# Programmable Microvesicles for Intracellular Macromolecule Delivery

> **NIH NIH R01** · UNIVERSITY OF COLORADO · 2023 · $232,000

## Abstract

Project Summary
Technologies to deliver macromolecules across the plasma membrane and bypass endosome
degradation are not only instrumental for elucidating gene function but also hold enormous potential
for therapeutics. Proteins, nucleic acids, and ribonucleoproteins (RNP) have become indispensable
tools for biomedical research, however, their applications in human therapeutics are largely limited to
modulating targets reside in the extracellular space. Only a few percent of exogenous macromolecules
can get through the cellular barriers and make it into the intracellular space. Extracellular vesicles
(EVs) are increasingly being explored as potential vehicles for intracellular therapeutics delivery since
they transport bioactive molecules natively between cells. Cell derived EVs are heterogeneous in size
and composition and, consequently, exhibit low specific activity for delivering cargo of interest. To
address these problems, we developed an innovative macromolecule delivery system based on
engineered extracellular vesicles called gectosomes (G protein ectosomes), designed to co-
encapsulate vesicular stomatitis virus G protein (VSV-G) with bioactive macromolecules via split GFP
complementation. The reversible tethering of cargo to VSV-G provides efficient cargo loading and
endosomal escape simultaneously. Gectosomes demonstrated efficient delivery of catalytic enzymes,
interference RNA, and Cas9 RNPs to the cytosol and nucleus and successful modifications of cellular
phenotypes. We aim to develop a versatile and broadly applicable platform technology that allows
rapid production of highly specific gectosomes capable of modulating intracellular targets in vitro and
in vivo. The objective of this application is to demonstrate the feasibility of our approach by improving
the homogeneity of gectosomes through CRISPR engineering of the producer cells and by creating
gectosomes that deliver engineered nanobodies or ubiquitin E3 ligase CRBN intracellularly to alter
protein aggregation or degradation. We will also examine host immune responses to gectosomes and
elucidate the efficacy window of gectosome delivery in vivo, which will help refine application areas.
This supplement application in response to PA-20-272 (NOT-GM-22-017) requests support to
purchase NanoAnalyzer, a new robust nano-flow cytometry analyzer for measuring the concentration
and size of very small particles according to the surface markers. NanoAnalyzer greatly increase the
speed, reliability and reproducibility of analysis of extracellular vesicles. The proposed purchase of
this cutting-edge instrument will overcome current limitations in analyzing extracellular vesicles and
enable us to develop the gectosome technology that aims to deliver biologics to the intracellular space
and accelerate research innovation for therapeutics development.

## Key facts

- **NIH application ID:** 10798752
- **Project number:** 3R01GM144749-02S2
- **Recipient organization:** UNIVERSITY OF COLORADO
- **Principal Investigator:** XUEDONG LIU
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $232,000
- **Award type:** 3
- **Project period:** 2022-02-01 → 2025-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10798752

## Citation

> US National Institutes of Health, RePORTER application 10798752, Programmable Microvesicles for Intracellular Macromolecule Delivery (3R01GM144749-02S2). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10798752. Licensed CC0.

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