# Defining the molecular interactions within nanoparticles that enable delivery of long nucleic acids

> **NIH NIH R01** · UT SOUTHWESTERN MEDICAL CENTER · 2020 · $364,500

## Abstract

Project Summary
Co-delivery of Cas9 mRNA and targeted sgRNA is a promising strategy to achieve CRISPR/Cas gene editing in
vivo with improved safety. Although great advances have been made in the delivery of short RNAs (siRNA,
miRNA), the ideal chemical and formulation composition is largely unknown for longer RNA cargo (mRNA,
sgRNA). We recently overcame this delivery challenge in reporting the first successful NP co-delivery of mRNA
and sgRNA in vivo. In this grant proposal, we aim to fundamentally understand why zwitterionic amino lipid (ZAL)
nanoparticles (ZNPs) are uniquely suitable and particularly efficacious for delivery of long RNAs. We hypothesize
that the molecular balance of zwitterionic and cationic groups within NPs is essential for delivery of long nucleic
acids, especially the noncovalent bonding forces at the interface between carrier molecules and RNAs. This
hypothesis is supported by computational modeling that indicates the role of phospholipids is to solubilize RNAs
inside of aqueous pockets within multi-component NPs. Indeed, we found experimentally that combining the
chemical and structural roles of zwitterionic lipids and cationic lipids into a single lipid compound (ZAL) greatly
improved delivery of long RNAs. In this proposal, we will develop (and fundamentally understand) improved
delivery carriers for co-delivery of Cas9 mRNA and targeted sgRNA to enable safe and efficacious CRISPR/Cas
gene editing in vivo. Completion of the proposed studies will: (1) Determine the functional roles of novel ZAL
headgroups, linkers, and hydrophobic domain tails for co-delivery of mRNA and sgRNA; (2) Identify the
physiochemical properties of efficacious long RNA (mRNA, sgRNA) lipid carriers that are correlated to the
mechanism of intracellular delivery; and (3) Determine how the chemical structure of ZALs mediate cell and
tissue specific CRISPR/Cas gene editing in vivo by utilizing a genetically engineered Lox-Stop-Lox tdTomato
mouse that can reveal editing in any organ or cell. Cumulatively, this will open new avenues for CRISPR/Cas-
based correction of genetic diseases by developing efficacious, safe, and clinically translatable nanoparticle
carriers. Defining the specific interactions is a critical goal that would greatly improve delivery of long RNAs by
numerous existing and future carriers. This broader impact may greatly accelerate the clinical development of
mRNA and CRISPR/Cas therapeutics.

## Key facts

- **NIH application ID:** 9939525
- **Project number:** 5R01EB025192-03
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Daniel John Siegwart
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $364,500
- **Award type:** 5
- **Project period:** 2018-08-01 → 2022-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9939525

## Citation

> US National Institutes of Health, RePORTER application 9939525, Defining the molecular interactions within nanoparticles that enable delivery of long nucleic acids (5R01EB025192-03). Retrieved via AI Analytics 2026-05-29 from https://api.ai-analytics.org/grant/nih/9939525. Licensed CC0.

---

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