Systematic comparison of nonviral gene delivery strategies for efficient co-expression of two transgenes in human mesenchymal stem cells.

BACKGROUND: Human mesenchymal stem cells (hMSCs) are being researched for cell-based therapies due to a host of unique properties, however, genetic modification of hMSCs, accomplished through nonviral gene delivery, could greatly advance their therapeutic potential. Furthermore, expression of multiple transgenes in hMSCs could greatly advance their clinical significance for treatment of multifaceted diseases, as individual transgenes could be expressed that target separate pathogenic drivers of complex diseases. Expressing multiple transgenes can be accomplished by delivering multiple DNA vectors encoding for each transgene, or by delivering a single poly-cistronic vector that encodes for each transgene and accomplishes expression through either use of multiple promoters, an internal ribosome entry site (IRES), or a 2A peptide sequence. These different transgene expression strategies have been used to express multiple transgenes in various mammalian cells, however, they have not been fully evaluated in difficult-to-transfect primary cells, like hMSCs. This study systematically compared four transgene expression and delivery strategies for expression of two reporter transgenes in four donors of hMSCs from two tissue sources using lipid- and polymer-mediate transfection, as follows: (i) delivery of separate DNA vectors in separate nanoparticles; (ii) delivery of separate DNA vectors combined in the same nanoparticle; (iii) delivery of a bi-cistronic DNA vector with an IRES sequence via nanoparticles; and (iv) delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence via nanoparticles. RESULTS: Our results indicate that expression of two transgenes in hMSCs, independent of expression or delivery strategy, is inefficient compared to expressing a single transgene. However, delivery of separate DNA vectors complexed in the same nanoparticle, or delivery of a bi-cistronic DNA vector with a dual 2A peptide sequence, significantly increased the number of hMSCs expressing both transgenes compared to other conditions tested. CONCLUSION: Separate DNA vectors delivered in the same nanoparticle and bi-cistronic DNA vectors with dual 2A peptide sequences are highly efficient at simultaneously expressing two transgenes in multiple donors of hMSCs from different tissue sources. The data presented in this work can guide the development of hMSC transfection systems for delivery of multiple transgenes, with the goal of producing clinically relevant, genetically modified hMSCs.

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

Human mesenchymal stem cells (hMSCs) are primary cells with high clinical relevance that could be enhanced through genetic modification. However, gene delivery, particularly through nonviral routes, is inefficient. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological "priming" of hMSCs with clinically approved drugs can increase transfection in hMSCs by modulating transfection-induced cytotoxicity. However, even with priming, hMSC transfection remains inefficient for clinical applications. This work takes a complementary approach to addressing the challenges of transfecting hMSCs by systematically investigating key transfection parameters for their effect on transgene expression. Specifically, we investigated two promoters (cytomegalovirus [CMV] and elongation factor 1 alpha), four DNA vectors (plasmid, plasmid with no F1 origin, minicircle, and mini-intronic plasmid), two cationic carriers (Lipofectamine 3000 and Turbofect), and four donors of hMSCs from two tissues (adipose and bone marrow) for efficient hMSC transfection. Following systematic comparison of each variable, we identified adipose-derived hMSCs transfected with mini-intronic plasmids containing the CMV promoter delivered using Lipofectamine 3000 as the parameters that produced the highest transfection levels. The data presented in this work can guide the development of other hMSC transfection systems with the goal of producing clinically relevant, genetically modified hMSCs.

Glucocorticoid Priming of Nonviral Gene Delivery to hMSCs Increases Transfection by Reducing Induced Stresses.

Human mesenchymal stem cells (hMSCs) are under study for cell and gene therapeutics because of their immunomodulatory and regenerative properties. Safe and efficient gene delivery could increase hMSC clinical potential by enabling expression of transgenes for control over factor production, behavior, and differentiation. Viral delivery is efficient but suffers from safety issues, while nonviral methods are safe but highly inefficient, especially in hMSCs. We previously demonstrated that priming cells with glucocorticoids (Gcs) before delivery of DNA complexes significantly increases hMSC transfection, which correlates with a rescue of transfection-induced metabolic and protein synthesis decline, and apoptosis. In this work, we show that transgene expression enhancement is mediated by transcriptional activation of endogenous hMSC genes by the cytosolic glucocorticoid receptor (cGR) and that transfection enhancement can be potentiated with a GR transcription-activation synergist. We demonstrate that the Gc-activated cGR modulates endogenous hMSC gene expression to ameliorate transfection-induced endoplasmic reticulum (ER) and oxidative stresses, apoptosis, and inflammatory responses to prevent hMSC metabolic and protein synthesis decline, resulting in enhanced transgene expression after nonviral gene delivery to hMSCs. These results provide insights important for rational design of more efficient nonviral gene delivery and priming techniques that could be utilized for clinical hMSC applications.

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

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 transfection-induced 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.

Surface- and Hydrogel-Mediated Delivery of Nucleic Acid Nanoparticles.

Gene expression within a cell population can be directly altered through gene delivery approaches. Traditionally for nonviral delivery, plasmids or siRNA molecules, encoding or targeting the gene of interest, are packaged within nanoparticles. These nanoparticles are then delivered to the media surrounding cells seeded onto tissue culture plastic; this technique is termed bolus delivery. Although bolus delivery is widely utilized to screen for efficient delivery vehicles and to study gene function in vitro, this delivery strategy may not result in efficient gene transfer for all cell types or may not identify those delivery vehicles that will be efficient in vivo. Furthermore, bolus delivery cannot be used in applications where patterning of gene expression is needed. In this chapter, we describe methods that incorporate material surfaces (i.e., surface-mediated delivery) or hydrogel scaffolds (i.e., hydrogel-mediated delivery) to efficiently deliver genes. This chapter includes protocols for surface-mediated DNA delivery focusing on the simplest and most effective methods, which include nonspecific immobilization of DNA complexes (both polymer and lipid vectors) onto serum-coated cell culture polystyrene and self-assembled monolayers (SAMs) of alkanethiols on gold. Also, protocols for the encapsulation of DNA/cationic polymer nanoparticles into hydrogel scaffolds are described, including methods for the encapsulation of low amounts of DNA (<0.2 µg/µl) and high amounts of DNA (>0.2 µg/µl) since incorporation of high amounts of DNA pose significant challenges due to aggregation.

Free Polyethylenimine Enhances Substrate-Mediated Gene Delivery on Titanium Substrates Modified With RGD-Functionalized Poly(acrylic acid) Brushes.

Substrate mediated gene delivery (SMD) is a method of immobilizing DNA complexes to a substrate via covalent attachment or nonspecific adsorption, which allows for increased transgene expression with less DNA compared to traditional bolus delivery. It may also increase cells receptivity to transfection via cell-material interactions. Substrate modifications with poly(acrylic) acid (PAA) brushes may improve SMD by enhancing substrate interactions with DNA complexes via tailored surface chemistry and increasing cellular adhesion via moieties covalently bound to the brushes. Previously, we described a simple method to graft PAA brushes to Ti and further demonstrated conjugation of cell adhesion peptides (i.e., RGD) to the PAA brushes to improve biocompatibility. The objective of this work was to investigate the ability of Ti substrates modified with PAA-RGD brushes (PAA-RGD) to immobilize complexes composed of branched polyethyleneimine and DNA plasmids (bPEI-DNA) and support SMD in NIH/3T3 fibroblasts. Transfection in NIH/3T3 cells cultured on bPEI-DNA complexes immobilized onto PAA-RGD substrates was measured and compared to transfection in cells cultured on control surfaces with immobilized complexes including Flat Ti, PAA brushes modified with a control peptide (RGE), and unmodified PAA. Transfection was two-fold higher in cells cultured on PAA-RGD compared to those cultured on all control substrates. While DNA immobilization measured with radiolabeled DNA indicated that all substrates (PAA-RGD, unmodified PAA, Flat Ti) contained nearly equivalent amounts of loaded DNA, ellipsometric measurements showed that more total mass (i.e., DNA and bPEI, both complexed and free) was immobilized to PAA and PAA-RGD compared to Flat Ti. The increase in adsorbed mass may be attributed to free bPEI, which has been shown to improve transfection. Further transfection investigations showed that removing free bPEI from the immobilized complexes decreased SMD transfection and negated any differences in transfection success between cells cultured on PAA-RGD and on control substrates, suggesting that free bPEI may be beneficial for SMD in cells cultured on bPEI-DNA complexes immobilized on PAA-RGD grafted to Ti. This work demonstrates that substrate modification with PAA-RGD is a feasible method to enhance SMD outcomes on Ti and may be used for future applications such as tissue engineering, gene therapy, and diagnostics.