# Mechanism and approach to inactivate mutant KRAS of lung metastatic colon cancer by RNA-ligand-displaying exosome to co-deliver dCas9--gRNA ribonucleoprotein complex and KRAS siRNA

> **NIH NIH R01** · OHIO STATE UNIVERSITY · 2024 · $497,002

## Abstract

PROJECT SUMMARY
 Targeted nanotechnologies have shown great promise in overcoming roadblocks in cancer therapeutics.
Tumor metastasis has a limited response or resistance to chemotherapeutics and a moderate response to new
antibody therapies. Our RNA nanotechnology has shown great promise in targeting metastatic disease to deliver
both gene silencing and chemical drug therapies.
 Our long goal is to overcome colon cancer, the second most common cause of cancer death primarily due
to the mutations to KRAS and subsequent lung metastasis with expected survival of months. KRAS, the gene
that codes for the K-Ras protein, is considered “undruggable”. KRAS mutations are found in up to 45% of
colorectal cancers. We have designed an epigenetic repressor to silence mutant K-Ras through epigenome
editing. We created a fusion protein consisting of nuclease-inactive dCas9 and the histone deacetylase HDAC1
and targeted dCas9-HDAC1 to the promoter of mutant KRAS. We can load the recombinant dCas9-HDAC1-
gRNA ribonucleoprotein (RNP) complex into exosomes and silence K-Ras; we designed RNA nanoparticles
carrying mutant K-Ras siRNA to inhibit KRAS mutant lung cancer; we also successfully constructed RNA 4WJ
carrying SN-38 to inhibit colon cancer lung metastasis.
 The goal of this proposal is to identify mechanisms that govern the high delivery platform to silence K-Ras in
colon cancer. We intend to deactivate mutant KRAS via RNA-ligand displaying exosomes loaded with dCas9-
HDAC1-gRNA ribonucleoprotein, siRNAs, and chemical drugs individually or in combination in colorectal cancer
primary and metastasis tumors, using orthotopic and PDX xenograft models. We will investigate the mechanism
of action in K-Ras inhibition, including the conditions for the integration and assembly of dCas9-HDAC1 and
gRNA or crRNA such as sequence and length requirement for silencing mutant KRAS and suppressing colon
cancer cells. We will apply RNA nanoparticle orientation to display targeting ligands on the surface of exosomes.
Instead of delivering dCas9 plasmids, we will deliver a ribonucleoprotein complex of dCas9-recombinant protein
and gRNA. Exosomes will display RNA nanoparticles with an aptamer to bind colon cancer cells specifically. We
will engineer RNA nanoparticles and increase the surface display density of the negatively charged RNA ligands
to enhance the negative zeta potential of exosomes for preventing binding to the vital organs and healthy cells
that normally have negatively charged lipid membranes. We will try to enhance therapeutic efficacy and reduce
toxicity by overcoming endosome trapping and non-specific cell entry through RNA ligand manipulation. Zonal
and density gradient ultracentrifugation or size exclusion columns will select exosomes smaller than 100 nm to
escape macrophage engulfment and improve biodistribution. This project with a multidisciplinary approach will
build a strong foundation from which researchers can deploy large protein complexes to treat ca...

## Key facts

- **NIH application ID:** 10979756
- **Project number:** 1R01CA293945-01
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** BIN GUO
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $497,002
- **Award type:** 1
- **Project period:** 2024-07-02 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10979756

## Citation

> US National Institutes of Health, RePORTER application 10979756, Mechanism and approach to inactivate mutant KRAS of lung metastatic colon cancer by RNA-ligand-displaying exosome to co-deliver dCas9--gRNA ribonucleoprotein complex and KRAS siRNA (1R01CA293945-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10979756. Licensed CC0.

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