspects.

In terms of antiviral effects, small nucleic acid drugs exhibit a direct anti-HBV effect. These drugs are capable of degrading or silencing all viral mRNAs transcribed from cccDNA and integrated HBV DNA. This mechanism enables them to exert a potent and highly specific inhibitory action on the synthesis of HBV antigens. Concurrently, they effectively impede the replication of HBV DNA.

Simultaneously, small nucleic acid drugs have an important indirect influence on modulating the host’s immune response. In contrast to therapeutic vaccines or interferons, which directly stimulate the immune system, small nucleic acid drugs primarily function by effectively reducing the viral antigen load, particularly HBsAg. This reduction helps alleviate antigen-mediated immune suppression in the host. Prolonged exposure to high levels of viral antigens can have a detrimental impact on HBV-specific immune cells, such as T cells and B cells. Specifically, it leads to a decrease in the number and a decline in the function of virus-specific T cell clones. Although the decline rate of B cells is not as pronounced as that of T cells, the effect of antigen levels on B cells remains substantial (Wooddell et al., 2021). Small nucleic acid drugs can effectively inhibit HBV viral antigens. This not only offers an opportunity for the recovery or reconstruction of the host’s immune response but also enhances the host’s anti-HBV immune activity to a certain degree (Hui et al., 2024). Michler et al. demonstrated in mice with ongoing HBV replication that small nucleic acid drugs have immunomodulatory effects (Michler et al., 2020). They combined siRNA with a therapeutic vaccine. The results showed that siRNA effectively decreased HBsAg levels, increased the quantity and improved the function of HBV-specific CD8⁺ T cells in mice, and raised the levels of HBV-neutralizing antibodies. The “unlocking” effect of small nucleic acid drugs on the host immune system is of fundamental importance for achieving a functional cure for CHB.

Compared to small-molecule and monoclonal antibody drugs, small nucleic acid drugs exhibit superior targeting ability and specificity. They can be designed for targets previously deemed “undruggable” by the other two drugs (Crooke et al., 2021). However, the research process for small nucleic acid drugs is fraught with numerous challenges (Whitehead et al., 2009). First and foremost, maintaining the integrity and stability of small nucleic acid drugs is a formidable task. Unmodified or naked small nucleic acid drugs, particularly ASOs, have a short half-life. In the in vivo environment, they are highly susceptible to enzymatic degradation, chemical degradation (such as oxidation and hydrolysis), or clearance by the kidneys (Wittrup and Lieberman, 2015). Moreover, exogenous small nucleic acid drugs are recognized by the immune system. This recognition triggers an immune response, which can destroy the nucleic acid structure (Zhang Z. et al., 2022). In addition, the off-target effects of small nucleic acid drugs pose a significant risk. They may lead to unintended gene activation or silencing, potentially resulting in severe adverse reactions. Insufficient targeting often leads to suboptimal drug concentrations at the target tissues. To achieve the desired therapeutic effect, higher doses are required. However, increased dosages raise more safety concerns (Jackson and Linsley, 2010; Sun et al., 2024). Finally, the high molecular weight and negative charge of small nucleic acid drugs present a major obstacle. The negatively charged lipid bilayer of the cell membrane acts as a barrier, making it difficult for these drugs to penetrate. Consequently, cells struggle to take up small nucleic acid drugs effectively (Stoddard et al., 2018). Even if the drug enters the cell successfully, it faces the problem of being captured by endosomes. Once captured, it is transported to lysosomes for degradation. Only drugs that successfully escape from endosomes can become active and exert therapeutic effects (Miao et al., 2024). All these factors impede the ability of small nucleic acid drugs to reach the nucleus and act on target genes. In response to these issues, two key technologies have emerged in recent years: chemical modification and delivery systems (Friedrich and Aigner, 2022; Ali Zaidi et al., 2023), which have promoted the development and clinical application of small nucleic acid drugs.

Given that HBV mRNA plays an important role in HBV viral replication, small nucleic acid drugs capable of either degrading mRNA or impeding its translation are regarded as the most promising contenders for attaining a functional cure. At present, several small nucleic acid drugs for CHB treatment have entered Phase II/III clinical trials (Table 1). It is reasonably expected that an increasing number of small nucleic acid drugs will commence their development journey in the foreseeable future.

The goal of small nucleic acid drugs is to improve functional cure rates; however, this remains unattainable with conventional monotherapy. Although these drugs can achieve significant reductions in viral antigens, sustained clearance is rarely achieved when used alone. Future therapeutic strategies will likely require combinations of agents targeting distinct mechanisms: (1) viral replication suppression (e.g., HBV entry inhibitors (Urban et al., 2014; Kang and Syed, 2020), NAs, capsid assembly modulators (Nijampatnam and Liotta, 2019; Yang et al., 2019; Kum et al., 2023)); (2) antigen burden reduction (e.g., siRNAs, ASOs); and (3) immune response modulation (e.g., IFN-α, TLR agonists (Kayesh et al., 2021; Roca Suarez et al., 2024), therapeutic vaccines (Lang-Meli et al., 2021), monoclonal antibodies (Beretta and Mouquet, 2022), or checkpoint inhibitors (Fisicaro et al., 2018)). Long-term viral replication inhibitors may diminish the cccDNA reservoir, thereby reducing antigen load and potentially reversing immune exhaustion (Cornberg et al., 2020). Concurrently, immune modulators could amplify cellular immunity and restore pathogen-specific responses. Such combinatorial approaches are anticipated to enable durable viral suppression (Chen et al., 2007; Loglio et al., 2021; Lim et al., 2023). Currently, multiple clinical trials are underway to evaluate small nucleic acid drugs in multidrug regimens (Table 2). According to the disclosed clinical trial data, monotherapy and combination therapy show different treatment effects (Table 3).

A phase 2 clinical trial (NCT03672188) has reported on the safety and antiviral activity of VIR-2218, either used alone or in combination with pegylated (PEG) IFN-α-2a, in patients suffering from CHB (Yuen et al., 2024). The participants in this trial were allocated into six groups. They received 200 mg of subcutaneous VIR-2218 Q4W. Some of these groups also received 180 μg of subcutaneous PEG IFN-α-2a on a weekly basis, and the treatment duration extended up to 48 weeks. Throughout the entire treatment period, all participants continued to undergo nucleoside or nucleotide reverse transcriptase inhibitor (NRTI) therapy. The trial results suggest that VIR-2218, whether administered as a monotherapy or in combination with PEG IFN-α-2a, is generally well-tolerated by the patients. When comparing the use of VIR-2218 or PEG IFN-α-2a alone with their combination, it was found that the combination therapy demonstrated higher rates of HBsAg seroclearance. Moreover, with a longer treatment duration, there was a more significant mean maximum reduction in HBsAg levels from the baseline. Specifically, among the participants who received the combination treatment of VIR-2218 and PEG IFN-α-2a, 11 achieved HBsAg seroclearance at some point during the trial. Of these 11 participants, 10 showed anti-HBsAg seropositivity. Additionally, 6 participants were able to maintain HBsAg seroclearance for up to 24 weeks after the completion of the treatment. In contrast, none of the participants treated with VIR-2218 monotherapy or those on the 12-week regimen of VIR-2218 plus PEG IFN-α-2a achieved HBsAg seroclearance. Overall, the findings of this phase 2 trial lend support to the continued development of VIR-2218 as a promising therapeutic option for patients with chronic HBV infection.

A Phase IIb clinical trial (NCT03982186) was carried out to assess the efficacy and safety of the siRNA JNJ-3989 and the capsid assembly modulator JNJ-6379 (bersacapavir) in combination with NAs. The REEF-1 trial recruited CHB patients aged between 18 and 65 years who were under continuous NA treatment throughout the treatment period. These patients were then subjected to different regimens combining JNJ-3989 and JNJ-6379 (Yuen et al., 2023a). Patients were randomly allocated to one of three groups: the JNJ-6379 (250 mg) dual-therapy group, the JNJ-3989 (40/100/200 mg) dual-therapy group, and the triple-therapy group (JNJ-6379 250 mg plus JNJ-3989 100 mg). By week 48, 47 patients fulfilled the criteria for discontinuing NA therapy. The highest proportion of patients meeting these criteria was observed in the JNJ-3989 (200mg) dual-therapy group, reaching 19%. The decline in HBsAg levels was dose-dependent on JNJ-3989. In the JNJ-3989 (200mg) dual-therapy group, as many as 75% of patients achieved HBsAg levels below 100 IU/mL. Although the mean HBsAg concentrations in all groups containing JNJ-3989 gradually rose during the follow-up period, their mean reductions from the baseline values at week 24 still exceeded 1 log₁₀ IU/mL. Ultimately, the researchers inferred that the antiviral treatment combinations employed were inadequate to attain a functional cure for CHB. Nevertheless, a significant decrease in HBsAg levels was witnessed in most patients treated with JNJ-3989, which might offer a solid basis for immune modulation. The REEF-2 trial is a Phase IIb, double-blind, placebo-controlled, randomized study (NCT04129554) that involved 130 HBeAg-negative CHB patients on NA therapy (Agarwal et al., 2024). The patients were randomly assigned to either the treatment group (receiving subcutaneous JNJ-3989 at 200 mg every 4 weeks, oral JNJ-6379 at 250 mg once daily, and oral NA once daily) or the control group (receiving oral NA once daily). The treatment lasted for 48 weeks, followed by a 48-week follow-up period. After 48 weeks of treatment, 71.1% of patients in the treatment group achieved an HBsAg level of less than 100 IU/mL, with a group mean HBsAg reduction of 1.89 log₁₀ IU/mL. At week 48 of the follow-up, 46.9% of patients in the treatment group still maintained HBsAg levels below 100 IU/mL, with a group mean HBsAg reduction of 1.46 log₁₀ IU/mL. Although the combined treatment regimen failed to achieve a functional cure ultimately, it significantly lowered HBsAg levels, increased the proportion of patients with suppressed HBV DNA, and led to fewer and less severe post-treatment HBV DNA rebounds and ALT flares. These outcomes are crucial steps towards achieving a functional cure for CHB.

As our understanding of the HBV replication process and the host’s immune response deepens, remarkable advancements have been made in the treatment of CHB, uncovering a plethora of potential therapeutic targets. Nevertheless, currently, only specific patient cohorts can attain high functional cure rates through NAs and PEG IFN-α therapy. For patients with high baseline HBsAg levels, monotherapy with PEG IFN-α or its combination with NAs still faces considerable hurdles in achieving a cure. The intricate HBV replication cycle, the persistent presence of cccDNA within the host cell nucleus, and the continuous synthesis of HBsAg from the integrated HBV DNA in the host genome present formidable challenges to attaining a functional cure for CHB. Moreover, the host’s immune response exerts a profound influence on treatment outcomes. With the rapid development of chemical modification and liver-targeted delivery systems, small nucleic acid drugs are now regarded as the most promising candidates for achieving a functional cure. Owing to their distinctive mechanism of action, these drugs can interfere with the transcription process of HBV cccDNA, rendering the cccDNA in a “silent” state, and thus potentially leading to a functional cure for hepatitis B. They can also precisely target HBV RNA, effectively suppress viral replication, and lower viral antigen levels, thereby breaking immune tolerance. Additionally, small nucleic acid drugs possess high target specificity. They can be rationally designed according to the target gene sequences, and multi-target siRNA cocktail therapy can significantly mitigate the risk of drug resistance. Nonetheless, several issues remain to be resolved in the future, such as how to maintain the in vivo stability of small nucleic acid drugs, avoid immune reactions, and minimize off-target effects. Small nucleic acid drugs are anticipated to play a key role in the next era of CHB treatment. Given the difficulty of achieving a functional cure through monotherapy, future treatment strategies will likely involve combination therapies. The ability of small nucleic acid drugs to efficiently inhibit viral replication and reduce antigen levels makes them the foundation of combination therapy. They can be used in combination with other antiviral drugs and immunomodulators to reach the ultimate goal. However, further research is required to determine the optimal timing of combination therapy, the sequence of different drugs, the duration of treatment, and personalized treatment approaches. Based on the current clinical and pre-clinical data for small nucleic acid drugs, as well as the emergence of innovative drugs and combination therapies, it is believed that this emerging therapeutic strategy will further boost the cure rate of CHB, decrease the risk of cirrhosis and hepatocellular carcinoma, and bring the dawn of a functional cure for CHB patients worldwide.