Nature Communications2020ResearchNon-viral Gene Delivery

In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

N.N. Parayath, S.B. Stephan, A.L. Koehne, P.S. Nelson, M.T. Stephan29 September 2026DOI 10.1038/s41467-020-19486-2

Summary

CAR T and TCR T cell therapies are powerful but face prohibitive costs and manufacturing complexity due to the need for leukapheresis, ex vivo transduction, expansion, and quality control—all under GMP conditions. An "off-the-shelf" injectable nanocarrier that can transiently reprogram circulating T cells in vivo would bypass these barriers, enabling scalable, cost-effective, and repeatable dosing for a wide range of diseases including cancer. ### NP Characterization & Ex Vivo Transfection | Parameter | Result | |---------------|------------| | NP size (PBAE/mRNA polyplex) | ~100 nm | | mRNA encapsulation efficiency | >90% | | T cell transfection efficiency |.

Keywords

mRNAT cellsNanocarriersPolymericTransfectionPoly(beta-amino ester)TCR signaling

Purpose: CAR T and TCR T cell therapies are powerful but face prohibitive costs and manufacturing complexity due to the need for leukapheresis, ex vivo transduction, expansion, and quality control—all under GMP conditions. An "off-the-shelf" injectable nanocarrier that can transiently reprogram circulating T cells in vivo would bypass these barriers, enabling scalable, cost-effective, and repeatable dosing for a wide range of diseases including cancer and chronic infections.

Hypothesis: Injectable biodegradable polymeric nanocarriers loaded with in vitro-transcribed (IVT) mRNA encoding disease-specific CARs or TCRs can transiently reprogram circulating T cells in vivo. Repeated infusions of these nanocarriers will generate sufficient functional T cells to induce disease regression in mouse models of leukemia, prostate cancer, and hepatitis B-induced hepatocellular carcinoma, achieving efficacy comparable to conventional ex vivo-engineered T cells.

Aims: 1. Develop and characterize T cell-targeted polymeric nanoparticles (PBAE/PGA) loaded with IVT mRNA encoding CARs or TCRs 2. Demonstrate ex vivo transfection of primary human T cells with CAR (CD19, ROR1) and TCR (HBcore) mRNA and confirm functional activity 3. Evaluate in vivo T cell targeting using Cre mRNA delivery to Ai6 reporter mice 4. Assess therapeutic efficacy in mouse models of: - B cell acute lymphoblastic leukemia (immunocompetent Eu-ALL01 model) with CD19 CAR mRNA - Prostate cancer (NSG model with human T cells) with ROR1 CAR mRNA - HBV-induced hepatocellular carcinoma (NSG model) with HBcore TCR mRNA 5. Perform safety and toxicity assessment using NCL assay cascade protocols and rat toxicology studies

Delivery system:

Component: Carrier Matrix; Description: PBAE 447 (poly(β-amino ester)) — biodegradable, endosomal escape via proton sponge effect, ester bond hydrolysis

Component: Shielding Layer; Description: Polyglutamic acid (PGA, 15 kDa) — negatively charged, reduces off-target binding

Component: Targeting Ligands; Description: • T cells: anti-CD3 (OKT3) or anti-CD8 (OKT8) antibodies • In vivo (immunocompetent): anti-CD3 MuIgG2a LALAPG (Fc-silenced)

Component: Antibody Modification; Description: • Deglycosylation (IgGZERO enzyme) • Conjugation to PGA via EDC/NHS chemistry • LALAPG mutations for Fc silencing

Component: Nucleic Acid; Description: IVT mRNA with modified nucleotides (pseudouridine + 5-methylcytidine), ARCA cap, codon-optimized

Component: Transgenes Tested; Description: • eGFP (reporter) • CD19-28z CAR (1928z; FDA-approved for B-cell lymphoma) • ROR1-28z CAR (prostate cancer) • HBcore18-27 TCR (hepatitis B virus core antigen) • Cre recombinase (in vivo targeting)

Component: NP Properties; Description: • Size: ~100 nm (PBAE/mRNA polyplex) • Final NP: antibody-functionalized with PGA • mRNA encapsulation: >90%

Component: Lyophilization; Description: Stable with 60 mg/mL sucrose cryoprotectant

Component: Cell Types; Description: • Primary human T cells (PBMC-derived) • Raji (CD19⁺ lymphoma) • LNCaP C42 (prostate cancer) • HepG2-HBcAg (hepatocellular carcinoma) • Eu-ALL01 (murine leukemia)

Approach:

Parameter: Ex Vivo Transfection; Details: Primary human CD8 T cells; 3 μg mRNA/10⁶ cells; 2 h exposure; Stemcell ImmunoCult-XF medium; >70% transfection efficiency

Parameter: CAR/TCR Expression Kinetics; Details: Flow cytometry at days 1-10 post-transfection; surface CAR/TCR detection

Parameter: In Vitro Function; Details: • Cytotoxicity: Nuclight Red-labeled target cells; IncuCyte live-cell imaging • Cytokine release: ELISA (IL-2, IFN-γ, TNF-α)

Parameter: In Vivo Targeting (Ai6 Mice); Details: Three daily doses of CD3-targeted Cre mRNA NPs (15 μg/dose); whole-organ fluorescence (IVIS); flow cytometry (spleen, blood)

Parameter: In Vivo Leukemia Model; Details: Eu-ALL01 (albino C57BL/6); CD19 CAR mRNA NPs (50 μg/dose, weekly ×3); IVIS bioluminescence

Parameter: In Vivo Prostate Cancer Model; Details: NSG mice; orthotopic LNCaP C42-luc; human T cell reconstitution; ROR1 CAR mRNA NPs (50 μg/dose, weekly); survival analysis

Parameter: In Vivo HBV-HCC Model; Details: NSG mice; intrahepatic HepG2-HBcAg-luc; human T cell reconstitution; HBcore TCR mRNA NPs (50 μg/dose, ×2); IVIS at endpoint

Parameter: Controls; Details: GFP mRNA NPs; non-targeted (isotype) NPs; untransfected T cells; PBS; adoptive transfer of ex vivo-engineered CAR/TCR T cells

Parameter: Safety Studies; Details: NCL assay cascade: hemolysis (ITA-1), complement activation (ITA-5.2), oxidative stress (ITA-32); Sprague Dawley rat toxicology (100 μg mRNA/dose; histopathology; serum chemistry; IL-6)

Parameter: Sample Sizes; Details: In vitro: n=3-4; In vivo: n=5-10 mice/group

Parameter: Statistical Tests; Details: Unpaired two-tailed Student's t-test; Log-rank test (survival); one-way ANOVA

Key methods:

Analysis Category: NP Characterization; Methods: DLS (size, zeta potential); Particle Tracking Analysis (Nanosite 300); Qubit RNA HS assay (encapsulation)

Analysis Category: Transfection Efficiency; Methods: Flow cytometry: eGFP; c-myc tag (CAR); MHC Pentamer (TCR)

Analysis Category: CAR/TCR Surface Expression; Methods: Flow cytometry: anti-c-myc (CAR); HLA-A201-HBVcore pentamer (TCR)

Analysis Category: In Vitro Cytotoxicity; Methods: IncuCyte Zoom (Nuclight Red target cells); normalized killing over 45 h

Analysis Category: Cytokine Secretion; Methods: ELISA: IL-2, IFN-γ, TNF-α (R&D Systems)

Analysis Category: In Vivo Imaging; Methods: IVIS Spectrum; D-luciferin i.p.; Living Image 4.3.1

Analysis Category: Flow Cytometry (In Vivo); Methods: Antibodies: mouse CD45, CD11b, CD11c, Ly6G, CD19, CD4, CD8, CD44, CD62L, CD69; Zombie Aqua live/dead

Analysis Category: Safety Assays; Methods: • Hemolysis (NCL ITA-1): cyanmethemoglobin, 540 nm • Complement activation (NCL ITA-5.2): C4d, Bb, iC3b ELISA (MicroVue) • Oxidative stress (NCL ITA-32): MitoSox, flow cytometry

Analysis Category: Toxicity Histopathology; Methods: H&E staining; board-certified pathologist (blinded); rat tissues: lung, liver, heart, brain, kidney, spleen, bone marrow, duodenum

Key results: ### NP Characterization & Ex Vivo Transfection

Parameter: NP size (PBAE/mRNA polyplex); Result: ~100 nm

Parameter: mRNA encapsulation efficiency; Result: >90%

Parameter: T cell transfection efficiency; Result: >70% (single application)

Parameter: CAR/TCR surface expression; Result: Peak at day 1-2

Parameter: CAR/TCR expression duration; Result: ~7 days (transient)

In Vivo T Cell Targeting (Ai6 Reporter Mice, CD3-Targeted Cre mRNA NPs):

Parameter: T cell transfection (spleen); Result: 8.1 ± 1.9%

Parameter: Macrophage transfection; Result: 3.2 ± 1.5%

Parameter: B cell transfection; Result: 1.1 ± 0.9%

Parameter: Neutrophil transfection; Result: 0.3 ± 0.2%

Parameter: Dendritic cell transfection; Result: 1.9 ± 0.8%

Antitumor Efficacy (Immunocompetent Eu-ALL01 Leukemia Model):

Group: CD19 CAR mRNA NPs; Tumor Burden Reduction vs. Untreated: 26-fold reduction (p < 0.0001)

Group: GFP mRNA NPs; Tumor Burden Reduction vs. Untreated: No reduction

Prostate Cancer (NSG, ROR1 CAR mRNA NPs vs. Adoptive Transfer):

Treatment: Untreated; Median Survival (days): 32 days; vs. Untreated: -

Treatment: Ex vivo ROR1 CAR T cells; Median Survival (days): 69 days; vs. Untreated: +37 days

Treatment: ROR1 CAR mRNA NPs; Median Survival (days): 72 days; vs. Untreated: +40 days (n.s. vs. adoptive)

Treatment: CAR⁺ T cells/mg tumor (day 4); Median Survival (days): 892 ± 295 (adoptive) vs. 648 ± 240 (NPs); vs. Untreated: Comparable

Treatment: Antigen escape; Median Survival (days): ROR1 low/negative variants emerged; vs. Untreated: Both groups

HBV-HCC Model (NSG, HBcore TCR mRNA NPs vs. Adoptive Transfer):

Treatment: Untreated; Tumor Reduction (photon count): Baseline; TCR⁺ T cells in liver: -

Treatment: GFP mRNA NPs; Tumor Reduction (photon count): No reduction; TCR⁺ T cells in liver: -

Treatment: Ex vivo HBcore TCR T cells; Tumor Reduction (photon count): 18.9-fold reduction; TCR⁺ T cells in liver: 2,040 ± 310 cells

Treatment: HBcore TCR mRNA NPs; Tumor Reduction (photon count): 13-fold reduction; TCR⁺ T cells in liver: 1,920 ± 280 cells (n.s.)

Safety Assessment:

Assay: Hemolysis (TPC); Result: 1.21 ± 0.26% (non-hemolytic; <2% threshold)

Assay: Complement iC3b; Result: No activation

Assay: Complement Bb; Result: No activation

Assay: Complement C4d; Result: Slightly above 2-fold threshold (2.3 ± 0.13-fold)

Assay: Oxidative stress; Result: 3.6 ± 0.2-fold (modest increase)

Assay: Rat toxicology; Result: No histologic lesions attributed to NPs

Assay: Rat platelet count; Result: Slight decrease (n.s.)

Assay: Rat IL-6; Result: 16.5 ± 5.9 pg/mL (moderate, considered safe)

Assay: Rat liver/kidney function; Result: Comparable to controls

Interpretation: The authors conclude that "repeated infusions of these polymer nanocarriers induce sufficient host T cells expressing tumor-specific CARs or virus-specific TCRs to cause disease regression at levels similar to bolus infusions of ex vivo engineered lymphocytes." They state: "Should engineered T cell therapy reach its promise of extending to diverse populations across a variety of cancer types, the challenges of economics and manufacturing will likely grow." The authors position this as a paradigm shift: "We demonstrate that a mRNA nanodrug can achieve the power of effective, side-effect-free cell therapy with the convenience of an off-the-shelf drug. Just as for a conventional drug, with this new treatment modality the patient could be easily re-dosed for as long as medically necessary."

10. Limitations (Explicitly Stated or Evident):

1. Transient expression requires repeated dosing: The ~7-day expression window necessitates weekly infusions, which may be burdensome for patients compared to a single adoptive transfer.

2. Modest in vivo T cell transfection efficiency: Only 8.1% of splenic T cells were transfected in vivo with CD3-targeted NPs; this may be sufficient for some indications but could limit efficacy in others.

3. No demonstration in fully immunocompetent models with human T cells: The solid tumor models used NSG mice with adoptive human T cell transfer, not truly autologous in situ reprogramming in immunocompetent mice.

4. Antigen escape observed: ROR1 low/negative variants emerged in both adoptive transfer and NP groups, highlighting the need for multi-targeting strategies.

5. C4d complement activation slightly elevated: While within acceptable limits, the slight complement activation (2.3-fold) suggests potential for infusion reactions at higher doses.

6. Rat toxicology lacked functional immune readouts: While histopathology was normal, the study did not assess whether NP-treated T cells were functional in vivo in rats.

7. CAR mRNA construct contains c-myc tag: The c-myc tag may be immunogenic in humans and could trigger clearance of CAR T cells.

8. All in vivo human T cell studies used T cells from healthy donors: Patient-derived T cells (especially from heavily pretreated patients) may not transfect as efficiently.

9. No direct comparison to FDA-approved CAR T cell products: The study compares to ex vivo-engineered T cells but not to clinical-grade products with defined manufacturing protocols.

10. Potential for anti-PEG/PGA antibodies: Repeated dosing with PEGylated/PGA-containing NPs could elicit immune responses that reduce efficacy over time.

11. PBAE polymer safety: While PBAE is biodegradable and well-tolerated in these studies, extensive clinical safety data in humans are lacking for this specific formulation.

12. No evaluation of endogenous TCR or MHC compatibility: The approach relies on the patient's own T cells and does not address potential alloreactivity or HLA restrictions for TCR-based therapies.

Report prepared based on the published Nature Communications article. For full experimental details, supplementary figures, and complete references, please refer to the original publication.

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In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

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