A novel CRISPR/Cas12 system targeting HBV DNA for gene therapy - A novel CRISPR/Cas12 system targeting HBV DNA for gene therapy SUMMARY Approximately 250 million people are chronically infected with hepatitis B virus (HBV) worldwide. In the United States, HBV infection remains a major public health problem. Current antiretroviral therapy using nucleotides analogs (NA) can restrict HBV replication, however, the treatment cannot eliminate the viral DNAs that are integrated into the host genome and cccDNA that is resistant to the current treatment. As such, HBV persist in NA-controlled individuals, and NA cessation readily leads to viral reactivation and disease progression. Therefore, there is an urgent medical need to develop a curative strategy that can eliminate the HBV (ccc)DNA without causing collateral cytotoxic effects. While the CRISPR/Cas9 system has been widely used for genome editing, recent studies indicated that a single guide RNA (gRNA) in the CRISPR/Cas9 system cannot completely inactivate the viral DNA, resulting in viral escape. The new RNA-guided endonuclease CRISPR/Cas12 (formerly Cpf1) system is a more promising tool for viral DNA engineering, with increased antiviral activity and specificity. A recent study compared Cas9 and Cas12 systems for editing of the viral DNA and revealed superior antiviral activity for Cas12, which could achieve full viral inactivation with only a single gRNA. Nevertheless, the current CRISPR/Cas12 delivery technologies often require viral vectors, which pose safety concerns for therapeutic applications in humans. Synthetic gRNA/Cas12 ribonucleoprotein (RNP) is an attractive non-viral formulation for the CRISPR/Cas12 system due to its rapid DNA cleavage activity, less off-target effects, low risk of insertional mutagenesis, easy production, and readiness for clinical application. To date, whether synthetic gRNA/Cas12 RNP can be used to edit integrated HBV DNA and its typical signature on the viral and host genomes remain largely unknown. Of note, highly potent and specific gRNAs targeting critical HBV DNA site(s), and the efficacy of gRNA/Cas12 RNP in inactivating viral DNA replication have yet to be evaluated. In this application, we propose to develop a novel synthetic CRISPR/Cas12 system targeting HBV DNA and evaluate its antiviral activity and cytotoxicity using HBV cellular models. We hypothesize that synthetic gRNA/Cas12 RNP will abolish HBV replication efficiently and will elicit minimal cytotoxic effects. We propose two specific aims to test our hypothesis. Aim 1 will design/synthesize a series of HBV gRNA/Cas12 RNPs and test their antiviral activity in HBV cellular models. Aim 2 will evaluate off-target effects and cytotoxicity of these HBV gRNA/Cas12 RNP in these cellular models. The goal of this project is to generate a proof of concept, collect key data, establish a new technology, and lay the foundation to achieve our long-term goal of developing an HBV cure. Our preliminary studies, experienced investigators, novel approach, and excellent environment ensure the likelihood of completing this clinically significant study in a timely manner. This research supplement will support an unrepresentative graduate student’s training and add diversity profile of PI’s research workforce.
