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| Self-amplifying mRNA Vaccines
Top considerations for generating self-amplifying
mRNA vaccines
BY VINAY MENON, PHD
The technology behind mRNA vaccines has been in develop
an immune response in the host. While these vaccines were
contain sequences for the antigen of interest, also known
ment
since the 1990s, but it has recently gained popularity
incredibly effective, this technology typically necessitates
as the subgenome, four non-structural proteins (nSP) that
since the COVID-19 vaccines became the first mRNA vaccines
booster shots and larger dosages to maintain host immunity.
form the replicase, a 5’ cap, untranslated regions (UTRs) at
approved for human use. In the shots developed by Pfizer
A new generation of self-amplifying RNA (saRNA) vac
both the 5’ and 3’ ends, and a 3’ poly(A) tail, resulting in a
BioNTech, Moderna, Johnson & Johnson, and others, patients
cines
aims to solve this problem by carrying the code for
molecule roughly 9 to 12 kilobases in length. Once delivered
receive an mRNA strand that encodes the SARS-CoV-2 spike
a replicase enzyme that amplifies the saRNA within cells.
into cells, the replicase catalyzes the production of many
protein, packaged within a lipid nanoparticle. Once inside cells,
Compared to conventional mRNA vaccines, saRNA vaccines
copies of the original saRNA molecule, which continues to
the mRNA is translated into the spike protein, which triggers
are significantly larger and more complex. They typically
produce the antigen of interest.
3. Delivery systems
To develop effective saRNA vaccines, researchers should consider
the following key aspects.
After researchers transcribe, process, and purify the saRNA molecule, they package it into an
appropriate delivery system. Because of saRNA’s large size and negative charge, delivering naked
saRNA has limited success. Researchers commonly deliver saRNA vaccines in vivo through both
1. saRNA design
viral and non-viral systems. However, delivery through viral replicon particles can trigger anti-vector
immunity (5). Alternative formulations are non-viral delivery systems like polymeric nanoparticles,
The RNA molecule itself is typically derived from the genome of single-stranded RNA viruses.
lipid nanoparticles, or cationic nanoemulsions, which impart a positive charge to protect the saRNA
Researchers most commonly use Venezuelan equine encephalitis virus and other negative strand
from degradation while not triggering immunogenic responses (2). In vivo delivery using non-viral
alphaviruses, although they have also used positive strand flaviviruses, such as West Nile virus
systems has successfully elicited robust host immunity against a wide range of viruses, parasites,
and Dengue virus, and other viral genera for saRNA development. The choice of the viral vector
and cancers with doses as low as 10 nanograms of saRNA.
impacts how the saRNA molecule will interact with the host cell. Researchers typically modify
Some clever innovations in this area have included the development of a temperature-controlla
the viral vector by replacing viral structural genes with genes encoding the antigen of interest to
ble
saRNA that replicates at 33ºC, ideal for intradermal administration. In another study, researchers
prevent the production of infectious viral particles (1).
used a synthetic, bioreducible lipid as the delivery vehicle for a SARS-CoV-2-targeting saRNA
In recent years, researchers have developed several design variations for the saRNA molecule.
vaccine, mitigating the biocompatibility issue surrounding other non-viral delivery systems such
One group identified six nsP mutations that could enhance subgenome expression, while another
as polyethylenimine. Scientists were also able to omit the delivery vehicle entirely by delivering
created a split replicon system, where the replicase and gene of interest are on separate RNA
DNA that encodes the saRNA in what is called a DNA-launched self-amplifying RNA replicon
molecules. Additionally, researchers introduced a new system using trans-amplifying RNA, where
(DREP). As a DNA-based technology, DREP is inherently more stable than naked saRNA, while
a second RNA molecule provides the missing replicase function, which demonstrated similar
producing a robust immune response (6).
efficiency to traditional systems in mouse models (2).
4. Improving saRNA stability and efficien
2. saRNA production
Since the saRNA technology is still in its infancy, many studies for improving stability and
Researchers can synthesize saRNA molecules from a linear DNA template in a cell-free in vitro
transcription and translation efficiency center around modifying the structural elements, such
transcription (IVT) reaction. Several commercial IVT kits are available to produce 0.1 to 8.5 mil
as UTRs, the poly(A) tail, and the 5’ cap. For example, researchers have replaced the 5’ cap
ligrams
of RNA within 30 minutes. Key components in an IVT reaction include a high affini
with an anti-reverse cap analog, a modified version of the traditional 5’ cap, which improved
promoter such as T7, a ribonucleotide triphosphate (rNTP) mix, RNAse inhibitors, pyrophosphatase,
RNA transcription four-fold, while adding
a-
or
ß
-globins to the UTR demonstrated improved
magnesium ions, and dithiothreitol. To protect the mRNA strand from enzymatic degradation in
RNA stability and translation efficiency. Researchers also found that the length of the poly(A)
the cytoplasm, researchers typically encode a 3’ poly(A) tail directly in the linear DNA template
tail (up to 120 residues) is directly correlated with translation efficiency and protein produc
or add it after RNA purification using enzymes like E. coli poly(A) polymerase (3,4). They also add
tion
(7), underscoring the importance of fine-tuning the poly(A) tail to optimize saRNA-based
the 5’ cap, which initiates transcription and protects from nuclease degradation, enzymatically
vaccines and therapies.
using a commercially available vaccinia virus-derived capping enzyme along with guanosine
triphosphate (GTP) and S-adenosylmethionine as a methyl source. These capping reactions are
Promising horizons
highly efficient and proceed in about an hour (3).
Following saRNA production via IVT, the next step is its purification. Due to its large size and
Despite facing challenges related to purification and immunogenic by-products, saRNA vac
polar nature, purifying saRNA molecules is challenging, especially at an industrial scale. For smaller
cines
have shown significant promise as therapeutics for a number of infectious diseases
molecules (= 3 megadaltons), conventional techniques such as size-exclusion chromatography
and cancers. Beyond improving current COVID-19 vaccines, researchers have tested saRNA
(SEC) and high-performance liquid chromatography (HPLC) might suffice. For larger molecules,
vaccines against other global health epidemics, such as human immunodeficiency virus 1
however, researchers typically precipitate the molecule out of cold ethanol with the addition of
(8). The recent approval of the first saRNA vaccine, ARCT-154, in Japan, which could elicit
lithium chloride. Scaling up saRNA purification for applications beyond routine preclinical studies
robust antibody responses at much lower doses compared to conventional mRNA vaccines,
remains an active area of research but could rely on techniques such as tangential flow filtration
highlights the exciting utility and possibilities of this technology for future vaccine and
SEC, affinity chromatography, or ion-exchange chromatography (2).
therapeutic development (9).
REFERENCES
1. Lundstrom, K. Self-amplifying RNA virus vectors for drug delivery. Expert Opinion on Drug Delivery.
5. Liu et al. Advances in saRNA Vaccine Research against Emerging/Re-Emerging Viruses. Vaccines
8. Bloom et al. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 28, 117 – 129 (2021)
1 – 15 (2025)
24, 1142 (2023)
9. Oda et al. Immunogenicity and safety of a booster dose of a self-amplifying RNA COVID-19 vaccine
2. Blakney et al. An Update on Self-Amplifying mRNA Vaccine Development. Vaccines 9, 97-123 (2021)
6. Silva-Pilipich et al. Self-Amplifying RNA: A Second Revolution of mRNA Vaccines against COVID-
(ARCT-154) versus BNT162b2 mRNA COVID-19 vaccine: a double-blind, multicentre, randomised,
19. Vaccines 12, 318 (2024)
controlled, phase 3, non-inferiority trial. Lancet 24, 351-360 (2024).
3. Brito et al. Self-Amplifying mRNA Vaccines. Advances in Genetics 89, 179 – 233 (2015)
7. Wadhwa et al. Opportunities and Challenges in the Delivery of mRNA-Based Vaccines.
4. Pourseif et al. Self-amplifying mRNA vaccines: Mode of action, design, development and
Pharmaceutics 12, 102 – 129 (2020)
optimization. Drug Discovery Today 27, 103341 (2022)
