# Non-Canonical Pathways for Electrogene Transfer

> **NIH NIH R01** · DUKE UNIVERSITY · 2021 · $312,336

## Abstract

Project Summary
The long-term goal of the project is to develop a general strategy for improving clinical applications of electrotransfection
(ET). The technology has been widely used for gene delivery in different applications, such as genome and epigenome
editing, cell and gene therapies, and vaccination for prevention of diseases. However, the technique is currently limited by
its low efficiency. Only a tiny fraction of plasmid DNA (pDNA) molecules in extracellular space can be delivered into the
nucleus of cells for target gene expression. As a result, ET requires to use buffers with high pDNA concentration and electric
pulses with high energy, which can cause cytotoxicity and induce undesired immune responses in cells. To improve the
efficiency, the overall objective of the proposed study is to understand molecular mechanisms of pDNA transport in cells.
Understanding the mechanisms is critical for development of a general strategy for improving the efficiency of ET, in which
intracellular pathways will be manipulated to enhance pDNA transport to the nucleus, and reduce its degradation in the
cytoplasm. The central hypothesis in the study is that intracellular transport of electrotransfected pDNA is mediated by
vesicles in noncanonical pathways that overlap with those for endocytosis and autophagy. To test the hypothesis, the study
will investigate specific pathways involved in cellular uptake and intracellular transport of electrotransfected pDNA (Aim
1). The investigation will be based on quantitative analysis of spatial and temporal distributions of pDNA in cells and its
associations with endocytic and autophagic markers. Meanwhile, components in intracellular pathways will be manipulated
to identify those that can be used to enhance ET efficiency and cell viability (Aim 2). The manipulation will include
treatment of cells with different electric pulses and pharmacological agents, or changing expression levels of specific genes
in cells prior to or post ET. The investigations in Aims 1 and 2 will use cells from two dimensional (2D) culture. To
understand how ET in 2D differ from that in 3D microenvironment, the proposed study will investigate mechanisms of ET
in 3D cell constructs (Aim 3). Results from the mechanistic study will be used, as a proof of principle, to enhance electrogene
transfer in solid tumors in vivo (Aim 3). Taken together, the proposed study will reveal new mechanisms of ET in both 2D
and 3D models, and develop a more general strategy for improving ET efficiency and cell viability for all cell types. The
increase in efficiency will also decrease the amount of pDNA required for ET, thereby reducing undesired innate immune
responses to ET. Compared to empirical, trial-and-error approaches used currently in the literature, the new strategy will be
more efficient, versatile, and practical, which is critical for improving ET in clinical applications.

## Key facts

- **NIH application ID:** 10246268
- **Project number:** 5R01GM130830-04
- **Recipient organization:** DUKE UNIVERSITY
- **Principal Investigator:** FAN YUAN
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $312,336
- **Award type:** 5
- **Project period:** 2018-09-20 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10246268

## Citation

> US National Institutes of Health, RePORTER application 10246268, Non-Canonical Pathways for Electrogene Transfer (5R01GM130830-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10246268. Licensed CC0.

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