Mechanisms of enhanced non-viral gene delivery to human mesenchymal stem cells induced by glucocorticoid priming

Background: Because of unique roles in wound healing, trophic tissue support, immunomodulation, differentiation ability, and immune privileged status, human mesenchymal stem cells (hMSCs), which can be easily derived from many adult tissues (e.g. bone marrow (BMSCs) and adipose tissue (AMSCs)), are under intense study for the applications of cell and gene therapeutics, as well as tissue engineering and regenerative medicine1. Genetic modification of hMSCs could allow for targeted delivery of transgenic therapeutic factors or genetically-guided differentiation. Non-viral gene delivery (i.e. cationic polymer- and lipid-mediated) is safer and more flexible than immunogenic and mutagenic viral vectors2, but it is less effective, especially in hMSCs (i.e. maximum 10-30% transfection)3. As part of an approach to understand molecular mechanisms of non-viral gene delivery4 and ‘prime’ cells to be more receptive to transfection5, our lab recently demonstrated that transgene expression from lipofected hMSCs can be increased about 10-fold by priming cells, 30 mins before plasmid DNA (pDNA) transfection, with 100 nM dexamethasone (DEX), a glucocorticoid (Gc) drug, relative to EtOH vehicle control (VC)6. This work investigates the mechanisms by which Gc priming enhances non-viral gene delivery, which are currently unknown. Studies provide insights into the biological processes of Gc priming and transfection to inform future gene delivery technologies, and characterize a simple protocol to significantly enhance non-viral gene delivery of therapeutic transgenes for future clinical applications. Please click Additional Files below to see the full abstract

Biomaterial substrate modifications that influence cell-material interactions to prime cellular responses to nonviral gene delivery

Gene delivery is the transfer of exogenous genetic material into somatic cells to modify their gene expression, with applications including tissue engineering, regenerative medicine, sensors and diagnostics, and gene therapy. Viral vectors are considered the most effective system to deliver nucleic acids, yet safety concerns and many other disadvantages have resulted in investigations into an alternative option, i.e. nonviral gene delivery. Chemical nonviral gene delivery is typically accomplished by electrostatically complexing cationic lipids or polymers with negatively charged nucleic acids. Unfortunately, nonviral gene delivery suffers from low efficiency due to barriers that impede transfection success, including intracellular processes such as internalization, endosomal escape, cytosolic trafficking, and nuclear entry. Efforts to improve nonviral gene delivery have focused on modifying nonviral vectors, yet a novel solution that may prove more effective than vector modifications is stimulating or “priming” cells before transfection to modulate and mitigate the cellular response to nonviral gene delivery. In applications where a cell-material interface exists, cell priming can come from cues from the substrate, through chemical modifications such as the addition of natural coatings, ligands, or functional side groups, and/or physical modifications such as topography or stiffness, to mimic extracellular matrix cues and modulate cellular behaviors that influence transfection efficiency. This review summarizes how biomaterial substrate modifications can prime the cellular response to nonviral gene delivery (e.g. integrin binding and focal adhesion formation, cytoskeletal remodeling, endocytic mechanisms, intracellular trafficking) to aid in improving gene delivery for future therapeutic applications

Controlled Release Systems for DNA Delivery

Adapting controlled release technologies to the delivery of DNA has the potential to overcome extracellular barriers that limit gene therapy. Controlled release systems can enhance gene delivery and increase the extent and duration of transgene expression relative to more traditional delivery methods (e.g., injection). These systems typically deliver vectors locally, which can avoid distribution to distant tissues, decrease toxicity to nontarget cells, and reduce the immune response to the vector. Delivery vehicles for controlled release are fabricated from natural and synthetic polymers, which function either by releasing the vector into the local tissue environment or by maintaining the vector at the polymer surface. Vector release or binding is regulated by the effective affinity of the vector for the polymer, which depends upon the strength of molecular interactions. These interactions occur through nonspecific binding based on vector and polymer composition or through the incorporation of complementary binding sites (e.g., biotin–avidin). This review examines the delivery of nonviral and viral vectors from natural and synthetic polymers and presents opportunities for continuing developments to increase their applicability

Incorporation of Polyethylene Glycol into Self-Assembled Monolayers Enhances Substrate-Mediated Gene Delivery by Nonspecifically- Bound Complexes

Developing systems capable of controlled and efficient gene transfer is a fundamental goal of biotechnology, with applications including functional genomics, gene therapy, and tissue engineering. Substrate-mediated delivery, also termed solid phase delivery, describes the immobilization of DNA, complexed with nonviral vectors, to a biomaterial or substrate through specific or nonspecific interactions. Cells cultured on the substrate are exposed to elevated DNA concentrations within the local microenvironment, which enhances transfection. We investigated transfection resulting from DNA complexes immobilized to a substrate through specific interactions introduced through complementary functional groups on the vector and surface or through nonspecific interactions. Self-assembled monolayers (SAMs) of alkanethiols on gold were used to provide a controlled surface to investigate transfection following specific and non-specific immobilization. DNA, complexed with polyethylenimine (PEI), was immobilized to SAMs through nonspecific mechanisms or covalently linked to SAMs presenting appropriate functional groups through a fraction of the functional groups available on the PEI present in the complex. Nonspecific immobilization of DNA complexes and subsequent transfection was mediated by the hydrophilicity and ionization of the substrate, while covalent tethering resulted in immobilized quantities similar to nonspecific conditions, but provided no transfection. Subsequent studies incorporated polyethylene glycol (PEG)-terminated alkanethiols into the SAMs to reduce nonspecific complex adsorption. Covalent tethering of complexes to PEG/carboxylic acid monolayers resulted in statistically less complex immobilization and no transfection. Nonspecific immobilization to monolayers containing 40% PEG resulted in statistically less DNA complexes immobilized, but substantially greater transfection. Cell adhesion was not affected at this percentage of PEG. Similarly, the addition of Pluronic block copolymers (of polyethylene oxide and polypropylene oxide) to surfaces also enhanced transfection. We hypothesize that the presence of PEG in the monolayer may better preserve complex conformation upon binding to substrates, thereby enhancing the activity of substrate-mediated delivery of DNA complexes

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

Temporal endogenous gene expression profiles in response to polymer-mediated transfection and profile comparison to lipid-mediated transfection

Background Design of efficient nonviral gene delivery systems is limited by the rudimentary understanding of specific molecules that facilitate transfection. Methods Polyplexes using 25-kDa polyethylenimine (PEI) and plasmid encoding green fluorescent protein (GFP) were delivered to HEK 293T cells. After treating cells with polyplexes, microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h of exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP–) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. Differentially expressed genes in polyplex-mediated transfection were compared with those differentially expressed in lipoplex transfection to identify DNA carrier-dependent molecular factors. Results Differentially expressed genes were RGS1, ARHGAP24, PDZD2, SNX24, GSN and IGF2BP1 after 2 h; RAP1A and ACTA1 after 8 h; RAP1A, WDR78 and ACTA1 after 16 h; and RAP1A, SCG5, ATF3, IREB2 and ACTA1 after 24 h. Pharmacologic studies altering endogenous levels for ARHGAP24, GSN, IGF2BP1, PDZD2 and RGS1 were able to increase or decrease transgene production. Comparing differentially expressed genes for polyplexes and lipoplexes, no common genes were identified at the 2-h time point, whereas, after the 8-h time point, RAP1A, ATF3 and HSPA6 were similarly expressed. SCG5 and PGAP1 were only upregulated in polyplex-transfected cells. Conclusions The identified genes and pharmacologic agents provide targets for improving transfection systems, although polyplex or lipoplex dependencies must be considered. Includes supplementary materials

Dynamic analysis of DNA nanoparticle immobilization to model biomaterial substrates using combinatorial spectroscopic ellipsometry and quartz crystal microbalance with dissipation

Gene expression within cells can be altered through gene delivery approaches, which have tremendous potential for gene therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery describes the delivery of plasmid DNA or DNA complexed with nonviral vectors to cells from a surface, with the DNA immobilized to a substrate through specific or nonspecific interactions. In this work, DNA-nanoparticle (DNA–NP) adsorption to substrates is evaluated using combinatorial, in situ spectroscopic ellipsometry and quartz crystal microbalance with dissipation (SE/QCM-D), to evaluate the basic dynamic processes involved in the adsorption and immobilization of DNA–NP complexes to substrates. The concentration of DNA–NP solutions influences the adsorbed DNA–NP surface mass, which increases by a factor of approximately 6 (detected by SE) and approximately 4.5-fold (detected by QCM-D), as the DNA concentration increases from 1.5 μg/mL to 15 μg/mL, with an increase in layer porosity. In addition, SE/QCM-D analysis indicates that DNA–NP adsorption rates, surface coverage densities, and volume fractions are dependent on the type of substrate: gold (Au) and silicon dioxide substrates, proteincoated and uncoated substrates, and surfaces modified with alkanethiol self assembled monolayers (SAMs). These studies also demonstrate that the influence of an adsorbed protein layer on resulting DNA–NP immobilization efficiency is substrate dependent. For example, Au surfaces coated with fetal bovine serum (FBS) resulted in two-fold greater mass of adsorbed DNA–NPs, compared to DNA–NP adsorption to FBS-coated SAM substrates. This investigation offers insights into dynamic DNA–NP surface adsorption processes, characteristics of the immobilized DNA–NP layer, and demonstrates substrate-dependent DNA–NP adsorption

Temporal endogenous gene expression profiles in response to lipid-mediated transfection

Background — Design of efficient nonviral gene delivery systems is limited as a result of the rudimentary understanding of the specific molecules and processes that facilitate DNA transfer. Methods — Lipoplexes formed with Lipofectamine 2000 (LF2000) and plasmid-encoding green fluorescent protein (GFP) were delivered to the HEK 293T cell line. After treating cells with lipoplexes, HG-U133 Affymetrix microarrays were used to identify endogenous genes differentially expressed between treated and untreated cells (2 h exposure) or between flow-separated transfected cells (GFP+) and treated, untransfected cells (GFP–) at 8, 16 and 24 h after lipoplex treatment. Cell priming studies were conducted using pharmacologic agents to alter endogenous levels of the identified differentially expressed genes to determine effect on transfection levels. Results — Relative to untreated cells 2 h after lipoplex treatment, only downregulated genes were identified ≥ 30-fold: ALMS1, ITGB1, FCGR3A, DOCK10 and ZDDHC13. Subsequently, relative to GFP– cells, the GFP+ cell population showed at least a five-fold upregulation of RAP1A and PACSIN3 (8 h) or HSPA6 and RAP1A (16 and 24 h). Pharmacologic studies altering endogenous levels for ALMS1, FCGR3A, and DOCK10 (involved in filopodia protrusions), ITGB1 (integrin signaling), ZDDHC13 (membrane trafficking) and PACSIN3 (proteolytic shedding of membrane receptors) were able to increase or decrease transgene production. Conclusions — RAP1A, PACSIN3 and HSPA6 may help lipoplex-treated cells overcome a transcriptional shutdown due to treatment with lipoplexes and provide new targets for investigating molecular mechanisms of transfection or for enhancing transfection through cell priming or engineering of the nonviral gene delivery system. Includes supporting materials

Dynamic analysis of DNA nanoparticle immobilization to model biomaterial substrates using combinatorial spectroscopic ellipsometry and quartz crystal microbalance with dissipation

Gene expression within cells can be altered through gene delivery approaches, which have tremendous potential for gene therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery describes the delivery of plasmid DNA or DNA complexed with nonviral vectors to cells from a surface, with the DNA immobilized to a substrate through specific or nonspecific interactions. In this work, DNA-nanoparticle (DNA–NP) adsorption to substrates is evaluated using combinatorial, in situ spectroscopic ellipsometry and quartz crystal microbalance with dissipation (SE/QCM-D), to evaluate the basic dynamic processes involved in the adsorption and immobilization of DNA–NP complexes to substrates. The concentration of DNA–NP solutions influences the adsorbed DNA–NP surface mass, which increases by a factor of approximately 6 (detected by SE) and approximately 4.5-fold (detected by QCM-D), as the DNA concentration increases from 1.5 μg/mL to 15 μg/mL, with an increase in layer porosity. In addition, SE/QCM-D analysis indicates that DNA–NP adsorption rates, surface coverage densities, and volume fractions are dependent on the type of substrate: gold (Au) and silicon dioxide substrates, proteincoated and uncoated substrates, and surfaces modified with alkanethiol self assembled monolayers (SAMs). These studies also demonstrate that the influence of an adsorbed protein layer on resulting DNA–NP immobilization efficiency is substrate dependent. For example, Au surfaces coated with fetal bovine serum (FBS) resulted in two-fold greater mass of adsorbed DNA–NPs, compared to DNA–NP adsorption to FBS-coated SAM substrates. This investigation offers insights into dynamic DNA–NP surface adsorption processes, characteristics of the immobilized DNA–NP layer, and demonstrates substrate-dependent DNA–NP adsorption

Use of a three-dimensional in vitro alginate hydrogel culture model to direct zonal formation of growth plate cartilage

Growth plate cartilage is found at the ends of long bones, and is responsible for the growth of the bones as a person is developing. The architecture of this growth plate is very specific and contributes to proper function to allow for bone growth. Although there are many factors known to be involved in the formation of the growth plate and its proper regulation, the exact mechanisms involved in these processes are not fully understood. So far, previous attempts to recapitulate a functioning growth plate in vitro have been unsuccessful. In this study, a new method to study the growth plate and the mechanisms involved in its formation was developed using an in vitro cell culture system made of alginate hydrogel scaffolds. Chondrocytes isolated from neonatal mouse growth plates were encapsulated within hydrogel beads and cultured. Please click Additional Files below to see the full abstract