Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection ...criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.
The study of Tau protein in disease-relevant neuronal cells in culture requires efficient delivery systems for transfection of exogenous Tau and also modulators and interactors of Tau. Transfection ...of cultivated cells using calcium phosphate precipitation is a simple and cost-effective approach, also for difficult-to-transfect and sensitive cells such as primary neurons. Because of its low cell toxicity and ease of use, the Ca
-phosphate transfection method is one of the most widely used gene transfer procedures in neuroscience. However, Ca
-phosphate transfection efficacy in neurons is poor, often in the range of 1-5%, limiting its use in functional investigations. Here, we outline our improved Ca
-phosphate transfection methodology for human iPSC-derived neurons that yields a reasonable efficiency (20-30% for bright volume markers) without apparent effects on cell health. We have used it to introduce wild-type and mutant human Tau with and without co-transfection of a volume marker (used here: tdTomato). In sum, our procedure can deliver neuronal genes (e.g., MAPT) using typical eukaryotic expression vectors (e.g., using CMV promoter) and is optimized for transfection of human iPSC-derived neurons.
Upstream production of AAV therapies is often driven by transient transfection of suspension HEK cells with multiple plasmid DNA constructs encoding essential virus proteins. AAV therapies require ...high-quality and high titers of viral genomes per dosage, prompting the need for a more efficient AAV manufacturing method to reduce overall costs. Improving AAV titers and quality hold significant implications for the economical manufacturing of high-dose assets and mainstream utilization of AAV gene therapy.
The RevIT™ AAV Enhancer is compatible with TransIT-VirusGEN® Transfection Reagent or conventional polymeric transfection reagents. RevIT™ AAV Enhancer has the potential to increase higher genome titers 2-4X in a range of suspension HEK 293 subtypes and cell culture media. When combined with the TransIT-VirusGEN® Transfection Reagent, this enhancer not only yields the highest titers but also allows for the use of lower amounts of plasmid DNA which represents a key cost-saving opportunity.
Additional features of RevIT™ include flexibility to produce multiple serotypes and transfection complex stability. RevIT™ AAV Enhancer produces high quality titers across a range of AAV serotypes, including AAV2, AAV5, AAV8, and AAV9. Complex stability is also a hallmark of TransIT-VirusGEN and RevIT with extended complex formation times required in large-scale AAV manufacture that do not negatively impact cell viability. We also implemented Design of Experiments (DOE) to optimize and determine the interactions of DNA dosage, the TransIT-VirusGEN:DNA ratio, and cell density. Optimized conditions were then tested in combination with a range of the RevIT™ AAV Enhancer to further increase genome titers by >2-fold. Productions with TransIT-VirusGEN® and RevIT™ AAV Enhancer are linearly scalable; small-scale shake flask model can be employed for DOE screening and be representative of results of stirred tank bioreactors. Through increasing recombinant AAV titers, RevIT™ AAV Enhancer paves the way for driving down upstream manufacturing costs, resulting in a lower cost per therapeutic dose.
Optimization of transfection processes is imperative to successful cellular drug product manufacturing. A key requirement for efficient transfection is utilization of an optimized buffer. ...Transfection buffer formulation is a multiplexed problem that can benefit from sophisticated optimization methods. After defining a base formulation via traditional experimental design principles (DOE), we implemented a custom, hybrid particle swarm optimization and differential evolution (PSODE) algorithm with archived memory to simultaneously screen and optimize efficacy of numerous buffer additives.
PSODE was implemented with adaptive social and cognitive parameters to balance design exploration and exploitation of information. Mutation and crossover of designs further leveraged the information gained from successful formulations. In this approach, additives were evaluated to improve delivery of GFP nanoplasmid DNA into Jurkats. Traditional DOE methods with 22 additives (i.e., 3k factorial design) results in a high-dimension space requiring >300E6 experimental iterations to characterize. PSODE identified optimal formulations after 5 iterations when combined with multi-objective optimization (MOO) (Figure 1) of transfection efficiency and cell viability. PSODE formulations demonstrated >75% transfection efficiency with >80% viability in Jurkats (Figure 2), a 3-fold increase over base formulation alone. PSODE formulations were subsequently tested in primary T cells, demonstrating >75% viability and >50% transfection efficiency. Transfections with various Flowfect® profiles identified synergistic buffer and energy effects, indicating further optimization is possible. PSODE implementation in Jurkats and T cells demonstrate the valuable coaction of Flowfect® technology and algorithm-based study designs, yielding high-throughput discovery capabilities to screen, optimize, and characterize transfection processes and parameters. In tandem, algorithmic buffer optimization methods can benefit cell and gene therapy applications, enhancing future development of specialized, optimal transfection processes for other cell type and payload combinations.
The development and mass adoption of cell and gene therapies has been limited by the lack of safe and effective delivery methods. The current widely accepted methods of delivery have significant ...limitations or side effects that prevent widespread application. Electroporation has unreliable dosage control regarding target cargo and can be significantly harmful to sensitive cell types. Viral vectors can cause unexpected downstream side effects and are unable to carry large payloads. Microinjections are time-consuming to an unscalable extent. Lipid and polymer-based methods are not reliable and cannot be used with all cell types. Such limitations have prevented the full potential of cell therapy development and CRISPR from widespread adoption, though they show promise in treating the previously untreatable.
Here we describe our novel cell engineering platform which utilizes an array of solid silicon nanoneedles to enable delivery of biomolecular payloads without the use of viral vectors or electroporation. Our method of silicon nanoneedle fabrication utilizes semiconductor technology which allows us to produce needles of consistent height and width across the array. This needle chip is loaded onto our device platform, along with cells that are first trapped using a microfluidic system capable of temporarily immobilizing the cells in an array, ensuring a single needle enters a single cell. This method, which causes minimal cell damage, unlocks unique capabilities for cell engineering, such as the ability to deliver high-risk payloads to cells with minimal loss of cell viability and an unprecedented level of dose control. Additionally, our method has been demonstrated to be independent of cell or cargo type and has been utilized to deliver a plethora of cargo (CRISPR-Cas reagents, nucleic acids) into a variety of cells (immortalized, primary, stem). With this, we show how our combination of unique technologies will revolutionize the future of cellular therapies with precision and scalability.
Effective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications. Pseudovirus-based systems and ...electro-transfection are the most popular strategies for genetic material transduction. Compared with viral-particle-mediated approaches, electro-transfection is safer, with a faster process time, and lower cost. However, this method is associated with low cell yields and is laborious due to multiple handling steps. Lipid nanoparticles (LNP) have emerged as a highly effective tool for nucleic acid delivery in vivo, yet have been difficult to demonstrate delivery to primary T-cells.
Here we demonstrate the development of a novel LNP composed of biodegradable COATSOME® SS-Series that provide high level of gene transfection into human primary T-cells ex-vivo with a rapid single step transfection protocol. Compared to electroporation, the expression of an mRNA luciferase reporter was 13 fold higher in CD3+ activated human primary T-cells. The COATSOME® SS-Series were also evaluated in vivo with single doses up to 124 mg./kg being well tolerated in mice.
We also describe efforts to lower the cellular toxicity of ionizable lipids through improvements in biodegradable characteristics. Additional improvements in the design were undertaken to reduce the immunogenicity of ionizable lipids. Together these approaches can enable the accelerated development and manufacturing of cell therapies.
Human bone marrow-derived mesenchymal stromal cells (MSCs) have been investigated as a therapy for a wide variety of disease indications, because of their powerful intrinsic properties of ...self-regeneration, immunomodulation and multi-potency, as well as being readily available and easy to isolate and culture. Nevertheless, MSCs therapy suffers limited efficacy because of unique challenges like unfavorable microenvironmental factors, immunocompatibility, and stability. To improve some of their abilities, many studies have employed genetic engineering to transfer key genes into MSCs. Genetic modification of MSCs, both viral and nonviral methods, can be used to overexpress therapeutic proteins that complement the inherent properties. Alternatively, messenger RNA (mRNA) transfection is an effective tool to temporarily engineer MSCs and improve their modulatory properties. One major obstacle is the transfection efficiency and viability of human MSCs.
Here, we evaluated two commercial RNA transfection reagents developed by Mirus Bio: TransIT-VirusGEN®, and TransIT®-mRNA by using CleanCap® EGFP mRNA as reporter mRNA.
MSCs were plated in 2D flask and grown until 80% confluent. MSCs were then transfected with mRNA encoding GFP by using TransIT-VirusGEN®, a lipid and polymer transfection formulation suitable for GMP applications and specifically designed to support Cell and Gene Therapies requiring enhanced nucleic acid delivery to cells, and TransIT®-mRNA transfection reagent, in combination with the mRNA boost reagent, specifically designed to deliver large RNA in a variety of cells. The transfection complex reaction, ratio of mRNA and transfection reagents, were altered to determine optimal conditions. Transfected MSCs were incubated overnight and then transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis.
TransIT-VirusGEN® was more effective in terms of transfection efficiency and cell viability compared to TransIT®-mRNA. The highest transfection efficiency was obteined when the mRNA complexes were generated with the lower amount of mRNA (1 μg/mL). GFP transfected MSCs fluoresced by 18 hours post-transfection and maintained fluorescence up to 1 week, as detected by fluorescence microscopy. By flow cytometry analysis, TransIT-VirusGEN® could transfect over 90% of human MSCs compared to the lower 75% efficiency of TransIT®-mRNA.
Current gene delivery technologies for cell therapy applications rely on mechanical or electrical mechanisms which can negatively impact cell quality and result in wide-spread unregulated gene ...expression. Retaining cell health and function following the delivery of cargo is a pivotal advancement in the manufacturing of next generation gene-modified cell therapies.
Solupore is an automated, reproducible closed physicochemical transfection system for the delivery of a wide variety of cargo (RNA, DNA, protein) to primary immune cells as well as CD34+ cells for use in the development of clinical cell therapies. Solupore yields cells with superior cell quality and yield compared with electroporation. As a result, cells transfected using Solupore possess enhanced in vitro and in vivo function which potentially translates to improved clinical outcomes.
By analyzing the health and function of T cells in various in vitro and in vivo assays (apoptosis, cytokines, phenotype, avidity, and NSG mice), Solupore has been evaluated against electroporation to determine the impact on the cell quality, function, and ultimately the therapeutic potential.
We demonstrate that Solupore efficiently delivers cargo for multiplexed as well as sequential editing of primary T cells. We use CRISPR-Cas9 RNP based knockout of TRAC, B2M, and CD7 as proof of concept. Transduction of the clinically approved anti-CD19 CAR was used to benchmark functionality of transfected and edited cell populations.
Apoptosis is reduced from 25% to 12% in Solupore transfected cells, retaining 16% more TSCM cells, post cargo delivery. Release of IFN-γ is increased 1.45x fold more per cell in Solupore compared to electroporation. Cellular avidity is significantly improved, demonstrating over 70% of cells binding to target cancer cells for Solupore transfected cells, compared to just 37% for electroporation. This leads to enhanced cytotoxicity of target cancer cells in vitro and performs as a more potent and significantly more effective therapy in vivo, with more than a 30x fold enhanced tumour growth inhibition (pval = <0.0001). Solupore transfected cells engrafted 5.4x fold more compared to electroporation in NSG mice and retained their gene KO profile at terminal endpoint. Taking the in vitro data together with the in vivo data ultimately highlights that the Solupore technology retains more functional and healthier cells compared to an electroporation standard and significantly inhibited tumor growth.
Levitation and controlled motion of matter in air have a wealth of potential applications ranging from materials processing to biochemistry and pharmaceuticals. We present a unique acoustophoretic ...concept for the contactless transport and handling of matter in air. Spatiotemporal modulation of the levitation acoustic field allows continuous planar transport and processing of multiple objects, from near-spherical (volume of 0.1–10 μL) to wire-like, without being limited by the acoustic wavelength. The independence of the handling principle from special material properties (magnetic, optical, or electrical) is illustrated with a wide palette of application experiments, such as contactless droplet coalescence and mixing, solid–liquid encapsulation, absorption, dissolution, and DNA transfection. More than a century after the pioneering work of Lord Rayleigh on acoustic radiation pressure, a path-breaking concept is proposed to harvest the significant benefits of acoustic levitation in air.
PDF
