Sunday, May 18, 2025
5/18/2025
Engineering exosomes for new gRNA/Cas therapeutics to eliminate HBV infection - SUMMARY – Engineering exosomes for new gRNA/Cas therapeutics to eliminate HBV infection Chronic hepatitis B virus (HBV) infection is a common public health problem 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 reservoir cells. CRISPR/Cas-mediated gene-editing is an appealing approach to tackle this problem. However, major hurdles in the application of this technology lie in selecting the most potent guide RNAs (gRNAs) to form a specific therapeutic regimen and delivering CRISPR/Cas therapeutics to the target cell, to elicit on-target gene-editing without causing off-target effects. Compared to Cas9 which often requires 2 or 3 gRNAs to avoid viral escape/resistance, Cas12 is a programmable and more potent DNA endonuclease and only requires a single gRNA for targeted gene-editing. Unlike Cas9 and Cas12 which primarily edit DNA, Cas13 edits RNA and can be used together with Cas9 and Cas12, for both DNA and RNA targeting. Additionally, the current CRISPR/Cas expression and delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. Synthetic ribonucleoproteins (RNPs) are a novel non-viral formula with excellent features, including rapid DNA cleavage activity, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for clinical use. We have designed and tested a series of gRNA/Cas9 gene-editing drugs targeting HBV cccDNA and selected the most specific and potent gRNA/Cas9 candidates to abolish HBV replication in HBV cellular models. We have also developed a novel exosome-based delivery platform engineered to specifically deliver these HBV gene-editing drugs to human hepatocytes. These engineered exosomes are designed to carry an HBsAg pre-S1-derived peptide (binds to HBV receptor) on the surface of exosomes so that they can specifically deliver our gRNA/Cas therapeutics to HBV reservoir cells. In this proposal, we will compare the capacity and specificity of gRNA/Cas12 and gRNA/Cas13 (with gRNA/Cas9 as a positive control) to eliminate HBV infection in HBV cellular models to select the most potent gRNA/Cas regimen in vitro (R21 phase). Then, we will evaluate the capability of our engineered exosomes to deliver our HBV gene-editing gRNA/Cas therapeutic (herein called Exo-HBV-Eliminator) for eliminating HBV infection using an HBV-infected, liver-humanized animal model (R33 phase). We hypothesize that our Exo-HBV-gRNA/Cas therapeutics will specifically and efficiently eliminate HBV infection and elicit minimal cytotoxic effects both in vitro and in vivo. We propose two specific aims to test our hypothesis. Aim 1 will select the most specific and potent gRNA/Cas9/12/13 candidates and test a combination regimen in vitro. Aim 2 will evaluate the antiviral efficacy and off-target effects of the Exo-HBV-Eliminator in HBV-infected, liver-humanized mice in vivo. The objective of this study is to develop a novel gene therapy capable of targeting HBV cccDNA, which is incurable by the current NA treatment, and thus this research will lay the foundation for achieving our long-term goal of curing chronic HBV infection.
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