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Original Article
Optimizing Cardiac Delivery of Modified mRNA
Nishat Sultana,
1,2,5
Ajit Magadum,
1,2,5
Yoav Hadas,
1,2,5
Jason Kondrat,
1,2,5
Neha Singh,
1,2,5
Elias Youssef,
1,2,5
Damelys Calderon,
3,4,5
Elena Chepurko,
1,2,5
Nicole Dubois,
3,4,5
Roger J. Hajjar,
1
and Lior Zangi
1,2,5
1
Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA;
2
Department of Genetics and Genomic Sciences, Icahn School of
Medicine at Mount Sinai, New York, NY 10029, USA;
3
Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York,
NY 10029, USA;
4
Mindich Child Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA;
5
Black Family Stem Cell
Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
Modified mRNA (modRNA) is a new technology in the field of
somatic gene transfer that has been used for the delivery of genes
into different tissues, including the heart. Our group and others
have shown that modRNAs injected into the heart are robustly
translated into the encoded protein and can potentially improve
outcome in heart injury models. However, the optimal composi-
tions of the modRNA and the reagents necessary to achieve
optimal expression in the heart have not been characterized
yet. In this study, our aim was to elucidate those parameters by
testing different nucleotide modifications, modRNA doses, and
transfection reagents both in vitro and in vivo in cardiac
cells and tissue. Our results indicate that optimal cardiac deliv-
ery of modRNA is with N1-Methylpseudouridine-50-Triphos-
phate nucleotide modification and achieved using 0.013 mg
modRNA/mm
2
/500 cardiomyocytes (CMs) transfected with
positivelycharged transfection reagentin vitro and 100 mg/mouse
heart (1.6 mg modRNA/mL in 60 mL total) sucrose-citrate buffer
in vivo. We have optimized the conditions for cardiac delivery of
modRNA in vitro and in vivo. Using the described methods and
conditionsmay allow for successful gene delivery using modRNA
in various models of cardiovascular disease.
INTRODUCTION
Somatic gene transfer approaches have been used extensively in
experimental models, and they may provide a new treatment option
for several diseases, including chronic or acute cardiovascular dis-
eases.
1–3
Gene delivery systems can be classified into two main
groups: non-viral physio-chemical systems and recombinant viral
systems. The advantages of non-viral systems include the ease of
vector production, greater expression cassette size, and relatively
minimal biosafety risks.
4–7
The limitations include lower transfection
efficiency and transient effect due to clearance and degradation of the
transfer vector.
8
Non-viral vectors include plasmid DNA, mRNA, and
polymer/liposome-DNA/mRNA complexes. Compared with DNA
plasmids, mRNA has several advantages as a gene delivery tool.
One advantage is that mRNA does not require nuclear localization
or transcription prior to translation of the gene of interest. An addi-
tional advantage is the negligible risk of genomic integration of the
delivered sequence.
In the early 1990s, mRNA was successfully delivered into brain and
skeletal muscle.
9, 10
However, the use of mRNA as a method to trans-
fer genes into mammalian tissue has been very limited since. This is
mostly due to mRNA activation of the innate immune response via
stimulation of Toll-like receptors (TLRs).
11, 12
In addition, mRNA
is prone to cleavage by RNase when delivered in vivo.
11, 13, 14
Kariko
et al.
14
showed that modified mRNA (modRNA), produced by the
replacement of uridine with pseudouridine, resulted in changes to
the mRNA secondary structure that prevented innate immune system
recognition and RNase degradation. In addition, the replacement
nucleotide, pseudouridine, occurs naturally in the body,
15–17
and it
enhanced translation of the modRNA compared to the unmodified
version.
14, 18
We
12, 19–21
and others
22, 23
have shown that modRNA
drives transient and robust expression of genes of interest in cardio-
myocytes (CMs) in vitro and in the hearts of mice.
12, 19, 21, 22
Addi-
tionally, published results indicate that modRNA can be delivered
into CMs of rat or porcine hearts in vivo.
23
In a murine experimental
myocardial infarction (MI) model, vascular endothelial growth factor
(VEGF-A) modRNA delivery, immediately upon the induction of
coronary ischemia, markedly improved heart function and enhanced
long-term survival of treated animals.
12
This improvement was, in
part, due to the mobilization of epicardial progenitor cells and re-
direction of their differentiation toward other cardiovascular cell
types.
12
Others have shown that IGF1 modRNA delivery had a cytoprotective
effect on CMs in vitro and in vivo.
22
Recently, our group showed that
IGF1 expression in the myocardium after MI induces the formation of
epicardial fat.
19
The formation of epicardial fat was reduced using
IGF1R dominant-negative modRNA that was applied in a biocompat-
ible gel on the surface of the heart immediately after injury.
19
More
recent studies have used modRNA in the cardiovascular system.
ModRNAs encoding for VEGF-A, IGF1 EGF, HGF, TGFb1, TGFb2,
SDF-1, FGF-1, GH, SCF, and different reporter genes have been inves-
tigated in cardiac cells and tissue.
12, 19–23
In these latter studies, the
nucleotide modifications used to generate the modRNAs consisted
of a 100% replacement of uridine with pseudouridine and cytidine
with 5-methylcytidine (cU + 5mC).
12, 19, 20, 23, 24
Over the past few
Received 18 January 2017; accepted 9 March 2017;
http://dx.doi.org/10.1016/j.ymthe.2017.03.016.
Correspondence: Lior Zangi, Icahn School of Medicine at Mount Sinai, One
Gustave L. Levy Place, Box 1030, New York, NY 10029, USA.
E-mail: lior.zangi@mssm.edu
Molecular Therapy Vol. 25 No 6 June 2017 ª2017 The American Society of Gene and Cell Therapy. 1
Please cite this article in press as: Sultana et al., Optimizing Cardiac Delivery of Modified mRNA, Molecular Therapy (2017), http://dx.doi.org/10.1016/
j.ymthe.2017.03.016
years, several groups have shown that other nucleotide modifications,
such as 100% replacement of uridine by 2-Thiouridine-50-Triphos-
phate (2-thio cU)
18
or by 1-Methylpseudouridine-50-Triphosphate
(1-mcU),
25, 26
yield high modRNA translation efficiency in non-
cardiac tissue, with no immunogenicity and resistance to RNase.
Early experiments with modRNA have used standard reagents (e.g.,
RNAiMAX) without systematic optimization of nucleotide composi-
tion or gene delivery protocol. In this study, we optimized modRNA
composition, concentration, and mode of delivery to achieve maximal
efficacy of gene transfer in cardiac cells and in murine myocardium.
RESULTS
We have used cardiac cells and tissues to test different modRNA
nucleotide modifications for their immunogenicity, their stability in
mouse blood plasma, and translation to protein efficiency. All tested
nucleotide modifications of modRNAs had a significantly reduced
activation of hallmark innate immunity genes, such as INFaor -b
and RIG-1, in comparison to unmodified mRNA (Figure S1A). Addi-
tionally, the tested modRNA modifications were more stable, with a
higher RNA integrity for a longer time in the presence of mouse blood
plasma, compared to unmodified mRNA (Figure S1B). Moreover, all
nucleotide modifications yielded higher protein translation compared
to unmodified mRNA (Figure 1). Importantly, of all the tested mod-
ifications, a complete replacement of uridine with 1-mcU yielded a
significantly higher (3.9-fold increase in vitro and 4.5-fold increase
in vivo, p < 0.001) modRNA translation in comparison to a modRNA
that was previously used in cardiac cells and organs
12, 19–24, 27
(cU+
5mC; Figures 1E and 1F). This higher translation efficiency also
affected modRNA kinetics in vitro and pharmacokinetics in vivo:
1-mcU modRNA modification prolonged kinetics by 24 hr in vitro
and pharmacokinetics by 96 hr in vivo compared to modRNA having
cU + 5mC nucleotide modification (Figures 1C and 1D).
Using this superior modRNA modification (1-mcU), we evaluated the
optimal conditions of modRNA transfection into CMs in vitro (Fig-
ure 2). For this, we isolated rat neonatal or human pluripotent stem
Optimizing cardiac delivery of modified mRNA
ψU + 5mC 1-mψU 2-thio ψU
Low
High
In vitro
Unmodified
In vitro
In vivo
Time (hours)
Luc signal (Log scale)
p/sec/cm2/sr
0
AC
D
Time (hours)
In vitro
In vivo
0
1X10
2X10
3X10
4X10
5X10
Total Luc signal Total Luc signal
Unmodified
2-thio ψU
ψU + 5mC
1-mψU
1X108
1X107
1X106
1X105
1X104
1X107
1X106
1X105
1X104
1X103
E
F
****
Unmodified
2-thio ψU
ψU + 5mC
1-mψU
Unmodified
In vivo
Luc signal (Log scale)
24 48 72 96 120 144
0 48 96 144 192 240
0
1X10
2X10
3X10
4X10
5X10
6
6
6
6
6
7
7
7
7
7
1-mψU
ψU + 5mC
2-thio ψU
Unmodified
1-mψU
ψU + 5mC
2-thio ψU
****
ψU + 5mC 1-mψU 2-thio ψU
Unmodified
B
Figure 1. Optimizing ModRNA Modification to Increase Translation and Expression Kinetics in Vitro and In Vivo
Luc mRNA expression was compared between mRNA with or without nucleotide modifications (unmodified), using the IVIS imaging system, in rat neonatal CMs in vitro or
CFW mouse heart in vivo. The nucleotide modifications tested were as follows: 100% replacement of uridine by 2-Thiouridine-50-Triphosphate (2-thio cU) or by 1-Meth-
ylpseudouridine-50-Triphosphate (1-mcU) or 100% replacement of uridine by Pseud ouridine-50-Triphosphate and cytidine by 5-Methylcytidine-50-Triphosphate (cU + 5mC).
(A and B) Representative images of transfected (with RNAiMAX) neonatal CMs or mice. Images were taken 24 hr post-transfection with 2.5 mg/well in a 24-well plate or
intramyocardial injection with 100 mg modified or non-modified Luc mRNA, respectively. (C and D) Kinetics or pharmacokinetics of modified or non-modified Luc mRNA
in vitro or in vivo, respectively. (E and F) Efficient translation of Luc modRNA with different nucleotide modifications. Total Luc signal was measured over 5 or 10 days post-
transfection in vitro or in vivo, respectively. Results represent two independent experiments with n = 2 or n = 3 (total n = 5 wells or mice; ****p < 0.0001, one-way ANOVA with
Bonferroni post hoc test).
Molecular Therapy
2 Molecular Therapy Vol. 25 No 6 June 2017
Please cite this article in press as: Sultana et al., Optimizing Cardiac Delivery of Modified mRNA, Molecular Therapy (2017), http://dx.doi.org/10.1016/
j.ymthe.2017.03.016
Optimizing cardiac delivery of modified mRNA
nGFP/Actinin /DAPI
PBS RNAiMax
Rats neonatal CMs Human PSC derived CMs
PBS RNAiMax
0μg
0.5μg
1μg
2.5μg
5μg
10μg
modRNA dose (μg)
AB
CD
EF
RNAiMax
TransIT
jetMESSENGER
MessengerMAX
jetPEI
X-tremeGENE
DharmaFECT
INTERFERin
Sucrose - Citrate
buffer
PBS
% of nGFP+ cells
0
20
60
40
80
RNAiMax
TransIT
jetMESSENGER
MessengerMAX
jetPEI
X-tremeGENE
DharmaFECT
INTERFERin
PBS
% of nGFP+ cells
100
0
20
60
40
80
100
0
20
60
40
80
100
0
20
60
40
80
100
% of nGFP+ cells
% of nGFP+ cells
Median GFP Intensity
G
J
I
H
0μg
0.5μg
1μg
2.5μg
5μg
10μg
modRNA dose (μg)
0μg
0.5μg
1μg
2.5μg
5μg
10μg
modRNA dose (μg)
0μg
0.5μg
1μg
2.5μg
5μg
10μg
0μg
0.5μg
1μg
2.5μg
5μg
10μg
modRNA dose (μg)
0μg
0.5μg
1μg
2.5μg
5μg
10μg
modRNA dose (μg)
0
500
1000
1500
0
20
60
40
80
0
2000
6000
4000
8000
10000
Median GFP Intensity
% Annexin V+ of viable cells
0
60
% Annexin V+ of viable cells
Sucrose - Citrate
buffer
nGFP/Actinin /DAPI
****
40
20
***
(legend on next page)
www.moleculartherapy.org
Molecular Therapy Vol. 25 No 6 June 2017 3
Please cite this article in press as: Sultana et al., Optimizing Cardiac Delivery of Modified mRNA, Molecular Therapy (2017), http://dx.doi.org/10.1016/
j.ymthe.2017.03.016
cell (hPSC)-derived CMs and plated them separately onto 24-well
plates in sub-confluent conditions. We transfected the different types
of CMs with nuclear GFP (nGFP), naked modRNA, or mixed with
different commercially available transfection reagents (Figures 2A–
2D). We found that positively charged transfection reagents are neces-
sary to achieve a high level of CM transfection. Additionally, we found
that naked nGFP modRNA, delivered in sucrose-citrate buffer or
saline, yielded very low transfection levels in neonatal rat (1.3%
or 0.4%, respectively) and in hPSC-derived CMs (2.1% or 0.5%,
respectively). In contrast, the use of positively charged transfection
reagents, such as RNAiMAX, resulted in a very high transfection
level in neonatal rat (98.3%) and in hPSC-derived CMs (98.9%). The
use of other reagents for modRNA transfection, such as TransIT,
jetMESSENGER, or MessengerMAX, yielded high transfection levels
of neonatal rats (95.3%, 93.4%, or 77.6%, respectively) or hPSC
CMs in vitro (94.2%, 90.2%, or 97.3%, respectively). We selected
RNAiMAX for further investigation.
We titrated modRNA doses in order to obtain the optimal dose that
yields the highest number of transfected cells (GFP+ cells) and GFP
intensity with low cell death (Annexin V+ cells). We found that trans-
fection of 2.5 mg/1 !105 cells/well (sub-confluent condition) in a
24-well plate (0.013 mg/mm
2
) resulted in >95% GFP+ cells in both
types of CMs (Figures 2E–2J). In neonatal rat CMs, 5 or 10 mg per
well/10
5
cells resulted in higher GFP intensity compared to 2.5 mg;
however, the percentage of cell death also increased significantly
with the higher modRNA doses (p < 0.01). In hPSC CMs, the highest
GFP intensity accompanied by low cell death was achieved with 2.5 mg
nGFP modRNA transfected into CMs plated in sub-confluent
conditions.
We then tested various modRNA compositions and deliveries in
vivo. We developed a mouse model of open-chest surgery that allows
direct injection of modRNA into the myocardium (100 mg/heart
[1.6 mg modRNA/mL in 60 mL total volume]; Figure S2). Using this
model, we directly delivered luciferase (Luc) modRNA into the
myocardium with one of the following vehicles/reagents: (1) naked
(saline or sucrose-citrate buffer), (2) positively charged particles
(such as RNAiMAX or calcium phosphate), or (3) encapsulated in
nanoparticles (in vivo fectamine or in vivo jetPEI) (Figure 3). We de-
tected protein expression in the heart post-direct injection into the
myocardium using most delivery approaches (Figure 3A; Figure S3A).
The use of calcium phosphate resulted in a very low expression in the
heart. The use of in vivo jetPEI resulted in modRNA expression in the
lung after direct myocardial injection. Luc signal quantifications indi-
cated that naked delivery (sucrose-citrate buffer or saline) increased
protein translation by 53- to 226-fold compared to delivery of
modRNA encapsulated in nanoparticles (in vivo JetPEI or in vivo
fectamine, respectively) (Figures 3B and 3C). The use of RNAiMAX
for Luc modRNA yielded a high expression (4.4 !107 total Luc
signal); however, it was associated with increased cell death in the
injected area compared to naked Luc modRNA delivery (Figure S3B).
Importantly, delivery of Luc modRNA in sucrose-citrate buffer
yielded a higher expression of Luc compared to saline (11.3 !107
versus 6.4 !107 total Luc signal, respectively) (Figure 3C).
We next sought to optimize the modRNA dose to achieve the high-
est protein translation and biodistribution in vivo. Delivery of
100 mg/heart of Luc modRNA with sucrose-citrate buffer resulted
in the highest expression in vivo compared to delivery of 50 or
200 mg/heart (11.4 !10
7
versus 8.95 !10
7
or 8.06 !10
7
total
Luc signal, respectively) (Figures 4A–4C). Notably, injection of a
volume of 60 mL (modRNA mix) with different doses of Cre
modRNA (50, 100, and 200 mg) directly into the myocardium of
Rosa26
mTmG
mice resulted in a similar biodistribution, covering
more than 20% of the left ventricle (Figures 4D and 4E). Lastly,
we tested the dynamics of modRNA translation into protein after
direct injection into skeletal or cardiac muscles in vivo. For this pur-
pose, mice were injected with luciferin (150 mg/g body weight) and
anesthetized prior to Luc modRNA injections into the quadriceps
femoris (Figures S4A and S4B) or heart muscles (Figures S4C
and S4D). The IVIS system was used to monitor translation every
2–5 min. We detected protein translation that was significantly
above baseline reading in the quadriceps femoris or the heart 13
or 10 min post-injection, respectively (Figure S4).
DISCUSSION
ModRNA is a novel gene transfer platform that allows for efficient,
local, fast (in minutes), and transient (a few days in the adult heart)
gene delivery that is non-immunogenic and with good biodistribution
(>20% of the left ventricle). The platform can be used for the delivery
of a single gene or gene combinations in a variety of organs, including
the heart.
12, 18–21, 23, 27, 28
In this work, we describe the optimization of
modRNA delivery into cardiac cells and tissue. We found, in line with
others,
14, 18, 25, 26, 29
that modRNA delivery yields a higher expression
with reduced immunogenicty and better resistance to RNase activity
when compared to unmodified mRNA delivery (Figure 1;Figure S1).
We show that 100% replacement of uridine with 1-mcU results in
Figure 2. Optimizing Vehicle and ModRNA Dose for CM Transfection In Vitro
Rat neonatal or hPSC-derived CMs were isolated and plated onto 24-well tissue culture plates. Different transfection regents were used to transfect the CMs with 2.5 mg
nGFP modRNA (1-mcU)/per well. Then 18 hr post-transfection, cells were fixed and stained for actinin (red) and GFP (green). ImageJ was used to calculate the number of
nGFP
+
and Actinin
+
cells in each well. (A and B) Representative images of transfected rat neonatal CMs (A) or hPSC- derived CMs (B) with RNAiMAX-nGFP modRNA complex
18 hr post-transfection. (C and D) Quantification of transfection efficiency of rat neonatal (C) or hPSC-derived CMs (D) using different transfection reagents. (E and F) Optimal
doses per well were tested using optimal transfection reagent (RNAiMAX) using different doses of nGFP in rat neonatal CMs (E) or in hPSC-derived CMs (F). (G–J) FACS
analysis was used 18 hr post-transfection with RNAiMAX and different nGFP modRNA doses to measure median GFP intensity and cell death (percentage Annexin V of viable
cells) for rat neonatal CMs (G and I) or hPSC-derived CMs (H and J), respectively. Results represent two indepe ndent experiments with n = 3 wells (*p < 0.05 and ***p < 0.001,
one-way ANOVA with Bonferroni post hoc test; scale bar, 10 mm).
Molecular Therapy
4 Molecular Therapy Vol. 25 No 6 June 2017
Please cite this article in press as: Sultana et al., Optimizing Cardiac Delivery of Modified mRNA, Molecular Therapy (2017), http://dx.doi.org/10.1016/
j.ymthe.2017.03.016
Optimizing cardiac delivery of modified mRNA
Control Saline
Sucrose- Citrate buffer RNAiMAXControl Control
Control
Control
in vivo JetPEI in vivo fectamine
Control
Calcium Phosphate
Sucrose- Citrate buffer
Saline
RNAiMax
Invivo-JetPEI
Invivofectamine
Calcium Phosphate
B
A
Low
High
p/sec/cm2/sr
1X10
8
1X10
7
1X10
6
1X10
5
1X10
4
1X10
3
0
1.5X108
Total Luc signal
0
16 24
Time (hours)
4812 20 32 4028 36 4844
C
***
****
Sucrose - Citrate
Buffer
Saline
i
RNA MAX
Invivo-JetPEI
Invivofectamin
Calcium Phosphate
e
1.0X108
0.5X107
Luc signal (Log scale)
****
Figure 3. Vehicle Optimization for ModRNA Cardiac Tissue Transfection In Vivo
Luc modRNA at a dose of 100 mg (1-mcU)/heart, complexed with different commercially available RNA transfection reagents, was injected directly into myocardium of CFW
mice in an open-chest surgery. The IVIS imaging system was used to calculate gene expression at different time points (4, 24, and 48 hr). (A) Representative images of
transfected mice 24 hr post-injection directly into myocardium with different commercially available RNA transfection reagents. (B and C) Pharmokinetics (B) or efficient
translation (C) of Luc modRNA transfected with different RNA transfection reagents and measur edat different time points (4, 24, and 48 hr) using IVIS. Results represent three
independent experiments with n = 2 or 3 mice (****p < 0.0001 and ***p < 0.001, one-way ANOVA with Bonferroni post hoc test).
www.moleculartherapy.org
Molecular Therapy Vol. 25 No 6 June 2017 5
Please cite this article in press as: Sultana et al., Optimizing Cardiac Delivery of Modified mRNA, Molecular Therapy (2017), http://dx.doi.org/10.1016/
j.ymthe.2017.03.016
higher protein translation, in vitro and in vivo, compared to other
tested modifications. These findings are aligned with the data from
other laboratories
25, 26
showing that 1-mcU-incorporated mRNA
outperforms cU-incorporated mRNA by providing enhanced protein
expression and reduced immunogenicity in mammalian cell lines and
after intradermal or intramuscular injections in mice.
25
Optimizing cardiac delivery of modified mRNA
Control 50μg 100μg 200μg
Transfected cells /Non-transfected cells/DAPI
LV
RV
A
BCE
0
10
20
30
40
%Transfect
edLeftVentricle
Pharmacokinetics of modRNA
Luc signal (Log scale)
0 482412 36
Time (hours)
Biodistribution of modRNA
D
1X108
1X107
1X106
1.5X108
Total Luc signal
0
50μg
100μg
200μg
Total translation
1.0X108
5X10 7
50μg100μg200μg
*
**
50μg100μg200μg0μg
N.S
N.S
Figure 4. Optimizing ModRNA Dose for Cardiac Tissue Transfection In Vivo
Different doses (50, 100, and 200 mg) of Luc or Cre modRNA (1-mcU)/heart, delivered in sucrose-citrate buffer, were injected directly into the myocardium of CFW or
Rosa26
mTmG
mice, respectively, in an open-chest surgery. (A) Representative image of transfected CFW mice 24 hr post-intramyocardial injection with differentLuc modRNA
doses. (B and C) Pharmokinetics (B) or efficient translation (C) of Luc modRNA transfected with different doses and measured at different time points (4, 24, and 48 hr) using IVIS.
(D) Representative images of a transfected heart (transfected cells are green and non-transfected cells are red) or heart cross-sections(short axis view) of Rosa26
mTmG
mouse
24 hr post-injection of 100 mg Cre modRNA directly into myocardium. (E) Quantification of the biodistribution of different doses of Cre modRNA post-transfection in vivo. Results
represent two independent experiments with n = 2 or 3 mice (total n =3–5 mice; **p < 0.01 and *p < 0.05; N.S., not significant; one-way ANOVA with Bonferroni post hoc test).
Molecular Therapy
6 Molecular Therapy Vol. 25 No 6 June 2017
Please cite this article in press as: Sultana et al., Optimizing Cardiac Delivery of Modified mRNA, Molecular Therapy (2017), http://dx.doi.org/10.1016/
j.ymthe.2017.03.016
We further show that positively charged transfection reagents are
necessary for in vitro modRNA transfection of neonatal rat or
hPSC-derived CMs (Figure 2). We hypothesize that the transfection
reagent masks the negatively charged mRNA with positively charged
polymers or lipids, allowing electromagnetic attachment and endocy-
tosis to the negatively charged cell membrane, while also creating a
dense modRNA complex that gravitates toward the cell surface
when placed in culture media. When saline or sucrose-citrate buffer
are used, a modRNA complex does not form. The result is a signifi-
cantly reduced transfection efficiency in various types of CMs. Impor-
tantly, we show that positively charged transfection reagents, such as
RNAiMAX, increase cell death in vitro (Figures 2I and 2J). Therefore,
the amount of modRNA that can be transfected per squared milli-
meter culture dish (contains about 2,500 cells) with different types
of CMs is limited. Therefore, we show that the primary CM cell
type optimal transfection is 0.013 mg/mm
2
and using a higher amount
is associated with increased cell death (Figure 2). We have shown, us-
ing a mouse model (Figure S2), that naked modRNA (delivered with
sucrose-citrate buffer or saline) resulted in significant local protein
translation in the heart (Figure 3A; Figure S3A). The local expression
may be related to the short half-life of modRNA in the plasma that
may limit modRNA travel to different organs away from the site of
injection. We now show, for the first time, that naked modRNA de-
livery with sucrose-citrate buffer or saline yields a higher protein
translation compared to encapsulation of modRNA in nanoparticles,
such as in vivo fectamine or in vivo jetPEI. These results indicate that
the beneficial protective role nanoparticles may have on modRNA
hinders its translation into protein. Additionally, our data show
that modRNA delivery in a mixture with transfection reagents
(RNAiMAX) is detrimental to the heart and may increase apoptosis
in cardiac cells in vivo (Figure S3B). Our results strongly support
naked modRNA delivery as the optimal approach in direct intramus-
cular injection (Figures 3B and 3C). Importantly, sucrose-citrate
buffer delivery is superior to saline delivery (11.3 !107 versus
6.4 !107 total Luc signal), and it yielded more protein translation
in vivo (Figure 3C). This may be explained by the fact that sucrose
is an available energy source and may generally increase mRNA
translation and endocytosis.
30–32
Moreover, sucrose increases the
viscosity of the modRNA solution and prevents clumping of single-
stranded modRNAs in the mixture, clumping that may result
in untranslatable double-stranded modRNA that may also elicit
an immune response via Toll-like receptor 3.
33
Additionally,
citrate, is a chelating agent for mRNA and, thus, may increase its
preservation.
34
As modRNA delivery to the heart is limited by the amount of volume
that can be injected into the mouse myocardium (60 mL/heart), we
evaluated the optimal dose of modRNA per microliter for heart deliv-
ery. Our results show that 100 mg/heart (1.6 mg/mL) is the optimal
dose to achieve the highest translation efficiency (Figure 4A). While
the dose-response ratio was kept when 50 or 100 mg was delivered, de-
livery of 200 mg modRNA resulted in a reduced or similar translation
compared to 100 mg(Figure 4B). It is plausible that the high concen-
tration of modRNA (3.2 mg/mL) creates undesired clumping/attach-
ments of single-stranded modRNA, which result in low translation.
Notably, no significant differences were found in biodistribution
among the different doses of Cre modRNA delivered into heart
Rosa26
mTmG
mice (Figures 4C–4E), and all resulted in "20% trans-
fection of the left ventricle. Our unpublished data indicate that, in
rats, a minimum of 20% transduction of left ventricle is required to
alter cardiac function in vivo. Our results indicate that modRNA is
widely translated in the myocardium, beyond the needle track, and
that the level of protein translation is dependent on the modRNA
dose. From our studies and experience, we can attempt to extrapolate
our results and calculate the doses necessary for human heart trans-
duction. While a mouse heart weighs 0.18 g,
35
the normal human
heart weighs at least 300 g
36
(1,667-fold difference). If the modRNA
dose ratio is kept, the optimal dose in human would be 166.7 mg.
This is a very large dose that may be cost prohibitive. Clearly more
research is needed to improve modRNA translation (via new modifi-
cations, modRNA capping, or 50UTRs) and transfection (possibly
via the use of improved reagents) in order to move the field toward
a clinical RNA therapy phase in humans. Finally, we have shown
that modRNA delivery into skeletal or cardiac muscle tissues resulted
in rapid protein translation at about 10 min post-delivery. This
uniquely rapid pharmacokinetics strengthens the potential use of
the modRNA gene delivery method in conditions of acute cardiac dis-
ease, such as MI.
In conclusion, we have shown that modRNA modification using
1-mcU yields superior protein translation in cardiac cells and tissues.
The delivery of 0.013 mg modRNA/mm
2
mixed with positively
charged transfection reagent is optimized for in vitro transfection
into two types of CMs. Furthermore, the delivery of 1.6 mg/mL
(60 mL/100 mg per heart) in sucrose-citrate buffer is optimized for
in vivo transfection into the mouse heart (see summary in Figure 5).
This work sheds light on the conditions needed for improved cardiac
delivery of modRNA in vitro and in vivo, and it may promote
modRNA delivery use and accessibility in cardiac studies for both
basic and translational science.
MATERIALS AND METHODS
Mice
All animal procedures were performed under protocols approved
by the Icahn School of Medicine at Mount Sinai Institutional Care
and Use Committee. CFW or R26
mTmG
mice strains, male and female,
were used. Luc or Cre modRNAs (50, 100, and 200 mg/heart, as
mentioned in the text) were injected directly into the quadriceps
femoris muscle or into the myocardium in an open-chest surgery
(see Figure S2 for more details).
Synthesis of ModRNA
ModRNAs were transcribed in vitro from plasmid templates (se-
quences provided in previous publications
12, 19, 21
), using a custom
ribonucleoside blend of Anti Reverse Cap Analog, 30-O-Me-m7G(50)
ppp(50)G (6 mM, TriLink Biotechnologies), guanosine triphosphate
(1.5 mM, Life Technologies), adenosine triphosphate (7.5 mM, Life
Technologies), cytidine triphosphate (7.5 mM, Life Technologies), or
www.moleculartherapy.org
Molecular Therapy Vol. 25 No 6 June 2017 7
Please cite this article in press as: Sultana et al., Optimizing Cardiac Delivery of Modified mRNA, Molecular Therapy (2017), http://dx.doi.org/10.1016/
j.ymthe.2017.03.016
5-methylcytidine triphosphate (7.5 mM, TriLink Biotechnologies) and
uridine triphosphate (7.5 mM, Life Technologies), or pseudouridine
triphosphate, or 2-Thiouridine-50-Triphosphate, or N1-Methylpseu-
douridine-50-Triphosphate (7.5 mM, TriLink Biotechnologies), as
described previously.
12, 19
The mRNA was purified using a Megaclear
kit (Life Technologies) and was treated with Antarctic Phosphatase
(New England Biolabs); then it was purified again using the
Megaclear kit. The mRNA was quantitated by Nanodrop (Thermo
Scientific), precipitated with ethanol and ammonium acetate, and re-
suspended in 10 mM TrisHCl and 1 mM EDTA. For a detailed proto-
col please see our recent publication.
21
In Vitro Transfection
Different doses of Luc or nGFP mRNA with different nucleotide
modifications were transfected into neonatal rat or hPSC-derived
CMs using the following different transfection reagents: RNAiMAX
or MessengerMAX (Life Technologies), TransIT (Mirus Bio),
jetMessenger or JetPEI or Interferin (Polyplus), Dharmacon (GE
Healthcare), X-tremeGENE (Roche), or naked with sucrose-citrate
buffer. The sucrose-citrate buffer contains 20 mL sucrose in
nuclease-free water (0.3 g/mL) and 20 mL citrate (0.1 M, pH 7; Sigma)
mixed with 20 mL modRNA at different concentrations in saline or
only in saline (modRNA at different concentrations in 60 mL saline).
The transfection mixture was prepared according to the manufac-
turer’s protocol, and then it was added to cells cultured in basal me-
dium containing growth factors and 2% fetal bovine serum (FBS)
(Lonza). Then, 18 hr post-transfection, cells were fixed, stained, and
Rats neonatal CMs
Direct injection into myocardium
in mouse model
100μg per heart (1.6μg modRNA /μl in 60μl total)
in Sucrose- Citrate buffer
or
Human PSC derived CMs
modRNA
modRNA
0.013 g modRNA / mm2 well diameter
transfected with positivley charged
transfection reagent
Figure 5. Optimized Cardiac Delivery of ModRNA
Cardiac delivery of modRNA with 100% replacement
of uridine with 1-mcU yields the highest gene expression
and longest pharmacokinetics in comparison to other
nucleotide modifications, both in vitro and in vivo. CM
transfection in vitro (rat neonatal or hPSC) is optimal
with a positively charged transfection reagent, such as
RNAiMAX, TransIT, jetMESSENGER, or MessengerMAX,
in a concentration of 0.013 mg modRNA/1 mm
2
well
diameter. For in vivo cardiac delivery in mice, our data
show that 100 mg/heart (1.6 mg modRNA/mL in 60 mL total)
in sucrose-citrate buffer directly injected into myocardium
yields the highest gene expression and covers close to a
quarter of the left ventricle of mouse heart.
evaluated using florescence microscopy or fluo-
rescence-activated cell sorting (FACS).
In Vivo Transfection
Different doses of Luc or Cre modRNA or
mRNA with different nucleotide modifications
in a total volume of 60 mL were delivered via
direct injection to the myocardium in an open-
chest surgery (see Figure S2). The modRNA
was formulated with different transfection re-
agents according to the manufacturer’s recom-
mendation. The transfection reagents used were
in vivo JetPEI (Polyplus), invivofectamine 3.0, RNAiMAX (Life Tech-
nologies), or Calcium Phosphate (Sigma); naked modRNA with su-
crose-citrate buffer; and saline (see above [In Vitro Transfection] for
details). The transfection mixture was made according to the manufac-
turer’s protocol and was directly injected (two to three individual injec-
tions, 20 ml each) into the quadriceps femoris muscle or heart muscle.
Detection of Luciferase Expression Using the IVIS System
Different doses of Luc mRNA with different nucleotide modifications
were transfected into CMs in vitro or directly injected into the quadri-
ceps femoris muscle or the heart of CFW mice. Bioluminescence imag-
ing of the transfected cells or injected mice was taken at different time
points (4–240 hr). Each unit of Luc signal represents p/s/cm
2
/sr !106.
To visualize tissues expressing Luc in vivo, mice were anesthetized with
isoflurane (Abbott Laboratories), and luciferin (150 mg/g body weight;
Sigma) was injected intraperitoneally. Mice were imaged using an
IVIS100 charge-coupled device imaging system every 2 min until the
Luc signal reached a plateau.Imaging data were analyzedand quantified
with Living Image software. Cells or muscle tissues that were transfected
with saline or RNAiMAX served as a baseline reading for Luc expres-
sion. To account for Luc background signal, the signal from untrans-
fected mice (yields "1!104 Luc signal) was subtracted from the reads
of transfected mice.
hPSC Differentiation
The hPSCs (H9) were differentiated along a cardiac lineage as previ-
ously described.
37
Briefly, hPSCs were maintained in E8 media and
Molecular Therapy
8 Molecular Therapy Vol. 25 No 6 June 2017
Please cite this article in press as: Sultana et al., Optimizing Cardiac Delivery of Modified mRNA, Molecular Therapy (2017), http://dx.doi.org/10.1016/
j.ymthe.2017.03.016
passaged every 4–5 days onto matrigel-coated plates. To generate em-
bryonic bodies (EBs), hPSCs were treated with 1 mg/ml collagenase
B (Roche) for 30 min or until cells dissociated from plates. Cells
were collected and centrifuged at 1,300 rpm for 3 min, and they
were resuspended into small clusters of 50–100 cells by gentle pipet-
ting in differentiation media containing RPMI (Gibco), 2 mmol/L
L-glutamine (Invitrogen), 4 !10
4
monothioglycerol (MTG, Sigma),
50 mg/mL ascorbic acid (Sigma), and 150 mg/mL transferrin (Roche).
Differentiation media were supplemented with 2 ng/mL BMP4 and
3mmol Thiazovivin (Millipore) (day 0). EBs were maintained in
six-well ultra-low attachment plates (Corning) at 37#C in 5% CO
2
,
5% O
2
, and 90% N
2
. On day 1, media were changed to differen-
tiation media supplemented with 20 ng/mL BMP4 (R&D Systems)
and 20 ng/mL Activin A (R&D Systems). On day 4, media were
changed to differentiation media supplemented with 5 ng/mL
VEGF (R&D Systems) and 5 mmol/L XAV (Stemgent). After day 8,
media were changed every 5 days to differentiation media without
supplements.
Neonatal Rat CM Isolation
CMs from 3- to 4-day-old neonatal rat heart were isolated as previously
described.
22
Neonatal rat ventricular CMs were isolated from 3- to
4-day-old Sprague-Dawley rats (Jackson ImmunoResearch Labora-
tories). We used multiple rounds of digestion with 0.14 mg/mL collage-
nase II (Invitrogen). After each digestion, the supernatant was collected
in horse serum (Invitrogen). Total cell suspension was centrifuged at
1,500 rpm for 5 min.Supernatants were discarded and cells were resus-
pended in DMEM (Gibco) with 0.1 mM ascorbic acid (Sigma), 0.5%
Insulin-Transferrin-Selenium (100!), penicillin (100 U/mL), and
streptomycin (100 mg/mL). Cells were plated in plastic culture dishes
for 90 min until most of the non-myocytes attached to the dish
and myocytes remained in the suspension. Myocytes were then
seeded at 1 !10
5
cells/well in a 24-well plate. Neonatal rat CMs were
incubated for 48 hr in DMEM containing 5% horse serum. After incu-
bation, cells were transfected with Luc or nGFP modRNAs as described
above.
Real-Time qPCR Analyses
Total RNA was isolated using the RNeasy mini kit (QIAGEN)
and reverse transcribed using Superscript III reverse transcriptase
(Invitrogen), according to the manufacturer’s instructions. Real-
time qPCR analyses were performed on a Mastercycler realplex
4 Sequence Detector (Eppendoff) using SYBR Green (QuantitectTM
SYBR Green PCR Kit, QIAGEN). Data were normalized to Gapdh
expression where appropriate (endogenous controls). Fold changes
in gene expression were determined by the vvCT method and
were presented relative to an internal control. PCR primer sequences
were as follows: for INFa, forward primer ATGGCTAGRCTC
TGTGCTTTCCT and reverse primer AGGGCTCTCCAGAYTT
CTGCTCTG; for INFb, forward primer AAGAGTTACACTGC
CTTTGCCATC and reverse primer CACTGTCTGCTGGTGG
AGTTCATC; and for RIG-1, forward primer GGACGTGGC
AAAACAAATCAG and reverse primer GCAATGTCAATGCC
TTCATCA.
RNA Integrity Test in Plasma
Plasma of CFW mice was isolated as described before.
38
Plasma was
immediately frozen and kept at $80#C. On the day of the experiment,
diluted plasma (1:50 in PBS) was warmed to 37#C. Then 10 mL
modRNA or mRNA (0.25 mg/mL) with different nucleotide modifica-
tions was added at a 1:1 ratio to the diluted plasma and kept at 37#C
for 15 min. Samples were collected at different time points (1, 3, 10,
and 15 min) and snap frozen before sending for an RNA integrity
test using the bioanalyzer in the Genomics Core Facility in Icahn
Mount Sinai Medical School.
Immunostaining
Immunostaining was performed on cryosections of hearts fixed
by perfusion with 4% paraformaldehyde (PFA) and stained using
primary antibodies for GFP, TUNEL, or Actinin (all from Abcam).
Secondary antibodies were used for fluorescent labeling of the cells
(Jackson ImmunoResearch Laboratories). Cells and heart slides were
imaged using a Zeiss Slide Scanner Axio Scan. Quantification of immu-
nostaining in cardiac sections was performed using ImageJ software.
Statistical Analyses
Values are reported as mean ±SEM or in dot plot graphs (with me-
dian and quartiles indicated). Comparisons between groups were
made using one-way ANOVA with Bonferroni post hoc test.
SUPPLEMENTAL INFORMATION
Supplemental Information includes four figures and can be found
with this article online at http://dx.doi.org/10.1016/j.ymthe.2017.
03.016.
AUTHOR CONTRIBUTIONS
N.S. designed and carried out most of the experiments, analyzed most
of the data, and wrote the manuscript. A.M. designed and performed
in vitro transfection in CM experiments, analyzed data, and wrote the
manuscript. Y.H. performed and analyzed the TUNEL expression
experiment. J.K. prepared modRNA for this study. N.S. and E.Y.
carried out in vitro CM isolation and cell culture experiments. D.C.
prepared the hPSC-derived CMs used in this study. E.C. performed
all mouse open-chest surgery and intramyocardial injection of
modRNA. N.D. prepared the hPSC-derived CMs used in this study
and analyzed data. R.J.H. designed experiments, analyzed data, and
revised the manuscript. L.Z. designed and carried out experiments,
analyzed data, and wrote the manuscript.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Talha and Irsa Mehmood for
their help with this manuscript. This work was funded by a cardiology
start-up grant for the Zangi lab; by NIH R01 HL117505, HL 119046,
HL129814, 128072, HL131404, HL135093, and P50 HL112324; and
by a Transatlantic Fondation Leducq grant.
REFERENCES
1. del Monte, F., and Hajjar, R.J. (2003). Efficient viral gene transfer to rodent hearts
in vivo. Methods Mol. Biol. 219, 179–193.
www.moleculartherapy.org
Molecular Therapy Vol. 25 No 6 June 2017 9
Please cite this article in press as: Sultana et al., Optimizing Cardiac Delivery of Modified mRNA, Molecular Therapy (2017), http://dx.doi.org/10.1016/
j.ymthe.2017.03.016
2. Del Monte, F., Ishikawa, K., and Hajjar, R.J. (2017). Gene Transfer to Rodent Hearts
In Vivo. Methods Mol. Biol. 1521, 195–204.
3. Ylä-Herttuala, S., Bridges, C., Katz, M.G., and Korpisalo, P. (2017). Angiogenic gene
therapy in cardiovascular diseases: dream or vision? Eur. Heart J. ehw547.
4. Andrieu-Soler, C., Bejjani, R.A., de Bizemont, T., Normand, N., BenEzra, D., and
Behar-Cohen, F. (2006). Ocular gene therapy: a review of nonviral strategies. Mol.
Vis. 12, 1334–1347.
5. Chaanine, A.H., Kalman, J., and Hajjar, R.J. (2010). Cardiac gene therapy. Semin.
Thorac. Cardiovasc. Surg. 22, 127–139.
6. Felgner, P.L. (1997). Nonviral strategies for gene therapy. Sci. Am. 276, 102–106.
7. Pahle, J., and Walther, W. (2016). Vectors and strategies for nonviral cancer gene
therapy. Expert Opin. Biol. Ther. 16, 443–461.
8. Michelfelder, S., Lee, M.K., deLima-Hahn, E., Wilmes, T., Kaul, F., Müller, O.,
Kleinschmidt, J.A., and Trepel, M. (2007). Vectors selected from adeno-associated
viral display peptide libraries for leukemia cell-targeted cytotoxic gene therapy.
Exp. Hematol. 35, 1766–1776.
9. Jirikowski, G.F., Sanna, P.P., Maciejewski-Lenoir, D., and Bloom, F.E. (1992).
Reversal of diabetes insipidus in Brattleboro rats: intrahypothalamic injection of
vasopressin mRNA. Science 255, 996–998.
10. Wolff, J.A., Malone, R.W., Williams, P., Chong, W., Acsadi, G., Jani, A., and Felgner,
P.L. (1990). Direct gene transfer into mouse muscle in vivo. Science 247, 1465–1468.
11. McIvor, R.S. (2011). Therapeutic delivery of mRNA: the medium is the message. Mol.
Ther. 19, 822–823.
12. Zangi, L., Lui, K.O., von Gise, A., Ma, Q., Ebina, W., Ptaszek, L.M., Später, D., Xu, H.,
Tabebordbar, M., Gorbatov, R., et al. (2013). Modified mRNA directs the fate of heart
progenitor cells and induces vascular regeneration after myocardial infarction. Nat.
Biotechnol. 31, 898–907.
13. Karikó, K., Buckstein, M., Ni, H., and Weissman, D. (2005). Suppression of RNA
recognition by Toll-like receptors: the impact of nucleoside modification and the
evolutionary origin of RNA. Immunity 23, 165–175.
14. Karikó, K., Muramatsu, H., Welsh, F.A., Ludwig, J., Kato, H., Akira, S., and
Weissman, D. (2008). Incorporation of pseudouridine into mRNA yields superior
nonimmunogenic vector with increased translational capacity and biological stability.
Mol. Ther. 16, 1833–1840.
15. Haas, J., and Engels, J.W. (2008). A convenient approach towards 20-O-modified
RNA-oligonucleotides on solid support using universal nucleosides. Nucleic Acids
Symp Ser (Oxf) 52, 331–332.
16. Limbach, P.A., Crain, P.F., Pomerantz, S.C., and McCloskey, J.A. (1995). Structures of
posttranscriptionally modified nucleosides from RNA. Biochimie 77, 135–138.
17. Pardi, N., Muramatsu, H., Weissman, D., and Karikó, K. (2013). In vitro transcription
of long RNA containing modified nucleosides. Methods Mol. Biol. 969, 29–42.
18. Kormann, M.S., Hasenpusch, G., Aneja, M.K., Nica, G., Flemmer, A.W., Herber-
Jonat, S., Huppmann, M., Mays, L.E., Illenyi, M., Schams, A., et al. (2011).
Expression of therapeutic proteins after delivery of chemically modified mRNA in
mice. Nat. Biotechnol. 29, 154–157.
19. Zangi, L., Oliveira, M.S., Ye, L.Y., Ma, Q., Sultana, N., Hadas, Y., Chepurko, E., Später,
D., Zhou, B., Chew, W.L., et al. (2017). Insulin-Like Growth Factor 1 Receptor-
Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial
Injury. Circulation 135, 59–72.
20. Lui, K.O., Zangi, L., Silva, E.A., Bu, L., Sahara, M., Li, R.A., Mooney, D.J., and Chien,
K.R. (2013). Driving vascular endothelial cell fate of human multipotent Isl1+ heart
progenitors with VEGF modified mRNA. Cell Res. 23, 1172–1186.
21. Kondrat, J., Sultana, N., and Zangi, L. (2017). Synthesis of Modified mRNA for
Myocardial Delivery. Methods Mol. Biol. 1521, 127–138.
22. Huang, C.L., Leblond, A.L., Turner, E.C., Kumar, A.H., Martin, K., Whelan, D.,
O’Sullivan, D.M., and Caplice, N.M. (2015). Synthetic chemically modified mrna-
based delivery of cytoprotective factor promotes early cardiomyocyte survival post-
acute myocardial infarction. Mol. Pharm. 12, 991–996.
23. Turnbull, I.C., Eltoukhy, A.A., Fish, K.M., Nonnenmacher, M., Ishikaw a, K., Chen, J.,
Hajjar, R.J., Anderson, D.G., and Costa, K.D. (2016). Myocardial De livery of Lipidoid
Nanoparticle Carrying modRNA Induces Rapid and Transient Expression. Mol.
Ther. 24,66–75.
24. Wang, G., McCain, M.L., Yang, L., He, A., Pasqualini, F.S., Agarwal, A., Yuan, H.,
Jiang, D., Zhang, D., Zangi, L., et al. (2014). Modeling the mitochondrial cardiomy-
opathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip tech-
nologies. Nat. Med. 20, 616–623.
25. Andries, O., Mc Cafferty, S., De Smedt, S.C., Weiss, R., Sanders, N.N., and Kitada, T.
(2015). N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouri-
dine-incorporated mRNA by providing enhanced protein expression and reduced
immunogenicity in mammalian cell lines and mice. J. Control. Release 217, 337–344.
26. Pardi, N., Tuyishime, S., Muramatsu, H., Kariko, K., Mui, B.L., Tam, Y.K., Madden,
T.D., Hope, M.J., and Weissman, D. (2015). Expression kinetics of nucleoside-modi-
fied mRNA delivered in lipid nanoparticles to mice by various routes. J. Control.
Release 217, 345–351.
27. Lui, K.O., Zangi, L., and Chien, K.R. (2014). Cardiovascular regenerative therapeutics
via synthetic paracrine factor modified mRNA. Stem Cell Res. (Amst.) 13 (3 Pt B),
693–704.
28. Chien, K.R., Zangi, L., and Lui, K.O. (2014). Synthetic chemically modified mRNA
(modRNA): toward a new technology platform for cardiovascular biology and med-
icine. Cold Spring Harb. Perspect. Med. 5, a014035.
29. Boros, G., Miko, E., Muramatsu, H., Weissman, D., Emri, E., Rózsa, D., Nagy, G.,
Juhász, A., Juhász, I., van der Horst, G., et al. (2013). Transfection of pseudouri-
dine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage
in human keratinocytes: a new approach with future therapeutic potential.
J. Photochem. Photobiol. B 129, 93–99.
30. Etxeberria, E., Baroja-Fernandez, E., Muñoz, F.J., and Pozueta-Romero, J. (2005).
Sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic
plant cells. Plant Cell Physiol. 46, 474–481.
31. Gamm, M., Peviani, A., Honsel, A., Snel, B., Smeekens, S., and Hanson, J. (2014).
Increased sucrose levels mediate selective mRNA translation in Arabidopsis. BMC
Plant Biol. 14, 306.
32. Nicolaï, M., Roncato, M.A., Canoy, A.S., Rouquié, D., Sarda, X., Freyssinet, G., and
Robaglia, C. (2006). Large-scale analysis of mRNA translation states during sucrose
starvation in arabidopsis cells identifies cell proliferation and chromatin structure
as targets of translational control. Plant Physiol. 141, 663–673.
33. Alexopoulou, L., Holt, A.C., Medzhitov, R., and Flavell, R.A. (2001). Recognition of
double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature
413, 732–738.
34. Koff, M.D., and Plaut, K. (1995). Expression of transforming growth factor-alpha-like
messenger ribonucleic acid transcripts in the bovine mammary gland. J. Dairy Sci. 78,
1903–1908.
35. Hoit, B.D., Ball, N., and Walsh, R.A. (1997). Invasive hemodynamics and force-
frequency relationships in open- versus closed-chest mice. Am. J. Physiol. 273,
H2528–H2533.
36. Paulsen, S., Vetner, M., and Hagerup, L.M. (1975). Relationship between heart weight
and the cross sectional area of the coronary ostia. Acta Pathol. Microbiol. Scand. [A]
83, 429–432.
37. Yang, L., Soonpaa, M.H., Adler, E.D., Roepke, T.K., Kattman, S.J., Kennedy, M.,
Henckaerts, E., Bonham, K., Abbott, G.W., Linden, R.M., et al. (2008). Human car-
diovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived pop-
ulation. Nature 453, 524–528.
38. Christensen, S.D., Mikkelsen, L.F., Fels, J.J., Bodvarsdóttir, T.B., and Hansen, A.K.
(2009). Quality of plasma sampled by different methods for multiple blood sampling
in mice. Lab. Anim. 43, 65–71.
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YMTHE, Volume 25
Supplemental Information
Optimizing Cardiac Delivery of Modified mRNA
Nishat Sultana, Ajit Magadum, Yoav Hadas, Jason Kondrat, Neha Singh, Elias
Youssef, Damelys Calderon, Elena Chepurko, Nicole Dubois, Roger J. Hajjar, and Lior
Zangi
Supplemental materials
Supplemental Figure 1. modRNA has reduced immunogenicity and RNase
sensitivity compared to unmodified mRNA. a. Expression of innate immune genes
(INF-α, INF-β or RIG-1) was measured by qRT-PCR in rat neonatal CMs 10 hours post
transfection with Luc mRNA with or without (unmodified nucleotide modification such
as: 100% replacement of Uridine by 2-Thiouridine-5'-Triphosphate (2-thio ψU) or by N1-
Methylpseudouridine-5'-Triphosphate (1-mψU) or 100% replacement of Uridine by
Pseudouridine-5'-Triphosphate and Cytidine by 5-Methylcytidine-5'-Triphosphate (ψU +
5mC). b. Representative images of RNA integrity with or without one minute incubation
in plasma of mRNA with similar modification as in a. c. Kinetics of RNA integrity were
measured using bio-analyzer at different time points in incubation with plasma (1, 3, 10,
15 minutes) in vitro. Results represent 2 independent experiments with n= 3 (wells or
RNA batches). ***, P<0.001, *, P<0.05, One-way ANOVA with Bonferroni post hoc
test.
Supplemental Figure 2. modRNA intramuscular delivery in vivo. Different doses of
Luc or Cre modRNAs with different nucleotide modifications or with different vehicles
were directly intramuscularly injected into hearts of CFW or Rosa26mTmG mice. Mice (6-
8 weeks old) were anesthetized with isoflurane and intubated. Left thoracic region was
shaved and sterilized, and the heart was exposed through a left thoracotomy. A direct
injection using insulin syringe (31G) was used for modRNA delivery to the myocardium.
After injection, thoracic region and skin were sutured closed in layers. Excess air was
removed from the thoracic cavity, and the mouse was removed from ventilation as soon
as normal breathing was established. Above, a representative image of modRNA direct
injection into the myocardium.
Supplemental Figure 3. Cardiac delivery of naked modRNA results in local
expression and reduced toxicity in vivo. a. 100μg Luc modRNA (1-mψU)/heart diluted
in sucrose-citrate buffer was injected directly into the myocardium of CFW mice in an
open chest surgery. IVIS imaging system was used to show expression in whole mice or
in different organs. b. 100μg nGFP modRNA (1-mψU)/heart, complexed with
RNAiMAX or naked (with sucrose-citrate buffer or saline), was intramyocardialy
injected into CFW mice in an open chest surgery. 24 hours post-injection hearts were
collected, and heart cross-section (short axis view) were stained for TUNEL (red), GFP
(green) and DAPI (blue). Yellow, light blue and white boxes represent TUNEL staining
in area of nGFP modRNA injection mixed with RNAiMAX, sucrose-citrate buffer or
saline, respectively. Results represent 2 independent experiments with n=3 or n=4 mice.
Supplemental Figure 4. Intramuscular delivery of naked modRNA results in its
translation into protein within minutes post-delivery in vivo. 100μg Luc modRNA (1-
mψU)/ quadriceps femoris muscle (a) or heart muscle (b) diluted in sucrose-citrate buffer
was intramusculary injected to CFW mice pre injected with luciferin. a. Immediately
after modRNA injection to the quadriceps femoris muscle Luc signal was measured
every 2 minutes for 60 minutes using IVIS. b. 0, 5, 10 or 15 minutes post cardiac
injection, hearts were collected and Luc signal was measured ex vivo using IVIS. Results
represent 2 independent experiments with n=2 or n=3 mice.
