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

Abstract

Background: Human mesenchymal stem cells (hMSCs) are intensely researched for applications in cell therapeutics due to their unique properties, however, intrinsic therapeutic properties of hMSCs could be enhanced by genetic modification. Viral transduction is efficient, but suffers from safety issues. Conversely, nonviral gene delivery, while safer compared to viral, suffers from inefficiency and cytotoxicity, especially in hMSCs. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological 'priming' of hMSCs with the glucocorticoid dexamethasone can significantly increase transfection in hMSCs by modulating transfectioninduced cytotoxicity. This work seeks to establish a library of transfection priming compounds for hMSCs by screening 707 FDA-approved drugs, belonging to diverse drug classes, from the NIH Clinical Collection at four concentrations for their ability to modulate nonviral gene delivery to adipose-derived hMSCs from two human donors. Results: Microscope images of cells transfected with a fluorescent transgene were analyzed in order to identify compounds that significantly affected hMSC transfection without significant toxicity. Compound classes that increased transfection across both donors included glucocorticoids, antibiotics, and antihypertensives. Notably, clobetasol propionate, a glucocorticoid, increased transgene production 18-fold over unprimed transfection. Furthermore, compound classes that decreased transfection across both donors included flavonoids, antibiotics, and antihypertensives, with the flavonoid epigallocatechin gallate decreasing transgene production − 41-fold compared to unprimed transfection. Conclusions: Our screen of the NCC is the first high-throughput and drug-repurposing approach to identify nonviral gene delivery priming compounds in two donors of hMSCs. Priming compounds and classes identified in this screen suggest that modulation of proliferation, mitochondrial function, and apoptosis is vital for enhancing nonviral gene delivery to hMSCs.

Comparison of promoter, DNA vector, and cationic carrier for efficient transfection of hMSCs from multiple donors and tissue sources

“…However, gene delivery, particularly through nonviral routes, is inefficient. 8,[12][13][14][15][16]24 We have previously reported enhanced nonviral gene delivery to hMSCs by priming with pharmacologic agents, 16 specifically with the glucocorticoid dexamethasone; [13][14][15] however, work remains to improve nonviral gene delivery to hMSCs, in particular through evaluation of key variables of the nonviral gene delivery system. This work systematically compares the key variables of nonviral gene delivery systems (e.g., DNA vector type, DNA vector bacterial elements, promoter, cationic carrier, and donor and tissue source) and the effects of these variables on transfection outcomes.…”

“…Here, two commercially available transfection reagents were used, which represent both lipid-and polymer-based systems: Lipofectamine 3000 and Turbofect, respectively. These commercially available transfection reagents were chosen as they are readily available, widely used, and have shown moderate levels of transfection in hMSCs [14][15][16]24 (T.K., A.H., and L.S., unpublished data). This study did not include other modes of delivery (e.g., electroporation) given the complexities involved in comparing carrier-based systems to physical methods, but future studies should investigate other delivery strategies in the context of the parameters investigated here.…”

“…12 To address the shortcomings of nonviral gene delivery to hMSCs, our group has demonstrated that pharmacological "priming" of cells prior to or simultaneously with application of nonviral DNA complexes can improve transfection in hMSCs. [13][14][15][16] Specifically, we have shown that the glucocorticoid, dexamethasone, can significantly increase transfection efficiency and transgene expression in multiple donors of hMSCs from different tissues, relative to a vehicle control (VC), 13 by modulating transfection-induced stress pathways and apoptosis. 14,15 Additionally, we have expanded our hMSC transfection priming library by screening a collection of 707 FDA approved drugs for drug repurposing 17 and identified new candidate priming agents that can significantly improve transfection compared to a VC.…”

“…14,15 Additionally, we have expanded our hMSC transfection priming library by screening a collection of 707 FDA approved drugs for drug repurposing 17 and identified new candidate priming agents that can significantly improve transfection compared to a VC. 16 However, even with priming, nonviral gene delivery to hMSCs remains inefficient for clinical applications.…”

Programmed Cell Death Ligand 1-Transfected Mouse Bone Marrow Mesenchymal Stem Cells as Targeted Therapy for Rheumatoid Arthritis

“…A primary study compared the therapeutic effects of different mesenchymal stem cells on rheumatoid arthritis in mice, and the authors found that both bone marrow mesenchymal stem cells and umbilical cord mesenchymal stem cells significantly alleviated RA [ 18 ]. Increasingly, recent developments in transfection methods and gene delivery have facilitated the delivery of exogenous DNA or RNA to MSCs to alter gene expression by viral gene delivery systems and gene therapy technologies [ 19 , 20 ].…”