# Suppression of disease causingnonsense mutations by targeted mRNA pseudouridylation

> **NIH NIH R21** · UNIVERSITY OF ROCHESTER · 2020 · $200,970

## Abstract

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.

## Key facts

- **NIH application ID:** 9968200
- **Project number:** 5R21CA241111-02
- **Recipient organization:** UNIVERSITY OF ROCHESTER
- **Principal Investigator:** YI-TAO YU
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $200,970
- **Award type:** 5
- **Project period:** 2019-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9968200

## Citation

> US National Institutes of Health, RePORTER application 9968200, Suppression of disease causingnonsense mutations by targeted mRNA pseudouridylation (5R21CA241111-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9968200. Licensed CC0.

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