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Original Article
Optimizing Modified mRNA
In Vitro Synthesis Protocol
for Heart Gene Therapy
Yoav Hadas,
1,2,3,4
Nishat Sultana,
1,2,3,4
Elias Youssef,
1,2,3
Mohammad Tofael Kabir Sharkar,
1,2,3
Keerat Kaur,
1,2,3
Elena Chepurko,
1,2,3
and Lior Zangi
1,2,3
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
Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
Synthetic modified RNA (modRNA) is a novel vector for gene
transfer to the heart and other organs. modRNA can mediate
strong, transient protein expression with minimal induction
of the innate immune response and risk for genome integra-
tion. modRNA is already being used in several human clinical
trials, and its use in basic and translational science is growing.
Due to the complexity of preparing modRNA and the high cost
of its reagents, there is a need for an improved, cost-efficient
protocol to make modRNA. Here we show that changing
the ratio between anti-reverse cap analog (ARCA) and
N1-methyl-pseudouridine (N1mJ), favoring ARCA over
N1mJ, significantly increases the yield per reaction, improves
modRNA translation, and reduces its immunogenicity in vitro.
This protocol will make modRNA preparation more accessible
and financially affordable for basic and translational research.
INTRODUCTION
Gene therapy treats disease by using genetic material to change or
correct gene expression to cure disease. The concept of gene therapy
emerged in the early 1970s as a consequence of both increased under-
standing of the role of genes in human disease and the development of
genetic engineering techniques.
1
First attempts to treat human pa-
tients with exogenous genes were conducted in the 1980s and
1990s; however, safety issues hampered their progress.
2,3
Extensive
research in recent years has yielded better and safer gene delivery stra-
tegies that reduce vector immunogenicity; consequently, the gene
therapy field is flourishing again, with exciting successes in treating
rare genetic diseases, including a first US Food and Drug Administra-
tion (FDA)-approved gene therapy for inherited retinal dystrophy,
4
and thousands of gene therapy clinical trials worldwide.
5
In pursuit
of improved treatments for cardiovascular disease, including compli-
cations of myocardial infarction (MI) and heart failure (HF), exten-
sive gene therapy preclinical research resulted in numerous clinical
trials aiming to promote angiogenesis
6
and improve calcium homeo-
stasis.
7
Although proving safety, all cardiac gene therapy trials failed
to demonstrate significant therapeutic efficacy.
7
It has been suggested
that inefficient gene transfer that resulted in poor expression of the
target gene is a potential reason for these neutral results.
7
Gene transfer can be achieved by using viral vectors including
adenovirus, retrovirus, adeno-associated virus (AAV), lentivirus,
vaccinia virus, poxvirus, and herpes simplex virus or non-viral vectors
including naked plasmid DNA (pDNA) or lipofection.
5
Modified
RNA (modRNA) is a novel vector for gene transfer to both dividing
and non-dividing mammalian cells that mediate fast, robust, transient
expression of proteins in the targeted cells or tissue.
8
Pre-clinical at-
tempts to use mRNA as a gene transfer vector showed successful
expression of the encoded proteins
9
and improved symptoms in a
rodent model of diabetes insipidus.
10
Yet the high immunogenicity
of exogenous mRNA
11–15
and high susceptibility to degradation by
RNase have hampered progress in developing mRNA as a vector
for treating humans.
16,17
Since the first description of a biologically
active and translatable in vitro transcription (IVT) product by Krieg
and Melton
18
in 1984, however, immense technological advances
have led to synthetic mRNA’s emergence as a promising vector for
gene delivery.
19
Two important milestones in developing IVT tech-
nology turned synthetic mRNA from a scientific tool to a potential
platform for gene therapy. First, pioneering work by Karikó et al.
20
demonstrated that incorporating chemically modified nucleotides
in the synthetic mRNA results in a more stable and less immunogenic
mRNA that mediates rapid, high expression of the encoded
protein.
21,22
Second, the discovery of a stable cap analog, anti-
reverse cap analog (ARCA), increases synthetic mRNA’s stability
and translatability.
23
We and others have recently shown that modRNA can be used to
express reporter proteins in rodent and porcine myocardium,
8,24,25
and that functional genes can be used to promote angiogenesis,
26
cardiomyocyte proliferation,
27
and cardiac cell survival.
28
Previously,
we described a detailed protocol for synthesizing modRNA capped
with ARCA 30-O-Me-m7G(50)ppp(50)G and substituting 100%
Received 23 June 2019; accepted 21 July 2019;
https://doi.org/10.1016/j.omtm.2019.07.006.
4
These authors contributed equally to this work.
Correspondence: Lior Zangi, Cardiovascular Research Center, Icahn School of
Medicine at Mount Sinai, New York, NY 10029, USA.
E-mail: lior.zangi@mssm.edu
300 Molecular Therapy: Methods & Clinical Development Vol. 14 September 2019 ª2019 The Authors.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
cytidine (C) with 5-methylcytidine (m5C) and 100% uridine (U) with
pseudouridine (J),
29
or substituting 100% uridine with N1-methyl-
pseudouridine (N1mJ), which has been shown to be superior over
the combination of Jand m5C.
8,30
Here we describe an optimized
protocol that indicates that a change in ARCA and N1mJratios
leads to cost-effective modRNA with higher protein expression and
lower immunogenicity in vitro.
RESULTS
modRNA Yield and Quality
Currently, there are two strategies for mRNA capping in an IVT re-
action: (1) post-transcriptional capping and (2) chemical capping.
In the post-transcriptional approach, the vaccinia virus capping
enzyme is used to cap the enzyme after the mRNA synthesis is
completed.
31,32
Although effective, this procedure is time-consuming
and expensive. In chemical capping, a cap analog is added directly to
the IVT reaction, after which the cap structure is incorporated at the
50end of the mRNA. To achieve a high percentage of capped mRNA
using this method, the ratio between the concentrations of the cap
analog and the guanosine triphosphate (GTP) in the reaction are
kept high. For IVT reactions, the ratio of ARCA to GTP should always
be 1:3.7. In order to reduce the synthetic mRNA’s immunogenicity
and increase its translatability, we replace 100% U with N1mJ. Pre-
viously, for the IVT reaction, we used a composition of 5 nM ARCA,
1.35 mM GTP, 7.5 mM N1mJTP, and 7.5 mM MTP (composition 1
in Table 1, hereafter termed ARCA 5 protocol). To achieve our aim of
increasing the translation capacity and lowering the cost of modRNA
production, we reduce the amount of template DNA by 85% (Fig-
ure S1). We then explored different nucleotide compositions and
desalting methods for making and cleaning the modRNA (Table 1).
Out of the various nucleotide stoichiometries tested, composition 5,
comprising higher concentrations of ARCA, GTP, ATP, and cytidine
triphosphate (CTP) and less N1mJTP, another costly element in
modRNA production, resulted in the greatest modRNA yield
compared with the previously used protocol (ARCA 5 protocol),
keeping the amount of T7 RNA Polymerase consistent (Table 1).
To desalt the modRNA in the first cleaning process, we compared
the MEGAclear RNA purification kit (used previously) with the
more cost-efficient Amicon filter. We found that the nuclear GFP
(nGFP) modRNA yield using the Amicon filter was equivalent to
the amount generated by the MEGAclear RNA purification kit.
Thus, we established that the most cost-effective protocol for IVT re-
action uses nucleotide concentrations of 10 mM ARCA, 2.7 mM GTP,
8.1 mM ATP, 8.1 mM CTP, and 2.7 mM N1mJTP followed by de-
salting and nucleotide clearing using the Amicon filter (this protocol
will now be referred to as the ARCA 10 protocol).
We also evaluated modRNA quality using a bioanalyzer. We found no
differences in the integrity of the modRNA generated using the
ARCA 10 protocol compared with that produced via the ARCA 5
protocol (Figures 1A and 1B). Interestingly, when using different
nucleotide compositions in the reaction, we observed high levels of
uncompleted IVT products (Figure 1F).
Translational Capacity of modRNA In Vitro
Next, we analyzed the translational capacity of modRNA generated
with the ARCA 10 protocol by transfecting several human cell
lines and primary cardiac cells isolated from neonatal rat hearts
and measuring the expression levels of two different reporter
genes: nGFP and firefly luciferase (Luc) (Figure 2). We observed
significantly increased nGFP expression in HeLa cells (data not
shown), primary rat cardiac cells, and neonatal rat cardiomyocytes
Table 1. Nucleotide Composition and Cleaning Methods
Nucleotide
Composition 1
(ARCA 5)
Nucleotide
Composition 2
Nucleotide
Composition 3
Nucleotide
Composition 4
Nucleotide
Composition 5
(ARCA 10)
Nucleotide
Composition 6
ARCA 5 mM 10 mM 10 mM 10 mM 10 mM 7.5 mM
GTP 1.35 mM 2.7 mM 2.7 mM 2.7 mM 2.7 mM 2.25 mM
ATP 7.5 mM 8.1 mM 15 mM 15 mM 8.1 mM 10 mM
CTP 7.5 mM 2.7 mM 15 mM 15 mM 8.1 mM 10 mM
N1mJTP 7.5 mM 2.7 mM 15 mM 2.7 mM 2.7 mM 2.25 mM
Total 28.85 mM 26.2 mM 57.7 mM 45.4 mM 31.6 mM 32 mM
Desalting with MEGAclear
Total mg nGFP modRNA per
1-mL reaction 1.33 3.12 1.19 2.13 2.94 1.93
% of ARCA 5 protocol 100 235 89 160 221 145
Desalting with Amicon Filter
Total mg nGFP modRNA per
1-mL reaction 2.12 1.04 1.92 2.94 1.74
% of ARCA 5 protocol 159 78 144 221 131
Summary of nucleotide composition and desalting (first cleansing) methods used in this study and their effects on the final modRNA yield. For modRNA integrity, see Figure 1.
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Molecular Therapy: Methods & Clinical Development Vol. 14 September 2019 301
(nrCM) transfected with nGFP modRNA generated with the
ARCA 10 protocol compared with the ARCA 5 protocol (Figures
2C and 2D). Similar results were observed in primary rat cardiac
cells, nrCM, human umbilical vein endothelial cells (HUVECs),
HeLa cells, and HEK293 cells when transfected with Luc modRNA
(Figures 2E–2H). We did not observe any difference in cell death
levels post transfection in vitro as measured by DAPI or propi-
dium iodide (PI)-positive cells (data not shown).
Immunogenicity of modRNA
Evaluating immunogenicity is critical to defining the safety profile
of mRNA therapeutics and their use in humans. To evaluate the
innate immune response in vitro, we measured the expression
levels of three innate immune-related genes, retinoic acid-induc-
ible gene I (RIG1), interferon alpha (IFNa), and interferon beta
(IFNb), in nrCM 8 h post transfection with Luc modRNA
(made by using either the ARCA 5 protocol or the ARCA 10
protocol) or unmodified mRNA (Figure 3). Importantly, Luc mod-
RNA made via both the ARCA 5 and ARCA 10 protocols has
significantly lower levels of RIG1, IFNa,andIFNbwhen compared
with unmodified mRNA (Figures 3B–3D). However, Luc made
using the ARCA 5 protocol was significantly more immunogenic
than Luc modRNA made using the ARCA 10 protocol (Figures
3B–3D).
DISCUSSION
Synthetic modRNA is now emerging as a promising vector for gene
delivery.
19
However, synthesizing modRNA in the high quantities
required by pre-clinical and clinical trials is expensive and time-
consuming. In light of recent successes in promoting angiogenesis,
26
CM proliferation,
27
and cardiac cell survival
28
in rodents, there in an
urgent need to reduce the cost and improve the quality of synthesizing
modRNA.
At present, our capping strategy is chemical capping; in this capping
method, the IVT reaction includes a cap analog, resulting in a cap
structure at the mRNA’s5
0end. This capping method requires a
high ratio of cap analog and GTP concentrations to produce a high
percentage of capped mRNA.
This study aimed to optimize the protocol for synthesizing mRNA
using ARCA and N1mJ. Previously published protocols
8,26,28,29
maintained the correct ratio between ARCA and GTP by keeping
the GTP concentration low (1.35 mM) and the nucleotides high
(7.5 mM). In this composition, the GTP becomes the limiting factor
in the reaction, leading the reaction to terminate without incorpo-
rating the other nucleotides present in excess. The unincorporated
nucleotides are later washed away during the cleaning process.
Although the ratio between ARCA and GTP must be kept high,
we realized that the composition of the other nucleotides in the re-
action can be modified, leading us to explore the effect of different
nucleotide compositions on the final yield and quality of the mod-
RNA. Interestingly, we found that when doubling the concentration
of all nucleotides to increase the final concentration to 57.7 mM, the
final yield is lower than the original concentration of 28.85 mM. The
optimal final nucleotide concentration in our study was 31.6 mM
(ARCA 10 mM, GTP 2.7 mM, CTP 8.1 mM, ATP 8.1 mM, and
N1mJ2.7 mM). Using this concentration, we were able to increase
the final modRNA yield by 290%. Combining this protocol with
improved cleaning methods results in modRNA with increases
translatability in both human cell lines and primary cardiac cells
(Figure 2), as well as reduced immunogenicity (Figure 3). A possible
explanation for this result is that optimizing nucleotide composition
in the IVT reaction raises the percentage of successfully capped
modRNA.
Here we developed an improved protocol for large-scale production
of modRNA. By increasing the final nucleotide concentrations while
Figure 1. Effect of Nucleotide Composition in IVT Reaction on modRNA Integrity
(A–F) modRNA integrity was evaluated using a bioanalyzer (Tap Station 2200) of compositions 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), and 6 (F).
Molecular Therapy: Methods & Clinical Development
302 Molecular Therapy: Methods & Clinical Development Vol. 14 September 2019
reducing the N1mJmolarity and using the same amount of T7 RNA
polymerase, we established a new method that is more cost-effective
than the previously published approach.
29
Our novel protocol
generated improved modRNA with higher translatability and lower
immunogenicity in vitro, and will allow basic and translational
research labs to overcome the high cost of modRNA preparation
and attain more effective modRNA for their research.
MATERIALS AND METHODS
Synthesizing modRNA
In brief, clean PCR products generated with plasmid templates
purchased from GenScript were used as the template for mRNA.
Figure 2. Effect of Nucleotide Composition in IVT
Reaction on Gene Expression in Human Cell Lines
and Rat Cardiac Cells
(A) GFP expression in rat cardiac cells 18 h post trans-
fection with or without nGFP modRNA generated with the
ARCA 5 protocol and ARCA 10 protocol. (B) Represen-
tative image of Luc activity level in neonatal rat car-
diomyocytes 24 h post transfection with or without Luc
modRNA generated using the ARCA 5 protocol and
ARCA 10 protocol. (C and D) Quantification of GFP
expression in neonatal rat cardiac cells (C) or car-
diomyocytes (D) 18 h post transfection with nGFP
modRNA generated using the ARCA 5 protocol and
ARCA 10 protocol. (E–H) Quantification of Luc activity
level in HUVECs (E), rat neonatal cardiomyocytes (F), HeLa
(G), or Hek293 (H) cells 18 h post transfection with or
without Luc modRNA generated using the ARCA 5
protocol and ARCA 10 protocol. One-way ANOVA,
Tukey’s multiple comparison test; ***p < 0.001, **p < 0.01,
*p < 0.05. n = 5 (A and B); n = 4 (C–F).
modRNAs were generated by transcription
in vitro with a customized ribonucleoside blend
of ARCA; 30-O-Me-m7G(50) ppp(50)G
(catalog no. [cat. #] N-1081; Trilink Biotechnol-
ogies); GTP (cat. #am1334-5; Life Technolo-
gies); ATP (cat. #am1334-5; Life Technologies);
cytidine triphosphate (cat. #am1334-5; Life
Technologies), and N1-methylpseudouridine-
50-triphosphate (cat. #N-1081; Trilink Biotech-
nologies). The mRNA was purified with the
MEGAclear kit (cat. #AM1908; Life Tech-
nologies) according to the manufacturer’s
instructions or using Amicon Ultra-4 Cen-
trifugal Filter Unit 4 mL,10 kDa (cat.
#UFC801024; MilliporeSigma) and treated
with Antarctic Phosphatase (cat. #M0289L;
NEB). It was then re-purified with the
MEGAclear kit. The mRNA was quantified
using a NanoDrop spectrometer (Thermo
Scientific), precipitated with ethanol and
ammonium acetate, and re-suspended in
10 mM Tris-HCl and 1 mM EDTA. The open reading frame for
the modRNA used is listed in Table S1. This protocol has been
described in detail elsewhere.
29
Desalting and Nucleotide Clearing
Amicon Ultra-4 Centrifugal Filter Unit 4 mL, 10 kDa
(cat. #UFC801024; MilliporeSigma) was washed with 4 mL
nuclease-free water two times. A total of 400 mLIVTreaction
was loaded to the filter and diluted with 3.5 mL nuclease-free
water. The filter was centrifuged at 3,200 gfor 20 min until
thevolumereducesto100mL. A total of 3.9 mL water was
added to the filter and centrifuged at 3,200 gfor 20 min two
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Molecular Therapy: Methods & Clinical Development Vol. 14 September 2019 303
times. The purified modRNA was incubated at 70Cfor10min(to
inactivate T7).
Neonatal Rat Cardiac Cells Isolation
Neonatal rat ventricular CMs were isolated from 3- to 4-day-
old Sprague-Dawley rats (Jackson ImmunoResearch Laboratories)
by multiple rounds of digestion with 0.1% collagenase II (In-
vitrogen) in PBS. After each digestion, the supernatant was
collected in horse serum (Invitrogen). The total cell suspension
was centrifuged at 300 gfor 5 min. The supernatants were
discarded and cells were resuspended in DMEM (GIBCO)
supplemented with 0.1 mM ascorbic acid (Sigma), 0.5% insulin-
transferrin-selenium (100), penicillin (100 U/mL), and strepto-
mycin (100 mg/mL). Cells were plated in plastic culture dishes
for 90 min until most of the non-myocytes were attached to
the dish while the myocytes remained in suspension. Myocytes
were then used to seed 24-well plates at a density of 1 10
5
cells/well. nrCMs were incubated for 48 h in DMEM supple-
mented with 5% horse serum before transfection with modRNAs.
Twenty-four hours post isolation, the non-myocytes were used
to seed 24-well plates at a density of 1 10
5
cells/well.
The non-myocytes were transfected with modRNA 24 h post
plating.
In Vitro Transfection with modRNA
Using a 24-well plate, we complexed 2.5 mg/well mRNA with Lipo-
fectamine RNAiMAX Transfection Reagent (cat. #13778030; Life
Technologies) and used the resulting complex to transfect either
neonatal rat cardiac cells or human cell lines according to the manu-
facturer’s instructions.
Figure 3. Immunogenicity of modRNA Compared
with Unmodified mRNA
(A) Luc mRNA or modRNA transfected into neonatal rat
cardiomyocytes 8 h after transfection cells were collected.
qPCR was performed to evaluate the gene expression
of innate immune response markers. (B–D) Expression of
innate immune response markers: RIG1 (B), IFNa(C), or
IFNb(D) 8 h post transfecting unmodified mRNA, Luc
modRNA (ARCA 5 protocol), or Luc modRNA (ARCA
10 protocol). One-way ANOVA, Tukey’s multiple com-
parison test; ****p < 0.0001. n = 5.
Assessing Number of nGFP-Expressing
Cells
Eighteen hours post transfection, cells were
imaged using a Zeiss Slide Scanner Axio Scan.
Quantification of nGFP-positive cells was per-
formed using ImageJ software.
Detection of Luciferase Activity
Bioluminescence of the transfected cells was
measured 18 h post transfection. Each unit
of Luc signal represents p/s/cm
2
/sr 10
6
.
Luciferin (cat. #L9504; Sigma) (1.5 mg/mL culture media) was added
to cell culture 10 s before imaging. Cells were imaged using an
IVIS100 charge-coupled device imaging system every 20 s until the
Luc signal reached a plateau. Imaging data were analyzed and quan-
tified with Living Image software.
qRT-PCR
Total RNA was reverse transcribed with Superscript III reverse
transcriptase (cat. #LS18080044; Invitrogen), according to the manu-
facturer’s instructions. qRT-PCR analyses were performed on a
Mastercycler Realplex 4 Sequence Detector (Eppendorf) with the
HotStart-IT SYBR Green qPCR master mix (2) (cat. #75762; Affy-
metrix). Data were normalized relative to GAPDH. Fold-changes in
gene expression were determined by the delta-delta-Ct (ddCT)
method. The PCR primer sequences used are listed in Table S2.
Statistical Analyses
Statistical analyses were performed with GraphPad Prism software.
Values are reported as means ±SD. Two-tailed Student’s t tests
(*p < 0.05 considered significant) or one-way ANOVA with Bonfer-
roni correction (*p < 0.05 considered significant) were used for
comparisons between groups.
SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.
1016/j.omtm.2019.07.006.
AUTHOR CONTRIBUTIONS
Y.H. designed most of the experiments, performed experiments,
analyzed most of the data, and wrote the manuscript. N.S. performed
Molecular Therapy: Methods & Clinical Development
304 Molecular Therapy: Methods & Clinical Development Vol. 14 September 2019
most of the experiments, analyzed data, and revised the manuscript.
E.Y. performed experiments. M.T.K.S. prepared modRNAs and per-
formed experiments. K.K. revised the manuscript. E.E. performed
CM isolation method. L.Z. designed experiments, analyzed data,
and revised the manuscript.
ACKNOWLEDGMENTS
This work was funded by a cardiology start-up grant awarded to the
Zangi laboratory and also by NIH grant R01 HL142768-01.
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