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Review
mRNA-Based Protein Replacement
Therapy for the Heart
Ajit Magadum,
1,2,3
Keerat Kaur,
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
Myocardial infarction (MI) and heart failure (HF) are the lead-
ing causes of death in the United States and in most other
industrialized nations. MI leads to a massive loss of cardiomyo-
cytes (CMs), which are replaced with non-CM cells, leading to
scarring and, in most cases, HF. The adult mammalian heart
has a low intrinsic regenerative capacity, mainly because of
cell-cycle arrest in CMs. No effective treatment promoting
heart regeneration is currently available. Recent efforts to use
DNA-based or viral gene therapy approaches to induce cardiac
regeneration post-MI or in HF conditions have encountered
major challenges, mostly because of the poor and uncontrolled
delivery of the introduced genes. Modified mRNA (modRNA)
is a safe, non-immunogenic, efficient, transient, local, and
controlled nucleic acid delivery system that can overcome the
obstacles to DNA-based or viral approaches for cardiac gene
delivery. We here review the use of modRNA in cardiac
therapy, to induce cardioprotection and vascular or cardiac
regeneration after MI. We discuss the current challenges in
modRNA-based cardiac treatment, which will need to be over-
come for the application of such treatment to ischemic heart
disease.
Ischemic Heart Disease
Despite advances in curative and preventive medicine, heart failure
(HF) remains the leading cause of mortality and hospitalization
worldwide.
1,2
Almost 300,000 individuals each year experience recur-
rent heart attacks in the United States alone,
3,4
and the prevalence of
ischemic heart disease is projected to rise to about 40.5% of the USA
population by 2030.
4
Traditional approaches for dealing with end-
stage HF are often not feasible, due to the limited number of hearts
available for transplantation. Preclinical trials have reported improve-
ments in patient outcomes,
5,6
but prognosis remains poor, and there
is, therefore, an urgent need for new approaches to the prevention and
treatment of HF.
During HF, billions of cardiomyocytes (CMs) are progressively lost,
and fibrotic non-functional scar tissue develops, significantly reducing
the pumping capacity of the heart muscle. The remaining CMs have a
limited intrinsic regenerative capacity and cannot, therefore, replace
the lost CMs. Cardiac regeneration studies have shown that dividing
CMs are abundant in the fetus, but rapidly lost after birth.
7
Cell-based
therapies with exogenous cells, such as bone marrow cells, cardiac pro-
genitor cells, and other self-renewing stem cells, have been developed
to improve heart function. However, little meaningful improvement
has been reported for these treatments, owing to the limited interac-
tion between the various progenitor cells and the myocardium envi-
ronment during myocardial infarction (MI).
In the last two decades, our understanding of the molecular pathways
and genes involved in the disease has improved, and gene therapy has
emerged as a possible treatment for HF. Given the limited site spec-
ificity of pharmacological inhibitors, gene therapy is an exciting pros-
pect for more precise targeting of the signaling pathways involved in
disease progression. The gene therapy approaches currently being
developed for HF aim: (1) to increase the proliferation or contractility
of endogenous CMs; (2) to reprogram cardiac fibroblasts to develop
into beneficial cardiac cell types, such as endothelial cells (ECs) or
CMs; and (3) to increase capillary density by activating endogenous
ECs or progenitors. Recent studies have reported reactivation of the
CM cell cycle following protein delivery to the myocardium, either
by direct injection or via patch delivering the protein to the epicar-
dium. CM proliferation has been reported following the delivery of
NRG1 protein via intraperitoneal (i.p.) injections,
8
intramyocardial
(IM) injections of agrin,
9
or the delivery of follistatin-like 1 to the
epicardium.
10
Furthermore, the proliferation of adult CMs has been
observed following transfection with an adenovirus encoding a domi-
nant-negative p38 mitogen-activated protein kinase (MAPK)
11
or an
extracellular matrix component, periostin.
12
Also, Hajjar and co-
workers
13,14
have shown that the adeno-associated virus (AAV)-
mediated delivery of Serca2a
13
or SUMO
14
improves cardiac function
post-MI and in HF condition via elevation of endogenous CMs
contractility.
Another successful avenue of gene therapy for heart repair is the ge-
netic in situ reprogramming of cardiac fibroblasts into CMs. Pioneer-
ing work by Srivastava and coworkers
15
showed that fibroblasts could
undergo cardiac reprogramming to become beating CMs following
direct virus-mediated IM delivery and overexpression of the cardiac
myocyte transcription factors Gata4, Mef2c, and Tbx5 (GMT). This
approach is promising as an alternative to cell-based regeneration
https://doi.org/10.1016/j.ymthe.2018.11.018.
Correspondence: Lior Zangi, Department of Genetics and Genomic Sciences,
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. 27 No 4 April 2019 ª2018 The Author(s). 1
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Magadum et al., mRNA-Based Protein Replacement Therapy for the Heart, Molecular Therapy (2018), https://doi.org/
10.1016/j.ymthe.2018.11.018
therapies, but the efficiency of in vivo reprogramming remained low,
and there is also a potential risk of viral genome insertions associated
with the use of viral vectors. Olson and coworkers
16
improved in vivo
reprograming by using Hand2 as an additional reprogramming fac-
tor, together with GMT. In addition to its use to stimulate CM prolif-
eration and reprogramming, gene therapy has also been used to
induce myocardial repair by enhancing angiogenesis and inducing
cardiovascular regeneration. Over the last decade, preclinical studies
have reported revascularization in ischemic heart following the direct
delivery of vascular endothelial growth factors (VEGFs) by various
methods.
17–23
Widely used vectors provided robust and consistent
gene expression leading to neovascularization, but this expression
was often accompanied by undesirable effects, such as the induction
of edema or angioma due to the prolonged expression of VEGF, the
elicitation of an immune response against the vector, or a potential
risk of genomic integration.
20–22
Nevertheless, these studies have pro-
vided useful insight and support for the use of gene therapy to repair
the injured myocardium, although a number of hurdles remain to be
overcome for this therapeutic approach to be considered successful.
Current Approaches in Cardiac Gene Therapy
Gene therapy can be defined as the transplantation of normal genes
into cells to replace missing or defective genes, with the aim of correct-
ing genetic disorders or promoting inactive beneficial mechanisms or
pathways. With improvements in our understanding of cardiac dis-
ease, interest is growing in the use of gene therapies to treat coronary
heart disease. The ultimate goal of gene therapies is the expression of
protein of interest, and the most feasible way of achieving this goal is to
introduce the corresponding protein directly into the myocardium.
Direct protein delivery overcomes the difficulties of translation within
the cell, thereby offering a potential advantage in terms of higher levels
of expression, dose regulation, and control over a viral-based gene
therapy approach. However, the short half-life and instability of
injected proteins, the lack of use of this approach for intracellular
proteins (e.g., transcription factors), and possible immunogenicity
due to minor histocompatibility antigens are problems that must be
overcome for this approach to be therapeutically successful.
However, approaches based on the insertion of nucleic acids, which
can be translated into proteins within the cardiac cells, can circum-
vent the challenges of protein therapy. In recent decades, considerable
advances have been made toward the delivery of nucleic acids into the
heart by viral and non-viral vectors. Lentiviral vectors are favored for
cardiac gene therapy by many researchers because they can transduce
non-dividing CMs. However, their chief advantage—their integration
into the host genome, ensuring sustained gene expression—also en-
tails a risk of compromising the genome and tumorigenesis. The
exceptional transduction efficiencies of adenoviruses and AAVs
have resulted in these vectors being the most widely used for cardio-
vascular applications. Adenoviruses transfer genes efficiently into the
myocardium in large animals,
24
but expression is transient, and these
viruses trigger a strong immune response.
25
AAVs have low immuno-
genicity and are a widely used alternative for gene delivery to the
heart. These nonpathogenic vectors ensure cardiac tropism without
integration into the host genome and have been used in a recent
study
26
in which persistent Yap-associated protein expression re-
sulted in CM proliferation and regeneration post-MI. AAV gene
delivery peak levels of expression are reached about 4 weeks after de-
livery
27
and continue for up to 11 months.
28
Despite the predomi-
nance of AAVs over other gene delivery vectors, the production of
neutralizing antibodies against the AAV capsid, delayed pharmacoki-
netics, and limited gene packaging capacity of these vectors restrict
their use in cardiac gene therapy.
29
The delivery of naked plasmid DNA overcomes the risk of immune
responses and oncogenesis, because of the absence of the viral vector.
Plasmid DNA displays impressive organ specificity, but transfection
efficiency is low. The recent elucidation of the role of microRNAs
(miRNAs) and long noncoding RNAs in cardiac repair and regener-
ation has provided new hope for innovative therapy.
30
A recent re-
view by Hermans-Beijnsberger et al.
31
summarized newly found
long non-coding RNAs involved in the cellular process during devel-
opment of cardiovascular disease (CVD). These non-coding RNAs
can efficiently suppress the target mRNA post-transcriptionally by
promoting mRNA degradation or inhibiting translation. Despite
the successful results obtained in vitro, systems for delivering them
to the heart in vivo have yet to be optimized. Furthermore, therapeutic
miRNAs may have off-target effects, resulting in potential risk of
oncogenesis.
32,33
There is, therefore, an urgent need to explore clini-
cally relevant approaches for enhancing cardiac regeneration and
maintaining correct heart function both during and immediately after
ischemic injury.
Modified mRNA Therapy
Different gene therapies have proved inefficient due to a short half-
life, production of neutralizing antibodies, or a poor transduction
capacity. By contrast, mRNA-based therapies are highly promising
for the treatment of various human disorders. The delivery of
mRNA to the cell has significant advantages over the use of protein
or DNA-based delivery systems: (1) the use of mRNA transfection
overcomes the need for nuclear localization or for transcription of
the gene of interest in the patient’s cells; (2) the introduction of
mRNA into cells is safe under physiological conditions, because
mRNA does not integrate into the host genome; and (3) the effect
of the mRNA is transient, minimizing the risk of mutagenesis after
mRNA therapy (Figure 1).
Successful direct mRNA transfer was first reported about three
decades ago, when Wolff et al.
34
demonstrated the delivery of
mRNA and its translation into protein in mouse skeletal muscle. After
a few initial successes with mRNA therapy, research into mRNA
structure and delivery methods continued, but the use of mRNA ther-
apy was limited to vaccine development, because of problems of insta-
bility and immunogenicity. Within cells, mRNA is prone to cleavage
by RNase and can trigger the innate immune system via Toll-like
receptors (TLRs) 7 and 8 (which recognize single-stranded RNA)
or TLR3 (which recognizes double-stranded RNA), leading to an in-
crease in cytokine levels and associated toxicity. In 2008, pioneering
2 Molecular Therapy Vol. 27 No 4 April 2019
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Please cite this article in press as: Magadum et al., mRNA-Based Protein Replacement Therapy for the Heart, Molecular Therapy (2018), https://doi.org/
10.1016/j.ymthe.2018.11.018
work by Karikó et al.
35
addressed these issues and provided a platform
for mRNA therapy in genetic, regenerative medicine, immunothera-
peutics, and cancer. The study showed that replacing the uridine
residues in mRNA with the naturally occurring modified nucleoside
pseudouridine (hence the name modified mRNA [modRNA])
enhanced translation, due to changes in the secondary structure of
the mRNA limiting its recognition by the TLRs and nucleases.
35,36
The use of modRNA has since been on the increase in genetic
medicine, for protein-replacement therapies and the treatment of ge-
netic diseases. The efficiency of modRNA delivery in vivo has been
increased by enhancing the stability of the mRNA and increasing
translational efficiency by capping the molecule with the 30-O-Me-
m7G(50)ppp(50)G Anti Reverse Cap Analog (ARCA) at its 50
end.
37,38
The uses of modRNA technology as a model for cardiac
repair are listed in Table 1.
Immediately after MI, CMs and other cardiac cells such as ECs are
lost due to occlusion of the coronary artery. A chain of events down-
stream leads to oxidative stress and inflammation, resulting in
impaired pump function and, ultimately, HF. The remaining CMs
in the heart have a very limited proliferative potential and are there-
fore unable to replace the lost cells. The damaged coronary vascula-
ture also creates a hostile environment in which it is difficult for
the remaining CMs to survive. Various strategies have been developed
Figure 1. Methods of Gene Delivery to the Heart
In vivo gene expression profiles for various methods of gene delivery to the heart. (A) Recombinant protein. (B) Modified mRNA (modRNA). (C) Adeno-associated viruses
(AAVs).
Molecular Therapy Vol. 27 No 4 April 2019 3
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to try to reverse the situation by inducing regeneration of cardiac neo-
vasculature and encouraging CMs to proliferate.
A few independent clinical trials over the last 20 years have assessed
the therapeutic potential of a potent angiogenic factor, VEGF-A,
after ischemic injury. VEGF-A was delivered by intracoronary,
intravenous, or IM injection, in the form of a recombinant
protein,
17–19
adenoviral plasmid,
20,22
naked cDNA, or non-viral
plasmid.
21,23
A moderate improvement in ejection fraction and left
ventricular (LV) function was reported, but findings differed between
trials.
39–41
These differences can be explained by the short half-life of
VEGF-A in plasma (about 30–45 min in humans), degradation by
proteases, off-target effects associated with systemic delivery,
and the lack of an efficient delivery platform. Attempts to retain
VEGF-A in the infarcted heart for therapeutic purposes have been
made, based on the implantation of biodegradable scaffolds including
hydrogel,
42
collagen,
43
or self-assembling peptide nanofibers,
44
but
the success of these approaches was limited.
42–44
Zangi et al.
45
introduced a modRNA encoding VEGF-A into mouse
hearts, and reported a decrease in infarct size and an improved
myocardial outcome with higher survival rates (80% survival with
VEGF-A modRNA versus only 20% for the group receiving DNA).
They observed that VEGF-A protein secretion levels were much
higher following treatment with modRNA than with unmodified
mRNA, with no reported apoptosis or increase in the expression of
immune response genes, such as retinoic acid-inducible gene
(RIG)-1, interferon (IFN)-a, and IFN-b. Both VEGF-A modRNA
and VEGF-A DNA increased capillary density and reduced infarct
size and apoptotic cell frequency in MI mice, but the prolonged expo-
sure to VEGF-A in DNA-treated hearts increased vessel permeability,
a sign of abnormal vessel function. The study also showed that the
favorable outcome achieved with pulse-like VEGF-A overexpression
was associated with better vessel formation in the peri-infarct area
because of the presence of larger numbers of WT1 epicardial progen-
itor cells activated via the kinase insert domain receptor (KDR) under
stress conditions. These activated progenitor cells remain confined to
the epicardial layer in the absence of VEGF-A, which induces their
mobilization to the myocardial layer. Stimulation of the endogenous
epicardial progenitor pool by the right paracrine factor (VEGF-A),
time, and place enhances the differentiation of these cells into ECs
and, to some extent, into CMs. Therefore, VEGF-A modRNA is an
Table 1. Published Studies for the Use of modRNA Technology as a Model of Cardiac Repair: modRNA as a Therapeutic Strategy for Cardiac Vascularization
and Regeneration
No. Publication Gene(s) Role Cellular Process or Disease Delivery Material Animal
1 Zangi et al.
45
VEGFa
directs the fate of heart progenitor
cells and induces vascular regeneration
after MI
cellular fate switch post-MI RNAiMAX mice
2 Lui et al.
46
VEGFa
VEGF-A promotes not only the endothelial
specification but also engraftment,
proliferation, and survival (reduced
apoptosis) of the human Isl1
+
progenitors
in vivo
VEGFa promotes Isl1
+
to
endothelial cell fate, proliferation
and survival of Isl1
+
progenitors
RNAiMAX mice
3 Huang et al.
52
IGF-1
anti-apoptosis, cardiomyocyte survival,
augmented Akt phosphorylation, and
decreased caspase-9 activity
anti-apoptosis, cardiomyocyte
survival post-MI
polyethylenimine-based
nanoparticle mice
4 Turnbull et al.
58
EGFP
modRNA delivery (direct myocardial or
intracoronary administration) into rat
and pig heart
modRNA expression in heart formulated lipidoid
nanoparticles (FLNP) rat and pig
5 Turnbull et al.
59
EGFP protocol lipidoid mRNA nanoparticles
protocol
formulated lipidoid
nanoparticles (FLNP) rodents
6 Kondrat et al.
37
protocol modified mRNA synthesis RNAiMAX mice
7 Sultana et al.
51
modRNA delivery
optimization
modRNA delivery optimization, modRNA
amount and time optimization optimal modRNA expression sucrose-citrate buffer mice
8 Zangi et al.
53
DN-IGF-1R, IGFR inhibition of adipogenic differentiation
post-MI
inhibition of adipogenic
differentiation post-MI RNAiMAX mice
9 Carlsson et al.
47
VEGFa increased angiogenesis, improved heart
function post-MI, reduced fibrosis
increased angiogenesis, improved
heart function post-MI, reduced
fibrosis
sucrose-citrate buffer pig, monkey
10 Singh et al.
57
EGFP, mCherry, Fluc modRNA delivery optimization optimal modRNA expression in
heart
alginate, nanomaterial
encapsulated mice and pig
11 Magadum et al.
48
mutated FSTL1
ablation of N180Q, N-glycosylation site of
hFSTL1 by modRNA delivery increased
CM proliferation, improved cardiac
output, and reduced scar size post-MI
CM proliferation, decreased scar
size, improved heart function sucrose-citrate buffer mice
4 Molecular Therapy Vol. 27 No 4 April 2019
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excellent clinical approach to repair of the damaged vasculature and
can further improve myocardial outcome and survival after injury.
Moreover, VEGF-A modRNA also can promote the engraftment,
proliferation, and survival (reduced apoptosis) of transplanted
human Isl1-positive cells.
46
Carlsson et al.
47
recently reported effi-
cient intracardiac transfection and protein expression from a
VEGF-A modRNA in a pig model of MI. They reported improve-
ments in % ejection fraction, inotropic function and compliance,
border zone capillary and arteriole density, and a decrease in myocar-
dial fibrosis 2 months after the treatment of MI with VEGF-A mod-
RNA. These improvements in cardiac systolic parameters were
observed following the delivery of 1 or 10 mg modRNA via intracar-
diac injections post-MI.
Several attempts have been made to use conventional viral proteins to
upregulate cell-cycle promoters or to downregulate the brakes on the
cell cycle, with the aim of promoting the re-entry of post-mitotic CMs
into the cell cycle. However, the long-term uncontrolled expression of
pro-proliferative genes can be detrimental to heart function. For this
reason, modRNA technology has been tested in the field of cardiac
regeneration. Magadum et al.
48
recently investigated the role of
hFSTL1 glycosylation in CM proliferation and showed that the
myocardial injection of a mutated hFSTL1 modRNA with a single
asparagine-to-arginine (N-Q) substitution in the glycosylation site
(N180Q) was necessary and sufficient to increase the proliferation
of neonatal rat or mouse adult CMs in vitro or after MI, respectively,
with no signs of cardiac hypertrophy. This finding can be explained
by changes in the glycosylation pattern of hFSTL1 upon N180 site
ablation, leading to activation of CM proliferation and regeneration.
Interestingly, a single administration of N180Q modRNA in the
mouse MI model was sufficient to increase cardiac function signifi-
cantly, with a decrease in scar size and an increase in capillary density
28 days post-MI, showing modRNA to be an efficient tool for induc-
tion of control CM proliferation and cardiac regeneration post-MI.
Our studies of the use of modRNA technology have yielded promising
results, showing that it is possible to create mutated constructs or pro-
teins for investigations of their role in heart disease and, potentially, to
introduce therapeutic constructs for cardiological treatments.
Cardioprotective Role of modRNA
modRNA-based gene delivery has several advantages over other
intracardiac therapies. Viral vectors and plasmid DNA delivery
methods have spatiotemporal shortcomings, whereas modRNA al-
lows rapid, transient, and efficient gene expression to a specific
time window after cardiac injury. In this respect, modRNA is an ideal
tool for delivering factors targeting the signaling pathways altered in
the first few hours of infarction.
A series of events takes place after MI, leading to a massive sudden
loss of CMs, beginning within an hour of occlusion. The stress to
which CMs are subjected post-MI leads to the induction of pro-
inflammatory cytokines and chemokines, and an accumulation of
inflammatory cells in the heart.
49
This chain of events occurs rapidly,
within 2–3 days of ischemia injury. These days thus constitute the
time frame in which desirable gene combinations could be delivered
to prevent CM apoptosis.
50
Sultana et al.
51
have shown that luciferase
modRNA can be detected in the heart 10 min after injection, and that
its expression peaks at 24 hr but remains detectable for up to 10 days.
Thus, based on its expression dynamics, modRNA has been used in
various studies to deliver genes or gene combinations for cytoprotec-
tion and to induce cellular reprogramming in a desired time frame
after cardiac injury.
Consistent with this approach, Huang et al.
52
delivered insulin growth
factor 1 (IGF-1) modRNA to the area of mouse hearts at high risk af-
ter injury, and extended the temporal window for the cytoprotection
of CMs against apoptosis after hypoxia and MI. The delivery of IGF-1
modRNA, with a polyethylenimine-based nanoparticle, resulted in
efficient transient protein expression within cells. IGF-1 was ex-
pressed rapidly, within 2 hr of injection, and its levels peaked 24 hr
post-injection, decreasing thereafter to 48 hr, and about 25% of cells
in the border zone were transfected. The delivery of IGF-1 modRNA
promoted CM survival and decreased cell apoptosis by more than
50% post-hypoxia in vitro and post-MI. The increase in IGF-1 levels
was shown to be associated with CM survival and a decrease in the
number of TUNEL-positive cells post-hypoxia. The decrease in
apoptosis rates was accompanied by higher levels of Akt and Erk
phosphorylation and a downregulation of IGF-1-specific miRNAs.
Despite this demonstration of the cardioprotective role of IGF-1,
Zangi et al.
53
found that the activation of IGF-1 signaling pathways
in the heart post-MI also had negative consequences. In addition to
its cardioprotective action, IGF-1 expression can lead to the forma-
tion of epicardial adipose tissue (EAT) post-MI. EAT is an active
tissue located between the myocardium and the visceral pericardium,
and contributes to the pathological mechanisms of coronary artery
disease. Excessive epicardial fat deposition around the heart may
trigger the production of several adipocytokines and chemokines
through the activation of various paracrine and vasocrine signaling
pathways, resulting in the development of atherosclerotic plaques in
the coronary vessels.
54
Hence the group evaluated the role of para-
crine contributors in the development of EAT under normal and
pathological conditions.
The study showed that IGF-1 delivered to post-MI stressed hearts by
modRNA contributed to the differentiation of epicardial progenitor
cells into adipogenic cells and the formation of EAT.
53
It was demon-
strated that WT1 expression was essential for epicardium-derived cells
(EPDCs) differentiation into adipocytes, by delivering a Cre modRNA
by gel application onto the surface of WT1
flx/flx
;Rosa26
Tomato
hearts
for local WT1 inactivation and EPDC labeling. This study provided
unique insight into the modRNA gene delivery method, in which
a gene can be delivered locally through the application of a biocom-
patible gel directly onto the cardiac tissue in situations in which the
development of knockout animals is not possible. This mode of gene
transfection was also used to deliver dominant-negative IGF-1 recep-
tor antagonists to the injury-exposed epicardial cells shortly after MI,
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during the brief time window in which the IGF-1-induced differenti-
ation of progenitor cells into adipocytes appears to occur. The tran-
sient inhibition of IGF-1 receptors significantly decreased EAT
formation, confirming our hypothesis that IGF-1 receptor signaling
is required to stimulate the adipogenic differentiation of EPDCs in
the context of MI. This study provides an illustration of the ability
of modRNA techniques to deliver a gene transiently at the appropriate
time and place to block an undesired signaling pathway in one cell type
(EPDCs), but not on another (CMs).
Challenges in the Cardiac Delivery of modRNA
Improvements in our understanding of the pathology of HF over time
have led to novel gene therapy targets being identified, although inef-
ficient delivery to the target tissue remains a substantial problem. For
efficient gene therapy in the heart, the delivery systems carrying the
nucleic acid must ensure: (1) the uptake of the nucleic acid by cardiac
cells; (2) escape from the immune response; and (3) efficient transla-
tion and biodistribution of the genes in the post-ischemic, peri-
ischemic, or non-ischemic areas of the myocardium.
Cells typically take up modRNA via endocytosis, a process in which
foreign molecules or ligands (in this case, modRNA) are engulfed
by an area of plasma membrane, which then buds off intracellularly,
leading to the formation of modRNA-containing endosomes. These
endosomes later disassemble to deliver the mRNA to the cytoplasm,
in which it is immediately translated into protein. However, human
TLR8 (hTLR8) and mouse TLR7 (mTLR7), which are expressed
only on endosomal membranes, recognize single-stranded RNA
[particularly poly(U) and poly(U/G) motifs in the case of hTLR8],
and this recognition triggers the innate immune response. TLR3 is
also expressed on endosomal membranes and can elicit an innate
immune response following its recognition of unmethylated CpG
motifs in double-stranded RNA.
55
Another obstacle to the translation
of the imported mRNA is its degradation by RNase. Over a decade
ago, a revolutionary study by Karikó et al.
35
demonstrated that the
replacement of uridine residues in the mRNA with naturally pro-
duced pseudouridine resulted in much lower levels of TLR-mediated
immunogenicity and prevented degradation by RNase. Subsequent
studies, including studies by our group, have used such modified
RNA to achieve high translation efficiencies without immunogenicity
in non-cardiac tissues.
45,51
In 2015, Andries et al.
56
showed that natu-
rally produced 1-mJU incorporation into mRNA reduced immuno-
genicity in mammalian cells lines by preventing endosomal TLR3
activation and downstream innate immune signaling. Consistent
with these findings, our modRNA, containing 1-mJU in place of uri-
dine residues, resulted in significantly lower levels of activation for
innate immunity genes, such as those encoding IFN-aor IFN-b
and RIG, in cardiac cells and tissues than were observed with unmod-
ified mRNA. We were also able to show that modRNA with 1-mJU
was less likely to be degraded by RNase compared with unmodified
mRNA (Figure 2). Thus, the choice of an appropriate delivery method
is critical for transfection with a large modRNA that cannot simply
diffuse into the negatively charged CMs.
Maximum transfection efficiency and modRNA stability in vitro can
be ensured by complexing modRNA with transfection reagents, to
Figure 2. Comparing Uptake of RNA and modRNA by the Cell
(Left) modRNA delivery does not cause any activation of immune response and escapes RNase degradation. (Right) mRNA triggers activation of TLR7/8 and is prone to
degradation by RNase.
6 Molecular Therapy Vol. 27 No 4 April 2019
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encapsulate the modRNA with positively charged polymers or lipids.
These spherical vesicles containing polar head groups and nonpolar
tails promote the electromagnetic attachment and subsequent endo-
cytosis of the complex. The efficient transfection of isolated CMs can
be achieved by delivering 0.013 mg/mm
3
modRNA in complex with
the positively charged transfection reagent RNAiMAX. However,
despite the efficient delivery of modRNA to cardiomyocytes in vitro
reported with RNAiMAX, the use of this agent is associated with
higher rates of cell death around the injection site in the myocardium,
suggesting it may not be an ideal vehicle for in vivo transfection.
51
Microencapsulated modRNA in nanoparticles was recently tested as a
way of delivering modRNA to the heart. Expression of the protein was
observed in multiple cell lines and primary CMs, within 2–4hrof
transfection, and persisted for up to 7 days without altering the struc-
tural and functional properties of the cells.
57
This study demonstrated
the simultaneous delivery of multiple genes to mouse hearts and
showed that the reporter gene was efficiently delivered by an alginate
gel in the pig MI model. Similarly, Turnbull et al.
58,59
used formulated
lipidoid nanoparticles
59
(FLNPs) and assessed modRNA transfer into
the heart. They demonstrated that FLNPs delivered mRNA much
more efficiently, within 20 min, to rat and pig myocardium than sa-
line containing naked modRNA. In contrast, Sultana et al.
51
found
that encapsulating the modRNA with nanoparticles hindered its
effective translation, whereas naked modRNA in sucrose-citrate
buffer was translated very efficiently, with the protein corresponding
to the reporter gene detected within 10 min in cardiac muscle. These
findings were recently confirmed by Carlsson et al.,
47
who reported
efficient intracardiac transfection and protein expression following
the delivery of modRNA in saline citrate buffer. Translation efficiency
in mouse heart was highest for 100 mg of naked modRNA delivered in
sucrose-citrate buffer.
51
Achieving the desired biodistribution of the
therapeutic gene in the heart remains one of the largest challenges fac-
ing us, but the modRNA used in this case was expressed in more than
20% of the LV, demonstrating the potential utility of this approach for
delivering disease-specific genes to the heart in cases of injury. The
use, in most cases, of intracardiac injection to deliver modRNA to
the heart may limit the biodistribution of the modRNA. A better, sys-
temic, non-invasive cardiac delivery method is therefore required.
With the increasing use of modRNA in preclinical studies (Table 1),
many researchers are now trying to increase its translational capacity
Figure 3. Use of Modified mRNA Therapy in
Prevention of Cardiac Remodeling
modRNA can be used to improve the condition of ischemic
injury by inducing cardiac and cardiovascular regeneration
and cardiac proliferation.
of modRNA in vivo. Conventional mRNA, con-
taining uridine, is associated with low translation
rates due to activation of the RNA-dependent
protein kinase (PKR), which then phosphorylates
translation initiation factor 2-alpha (elF2a). The phosphorylated
form, elf-2, binds to elF2B with a higher affinity, preventing the for-
mation of the elF2,GTP,Met-tRNA
i
tertiary complex required to
deliver mRNA to the ribosome, limiting the translation capacity of
the mRNA. However, we have shown that this process can be altered
by the complete replacement of uridine with 1-mJU, which results in
maximal translation and optimal expression kinetics for modRNA in
the heart. The 1-mJU modRNA displayed significantly higher levels
of reporter mRNA expression in rat CMs in vitro and in mouse hearts
in vivo than the modRNAs used in previous studies. In a complemen-
tary study, Svitkin et al.
60
showed that mRNA with the 1-mJU modi-
fication resulted in much higher levels of reporter protein production,
due to attenuation of the elF2 phosphorylation-dependent inhibition
of translation and an increase in ribosome density on the mRNA.
Given the large amounts of modRNA needed to transfect large-size
heart, such as human heart, and the detrimental nature of the trans-
fection achieved by IM injection, the use of modRNA as a therapeutic
option in cardiac disease patients would require improvements in
modRNA translation and non-invasive transfection methods.
Future Directions in Cardiac modRNA Therapy
modRNA is a promising approach for the treatment of cardiovascular
disorders because it circumvents the key difficulties presented by con-
ventional protein- and DNA-based gene therapy. Figure 3 summa-
rizes the ideal use of modRNA in prevention of cardiac remodeling.
Transfection with modRNA results in transient protein expression
and is, therefore, an attractive tool for therapeutic purposes for the
correction of cellular processes that do not require long-term protein
expression, such as cardiac regeneration, CM proliferation, and re-
programming. However, current modRNA approaches have no
inherent tissue- or cell-type-specific targeting capability in vivo,
whereas AAV gene therapy vectors can include tissue-specific pro-
moters.
61–63
Improvements in targeting are, therefore, required,
because the activation of intracellular genes (e.g., transcription fac-
tors) in the wrong cell type can be detrimental. In addition, because
the IM injections may be stressful to the tissue, further research is
needed in development of non-invasive delivery methods. To ensure
targeted and non-invasive delivery of RNA, RNA aptamers, which
have great affinity to bind specific cell markers and are widely used
in cell-type-specific delivery of other RNA therapeutics like small
Molecular Therapy Vol. 27 No 4 April 2019 7
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interfering RNA (siRNA), can be used in conjunction with modRNA
to ensure its target-specific delivery.
64,65
Moreover, the transient
expression of modRNA may have made this tool ideal for approaches
targeting regeneration, but it is also the principal obstacle to the
replacement of long-term protein therapy by modRNA therapy in
the heart. Long-term controlled protein expression, with a method
of repeated systemic modRNA delivery, would make it possible to
use the modRNA delivery system to promote cardiac function in pre-
clinical or in clinical HF settings.
In our view, as research effects increase safety and scalability, and lead
to the development of cost-effective clinical-grade materials, robust
delivery methods, and lower treatment costs, modRNA technology
will become an excellent therapeutic agent to address experimental
and clinical needs to induce cardiac regeneration and promote car-
diac function in ischemic heart disease.
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