# Enhancing the Gene Ride vector for Liver Gene Therapy

> **NIH NIH F32** · OREGON HEALTH & SCIENCE UNIVERSITY · 2020 · $67,446

## Abstract

Project Summary
Recombinant adeno-associated viruses (rAAVs) are promising for use in liver targeted human gene therapy.
However, for rAAV vectors to be useful for therapies of inborn errors affecting the liver, these vectors must be
able to have continuous long-term and regulated expression of the transgene. Currently, standard rAAV vectors
have strong promoters that can lead to hepatocellular carcinoma if random integration occurs in proximity to
an oncogene. rAAVs have a limited payload capacity and would be unsuitable for gene therapy of mutations
occurring in large genes. Additionally, rAAVs are mainly non-integrating and function therapeutically by
episomal expression which can be lost during cell replication. One method to address these limitations is to
directly repair the mutation in hepatocytes. However, efficiency of in vivo gene repair with currently available
methods is low. Selection of hepatocytes with the corrected mutation is one way to increase the efficiency of
in vivo gene correction. One way to achieve this is to use the selectable Gene Ride vector that has been
described by our lab. The selectable Gene Ride vector provides a selectable advantage to correctly target
hepatocytes, allowing their expansion over untargeted hepatocytes.
In this proposal, we aim to develop the promoterless selectable Gene Ride vector to have broader clinical
potential. To allow the Gene Ride vector to be functional in any gene, we propose to utilize the CRISPR/Cas9
gene editing system in place of shRNAs to overcome the limitations of shRNAs. shRNAs typically knock down
but do not knock out a gene, while mutations caused by gRNAs can lead to gene knockout. Furthermore, high
expression levels of shRNAs are required for gene knockdown. We hypothesize that lower levels of gRNAs will
be sufficient for gene knockouts. In order to allow the gRNA to be expressed from an endogenous polymerase
2 promoter, we will flank the gRNA with ribozymes. These ribozymes are self-cleaving and allow release of the
active gRNA.
The current selectable Gene Ride vector utilizes the small molecule inhibitor CEHPOBA for selection.
CEHPOBA is not an FDA approved drug, making translation into the clinic difficult. We propose to examine
more clinically relevant gene knockout and drug selection combinations. This includes NTCP, the main bile
acid transporter in hepatocytes. In combination with a cholic acid diet, knockout of NTCP can be used to select
for correctly targeted hepatocytes. Cytochrome p450 reductase (Cypor), is a cofactor for all cytochrome p450s.
A knockout of Cypor in combination with the hepatotoxic drug retrorsine is an alternative method for selection.
Development of the self-cleaving ribozyme gRNA-selectable Gene Ride vector provides a potential gene
therapy approach for treating genetically inherited disorders.
!

## Key facts

- **NIH application ID:** 9867724
- **Project number:** 5F32DK117516-03
- **Recipient organization:** OREGON HEALTH & SCIENCE UNIVERSITY
- **Principal Investigator:** Amita Tiyaboonchai
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $67,446
- **Award type:** 5
- **Project period:** 2018-03-01 → 2021-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9867724

## Citation

> US National Institutes of Health, RePORTER application 9867724, Enhancing the Gene Ride vector for Liver Gene Therapy (5F32DK117516-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9867724. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*