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

Abstract

BackgroundMesenchymal 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 textTransfection 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.ConclusionRecent 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.

Smurf1 Silencing Using a LNA-ASOs/Lipid Nanoparticle System to Promote Bone Regeneration

“…Our group has previously shown that silencing of the ubiquitin ligase Smurf1 using a lentiviral vector carrying a specific siRNA leads to a significant improvement of bone formation . Although genetic modification of MSCs by viral transfection is highly efficient, designing clinical applications based on this system is restricted due to important safety concerns . We have managed to overcome this problem by transiently modifying MSCs using GapmeRs, a specific type of LNA‐ASOs with enhanced stability that represent a clinically safe way to modify MSC gene expression .…”

<p>Growth Factor Gene-Modified Mesenchymal Stem Cells in Tissue Regeneration</p>

“…The most common vectors used for such approaches are compiled in Table 2. [31][32][33][34][35][36][37][38][39] Using viral vectors to insert genes into MSCs is a high transduction efficiency approach that has the potential to induce off-target effects owing to insertional mutagenesis. 32,35,40,41 Viral systems are also limited by relatively small transgene cargo capacity, high production cost, difficulties in production and scale-up, and adverse immune reactions.…”

“…These alternative delivery strategies are more scalable and flexible, easier to synthesize and target to tissues, less likely to drive immune stimulation, and more amenable to scale-up manufacturing. 37 However, the disadvantages of non-viral vectors can include their transient expression with low efficiencies, and their potential for associated toxicity. He et al 42 utilized the cationic polymer pullulan-spermine to overexpress HGF encoded in the pMEX vector in MSCs, resulting in high in vitro HGF expression.…”

Improving Cell Therapy Survival and Anabolism in Harsh Musculoskeletal Disease Environments

“…64,65 An option for overcoming this concern would be the use of nonviral gene delivery approaches. Nonviral gene delivery has historically had low delivery efficacy; however, in recent years, there have been strides made in this field that have increased the efficacy of nonviral gene delivery, 66 improving the likelihood that it may be an effective delivery option moving forward.…”