# Cas9 RNP delivery to immune cells in vivo via molecular targeting

> **NIH NIH UG3** · UNIVERSITY OF CALIFORNIA BERKELEY · 2020 · $781,190

## Abstract

PROJECT SUMMARY / ABSTRACT
CRISPR-Cas9 has demonstrated incredible potential to provide clinical benefit, but the challenge of delivery
currently hinders therapeutic use of genome editing in vivo. Use of viral vectors and lipid nanoparticles has
established the viability of in vivo genome editing, but these technologies have substantial drawbacks. Viral
vectors are immunogenic, difficult to manufacture, and have been associated with increased risks of off-target
editing. Lipid nanoparticles are unsuitable for systemic administration if targeting organs other than the liver. Ex
vivo therapies relying on autologous transplantation have shown the immense value in genetic manipulation of
immune cells, but the procedures remain risky, resource-intensive, and prohibitively expensive.
An ideal method to deliver therapeutic genome editing enzymes would be non-toxic, compatible with
intravenous administration, amenable to large-scale manufacture, and targeted to the cell type in need of
genetic correction. With all this in mind, we propose delivery of CRISPR-Cas9 in the form of an RNA-protein
(RNP) complex. Cas9 RNP has been shown to be safe and effective in vivo following local administration, and
we have established a strategy to enable cell type-specific delivery of Cas9 RNP tethered to a molecular
targeting agent (MTA) such as a receptor-binding ligand, antibody, or aptamer.
Our proposal aims to use MTA-tethered Cas9 RNP for targeted editing of T cells in vivo. We will rely on
established and novel MTAs to promote efficient and specific uptake of Cas9 RNP into T cells. Well-
characterized antibody MTAs will direct specific editing in human, mouse, and primate T cells. Novel aptamer
MTAs will be screened with a focus on cross-species reactivity to streamline the transition from pre-clinical to
clinical development. Because the Cas9 RNP has no inherent ability to cross cellular membranes, it will be
augmented with the ability to escape the endosome to avoid lysosomal degradation following MTA-induced
endocytosis. We have established a novel modular approach to functionalize Cas9 for endosomal escape,
facilitating re-optimization for specific cell types as needed.
In the UG3 phase, we will complete the following three aims: (1) Enable in vivo-compatible genome editing of
immune cells using targeted Cas9 RNP; (2) Identify robust molecular targeting agents for T cell-specific
editing; (3) Use targeted Cas9 RNP for in vivo genome editing of T cells. Following independent validation of
editing in mice, the UH3 phase will perform the following: (1) Scale up production of targeted Cas9 RNP for
large animal testing; (2) Validate targeted Cas9 RNP for in vivo genome editing in non-human primates.
The intersection of MTA-based cell targeting and the efficient endosomal escape of Cas9 RNP will generate a
versatile genome editing platform suitable for intravenous administration. Successful completion of the
proposed work will result in an engineered Cas9 RNP system...

## Key facts

- **NIH application ID:** 10003948
- **Project number:** 5UG3AI150552-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** JENNIFER A DOUDNA
- **Activity code:** UG3 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $781,190
- **Award type:** 5
- **Project period:** 2019-08-28 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10003948

## Citation

> US National Institutes of Health, RePORTER application 10003948, Cas9 RNP delivery to immune cells in vivo via molecular targeting (5UG3AI150552-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10003948. Licensed CC0.

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