RNA interference (RNAi) technology has shown great potential in the treatment of genetic diseases and viral infections. It is also considered an attractive therapeutic approach to achieve functional cure of hepatitis B by inducing antigen suppression, reducing viremia, and silencing covalently closed circular DNA (cccDNA). siRNA-based therapies can also alleviate immune tolerance induced by high viral antigens, providing opportunities for subsequent immune stimulation to gain immune control of the virus.
However, designing functional siRNAs for complex viruses like HBV remains challenging due to the extremely high genetic diversity among the 10 different genotypes of HBV. It is feasible to design siRNAs targeting conserved regions among HBV genotypes through computational prediction, which is expected to resist potential viral mutation escape. However, siRNAs targeting common 3'-end sequences in cccDNA-derived transcripts may lose their targets for intHBV-derived transcripts. Further research is expected to use siRNA triggers targeting cccDNA and intHBV-driven synthesis to surpass previous drugs in terms of efficacy and functionality.
The clinical application of RNAi therapy is hampered by the challenges posed by liver-targeted delivery systems. With the development of novel GalNAc technology and lipid nanoparticles (LNPs), siRNA-based drugs such as Partisiran and Inclisiran have been approved for clinical use.
Lipid nanoparticles (LNPs) are usually composed of ionizable cationic lipids, cholesterol, PEG lipids, and auxiliary lipids. PEGylated lipids provide a neutral hydrophilic outer layer, stabilize the nanoparticles, and prevent rapid clearance in the blood circulation. However, there is also a "PEG dilemma", and the PEG ratio affects the performance of LNPs in vivo. Although PEG has weak immunogenicity, some people may still produce low levels of PEG-specific antibodies, resulting in accelerated clearance of PEGylated nanomedicines and reduced efficacy.
Recent studies have found that hydroxy-PEG has weaker antigenicity and can avoid binding to anti-PEG antibodies present in human blood, thereby reducing complement activation and enabling LNP to escape antibody recognition and rapid clearance. Although RNAi and antibody-mediated HBsAg clearance can eliminate immune dysfunction by reducing HBV viral load, they cannot activate virus-specific T cells to achieve long-term viral control. Clinically, only a few patients produce anti-HBsAg antibodies, and viral antigens will recover after drug discontinuation.
Recently, some preclinical and clinical studies are exploring new combination strategies to achieve better therapeutic effects, such as combining RNAi therapy with therapeutic vaccines, interferon and anti-HBs antibodies. Recently, researchers from Fudan University in China and others published a research paper entitled "Optimized RNA interference therapeutics combined with interleukin-2 mRNA for treating hepatitis B virus infection" in the journal Signal Transduction and Targeted Therapy.
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Studies have shown that IL-2 is a key regulator of appropriate viral antigen presentation and activation and differentiation of virus-specific CD4+ and CD8+ T cells. Sequential low-dose IL-2 combined with IFN-α increased the frequency and restored the function of HBV-specific CD8+T cell responses in patients, suggesting the potential for building virus-specific immune control to complement RNAi.
The research team believes that suppressing HBV viral load through RNAi therapy and using IL-2 to enhance host immunity may be sufficient to break immune tolerance and rebuild antiviral immunity. The research team screened and chemically modified a pan-genotypic, multifunctional siRNA combination (siHBV) that targets all forms of cccDNA and intHBV-derived transcripts and verified its efficacy and safety in multiple cell culture and mouse models. Using HO-PEG2000-DMG lipids and optimizing the molar ratio of traditional PEGylated lipids in LNP formulations, they developed an optimized LNP platform, tLNP, which is low antigenic and highly efficient, to determine the role of siHBV in controlling HBV transcription and replication and evaluate its safety characteristics.
Figure 1. Schematics of the preparation procedures of LNPs. (Zai W, et al., 2024)
Taking advantage of the feasibility of tLNP encapsulation of different forms of nucleic acids, the research team co-encapsulated siHBV and mouse IL-2 (mIL-2) mRNA in a single tLNP formulation - tLNP/siHBVIL2, to achieve both antigenic and immune control of the virus. It is expected that the combined delivery of siHBV and mIL-2 mRNA based on tLNP may provide a feasible method for the treatment of chronic hepatitis B.
Figure 2. Additive antiviral efficacy of tLNP/siHBVIL2 in rAAV-HBV1.3 mouse model. (Zai W, et al., 2024)
The experimental results showed that tLNP/siHBV significantly reduced the expression of HBV viral antigens and DNA, and was dose- and time-dependent at single or multiple dosing frequencies, with good safety. tLNP/siHBVIL2 introduced a strong HBsAg clearance ability through RNAi technology, and triggered a strong HBV-specific CD4+ and CD8+ T cell response by expressing mIL-2 protein, thereby achieving additive antigenicity and immune control of HBV. These results suggest that the use of tLNPs as nucleic acid nanocarriers for the coordinated delivery of siHBV and mIL-2 mRNA can achieve synergistic control of HBV antigens and immunity, providing a promising translational therapeutic strategy for the treatment of chronic hepatitis B.
Reference
Zai W, et al. Optimized RNA interference therapeutics combined with interleukin-2 mRNA for treating hepatitis B virus infection. Signal Transduction and Targeted Therapy, 2024, 9(1): 150.