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Principal Investigator: Ju-Tao Guo
Organization: GENELANCET BIOSCIENCES INC
Fiscal Year: 2022
Award: $300,000
Funding agency: National Institute of Allergy and Infectious Diseases
Abstract
This phase 1 SBIR project is to investigate the feasibility of an innovative CRISPR-Cas9 scar-less editing
technology to permanently inactivate both hepatitis B virus (HBV) covalently closed circular DNA (cccDNA)
and integrated DNA for the cure of chronic hepatitis B (CHB). Although the currently available antiviral agents,
including nucleos(t)ide analogue viral DNA polymerase inhibitors and pegylated alpha-interferon, can
efficiently inhibit HBV replication and prevent disease progression in the majority of treated patients, the cure
of chronic HBV infection is rarely achieved and life-long antiviral therapy is thus required. The failure of a cure
is due to the current antiviral regimens cannot eliminate cccDNA from the nuclei of infected hepatocytes.
cccDNA, the transcription template of viral RNA, is the most stable HBV replication intermediate and the
resource of viral replication rebound after disruption of antiviral therapies. Moreover, despite not being
essential for viral replication, the transcripts from integrated HBV DNA in cellular chromosomes have recently
been proven to support the secretion of majority of HBV surface antigen (HBsAg) in HBeAg-negative CHB
patients. Prolonged excessive expression of HBsAg induces the exhaustion of viral antigen-specific T and B
cells and favors the persistent infection of HBV. Apparently, elimination or permanent inactivation of cccDNA
as well as integrated HBV DNA is essential to achieve the cure of CHB. CRISPR-Cas9 gene editing
technology is thus far the most promising approach to achieve this therapeutic goal. However, although the
classic CRISPR-Cas9 gene editing technologies had been proven to cleave and edit cccDNA in cultured cells
and animal models, their cleavage of integrated HBV DNA may lead to chromosome break and unintended
mutations, causing genetic instability and genotoxicity. Recently, CRISPR-Cas9 base editor technology had
been developed to overcome this limitation. Unfortunately, the low editing efficiency, high rate of guide-
independent editing and usage of lentiviral vector for delivery hampered its further development. In this
project, we propose to develop our proprietary ligated-guide RNA (lgRNA)-based STAR (Seek-Tag-Amend-
Release) editor technology for efficient and accurate inactivation of both forms of nuclear HBV DNA by
introducing stop codons into overlapping HBsAg and viral DNA polymerase genes. Specifically, we will first
verify the cleavage activity and specificity of STAR editors in vitro to optimize the structures of lgRNA as well
as the conjugation sites of single strand DNA (ssDNA) (the template of editing) (Aim 1). We will then evaluate
the editing efficiency and specificity of STAR editors in human hepatoma cell line harboring both integrated
HBV DNA and cccDNA (Aim 2). Successful completion of this Phase 1 project will enable us to apply for a
Phase 2 study to evaluate selected multiplexing STAR editors for their inactivation activity of integrated HBV
DNA in HBV transgenic mice and cccDNA in HBV infected humanized uPA-SCID mice in vivo.
Terms: <(IFN) α><(IFN)-α><(IFN)α><Address><Affect><Alferon><Animal Model><Animal Models and Related Studies><Antiviral Agents><Antiviral Drugs><Antiviral Therapy><Antivirals><Assay><B blood cells><B cell><B cells><B-Cells><B-Lymphocytes><B-cell><Binding><Bioassay><Biochemical><Biologic Assays><Biological Assay><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Cas nuclease technology><Cell Body><Cell Line><Cell Nucleus><Cell Surface Antigens><Cell model><CellLine><Cells><Cellular model><Cessation of life><Chemicals><Chemistry><Chromatin><Chromosomal Breaks><Chromosome Break><Chromosomes><Chronic><Chronic Hepatitis B><Cicatrix><Circular DNA><Clinical Research><Clinical Study><Clinical Trials><Closure by Ligation><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Cultured Cells><DNA><DNA Damage Repair><DNA Double Strand Break><DNA Polymerase Inhibitor><DNA Repair><DNA Replication><DNA Sequence><DNA Synthesis><DNA biosynthesis><Death><Deoxyribonucleic Acid><Development><Disease><Disease Progression><Disorder><Drugs><FDA approved><Failure><Formulation><Gene Transcription><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Genome><Goals><Grant><Guide RNA><HBV><HBeAg><Hepatic Cells><Hepatic Parenchymal Cell><Hepatitis B><Hepatitis B Virus><Hepatitis B e Antigens><Hepatitis Be Antigens><Hepatocyte><Homologous Serum Hepatitis Virus><Human><IFN Alpha><IFN α><IFN-α><IFNa><IFNα><Immunologic Surface Markers><Immunological Surface Markers><In Vitro><Infection><Interferon Alfa-n3><Interferon-alpha><Interferon-α><Intermediary Metabolism><Interruption><Investigation><Lead><Length><Lentiviral Vector><Lentivirus Vector><Leukocyte Interferon><Life><Ligation><Liver Cells><Lymphoblast Interferon><Lymphoblastoid Interferon><Medication><Messenger RNA><Metabolic Processes><Metabolism><Modern Man><Molecular><Molecular Interaction><Multi-Drug Resistance><Multidrug Resistance><Multiple Drug Resistance><Multiple Drug Resistant><Mutation><NHEJ><Non-Homologous End Joining><Non-Polyadenylated RNA><Non-homologous DNA End Joining><Nonhomologous DNA End Joining><Nonhomologous End Joining><Nuclear><Nucleotides><Nucleus><ORFs><Open Reading Frames><Outcome><Patients><Pb element><Persons><Pharmaceutic Preparations><Pharmaceutical Preparations><Phase><Polymerase Gene><Position><Positioning Attribute><Production><Protein Biosynthesis><Protein Coding Region><RNA><RNA Expression><RNA Gene Products><Regimen><Research Resources><Residual><Residual state><Resistance to Multi-drug><Resistance to Multidrug><Resistance to Multiple Drug><Resistant to Multiple Drug><Resistant to multi-drug><Resistant to multidrug><Resources><Reverse Transcription><Ribonucleic Acid><Ribosomal Peptide Biosynthesis><Ribosomal Protein Biosynthesis><Ribosomal Protein Synthesis><Risk><SBIR><SCID Mice><Scars><Severe Combined Immunodeficient Mice><Single-Stranded DNA><Site><Small Business Innovation Research><Small Business Innovation Research Grant><Specificity><Stop Codon><Strains Cell Lines><Structure><Surface Antigens><T-Cells><T-Lymphocyte><Technology><Termination Codon><Terminator Codon><Testing><Therapeutic><Transcript><Transcription><Transgenic Mice><Translation Stop Signal><Unscheduled DNA Synthesis><Viral><Viral Antigens><Viral Gene Products><Viral Gene Proteins><Viral Hepatitis B><Viral Proteins><Virus><Virus Replication><adaptive immune response><analog><anti-viral agents><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-viral therapy><anti-virals><antigen-specific T cells><antiviral compound><antiviral medication><antiviral therapeutic><arm><base><base editor><chemical synthesis><chronic HBV infection><chronic hepatitis B virus infection><chronic infection><cultured cell line><delivery vector><delivery vehicle><developmental><drug/agent><e Antigens><efficacy study><exhaustion><gRNA><genome mutation><genotoxicity><heavy metal Pb><heavy metal lead><hepatoma cell><improved><in vivo><indel><innovate><innovation><innovative><insertion-deletion><insertion-deletion mutation><insertion/deletion><insertion/deletion mutation><lipid based nanoparticle><lipid nanoparticle><mRNA><model of animal><model organism><mouse model><multi-drug resistant><multidrug resistant><murine model><novel><persistent infection><phase 2 study><phase II study><prevent><preventing><protein synthesis><repair><repaired><serum hepatitis><side effect><standard of care><thymus derived lymphocyte><viral DNA><viral RNA><viral infectious disease treatment><viral multiplication><viral replication><virus DNA><virus RNA><virus antigen><virus multiplication><virus protein>