Description

The Problem

Hepatitis B is the most common serious liver infection in the world — and yet, one of the most overlooked. According to the World Health Organization, over 254 million people are living with chronic HBV infection globally. Caused by a small DNA virus that targets the liver, HBV spreads silently and slowly, often showing no symptoms until it has already caused irreversible liver damage such as cirrhosis or hepatocellular carcinoma — one of the deadliest forms of cancer. In 2022 alone, 1.1 million people died from hepatitis B-related complications.

Despite the existence of a safe and effective vaccine, the virus continues to spread — especially in regions with limited access to medical infrastructure. But vaccination only protects the uninfected. What about the 254 million people already living with chronic hepatitis B?

For them, there is no cure. Current treatments can suppress the virus, but they cannot eliminate it. HBV hides inside liver cells in the form of cccDNA, which allows the infection to persist for life and evade most available therapies.

Because of this, hepatitis B has been called a "forgotten pandemic" — a global crisis that persists in silence. Without intervention, 1 in 4 chronically infected individuals will eventually die from liver failure or cancer.

Hepatitis B is not just an infection. It is a problem of access, of awareness, of inequality — and, most of all, of time. Every 30 seconds, someone dies from it.

Why Current Solutions Are Not Enough

Today's standard treatments for chronic hepatitis B — like tenofovir and entecavir — help control the virus but fall far short of a cure. These drugs suppress viral replication and reduce the risk of liver damage, but they do not eliminate the cccDNA reservoir that allows the virus to persist inside liver cells.

As a result, patients require lifelong therapy, facing years or decades of daily medication — and the current options come with significant limitations.

Tenofovir, in particular, has been associated with both renal toxicity and loss of bone mineral density (BMD), especially with long-term use. Although these side effects are widely recognized in older patients or those at elevated clinical risk, recent studies have shown that younger patients with chronic hepatitis B may be more vulnerable to these aftereffects. However, younger patients are often overlooked in risk assessments and treatment decisions, due to the prevailing assumption that they are less vulnerable to such complications and the absence of early screening for subclinical bone or kidney changes in this age group. In a 96-week study of 154 patients on tenofovir monotherapy (median age 36.75), 35.1% developed elevated urinary β2-microglobulin, indicating proximal tubular dysfunction, and 20.1% showed reduced hip BMD. Notably, even among those with normal baseline BMD, sustained tenofovir use led to measurable decreases in bone density and renal function over time.

Entecavir is generally considered well-tolerated and has a high barrier to resistance. That said, long-term treatment with entecavir still presents notable limitations. Clinical studies have reported rare but serious side effects, including lactic acidosis and mitochondrial toxicity. In addition, while genotypic resistance to entecavir remains relatively uncommon, the risk increases marginally with prolonged therapy, particularly in patients with poor adherence or high baseline viral load. In one 48-week study of 227 patients, virologic breakthrough occurred in 2% of patients by week 96 and 6% by week 144. Among patients with partial virologic response (PVR) and a high viral load at week 48, the cumulative rate of breakthrough rose dramatically to 28.4% by week 144 — nearly five times higher than average — highlighting a vulnerable subgroup. Although genotypic resistance remained rare overall, this study challenges the long-held assumption that resistance under entecavir therapy is negligible. This shows that even without detectable mutations, the virus can resurface — especially if treatment doesn't fully suppress it or if patients struggle to follow the regimen — highlighting the need for close monitoring and tailored care.

Moreover, lifelong daily therapy poses a significant burden. While the price of antiviral medications has decreased substantially — with generic tenofovir dropping from $208 in 2004 to just $32 in 2016 — the overall cost of care remains a challenge in many low- and middle-income countries (LMICs). In 2017, nearly all LMICs were legally permitted to procure generic versions of entecavir and tenofovir, yet global treatment coverage remained strikingly low. According to WHO estimates, only about 17% of diagnosed individuals were receiving treatment in 2016, and just 16.7% of the estimated 27 million people aware of their HBV infection were on therapy. Access to these drugs is limited not only by cost, but also by gaps in diagnostic infrastructure, shortages of trained healthcare workers, and insufficient public awareness.

Many of the countries with the highest burden of HBV infection — including those in sub-Saharan Africa — are the least equipped to support long-term treatment access. Even in countries with strong economies, like China, access to antiviral therapy for HBV was limited until recent years. Before the inclusion of antivirals into public insurance schemes and substantial price reductions, the cost of treatment posed a serious barrier, particularly in rural and underinsured populations.

Finally, and most critically, none of these treatments eliminate the cccDNA reservoir in infected liver cells — meaning that the virus can rebound if therapy is paused. There is currently no functional cure for chronic HBV, and patients remain on therapy indefinitely.

Societal Relevance

China bears the largest burden of hepatitis B virus (HBV) infection globally, accounting for approximately one-third of all chronic HBV cases. According to a 2024 national epidemiological review, around 75 million people in China are chronically infected with HBV — a prevalence of 5.86%, which is significantly higher than the global average of 3.2%.

Despite significant progress in reducing new infections through universal neonatal vaccination — with three-dose coverage at 99.6% and timely birth dose coverage at 95.6% — the burden of chronic infections remains high, particularly in older cohorts born before immunization programs were established (Yan et al., 2025). In 2020, HBV led to more than 380,000 liver cancer-related deaths in China, representing over 30% of global HBV mortality (WHO Global Hepatitis Report 2024).

One of the biggest challenges in China's HBV response is the gap in diagnosis and treatment. As of 2020, only 58.78% of HBsAg-positive individuals were aware of their infection, and just 17.33% of those eligible were receiving antiviral therapy (Yan et al., 2025). That means at least 30 million people remain undiagnosed and ~14 million are untreated despite medical eligibility. These numbers fall well short of the WHO's 2030 targets of 90% diagnosis and 80% treatment coverage.

HBV prevalence is unevenly distributed across regions, with higher rates found in rural and resource-limited provinces such as Tibet and parts of western China. This regional disparity reflects differences in healthcare access, screening infrastructure, and public awareness.

A major obstacle in China's HBV response is the continued virus transmission despite vaccination programs. Mother-to-child transmission (MTCT) remains critical: transmission rates among HBV-exposed infants still range from 3–5%, resulting in approximately 50,000 new pediatric infections annually (Qiao et al., 2019).

In addition to vertical transmission, unsafe medical, cosmetic, and body-modification procedures contribute significantly to HBV spread. Studies have identified reused or improperly sterilized needles, tattoos, ear piercings, pedicures, and cosmetic body care services —especially in rural areas—as independent risk factors for acute HBV infections (Yan YP et al., 2014). Though vaccination has reduced blood-borne spread substantially, cultural norms and limited oversight in beauty clinics and personal service sectors still pose significant risks.

Finally, the socioeconomic and geographic inequalities in access to diagnosis and treatment services must be addressed to make elimination feasible. While the price of first-line antiviral drugs has dropped significantly through national procurement programs (e.g., tenofovir now costs <10 CNY/month), uptake is still limited by lack of awareness, stigma, and systemic barriers.

Our Solution

Persistent hepatitis B virus (HBV) infection is maintained by a stable, episomal DNA form known as covalently closed circular DNA (cccDNA), which resides in the nucleus of hepatocytes and resists current therapies. Our project proposes a novel strategy to selectively suppress transcriptional activity from this reservoir using a dual-guide RNA system designed to enhance CRISPR-dCas targeting precision. By reducing off-target effects, this approach aims to enable safer and more effective epigenetic silencing of HBV cccDNA.

To implement this strategy, we will design and screen RNA sequences that promote stable dimerization of two dCas-bound gRNAs. Several well-characterized RNA dimerization domains are known to form kissing-loop interactions and will serve as a basis for our constructs. We will employ a genetic selection platform based on the Ptet/TetR regulatory system to identify effective RNA-RNA interacting gRNA pairs. This screening system will rely on a reporter circuit where cooperative binding of two dCas complexes induces DNA looping, thereby repressing expression of the TetR repressor. Repression of TetR in turn activates a pTet-controlled reporter gene, allowing us to quantify interaction efficiency.

As CRISPR targets, we will use DNA fragments from the HBV genome that exhibit the highest rates of cytosine-to-thymine (C-to-T) or guanine-to-adenine (G-to-A) deamination conversion. These regions will be inserted into the modified promoter driving TetR expression, ensuring that only highly cooperative RNA-RNA complexes capable of stable dCas positioning on HBV DNA will trigger reporter activation. To enhance screening robustness, we supplemented the library of known kissing-loop variants with randomized sequences at the 3' end of the gRNA, enabling the discovery of novel RNA–RNA interaction motifs.

Candidate gRNA pairs selected through the genetic screen will subsequently be validated in vitro using minicircle-derived recombined cccDNA (rcccDNA), which closely mimics native HBV cccDNA in both structure and function. This model provides an accurate and scalable platform to test CRISPR interference or epigenetic silencing effects on persistent HBV templates.

Together, this system increased the specificity of epigenetic editing platforms by over an order of magnitude compared to standard dCas-based designs, offering a powerful new approach for functional suppression of cccDNA without introducing double-stranded breaks.

Objectives

In our project, we aim to develop a strategy for suppressing the expression of HBV genes transcribed from covalently closed circular DNA (cccDNA) — the persistent viral reservoir — in order to block the production and replication of viral particles using a CRISPR-based approach. Rather than simply cutting the viral genome, we aimed to build a system capable of turning off its transcriptional activity at the epigenetic level.

• Engineering a cooperative dual-guide RNA system.

We engineered a dual-guide RNA system capable of bringing two dCas proteins together through programmed RNA–RNA interactions. This design aimed to achieve high targeting precision through molecular cooperation rather than DNA cleavage.

• Developing a functional selection platform.

To identify the most effective gRNA pairs, we constructed a genetic selection system based on the Ptet/TetR regulatory circuit. This platform converts successful RNA–RNA dimerization into a measurable output, allowing us to screen for the most stable and specific interactions directly in living cells.

• Validating selected gRNA pairs in vitro.

The top-performing candidates from the screen were validated using a minicircle-derived recombined cccDNA (mcccDNA) model, which faithfully reproduces the structure and behavior of native cccDNA. This setup allowed us to assess the efficiency of CRISPR interference and epigenetic silencing under realistic conditions.

• Demonstrating functional suppression of cccDNA.

Through this iterative process, we aimed to show that precise RNA-guided cooperation can be engineered into CRISPR systems to achieve long-term transcriptional suppression of HBV cccDNA without introducing double-strand breaks.