Oral Presentation 12th Australasian Virology Society Meeting 2024

Using CRISPR-Cas13b as a novel antiviral to target the hepatitis B RNAs to suppress hepatitis B virus replication and protein expression in vitro and in vivo (#107)

Laura C McCoullough 1 2 3 , Mohamed Fareh 4 5 , Wenxin Hu 4 5 , Vitina Sozzi 1 , Christina Makhlouf 1 , Yianni Droungas 1 2 , Chee Leng Lee 1 6 , Mina Takawy 1 3 , Stewart A Fabb 6 , Thomas J Payne 6 , Colin W Pouton 6 , Hans J Netter 1 , Sharon R Lewin 3 7 8 , Damian FJ Purcell 2 , Jacinta A Holmes 9 , Joe A Trapani 4 5 , Margaret Littlejohn 1 3 , Peter A Revill 1 3
  1. Victorian Infectious Disease Reference Laboratory, Royal Melbourne Hospital, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  2. Department of Microbiology and Immunology, The University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  3. Department of Infectious Diseases, The University of Melbourne, Melbourne, Victoria, Australia
  4. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
  5. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Victoria, Australia
  6. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
  7. Victorian Infectious Disease Service, Royal Melbourne Hospital at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
  8. Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, Victoria, Australia
  9. Department of Gastroenterology, St. Vincent's Hospital, Melbourne, Victoria, Australia

Background: Bacterial CRISPR-Cas13b endonuclease has been repurposed to target RNA in mammalian cells by designing highly specific 30 nucleotide CRISPR RNAs (crRNAs) complementary to the target RNAs of interest, which reduces the possibility of off-target effects. Recent preclinical studies have used CRISPR-Cas13b as a novel antiviral to target viral RNAs such as SARS-CoV-2 and influenza RNAs to reduce viral replication.

 

Hepatitis B virus (HBV) is a DNA virus that replicates through an RNA intermediate known as the pregenomic RNA (pgRNA). The pgRNA and viral mRNAs represent novel antiviral targets, which may be targeted by CRISPR-Cas13b. Here, in a world first study, we used CRISPR-Cas13b to target the HBV RNAs to reduce HBV replication and protein expression in vitro and in vivo.

 

Methods: Cas13b crRNAs were designed to target the HBV RNAs. Hepatoma cells were transfected with wildtype (WT) HBV of multiple genotypes, Cas13b and crRNA plasmids. A HBV stable cell line and HBV infection model were transfected with Cas13b and crRNA plasmids. The impact on HBV replication and protein expression was determined. WT HBV, Cas13b and crRNA plasmids were hydrodynamically co-injected into CBA mice and sera hepatitis B surface antigen (HBsAg) was measured. Cas13b mRNA and crRNA were delivered by lipid nanoparticles (LNPs) in a HBsAg-expressing stable cell line and secreted HBsAg was measured. 

 

Results: Cas13b strongly suppressed HBV replication and protein expression in all cell lines tested. The effect was pan-genotypic. Sera HBsAg was reduced by ~50% in vivo. LNP-encapsulated Cas13b mRNA reduced secreted HBsAg by 87% in a HBsAg-expressing stable cell line.

 

Conclusion: CRISPR-Cas13b successfully targeted the HBV RNAs to significantly reduce HBV replication and protein expression in vitro and in vivo which, together with other studies that have used CRISPR-Cas13b to target viral RNAs, further demonstrates its potential as a novel antiviral.