Nature Communications2017ResearchNon-viral Gene Delivery

Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers

H.F. Moffett, M.E. Coon, S. Radtke, S.B. Stephan, L. Mcknight, A. Lambert, B.L. Stoddard, H.P. Kiem, M.T. Stephan29 September 2026DOI 10.1038/s41467-017-00505-8

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Summary

Therapies based on genetically engineered immune cells (CAR T cells, HSCs) are transforming medicine, but the viral and electroporation methods used to create these cytoreagents are complex, expensive, and time-consuming. There is a growing need for transient "hit-and-run" genetic programming (transcription factors, genome-editing nucleases) where permanent transgene integration is undesirable or dangerous. A simple, non-viral method to. ### NP Characterisation & Transfection Efficiency | Parameter | Result | |---------------|------------| | NP size | 109.6 ± 26.6 nm | | Zeta potential | 1.1 ± 5.3 mV (near-neutral) | | T cell transfection efficiency |.

Keywords

mRNA Nanocarriers CAR-T cells T cells Transfection Poly(beta-amino ester)TCR signalling

Purpose: Therapies based on genetically engineered immune cells (CAR T cells, HSCs) are transforming medicine, but the viral and electroporation methods used to create these cytoreagents are complex, expensive, and time-consuming. There is a growing need for transient "hit-and-run" genetic programming (transcription factors, genome-editing nucleases) where permanent transgene integration is undesirable or dangerous. A simple, non-viral method to transiently programme therapeutic cells without complex handling or reduced viability is needed.

Hypothesis: Targeted mRNA nanocarriers—simply mixed with cells in culture—can achieve efficient, dose-controlled transient gene expression in primary T lymphocytes and haematopoietic stem cells without compromising cell viability or function. This platform will enable: (1) efficient genome editing via megaTAL nuclease mRNA delivery; (2) reprogramming of CAR T cells toward a central memory phenotype via Foxo1 mRNA; and (3) improved self-renewal of HSCs via Musashi-2 mRNA, all without adding complexity to existing manufacturing protocols.

Aims: 1. Design and characterise targeted mRNA nanocarriers with four functional components: targeting antibodies, PGA shielding, PBAE carrier matrix, and modified mRNA payload 2. Demonstrate efficient transfection of primary T cells and HSCs with minimal impact on viability, proliferation, or function 3. Validate genome editing via delivery of mRNA encoding megaTAL nuclease to knockout TCR expression in CAR T cells 4. Reprogram T cell differentiation toward central memory phenotype via Foxo1 mRNA delivery and assess functional improvement in vivo 5. Enhance HSC self-renewal via Musashi-2 mRNA delivery and demonstrate improved clonogenic potential 6. Compare NP approach to electroporation in terms of simplicity, viability, and cell yield

Delivery system:

Component: Carrier Matrix; Description: PBAE 447 (poly(β-amino ester)) — biodegradable, half-life 1-7 h in aqueous conditions; condenses and protects mRNA

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

Component: Targeting Ligands; Description: • T cells: anti-CD3 (OKT3), anti-CD8 (OKT8) • HSCs: anti-CD105 • Control: non-specific IgG

Component: Nucleic Acid; Description: Synthetic mRNA with modified nucleotides (pseudouridine + 5-methylcytidine) and ARCA cap; codon-optimised

Component: Transgenes Tested; Description: • eGFP (reporter) • TRAC-megaTAL nuclease + TREX2 (genome editing) • Foxo1₃A (AKT-insensitive, nuclear-retaining transcription factor) • Musashi-2 (MSI2; stem cell self-renewal factor)

Component: NP Properties; Description: • Size: 109.6 ± 26.6 nm • Zeta potential: 1.1 ± 5.3 mV (near-neutral) • Lyophilisable (stable with sucrose)

Component: Particle Formation; Description: Two-step self-assembly: (1) mRNA + PBAE (charge-driven condensation), (2) PGA-antibody addition

Component: Cell Types; Description: • Primary human T cells (PBMC-derived) • CD34⁺ HSCs (mobilised peripheral blood) • Jurkat T cell line

Approach:

Parameter: mRNA Transfection; Details: NP:T cell ratio = 2×10⁴:1; 2.5 µg mRNA/10⁶ cells; 2 h exposure at 37°C in XFSFM; cells diluted 4× with TCM + IL-2

Parameter: T Cell Stimulation; Details: CD3/CD28 Dynabeads (1:1 bead:cell ratio); beads removed 24 h before NP addition

Parameter: Genome Editing; Details: NP containing TRAC-megaTAL + TREX2 + eGFP mRNA (42:42:16 w:w:w); 30°C cold shock for 40 h post-transfection

Parameter: CAR T Cell Manufacturing; Details: Day 0: stimulation; Days 1-2: NP transfection; Day 3: lentiviral transduction (19-41BBζ CAR); expansion with TM-LCL feeders

Parameter: Foxo1 Reprogramming; Details: Foxo1₃A + eGFP mRNA (84:16 w:w); CD8-targeted NPs; transfection during primary and secondary expansion

Parameter: HSC Transfection; Details: CD34⁺ cells; 1 µg mRNA/well (96-well); 1 h exposure; CD105-targeted NPs

Parameter: In Vivo Model; Details: NSG mice; Raji-luc lymphoma (5×10⁵ cells i.v.); CAR T cells (2.5×10⁶ CD8⁺); IVIS bioluminescence imaging

Parameter: Controls; Details: eGFP mRNA NPs; non-targeting IgG NPs; untransfected cells; electroporation (Lonza Nucleofector)

Parameter: Replicates; Details: ≥3 independent experiments; n=9 mice/group for in vivo

Parameter: Statistical Tests; Details: Unpaired two-tailed Student's t-test; Log-rank test (survival); GSEA for RNAseq

Key methods:

Analysis Category: NP Characterisation; Methods: DLS (size, zeta potential); TEM (morphology); lyophilisation stability

Analysis Category: Transfection Efficiency; Methods: Flow cytometry (eGFP, cy5-labelled mRNA); confocal microscopy (internalisation)

Analysis Category: Genome Editing; Methods: Surveyor assay (indel detection); flow cytometry (CD3 surface loss); TCRα PCR

Analysis Category: T Cell Phenotype; Methods: Flow cytometry: CD62L, CD45RO, CD28, S1PR1; RNASeq (Illumina HiSeq); GSEA

Analysis Category: T Cell Function; Methods: Proliferation (TM-LCL co-culture); Cytotoxicity (CD19+ K562, CFSE/7AAD); IFN-γ ELISA

Analysis Category: HSC Function; Methods: Flow cytometry: CD34, CD133, CD105; CFU assays (MethoCult H4435); qPCR (MSI2 mRNA)

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

Analysis Category: Gene Expression; Methods: qPCR (codon-optimised Foxo1₃A, MSI2); PrimeTime qPCR assays; QuantStudio5

Analysis Category: Cell Viability; Methods: 7AAD staining; trypan blue exclusion

Key results: ### NP Characterisation & Transfection Efficiency

Parameter: NP size; Result: 109.6 ± 26.6 nm

Parameter: Zeta potential; Result: 1.1 ± 5.3 mV (near-neutral)

Parameter: T cell transfection efficiency; Result: >80% (single NP application)

Parameter: Transgene expression onset; Result: As early as 5 h post-transfection

Parameter: Lyophilisation; Result: No change in properties or efficacy

Parameter: CD3-targeted NP selectivity; Result: Low off-target binding

T Cell Viability & Expansion vs. Electroporation:

Parameter: Handling complexity; mRNA NPs: Simple (mix with cells); Electroporation: Complex (multiple steps)

Parameter: Cell viability; mRNA NPs: Maintained; Electroporation: Compromised

Parameter: Cell yield; mRNA NPs: 60× higher than electroporation; Electroporation: Reduced

Parameter: Expansion with CD3/CD28 beads; mRNA NPs: Unimpaired even with repeated transfections; Electroporation: Not tested

TCR Knockout (Genome Editing):

Parameter: TCR knockout efficiency; Result: 60.8 ± 17.7% (day 14)

Parameter: Indel frequency; Result: Confirmed by Surveyor assay

Parameter: CAR transduction efficiency; Result: Equal in edited vs. control T cells

Parameter: T cell function post-editing; Result: Fully maintained (proliferation, killing, IFN-γ)

Foxo1₃A Reprogramming (T Cell Memory):

Parameter: Foxo1₃A mRNA expression; Result: Peak at day 1; near baseline by day 8

Parameter: Foxo1 protein levels; Result: Maintained near-physiological levels

Parameter: CD62L induction; Result: Rapid increase (within 24 h)

Parameter: CD62L persistence; Result: Maintained at days 8 and 20

Parameter: RNASeq; Result: Strong concordance with TCM gene signature

Parameter: Key memory genes upregulated; Result: KLF2, SELL, CD28, S1PR1

Parameter: Gene set enrichment; Result: Strong connection to TCM gene expression

In Vivo CAR T Cell Efficacy (NSG, Raji Lymphoma):

Parameter: Median survival; Control CAR T cells: 29 days; Foxo1₃A-NP CAR T cells: 43 days (14-day improvement)

Parameter: Tumour burden (day 14 post-therapy); Control CAR T cells: Baseline; Foxo1₃A-NP CAR T cells: 11.3× reduced vs. control CAR T cells

Parameter: Disease progression; Control CAR T cells: Temporary delay; Foxo1₃A-NP CAR T cells: Substantial regression

HSC Transfection & Expansion:

Parameter: HSC transfection efficiency (CD105-NPs); Result: 49.0 ± 6.9%

Parameter: HSC transfection (control IgG-NPs); Result: 4.9 ± 0.9%

Parameter: MSI2 mRNA expression; Result: Peak at day 1; near baseline by day 7

Parameter: CD34⁺CD133⁺ cells (day 8); Result: 2.6× increase (frequency)

Parameter: Total CD34⁺CD133⁺ cells; Result: 2.3× increase

Parameter: Differentiated CD34⁺ cells; Result: 2× reduction

Parameter: Colony forming units (CFC); Result: 30% overall increase

Parameter: BFU-E (erythroid); Result: 67% increase

Parameter: CFU-MIX (primitive); Result: 41% increase

Interpretation: The authors conclude that "exposing pharmaceutical cells to an NP reagent can improve their therapeutic value by accomplishing 'hit-and-run' gene modification." The platform "does not add complexity to manufacturing because it involves no special equipment or training," and "can substantially streamline the manufacture of cell-based therapies at clinical scales." Three examples—genome editing in CAR T cells (60.8% TCR knockout), memory phenotype reprogramming (14-day survival improvement in lymphoma model), and HSC expansion (30% increase in colony formation)—demonstrate that the approach is "simple and generalizable." The authors state: "Our project demonstrates that this platform can substantially streamline the manufacture of cell-based therapies at clinical scales, which means that treating patients with genetically engineered cells could become less expensive, and more effective as a disease-fighting intervention."

10. Limitations (Explicitly Stated or Evident):

1. Transient expression only: By design, the approach is "hit-and-run" and does not provide permanent transgene expression; this is an advantage for safety but limits applications requiring sustained expression.

2. In vitro HSC expansion not translated to in vivo engraftment: The study demonstrates improved HSC expansion and colony formation in vitro but does not show enhanced long-term engraftment or repopulation in vivo.

3. Cold shock required for genome editing: The authors used a transient cold shock (30°C for 40 h) to enhance genome editing, which adds complexity to the manufacturing process.

4. No direct comparison to viral transduction for permanent expression: The study compares NP transfection to electroporation but does not extensively benchmark against viral transduction for the same applications.

5. In vivo efficacy in NSG mice only: Immunodeficient NSG mice were used for in vivo CAR T cell studies; efficacy in immunocompetent models was not demonstrated.

6. PBAE polymer half-life: The PBAE formulation has a half-life of 1-7 h in aqueous conditions, requiring careful handling and potentially limiting in vivo stability.

7. Limited characterisation of Foxo1 stability in vivo: While in vitro Foxo1 expression kinetics were measured, in vivo transgene expression kinetics after adoptive transfer were not tracked.

8. No evaluation of off-target effects: Genome editing specificity was not fully characterised; off-target nuclease activity was not systematically assessed.

9. CD105 targeting for HSCs: CD105 is expressed on primitive HSCs but also on other cell types; the specificity of targeting was not tested in a mixed cell population.

10. mRNA manufacturing complexity: While NP transfection is simple, manufacturing of modified mRNA at clinical scale remains complex and expensive.

11. No comparison to other non-viral delivery methods: The study compares NPs to electroporation but not to other emerging methods (e.g., lipid nanoparticles, cell-penetrating peptides).

12. Immunogenicity of modified mRNA: While modified nucleotides reduce immunogenicity, the authors did not assess whether repeated NP transfection elicits anti-mRNA or anti-NP immune responses.

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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