Efficient Transfection of In vitro Transcribed mRNA in Cultured Cells Using Peptide-Poloxamine Nanoparticles

Podsumowanie

A self-assembled peptide-poloxamine nanoparticle (PP-sNp) is developed using a microfluidic mixing device to encapsulate and deliver in vitro transcribed messenger RNA. The described mRNA/PP-sNp could efficiently transfect cultured cells in vitro.

Streszczenie

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

Wprowadzenie

Vaccination has been heralded as one of the most efficient medical interventions for reducing the morbidity and mortality caused by infectious diseases¹. The importance of vaccines has been demonstrated since the outbreak of coronavirus disease 2019 (COVID-19). As opposed to the traditional concept of injecting inactivated or live-attenuated pathogens, state-of-the-art vaccine approaches, such as nucleic acid-based vaccines, concentrate on preserving the immune-stimulatory properties of the target pathogens while avoiding the potential safety issues associated with the conventional whole-microbial virus- or in bacteria-based vaccines. Both DNA- and RNA (i.e., in vitro transcribed messenger RNA, IVT mRNA)-based vaccines exhibit prophylactic to therapeutic potential against a variety of diseases, including infectious diseases and cancers^2,3. In principle, the potential of nucleic acid-based vaccines relates to their production, efficacy, and safety⁴. These vaccines can be manufactured in a cell-free manner to allow cost-effective, scalable, and rapid production.

A single nucleic acid-based vaccine can encode multiple antigens, enabling the target of numerous viral variants or bacteria with a reduced number of inoculations and strengthening the immune response against resilient pathogens^5,6. Besides, nucleic acid-based vaccines could mimic the natural invasion process of virus or bacterial infection, bringing both B cell- and T cell-mediated immune responses. Unlike some virus- or in DNA-based vaccines, IVT mRNA-based vaccines offer a huge advantage in terms of safety. They can rapidly express the desired antigen in the cytosol and are not integrated into the host genome, obviating concerns about insertional mutagenesis⁷. IVT-mRNA is automatically degraded after successful translation, so its protein expression kinetics can be easily controlled^8,9. Catalyzed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, efforts from companies/institutions worldwide have enabled the release to the market of many types of vaccines. IVT mRNA-based vaccine technology shows great potential and, for the first time, has demonstrated its previously anticipated success, owing to its rapid design and flexible capacity to adapt to any target antigens within several months. The success of IVT mRNA vaccines against COVID-19 in clinical applications not only opened up a new era of IVT mRNA vaccine research and development but also accumulated valuable experience for the rapid development of effective vaccines for dealing with outbreaks of infectious diseases^10,11.

Despite the promising potential of IVT mRNA vaccines, the efficient intracellular delivery of IVT mRNA to the site of action (i.e., cytoplasm) continues to pose a major hurdle¹², especially for those administered via the airways⁴. IVT mRNA is inherently an unstable molecule with an extremely short half-life (~7 h)¹³, which renders IVT mRNA highly prone to degradation by the ubiquitous RNase¹⁴. The lymphocytes of the innate immune system tend to engulf the recognized IVT mRNA in cases of in vivo application. Moreover, the high negative charge density and large molecular weight (1 x 10⁴-1 x 10⁶ Da) of IVT mRNA impair its effective permeation across the anionic lipid bilayer of cellular membranes¹⁵. Therefore, a delivery system with certain bio-functional materials is required to inhibit the degradation of the IVT mRNA molecules and facilitate cellular uptake¹⁶.

Apart from a few exceptional cases in which naked IVT mRNA was directly utilized for in vivo investigations, various delivery systems are used to carry IVT mRNA to the therapeutic site of action^17,18. Previous studies have revealed that only a few IVT mRNAs are detected in cytosol without the assistance of a delivery system¹⁹. Numerous strategies have been developed to improve RNA delivery with continuous efforts in the field, ranging from protamine condensation to lipid encapsulation²⁰. Lipid nanoparticles (LNPs) are the most clinically advanced among the mRNA delivery vehicles, as proved by the fact that all the approved mRNA COVID-19 vaccines for clinical use employ LNP-based delivery systems²¹. However, LNPs cannot mediate effective mRNA transfection when the formulations are delivered via the respiratory route²², which remarkably limits the application of these formulations in inducing mucosal immune responses or addressing pulmonary-related diseases such as cystic fibrosis or α1-antitrypsin deficiency. Therefore, developing a novel delivery system to facilitate the efficient delivery and transfection of IVT mRNA in airway-related cells is required to solve this unmet need.

It has been confirmed that the peptide-poloxamine self-assembled nanoparticle (PP-sNp) delivery system can mediate the efficient transfection of nucleic acids in the respiratory tract of mice²³. The PP-sNp adopts a multifunctional modular design approach, which can integrate different functional modules into the nanoparticles for rapid screening and optimization²³. The synthetic peptides and electrically neutral amphiphilic block copolymers (poloxamine) within the PP-sNp can spontaneously interact with IVT mRNA to generate uniformly distributed nanoparticles with a compact structure and smooth surface²³. PP-sNp can improve the gene transfection effect of IVT mRNA molecules in cultured cells and the respiratory tract of mice²³. The present study describes a protocol for generating PP-sNp containing IVT mRNA that encodes Metridia luciferase (MetLuc-mRNA) (Figure 1). Controlled and rapid mixing via a microfluidic mixing device, which employs the staggered herringbone mixing design, is utilized in this protocol. The procedure is easy to execute and allows the generation of PP-sNp with more uniform sizes. The general goal of PP-sNp production using the microfluidic mixer is to create PP-sNp for mRNA complexation in a well-controlled manner, thus allowing efficient and reproducible cell transfection in vitro. The present protocol describes the preparation, assembly, and characterization of PP-sNp containing MetLuc-mRNA.

Protokół

1. In vitro transcription of chemically modified mRNA

NOTE: It is required to use nuclease-free tubes, reagents, glassware, pipette tips, etc., because RNases are ubiquitous in the environment, such as laboratory solutions, instrument surfaces, hair, skin, dust, etc. Clean the bench surfaces and pipettes thoroughly before use, and wear gloves to avoid RNase contamination.

1. Perform linearization of the DNA template.
   1. Synthesize the open reading frame (ORF) of Metridia luciferase (MetLuc) flanked by a T7 polymerase promoter, 5' untranslated region (UTR), 3'UTR, and poly (A) tails and clone it into the PUC57 vector, which is expressed in bacteria (see Table of Materials).
      NOTE: The coding sequence of the DNA template is provided in Supplementary File 1.
   2. Culture bacteria, lyse the bacteria, and purify the plasmid DNA by the corresponding column (see plasmid DNA extraction kit in the Table of Materials).
   3. Perform a single 50 µL reaction that contains 2 µL of BamHI, 2 µL of KpnI (see Table of Materials), and 2 µg of plasmid DNA at 37 °C for 1 h, and achieve linearization of the DNA template.
2. Perform in vitro transcription to generate uncapped-IVT mRNA.
   1. Perform mRNA synthesis by a single 20 µL reaction using a T7 transcription kit and pseudouridine (see Table of Materials): 10 µL (0.8-1 µg) of linearized DNA template, 1.5 µL (150 mM) of ATP, pseudo-UTP, GTP, and CTP, 2 µL of 10x transcription buffer, 1 µL of T7 enzyme, and 1 µL of nuclease-free water (NF-water). Mix the abovementioned components thoroughly and incubate at 37 °C for 3 h.
3. Remove the template DNA.
   1. Add 1 µL (1 U) of DNase (RNase-free) after the transcription process and incubate at 37 °C for 15 min.
4. Perform uncapped-IVT mRNA purification using lithium chloride precipitation.
   1. Purify the uncapped-IVT mRNA using lithium chloride (see Table of Materials) with a working concentration of 10 mM. Add 50 µL of 10 mM lithium chloride to 20 µL of uncapped-IVT mRNA solution.
   2. Mix the complete volume thoroughly and chill at −20 °C for 1 h. Collect the uncapped-IVT mRNA for 12 min at 12,000 x g, which routinely produces 80-120 µg of uncapped-IVT mRNA from each single 20 µL reaction.
5. Perform IVT mRNA capping.
   1. Perform the IVT mRNA capping using the cap 1 capping system (see Table of Materials). Briefly, remove the secondary structure of 50 µg of uncapped-IVT mRNA by heating at 65 °C for 10 min, then link the 5' end of uncapped-IVT mRNA with cap 1, which is prepared by modifying the m7G cap structure using 2.5 µL of s-adenosylmethionine (SAM) (20 mM) and 4 µL of 2'-O-methyltransferase (100 U) in a 100 µL reaction at 37 °C for 30-60 min.
   2. Purify 100 µL of the capped IVT mRNA using 250 µL of 10 mM lithium chloride and dilute in 50 µL of NF-water.
6. Determine the purity and molecular size of capped-IVT mRNA.
   1. Measure the concentration of capped-IVT mRNA with a UV-visible spectrophotometer. Using an RNA marker (see Table of Materials), analyze the molecular size of capped-IVT mRNA in a 1% formaldehyde denaturing agarose gel containing 18% formaldehyde (voltage is 120 V). Ensure the uncapped-IVT mRNA and capped-IVT mRNA are stored at −80 °C.
      ​NOTE: The IVT mRNA was considered to have good purity in an A₂₆₀/A₂₈₀ ratio of 1.8-2.1 and an A₂₆₀/A₂₃₀ ratio of 2.0 or slightly higher.

2. Generation of IVT mRNA/PP-sNp

1. Prepare poloxamine 704 (T704) stock solution by solubilizing the T704 (see Table of Materials) in NF-water to obtain a 10 mg/mL stock solution. Store the prepared solution at 4 °C.
   NOTE: T704 contains an X-shaped structure made of an ethylenediamine central group bonded to four chains of poly(propylene oxide) (PPO) blocks and poly(ethylene oxide) (PEO) blocks²⁴. The molecular weight (Mw) of T704 is 5500.
2. Prepare the synthetic peptide (sPep, see Table of Materials) stock solution by solubilizing the sPep (sequence: KETWWETWWTEWWTEWKKKKRRRRRKKKKGACSE
   RSMNFCG) in NF-water to obtain a 2 mg/mL stock solution and store at 4 °C.
3. Prepare the IVT mRNA solution by thawing the IVT mRNA (step 1) on ice and centrifuging shortly for 3 s at 300 x g at room temperature before opening the tube. Dilute the IVT mRNA solution to 0.04 µg/µL with NF-water.
   NOTE: It is recommended to work in a biosafety cabinet whenever possible with the IVT mRNA.
4. Prepare T704 and sPep mix solution by diluting the sPep solution to 0.555 µg/µL and the T704 solution to 8 µg/µL with NF-water. Incubate the mixed solution for 15 min at room temperature prior to further use.
   NOTE: Calculate the required sPep component based on the desired N/P ratio. The N/P ratio is the total number of nitrogen residues (N) within the sPep to the total number of negatively charged phosphate groups (P) within the IVT mRNA. Calculate the required T704 based on the weight/weight (w/w) ratio between T704 and IVT mRNA. It is necessary that the N/P ratio is 5 and the w/w ratio is 100.
5. Prepare the IVT mRNA/PP-sNp formulation following the steps below.
   1. Draw the IVT mRNA solution (step 3) into a 1 mL syringe, ensuring no air gaps or bubbles in the syringe tip. Load the syringe into one side of the cartridge next to the rotating block.
   2. Fill a 1 mL syringe with the T704 and sPep mix solution (step 4). Remove any bubbles or air gaps at the syringe tip and put the syringe into the other inlet of the pump (see Table of Materials).
   3. Set the pump with a flow ratio of 1:1 and a total flow rate of 4-10 mL/min.
      NOTE: A total flow rate of 6 mL/min is optimal in the studies presented here.
   4. Place a 10 mL RNase-free conical tube (see Table of Materials) to collect the mixed IVT-mRNA/PP-sNp solution at the end of the flow path of the mixing device.
   5. Run the pump to start the mixing, ensuring the parameters are input correctly. After the pump is finished running for 6 s, collect the IVT-mRNA/PP-sNp sample from the conical tube.
      NOTE: PP-sNp were generated using a microfluidic mixer (Supplementary Figure 1). The device comprises a constant flow pump, link device, chip, and fixed device. In the mixing process, the constant flow pump connected to the chip delivers liquid to the chip according to the preset flow rate. The connected constant flow pump can be connected to multiple input channels of the chip with a single output channel. The components of the device, including the chip, were obtained from commercial sources and rationally assembled (see Table of Materials). The parameters, such as flow rate and volume of the IVT mRNA solution and T704-sPep mixed solution, may differ from those displayed in the current protocol if some different setup is used and must be optimized accordingly.

3. Measurement of the hydrodynamic diameter and polydispersity of IVT-mRNA/PP-sNp

1. Dilute an aliquot of the IVT mRNA/PP-sNp solution (step 2) with NF-water to obtain a final volume of 1 mL.
2. Measure the hydrodynamic size and polydispersity index (PDI) using a semi-micro cuvette²⁵. Add the IVT mRNA/PP-sNp solution into the cuvette and place it into the particle size meter (see Table of Materials). Set up a standard operating procedure and click on Start to begin the data acquisition.

4. Preparation of the cells for transfection

1. Plate human bronchial epithelial cells (16HBE) and a dendritic cell line (DC2.4) in 96-well-plates at a density of 3.5 x 10⁴ cells/well 24 h prior to transfection. Grow the cells in a culture medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin.
   NOTE: The cells were obtained from a commercial source (see Table of Materials).
2. Incubate the cells in an incubator (37 °C and 5% CO₂ atmosphere) for 24 h to ensure the cells are 60%-80% confluent before the transfection.

5. Transfection of the cultured cells

1. After removing the growth medium, wash the plated cells with 0.2 mL/well 1x PBS.
2. Add 170 µL of serum-free culture medium to each well containing the plated cell cultures.
3. Add the IVT mRNA/PP-sNp formulation containing 0.4 µg of MetLuc mRNA (prepared in step 2) dropwise with an amount of 30 µL/well.
4. Incubate the cells with the IVT mRNA/PP-sNp formulation at 37 °C in a humidified 5% CO₂-enriched atmosphere for 4 h.
5. Replace the transfection medium with 0.2 mL of fresh culture medium supplemented with 10% heat-inactivated fetal bovine serum and 1% (v/v) penicillin/streptomycin.
   NOTE: The fetal bovine serum was heated at 56 °C for 30 min for inactivation.
6. Incubate the transfected cells at 37 °C and in a 5% CO₂-enriched atmosphere for 24 h and collect the supernatants to be detected from each well.

6. Analysis of cell transfection efficacy using Metridia luciferase (MetLuc) assay

1. Assay the supernatant from each well for MetLuc activity using coelenterazine substrate (see Table of Materials) following the steps below.
   1. Prepare a fresh assay solution by adding 1x PBS to the coelenterazine substrate (the concentration of coelenterazine is 15 mM).
   2. Vortex the coelenterazine solution for 10 s for thorough mixing.
   3. Add 50 µL of supernatant (collected in step 5) to a 96-well plate.
   4. Set up the microplate reader (see Table of Materials) with a 1,000 ms reading time before adding 30 µL of coelenterazine solution (15 mM) to each well manually or by automated injection.
   5. Click on Start to measure the luminescence signal immediately after adding the coelenterazine solution to the supernatant.
      NOTE: The luminescence signal is measured using a microplate reader, and its activity is expressed in relative light units. The values obtained from PBS-transfected wells will be used as blank controls.

Wyniki

The recombinant plasmid was digested to produce the linearized DNA template (Figure 2A). Using the protocol described, The T7 in vitro transcription kit can produce up to 80-120 µg of uncapped MetLuc-mRNA per 20 µL reaction and 50-60 µg of capped MetLuc-mRNA per 100 µL reaction. When analyzed with electrophoresis, intact MetLuc-mRNA with high quality should show a single and clear band, as displayed in Figure 2B. Contaminants introdu...

Dyskusje

The protocol described here not only allows the cost-effective and rapid production of IVT mRNA vaccine formulations with defined properties, but it also offers the possibility to customize the PP-sNp formulation according to specific therapeutic purposes, such as gene therapy. In order to ensure the successful generation of IVT mRNA/PP-sNp, it is suggested to pay extra attention to some critical steps. When working with mRNA, always remember that RNase-free conditions should be maintained throughout the process because ...

Materiały

Name	Company	Catalog Number	Comments
BamHI	Takara	1010
cap 1 capping system	Jinan	M082
Dendritic cell-line	Sigma	SCC142
DNA sequence	Genescript
Human bronchial epithelial cells	Sigma	SCC150
KpnI	Takara	1068
LP	Beyotime	C0533
Lithium chloride	APEXBio	B6083
Malvern Zetasizer Nano ZS90	Malvern	NB007605
Microfluidic chip	ZHONGXINQIHENG	Standard PDMS chip
Microplate readers	ThermoFisher	Varioskan lux
NanoDrop One	ThermoFisher	ND-ONE-W (A30221)
Nuclease-free water	ThermoFisher	AM9932
OptiMEM	Gibco	31985070
Penicillin-streptomycin	Gibco	15140122
Pseudouridine	APE×Bio	B7972
QIAprep Spin Miniprep Kit	Qiagen	27106
Quanti-Luc	InvivoGen	Rep-qlc2
RiboRuler High Range RNA Ladder	ThermoFisher	SM1821
RNase-free conical tube	Biosharp	BS-100-M
RPMI Medium 1640	ThermoFisher	C11875500BT
Syringe pump	Chemyx	Fusion 101
T7 transcription Kit	Jinan	E131