In this exploratory grant application, we propose to fully develop a novel approach, namely, targeted RNA pseudouridylation, to suppress nonsense mutations in genes that cause diseases (e.g., nonsense mutations in the CFTR gene that cause Cystic Fibrosis, and nonsense mutations in p53 gene that cause various types of cancer). This project, if successful, will constitute a giant step toward our ultimate goal of developing novel therapeutic treatment for a number of genetic disorders and certain types of cancer caused by nonsense mutations. We propose to carry out this project under two specific aims.
Aim 1. To improve the efficiency of RNA-guided pseudouridylation in human cells. We will improve the efficiency of targeted mRNA pseudouridylation by identifying a better modifying enzyme (specifically, a better box H/ACA guide RNA). We will focus on the three elements within the guide RNA that have been identified (by our lab and other labs) to be important for directing pseudouridylation, and construct a perfect box H/ACA guide RNA. We will also engineer the guide RNA by adding a nucleoplasmic localization signal, thus targeting the guide RNA (and RNP) to the nucleoplasm where mRNA is synthesized and matures. Finally, we will increase box H/ACA RNA expression level, thus raising its concentration in the nucleoplasm of human cells. In doing so, we believe we will be able to create a super guide RNA that can efficiently direct site-specific mRNA pseudouridylation.
Aim 2. To directly target the PTC within a disease gene in human disease cell lines. Using an designer box H/ACA RNA (especially when the improved super box H/ACA RNA identified in Aim 1 is available), we will directly target the premature termination codon (PTC, resulting from nonsense mutations) within the CFTR mRNA in a CF cell line and within the p53 mRNA in a human cancer cell line. The efficiency of guide RNA transfection and its expression level in transfected cells will be measured. Site-specific pseudouridylation at the PTC of mRNAs will also be quantified. Furthermore, nonsense suppression, including the suppression of NMD (nonsense-mediated mRNA decay) and PTC read-through, will be monitored. In short, this proposal presents a potential novel approach to treat many genetic diseases and certain types of cancer associated with nonsense mutations.

Public Health Relevance

In this application, we propose to fully develop a novel approach, namely, targeted RNA pseudouridylation, to suppress disease-causing nonsense mutations, focusing on CFTR, the Cystic Fibrosis gene, and p53, a gene associated with many types of cancer. Our preliminary results indicate that targeted pseudouridylation at premature termination (or nonsense) codons specifically suppresses premature translation termination, thus restoring the production of full-length functional protein. From the clinical perspective, a large number of human genetic diseases are associated with codon mutations, especially those that create a premature termination codon (nonsense mutations). Thus, the current proposal (to fully develop the targeted RNA pseudouridylation approach), if successful, will constitute a giant step toward our ultimate aim of developing novel therapeutic treatment for a number of genetic diseases and certain types of cancer caused by nonsense mutations. Our application is thus of clinical relevance (especially has relevance to cancer).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA241111-02
Application #
9968200
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Strasburger, Jennifer
Project Start
2019-07-01
Project End
2021-06-30
Budget Start
2020-07-01
Budget End
2021-06-30
Support Year
2
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
School of Medicine & Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627