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

Abstract

Therapies based on immune cells have been applied for diseases ranging from cancer to diabetes. However, the viral and electroporation methods used to create cytoreagents are complex and expensive. Consequently, we develop targeted mRNA nanocarriers that are simply mixed with cells to reprogram them via transient expression. Here, we describe three examples to establish that the approach is simple and generalizable. First, we demonstrate that nanocarriers delivering mRNA encoding a genome-editing agent can efficiently knock-out selected genes in anti-cancer T-cells. Second, we imprint a long-lived phenotype exhibiting improved antitumor activities into T-cells by transfecting them with mRNAs that encode a key transcription factor of memory formation. Third, we show how mRNA nanocarriers can program hematopoietic stem cells with improved self-renewal properties. The simplicity of the approach contrasts with the complex protocols currently used to program therapeutic cells, so our methods will likely facilitate manufacturing of cytoreagents.Current widely used viral and electroporation methods for creating therapeutic cell-based products are complex and expensive. Here, the authors develop targeted mRNA nanocarriers that can transiently program gene expression by simply mixing them with cells, to improve their therapeutic potential.

Fig. 1

Creating mRNA nanoparticles to program therapeutic T-cells. a Schematic explaining how cultured T-cells can be programmed to express therapeutically relevant transgenes carried by polymeric nanoparticles (NPs). These particles are coated with ligands that target them to specific cell types, enabling them to introduce their mRNA cargoes and cause the targeted cells to express selected proteins (like transcription factors or genome-editing agents). b Design of targeted mRNA-carrying NPs. The inset shows a transmission electron micrograph of a representative NP; scale bar, 50 nm. Also depicted is the synthetic mRNA encapsulated in the NP, which is engineered to encode therapeutically relevant proteins

Fig. 2

mRNA nanoparticle transfection choreographs robust transgene expression by lymphocytes. a Primary T-cells were mixed with CD3-targeted polymeric nanoparticles (NPs) carrying Cy5-labeled mRNA. Confocal microscopy establishes that these particles are rapidly internalized from the cell surface. The images are representative of 15 randomly chosen fields. Scale bars, 2 μm. b Flow cytometry of preactivated PBMCs 24 h after incubation with CD3-targeted or isotype control antibody-targeted nanoparticles bearing eGFP-encoding mRNA. c Bar graph summarizing transfection efficiencies from three independent experiments conducted in duplicate. d, e Comparison of the effects electroporation and NP gene delivery have on cell expansion. Left panels show the workflow for transfection with NPs (top) and electroporation (bottom). Right panels show the -fold expansion of PBMC cultures from three independent donors treated with stimulatory beads on days 0 and 12. Matched cultures from each donor were not treated, or transfected using CD3/CD28-targeted NPs (d, right) or electroporation (e, right) on days 5 and 17. Every line represents one donor and each dot reflects the -fold T-cell expansion. Pairwise differences between groups were analyzed with the unpaired, two-tailed Student’s t test; n.s., non-significant; *, significant, n = 3). f Relative viability of NP-transfected and electroporated T-cells. Samples of 2 × 10⁶ activated T-cells per condition were untreated, transfected with NPs, or electroporated. 18 h after treatment, cells were labeled with fluorescent dyes to assess viability. Results from three separate experiments conducted in duplicate are summarized in the bar graph shown in g. Statistical analysis between groups was performed using the unpaired, two-tailed Student’s t Test. *P < 0.0001

Fig. 3

Nanoparticles can knockout T cell receptors in CAR-programmed lymphocytes. a Integration of nanoparticle (NP) transfection into normal manufacturing of CAR-T-cells. After stimulation with anti-CD3/CD28-coated beads (day 0), CD8-targeted mRNA NPs were introduced on days 1 and 2, then lentiviral transduction with a vector encoding the leukemia-specific 19-41BBz CAR was performed on day 3. We added either NPs carrying mRNAs encoding megaTAL nuclease plus eGFP, or control particles loaded with eGFP mRNA alone. b Flow cytometry of NP transfection efficiencies (based on eGFP signals) correlated with surface expression levels of TCRs (based on CD3 signals) by T-cells following NP treatments. c Summary plot showing editing efficiency as measured by loss of CD3 surface expression at day 14 (n = 6). d Surveyor assay confirming TCRα chain gene locus disruption. e Flow cytometry of lentiviral transduction in genome-edited versus control T-cells. f Bar graph showing mean viral transductions and SE of three independent experiments conducted in duplicate; n.s., not significant g, h Proliferation and cytolytic activity of TCR+ (FACS sorted TCR-positive, unedited 19-41BBz CAR-T-cells) and TCR- (FACS sorted TCR-negative, genome edited) 19-41BBz CAR-T-cells. To measure proliferation, T-cells were co-cultured on irradiated TM-LCL leukemia cells. Cytolytic assays were performed with CD19-expressing K562 target cells. i T cell IFN-γ release was measured with ELISA 48 h after stimulation on CD19+ TM-LCL leukemia cells or control LNCaP C4-2 prostate adenocarcinoma cells. Data from two experiments run in triplicate are shown

Fig. 4

Nanoparticles can induce markers and transcriptional patterns characteristic of memory T-cells. a Expression of total Foxo1 protein measured by intracellular labeling in Jurkat and primary T-cells treated with CD3-targeted control (GFP+) or Foxo1_3A-GFP NPs. Mean Foxo1 fluorescence intensities (MFI) for cells transfected with control NPs (shown in blue) compared to Foxo1_3A-NPs (shown in red) are indicated above the respective histograms. b qPCR measurements of relative Foxo1_3A mRNA expression over time after cells were exposed to Foxo1_3A-eGFP nanoparticles (NPs). Data are representative of two independent experiments. Graphs show mean ± SE. c Effect of CD8-targeted Foxo1_3A-GFP NPs on CD62L expression after 24 h of particle treatment. d Percentage of CD62L⁺ cells in sorted CD8⁺ eGFP⁺ cells treated with CD8-targeting control or Foxo1_3A/eGFP-encoding NPs at 1, 8, and 20 days of culture after the particles were introduced. These results are from three independent donors. *P < 0.05; **P < 0.01 between the indicated conditions as calculated from a ratio-paired t-test. e, f Heat maps of T_CM signature gene expression in T_CM, naive, and control cells 8 days after treatment. g Volcano plot of differential gene expression in Foxo1_3A-NP-treated cells after 8 days. T_CM signature genes and selected memory phenotype genes are indicated. P value of overlap between Foxo1_3A and the T_CM signature gene set was determined by GSEA (via analysis shown in h)

Fig. 5

Foxo1_3A-NP-transfection improves the anti-cancer activities of CAR T-cells. NSG mice were inoculated with CD19 + Raji-luc tumor cells. After 7 days the mice were injected with luciferin and imaged on an IVIS before being randomly sorted into groups (n = 9) with representative tumor burden. Next 2.5 × 10⁶ CD8+ 19-41BBζ CAR + T-cells (transfected with NPs loaded either with Foxo1_3A mRNA or GFP mRNA) were infused intravenously. Control mice received no treatment. a Representative IVIS imaging depicting five mice per cohort. b Quantified tumor burden (as mean radiance from luciferase activity from each mouse from a ± SE). Pairwise differences between groups were analyzed with the unpaired, two-tailed Student’s t Test; n.s., non-significant; *, significant. c Kaplan–Meier survival curves for treated and untreated control mice. Shown are nine mice per treatment group pooled from two independent experiments. ms, median survival. Statistical analysis between the treated experimental and the untreated control group was performed using the Log-rank test; P < 0.05 was considered significant

Fig. 6

Transfection of stem cells with mRNA nanoparticles can promote their expansion and self-renewal. a Targeting of CD105 enables specific transfection of HSC CD34⁺ cells. Cells were left untreated, or transfected with eGFP-encoding mRNA in nanoparticles (NPs) coated with PGA coupled to a control antibody or anti-CD105. Transfection efficiency was assayed by flow cytometry after 24 h. b NP transfection efficiency in CD34⁺ samples from three independent donors. Viability is shown in c. d Expansion of CD34 + PBSCs after NP-transfection. e Phenotypical characterization of PBSC-derived CD34⁺ subpopulations after 2 days in culture. Cells were either transfected with eGFP NPs on day 1 or left unmodified. Gating is indicated in brackets on top of each column. f Summary bar graph showing mean frequencies and SE of primitive Hematopoietic Stem Cells (HSCs), Multipotent Progenitors (MPPs), Lymphoid-primed Multipotent Progenitors (LMPs), and Early Myeloid Progenitors (EMPs). PBSCs from four independent donors were analyzed. Error bars represent mean ± SE. g Colony output of sort-purified GFP-NP transfected versus unmodified CD34⁺ cells from day 7 cultures (n = 3 cultures from independent donors); n.s., non-significant. Arising colonies were identified as colony forming unit (CFU) granulocyte (CFU-G), macrophage (CFU-M), granulocyte-macrophage (CFU-GM) and burst forming unit-erythrocyte (BFU-E). Colonies consisting of erythroid and myeloid cells were scored as CFU-MIX; n.s., non-significant. Error bars represent mean ± SE. h Representative images of CFU-MIX colonies from untransfected and GFP-NP-transfected CD34⁺ cells (×4-magnification; scale bar 1000 µm). i qPCR measurements of NP-delivered Musashi-2 (MSI2) mRNA expression over time. Error bars represent mean ± SE. j Comparison of CD133 and CD34 expression in HSCs transfected with control GFP mRNA-NPs versus MSI2 mRNA-NPs, assessed by flow cytometry 8 days after NP exposure and cell expansion. Data represent two independent experiments conducted in triplicate. k Cellular fold expansion of CD34− (differentiated) and CD34⁺ CD133⁺ (progenitor) cells. Bar graphs show mean and SE of three independent experiments. Data represent two independent experiments conducted in triplicate. l Colony forming unit outputs of untransfected versus MSI2-NP-transfected HSCs (n = 3 cultures from independent donors); Pairwise differences between groups were analyzed with the unpaired, two-tailed Student’s t Test. *P = 0.049, **P = 0.012, ***P = 0.011; n.s., non-significant. Error bars represent mean ± SE. m Representative images of colonies from untransfected and MSI2-NP-transfected CD34⁺ cells (scale bar, 300 µm)