Chemically ligated-guide RNA (lgRNA)-based CRISPR/Cas9 gene editing for elimination of hepatitis B virus cccDNA - 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 preclinical/clinical development for treatment of CHB.
