SUPPLEMENTARY MATERIALS AND METHODS

Isolation and culture of primary bovine Müller cells

To obtain primary bovine Müller cells (pMC), fresh bovine eyes were obtained from the

local abattoir and kept in 4°C CO2 independent medium until dissection. Extra-ocular tissue

was removed, followed by eye disinfection using 20% ethanol. Using sharp curved scissors

the eye was bisected, vitreous was carefully removed and the posterior eye cup was

transferred to a culture dish containing PBS buffer with 5% penicillin-streptomycin mixture.

Subsequently, the eye cup was cut into 4 flaps, of which the retinal tissue was removed and

transferred to a tissue grinder containing separation medium, consisting of advanced D-

MEM medium (Gibco-Invitrogen, Merelbeke, Belgium), supplemented with 1% Penicillin-

Streptopmycin and 1% Glutamax (Gibco-Invitrogen). After thoroughly grinding of the retinal

tissue the cell suspensions were poured into a 40 µm cell strainer mounted on a 50 ml falcon

tube and spun down at 300 g for 5min at RT. The supernatant of each falcon tube was

discarded and the cell pellets were washed with separation medium. After 3 washing steps,

the cell pellets were re-suspended in separation medium supplemented with 10% heat-

inactivated FBS (Hyclone, Cramilton, UK) and 4 ng ml-1 epidermal growth factor (Sigma-

Aldrich, Bronem, Belgium). The retinal tissue of two eye flaps was transferred into one T75

Cellbind flask (Corning®). The cells were cultured in a 5% CO2 incubator at 37°C and the

medium was renewed once a week. After 2-3 weeks the cells were passaged and at passage

3 they were seeded. Five days prior to transfection, 2x104 cells were plated per well in 24-

well plates.

MTT-assay

The viability of MIOM1 Müller cells was evaluated 24 h after addition of the

MessengerMAX lipoplexes, which were prepared as described above at different v/w

cationic lipid-to-mRNA ratios. After removal of the lipoplexes, fresh cell medium containing 5

mg/ml of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent

(Sigma-Aldrich, USA) was added to the cells. After 3 h incubation at 37°C, cells were washed

with PBS and the newly formed formazan crystals were dissolved by addition of 100% DMSO.

The plates were covered in aluminum foil and placed on an orbital shaker (Rotamax 120,

Heidolph, Germany) for 45 min at 1200 rpm. Finally, the absorbance was measured at 590

nm and 690 (background) with an Envision plate reader (Perkin Elmer, Zaventem, Belgium).

Cells treated with 50 µl Opti-MEMTM alone were used as positive controls, representing 100%

viability.

Evaluation of uptake and expression using confocal microscopy

Five days prior to transfection, MIOM1 Müller cells were seeded in 35 mm CELLview

microscopy dishes with glass bottom (Greiner Bio-One, Vilvoorde, Belgium) at a density of

5x104cells in 1.5 ml. Cells were transfected with naked or MessengerMAX-complexed Cy5-

labeld m1ΨU(1.0) mRNA as described before. After 24 h incubation at 37°C, cell nuclei were

stained with Hoechst 33342 staining (1 mg/ml in PBS; 1000x diluted) and incubated for 15

min at 37°C. Next, cells were washed with PBS and provided with fresh cell culture medium.

Live-cell imaging was performed using a confocal laser scanning microscope (C1si, Nikon,

Japan) with a Plan Apo VS 60x 1.4 NA oil immersion objective lens (Nikon, Japan). Image

processing was performed using ImageJ software.

SUPPLEMENTARY FIGURES

Figure S1 │ Cytotoxicity of different v/w ratios of MessengerMAX lipoplexes. Cell viability of MIOM1 cells 24h after incubation with MessengerMAX-complexed mRNA at different v/w ratios as determined by MTT assay. Cells treated with

Opti-MEMTM alone served as a blank. Data reflect mean ± SD (n=1).

Figure S2 │ Transfection efficiency of mRNA versus pDNA containing lipoplexes in primary bovine Müller cells. (A) Percentages of eGFP transfected Müller cells, 24 h after incubation with Lipofectamine and MessengerMAX containing

unmodified mRNA or pDNA. Data represent mean ± SD (n=1). Representative flow cytometry histograms are shown in (B). ***, p < 0.001 mRNA versus pDNA by one-way ANOVA.

Figure S3 │ eGFP expression after transfection of chemically modified mRNAs with MessengerMAX in MIOM1 cells. (A) Percentage eGFP positive cells 24 h after incubation with the lipoplexes in serum-containing medium. Mean fluorescence intensity (MFI) and corresponding percentage of viable cells as determined by flow cytometry are shown in (B). % viable

cells was gated as DiIC1(5)-/DAPI-. m5C: 5-methylcytidine; ψU: pseudouridine; s2U: 2-thiouridine and m1ψU: N1-methylpseudouridine; 0.25 symbolizes mRNA with replacement of 25% of total uridine or cytidine by the corresponding

modified nucleoside; 1.0 symbolizes mRNA with complete replacement of uridine or cytidine by the corresponding modified nucleoside. Cells treated with eGFP encoding pDNA, naked (i.e. unpackaged) mRNA, unmodified mRNA, CleanCap™ Cyanine

5 EGFP mRNA (5moU) purchased from Trilink (San Diego, CA) and mRNA only modified by polyadenylation and ARCA capping were used as control transcripts. Data represent mean ± SD (n=3). ns: not significant, p > 0.05 versus unmodified

eGFP mRNA by one-way ANOVA.

Figure S4 │ Comparison of eGFP expression after transfection of unmodified or modified (ψU and m1ψU) mRNAs with MessengerMAX in ARPE-19 cells. Percentage eGFP positive cells (A) and mean fluorescence intensity (MFI) (B) 24 h after incubation with the lipoplexes in serum-containing medium. m1ψU(0.25) symbolizes modified mRNA with replacement of

25% of total uridine by N1-methylpseudouridine; ψU(1.0) and m1ψU(1.0) symbolizes complete replacement of total uridine by pseudouridine and N1-methylpseudouridine, respectively. Data represent mean ± SD (n=2). ***, p < 0.001 versus

unmodified eGFP mRNA by one-way ANOVA.

Figure S5 │ Transfection efficiency and uptake of m1ΨU(1.0)-mRNA in its naked versus MessengerMAX-complexed form. Percentage of eGFP positive cells (A) and MFI (B) 24h after incubation of MIOM1 cells with naked or MessengerMAX-

complexed mRNA in serum-containing medium. Data represent mean ± SD (n=1). Representative confocal images showing uptake (red) and eGFP expression (green) of Cy5-labeled m1ΨU(1.0)-mRNA administered as such (C) or complexed with the

MessengerMAX carrier (D). All nuclei are stained with Hoechst (blue), scale bar: 30 µm.

Figure S6 │ Expression of cy5-labeld m1ψU-fLuc mRNA after application to a conventional bovine retinal explant. Bioluminescence obtained 4h after administration of naked or MessengerMAX-complexed fLuc mRNA to the photoreceptor

segment of the explant (mimicking subretinal administration) (A) and to the vitreal side of the explant (mimicking intravitreal administration) (B) . Individual values represent different retinal explants. Explants treated with OptiMEM were

used as non-treated control (NTC). Results were obtained by 3 independent experiments (n=1). *, p < 0.05 by an unpaired t-test. Corresponding representative bioluminescence images are displayed above the graph.

Figure S7 │ Degradation of mRNA in fetal bovine serum (FBS) and adult bovine vitreous (ABV). Gel electrophoresis on naked mRNA and MessengerMAX-complexed mRNA demonstrates degradation of uncomplexed mRNA at all studied v/w ratios

when incubated for 30 min in ABV or FBS, an essential component of serum-containing culture medium.

Figure S8 │ Retinal distribution of Cy-5 labeled m1ψU-mRNA after IVT injection in the VR bovine explant. Figure represents representative confocal microscope images of vertical frozen sections showing the transport of messengerMAX-complexed mRNA through the VR interface 24 h after IVT injection. Locations with comprised ILM show penetration of lipoplexes in the retina. ILM is stained by anticollagen antibodies (green), which also stains blood vessels. All nuclei are stained with Hoechst

(blue), scale bar: 30 µm.