Article
Cy5-mRNA JETPEI123(^124)
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acccagtccgcgcgaggcgcccgccaccgtcgccgcatgcacttgctgtgcttcttgtctctggcgtgttccctgccttcgagtcgggatcgccgctgcgctgatc-ctggg
cttctcggaagcggagcccgacgggggcgaggtcaaggcgacctggaggagcagttgcggtctgtgtccagcgtagatgagttttgaaggcaaactgatgtctgtcc
tgtacccagaccagcagcccaccctacttcaataccaggacaggggacagtgtaaaatttgcggaaaatgtacaagtgccagctgcggaaaggcggctgctgcatgg
cattataacacagagatcgcatgccacgtgaggtgtgtatagatgtggggaaggagtttggagcagccacactgaaaagtattgataatgagtggagaaagactcaat
aacacagcgaggggccttctttaaacctgcagtgcatgaacaccagcacaggttactccatgtgtgtccgtctacagatgtgggggttgcctcagcaagacgtttgcaac
gtttgaaattacagtgccaatcacacttcctgccggtgcatgtctctctcacaaggccccaaaccagtcacaatcagttttgcctaaactggatgtttacagacaagttcattc
aattattagacgttcatgtccaacaaacctctgccagcaacattaccacagtgtcaggcagcttatgtgtggaataactacatgtgccgatgcctggcaacaagatcagca
ggattttatcgtggacccaacaaggagcttttattcaaatgttgaagatgatggatgaagacacctcaaccaatggattccatgatgtctctgtcagtgtgtctgcaagggg
gggcagtgtgtttcggccatcctgtaaaaacaaactagttgtggaccccacaaagaacttttccctaattcatgtggagccaacagggaattttagatagagactcatgtcg
atgagaatacatgtcagtgtgtatgtaaaagaacgtgtccaagaaatcagccccgaatcctgggaaatgtgcctgtgaatgtacagaaaacacacagaagtgcttccttt
aaagggaagaagttccaccatcaaacatgcagttgttacagaagaccgtgtgcgaatcgactgaagcattgtgatccaggactgtcctttagtgaagaagtatgccgctg
tgtcccatcgtattggaaaaggccacatctgaactaa
b c
Cytosine 5-methyl-cytosine Uridine Pseudo Uridine
Contro
l
α-VEGF-C
Cy5-GFPVEGF-CContro
l
Cy5-GFPVEGF-C
Cell lysate 6hCell lysate 24h
Contro
l
Cy5-GFPVEGF-C
Media 24h
50kDa37kDa
25kDa
Full lengthIntermediate
Processed
(1-6) HEK293T In Vitro (1-3) HEK293T In Vitro Supernatant(1-6) Mouse CSF
(^123456123123456789)
(7-9) recombinant VEGF-C
20kDa15kDa
α-VEGF-C
GFP-mRNA VEGF-C-mRNA
CSF 6h
c
c
d
e
f
Single Cells98.1
Lymphocytes89.6
CD45neg CD45+93.3
6.66
CD45neg CD45+51.5
46.9
CD45neg CD45+15.6
81.5
Control VEGFC-mRNA
Empty
mRNA-Cy5
Empty
CD31-BV421
Endothelialcells
Other cells
Immune cells
CD45 negative
CD45-PE
Empty
Brain
Meninges
Lymph Node
FSC-A
SSC-A
FSC-A
FSC-H CD45 neg
CD45+ CD45- CD31+CD45- CD31-
Brain
Meninges
Lymph node
control
Cy5-mRN
A
-1
0
1
2
3
4
(^5) P = 0.09
control
Cy5-mRNA
-1
0
1
2
3
4
5 P = 0.14
0
2
4
6
8
10
% Cy5 + cells
**
0
2
4
6
8
10
12 P = 0.29
control
Cy5-mRNA
-1
0
1
2
3
4
5
% Cy5 + cell
s
0
2
4
6
8
10
12
% Cy5 + cells
0
2
4
6
8
10
12
P = 0.20
0
2
4
6
8
10
P = 0.28
0
2
4
6
8
10
P = 0.07
P = 0.00 8
P < 0.0001
P = 0.0096
AAV-CTRLAAV-VEGF-
C
GFP-mRNA
VEGF-C-mRNA
d7 tumo
r
d28 tumor
0
20
40
60
80
pg/mg of brain tissue
ELISA (Brain)
AAV- CTRLAAV-VEGF-CGFP-mRNA
VEGF-C-mRNA
d7 tumord28 tumor
0
20
40
60
80
pg/m
L
ELISA (Serum)
AAV-CTRLAAV-VEGF-
C
GFP-mRNAVEGF-C-mRNAd7
tumor
d28 tumo
(^0) r
20
40
60
80
detection limit
pg/mL
E(CSF)LISA
AAV-CTRLAAV- VEGF-CGFP-mRN
A
VEGF-C-m
RNA
d7 tumo
r
d28 tumo
(^0) r
20
40
60
80
pg/mg of meningeal tissue
ELISA(Meninges)
P = 0.004 P = 0.053
P = 0.006
P = 0.02
P = 0.02
P = 0.03
P = 0.007
g
h
i
Extended Data Fig. 3 | Validation of VEGFC mRNA in vitro, and its in vivo
uptake and expression tropism. a, The complementary DNA (cDNA) sequence
of VEGFC that was used for mRNA production. b, 5-Methylcytosine and
pseudouridine were used to substitute all of the cytosine and uridine bases in
the mRNA. c, The VEGFC mRNA construct was transfected into HEK293T cells
and cell lysate and the medium was collected to detect full-length and secreted
intermediate and processed peptides. d, The VEGFC mRNA construct (5 μg)
was delivered in vivo into the cisterna magna of mice using a JETPEI polyplex
system. Six hours later, mouse CSF was collected using a capillary tube and
filtered with an amicon filter, and the wash-through was used to run a western
blot (each lane is n = 10 mice pooled). e, Raw western blot images for Extended
Data Fig. 4c (gel 1: column 1, control; column 2, Cy5–GFP mRNA; column 3,
VEGFC mRNA; column 4, control; column 5, Cy5–GFP mRNA; column 6,
VEGFC mRNA; gel 2: column 1, control; column 2, Cy5–GFP mRNA; column 3,
VEGFC mRNA) and Extended Data Fig. 4d (gel 3: columns 1–3, Cy5–GFP mRNA;
columns 4–6, Cy5–GFP mRNA; columns 7-9, recombinant human VEGF-C in
increasing concentration). The experiments were repeated twice
independently with similar results. f, VEGFC mRNA and Cy5-labelled GFP mRNA
were mixed at a 1:1 ratio and delivered to mice in vivo using JETPEI. Fifteen
minutes later, mice were euthanized and the whole skull cap was imaged to
observe the distribution of mRNA particles. The experiments were repeated
independently twice with similar results. g, h, VEGFC mRNA and Cy5-labelled
GFP mRNA were mixed at a 1:1 ratio and delivered to mice in vivo using JETPEI.
After 24 h, the brains, meninges and lymph nodes of treated mice were
collected for f low cytometry to measure the percentage of Cy5-positive cells in
each compartment (control, n = 6; Cy5-labelled mRNA, n = 9; data are pooled
from two independent experiments). i, The CSF, meninges, brain and serum
were collected after two months (A AV-CTRL, A AV-VEGF-C), after 24 h
(GFP mRNA, VEGFC mRNA) or at days 7 and 28 after tumour inoculation, and an
ELISA was performed to detect VEGF-C (CSF: A AV-VEGF-C, n = 6; other groups,
n = 3; 5 mice were pooled for each sample; meninges: A AV-VEGF-C, n = 6; 7-day
tumour, n = 3; other groups, n = 5; brain: A AV-CTRL, GFP mRNA, n = 6; A AV-
VEGF-C, VEGFC mRNA, n = 5; 7-day tumour, n = 3; 28-day tumour, n = 7; serum:
n = 3). Data are mean ± s.d. *P < 0.05; *P < 0.01; P < 0.001; ****P < 0.0001 (t wo-
tailed unpaired Student’s t-test).