# Enabling Nanoplatforms for Targeted in vivo Delivery of CRISPR/Cas9 Ribonucleoproteins in the Brain

> **NIH NIH UG3** · UNIVERSITY OF WISCONSIN-MADISON · 2020 · $750,461

## Abstract

Project Summary:
CRISPR/Cas9 is a revolutionary and versatile genome editing technique with wide-ranging utility. In vivo genome
editing is anticipated to be the next wave of therapeutics for various major health threats, including neurode-
generative diseases. However, there is an urgent need to develop efficient, non-viral delivery vehicles for safe
and efficient in vivo CRISPR genome editing. Furthermore, delivering CRISPR genome editing machinery to the
brain/neuron represents a major hurdle due to its dense structure and the blood–brain barrier (BBB).
The objective of this project is to engineer a family of versatile, novel, non-viral Cas9-gRNA ribonucleoprotein
(RNP) delivery nanocapsules (NCs) that can robustly and safely generate targeted gene edits in neurons within
the brain. We envision that our robust and universal RNP delivery nanoplatforms will enable innovative
treatments for devastating neurodegenerative diseases. Towards this goal, we will evaluate the feasibility of our
approach, in a demonstration, by targeting the amyloid precursor protein (APP) – relevant to Alzheimer's disease
(AD) in healthy mice and monkey models.
During our preliminary studies, we developed a PEGylated NC with a high RNP loading content (68 wt.%),
versatile surface chemistry, ultrasmall size (dH~13 nm), controllable stoichiometry, excellent biocompatibility, and
high genome editing efficiency in vitro and in vivo. In UG3 Aim 1, we will further optimize the design of the NC
for brain/neuron-targeted genome editing. In particular, we will investigate the synergistic effects of hybrid
targeting ligands, including (1) glucose+RVG peptide for intravenous (i.v.) injection to enhance the crossing of
the BBB and neuron-specific editing, and (2) CPP+RVG peptide for intracerebral injection to enhance uptake
and neuron-specific genome editing. The effects of different types/amounts of targeting ligands on the cellular
uptake, biocompatibility, genome editing efficiency, and functional consequences of the NCs in both Neuro2a
and primary neuron cells will be investigated. In UG3 Aim 2, we will evaluate the brain/neuron targeting
specificity, genome editing efficiency, and potential immune response and systemic toxicity of the i.v. or
intracerebrally administered NCs conjugated with various targeting ligands in healthy mice. In UH3 Aim 1, we
will develop the set up and synthesis process to scale up the production of NCs. In UH3 Aim 2, we will further
evaluate the genome editing efficiency and biocompatibility of the brain/neuron-targeted NCs in healthy rhesus
macaques.
Our uniquely designed NCs are expected to achieve high brain accumulation, high penetration depth, and high
neuron-specific genome editing efficiency due to their desirable characteristics. Given the modularity and ease
of targeting different genes by the CRISPR system, we anticipate that the resulting NCs will be useful for a wide
range of human diseases, including debilitating neurodegenerative disease...

## Key facts

- **NIH application ID:** 9999694
- **Project number:** 5UG3NS111688-03
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** MARINA EMBORG
- **Activity code:** UG3 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $750,461
- **Award type:** 5
- **Project period:** 2018-09-30 → 2021-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9999694

## Citation

> US National Institutes of Health, RePORTER application 9999694, Enabling Nanoplatforms for Targeted in vivo Delivery of CRISPR/Cas9 Ribonucleoproteins in the Brain (5UG3NS111688-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9999694. Licensed CC0.

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