High‐throughput screening of clinically approved drugs that prime polyethylenimine transfection reveals modulation of mitochondria dysfunction response improves gene transfer efficiencies

Abstract

Nonviral gene delivery methods are advantageous over viral vectors in terms of safety, cost, and flexibility in design and application, but suffer from lower gene transfer efficiency. In addition to modifications to nucleic acid design and nonviral carriers, new tools are sought to enhance transfection. Priming is the pharmacological modulation of transfection efficiency and transgene expression, and has demonstrated transfection increase in several compounds, for example, chloroquine and glucocorticoids. To develop a library of transfection priming compounds, a high‐throughput screen was performed of the NIH Clinical Collection (NCC) to identify clinical compounds that prime polyethylenimine (PEI) transfection. HEK293T cells were treated with priming compounds, then transfected with enhanced green fluorescent protein (EGFP)‐encoding plasmid by PEI. After 48‐hr culture, primed and transfected cells were assayed for transfection, cell proliferation, and cell viability by fluorescence measurement of EGFP reporter, Hoechst 33342 nuclei stain, and resazurin metabolic assay. From the microscope image analysis and microplate measurements, transfection fold‐changes were determined, and compounds resulting in statistically significant transfection fold‐change were identified. NCC compounds were clustered using PubChem fingerprint similarity by Tanimoto coefficients in ChemmineTools. Fold‐changes for each compound were linked to drug clusters, from which drug classes that prime transfection were identified. Among the identified drugs classes that primed transfection increases were antioxidants, GABAA receptor modulators, and glucocorticoids. Resveratrol and piceid, stilbenoid antioxidants found in grapes, and zolpidem, a GABAA modulator, increased transfection nearly three‐fold. Literature indicate interaction of the identified transfection priming drug clusters with mitochondria, which may modulate mitochondrial dysfunction known to be associated with PEI transfection.

Mechanisms of unprimed and dexamethasone‐primed nonviral gene delivery to human mesenchymal stem cells

“…Our previous work has sought to improve and better understand the biology of nonviral gene delivery by pharmacologically 'priming' cells for increased transgene expression (Nguyen, Beyersdorf, Riethoven, & Pannier, 2016) through modulation of relevant molecular pathways that are important to the biological processes involved in gene delivery (Martin, Plautz, & Pannier, 2015a;Martin, Plautz, & Pannier, 2015b). Specifically, we investigated anti-inflammatory glucocorticoid (Gc) drugs as priming candidates in hMSCs due to reports in other cell types that Gc can increase transfection efficiency by increasing plasmid DNA (pDNA) nuclear internalization or reducing the inflammatory response to transfection (Braun et al, 1999;Kim, Kim, Bae, Choi, & Lee, 2009).…”

High-throughput screening of clinically approved drugs that prime nonviral gene delivery to human Mesenchymal stem cells

“…An efficient nonviral gene delivery system for ex vivo genetic modification of clinically relevant hMSCs is lacking, however, our lab has previously demonstrated that pharmacological priming, or the addition of compounds to the culture media to modulate the cellular response to transfection, is a simple and effective method to enhance transfection in multiple cell types [15][16][17][23][24][25][26]. The idea of priming was developed after our studies using microarray analysis of transfected versus treated, but untransfected cells, where differentially expressed endogenous genes were identified between each condition in HEK 293 T cells and the expression of these identified genes were perturbed pharmacologically (i.e.…”

“…primed) in order to gain insight into the role of endogenous molecular factors in the transfection process [23][24][25][26]. Furthermore, our lab sought to expand our transfection priming library by screening the National Institutes of Health Clinical Collection (NCC), a collection of over 700 small molecules made available for drug repurposing [21], for compounds that could prime polymer-mediated transfection to HEK 293 T cells [17].…”

“…Our previous work has established chemical priming as a simple method to enhance gene delivery to hMSCs, however, few compounds have been identified that can prime nonviral gene delivery to hMSCs [15][16][17][18][19][20], and the underlying biological processes that are necessary for successful transfection are not well understood. To address these issues, a drug repurposing approach similar to our previous screen for compounds that prime polymer-mediated gene delivery to human embryonic kidney cells (HEK 293 T) [17] was used in this current work to generate a library of compounds from the National Institutes of Health Clinical Collection (NCC), a collection of over 700 clinically approved drugs, belonging to diverse drug classes, made available for drug repurposing [21], which prime lipid-mediated nonviral gene delivery to adipose-derived hMSCs (hAMSCs) from two human donors at four concentrations spanning four orders of magnitude. This work constitutes the first large-scale screen of priming compounds for nonviral gene delivery to hAMSCs and proposes molecular mechanisms that could be exploited for the rational design of new delivery systems to clinically relevant cells.…”

Introduction to the BioTM special issue “Nucleic Acid Delivery: Enabling the Drugs of Tomorrow”

“…Angela Pannier and coworkers describe the identification of small molecule drugs that enhanced DNA transfection efficacy using a high throughput screening approach . Stephanie Seidlits, Lonnie Shea, and their colleagues employ gene therapy to reduce neuroinflammation following spinal cord injury .…”