Invariance Mapping of the Complete HBV Genome and Testing of Mre11 Repair Nuclease Toward the Improvement of CRISPR/Cas Therapy for Hepatitis B
Abstract
A quarter of a billion people worldwide are chronically infected with hepatitis B virus (HBV). The virus can be lethal (killing 800,000 people each year), resulting from complications such as acute hepatitis, liver cirrhosis, or liver cancer. An effective vaccine is available, but it does not help those already infected with the virus. Anti-HBV drugs are available, but they have low effectiveness, do not permanently eradicate the virus, and must be given for life, can engender drug resistance, and/or do not reduce the probability of developing liver cancer. In addition to the lack of effective therapies, the viral genetic material has proven to be difficult to completely eliminate from infected cells, thereby perpetuating the disease. CRISPR/Cas, a revolutionary new gene editing technology, has been utilized in experiments to address the issue of persistent HBV by cleaving its DNA genome. However, this methodology has much room for improvement: (1) HBV mutates rapidly, and since CRISPR recognizes specific target sequences and cleaves them, its targets can change, making the virus resistant to CRISPR; and (2) oftentimes after CRISPR cleaves, the two fragments come together again without introducing destructive mutations, thereby restoring viral function. Our project, which aligns with the FY21 PRMRP Hepatitis B Topic Area, aims to improve CRISPR treatment of HBV by addressing these two limitations. First, we will characterize the complete HBV genome by quantifying the effect of all possible mutations at every possible point along the genome. A given mutation can be (a) lethal, resulting in a dead virus, (b) neutral, having no effect on virus, or (c) attenuating, reducing the viability of the virus without killing it. These are broad categories, but we have devised a protocol to assign a quantifying number to each mutation, which we have termed invariance ratio. The output of our first aim will be a complete map of the HBV genome showing the invariance ratios of all the possible mutations. This will allow us to focus in on short invariant stretches where all mutations are lethal to the virus. If we designate these as the targets of our CRISPR therapy, the virus cannot mutate within these targets so as to evade the therapy. In our second aim, we will test an enzyme that acts on DNA in a specialized way in conjunction with the CRISPR method. We predict that this enzyme will prevent the two cleaved DNA fragments from coming together again without the introduction of a mutation, so that the cleavage will be permanent. In our third aim, we will test these invariant HBV targets to see if CRISPR can destroy HBV in cells in a petri dish. In our fourth aim, we will test whether with these invariant targets, CRISPR can destroy HBV in the liver in a mouse model of hepatitis B. This work will be of significant benefit to the U.S. military, where many Service people still carry and transmit HBV, and new infections occur largely because of overseas deployment, the need for blood transfusions, and personnel sharing close living quarters. The short-term impact of the project will be the development of a novel and far-more-effective therapy for those infected with HBV. The long-term impact will extend from making CRISPR cleavage permanent with our proprietary enzyme. This will make gene editing, where a gene needs to be disrupted in a majority of cells in the body to effect treatment, much more efficient. Treatments for dominant genetic disorders by CRISPR can then be a possibility, addressing a major unmet medical need with far-reaching possibilities.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210994
Entities
People
- Arthur Young
Organizations
- United States Army