International Journal of

Molecular Sciences

Review
Self-Amplifying RNA Approach for Protein
Replacement Therapy
Dimitri Papukashvili † , Nino Rcheulishvili † , Cong Liu, Yang Ji, Yunjiao He * and Peng George Wang *

Department of Pharmacology, School of Medicine, Southern University of Science and Technology,

Shenzhen 518000, China
* Correspondence: [email protected] (Y.H.); [email protected] (P.G.W.);
Tel.: +86-135-3765-7996 (Y.H.); +86-0755-8801-5584 (P.G.W.)
† These authors contributed equally to this work.

Abstract: Messenger RNA (mRNA) technology has already been successfully tested preclinically and
there are ongoing clinical trials for protein replacement purposes; however, more effort has been put
into the development of prevention strategies against infectious diseases. Apparently, mRNA vaccine
approval against coronavirus disease 2019 (COVID-19) is a landmark for opening new opportunities
for managing diverse health disorders based on this approach. Indeed, apart from infectious diseases,
it has also been widely tested in numerous directions including cancer prevention and the treatment
of inherited disorders. Interestingly, self-amplifying RNA (saRNA)-based technology is believed
to display more developed RNA therapy compared with conventional mRNA technique in terms
of its lower dosage requirements, relatively fewer side effects, and possessing long-lasting effects.
Nevertheless, some challenges still exist that need to be overcome in order to achieve saRNA-based
drug approval in clinics. Hence, the current review discusses the feasibility of saRNA utility for
protein replacement therapy on various health disorders including rare hereditary diseases and also
provides a detailed overview of saRNA advantages, its molecular structure, mechanism of action,
Citation: Papukashvili, D.; and relevant delivery platforms.
Rcheulishvili, N.; Liu, C.; Ji, Y.; He, Y.;
Wang, P.G. Self-Amplifying RNA Keywords: saRNA; protein replacement; protein deficiency; single-gene disorders; alpha-1 antitrypsin
Approach for Protein Replacement deficiency; AATD; mRNA; taRNA
Therapy. Int. J. Mol. Sci. 2022, 23,
12884. https://doi.org/10.3390/
ijms232112884

Academic Editors: Claudia Ghigna, 1. Introduction

Laura Cerchia and Laura Poliseno Despite significant progress in science and medicine, until now, there are a number
Received: 19 September 2022
of diseases for which the development and improvement of new strategies are emerging.
Accepted: 21 October 2022
The potential of messenger RNA (mRNA) application is enormous for the well-being
Published: 25 October 2022
of humans [1]. Compared with the traditional vaccine, making the mRNA vaccine has
numerous advantages. In the case of mRNA vaccine administration, the human body
Publisher’s Note: MDPI stays neutral
makes proteins itself, and huge bioreactors are no longer needed for vaccine production.
with regard to jurisdictional claims in
Therefore, the human body becomes a bioreactor itself, and the time for making a vaccine
published maps and institutional affil-
is markedly reduced [2,3]. There are two types of mRNA, conventional–non-replicating
iations.
RNA and self-replicating, in other words, self-amplifying RNA (saRNA). saRNA has many
structural similarities to conventional mRNA: it is a linear, single-stranded RNA molecule
that is synthesized with a 50 cap, 30 polyA tail, and 50 and 30 untranslated regions (UTRs).
Copyright: © 2022 by the authors.
The main structural difference is that saRNA is a much larger molecule as it encodes four
Licensee MDPI, Basel, Switzerland. extra proteins in addition to the protein of interest or vaccine antigen. These non-structural
This article is an open access article proteins (nsPs) that encode a replicase are usually derived from an alphavirus [4]. saRNA
distributed under the terms and vaccine is proposed to have features of an updated version of the conventional mRNA
conditions of the Creative Commons vaccine with multiple advantages, especially its low dosage [5], reduced side effects [4],
Attribution (CC BY) license (https:// and long-lasting outcomes [6]. It is shown that 64-fold less material is needed in case of
creativecommons.org/licenses/by/ saRNA vaccine development compared with the conventional mRNA to achieve the same
4.0/). result for producing influenza virus antigens [6]. Hence, it also makes the approach less

Int. J. Mol. Sci. 2022, 23, 12884. https://doi.org/10.3390/ijms232112884 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2022, 23, 12884 2 of 18

costly. Meanwhile, mRNA technology is also preclinically tested effectively for protein
replacement therapy in certain health complications such as heart disease [7] and alpha-1
antitrypsin deficiency (AATD) [8]. In addition, it is hypothesized that saRNA can be used
for preventing Alzheimer’s disease (AD) by raising adenosine triphosphate (ATP) levels in
the brain via reducing amyloid beta (Aβ), pyroglutamate-modified Aβ, and cyclophilin-
D [9]. saRNA has great potential to open a new platform in medicine to produce a drug in
simple and inexpensive ways. Therefore, it might alleviate the process and save millions
of lives that are suffering not only from infectious diseases but also from hereditary or
metabolic disorders as well. In case of infectious diseases, for fighting the coronavirus
disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), the use of the saRNA vaccine has also been suggested [10].
Indeed, saRNA vaccine use has already been widely assessed against COVID-19, and
there are a number of preclinical [11] and ongoing clinical studies. Remarkably, Arcturus
Therapeutics is working on saRNA vaccine development for COVID-19, and the current
stage of clinical development is Phase 3 in Vietnam (NCT05012943) [12,13]. saRNA strategy
seems to be promising for protein replacement in certain diseases. This approach should
also be considered for cosmetic uses such as alopecia treatment. In case of androgenetic
alopecia (AGA), it has been proposed that microRNAs (miRNAs) have the ability to inhibit
certain proteins to activate signaling pathways involved in this process. On the other
side, the saRNA approach also has a huge potential to activate certain signaling pathways
implicated in the hair growth process (such as Wnt/β-catenin) by an augmentation of
certain peptides (Wnt ligand, β-catenin, etc.) into dermal papilla cells and initiate hair
growth [14].
Apparently, the mRNA vaccine approach has multiple benefits compared with the
conventional way of vaccine development [2,15]. Indeed, the saRNA approach allows us to
use the advantage to produce even more proteins inside the body when it is needed for a
longer period of time compared with the conventional mRNA technique. Therefore, the
saRNA approach appears to have promising outcomes compared with the other techniques
of vaccines and therapies. However, despite the auspicious results of the mRNA approach,
more preclinical and clinical studies are warranted on mRNA and saRNA-based therapies
for protein replacement. Therefore, here, the need for the initiation of studies directed at
protein replacement therapy based on accurate designing and administration in precise
doses of saRNA constructs is first proposed. This review summarizes the recent and
relevant studies on protein replacement therapy using saRNA technology. Moreover,
in order to facilitate this direction and solve numerous problems that currently exist,
considering treating various health disorders including the rare congenital diseases saRNA
approach seems to be the right target. In addition, the mechanism of action of saRNA, as
well as current challenges and limitations of this approach, are also reviewed.

2. Clinical Trials of mRNA Approach for Protein Replacement Therapy

saRNA technology indeed represents a promising approach for advancing the field
of vaccinology and protein replacement therapy for various diseases. The manufacturing
process of the vaccine based on saRNA technology is entirely synthetic. It does not require
a viral culture or any complicated process that is usually related to the manufacturing
process of conventional licensed vaccines [16]. Basically, all the existing evidence about
mRNA vaccine use can be substituted with an saRNA approach that might even overcome
certain difficulties of conventional mRNA application [17]. Indeed, saRNA is currently in
clinical development for SARS-CoV-2 intensively (the trials are registered as NCT05012943;
NCT05037097; ISRCTN17072692, NCT04758962, EudraCT 2020-001646-20). Apart from
clinical trials, there are a number of saRNA approaches that undergo preclinical testing for
various infectious diseases such as influenza, rabies, etc. Besides the infectious diseases,
it can also be used as a powerful tool for cancer treatment as there are studies on the
mRNA vaccine approach for cancer therapy [17–21]. Using the mRNA vaccine for cancer
treatment is a new approach compared to its initial use, which is the prevention of infectious
Int. J. Mol. Sci. 2022, 23, 12884 3 of 18

diseases [22]. Undoubtedly, there will be obstacles that need to be overcome related to
saRNA use for cancer immunotherapy. However, unlike conventional mRNA vaccines,
this way of treatment has greater potential with its self-amplifying function, hence it may
facilitate the developing process for generating the finest mRNA-based therapy against
cancer. Interestingly, there are a number of studies in clinical trials using the mRNA
approach for protein replacement for the treatment of certain diseases (Table 1).

Table 1. Clinical trials that use mRNA technology for protein replacement in various diseases
(accessed in September 2022). T2DM, type 2 diabetes mellitus; OTD, ornithine transcarbamylase
deficiency.

ClinicalTrials.gov Delivery Administration Completion

Condition Sponsor Drug Name Status
Identifier Platform Route Date
Epicardial
Heart failure NCT03370887 AstraZeneca AZD8601 Naked mRNA Phase 2 30 June 2021
injection
Ulcers associated
NCT02935712 AstraZeneca AZD8601 Naked mRNA Intradermal Phase 1 8 January 2018
with T2DM
Propionic In vivo/Lipid
NCT04159103 ModernaTX, Inc. mRNA-3927 Intravenous Phase 1 and 2 6 January 2027
acidemia nano-systems
Methylmalonic In vivo/Lipid
NCT03810690 ModernaTX, Inc. mRNA-3704 Intravenous Withdrawn 18 August 2020
Acidemia nano-systems
In vivo/Lipid
OTD NCT03767270 Translate Bio, Inc. MRT5201 Intravenous Withdrawn July 2022
nano-systems
In vivo/Lipid
Cystic Fibrosis NCT03375047 Translate Bio, Inc. MRT5005 Nebulization Phase 1 and 2 December 2021
nano-systems

3. Major Differences between saRNA & mRNA Technologies

While plasmid DNA has advantages compared to direct protein therapy for protein
substitution, mRNA is superior to the two strategies mentioned [23,24]. Most importantly,
mRNA vaccine therapy saves a lot of time, which is equal to saving millions of lives.
Compared with traditional ways of vaccine preparation, which require big bioreactors, in
case of the mRNA vaccine approach, antigens of interest are autologously produced by the
cell’s machinery itself [2]. The advantages of mRNA are conditioned by several facts. First
of all, mRNA, which is delivered into the host cell, does not need to be transported into the
nucleus as happens in the case of DNA. It is translated into protein in the cytoplasm [25]
that does not alter the physiological state of the cell. This averts any possible interven-
tion in the human genome and makes it a safe and feasible strategy [26]. On the other
hand, saRNA provides advancement for protein replacement [27]. The main differences
between conventional mRNA and saRNA vaccine technologies are as follows: in case of
saRNA, a lower dose of vaccine is required compared with the conventional mRNA-based
immunization strategy. This characteristic is conditioned by the fact that only a small
amount of saRNA is necessary for producing large amounts of antigens in the body. For
example, Vogel et al. assessed the difference between the effects of mRNA and saRNA
vaccines against influenza virus in BALB/c mice and revealed the protective efficiency of
both approaches; however, the same level of protection was observed via 80 mg mRNA
and 1.25 mg saRNA [6]. Furthermore, if comparing the doses of the currently available two
SARS-CoV-2 mRNA vaccines by Moderna (mRNA-1273) and BioNTech/Pfizer (BNT162b2)
that are 100 µg and 30 µg, respectively [2], in a clinical trial (Phase 3) on saRNA vac-
cine (ARCT-154) against COVID-19 by Arcturus Therapeutics, only 5 µg saRNA are used
(NCT05012943). In addition, the saRNA vaccine has long-lasting efficiency, comparatively
fewer side effects, and is more cost-effective per dose. The length of the two types of RNA
also differs—saRNA construct is longer (~10,000 nt) unlike mRNA (~2000 nt) [28]. Accord-
ing to the abovementioned, the application of saRNA approach seems to be rational not
only for vaccine development against infections but also for protein replacement therapy
(Figure 1).
Int.
Int.J.J.Mol.
Mol.Sci.
Sci.2022,
2022,23,
23,12884
12884 4 4ofof18
18

Figure 1. Protein production via mRNA and saRNA approaches. UTR, untranslated region; GOI, gene
Figure 1. Protein production via mRNA and saRNA approaches. UTR, untranslated region; GOI,
of interest; ORF, open reading frame; nsP1-4, non-structural protein 1-4; sgPr, subgenomic promoter.
gene of interest; ORF, open reading frame; nsP1-4, non-structural protein 1-4; sgPr, subgenomic
promoter.
4. saRNA—Mechanism of Action
The saRNA vector is a positive-strand RNA (+saRNA) containing the genes coding for
4. saRNA—Mechanism of Action
the viral replicase and the gene of interest (GOI) downstream of a subgenomic promoter
TheThe
(sgPr). saRNA vector
replicon is a positive-strand
is based on the alphavirus RNAnsPs. (+saRNA)
There are containing
three types theofgenes coding
alpha-viruses
for the viral replicase and the gene of interest (GOI)
that are used in the saRNA approach: Venezuelan Equine Encephalitis Virus—VEEVdownstream of a subgenomic pro-
moter (sgPr). The replicon is based on the alphavirus nsPs.
(commonly used), Semliki Forest Virus—SFV, and Sindbis Virus. By deleting the viral There are three types of alpha-
viruses
structuralthatproteins,
are usedthe in RNA
the saRNAcannotapproach:
produce an Venezuelan
infectious Equine Encephalitis
virus. After Virus—
being delivered
VEEV
to the cytoplasm, the nsPs form an RNA-dependent RNA polymerase (RDRP). Eachthe
(commonly used), Semliki Forest Virus—SFV, and Sindbis Virus. By deleting of
viral structural
the nsPs plays proteins,
a role in thethe RNA cannotofproduce
formation the RDRP: an infectious virus. After
nsP1 is required 0 capping
for 5being deliv-of
ered
viraltoRNA.
the cytoplasm, the nsPs and
nsP2 has helicase formprotease
an RNA-dependent
activity. OnRNA the onepolymerase
hand, it(RDRP).
unwinds Each
the
of
RNAthe duplex
nsPs plays a role
during in the formation
replication, while, on of the
theother
RDRP: hand,nsP1itiscleaves
required thefor 5′ cappinginto
polyprotein of
viral RNA. nsP2 has helicase and protease activity. On the one hand,
individual nsPs [29]. nsP3 plays an essential role in the interactions between viral and host it unwinds the RNA
duplex
proteinsduring replication,
[30]. nsP4 is an RDRP while,
andon thefirst
is the othernsPhand,
which it is
cleaves
cleaved the polyprotein
from into indi-
the polyprotein [29].
vidual
As saRNA nsPscontains
[29]. nsP3the plays
replicon an of essential role innsPs,
alphaviruses’ the interactions between viralinand
they can be self-amplified thehost
host
proteins
cell; thus,[30]. nsP4
a huge is an RDRP
number of desiredand proteins
is the first
arensP which is
produced cleaved
[31,32]. fromreplicates
RDRP the polyprotein
both the
entire
[29]. AsRNA
saRNAstrand and sgRNA
contains [32]. This
the replicon RNA replication
of alphaviruses’ leads
nsPs, theyto acan
higher and long-lasting
be self-amplified in
antigen
the host expression
cell; thus, acompared
huge number to theofnon-replicating
desired proteins mRNA [9,33]. The
are produced mechanism
[31,32]. RDRPofrepli-how
saRNA
cates bothworks after the
the entire RNA delivery
strand is and based
sgRNAon the
[32].following:
This RNAsaRNA enters
replication the to
leads cells where
a higher
and long-lasting antigen expression compared to the non-replicating mRNA [9,33]. Thea
the replicase can be directly translated, being able to use saRNA as a template to make
complementary
mechanism of how negative
saRNA works(−
saRNA saRNA)
after strand. Replicase
the delivery is based on canthe use this −saRNA
alsofollowing: saRNA
as a template
enters the cellsto makethe
where more +saRNA,
replicase can allowing
be directly itstranslated,
self-amplification.
being able On tothe
useother
saRNA hand,
as
areplicase
templatecan to recognize the sgPr in the negative strand
make a complementary saRNAfrom which strand.
(−saRNA) a subgenomic
Replicase mRNA can
(+sgRNA)
also use thisof−saRNA
positiveaspolarity
a template is synthesized.
to make moresgRNA +saRNA, canallowing
be translated to produce the
its self-amplification.
desired protein at very high levels, which will be secreted
On the other hand, replicase can recognize the sgPr in the negative strand from which if having a corresponding signala
peptide [31,34,35]. The schematic illustration is given in
subgenomic mRNA (+sgRNA) of positive polarity is synthesized. sgRNA can be trans- Figure 2.
lated After the delivery
to produce the desiredto the host at
protein cells,
verysaRNA as well
high levels, whichas thewilldouble-stranded
be secreted if having RNA
aintermediates
corresponding produced during [31,34,35].
signal peptide the self-replication [36] stimulate
The schematic illustrationan innate
is givenimmune
in Figuresystem
2.
as they are recognized as non-self-molecules [37]. This recognition leads to the secretion
of interferon (IFN) leading to impeding the successful translation of saRNA. First, pattern
recognition receptors (PRRs) are stimulated. According to the cellular location, saRNA
can activate the retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated
protein 5 (MDA5), protein kinase R (PKR), 20 -50 -oligoadenylate synthetase (OAS), and
possibly activate other pathways too that are present in the cytosol [3]. On the other hand,
via endosomal sensing, saRNA and its replication intermediates can induce the activation
of toll-like receptors (TLRs) which are present in the endosomes [3]. The saRNA sensing
 Int. J. Mol. Sci. 2022, 23, 12884 5 of 18

leads to the production of type I IFN and other cytokines [38] that ultimately induce the
maturation of dendritic cells (DCs), activation of T helper cells, and T cell-dependent B
cells [37]. For escaping cytosolic and endosomal sensing of in vitro transcribed mRNA
and, thus, avoiding the undesirable innate immune response, nucleoside modification can
be performed as developed by Kariko and coworkers [39]. However, the same cannot be
completed in case of saRNA as its replication is based on the host-cell factors, making the
addition of modified nucleosides not reasonable as these modifications would vanish after
the first round of amplification. To overcome this obstacle, Blakney et al. have proposed
the potential strategy of diminishing the outcome of type I IFN activation via including the
ORF of innate inhibiting proteins (IIPs) directly in saRNA construct. For that, they have
Int. J. Mol. Sci. 2022, 23, 12884 used the same mechanism that allows the RNA viruses escape innate immune recognition.
5 of 18
The library of saRNA constructs encoding IIPs was screened, and the study demonstrated
that IIPs enhance the saRNA expression [40].

Figure 2. saRNA
Figure 2. saRNAmechanism
mechanism of action. 1. saRNA
of action. enters
1. saRNA the the
enters cells; 2. Replicase
cells; is translated
2. Replicase which
is translated which
usesuses
saRNA as a as
saRNA template to make
a template a complementary
to make negative
a complementary saRNAsaRNA
negative (-saRNA) strand;strand;
(-saRNA) 3. Self-am-
3. Self-
plification takes place:
amplification replicase
takes place: also uses
replicase also this
uses-saRNA as a as
this -saRNA template
a templateto make more
to make more+saRNA;
positive4.saRNA
In
addition, replicase can recognize the sgPr in the negative strand from which a sgRNA
(+saRNA); 4. In addition, replicase can recognize the sgPr in the negative strand from which a of positive
polarity (+sgRNA) is synthesized; 5. sgRNA is then translated into desired antigen or protein at very
sgRNA of positive polarity (+sgRNA) is synthesized; 5. sgRNA is then translated into desired antigen
high levels; 6. Protein is released from the cell. RDRP, RNA-dependent RNA polymerase; GOI, gene
or protein at very high levels; 6. Protein is released from the cell. RDRP, RNA-dependent RNA
of interest; saRNA, self-amplifying RNA.
polymerase; GOI, gene of interest; saRNA, self-amplifying RNA.

5.After the delivery

Potential to theof
Application host cells, for
saRNA saRNA as well as the
Non-Infectious double-stranded
Health Disorders RNA inter-
mediates produced during the self-replication [36] stimulate an innate immune system as
In terms of infectious diseases, the COVID-19 outbreak already showed us how
they are recognized as non-self-molecules [37]. This recognition leads to the secretion of
important preparedness is for future pandemics, therefore, along with the conventional
interferon (IFN) leading to impeding the successful translation of saRNA. First, pattern
mRNA approach, saRNA indeed represents one of the main targets for quick design and
recognition receptors (PRRs) are stimulated. According to the cellular location, saRNA
development of vaccines for any outbreaks in the future. It is already evidenced that the
can activate the retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associ-
mRNA vaccine works effectively against the COVID-19 pandemic; hence, research on
ated protein 5 (MDA5), protein kinase R (PKR), 2′-5′-oligoadenylate synthetase (OAS), and
possibly activate other pathways too that are present in the cytosol [3]. On the other hand,
via endosomal sensing, saRNA and its replication intermediates can induce the activation
of toll-like receptors (TLRs) which are present in the endosomes [3]. The saRNA sensing
leads to the production of type I IFN and other cytokines [38] that ultimately induce the
Int. J. Mol. Sci. 2022, 23, 12884 6 of 18

advancing the mRNA approach has also started. There are a number of clinical trials based
on mRNA technology for protein replacement. However, it would be more rational to
use the RNA technology for protein replacement, which would result in the production of
more proteins with a lower dose while eliciting a long-lasting effect. More importantly, the
administration of a lower dose is associated with reduced side effects and the approach
itself is cost-effective. However, it is also noteworthy that there is no saRNA-based drug
approved in clinics so far, although the promising results are demonstrated by Arcturus
Therapeutics and they currently are in clinical trial Phase 3 for saRNA vaccines against
COVID-19. Currently, most of the scientific attention is paid to infectious diseases, while
there are a number of other diseases that deserve a proper fight and should not be forgotten.
Since the start of the current pandemic, fighting infectious diseases became the main target
for researchers and more efforts are put in place against SARS-CoV-2 and other infectious
diseases (Table 2), while other health disorders should not be forgotten as well.

Table 2. List of the interventional clinical trials of self-amplifying RNA as a vaccine (accessed in
September 2022). COVID-19, coronavirus disease 2019.

Sponsors and Estimated NCT

Condition Location Status Start Date
Collaborators Enrollment Number/Phase
Vinbiocare
Biotechnology Joint
Active, not NCT05012943
COVID-19 Vietnam Stock Company 19,400 August 2021
recruiting Phase 2/3
Arcturus
Therapeutics, Inc.
NCT05227001
Influenza United States Pfizer 468 Recruiting February 2022
Phase 1
United States, Arcturus NCT05037097
COVID-19 72 Recruiting September 2021
Singapore Therapeutics, Inc. Phase 1/2
MRC/UVRI and
NCT04934111
COVID-19 Uganda LSHTM Uganda 42 Recruiting June 2021
Phase 1
Research Unit
NCT05370040
COVID-19 South Africa ImmunityBio, Inc. 180 Recruiting May 2022
Phase 1/2
United States, Arcturus NCT04668339
COVID-19 600 Terminated December 2020
Singapore Therapeutics, Inc. Phase 2
HDT Bio
Kaiser Permanente
Rainier Clinical NCT05132907
COVID-19 United States 63 Recruiting November 2021
Research Center Phase 1
C3 Research
Associates
February NCT04758962
COVID-19 United States GlaxoSmithKline 10 Completed
2021 Phase 1

Therapeutic in vitro transcribed-saRNA can be used for encoding the downregulated

proteins that are responsible for diseases. These genomic defects and other diseases include
Fabry disease, which is associated with the deficiency of alpha-galactosidase A, propionic
acidemia, which is manifested with the deficiency of an enzyme propionyl-CoA carboxy-
lase, hemophilia B, which is a condition of blood coagulation defects and is caused by the
deficit of coagulation factor IX, ornithine transcarbamylase (OTC) deficiency, cystic fibrosis
caused by the defect of cystic fibrosis transmembrane conductance regulator, diabetes
mellitus (DM) caused by the deficiency of insulin, etc. Moreover, the first preclinical study
conducted in 1992 has already demonstrated the positive effect of mRNA application as
a protein replacement. In the first study, a temporary reversal of diabetes insipidus (DI)
took place in mice that were injected with the mRNA of vasopressin in the hypothala-
mus [41]. Interestingly, saRNA has also been suggested for preventing AD to raise ATP
levels in the brain by reducing Aβ [9]. Notably, DM (both type 1 and type 2) remains an
unsolvable global health disorder that still has insulin therapy as the main treatment. DM
is an extremely common metabolic disorder that is characterized by abnormally elevated
blood glucose levels. There are two main types of DM—type 1 DM (T1DM) and type
Int. J. Mol. Sci. 2022, 23, 12884 7 of 18

2 DM (T2DM). In T1DM, insulin is not produced by the pancreatic cells as the immune
system destroys the insulin-producing β cells, while, in T2DM, either insulin secretion is
impaired, or resistance to peripheral actions of insulin occurs and the body does not react to
insulin [42]. The reasons for the development of T1DM include genes, and environmental
factors like viruses while T2DM can be conditioned by obesity, physical inactivity, stress,
etc. [43,44], and, as a result of T1DM and T2DM, hyperglycemia takes place. DM, on the
other hand, increases the risk of many other diseases such as chronic kidney diseases,
heart diseases, mental health, etc. [42,45]. Unlike DM, DI is a rare disease conditioned
via the endocrine condition affecting approximately 0.004% of the world population. In
this disease, the levels of the antidiuretic hormone vasopressin, which regulate the water
and salts in the body, are downregulated and induce the consumption of extremely high
amounts of water [46,47]. As the thirst is dramatically increased, the water intake reaches
up to 20 L/day [48]. Currently, the only treatment is the use of the synthetic hormone
desmopressin as a replacement for the deficient anti-diuretic hormone [48]. In order to
overcome these health complications, the saRNA approach is hypothesized to replace
insulin injections or induce vasopressin synthesis in the body by inserting the appropriate
encoding gene into the saRNA construct and allowing the body to produce the necessary
protein for long-lasting effects. Obviously, there will be difficulties to accomplish this
hypothesis; however, this theory seems promising. An et al. have used mRNA-based
technology with the delivery of biodegradable LNPs for protein replacement in mouse
models of methylmalonic acidemia and demonstrated remarkably improved survival of the
mouse models without manifesting liver toxicity [49]. Apart from that, AATD represents
a hereditary disease that also might be a noteworthy target for saRNA-based therapeu-
tics. AATD affects the liver and the lungs, and it is the main cause of chronic obstructive
pulmonary disease (COPD) emphysema.
Besides the abovementioned, the saRNA approach might be considered for cosmetic
uses such as alopecia treatment. In case of AGA, it has been proposed that miRNAs
have the ability to inhibit certain proteins to activate signaling pathways involved in
this process [14]. On the other side, the saRNA approach also has a huge potential to
activate certain signaling pathways implicated in the hair growth process (such as Wnt/β-
catenin) by an augmentation of certain peptides (Wnt ligand, β-catenin, etc.) into dermal
papilla cells and initiate hair growth. Therefore, the current study suggests designing and
developing saRNA approaches for protein replacement therapy that might become a new
milestone for the treatment of numerous diseases.
All the abovementioned information supports the concept of saRNA application for
protein replacement. In order to achieve the desired outcomes, the right strategy is the key.
Here, we discuss the steps for saRNA research design in vivo. The first step is to design and
synthesize the plasmid vector containing the GOI. The sequence of sgPr varies and depends
on the selected alphavirus for the saRNA approach [50]. After the plasmid is transformed
into the competent cells and amplified, the following step is to extract it and validate the
expression of desired protein via in vitro analysis. In case the expression of the protein
is satisfactory, the plasmid should be linearized and in vitro transcribed. Consequently,
the obtained RNA construct will be packed with lipid nanoparticles (LNPs) [51] or other
delivery formulations [52,53] and, eventually, administered to experimental animals with
the appropriate administration route [54]. This is conditioned by the research purposes. For
example, in case of immunization against the influenza virus, intradermal (I.D.), intramus-
cular (I.M.), or subcutaneous (S.C.) routes [55,56] can be used according to the designed
experiment. Ultimately, the evaluation of in vivo analysis can be carried out. In vitro tran-
scription (IVT) of the large-sized saRNA, however, is a challenge as typical IVT protocols
are developed for smaller-size RNAs. IVT reaction comprises the following: linearized
plasmid DNA with T7 promoter, nucleoside triphosphates (NTPs), ribonuclease inhibitor,
magnesium ions as cofactor for T7 polymerase, pH buffer providing the optimal condition
for the reaction. There are studies on making improvements in saRNA IVT. Samnuan et al.
have demonstrated that several important reaction components can aid in obtaining a high
 be carried out. In vitro transcription (IVT) of the large-sized saRNA, however, is a chal-
lenge as typical IVT protocols are developed for smaller-size RNAs. IVT reaction com-
prises the following: linearized plasmid DNA with T7 promoter, nucleoside triphosphates
(NTPs), ribonuclease inhibitor, magnesium ions as cofactor for T7 polymerase, pH buffer
providing the optimal condition for the reaction. There are studies on making improve-
Int. J. Mol. Sci. 2022, 23, 12884 8 of 18
ments in saRNA IVT. Samnuan et al. have demonstrated that several important reaction
components can aid in obtaining a high yield of functional saRNA during the IVT. For
example, saRNA with a size ranging from 1.8 to 12.4 kb can be produced via the properly
yield of balance
adjusted functional saRNA during
of magnesium ionsthe
andIVT. For example,
NTPs, the use ofsaRNA
acetatewith
ions,aetc.
size[57].
ranging from
The line
1.8 to 12.4 kb can be produced via the properly adjusted balance of magnesium
of studies demonstrates that the IVT of saRNA is performed successfully [58–60]. The cap- ions and
ping can be performed during the IVT as well as post-transcriptionally. Remarkably, forof
NTPs, the use of acetate ions, etc. [57]. The line of studies demonstrates that the IVT
saRNAcapping,
saRNA is performed
the use successfully
of the AU cap[58–60]. The capping
is suggested as it iscan be performed
advantageous during the
compared IVT
to the
as well as post-transcriptionally. Remarkably, for saRNA capping, the
AG cap, as the AU cap preserves the authentic alphavirus 5′ end, resulting in efficient use of the AU cap
is suggested as it is advantageous
capping and high yield 0[38]. compared to the AG cap, as the AU cap preserves the
authentic alphavirus
The schematic 5 end, resulting
illustration in efficient capping
of the experimental workflow andfor
high
theyield [38].
development of the
The schematic illustration of the experimental workflow
saRNA approach for protein replacement therapeutic use is given in Figure 3. for the development of the
saRNA approach for protein replacement therapeutic use is given in Figure 3.

Figure 3. Schematic illustration of experimental design of saRNA treatment for protein deficiency
Figure 3. Schematic
disorders. illustrationprotein;
nsP, non-structural of experimental design
GOI, gene of saRNA
of interest; treatment
IVT, in for protein deficiency
vitro transcription.
disorders. nsP, non-structural protein; GOI, gene of interest; IVT, in vitro transcription.
6. saRNA for Single-Gene Disorders—Special Focus on AATD
6. saRNA forfrom
Apart Single-Gene Disorders—Special
its application Focus
as an immunization on AATD
approach for infectious diseases, mRNA
technology
Apart fromis thought to be a next-generation
its application as an immunizationtherapeutic strategyfor
approach for infectious
single-genediseases,
disorders
that aretechnology
mRNA caused by is thethought
DNA changeto be ainnext-generation
a single gene [26]; hence, it is
therapeutic the factfor
strategy that these disor-
single-gene
ders are inherited
disorders that is by
that are caused thethe
reason
DNAfor morbidity
change and premature
in a single gene [26];mortality
hence, it is inthe
families that
fact that
are affected with these diseases [61]. Common single-gene disorders
these disorders are inherited that is the reason for morbidity and premature mortality in include hemophilia
B—a bleeding
families that aredisorder
affectedthat withisthese
characterized
diseases by[61].the deficiency
Common of clottingdisorders
single-gene factor IX, include
meaning
that, in thisB—a
hemophilia condition,
bleeding blood coagulation
disorder does not takeby
that is characterized place normally [62].
the deficiency Another
of clotting com-
factor
mon
IX, single-gene
meaning that, in disorder is a rare blood
this condition, lipoprotein metabolism
coagulation does not condition lecithin-cholesterol
take place normally [62].
acyltransferase
Another commondeficiency
single-gene (LCATD)
disorder which results
is a rare in severely
lipoprotein reduced high-density
metabolism lipopro-
condition lecithin-
cholesterol acyltransferase deficiency (LCATD) which results in severely reduced failure,
tein (HDL) cholesterol and the clinical manifestation by corneal opacities, renal high-
and hemolytic
density lipoprotein anemia
(HDL) [26]. Interestingly,
cholesterol and the AATD also
clinical belongs to single-gene
manifestation disorders
by corneal opacities,
and failure,
renal is a typeandof lung and liver
hemolytic disease
anemia [26].conditioned
Interestingly, by AATD
hereditary alsometabolic
belongs tocondition
single-gene [63].
The mutation arises in the gene SERPINA1, which encodes the
disorders and is a type of lung and liver disease conditioned by hereditary metabolic con-serine protease inhibitor
(serpin)—AAT.
dition As a result,
[63]. The mutation thein
arises serum levels
the gene of AAT decrease
SERPINA1, well below
which encodes thethe normal
serine range.
protease
AAT is a protein that protects the lungs from the damage caused
inhibitor (serpin)—AAT. As a result, the serum levels of AAT decrease well below the by inflammation that can
lead to emphysema. AAT controls chemical reactions via inhibiting
normal range. AAT is a protein that protects the lungs from the damage caused by inflam- the activity of certain
enzymes
mation thatincluding
can leadneutrophil
to emphysema. elastaseAAT(NE) in the lungs.
controls chemicalIn case of insufficient
reactions circulating
via inhibiting the
levels of AAT, the levels of NE are drastically elevated, and this causes
activity of certain enzymes including neutrophil elastase (NE) in the lungs. In case of in- over-inhibition of a
peptide called elastin—the main component of alveoli. Elastic tissue disruption is a major
sufficient circulating levels of AAT, the levels of NE are drastically elevated, and this
factor of pressure loss in alveoli and destruction of alveolar membranes, which results
in the reduction of airflow and hence reduction of oxygen, further damage to the lungs,
and, eventually, loss of function. Therefore, when the AAT is not going to the bloodstream
and hence, in the lungs, circulating levels are decreased and the accumulation takes place
within the liver where its synthesis occurs, lungs, as well as the liver become vulnerable.
On the one hand, it cannot regulate the activity of elastase that makes NE free to attack the
lungs and leads to emphysema, COPD, chronic bronchitis, etc., while on the other hand,
it may lead to cirrhosis and increased risk of liver cancer. AATD is not a rare disease but
is rarely diagnosed, although it can be simply and accurately detected via a blood test
that measures the AAT levels. Notably, very often, underdiagnosis takes place as, when
Int. J. Mol. Sci. 2022, 23, 12884 9 of 18

the patient has COPD or emphysema, AATD is often not considered while sometimes
it is overdiagnosed [64]. Indeed, a misdiagnosis-related burden is a great challenge for
understanding and managing the disease [65]. Often, many people who have AATD are
misdiagnosed with asthma [66]. It happens as these two health conditions share the symp-
toms. In addition, people who have AATD respond well to asthma medicines. AATD was
first described by Laurell and Erikson. Their case studies revealed the patients with very
pronounced AATD, which was the new type of dysproteinemia in 1963 [67]. The healthy
range of AAT serum levels is considered 20–53 µmol/L (100–220 mg/dL) [68], while people
with serum AAT levels below 11 µmol/L (80 mg/dL) are at risk of developing COPD or
liver diseases [26]. The main therapy for AATD is AAT pooled from the sera of the donors
or the liver and lung transplantation that are very costly. Therefore, saRNA-based peptide
replacement therapy is proposed for AATD. In addition, the preclinical study of modified
systemic mRNA therapy for AATD already showed promising results [8].

7. saRNA for Cancer Immunotherapy

Recently, the development of cancer vaccines is the main focus of cancer research.
Indeed, a number of methods are being developed to elicit a strong anti-tumor immune
response. For designing the vaccine for cancer, the identification of tumor-associated
antigens is of great importance. In addition, knowing the tumor microenvironment that
allows the progression of the tumor and escaping from the host’s immune system is
essential for the design of a cancer vaccine [69,70]. Cancer research focuses on several types
of vaccines—antigen-based cancer vaccines that are based on tumor-associated proteins
with different delivery systems [71]; peptide-based vaccines; cellular vaccines that are
based on dendritic cells loaded with tumor antigens, allogenic tumor cell lines, autologous
cancer cells; and nucleic acid vaccines including DNA and RNA-based approaches [72].
Considering its advantages including efficacy, safety, time, and cost-effectiveness, the
mRNA vaccine strategy merits the most attention. mRNA vaccine against infectious
diseases has already proved its benefits of application, while it is still in the clinical trials for
cancer therapy [73]. On the other hand, saRNA technology has not been approved by the
Food and Drug Administration (FDA) until now, but its promising results from clinical trials
and superior characteristics compared with the conventional mRNA approach give rise to
a new era of vaccinology [74]. Using the saRNA vaccine for cancer immunotherapy can
achieve the robust expression of antigens in a short time. After the saRNA injection, desired
candidate antigens are translated and presented via a major histocompatibility complex
(MHC) I and II that induces a cellular and humoral immune response and results in tumor
eradication [74]. Remarkably, there are a number of mRNAs in different phases of clinical
development, e.g., mRNA-4157, mRNA-5671, and mRNA-4359 designed by Moderna as
personalized cancer vaccine (PCV), Kirsten rat sarcoma (KRAS), and checkpoint vaccine,
respectively [75]. According to the superiority of saRNA technique, basically, it can be used
for every cancer vaccine development where a conventional mRNA approach is applied.

8. Delivery Systems for saRNA Therapeutics

The major challenge in developing saRNA vaccines is a delivery system that can carry
saRNA to the target cells [4]. Moreover, the large structure of saRNA which can reach
15,000 nucleotides in length, makes cellular uptake more difficult. Until now, nanopar-
ticles and nanoemulsions are used as delivery platforms in saRNA studies. These de-
livery platforms protect saRNA from degradation and facilitate uptake by the target
cells [4]. There are degradable and non-degradable polymeric nanoparticles. For example,
polyethylenimine (PEI), which is a cationic non-degradable polymer, has successfully de-
livered saRNA expressing influenza hemagglutinin of H1N1 A/Puerto Rico/8/1934 and
A/California/7/2009 strains. Moreover, the saRNA-PEI induced remarkable protection
in mice. A 64-fold less dose was enough to induce the same protection as non-replicating
mRNA [6]. Apparently, the carrier systems that are applicable for mRNA or small in-
terfering RNA (siRNA) delivery are not quite suitable for saRNA carriage as they are
Int. J. Mol. Sci. 2022, 23, 12884 10 of 18

intended for the delivery of smaller size molecules—~2000–5000 nt (mRNA) and ~20 nt
(siRNA), respectively. Blakney et al. have engineered a linear, cationic bioreducible polymer
pABOL for influenza hemagglutinin saRNA delivery in mice. The study demonstrated that
intramuscular and intradermal injection of saRNA with this delivery system resulted in
enhanced protein expression. The transfection with a higher molecular weight of pABOL
was efficient and protected the mice from challenge [76]. Another study has consolidated
the abovementioned results in terms of high protein expression but also showed that LNP
formulations are more immunogenic [77]. Indeed, LNPs are currently the most prevalent
non-viral delivery for saRNAs as they require a minimal quantity of saRNA to evoke
a strong immune response [78,79]. These systems have come to the fore of therapeutic
platforms by pharmaceutical companies, especially since the successful development of
SARS-CoV-2 mRNA LNP vaccines mRNA-1273 and BNT162b2 that are currently used
worldwide [80,81].
The application of LNPs for the delivery of genetic materials originated from the
development of liposomal drug carrier systems for small molecules. The principle of
LNPs is to encapsulate the nucleic acid molecule into the system containing lipids that
are organized in a bilayer form to deliver it into the target cells [21,82]. LNPs used for
currently available COVID-19 vaccines contain four components with distinct functions:
the ionizable lipids with the positive charge bind to the negatively charged backbone of the
mRNA and enable RNA complexation; PEGylated lipids allow the stabilization and the
longer systemic circulation of the particle via reducing antibody association and clearance
by phagocytes; molecules of cholesterol and phospholipids contribute to the structure of
the particle– allowing for packing the mRNA cargo into the LNPs. After the mRNA is
packed in these components, it is protected from the destructive enzymes, transported, and
successfully unloaded into the target cells to be translated into proteins [83]. These LNPs
contain about 100 mRNA molecules per LNP [84] and are 80–100 nm in diameter [85,86].
Blakney et al. have formulated cationic LNPs and compared the LNP formulations with
ionizable and cationic lipids with a diameter of 100–200 nm packing human immunodefi-
ciency virus (HIV)-1 Env gp140 saRNA on the interior or adsorbed on the exterior of the
particle. The results showed that both formulations induced similar antibody responses
against the antigen. Moreover, LNPs containing cationic lipids protected saRNAs from
nuclease degradation even when they were present on the surface [87]. These and the
number of other preclinical [18,19,87,88] and clinical studies [12,78,89] demonstrate the
feasibility and flexibility of LNPs for the saRNA approach [59,78,87,90–97]. On the other
hand, the challenge of LNP formulations developed by Moderna and BioNTech/Pfizer is
the requirement of the cold chain storage −20 ◦ C and −70 ◦ C, respectively. In response to
this, Gerhardt et al. have studied the effectiveness of delivery of Zika saRNA with ther-
mostable nonstructured lipid carrier (NLC). The RNA-NLC complex demonstrated stability
for more than 8 months and more than 21 months at room temperature and 4 ◦ C, respec-
tively [98]. Alternatively, cationic nano-emulsions (CNEs) are water-in-oil emulsions that
are also applicable for saRNA delivery [4,99–103]. They have already been demonstrated
to elicit immune responses against influenza in mice and ferrets [100], HIV-1 in rhesus
macaques [104], rabies in mice [105], etc. [4]. Besides the LNPs, polymeric nanoparticles,
and nanoemulsions, adjuvanted saRNAs should also be considered. Manara et al. have
studied the effect of saRNA encoding nucleoprotein of influenza A virus adjuvanted with a
murine granulocyte–macrophage colony-stimulating factor (GM-CSF). The results demon-
strated the induced nucleoprotein-specific immune response and enhanced recruitment of
antigen-presenting cells (APCs) at the injection site [106]. Remarkably, immunization with
naked saRNA has also been studied [107–110] and demonstrated efficiency against Zika
virus, HIV-1, and influenza [4]. Electroporation has been successfully used to increase the
delivery of saRNA after intramuscular administration with the broad immune response
against HIV envelope protein in Balb/c mice [111] and moderate immune responses against
Zika virus and protected the IFNAR1−/− mice from the viral challenge [110].
 phage colony-stimulating factor (GM-CSF). The results demonstrated the induced nucleopro-
tein-specific immune response and enhanced recruitment of antigen-presenting cells (APCs)
at the injection site [106]. Remarkably, immunization with naked saRNA has also been studied
[107–110] and demonstrated efficiency against Zika virus, HIV-1, and influenza [4]. Electro-
poration has been successfully used to increase the delivery of saRNA after intramuscular ad-
Int. J. Mol. Sci. 2022, 23, 12884 ministration with the broad immune response against HIV envelope protein in Balb/c11mice of 18
[111] and moderate immune responses against Zika virus and protected the IFNAR1−/− mice
from the viral challenge [110].
A number
A number of of other
other approaches
approaches forfor saRNA
saRNA delivery
delivery have
have also
also been
been studied.
studied. These
These
delivery systems include chitosan nanogel alginate (Chitosan NGA)
delivery systems include chitosan nanogel alginate (Chitosan NGA) [112], dendrimer [112], dendrimer
[113],
[113],
or or modified
modified dendrimer
dendrimer nanoparticle
nanoparticle (MDNP) (MDNP) [113,114],
[113,114], cell-penetrating
cell-penetrating peptidespeptides
(CPP)
(CPP)
PEI PEIcationic
[115], [115], cationic lipids polyplexes
lipids [116], [116], polyplexes [117], natural
[117], natural lipopolyplexes
lipopolyplexes [118], [118],
NLC NLC[119],
[119],inorganic
lipid lipid inorganic nanoparticle
nanoparticle emulsion
emulsion (LION(LION emulsion)
emulsion) [120], lipoplex
[120], lipoplex [121], cati-
[121], cationic ad-
onic adjuvant
juvant formulation
formulation (CAF)virus-like
(CAF) [122], [122], virus-like particles
particles [123],gun
[123], gene gene[92,124],
gun [92,124], and
and other
other techniques
techniques [40,125–127].
[40,125–127].

9.
9. Challenges
Challenges
As
Asfor
forall
allof
ofthe
thenovel
novelapproaches,
approaches,the thesaRNA
saRNA technique
technique is is
also characterized
also characterized byby
certain
cer-
limitations that need
tain limitations to be to
that need overcome. The main
be overcome. The obstacles are theare
main obstacles lacktheoflack
studies regarding
of studies re-
the immunogenicity of RDRP complex, limited clinical data, the
garding the immunogenicity of RDRP complex, limited clinical data, the necessity of necessity of prime-boost
administration [5], large size
prime-boost administration [5],and complex
large size andsequence
complex of the saRNA
sequence of themolecule [33], the
saRNA molecule
challenge of efficient
[33], the challenge of delivery, the largerthe
efficient delivery, size of the
larger delivery
size system, shorter
of the delivery system,half-life
shorter [128],
half-
the
lifeproneness
[128], the to degradation
proneness by nucleasesby[5],
to degradation and induction
nucleases [5], andof induction
strong innate host immune
of strong innate
responses
host immune [31,37]. There is
responses no approved
[31,37]. There issaRNA-based
no approved vaccinesaRNA-basedtill now, whichtill
vaccine is also
now,a
burden. Nevertheless, advantages prevail over challenges. Remarkably,
which is also a burden. Nevertheless, advantages prevail over challenges. Remarkably, the challenge
of
thethe large size
challenge of saRNA
of the can
large size of be partially
saRNA can beovercome
partiallyvia splitting
overcome viathe wholethe
splitting sequence
whole
into nsPs and GOI in separate molecules and the application of trans-amplifying
sequence into nsPs and GOI in separate molecules and the application of trans-amplifying RNA
(taRNA) [32,33]. taRNA has been demonstrated to be as effectively
RNA (taRNA) [32,33]. taRNA has been demonstrated to be as effectively expressed as expressed as saRNA,
pointing
saRNA, out the promising
pointing outcome of
out the promising taRNA application
outcome [109]. The schematic
of taRNA application [109]. The illustration
schematic
of saRNA and
illustration taRNA and
of saRNA comparison is given in Figure
taRNA comparison is given4. in Figure 4.

Figure4.4. Comparison
Figure Comparison of of saRNA
saRNA andand taRNA.
taRNA. (A)
(A) saRNA
saRNA is
is aa single
single large
large RNA
RNA molecule
molecule encoding
encoding
nsP1-4and
nsP1-4 andGOI.
GOI.As
Asa aresult,
result,protein
proteinofof interest
interest is produced
is produced in augmented
in augmented levels.
levels. (B) (B) taRNA
taRNA repre-
represents
sents two RNA molecules encoding GOI and nsP1-4 separately. As a result, protein of interest is
two RNA molecules encoding GOI and nsP1-4 separately. As a result, protein of interest is produced
produced in augmented levels. CSE, conserved sequence elements; nsP1-4, non-structural protein
in augmented levels. CSE, conserved sequence elements; nsP1-4, non-structural protein 1-4; GOI,
1-4; GOI, gene of interest.
gene of interest.

10. Conclusions and Future Perspectives

The current review provides insights into the saRNA approach for protein replacement
therapy. saRNA indeed has a huge potential to display a better RNA approach compared
to a conventional mRNA technique, especially for those disorders which necessitate protein
replacement therapy. Till now, all the clinical studies on saRNA have been performed in
order to develop prevention strategies against infectious diseases (Table 1). This is mainly
conditioned by the outbreak of COVID-19. Indeed, it is crucial for pandemics/epidemics
preparedness to develop vaccines against the existing viruses to save lives. In the meantime,
certain disorders that might be single-gene or non-heritable diseases that require protein
replacement therapy should not be forgotten for the saRNA approach employment. There-
fore, in terms of protein replacement therapy, saRNA certainly seems to exhibit distinctly
Int. J. Mol. Sci. 2022, 23, 12884 12 of 18

advantageous benefits. For protein replacement purposes, the main advantage of the
saRNA is its plausible long-lasting effects, the requirement of a much lower dosage that
reduces the side effects markedly. Moreover, as the saRNAs allow for conferring immune
response at low doses, a single-dose regimen might also be used in the future. However, the
difficulties in this direction are noteworthy. The main challenge is the size of saRNA. The
construct of saRNA appears to have a much larger size than conventional mRNA which
also requires a suitable delivery vehicle for efficient uptake. In order to avoid this difficulty,
taRNA is also proposed that represents two molecules instead of a single large-sized saRNA
molecule. Nevertheless, there are substantially more data on saRNA itself compared to
taRNA till now; thus, the latter approach requires more studies for further development.
Fortunately, along with developing RNA technologies, carrier platforms also continue to
advance, which results in the successful delivery of molecules as large as saRNA [129]. Evi-
dently, the saRNA-based vaccine for COVID-19 (NCT05012943) is in clinical study Phase 3,
and, therefore, it is close to getting approval and becoming the first approved saRNA-based
vaccine. Here, we proposed the use of saRNA technique for protein replacement purposes
for a number of diseases. Proteins including receptors and extracellular proteins can be
generated via saRNA application without the risk of genome integration. This approach
seems to have enormous potential for single-gene disorders, also for hereditary and some
non-heritable diseases. As there is a vast number of pathologies in which certain genes are
down-expressed and the normal function of the body is dysregulated, a new approach is
emergent to overcome these huge obstacles. The proposed protein-replacing technique may
be beneficial to AATD, DM, DI, or other health disorders. Furthermore, in order to exhibit
satisfying results, studies on saRNA application for AD are also suggested [9]. Based on
the promising results of mRNA for designing cancer vaccines, the saRNA approach seems
even more promising owing to its superior characteristics.
There are crucial points to be addressed: determining the precise dosage of saRNA
is essential. It is also important to evaluate the time period of the saRNA replication into
the body. Time alike dosage might be dependent on the certainty of the disorder and the
species of mammals. Multiple studies are required for this reason in the future.
Taken together, saRNA is gaining momentum worldwide due to the number of advan-
tages that makes it superior to the conventional approaches [26]. On the other hand, looking
back and going forward, saRNA can be used similarly but more efficiently than mRNA. A
number of advantages of mRNA-based approaches [130] also indicate the promising future
of saRNA-based protein replacement therapeutics. The simplicity, cost, and time-effective
manufacturing technology of saRNA may allow the quick and successful development of
protein replacement therapeutics.

Author Contributions: Conceptualization, D.P. and Y.H.; data curation, N.R. and D.P.; writing—original
draft preparation, N.R. and D.P.; writing—review and editing, N.R., D.P., C.L., and Y.J.; visualization,
N.R. and D.P.; supervision, P.G.W. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Shenzhen Science and Technology Innovation Program
(Grant No. KQTD20200909113758004).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data are available in the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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