Wednesday, May 1, 2024
5/1/2024
SUMMARY - Engineered exosomes carrying synthetic gRNA/Cas9 targeting HBV-infected cells Despite universal vaccinations against hepatitis B virus (HBV), chronic HBV infection remains a public health threat in the United States and worldwide. The current antiviral treatments, using various nucleos(t)ide analogs (NAs), can block the HBV life cycle but cannot eliminate integrated HBV DNA and have little effect on HBV covalently closed circular DNA (cccDNA), which sustains viral replication. Thus, novel curative strategies are urgently needed to eliminate HBV cccDNA from infected hepatocytes. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated gene-editing is an appealing approach to tackle this problem. However, the major hurdle in the application of this technology is how to deliver gene-editing drugs to target cells and elicit specific antiviral activities without causing off-target effects. Notably, the current CRISPR/Cas9 delivery technologies often require viral vectors, which pose safety concerns for therapeutic application in humans. Synthetic guide RNA (gRNA)/Cas9 ribonucleoproteins (RNPs) represent a novel non-viral formula with excellent features, including rapid DNA cleavage activity, low off-target effects, no risk of insertional mutagenesis, easy production, and readiness for clinical use. We have designed and tested a series of gRNA/Cas9 gene-editing RNPs targeting HBV and selected the most specific and potent gRNA/Cas9 RNP candidates to abolish HBV replication in HBV cellular models. However, the existing viral and non-viral delivery systems for gRNA/Cas9 RNPs in vivo use face several challenges, such as non-specific (off-site) delivery, limited drug-loading capacity, low biocompatibility, poor stability, cytotoxicity, and potential of immunogenicity. These challenges are major bottlenecks, limiting the use of synthetic gRNA/Cas9 RNPs for in vivo applications. To address these limitations, we developed a novel exosome- based delivery platform engineered to deliver the HBV gene-editing RNPs specifically to human hepatocytes. These engineered exosomes are designed in such a way that they carry an HBV pre-S1-derived peptide (binding to HBV receptor) on the surface of exosomes so that they can more specifically deliver and intracellularly release our synthetic gRNA/Cas9 RNPs to HBV target cells. In this proposal, we aim to evaluate the capability of these exosome- based HBV gene-editing gRNA/Cas9 RNPs (herein called Exo-HBV-Eliminator) in targeting HBV-infected hepatocytes and investigate their antiviral efficacy and potential cytotoxicity using both cellular and animal models. We hypothesize that our Exo-HBV-Eliminator will specifically target HBV DNA and thus efficiently abolish HBV replication and elicit minimal cytotoxic effects both in vitro and in vivo. We propose two specific aims to test our hypothesis. Aim 1 will determine the biophysical and biological properties of our Exo-HBV-Eliminator in vitro. Aim 2 will evaluate the antiviral and off-target effects of our Exo-HBV-Eliminator in HBV-infected, liver-humanized mice in vivo. The objective of this study is to develop and test a novel gene therapy (engineered exosomes carrying synthetic gRNA/Cas9 RNPs) targeting HBV cccDNA in HBV-infected hepatocytes that are incurable by the current antiviral regimens, to lay the foundation for achieving our long-term goal of curing chronic HBV infection.
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