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Functionalized nanowires for miRNA-mediated therapeutic programming of naïve T cells

Nature Nanotechnology (2024)Cite this article

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Abstract

Cellular programming of naïve T cells can improve the efficacy of adoptive T-cell therapy. However, the current ex vivo engineering of T cells requires the pre-activation of T cells, which causes them to lose their naïve state. In this study, cationic-polymer-functionalized nanowires were used to pre-program the fate of primary naïve CD8+ T cells to achieve a therapeutic response in vivo. This was done by delivering single or multiple microRNAs to primary naïve mouse and human CD8+ T cells without pre-activation. The use of nanowires further allowed for the delivery of large, whole lentiviral particles with potential for long-term integration. The combination of deletion and overexpression of miR-29 and miR-130 impacted the ex vivo T-cell differentiation fate from the naïve state. The programming of CD8+ T cells using nanowire-delivered co-delivery of microRNAs resulted in the modulation of T-cell fitness by altering the T-cell proliferation, phenotypic and transcriptional regulation, and secretion of effector molecules. Moreover, the in vivo adoptive transfer of murine CD8+ T cells programmed through the nanowire-mediated dual delivery of microRNAs provided enhanced immune protection against different types of intracellular pathogen (influenza and Listeria monocytogenes). In vivo analyses demonstrated that the simultaneous alteration of miR-29 and miR-130 levels in naïve CD8+ T cells reduces the persistence of canonical memory T cells whereas increases the population of short-lived effector T cells. Nanowires could potentially be used to modulate CD8+ T-cell differentiation and achieve a therapeutic response in vivo without the need for pre-activation.

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Fig. 1: Functionalized nanowires deliver miRNA to naïve murine CD8+ T cells with high viability and delivery efficiency.
Fig. 2: Nanowires outperform conventional miRNA delivery methods and deliver larger biomolecules.
Fig. 3: Nanowire delivery of single miRNA into murine CD8+ T cell modulates the target expression, phenotype and function.
Fig. 4: Co-delivery of miR-29-ASO and miR-130-mimic oligonucleotides by functionalized nanowire platform toggles CD8+ T-cell fate switches.
Fig. 5: Delivery of dual miRNA components by functionalized nanowire enhances murine CD8+ T-cell effector function in vivo in influenza A.
Fig. 6: Delivery of dual miRNA enhances murine CD8+ T-cell effector function in vivo in LM infection.

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Data availability

The raw RNA-seq data on nanowires are available in the GEO database under accession code GSE255468. The naïve dataset used for comparison was the expression profiles of adult naïve CD8+ T cells from GSE97795 (ref. 57). The remaining data are available within the Article and its Supplementary Information. Due to the very large file sizes and volume of data, the remaining raw data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We acknowledge financial support from the Curci Foundation Award (A.S.); the National Science Foundation (EEC-1648035, seed funding awarded to A.S.); US National Institutes of Health NIH 5R01AI132738-06, 1R01CA266052-01, 1R01CA238745-01A1 and U01CA280984-01 (awarded to A.S.); and NIH R01AI110613 and U01AI131348 (awarded to B.D.R.). This work was performed in part at the Georgia Institute of Technology, Institute for Electronics and Nanotechnology, a member of the National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (ECCS-2025462). We further acknowledge support from the Cornell Nanoscale Science and Technology Facility under the NSF Grant ECCS-1542081 for the use of equipment. We acknowledge technical support on STEM from M. Tian at Georgia Institute of Technology, Institute for Electronics and Nanotechnology, IEN/IMAT material characterization facilities. We acknowledge technical support on Zetasizer from A. J. Heiler and S. N. Thomas at Georgia Institute of Technology. Figures 1a, 2i, 4a, 5a and 6a,e, and Extended Fig. 2c were created with BioRender.com.

Author information

Author notes

  1. These authors contributed equally: Kristel J. Yee Mon, Sungwoong Kim.

Authors and Affiliations

  1. Department of Microbiology and Immunology, Cornell University, Ithaca, NY, USA

    Kristel J. Yee Mon & Brian D. Rudd

  2. George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA

    Sungwoong Kim & Ankur Singh

  3. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA

    Sungwoong Kim, Zhonghao Dai, Ritika Jain & Ankur Singh

  4. Department of Materials Science and Engineering, Cornell University, Ithaca, NY, USA

    Sungwoong Kim

  5. Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY, USA

    Jessica D. West, Hongya Zhu & Andrew Grimson

  6. Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA

    Ankur Singh

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Contributions

A.S. conceived the nanowire fabrication, and B.D.R. and A.S. conceived and designed the T-cell studies. K.J.Y.M. and S.K. designed and performed the original experiments and analysis. Z.D. performed all the revision experiments and analysis with A.S. K.J.Y.M., S.K., B.D.R., Z.D. and A.S. wrote the manuscript. A.G. and J.D.W. generated the miRNA. A.G. and H.Z. performed RNA-seq analysis. R.J. generated the eGFP-encoding lentiviruses. All authors provided critical feedback on the research, analysis and manuscript.

Corresponding authors

Correspondence to Brian D. Rudd or Ankur Singh.

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Competing interests

A.S. receives research support from Genentech, Inc. A.S. has an intellectual property disclosure filed on the technology with US Patent and Trademark Office (USPTO) assigned U.S. Application No. 63/591,553. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 T cell characterization on nanowires.

a) Effect of Centrifugal g-Force on the survival of primary naïve CD8+ T cells on PEI-functionalized nanowires with 10 wt/v% PEI. Data are presented as mean ± s.e.m. One-way ANOVA with Tukey’s test (n = 3, each dot represents an independent nanowire chip). b-d) Representative SEM images of primary naïve murine CD8+ T cells seeded on the nanowires (B) and FIB-SEM etched T cells on the nanowires (C-D). e) Representative FIB-SEM image of melted nanowire inside a T cell.

Source data

Extended Data Fig. 2 Nanowire functionalization and characterization of T cell response.

a) Representative pseudocolor plots of small molecular weight dextran delivery into primary naïve murine CD8+ T cells using nanowires. b) Representative flow cytometry histograms of tbet and emoes proteins in naïve mouse CD8+ T cells incubated with no-nanowire (2D) and no-PEI coated nanowire delivery of miR-29-mimic to murine CD8+ T cells. c) Schematic of functionalization process of nanowires via covalent conjugation of PEI on nanowire surface. d) Representative flow cytometry gating of cell viability (top) and delivery efficacy (bottom) of 6-FAM-miR-29-mimic in primary naïve murine CD8+  T cells using 2D control, unmodified nanowires, and covalently conjugated (CC) or surface coated (SC) nanowires with PEI. T cells were incubated on nanowires for 96 hrs. e) Representative STEM images of blank nanowires showing the absence of a PEI layer. f) Representative EDX images of blank nanowires showing the minimal presence of Nitrogen (N), Carbon (C), and Oxygen (O) on Silicon (Si). g) Zeta potential measurement of bare silicon nanowires, PEI-functionalized nanowires, and miRNA-loaded PEI-functionalized nanowires. Data are presented as mean ± s.e.m. One-way ANOVA with Tukey’s test (n = 3, each dot represents an independent nanowire chip). h) Representative flow cytometry gating of cell viability (top) and delivery efficacy (bottom) of 6-FAM-miR-29-mimic in primary naïve murine CD8+ T cells at various miRNA doses and fixed PEI wt/v%. T cells were incubated on nanowires for 96 hrs.

Source data

Extended Data Fig. 3 Nanowire penetration in T cells.

a) 3D projection of confocal microscopy image of nanowires in T cells. PEI-functionalized nanowires were loaded with FITC-BSA (green), naïve mouse CD8+ T cells were seeded on nanowires, centrifuged, and cultured for 12 hrs. Fixed samples were stained with phalloidin (actin, orange) and DAPI (nucleus, blue). Data representative of 40 cells from 4 independent technical replicates, with 10 cells from each replicate. b) Orthogonal projection with nanowires (FITC), DAPI, and actin (orange) from the same sample as in (A). The inset on the right represents the maximum intensity projection (MIP) of the representative sample. Data representative of 40 cells from 4 independent technical replicates, with 10 cells from each replicate. c) 3D projection of representative confocal microscopy image of nanowires in T cells comparing PEI-functionalized nanowires to blank nanowires. Data representative of 40 cells from 4 technical replicates for each condition, with 10 cells from each replicate. d) Measurement of the height of nanowire penetration inside a naïve mouse CD8+ T cell. Data represents 10 samples randomly selected from 40 cells from 4 independent technical replicates. Data are presented as mean ± s.e.m. A two-tailed, unpaired t-test with Welch’s correction was performed. e) Quantification of the number of nanowires per cell from maximum intensity projection of confocal microscopy images. Each dot represents a cell and a total of 40 cells were analyzed across 4 independent technical replicates. Data are presented as mean ± s.e.m. A two-tailed, unpaired t-test with Welch’s correction was performed.

Source data

Extended Data Fig. 4 miRNA delivery and release in T cells.

a-b) Histogram overlays (A) and bar graph of 6-FAM-miR-29-mimic uptake or release in primary naïve mouse CD8+ T cells (B). Data are presented as mean ± s.e.m. One-way ANOVA with Tukey’s test (n = 3, each dot represents an independent nanowire chip). c) Representative confocal microscopy image of murine CD8+ T cell with delivered miR-29-mimic using PEI-functionalized nanowires. T cells were incubated on nanowires for 96 hrs. d) Bar graph of miR-29 delivery efficacy of CD8+ T cells upon 6-FAM-miR-29-mimic delivery of primary naïve murine CD8+ T cells with different nanowire tip sizes and covalently conjugated with 10 wt/v% PEI. T cells were incubated on nanowires for 96 hrs. Data are presented as mean ± s.e.m. One-way ANOVA with Tukey’s test (n = 3, each dot represents an independent nanowire chip).

Source data

Extended Data Fig. 5 Benchmarking PEI-functionalized nanowires against conventional methods of miRNA delivery.

a) Bar graph representing % viability of primary naïve murine CD8+ T cells exposed to PEI-functionalized nanowires, Lipofectamine, PEI complexation, lentivirus, and nucleofection. 2D soluble miRNA delivery was used as a positive control. T cells were incubated on nanowires for 96 hrs. b) Bar graph representing % viability of primary, pre-activated murine CD8+  T cells exposed to PEI-functionalized nanowires, Lipofectamine, PEI complexation, lentivirus, and nucleofection. 2D soluble miRNA delivery was used as a positive control. T cells were pre-activated using anti-CD28/anti-CD3 beads. T cells were incubated on nanowires for 96 hrs. c) Bar graph representing % viability of primary naïve human CD8+ T cells exposed to PEI-functionalized nanowires, Lipofectamine, PEI complexation, lentivirus, and nucleofection. 2D soluble miRNA delivery was used as a positive control. T cells were incubated on nanowires for 96 hrs. d) Bar graph representing % viability of primary, pre-activated human CD8+ T cells exposed to PEI-functionalized nanowires, Lipofectamine, PEI complexation, lentivirus, and nucleofection. 2D soluble miRNA delivery was used as a positive control. T cells were pre-activated using anti-CD28/anti-CD3 beads. T cells were incubated on nanowires for 96 hrs. e) Bar graph representing % naïvity after 96 hrs when naïve murine CD8+ T cells are exposed to PEI-functionalized nanowires, Lipofectamine, PEI complexation, lentivirus, and nucleofection. 2D soluble miRNA delivery was used as a positive control. T cells were incubated on nanowires for 96 hrs. f) Bar graph representing % naïvity after 96 hrs when naïve human CD8+ T cells are exposed to PEI-functionalized nanowires, Lipofectamine, PEI complexation, lentivirus, and nucleofection. 2D soluble miRNA delivery was used as a positive control. T cells were incubated on nanowires for 96 hrs. In all figures, data are presented as mean ± s.e.m. One-way ANOVA with Tukey’s test (n = 3, each dot represents an independent nanowire chip).

Source data

Extended Data Fig. 6 Delivery of eGFP-encoding lentivirus to primary naïve human CD8+ T cells using nanowires.

a-b) Standard curve of concentration versus absorbance of ELISA against p24 antigen with lentiviral particulates (A) and bar graph representing % loading efficiency of lentivirus on nanowires (B) at various concentrations. Data are presented as mean ± s.e.m. (n = 3, each dot represents an independent nanowire chip). c) Histograms of fold-change in CD8+ GFP+ T cells upon eGFP-encoding lentiviral delivery using nanowires and without nanowires, in the presence or absence of reverse transcriptase inhibitor Efavirenz. T cells were incubated on lentiviruses on nanowires or without nanowires for 24 hr to minimize toxicity with lentiviruses, followed by culture in hIL-2 till day 4.

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Extended Data Fig. 7 Co-delivery of miRNA antisense and mimic oligonucleotides by single functionalized nanowire platform toggles human CD8+ T cell fate switches.

a) % CD8+ CD62L- T cells in human CD8+ T cells. Primary naïve human CD8+ T cells were incubated on PEI-functionalized nanowires, loaded with miR-29-ASO, miR-130-mimic, or coloaded with miR-29-ASO and miR-130-mimic for 96 hrs, followed by TCR stimulation for the next 48 hrs. 2D culture of Primary naïve human CD8+ T cells with soluble miR-29-ASO and miR-130-mimic, followed by TCR stimulation for the next 48 hrs was used as a control. b) % viable CD8+ T cells in human CD8+ T cells in conditions defined in (A). c-d) % activated T cells expressing CD69 (C) and CD28 (D) markers in human CD8+ T cells in conditions defined in (A). e) % effector T cells in human CD8+ T cells in conditions defined in (A). f) % PD1+ T cells in human CD8+ T cells. Primary naïve human CD8+ T cells were incubated on PEI-functionalized nanowires, followed by TCR stimulation for the next 3 or 6 days. 2D culture of Primary naïve human CD8+ T cells with soluble miR-29-ASO and miR-130-mimic, followed by TCR stimulation for the next 48 hrs was used as a control. In all figures, data are presented as mean ± s.e.m. One-way ANOVA with Tukey’s test (n = 3 for A-D, F; n = 6 for E; each dot represents an independent nanowire chip).

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Extended Data Fig. 8 Delivery of two miRNAs by functionalized nanowire enhances murine CD8+ T-cell effector function in vitro.

a-b) Representative histograms (A) and quantification (B) of cell-death markers Fas and AnnexinV expression in murine CD8+ T cells post 72 hrs of gB peptide stimulation. Primary naïve murine CD8+ T cells from gBT-1Tg mice were incubated on nanowires for 96 hrs, and gB peptide stimulation was done for the next 72 hrs. Data are presented as mean ± s.e.m. Two-way ANOVA with Tukey’s test, (n = 4, each dot represents an independent nanowire chip). c) Representative histograms (left) and bar graph (right) representing the expression of CD62L (MFI) on CD8+ T cells post 72 hrs of gB peptide stimulation. Primary naïve murine CD8+ T cells from gBT-1Tg mice were incubated on nanowires for 96 hrs, and gB peptide stimulation was done for the next 72 hrs. Data are presented as mean ± s.e.m. One-way ANOVA with Tukey’s test, (n = 4, each dot represents an independent nanowire chip). d) Bar graph representing the % CD8+ CD62L- cells in CD8+ T cells post 72 hr of gB peptide stimulation. Primary naïve murine CD8+ T cells from gBT-1Tg mice were incubated on nanowires for 96 hrs, and gB peptide stimulation was done for the next 72 hrs. Data are presented as mean ± s.e.m. One-way ANOVA with Tukey’s test, (n = 4, each dot represents an independent nanowire chip). e) Representative activation markers CD44, CD69, and CD28 expression histograms post 72 hr of gB peptide stimulation. Primary naïve murine CD8+  T cells from gBT-1Tg mice were incubated on nanowires for 96 hrs, and gB peptide stimulation was done for the next 72 hrs. Data are presented as mean ± s.e.m. Two-way ANOVA with Tukey’s test, (n = 4, each dot represents an independent nanowire chip). f) Representative histograms of cytokine TNFα and perforin-producing CD8+ T cells post 72 hr of gB peptide stimulation. Primary naïve murine CD8+ T cells from gBT-1Tg mice were incubated on nanowires for 96 hrs, and gB peptide stimulation was done for the next 72 hrs. Data are presented as mean ± s.e.m. Two-way ANOVA with Tukey’s test, (n = 4, each dot represents an independent nanowire chip).

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Extended Data Fig. 9 Functionalized nanowire delivery of dual miRNA-related oligos influence the naïve CD8 T cell transcriptome.

a). Principal component analysis of naïve CD8+ T cells incubated for 96 hrs on nanowires coated with negative control miRNA (salmon) relative to miR-29-ASO/miR-130-mimic miRNA (blue). Data (each dot) represents a biological replicate (mice). b). Volcano plot of statistically significant, differentially expressed T cell-related genes comparing nanowires coated with negative control miRNA relative to miR-29-ASO/miR-130-mimic miRNA. The red dots represent select labeled genes that are upregulated, the blue dots represent select labeled genes that are downregulated. The colors green and magenta represent select genes from other biological processes. Data represent an average of two biological replicates (mice). c). Volcano plot of statistically significant, differentially expressed mechanoregulatory genes in naïve CD8+ T cells on nanowires coated with negative control miRNA (blue) relative to no nanowire control (red). Data represent an average of two biological replicates (mice).

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Extended Data Fig. 10 Delivery of dual miRNA components by functionalized nanowire enhances murine CD8+ T cell effector memory function in vivo in influenza A.

a-b) Representative contour plots (A) and quantification (B) of effector memory T cell (Tem) and central memory T cell (Tcm) phenotype of Lung and spleen CD8+ T cells at Day 8 Flu gB primary infection. Data are presented as mean ± s.e.m. Two-way ANOVA with Tukey’s multiple comparison test, (n = 6 mice).

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Yee Mon, K.J., Kim, S., Dai, Z. et al. Functionalized nanowires for miRNA-mediated therapeutic programming of naïve T cells. Nat. Nanotechnol. (2024). https://doi.org/10.1038/s41565-024-01649-7

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