In Situ Generation of Claudin18.2-Specific CAR-T Cells via Lipid Nanoparticle–Mediated mRNA Delivery

Summary

CAR T-cell application to solid tumors is limited by complex ex vivo manufacturing and safety concerns. This protocol describes a method for the in situ generation of transient CAR-T cells via LNP–mediated delivery of CAR-encoding mRNA, providing a reproducible platform adaptable to other mRNA-based immune cell engineering applications.

Abstract

Chimeric antigen receptor (CAR) T-cell therapy has achieved remarkable clinical success in hematological malignancies; however, its broader application is limited by high manufacturing costs and insufficient efficacy against solid tumors. To overcome these challenges, an in situ CAR-T cell generation strategy was developed based on lipid nanoparticle (LNP)-mediated delivery of CAR mRNA targeting Claudin18.2, enabling the transient production of CAR-T cells directly within tumor tissue. Following LNP transfection, activated T cells exhibited efficient surface expression of CARs. These transient CAR-T cells demonstrated potent cytotoxic activity against Claudin18.2-positive gastric carcinoma cell lines, including NUGC4 and NCI-N87, and produced significant antitumor efficacy in tumor-bearing mouse models. This protocol describes detailed procedures for in vitro transcription of CAR mRNA, LNP formulation, T-cell activation and transfection, CAR expression analysis, and in vitro and in vivo functional evaluation of CAR-T-mediated cytotoxicity. Taken together, this strategy provides a cost-effective and versatile platform for generating transient CAR-T cells and offers a promising approach for treating solid tumors.

Introduction

The incidence and mortality of cancer are rising rapidly worldwide. This poses a threat to human survival and societal development. Gastric cancer is the third leading cause of cancer-related deaths globally and is highly prevalent¹. Due to low rates of early diagnosis, a significant proportion of patients are diagnosed at advanced stages. Traditional treatments include surgery, radiotherapy, and chemotherapy. Surgical resection for patients with mid-to-late-stage or complex gastric cancer may be incomplete. Furthermore, the significant surgical trauma and prolonged postoperative recovery can adversely affect patients' quality of life and potentially lead to complications. Radiotherapy and chemotherapy severely compromise normal tissues, generating numerous side effects during treatment. In contrast, targeted and cell therapies offer precision and efficacy. These treatments have emerged as crucial contemporary care modalities and represent the prevailing trend in the selection of future therapeutic strategies.

The advent of chimeric antigen receptor T-cell (CAR-T) therapy has propelled cell therapy toward a landmark breakthrough. It has demonstrated potent efficacy in treating hematologic malignancies^2,3. CAR-T therapy utilizes the patient's own T cells, which are genetically modified in vitro by inserting a specific chimeric antigen receptor (CAR). This receptor recognizes specific antigens on the surfaces of tumor cells and activates T cells to attack cancer cells. In recent years, CAR-T therapy research for gastric cancer has also advanced. The selection of therapeutic targets is particularly critical as it determines the targeting precision and efficacy of CAR-T cells. Following HER2, Claudin18.2 has emerged as another prominent therapeutic target in gastric cancer, exhibiting expression rates of 40% to 87% among patients. Claudin18.2 is typically buried within the gastric mucosa. When malignant tumors disrupt the tight junctions responsible for intercellular molecular exchange, Claudin18.2 becomes exposed on the surface of tumor cells. This makes it a specific therapeutic target for these tumors⁴.

Typically, the standard manufacturing process for CAR-T cells involves cell collection, T-cell selection and enrichment, T-cell activation, CAR gene construction, gene transduction, expansion, quality control, storage and transportation, followed by patient infusion. The entire CAR-T cell preparation process is highly complex, requiring sterile conditions and demanding stringent environmental and equipment specifications. This results in the high cost of CAR-T therapy, limiting its clinical application. To address the complexities of ex vivo CAR-T production, the concept of in vivo CAR-T preparation has been proposed. This approach involves directly programming CAR-T cells within the patient's body, eliminating the need for ex vivo cell collection, modification, and expansion. This strategy aims to reduce costs, enhance safety and accessibility, and enable broader applications of disease treatment⁵.

The key to in vivo CAR-T preparation lies in selecting an optimal delivery vector. Currently, vectors are categorized as viral or non-viral. Among viral options, lentiviral (LV) and adeno-associated viral (AAV) vectors are predominantly utilized. However, the significant risks and limited payload capacity associated with viral vectors create major obstacles for clinical research⁶. A primary disadvantage of viral vectors for in vivo delivery is their potential for genomic integration, which may result in genetic toxicity and insertional mutations. Previous cases have documented fatal immune responses or cancer in a small portion of patients following treatment. Moreover, the inherent toxicity and immunogenicity of viruses severely restrict their application for repeated in vivo administration. Another challenge is the limited payload capacity of viral vectors, which prevents the simultaneous delivery of multiple genes.

Non-viral mRNA delivery systems effectively address these shortcomings. They avoid integrating into the host genome and thus circumvent many risks. Their transient action prevents off-target toxicity, enabling short-term, controllable CAR-T cell expression, thereby reducing side effects and safety risks. Among non-viral systems, lipid nanoparticles (LNPs) offer a safer approach. They overcome the size limitations of viral vectors. LNPs also resolve issues of insertion mutations and immunogenicity. This positions them as promising candidates in CAR-T cell therapy, with the potential to reduce toxicity, enhance safety, and lower costs⁷.

Current research on immunotherapy for solid tumors primarily focuses on LNP formulation optimization, targeted modification, and mRNA sequence engineering across various cancer models, including melanoma⁸, breast cancer, and colorectal cancer⁹. Efforts have also been directed toward constructing chimeric antigen receptor macrophages (CAR-M)¹⁰ and dendritic cells (CAR-DC)¹¹ via LNP delivery systems to breach the complex physical barriers and immunosuppressive tumor microenvironment (TME) of solid tumors. The platform presented herein provides a versatile, non-viral mRNA delivery system characterized by high modularity. By simply exchanging the mRNA sequence, this platform can be rapidly adapted to solid tumor models targeting other antigens or extended to non-oncology therapeutic areas such as autoimmune, cardiovascular, and fibrotic diseases.

However, a technical gap remains in gastric cancer treatments using these platforms: prior studies have predominantly focused on myeloid cell editing, while strategies for highly efficient in vivo T-cell reprogramming remain largely exploratory. To address this gap, this protocol details a method for generating transient CAR-T cells via LNP-mediated mRNA delivery. This approach aims to overcome the limitations inherent to conventional CAR-T cell therapies, including prohibitive costs, complex manufacturing processes, and systemic toxicities, thereby offering a more efficacious and accessible therapeutic alternative for patients. It is demonstrated that transfection of T cells with an optimized mRNA sequence encoding a specific CAR targeting Claudin18.2 enables robust cell-surface CAR expression, directing the modified T cells to exert targeted cytotoxic activity against specific tumor cells. This protocol is structured into five distinct sections: in vitro transcription of CAR mRNA, LNP preparation and characterization, T-cell activation and transfection, detection of CAR expression, and evaluation of CAR-T cell cytolytic efficacy in vitro and in vivo.

Compared with existing mRNA-LNP delivery technologies, the core methodological innovation of this protocol lies in the kinetic optimization of transient expression. A synergistic optimization was performed on the 5' UTR, 3' UTR, 5' Cap, and a 120-bp poly(A) tail. This tailored design modulates the mRNA half-life within the tumor microenvironment, achieving highly efficient translation within a defined therapeutic window followed by safe degradation. Characterized by rapid and transient CAR generation, this platform serves as an "off-the-shelf" formulation for immediate use, preventing disease progression during the prolonged manufacturing window required for conventional therapies. Furthermore, this transient expression profile offers a controllable safety advantage when targeting antigens with potential "on-target, off-tumor" toxicities, or for patients unable to tolerate severe cytokine release syndrome (CRS) associated with conventional CAR-T therapies.

Despite the profound advantages of in vivo in situ CAR-T cell generation, namely the elimination of complex ex vivo manufacturing and reduced economic burdens, certain limitations persist. In systemic circulation, LNPs are prone to non-specific accumulation in the liver and rapid internalization by macrophages and dendritic cells. Without specific targeting moieties directed toward T cells, the transfection efficiency of the target T-cell population is inevitably compromised. Furthermore, upon entering the tumor tissue, in situ generated CAR-T cells are subjected to the hostile TME; under hypoxic, acidic, and immunosuppressive conditions, these T cells are highly susceptible to immune exhaustion, thereby restricting their cytotoxic potency. Additionally, while the transient nature of mRNA expression enhances safety, it restricts the duration of the therapeutic window. To achieve sustained antitumor efficacy, intermittent and repeated administrations are often required. Consequently, future investigations will focus on addressing these limitations by refining LNP delivery efficiency to achieve precise, lineage-specific targeting.

Protocol

All animal experiments were approved by the Ethics Committee for Laboratory Animals at Shanghai Tenth People’s Hospital (Approval No.: SHDSYY-2025-P0085; Approval Date: January 1, 2025). This study complies with local laws, regulations, and institutional requirements. NSG female mice aged 6–8 weeks were used. The Peripheral Blood Mononuclear Cells (PBMCs) used in this study were obtained from a commercial source. The reagents and equipment used in this study are listed in the Table of Materials.

1. Preparation of CAR mRNA

1. Linearized DNA templates preparation
   ​NOTE: The plasmid containing the CAR sequence was linearized using the BspQI restriction enzyme. The selected restriction enzyme should insert the DNA fragment downstream of the promoter region on the right side and contain no recognition sites within the inserted DNA fragment.
   1. Add double-distilled water (ddH₂O), 10× buffer, DNA template, and BspQI enzyme in the order specified in Table 1. Incubate at 50 °C for 2 h, then inactivate the enzyme at 80 °C for 20 min. The linearized plasmid can be stored at 4 °C.
      1. Set up the restriction digestion reaction in a sterile microcentrifuge tube. Add double-distilled water, restriction enzyme buffer, plasmid DNA template, and BspQI restriction enzyme in the specific order and volumes indicated in Table 1.
      2. Mix the reaction mixture gently by pipetting up and down, then spin down briefly in a microcentrifuge. Incubate the mixture at 50 °C for 2 h in a thermal cycler to allow complete digestion. Inactivate the restriction enzyme by incubating the mixture at 80 °C for 20 min.
         ​NOTE: Optional - Store the linearized plasmid at 4 °C for short-term use or proceed immediately to quality control.
   2. Prepare a 1% agarose gel by weighing 0.25 g of agarose and dissolving it in 25 mL of 1× Tris–Acetate–EDTA (TAE) buffer. Add 2.5 µL of Gel Green and mix thoroughly. Bring to a boil, then shake vigorously. Repeat this process three times before pouring the mixture into the gel casting tray. Run at 110 V for 10 min, followed by 130 V for 20 min.
      1. Perform ultraviolet (UV) visualization to verify complete digestion. By comparing the band positions with those of DNA markers and the uncut plasmid, the linearized plasmid showed only two distinct bands corresponding to its predicted molecular weights: the target sequence was approximately 1,791 bp, and the remaining fragment was approximately 2,535 bp.
   3. Purify the digestion products using DNA isolation beads.
      1. Vortex the magnetic beads to ensure thorough mixing. Pipette the specified volume of magnetic beads into the DNA sample and mix gently. Incubate at Room Temperature (RT) for 10 min to allow DNA binding to the beads.
      2. Place the sample on a magnetic stand for 5 min. Carefully remove the supernatant. Add 200 µL of freshly prepared 80% ethanol for washing. Incubate at RT for 30 s and carefully remove the supernatant. Repeat this step twice.
      3. Keep the sample on the magnetic stand at room temperature with the lid open for 5 min to dry. Remove the sample from the magnetic stand. Add 11 µL nuclease-free ddH₂O. Mix thoroughly by pipetting up and down. Incubate at room temperature for 2 min. Allow the solution to settle on the magnetic stand for 5 min until clear.
      4. Carefully transfer the supernatant to a new nuclease-free centrifuge tube. Use a spectrophotometer to measure the DNA concentration; the resulting DNA must be used immediately for in vitro transcription. Calculate the recovery rate based on the amount of purified DNA divided by the initial amount of DNA added.
2. In Vitro Transcription (IVT)
   1. Thaw all kit components of a T7 RNA polymerase–based in vitro transcription and RNA capping kit on ice (except for the T7 RNA Polymerase Mix, which should be kept at -20 °C until use). Vortex the buffers briefly and spin down.
   2. Assemble the IVT reaction mixture in a sterile, nuclease-free microcentrifuge tube at room temperature according to the volumes specified in Table 2. Add the components in the listed order, ending with the T7 enzyme mix.
   3. Mix the reaction mixture thoroughly but gently by flicking the tube or pipetting up and down. Spin down briefly to collect the liquid at the bottom. Incubate the mixture at 37 °C for 2 h in a thermal cycler.
   4. Add 2 µL of DNase I enzyme directly to the IVT reaction to degrade the residual DNA template. Mix gently and incubate at 37 °C for an additional 15 min. Proceed immediately to the purification step.
3. Purify the resulting RNA product using RNA isolation beads following the same procedure as for DNA digestion product purification.
   1. Dissolve in 20 µL enzyme-free water. Use a NanoDrop spectrophotometer to measure mRNA concentration and the absorbance (OD) ratio. Use 1 µL of nuclease-free water as a blank control. The OD260/280 ratio should be between 1.8 and 2.1, and the OD260/230 ratio should be between 2.0 and 2.4.
      NOTE: If the ratios fall outside these ranges, this indicates contamination, and the sample must be purified again.
   2. Use an automated capillary electrophoresis system for nucleic acid and protein analysis to analyze the length and integrity of the synthesized CAR mRNA. The electrophoresis profile should show a single, sharp peak corresponding to the expected full-length CAR mRNA size (approximately 1800 nt), with no tailing and a flat, clean baseline. No significant low-molecular-weight diffuse bands or multiple peaks should be present; otherwise, this indicates RNA degradation.
   3. Aliquot the validated CAR mRNA to avoid repeated freeze-thaw cycles and store it at -80 °C for long-term preservation.

2. LNP preparation and characterization

1. Dissolve SM102, cholesterol, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), and DMG-PEG2000 in anhydrous ethanol to prepare a 10 mg/mL stock solution. Prepare the organic phase using a 1:50:38.5:10:1.5 ratio.
   1. Calculate the required mRNA amount based on the 8:1 ratio of positively charged amino N to negatively charged phosphate P groups. Dissolve mRNA in sodium citrate solution to prepare the aqueous phase. Maintain an organic: aqueous phase ratio of 1:3 with flow rates of 1.5 mL/min each. Prepare LNPs using a herringbone mixer microfluidic device.
2. First, rinse the tubing and microfluidic chip (herringbone mixer) once with anhydrous ethanol and a sodium citrate solution. After mixing the prepared organic and aqueous phases by purging, draw them up using a 1 mL syringe; the ratio of organic to aqueous phase is 1:3. Load them into the two channels of the syringe pump, respectively.
   1. Set the flow rates for the organic and aqueous phases. Add 5 mL of sodium citrate buffer (50 mM) to the centrifuge tube connected to the outlet. After flushing out any residual washing solution from the tubing, start the device and collect the eluate.
   2. Transfer the LNPs dispersed in the sodium citrate solution to a 100 kDa ultrafiltration tube, centrifuge at 4000 × g for 5–10 min, then add 2.5 mL of sodium citrate solution and 2.5 mL of 1× TBS solution to redisperse them, and repeat the centrifugation once.
   3. Add 1 mL of sodium citrate solution and 4 mL of 1× TBS solution to redisperse the particles, and centrifuge again. Store the concentrated LNPs in 1 mL of TBS solution at 4 °C for a short period.
3. Use a fluorescence-based RNA quantification kit to measure the mRNA content in the LNP before and after demulsification; the difference between the two values represents the encapsulation efficiency of the LNP.
   1. Prepare 1× TE buffer, probe working solution, and 2% Triton X-100. Dilute the mRNA standard (100 µg/mL) to 2 µg/mL using 1× TE buffer, and prepare a standard curve with blank, 10, 20, 100, 500, and 1000 ng/mL concentrations. Vortex 4 µL of LNP solution with 16 µL of 2% Triton X-100 solution, then let stand for 10 min.
   2. Add 5 µL of the demulsified LNP solution to 95 µL of 1× TE buffer. Add 100 µL of probe working solution to both the standard curve and sample wells, mix well, protect from light, and transfer to a black-bottom 96-well plate.
   3. After incubation, measure the fluorescence intensity (Ex480/Em520) using a microplate reader. Subtract the absorbance values of the blank wells from those of the standard curve wells and sample wells, respectively, to plot the standard curve.
   4. Use the standard curve to calculate the mRNA concentrations in the LNP solution before and after demulsification, multiply these values by 200 to obtain the pre-dilution concentrations, and substitute them into the formula for calculation:
      
4. Take 10 µL of LNP and dilute it in 1 mL of TBS solution. Determine the particle size, zeta potential, and Polydispersity Index (PDI) value using a Malvern particle size analyzer.

3. T cell activation and transfection

1. T cell sorting
   1. Take PBMCs at a concentration of 1 × 10⁷/mL, resuspend them at 37 °C, add 10 mL of an immunomagnetic cell separation buffer, mix thoroughly, centrifuge at 500 × g for 8 min, and count.
   2. Resuspend cells and adjust density to 5 × 10⁷ cells/mL, add 50 µL of Isolation Cocktail per mL of cell suspension and incubate at room temperature for 5 min; add 40 µL of magnetic particles for immunomagnetic cell isolation per mL of cell suspension and mix; place the tube on a magnetic stand and incubate at RT for 10 min.
      1. Carefully transfer the enriched cell suspension to a new centrifuge tube using a pipette. Remove the tube from the magnet, add 4 mL of fresh immunomagnetic cell separation buffer, and incubate for an additional 5 min for a second separation round.
   3. Wash cells with T-cell culture medium (RPMI 1640 GlutaMAX + 10% Fetal Bovine Serum + 1% Penicillin/Streptomycin + 100 IU/mL rhIL-2 + 5 ng/mL rhIL-7 + 5 ng/mL rhIL-15), then centrifuge and count. Add T-cell culture medium to adjust cell density to 1 × 10⁶/mL and add 12.5 µL human T cell activator CD3/CD28 magnetic beads. Incubate at 37 °C in a 5% CO₂ incubator for 48 h to activate cells.
2. T-cell activation parameter detection
   1. Transfer 200 µL of T cells activated for 48 h into a 1.5 mL microcentrifuge tube. Remove CD3/CD28 activation magnetic beads, add 1 mL of FACS buffer for washing, gently mix by pipetting, then centrifuge at 500 × g, 4 °C for 5 min. Carefully remove the supernatant and repeat 2–3 times.
   2. Resuspend cells in 100 µL FACS buffer. Add 1 µL each of anti-human CD3, anti-human CD25, and anti-human CD69 antibodies. Gently mix samples and incubate on ice in the dark for 30 min.
      1. Add 1 mL of FACS buffer to wash the cells, centrifuge, then carefully remove the supernatant, add 100 µL of FACS buffer and 0.1 µL of LIVE/DEAD fixable dead cell stain, and incubate on ice in the dark for 10 min.
   3. Resuspend the cells in 1 mL FACS buffer and centrifuge at 500 x g, 4 °C for 5 min, then carefully remove supernatant. Repeat 2–3 times. Discard supernatant and resuspend cells in 200 µL FACS buffer. Analyze cells using flow cytometry.
   4. Select target cells using FSC-A and SSC-A, isolate single cells using FSC-A and FSC-H, and identify live cells using LIVE/DEAD Cell Discrimination Dye (LIVE/DEAD). Compare the CD25 and CD69 ratios between inactive and activated T cells.
3. LNP Transfection of T cells
   1. Based on the encapsulation efficiency calculation, take the required volume of LNP according to the amount of mRNA. First, centrifuge the LNP dispersed in TBS to separate it, then transfer it to RPMI 1640 medium.
   2. After 48 h, use a magnetic rack to remove CD3/CD28 activation magnetic beads. Centrifuge at 500 × g for 8 min, count, and add T-cell culture medium to adjust density to 2 × 10⁶ cells/mL. Add 1 µg of LNP-mRNA per 1 × 10⁵ cells. Gently mix by pipetting and incubate at 37 °C in a 5% CO₂ incubator for 4–6 h.

4. CAR expression detection

1. 4–6 h post-transfection, gently pipette CAR-T cells to mix thoroughly. Transfer at least 2 × 10⁵ cells to a 1.5 mL microcentrifuge tube.
2. Add 1 mL FACS buffer to the 1.5 mL microcentrifuge tube. Pipette to mix, then centrifuge at 500 x g for 5 min at 4 °C. Carefully remove the supernatant. Repeat this step 2–3 times.
3. Resuspend the cell pellet in 100 µL of FACS buffer and divide the sample into a control group and an experimental group, each containing 50 µL. Add 1 µL of the CAR detection antibody G4S-linker to the cell suspension in the experimental group, gently mix the sample, and incubate on ice in the dark for 30 min. For the control group, do not add the antibody; incubate on ice.
4. Pipette 0.2 µL of LIVE/DEAD fixable dead cell stain (1000×) and dilute it in 100 µL of FACS buffer; then pipette 50 µL into each of the control and experimental groups, gently mix the sample, and incubate on ice in the dark for 10 min.
5. Wash cells with 1 mL FACS buffer. Centrifuge at 500 x g for 5 min at 4 °C. Carefully remove supernatant. Repeat 2–3 times.
6. Discard supernatant and resuspend cells in 200 µL FACS buffer. Analyze cells using flow cytometry.
7. Evaluate CAR expression using the following gating strategy: gate on the target cells using FSC-A and SSC-A plot, gate on the single cells using FSC-A and FSC-H plot, and gate on the live cells using LIVE/DEAD fixable cell stain.
   NOTE: Based on the selection of cells stained positive for the G4S-linker antibody in the far-right column of the control group’s live cell population, the percentage shown for the experimental group represents the CAR positivity rate.

5. In vitro cytotoxicity analysis of CAR-T cells

1. Digest NUGC4 and NCl-N87 tumor cells expressing luciferase with trypsin. Centrifuge at 300 x g for 5 min. Add 1 mL RPMI 1640 medium, count and adjust the cell density to 5 × 10⁴ cells/mL. Aliquot 100 µL into each well of a 96-well plate. Add an equal volume of Phosphate-Buffered Saline (PBS) to the remaining wells to prevent evaporation. Incubate in a 37 °C incubator with 5% CO₂ for 24 h.
2. Calculate the CAR positivity rate based on flow cytometry results. Add CAR-T or T cells at effector-to-target cell ratios of 10:1, 5:1, 3:1, 1:1, 1:3, and 1:5 to wells containing tumor cells (100 µL/well). Incubate at 37 °C with 5% CO₂ for 24 h.
3. Using the firefly luciferase reporter gene assay kit, detect the luciferase signal from remaining tumor cells according to the product manual and calculate the kill rate: (1 - (effector cell RLU value/target cell RLU value)) × 100%.

6. In vivo functional validation of CAR-T cells

1. After a 1-week acclimatization period prior to inoculation, inject each mouse subcutaneously in the axilla with 5 × 10⁶ NUGC4 cells. Measure the tumor size daily, and calculate the tumor volume using the formula: V (mm³) = L × W²/2 (where L is the longest diameter of the tumor and W is the shortest diameter) until the tumor reached 100 mm³.
2. Randomly divide the mice into four groups: three treatment groups receiving different doses and a control group receiving PBS, with three mice in each group.
   NOTE: The Blindness Act was not implemented.
3. After incubating 10 µg of mRNA with 1 × 10⁶ activated T cells for 4 h, prepare dosages of 5 × 10⁵ cells/mouse, 1 × 10⁶ cells/mouse, and 2 × 10⁶ cells/mouse. Administer the mixture of LNP and T cells to the mice via tail vein injection, with each mouse receiving a 100 µL volume of cells or PBS. Doses were administered on days 1, 4, 7, and 10, for a total of four doses.
4. Measure the tumor size of the mice on days 0, 2, 4, 6, 8, 10, 12, and 14, calculate the tumor volume, and plot a growth curve. Express the data as mean ± SD. Analyze the differences between groups using two-way analysis of variance (ANOVA) with a statistical and graphing software, combined with Tukey’s multiple comparison test. ns: not statistically significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Results

A CAR sequence targeting Claudin18.2 was employed, composed of the single-chain variable fragment (scFv) from a humanized anti-Claudin18.2 monoclonal antibody, the CD8 hinge region, the CD28 transmembrane region, the co-stimulatory domain, and the CD3ζ activation domain. A DNA template containing the T7 promoter and poly A tail was constructed based on this sequence for in vitro CAR mRNA transcription (Figure 1A). The CAR mRNA was approximately 1800 bp in length, as verified by capi...

Discussion

This protocol provides a non-viral mRNA delivery system that avoids integration into the host genome, thereby circumventing numerous risks, including immune rejection. Its transient functionality also prevents off-target toxicity, enabling short-term, controllable expression in CAR-T cells to reduce side effects and safety risks.

Optimization of the CAR mRNA sequence design was performed to regulate its translation efficiency and expression duration. The 5'UTR, 3'UTR, 5'-Cap, and p...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absolute Ethanol	Sigma-Aldrich	459836	Organic phase
Agarose	Beyotime	ST004L	Gel electrophoresis
anti-G4S linker (B02H1) mAb PE	Hycells	GS-ARPE100	CAR detection
BspQI Restriction Enzyme	NEB	R0712	Plasmid linearization
BV421 Mouse Anti-Human CD25	BD Biosciences	564033	Activation marker
CD3/CD28 Activation Beads	Thermo Fisher Scientific	11161D	T cell activation
Cholesterol	Sigma-Aldrich	C8667	Structural lipid
CO2 Cell Culture Incubator	Thermo Fisher / equivalent	250i	For T cell and tumor cell culture
DMG-PEG2000	Avanti Polar Lipids	880151	PEGylated lipid
DNA Isolation Magnetic Beads	SYNTHGENE	10501-005	DNA purification
DNase I (RNase-free)	NEB	M0303L	Removal of DNA template
DSPC	Avanti Polar Lipids	850365	Phospholipid
Dynamic Light Scattering Analyzer	Malvern Panalytical	Zetasizer	For LNP size, PDI, zeta potential
EasySep Buffer	STEMCELL Technologies	20144	Magnetic separation
Fetal Bovine Serum (FBS)	Gibco	10099-141	Culture supplement
FITC Mouse Anti-Human CD3	BD Biosciences	555332	T cell identification
Flow Cytometer	BD Biosciences / equivalent	FACSCelesta / LSRFortessa	For CAR and activation marker
GelGreen Nucleic Acid Stain	Biotium	41005	DNA visualization
LIVE/DEAD Fixable Dye	Invitrogen	L34955	Viability staining
Luciferase Assay Kit	Promega	E1500	Cytotoxicity readout
Magnetic Separation Stand	STEMCELL Technologies / equivalent	18000	For PBMC and T cell isolation
Nanodrop Spectrophotometer	Thermo Fisher Scientific	NanoDrop 2000	For DNA/RNA quantification
NSG Female Mice (6–8 weeks)	Jackson Laboratory	5557	In vivo model
PBMCs	Shanghai Saili Biotechnology Company
PCR thermal cycler	Bio-Rad / equivalent	T100	For DNA amplification
PE Mouse Anti-Human CD69	BD Biosciences	557050	Early activation
Penicillin-Streptomycin	Gibco	15140-122	Antibiotics
Qsep Nucleic Acid Analyzer	BiOptic Inc.	Qsep100	For mRNA size/integrity analysis
Quant-iT RiboGreen RNA Kit	Invitrogen	R11490	Encapsulation efficiency
RapidSpheres	STEMCELL Technologies	15061	Magnetic labeling
Recombinant Human IL-15	PeproTech	200-15	Memory phenotype support
Recombinant Human IL-2	PeproTech	200-02	T cell expansion
Recombinant Human IL-7	PeproTech	200-07	T cell survival
Refrigerated Centrifuge	Eppendorf / equivalent	5425R	For LNP concentration and cell processing
RNA 6000 Ladder	Thermo Fisher Scientific	AM7152	RNA characterization
RNA Cartridge Kit(1pc)	Bioptic	C105110	RNA quality control
RNA Isolation Magnetic Beads	SYNTHGENE	10511-005	mRNA purification
RNase-free Water	Thermo Fisher Scientific	AM9937	IVT and RNA handling
RPMI 1640 GlutaMAX	Gibco	61870-036	T cell culture
SM-102	MedChemExpress / equivalent	HY-112563	Ionizable lipid
T Cell Isolation Cocktail	STEMCELL Technologies	15061	CD3+ T cell isolation
T7 Co-Transcription and Cap-Addition Kit	SYNTHGENE	10111-3011	IVT mRNA synthesis
TBS Buffer	Gibco	3005	LNP storage
Trypsin-EDTA	Gibco	25200-056	Cell detachment
UV Gel Imaging System	Syngene / equivalent	NuGenius/NuGenius+	For DNA digestion verification