RESUMEN
BACKGROUND & AIMS: New antiviral approaches are urgently required that target multiple aspects of the hepatitis B virus (HBV) replication cycle to improve rates of functional cure. HBV RNA represents a novel therapeutic target. Here, we programmed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas13b endonuclease, to specifically target the HBV pregenomic RNA (pgRNA) and viral mRNAs in a novel approach to reduce HBV replication and protein expression. METHODS: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pgRNA. Mammalian cells with replication competent wildtype HBV DNA of different genotypes, a HBV stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-blue fluorescent protein (BFP) and crRNAs plasmids and the impact on HBV replication and protein expression was measured. WT HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and sera HBsAg was measured. PspCas13b mRNA and crRNA were also delivered by lipid nanoparticles (LNP) in a HBsAg-expressing stable cell line and the impact on secreted HBsAg determined. RESULTS: Our HBV targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p<0.0001). HBV protein expression was also reduced in an HBV stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p<0.0001) in vivo. LNP-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p=0.0168) in a HBsAg-expressing stable cell line. CONCLUSIONS: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection. IMPACT AND IMPLICATIONS: There is an urgent need for new treatments that target multiple aspects of the HBV replication cycle. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B.
RESUMEN
Elevated expression of the Zinc finger E-box binding homeobox transcription factor-2 (ZEB2) is correlated with poor prognosis and patient outcome in a variety of human cancer subtypes. Using a conditional gain-of-function mouse model, we recently demonstrated that ZEB2 is an oncogenic driver of immature T-cell acute lymphoblastic leukemia (T-ALL), a heterogenic subgroup of human leukemia characterized by a high incidence of remission failure or hematological relapse after conventional chemotherapy. Here, we identified the lysine-specific demethylase KDM1A as a novel interaction partner of ZEB2 and demonstrated that mouse and human T-ALLs with increased ZEB2 levels critically depend on KDM1A activity for survival. Therefore, targeting the ZEB2 protein complex through direct disruption of the ZEB2-KDM1A interaction or pharmacological inhibition of the KDM1A demethylase activity itself could serve as a novel therapeutic strategy for this aggressive subtype of human leukemia and possibly other ZEB2-driven malignancies.
