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Principal Investigator: Ju-Tao Guo
Organization: BARUCH S. BLUMBERG INSTITUTE
Fiscal Year: 2024
Award: $240,000
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <Achievement><Achievement Attainment><Address><Adeno-Associated Viruses><Anti-viral Agents><Assay><Binding><Bioassay><Biological Assay><CRISPR><CRISPR/Cas system><Candidate Disease Gene><Candidate Gene><Cell Nucleus><Cessation of life><Chemicals><Chemistry><Chromatin><Chronic><Chronic Hepatitis B><Circular DNA><Clinical Treatment><Clinical Trials><Closure by Ligation><Clustered Regularly Interspaced Short Palindromic Repeats><Complex><DNA><DNA Integration><DNA Polymerase Inhibitor><DNA Replication><DNA Sequence><DNA Synthesis><DNA biosynthesis><Death><Deoxyribonucleic Acid><Dependoparvovirus><Dependovirus><Detection><Dose><Drug Targeting><Drugs><Evaluation><FDA approved><Formulation><Gene Expression><Genes><Goals><Guide RNA><HBV><Hepatic Cells><Hepatic Parenchymal Cell><Hepatitis B Virus><Hepatocyte><Homologous Serum Hepatitis Virus><Human><Immune><Immunes><In Vitro><Laboratories><Length><Ligation><Liver><Liver Cells><Medication><Messenger RNA><Metabolic><Methods><Modern Man><Modification><Molecular><Molecular Interaction><Non-Polyadenylated RNA><Nucleus><PEG-IFN-a><PEG-interferon alfa><Pathology><Pegylated Interferon Alfa><Persons><Pharmaceutical Preparations><Phase><Position><Positioning Attribute><RNA><RNA Gene Products><Regimen><Residual><Residual state><Ribonucleic Acid><Ribonucleoproteins><Role><Specific qualifier value><Specified><Structure><Supporting Cell><Technology><Therapeutic><Therapeutic Gene Editing><Transcription Regulation><Transcriptional Control><Transcriptional Regulation><Treatment Efficacy><Viral><Viral Burden><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Genes><Viral Genome><Viral Load><Viral Load result><Viral Proteins><Virus Diseases><Virus Replication><Work><adeno associated virus group><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><chemical synthesis><chronic HBV infection><chronic hepatitis B virus infection><chronic infection><clinical development><cost effective><delivery vector><delivery vehicle><determine efficacy><develop therapy><drug discovery><drug/agent><efficacy analysis><efficacy assessment><efficacy determination><efficacy evaluation><efficacy examination><evaluate efficacy><examine efficacy><gRNA><gene editing platform><gene editing system><gene editing technology><gene editing tools><gene-editing therapy><gene-editing toolkit><genome editing based therapy><genome editing therapy><genome editing treatment><genome editing-based therapeutics><hepatic body system><hepatic organ system><hepatoma cell><immunogenicity><improved><in vivo><intervention development><intervention efficacy><lipid based nanoparticle><lipid nanoparticle><mRNA><manufacture><mouse model><murine model><new technology><novel technologies><persistent infection><pre-clinical><pre-clinical development><preclinical><preclinical development><site targeted delivery><social role><standard of care><targeted delivery><therapeutic candidate><therapeutic editing><therapeutic efficacy><therapeutic evaluation><therapeutic genome editing><therapeutic testing><therapy development><therapy efficacy><timeline><treatment development><trial regimen><trial treatment><viral DNA><viral infection><viral multiplication><viral replication><virus DNA><virus genome><virus infection><virus multiplication><virus protein><virus-induced disease>