# Engineered Exosomes for Targeted Delivery of the CRISPR/Cas9 Genome-editor

> **NIH NIH R41** · 3P BIOTECHNOLOGIES, INC. · 2022 · $264,507

## Abstract

Technical Abstract
 Genetic mutations have been identified as a causative factor in numerous diseases. The genome editing
system CRISPR/Cas9 is a recent development in gene therapy. Both viral and non-viral vectors have been used
in attempts to direct delivery of Cas9 to specific locations with advantages and limitations similar to those known
for other nucleic acid-based therapeutics. These challenges have limited the current clinical progress of this
genome-editing tool. The goal of this project is to develop an effective targeted delivery system for Cas9-mediated
genome editing. The investigators take advantage of a novel technology for delivery of plasmid DNA (pDNA)
based on bovine milk/colostrum exosomes developed in the PI's laboratory. In this project, we will apply our
knowledge and extensive experience in exosomes for efficient targeted delivery of the Cas9-mediated genome-
editing tool. To establish feasibility, we have used pDNA to deliver the coding sequences for Cas9-mediated
knockout of NFκB as a model gene. This single plasmid, pKO-NFκB, contains the mammalian-optimized Cas9
coding sequence, the single-guide RNA (sgRNA) specific to NFκB, as well as sequences to derive a guide RNA
(gRNA) scaffold to assist in the binding of Cas9 to the target DNA. We hypothesize that pKO-NFκB, ionically
entrapped in a novel exosome matrix, formulated by complexing exosomes and polycationic polyethyleneimine
(PEI), will serve as an effective genome-editing tool of NFκB. Furthermore, use of engineered exosomes,
prepared by loading milk lactoferrin (LF) onto exosomes, will target bronchial epithelium overexpressing LF
receptors. Thus, LF-EPM-pKO-NFκB administered intranasally (i.n.) will target lung with minimal off-target effects
for delivery of this genome-editing tool. Our hypothesis is supported by compelling preliminary data: high loading
of nucleic acid onto EPM and protection from degradation, functionalization of exosomes by surface-bound LF
loading, inhibition of NFκB expression in H2030 lung cancer cells by LF-EPM delivered pKO-NFκB,
overexpression of the LF receptor intelectin (also called omentin) in the mouse lung, and predominant delivery of
LF-functionalized exosomes to the mouse lung by intranasal delivery. Investigators experienced in exosomes,
drug delivery, and biological sciences will pursue the following specific aims: Aim 1. Optimize targeted delivery
of CRISPR/Cas9 genome-editing tool using engineered exosomes in vitro. Aim 2. Determine potential
toxicity, and biodistribution and efficacy of engineered exosomes for targeted delivery of CRISPR/Cas9
genome-editing tool. If we are successful in achieving these milestones, we will move to Phase II. Results from
this project will provide feasibility data for advancing this genome-editing tool delivery `platform' in a disease
model. Cost-effective isolation of exosomes from a biocompatible source, combined with ultracentrifugation-
independent methods currently being developed in PI's ...

## Key facts

- **NIH application ID:** 10383110
- **Project number:** 1R41OD031942-01A1
- **Recipient organization:** 3P BIOTECHNOLOGIES, INC.
- **Principal Investigator:** RAMESH C GUPTA
- **Activity code:** R41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $264,507
- **Award type:** 1
- **Project period:** 2022-06-01 → 2024-05-01

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10383110

## Citation

> US National Institutes of Health, RePORTER application 10383110, Engineered Exosomes for Targeted Delivery of the CRISPR/Cas9 Genome-editor (1R41OD031942-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10383110. Licensed CC0.

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