Modified mRNA as a Therapeutic Tool for the Heart

Abstract

Despite various clinical modalities available for patients, heart disease remains among the leading causes of mortality and morbidity worldwide. Genetic medicine, particularly mRNA, has broad potential as a therapeutic. More specifically, mRNA-based protein delivery has been used in the fields of cancer and vaccination, but recent changes to the structural composition of mRNA have led the scientific community to swiftly embrace it as a new drug to deliver missing genes to injured myocardium and many other organs. Modified mRNA (modRNA)–based gene delivery features transient but potent protein translation and low immunogenicity, with minimal risk of insertional mutagenesis. In this review, we compared and listed the advantages of modRNA over traditional vectors for cardiac therapy, with particular focus on using modRNA therapy in cardiac repair. We present a comprehensive overview of modRNA’s role in cardiomyocyte (CM) proliferation, cardiac vascularization, and prevention of cardiac apoptosis. We also emphasize recent advances in modRNA delivery strategies and discuss the challenges for its clinical translation.

Full text

INVITED REVIEW ARTICLE
Modified mRNA as a Therapeutic Tool for the Heart
Keerat Kaur
1,2,3
&Lior Zangi
1,2,3
Accepted: 1 August 2020
#The Author(s) 2020
Abstract
Despite various clinical modalities available for patients, heart disease remains among the leading causes of mortality and
morbidity worldwide. Genetic medicine, particularly mRNA, has broad potential as a therapeutic. More specifically, mRNA-
based protein delivery has been used in the fields of cancer and vaccination, but recent changes to the structural composition of
mRNA have led the scientific community to swiftly embrace it as a new drug to deliver missing genes to injured myocardium and
many other organs. Modified mRNA (modRNA)–based gene delivery features transient but potent protein translation and low
immunogenicity, with minimal risk of insertional mutagenesis. In this review, we compared and listed the advantages of
modRNA over traditional vectors for cardiac therapy, with particular focus on using modRNA therapy in cardiac repair. We
present a comprehensive overview of modRNA’s role in cardiomyocyte (CM) proliferation, cardiac vascularization, and pre-
vention of cardiac apoptosis. We also emphasize recent advances in modRNA delivery strategies and discuss the challenges for
its clinical translation.
Keywords Modified mRNA .Gene therapy .Myocardial infarction .Cardiovascular regeneration .Cardiac protection .
Cardiomyocyte proliferation
Genetic Medicine in Heart Disease
Completed in 2003, the overwhelming success of the Human
Genome Project enabled researchers across the globe to iden-
tify and sequence all genes present in human DNA. The abil-
ity to analyze target genes and related signaling pathways
made gene therapy a novel form of molecular medicine.
Conceptually, gene therapy is quite straightforward: introduc-
ing a normal gene into a cell involved in a disease process
should enable that gene’s protein product to correct or slow
the disorder’s advancement. Based on this idea, gene therapy
aims to deliver genetic material to manage both inherited and
acquired diseases.
Although pharmacological therapeutic approaches devel-
oped over the past 50 years have considerably improved qual-
ity of life for patients with heart disease, these common phar-
macological interventions (β-blockers, angiotensin-
converting enzyme inhibitors, angiotensin receptor II-antago-
nists, and diuretics) do not interact with or demonstrably cur-
tail the relevant underlying intracellular signal transduction
mechanisms that cause or intensify the development and pro-
gression of heart disease [1,2]. To date, there is no known
cure for heart failure (HF), and the limited numbers of hearts
available for transplantation are not a sufficient solution. As a
result, the morbidity and mortality rates of heart disease re-
main unacceptably high in Western industrialized countries
and are rising in others [3]. Thus, to fill this gap in modern
pharmacologic therapies and better assist patients with heart
failure patients, novel remedies are badly needed.
Gene therapy presents an ideal approach for manipulating
molecular mechanisms that cannot be altered pharmacologi-
cally. Also, gene therapy offers spatially and temporally ef-
fective gene delivery to the myocardium, an approach that can
provide lasting benefitsin organ-widechronic and progressive
HF. In the case of ischemic heart disease (IHD), gene delivery
ideally should (a) induce cardiac regeneration via cardiomyo-
cyte proliferation, (b) prevent cardiac cell death (CMs or non-
*Lior Zangi
lior.zangi@mssm.edu
1
Cardiovascular Research Center, IcahnSchool of Medicine at Mount
Sinai, New York, NY, USA
2
Department of Genetics and Genomic Sciences, Icahn School of
Medicine at Mount Sinai, New York, NY, USA
3
Black Family Stem Cell Institute, Icahn School of Medicine at Mount
Sinai, New York, NY, USA
https://doi.org/10.1007/s10557-020-07051-4
/ Published online: 21 August 2020
Cardiovascular Drugs and Therapy (2020) 34:871–880
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CMs), (c) target endothelial cells (ECs) to stabilize coronary
plaques and induce angiogenesis, (d) trigger cardiac
reprogramming through fibroblasts, and (e) reduce electro-
physiological abnormalities. Gene delivery vehicles should
have an appropriate packaging system to convey the genetic
material into a variety of cells, with high efficiency and for a
desirable period of time.
Current Approaches to Achieve Gene Therapy
Goals
Current gene delivery vehicles fall into two broad categories:
viral and non-viral gene delivery vectors. Conventional viral
vectors used in cardiovascular diseases, including retrovi-
ruses, lentiviruses, and adenovirus-associated virus (AAV),
can efficiently deliver genes into cardiac cells; however, every
known viral vector bears some risks and limitations. By con-
trast, non-viral vectors, which are comprised of 2 categories:
physical (plasmid DNA, electroporation, sonoporation, hy-
drodynamic, ultrasound, magnetofection, gene gun) and
chemical (cationic lipids, different cationic polymers,
Chitosan, dendrimers, lipid polymers, inorganic nanoparti-
cles, cell-penetrating peptides), are safer to use but have lim-
ited transfection efficiency and require proper structure with
specific absorption efficiency to provide effective means of
gene delivery [4,5]. In this regards, novel mRNA-based ther-
apy is a promising gene delivery platform for cardiac gene
therapy.
I. Gene packaging and delivery: The expression level of the
gene in the target cells is controlled by regulatory elements
(promoters and enhancers) packaged alongside the gene
inside the viral protein coat. Thus, the space available in
the capsid determines the size of the therapeutic gene to be
delivered. Adenoviruses (AV) and lentiviruses have a rel-
atively large insert capacity and contain a genome of ap-
proximately 36 kb and 14 kb, respectively, while smaller
viruses, like AAV, have a 25-nm diameter protein coat and
a much smaller insert capacity space (only ~5 kb), which
limits the size of the therapeutic gene [6–8]. In this con-
text, non-viral vectors like naked DNA plasmid or
modRNA do not have any size constraint and can be use-
ful in carrying and delivering a therapeutic gene of any
size directly to cardiac cells. Given the fact that gene ex-
pression reduces in correlation with an increase in the size
of mRNA, modRNA provides the flexibility for control-
ling the amount of gene delivery in the cells. Furthermore,
modRNA delivery is not influenced by the state of the
nuclear membrane and can thus transfect both dividing
and non-dividing cells, a trait most viral vectors lack.
II. Gene expression pharmocokinetics: The temporal ex-
pression patterns of therapeutic genes are critical to
whether the gene transfer system can be employed for
efficient and positive recovery. Because every disorder
requires unique temporal expression, it is desirable to
choose an optimal vector that can deliver genes within a
particular time frame for appropriate protein turnover.
Viral vectors like lentiviruses provide strong gene expres-
sion for an extended period of time and are popular
choices for treating pathophysiologies that need lifelong
expression of a missing protein. In a heart failure model,
prolonged expression of sarcoplasmic reticulum Ca
2+
ATPase via pump with lentivirus injection was reported
to improve myocardial function in mice [9]. Over the last
decade, various pre-clinical studies have explored using
AAV in prolonged replacement of genes involved in
inherited heart disorders. AAV-assisted Sumo-1 gene
transfer into pig hearts was shown to improve their car-
diac function post-injury [10], as AAV-assisted gene ex-
pression peaks after 4 weeks and continues up to
11 months [11]. However, uncontrolled and prolonged
gene delivery can pose unnecessary risks when only tran-
sient expression of an appropriate gene is needed to trig-
ger an underlying signaling pathway. Further, as signifi-
cant changes occur in cardiac cells as early as 24 h post-
infarction, early and quick interventions are needed to
prevent and protect the heart from further damage.
Accordingly, modRNA’s unique pulse-like, immediate
gene expression is highly favorable in preventing cardiac
remodeling post-MI. ModRNA gene therapy has now
been shown to prevent cardiomyocyte death [12,13]
and induce cardiomyocyte and vascular proliferation
without risking uncontrolled cell division or tumor for-
mation. In 2013, Zangi et al. successfully showed vascu-
lar regeneration after MI with modRNA-induced
VEGFA expression [14].
III. Gene transfer efficiency: Efficient gene transfer into the
cell is vital to successful gene translation and depends on
properties of the vector used for transfection. Viral vec-
tors depend on vector infectivity, promotor control over
the gene of interest, the viral vector’saffinitytomem-
brane receptors, receptor availability, and foreign gene
inactivation by the host cell. In the failing heart, endog-
enous molecular mechanisms in cardiac cells change,
which may result in the delivered gene being silenced
despite its active form, thereby substantially reducing
therapeutic gene expression. Effective gene therapy thus
requires a viral vector with high infection multiplicity
that can transfer a high number of viral particles to the
targeted cardiac cell in order to achieve the desired func-
tional effect. mRNA-based therapies are proven to be
successful in this difficult context, as therapeutic gene
amounts can be finely controlled. Moreover, this tech-
nology can deliver gene combinations with ratios tai-
lored to the targeted cell. In the case of gene delivery
872 Cardiovasc Drugs Ther (2020) 34:871–880
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assisted by viral vectors, the target gene must be
translocated to the cell nucleus, where it then interacts
with the array of nuclear proteins that regulate gene ex-
pression. Using mRNA transfection overcomes the need
for nuclear localization to induce transcription, enabling
mRNA therapy to efficiently translate the desired gene
without other influencers.
IV. Potential safety concerns: Gene delivery system
safety must be thoroughly determined before vehi-
cles can be selected for myocardial gene therapy.
Using viruses for gene therapy raises a number of
safety concerns. AVs can trigger a strong innate
immune response and toxicity due to viral gene
products. The use of AVs came into serious ques-
tion in 1999 after a patient with ornithine
transcarbamylase deficiency died due to a massive
immune response following the injection of an AV
vector [15]. Lentivirus vectors are of limited use in
cardiovascular disorders because these viruses ran-
domly integrate into the host with a preference for
targeting coding regions, thus creating a huge risk
of insertional mutagenesis and oncogenesis [16].
While AAVs are highly favored over other vectors
due to their lack of immunogenicity, a critical ob-
stacle in AAV gene therapy translation is the pres-
ence of preexisting neutralizing anti-AAV antibod-
ies that are present in 30 to 50% of the population
[17]. In addition to ease of vector production and
reduced limitations on expression cassette size,
non-viral gene delivery is also a safer option under
several physiological conditions. modRNA non-
viral gene delivery shows minimal biosafety risks,
as the mRNA does not integrate into the host ge-
nome. Moreover, mRNA offers transient gene ex-
pression, which minimizes the risk of mutagenesis
after mRNA therapy. A side-by-side comparison of
commonly used vectors for cardiac repair can be
foundinTable1.
The Popularity of Messenger RNA
mRNA, a naturally occurring molecule, holds revolutionary
medical potential as it can efficiently and accurately translate
the information from DNA into proteins, thus allowing the
host to generate their own personalized medicine. The concept
of mRNA-based gene transferin mammalian cells in vitro was
first introduced by Bhargava and Shanmugam in 1970 [18],
and almost two decades later, mRNA encoding for reporter
gene B galactosidase was successfully injected intramuscular-
ly in a mouse model [19]. Although its capacity to self-
amplify made mRNA therapy useful in vaccine development
[20], it was not considered a therapeutic entity and was be-
lieved to be highly therapeutic unsuitable due to the immune
response it elicited [21]. Upon entry into the cell, in opposed
from coming out from the nucleus, unmodified mRNAs are
recognized by the innate immune system via recognition re-
ceptors, known as toll-like receptors (TLRs) 7 and 8, located
in the endosome [22]. Activation of TLRs leads to the produc-
tion of pro-inflammatory cytokines and type I interferons that
activate the endonuclease (RNaseL), which ultimately de-
grades the imported mRNA [23,24], shutting downs its trans-
lation. This process meant synthetic mRNA had limitations in
gain-of-function studies.
Yet modifying mRNA’s secondary structure (Fig. 1), par-
ticularly changing uridine with naturally occurring
pseudouridine and 5-methyl-cytosine for cytosine [25], com-
bined with advances in in vitro transcription technologies led
to less recognition by TLRs and nucleases. Additionally, the
stability and in vivo translation efficiency of modRNA have
Table 1 Various gene delivery
vectors for cardiac repair Delivery
method
Vehicle
diameter
Gene
packaging
capacity
Expression kinetics Immunogenicity Major drawback
ModRNA Variable Unlimited Short term expression
up to 2–7days
Minimal Transient expression
Plasmid Variable Unlimited Expression up to
2 months
Minimal Low transfection
efficiency
Lentivirus 90 nm ~ 8 kb Long-term cardiac
expression
Moderate Risk of insertional
mutagenesis
AAV 25 nm ~ 5 kb Long-term cardiac
expression up to
11 months
Mild Risk of neutralizing
antibodies and T
cell responses
Adenovirus 100 nm ~ 36 kb Expression up to
2weeks
Strong High antibody and
inflammatory
response
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been increased by capping the molecule with the 3′-O-
Mem7G(5′)ppp(5′)G Anti Reverse Cap Analog (ARCA) at
its 5′end [26,27] and replacing 5′UTR with one from the fatty
acid metabolism gene carboxylesterase 1D (Ces1d) [28].
ModRNA has the capacity to target the gene of interest via
multiple mechanisms including: (i) gain of function by over-
expressing a target molecule [13,29], (ii) loss of function by
either using dominant negative molecules or introducing
miRNA [30], (iii) correcting gene deletions at the mRNA level
[31], and (iv) inhibiting intracellular signaling pathways by
introducing decoy receptors to dilute the ligands [30].
Modified mRNA Therapy in the Heart
During MI, the occlusion of the coronary artery leads to is-
chemia and subsequent loss of CMs and ECs, which are
quickly replaced by highly proliferating fibroblasts, resulting
in scarring and remodeling of heart tissue in the affected areas.
Due to their limited proliferation capacity, surviving CMs are
incapable of reversing the damage and replace the dead CMs.
Moreover, the damage in the coronary vasculature creates an
unfavorable milieu for CM survival. These irreversible chang-
es in the heart along with increased oxidative stress and in-
flammation lead to impaired pump function and, ultimately,
heart failure. ModRNA is a promising gene therapy approach
that can therapeutically target several mechanisms that may
protect MI survivors against HF. Studies have proven three
major strategies by which modRNA can be used to treat is-
chemic injury: (i) inducing CM proliferation, (ii) inhibiting
heart cell death and attenuating inflammation, and (iii)
supporting cardiovascular regeneration. The uses of
modRNA technology as a therapeutic approach for cardiac
repair are listed in Table 2.
I. CM proliferation by modRNA approach:Whendelivered
at the time of MI, modRNA is an effective vehicle for re-
awakening CM proliferation. In 2018, Magadum et al.
published the first report noting modRNA can induce
CM proliferation and regeneration by upregulating mutat-
ed human follistatin-like (hFSTL1). This work shows that
mutation at the N-glycosylation site, position 180 of as-
paragine (N) with glutamine (Q), was sufficient and nec-
essary to activate CM proliferation and reduce cardiac
remodeling post-MI. Post-translational modification, i.e.,
glycosylation of hFSTL1 upon N180 site ablation, by
allowing it to activate unknown receptors in the heart
was hypothesized to be responsible for CM regeneration
in vitro or neonatal rats or adult mice after MI with no
indications of cardiac hypertrophy. Further, a single dose
of N180Q hFSTL1 modRNA to the mouse myocardium
post-MI significantly improved cardiac function, de-
creased scar size, and increased capillary density after
28 days, showing the effectiveness of modRNA in trigger-
ing CM proliferation and cardiac regeneration [12].
In a subsequent study, Zangi and colleagues showed that
modRNA technology can induce the CM cell cycle by upreg-
ulating the glycolytic enzyme Pkm2 (pyruvate kinase muscle
isoenzyme 2) [13]. Pkm2 is primarily expressed at higher
levels in regenerative fetal heart and neonatal CMs but not
in adult CMs. This work established that Pkm2 can regulate
cell cycle progression by elevating anabolic metabolism in
CMs (via pentose phosphate pathway) interacting with β-
catenin and upregulating its downstream targets Cyclin, D1,
and C-Myc. Further, they demonstrated that Pkm2 plays a role
in regulating oxidative stress by reducing ROS production
post-MI. ModRNA-mediated Pkm2 elevation re-invigorated
the CM cell cycle, which led to CM cell division and subse-
quent cardiac regeneration. Using the lineage-tracing mouse
model and relabeling the CMs, the study showed that the
ectopic expression of modRNA encoding the Pkm2 gene in-
creased cardiomyocyte cell division in adult mice and sup-
pressed postnatal CM cell cycle arrest. Moreover, Pkm2
modRNA delivery led to significantly improved cardiac func-
tion and outcomes after acute or chronic myocardial infarc-
tion, thus rescuing cardiac remodeling.
II. ModRNA offers cardiac protection: After ischemic stress
in the mammalian heart tissue, progressive death of heart
cells in the left ventricle results in deteriorated cardiac
function. A powerful predictor of heart failure in IHD
patients is high concentrations of simple membrane
sphingolipids, known as ceramides, in the plasma. More
specifically, elevated ceramide levels in the blood are
associated with programmed cell death and higher prob-
ability of MI recurrence. Considering the role of
Fig. 1 Schematic illustration showing the modifications made to mRNA’s structure to increase its translation and stability
874 Cardiovasc Drugs Ther (2020) 34:871–880
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ceramides and the enzyme associated with their metabo-
lism in heart disease progression, they may also affect
CM death. As was recently reported, modRNA-
delivered acid ceramidase (AC) overexpression is
associated with lower CM death rates and increased cell
survival after hypoxia or MI. AC primarily frees fatty
acids and sphingosine in ceramide hydrolysis, and elevat-
ed AC has been reported to lessen the negative effects of
Table 2 Key studies identifying modRNA as cardiac repair therapy
Focus area Publication Protein target Experimental outcome Delivery vehicle Administration method Reference
Inducing CM
proliferation
Magadum
et al.
mutated
FSTL1
CM proliferation,
decreased scar size,
improved heart function
Sucrose-citrate buffer Intracardiac injection [12]
Magadum
et al.
Pkm2 Induced CM cell cycle,
reduced oxidative stress
Sucrose-citrate buffer Intracardiac injection [13]
Inhibiting cardiac
apoptosis/-
enhancing
survival
Huang et al. IGF-1 Reduced cell
apoptosis/promoted cell
survival
Polyethylenimine-based
nanoparticle
Intracardiac injection [32]
Zangi et al. DN-IGF-1R,
IGFR
Reduced cell
differentiation into
adipocytes post-MI
RNAiMAX Intracardiac injection/gel ap-
plication
[30]
Hadas et al. AC Increased cell survival,
improved cardiac
function and mice
survival
Sucrose-citrate buffer Intracardiac injection [29]
Chen et al. aYAP Decreased CM necrosis,
attenuated innate
immune responses
Saline Intracardiac injection [33]
Inducing
cardiovascular
regeneration
Zangi et al. VEGFA Induced angiogenesis,
improved myocardial
function and mice
survival
RNAiMAX Intracardiac injection [14]
Lui et al. VEGFA Endothelial specification
engraftment,
proliferation, and
reduced apoptosis of the
human Isl1+
progenitors in vivo
RNAiMAX Matrigel, subcutaneous
injection
[34]
Carlsson
et al.
VEGFA Increased capillary
density, decreased
fibrosis and improved
heart function post-MI
Sucrose-citrate buffer Intracardiac injection [35]
Moderna
Therapeu-
tics
VEGFA Not reported Citrate buffer saline Epicardial injection [36]
ModRNA delivery
and production
optimization
Turnbull
et al.
EGFP Efficient modRNA
delivery to the heart
Formulated lipidoid
nanoparticles (FLNP)
Intramyocardial/intracoronary
injection
[37]
Turnbull
et al.
EGFP Protocol Formulated lipidoid
nanoparticles (FLNP)
[38]
Kondrat
et al.
varies Protocol RNAiMAX [39]
Sultana et al. Luciferase Optimized modRNA
amount, time and
delivery
Sucrose-citrate buffer Intracardiac injection [40]
Singh et al. EGFP,
mCherry,
Fluc
Optimized modRNA
delivery into
myocardium
Alginate, nanomaterial
encapsulated
Intracardiac injection [41]
Hadas et al. GFP,
Luciferase
Improved modRNA yield
and translation
efficiency, reduced its
immunogenicity
Sucrose-citrate buffer Intracardiac injection [27]
Sultana et al. Luciferase Increased translation by
replacing 5’UTR
Sucrose-citrate buffer Intracardiac injection [28]
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elevated ceramides. In addition, elevated levels of this
enzyme reduced detrimental neutrophil levels in the LV,
thus decreasing the inflammation associated with MI and
promoting cell survival. Indeed, AC-modRNA–treated
mice showed significantly better heart function, smaller
LV scars, and longer survival in post-ischemic injury
[29].
ModRNA gene delivery has also been used to ease IR-
induced inflammation. As the first response to cardiac injury,
innate immune system activation recruits leukocytes, includ-
ing damage-associated molecular patterns, cytokines, and
chemokines, that help coordinate dead and damaged cell re-
moval, clear extracellular matrix debris, revascularize, and
form scars. On the downside, these recruited leukocytes also
activate the downstream signaling pathways, resulting in cell
necrosis [42]. Chen et al. showed that modRNA-mediated
transient activation of transcriptional co-activator yes-associ-
ated protein (aYAP), already known to promote cell prolifer-
ation and survival, attenuated the inflammatory innate im-
mune response by lowering neutrophil infiltration after IR
injury. This efficient expression of aYAP protein via
modRNA treatment led to improved heart function, reduced
CM necrosis, diminished scar size, and prevented hypertro-
phic cardiac remodeling [33].
Huang and group showed the role of modRNA-delivered
insulin-like growth factor-1 (IGF1) in providing
cardioprotection in CM after ischemia and MI [32]. Using
modRNA to deliver IGF1 to the at-risk area in mouse hearts
post-MI promoted CM survival and limited cell death under
hypoxia-induced apoptosis conditions. A polyethylenimine-
based nanoparticle-assisted modRNA delivery of IGF-1
showed rapid protein expression as early as 2 h after injection
and peaked after 24 h. Elevated IGF-1 protein levels led to
phosphorylation of downstream targets Akt and Erk, decreas-
ing cell apoptosis by 50%, as seen by lower levels of TUNEL-
positive cells and reduced caspase-9 activity in the mouse
model following ischemic injury and hypoxia. By contrast,
IGF-1 upregulation triggers epicardial progenitor cells to dif-
ferentiate into adipogenic cells, leading to epicardial adipose
tissue (EAT) formation post-MI. Further downregulating the
IGF-1 signaling pathway by delivering dominant-negative
IGF-1 receptor antagonists reversed EAT formation in the
heart [30]. Overall, these studies demonstrate that transient
protein expression driven by modRNA gene delivery has the
potential to enact an extended cytoprotective effect.
III. ModRNA therapy induces cardiovascular regeneration:
Post-MI, tissue ischemia develops around the infarction
site due to loss of ECs and subsequent reduction of vas-
cularization. One established way to repair this damage
is to regenerate the blood vessels supporting the CMs.
Various studies have shown the potency of VEGFA as
an angiogenic factor after ischemic injury. However, the
clinical trials addressing VEFGA delivered via adenovi-
ral plasmid or recombinant protein as a cardiac therapy
showed only moderate improvements in cardiac func-
tion. One explanation for this lack of vascular regenera-
tion is the inefficient delivery platform, which led to
either protein degradation by proteases or off-target de-
livery due to systemic injections [43]. Another issue with
prolonged VEGFA expression is its negative effects on
vascular permeability, as increased VEGFA is associated
with development of leaky, immature vessels with ele-
vated vessel permeability, and pool perfusion rate [44].
To overcome the problems related to sustained protein de-
livery with viral vectors, Zangi et al. [14] explored efficient
pulse-like delivery of VEGFA in a mouse MI model. This
study demonstrated that modRNA-induced VEGFA expres-
sion increased the levels of endogenous heart progenitors,
mobilized their migration into the myocardium, and redirected
their differentiation toward cardiovascular lineages. Further,
transient VEGFA expression derived from modRNA was su-
perior to plasmid DNA in reducing infarct size, enhancing
myocardial perfusion and improving survival. These findings
verified that modRNA therapy might be well suited to deliv-
ering paracrine factors that enhance cardiac regeneration.
Wide screening of angiocrine factors expressed in ECs de-
rived from the outflow tract of human fetal hearts compared
with ECs derived from human cord blood revealed that
VEGFA is the key factor involved in differentiating human
ESC-derived Isl1+ progenitors toward an EC fate. Lui et al.
reported that modRNA-driven pulse-like VEGF-A overex-
pression not only caused endothelial specification but also
engraftment, proliferation, and survival (reduced apoptosis)
of the human Isl1+ progenitors in vivo [34]. Promising results
from a study conducted in swine moved the field one step
closer to taking modRNA-delivered VEGFA intervention to
the clinic [35]. Intracardiac delivery of VEGFA mRNA im-
proved left ventricular ejection fraction, inotropy, and ventric-
ular compliance, with increased border zone arteriolar and
capillary density 2 months after permanent occlusion surgery.
This modRNA-based single-dose VEGFA delivery proved
sufficient to improve ventricular function and curtail myocar-
dial damage in mini pigs. In addition to these promising re-
sults, Moderna, an mRNA-based company, and its partner
AstraZeneca are examining the effect of modRNA-delivered
VEGFA delivery in patients with moderately impaired systol-
ic function undergoing coronary artery bypass grafting sur-
gery (clinical trial number NCT03370887) [36]. This phase
II clinical trial is investigating the safety and tolerability of
VEGFA modRNA epicardial injection in patients. Once pos-
itive results are received, modRNA-delivered VEGFA thera-
py may come to represent a new class of therapies for improv-
ing cardiac function after ischemic injury. Figure 2illustrates
876 Cardiovasc Drugs Ther (2020) 34:871–880
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the mechanisms by which modRNA offers cardioprotection to
the injured myocardium.
ModRNA Delivery and Translation Systems
Multiple methods and vehicles have been used for intracellular
modRNA delivery to cells in vitro and in vivo. Traditionally,
physical transfection methods like electroporation and gene gun
or microinjections have been shown to efficiently deliver
modRNA to cells; however, these methods were limited to
cancer therapy and, to a lesser extent, protein replacement ther-
apy due to their high costs and invasive nature [45]. As an
alternate option, cationic lipid or polymers, which make an
electrostatic bond with the negatively charged nucleic acid,
have shown considerable promise for modRNA delivery.
Other possible modRNA delivery methods are nanoparticles,
which ensure nucleic acid enters the cell membrane by their
spherical shape containing polar head groups and non-polar
tails. These chemical methods feature high versatility, reduced
costs, simpler use, and low toxicity. The common formulations
contain four components: an amine-containing lipid or lipid-
like material, a phospholipid, cholesterol, and lipid-anchored
polyethylene glycol. These compounds are flexible, as the
amount of each component can be adjusted to enhance targeted
delivery and protection of the conjugated nucleic acid from
nuclease degradation. Nanoparticles’reliability, efficacy, and
flexibility make them attractive for modRNA delivery.
Cationic lipoplex formulations, e.g., lipofectamine, have
proven to successfully deliver modRNA to isolated CMs
in vitro. Transfecting modRNA using positively charged lipofec-
tamine resulted in very high transfection levels in neonatal rat
(98.3%) and human pluripotent stem cell-derived CMs (98.9%).
Despite such convincing mRNA delivery results in vitro,
RNAimax use was limited for in vivo studies due to high cell
death rates around intracardiac injection sites [40].
In comparing different modRNA delivery modes, Sultana
et al. [40] reported that naked modRNA (with sucrose-citrate
buffer) was superior in modRNA translation to lipid nanoparti-
cles. This was associated with lower heart cell death associated.
This lead us to believe that naked modRNA is an optimal ap-
proach for cardiac gene delivery as the buffer solution may pro-
vide viscosity and act as a chelating agent for mRNA preserva-
tion [46]. With this strategy, delivering 25–100 μgofmodRNA
was able to widely translate the gene of interest, covering more
than 20% of the myocardium. The rapid expression element of
modRNA resulted in protein translation within 10 min of trans-
fection. Carlsson et al. later verified these findings in large animal
studies. VEGFA delivery into pig hearts with biocompatible
citrate-saline buffer showed tissue-specific protein expression
without stimulating any immune response [35]. Hence, deliver-
ing free or loosely bound mRNA in cytoplasm leads to higher
translation efficiency and opens the possibility of mRNA therapy
in acute cardiac diseases.
Another modRNA delivery option was explained by Turnbull
et al., who showed successful in vivo modRNA delivery to the
myocardium using formulated lipidoid nanoparticles (FLNP)
[37,38]. Intracardiac injection of FLNP carrying modRNA
showed reporter gene protein expression peaked 20 h after injec-
tion with minimal off-target expression. The authors show
modRNA has intracardiac stability, with gene expression detect-
ed up to 14 days post-delivery. Further, the group also success-
fully observed fluorescent signal as early as 20 min after FLNP/
eGFPmodRNA injection in the pig heart, confirming the rapid
Fig. 2 Direct cardiac repair using
modRNA system to deliver genes
involved in inducing
cardiomyocyte proliferation,
cardiomyocyte protection, and
cardiovascular regeneration
877Cardiovasc Drugs Ther (2020) 34:871–880
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

and efficient nature of modRNA-assisted gene delivery. Novel
microencapsulation technologyhasalsobeentestedfor
modRNA delivery into myocardium of small and large animals
[41]. The study reported that using ∼100 nm microencapsulated
modRNA (M3RNA) lead to protein detection as early as 2–4h
withstabilityforupto7daysinisolatedCMsand72hinmurine
hearts. M3RNA in a porcine model further indicated rapid,
targeted protein expression, thus providing an alternate approach
to deliver modRNA into the injured heart.
Although modRNA therapy is now undergoing clinical trials,
and numerous pre-clinical trials are also underway, researchers
are still finding ways to improve its translational capacity in vivo
and develop cost-effective protocols for modRNA production.
The 5′7-methylguanylate cap plays a role in stabilizing mRNA
by protecting it from exonucleases and promoting the translation
initiation by binding eukaryotic initiation factor 4E [47]. The
addition of Anti reverse cap analogue increases the translation
efficiency by ensuring capping occurs in the correct orientation
[48]. However, this chemical capping strategy requires a high
ratio of ARCA and GTP concentrations to produce a high per-
centage of capped mRNA. Hadas and colleagues provided an
amendment to the ratios of 5′ARCA cap and previously de-
scribed N1-methyl-pseudouridine [39], which can cut
modRNA production costs and enhance its protein expression.
They also optimized the nucleotide concentration to 31.6 mM
per reaction, resulting in a 290% higher modRNA yield. This
nucleotide optimization led to increased percentage of the capped
modRNA, thus reducing its immunogenicity in both human cell
lines and primary cardiac cells [27].
In an attempt to further enhance the translational efficiency
of modRNA, modifications of other mRNA structural domains
provide additional avenues for investigation. Eukaryotic gene
expression is regulated by 5′and 3′UTRs of modRNA, which
stabilize mRNA [49] by integrating with the translational ma-
chinery or serving as the binding site for micro RNA and
mRNA decay-promoting proteins, respectively [50]. In an at-
tempt to increase the half-life of IVT mRNA and its translation
into protein, various studies have selected 3′UTRs of α-andβ-
globin mRNAs and incorporated them into the 3′UTR of IVT
mRNA [51]. A recent study showed that replacing traditionally
used artificial 5′UTR with one from Ces1d doubled the trans-
lation of a modRNA-delivered reporter gene in the heart post-
MI. Mechanistically, Ces1d is involved in lipid metabolism,
and changes in this metabolic activity under MI conditions
trigger Ces1d mRNA, leading to better translation. Further, this
altered modRNA structure also enhanced translation in other
organs, including the liver, under ischemic conditions [28].
These promising studies thus indicate modRNA technology
can be used in future cardiac regenerative applications.
To date, cardiac gene therapy via modRNA has shown the
ability to repair injured hearts in various aspects including ex-
pressing angiogenic factors for vessel regeneration and
sphingolipid metabolic genes to limit and prevent cardiac
damage or to induce cell cycle-promoting genes. However, while
upregulating these genes can prove to be favorable for one cell
type, their overexpression in neighboring cells can produce un-
favorable outcomes. For instance, after cardiac injury, there is an
urgent need for CMs to undergo cell cycle. The global delivery of
cell cycle-promoting genes can also stimulate non-CMs in the
heart to undergo division, leading to increased scar formation or
eliciting an undesirable immune response. Thus, to overcome
these detrimental effects of non-specific delivery of modRNA
into cardiac cells, Magadum et al. demonstrated a novel mecha-
nism of desirable gene translation exclusively in CMs upon in-
tracardiac injection. They create a unique circuit modRNA based
on archaeal ribosomal protein L7Ae, which suppresses transla-
tion of the gene containing a kink-turn motif, a specific binding
site for L7Ae. Upon simultaneous transfections of L7Ae and a
gene containing kink-turn motif, L7Ae attaches to the binding
site and suppresses translation of the gene of interest. Using this
cell specific system, the study ensured CM specificity by adding
a CM-specific microRNA recognition element to the 3′UTR of
the L7Ae gene. This prevented L7Ae translation in CMs that
extensively and particularly express those microRNAs, so that
the gene of interest translated exclusively in CMs [13]. Thus, this
novel, first of its kind, in vivo modRNA model promotes gene of
interest homing to the desired cell in the infarcted myocardium.
Challenges and Future Directions of Cardiac
modRNA Therapy
Although modRNA applications in cardiology are progressing
quickly, hurdles remain that must be overcome to achieve trans-
lation. Various publications have shown that modRNA therapy
improves outcomes after MI [52]; however, taking this research
to the clinic has been hampered by poorly defined delivery sys-
tems. Currently, intracardiac injection is the most effective deliv-
ery method for delivering genes to the heart, but this direct gene
penetration into the myocardium causes stress and local injury to
the tissue. Thus, there is a real need to develop efficient mRNA
delivery systems that can ensure targeted, non-invasive gene
relay into the heart. There is growing interest in cell-penetrating
peptides (CPP) that can be used in conjunction with modRNA to
ensure its target-specific delivery. Recent studies have supported
the use of CPP to deliver small interfering RNA to inhibit target
gene expression in cancer cells [53]. Additionally, mRNA trans-
fection may be efficiently mediated by RNA aptamers, which
bind to specific cell markers and can be modified to target spe-
cific tissues [54]. Furthermore, it is crucial to optimize consistent
dosing across the myocardium and among all patients receiving
modRNA therapy. Both controlled modRNA release into the
cytoplasm following endocytosis and modRNA dose/protein ef-
fect relationships must be considered before modRNA can be
implemented in cardiac therapy.
878 Cardiovasc Drugs Ther (2020) 34:871–880
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Although modRNA’s transient gene delivery eliminates
the risk of malignancies associated with overexpression of
genes delivered for long periods, further research is needed
to improve modRNA’s translation efficiency in order to com-
pensate for its short expression pattern and mitigate the need
for repeated transfections. Further, plans to develop modRNA
as a therapeutic intervention must consider the cost of produc-
tion. Given the large amounts of modRNA needed to transfect
organs as large the human heart (between 3 and 30 mg), [36]
production costs must be reduced.
Regarding modRNA generation, the primary hurdle for
mRNA therapy—its instability—has been effectively ad-
dressed. We hope that in the coming years, as research into
modRNA targeting and delivery accumulates, reduced pro-
duction costs and improved expression kinetics will accelerate
the use of modRNA in numerous areas of medicine, including
protein replacement therapies and genetic disorders.
List of abbreviations
Abbreviation Full name
AC Acid ceramidase
AAV Adenovirus-associated virus
AV Adenoviruses
ARCA Anti-reverse cap analog
Ces1D Carboxylesterase 1D
CM Cardiomyocyte
CPP Cell-penetrating peptides
EC Endothelial cell
EAT Epicardial adipose tissue
FLNP Formulated lipidoid nanoparticles
HF Heart failure
hFSTL1 Human follistatin-like
IGF1 Insulin-like growth factor-1
IHD Ischemic heart disease
M3RNA Microencapsulated modified mRNA
ModRNA Modified mRNA
Pkm2 Pyruvate kinase muscle isoenzyme 2
TLRs Toll-like receptors
aYAP Yes-associated protein
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