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.

Innovative nonviral gene delivery strategies for engineering human mesenchymal stem cell phenotypes toward clinical applications.

Although human mesenchymal stem cells (hMSCs) have been used in many clinical trials, variable outcomes have resulted in no FDA-approved hMSC treatment. However, research into developing hMSC therapies for many diseases continues. An approach to manipulate hMSCs for therapeutic applications is gene delivery. Nonviral gene delivery is safer and more flexible than viral vectors, but much less efficient, especially in hMSCs. It is not understood why hMSCs are more difficult to transfect than cell lines, but innate features of hMSCs may present unique barriers to transfection. Recently, strategies to improve hMSC transfection have been developed by innovating nanocarriers, nucleic acid cargos, and by 'priming' hMSCs chemically and physically for more efficient transfection. These strategies aim to engineer hMSCs with new phenotypes mediated by transgenic secreted factors, receptors, transcription factors, and genome editing systems for clinical applications requiring enhanced immunomodulation and/or tissue regeneration, or for functions such as tumor-killing and tissue engineering.

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.

Nucleic acid delivery to mesenchymal stem cells: a review of nonviral methods and applications.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells that can be isolated and expanded from many tissues, and are being investigated for use in cell therapies. Though MSC therapies have demonstrated some success, none have been FDA approved for clinical use. MSCs lose stemness ex vivo, decreasing therapeutic potential, and face additional barriers in vivo, decreasing therapeutic efficacy. Culture optimization and genetic modification of MSCs can overcome these barriers. Viral transduction is efficient, but limited by safety concerns related to mutagenicity of integrating viral vectors and potential immunogenicity of viral antigens. Nonviral delivery methods are safer, though limited by inefficiency and toxicity, and are flexible and scalable, making them attractive for engineering MSC therapies. MAIN TEXT: Transfection method and nucleic acid determine efficiency and expression profile in transfection of MSCs. Transfection methods include microinjection, electroporation, and nanocarrier delivery. Microinjection and electroporation are efficient, but are limited by throughput and toxicity. In contrast, a variety of nanocarriers have been demonstrated to transfer nucleic acids into cells, however nanocarrier delivery to MSCs has traditionally been inefficient. To improve efficiency, plasmid sequences can be optimized by choice of promoter, inclusion of DNA targeting sequences, and removal of bacterial elements. Instead of DNA, RNA can be delivered for rapid protein expression or regulation of endogenous gene expression. Beyond choice of nanocarrier and nucleic acid, transfection can be optimized by priming cells with media additives and cell culture surface modifications to modulate barriers of transfection. Media additives known to enhance MSC transfection include glucocorticoids and histone deacetylase inhibitors. Culture surface properties known to modulate MSC transfection include substrate stiffness and specific protein coating. If nonviral gene delivery to MSCs can be sufficiently improved, MSC therapies could be enhanced by transfection for guided differentiation and reprogramming, transplantation survival and directed homing, and secretion of therapeutics. We discuss utilized delivery methods and nucleic acids, and resulting efficiency and outcomes, in transfection of MSCs reported for such applications. CONCLUSION: Recent developments in transfection methods, including nanocarrier and nucleic acid technologies, combined with chemical and physical priming of MSCs, may sufficiently improve transfection efficiency, enabling scalable genetic engineering of MSCs, potentially bringing effective MSC therapies to patients.

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

Human mesenchymal stem cells (hMSCs) are under intense study for applications of cell and gene therapeutics because of their unique immunomodulatory and regenerative properties. Safe and efficient genetic modification of hMSCs could increase their clinical potential by allowing functional expression of therapeutic transgenes or control over behavior and differentiation. Viral gene delivery is efficient, but suffers from safety issues, while nonviral methods are safe, but highly inefficient, especially in hMSCs. Our lab previously demonstrated that priming cells before delivery of DNA complexes with dexamethasone (DEX), an anti-inflammatory glucocorticoid drug, significantly increases hMSC transfection success. This work systematically investigates the mechanisms of hMSC transfection and DEX-mediated enhancement of transfection. Our results show that hMSC transfection and its enhancement by DEX are decreased by inhibiting classical intracellular transport and nuclear import pathways, but DEX transfection priming does not increase cellular or nuclear internalization of plasmid DNA (pDNA). We also show that hMSC transgene expression is largely affected by pDNA promoter and enhancer sequence changes, but DEX-mediated enhancement of transfection is unaffected by any pDNA sequence changes. Furthermore, DEX-mediated transfection enhancement is not the result of increased transgene messenger RNA transcription or stability. However, DEX-priming increases total protein synthesis by preventing hMSC apoptosis induced by transfection, resulting in increased translation of transgenic protein. DEX may also promote further enhancement of transgenic reporter enzyme activity by other downstream mechanisms. Mechanistic studies of nonviral gene delivery will inform future rationally designed technologies for safe and efficient genetic modification of clinically relevant cell types.