# Engineering exosomes for new gRNA/Cas therapeutics to  eliminate HBV infection

> **NIH NIH R21** · EAST TENNESSEE STATE UNIVERSITY · 2024 · $225,000

## Abstract

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...

## Key facts

- **NIH application ID:** 10783166
- **Project number:** 1R21AI179794-01
- **Recipient organization:** EAST TENNESSEE STATE UNIVERSITY
- **Principal Investigator:** Zhi Q. Yao
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $225,000
- **Award type:** 1
- **Project period:** 2024-01-01 → 2025-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10783166

## Citation

> US National Institutes of Health, RePORTER application 10783166, Engineering exosomes for new gRNA/Cas therapeutics to  eliminate HBV infection (1R21AI179794-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10783166. Licensed CC0.

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