# Chemically ligated-guide RNA (lgRNA)-based CRISPR/Cas9 gene editing for elimination of hepatitis B virus cccDNA

> **NIH NIH R21** · BARUCH S. BLUMBERG INSTITUTE · 2024 · $240,000

## Abstract

Abstract
This R21/R33 application is to develop our proprietary chemically ligated guide RNA (lgRNA)-based
CRISPR/Cas9 gene editing technology for therapeutic elimination of hepatitis B virus (HBV) covalently closed
circular DNA (cccDNA) and cure of chronic hepatitis B (CHB). Prior studies have already demonstrated in
hepatocyte cultures and in mice models that HBV cccDNA can be successfully edited by several clustered
regularly interspaced short palindromic repeats (CRISPR) gene editing technologies. Apparently,
achievement of CHB cure requires elimination and/or inactivation of the vast majority of cccDNA, if not all, in
the liver to allow the ultimate clearance or immune control of residual HBV infection. However, due to the
relatively low in vivo editing efficiency, multiple doses of current candidate CRISPR/Cas therapeutics under
preclinical development are most likely required for significant reduction of cccDNA pool in the liver, which
may be limited by the immunogenicity of the CRISPR ribonucleoprotein (RNP) complexes and of their delivery
vehicles, such as adeno-associated viruses (AAV). To improve the gene editing efficiency, accuracy and
durability, we are focusing on the chemical optimization of guide RNA. Particularly, for robust, convergent,
and scalable chemical synthesis and chemical modification of guide RNA, instead to synthesize a full-length
single guide RNA (sgRNA), the guide RNA was synthesized through ligation of two or three short RNA
segments via non-phosphoramidite chemistry, i.e., chemically ligated guide RNA (lgRNA). This new
technology not only makes the manufacture of long RNAs cost-effective but also gives access to high-quality
validated full-length products with much fewer synthetic errors at the critical spacer segment than classic
sgRNA. Obviously, it enables cost-effective global chemical modifications for better efficacy, selectivity and
stability as well as targeted delivery by molecular tagging and various formulation technologies. Thus far, we
have already developed state-of-the-art chemical methods for the synthesis of lgRNAs that support efficient
cleavage of target DNA in vitro by Cas9 and edit HBV cccDNA as well as integrated HBV DNA in human
hepatoma cells supporting HBV replication and gene expression. In R21 phase, we will further chemically
optimize lgRNA and identify at least three lgRNAs that can efficiently edit cccDNA in human hepatoma cells.
In R33 phase, Cas9 mRNA and lgRNA will be co-formulated into lipid nanoparticles (LNP) and their efficiency
on cccDNA editing and viral gene expression will be evaluated in HBV infected hepatoma cells and primary
human hepatocytes. The therapeutic efficacy and durability of optimized Cas9 mRNA-lgRNA LNP on HBV
infection will be evaluated in HBV infected FRG-human hepatocyte chimeric mice model, alone or in
combination with a HBV DNA polymerase inhibitor. Successful completion of the proposed work should well
position the candidate therapeutics for further p...

## Key facts

- **NIH application ID:** 10785441
- **Project number:** 1R21AI179930-01
- **Recipient organization:** BARUCH S. BLUMBERG INSTITUTE
- **Principal Investigator:** Ju-Tao Guo
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $240,000
- **Award type:** 1
- **Project period:** 2024-01-01 → 2025-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10785441

## Citation

> US National Institutes of Health, RePORTER application 10785441, Chemically ligated-guide RNA (lgRNA)-based CRISPR/Cas9 gene editing for elimination of hepatitis B virus cccDNA (1R21AI179930-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10785441. Licensed CC0.

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