ArticlePDF AvailableLiterature Review

Abstract

The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell reprograming has become increasingly popular in research and medicine. This method can be used in vitro (transfected in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA. In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the functionality of this synthetic transient genetic vector.
NCCR RNA & DiseAse CHIMIA 2019, 73, No. 5 391
doi:10.2533/chimia.2019.391 Chimia 73 (2019) 391–394 © Swiss Chemical Society
*Correspondence: PD Dr. S. Pascoloab
E-mail: steve.pascolo@usz.ch
aUniversity Hospital of Zürich; Department of Dermatology, Gloriastrasse 31, CH-8091 Zürich; bFaculty of Medicine, University of Zurich, Zurich; cCenter for Integrative
Genomics, University of Lausanne, Genopode, CH-1015 Lausanne; dDepartment of Dermatology and Allergy, University Hospital, LMU Munich, Munich, Germany
Design of in vitro Transcribed mRNA
Vectors for Research and Therapy
Marina Tusupab, Lars E. Frenchabd, Mara De Matosc, David Gatfieldc, Thomas Kundigab,
and Steve Pascolo*ab
Abstract: The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell repro-
graming has become increasingly popular in research and medicine. This method can be used in vitro (transfect-
ed in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA
to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or
immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading
frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA.
In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the func-
tionality of this synthetic transient genetic vector.
Keywords: Cap · Gene therapy · Globin UTR · in vitro transcribed mRNA · ivt RNA · Vaccination
Steve Pascolo studied biology/biochemistry at Ecole Normale
Supérieure (Paris, France) and obtained his PhD in 1998 from
the Pasteur Institute / University Paris 6, where he created an im-
munologically humanised mouse model called HHD (mice defi-
cient in the endogenous H-2 class I molecules and transgenic for
a human HLA class I molecule). As a post-doctoral fellow at the
University of Tübingen, Germany, he focused on immunisation
methods using WT and HHD mice, testing different vaccination
formats (e.g. peptides, plasmids, and ivt mRNAs), and discov-
ered that RNA is a ‘danger signal’ that primes innate and adaptive
immune responses. With the goal of translating this vaccination
method to humans, Steve Pascolo co-founded CureVac GmbH in
2000, implemented the first large scale and GMP production of
ivt mRNAs worldwide and initiated the first clinical studies in
which patients with cancer received direct ivt mRNA injections.
In 2006, Steve Pascolo moved to the University Hospital of Zurich
(Switzerland) and continued to develop innovative immunochem-
otherapies against cancer (vaccination, immunomodulation, and
chemotherapies). In 2017, the University of Zurich supported the
creation of an mRNA platform. This platform allows researchers
and clinicians to have access to high quality ivt mRNAs produced
on demand for their research and pre-clinical projects.
1. Introduction
Vaccination (induction of an immune response against a pro-
tein) and gene therapy (expression of a therapeutic protein) can be
performed using genetic vectors, such as plasmid DNA, in vitro
transcribed mRNA (ivt mRNA) or recombinant microorganisms
(modified viruses or bacteria). Because of this technology’s su-
perlative features (safety, efficacy, steerable immunostimulatory
properties, and ease of production and preservation), ivt mRNA
is emerging as the optimal vector format.[1,2] We have initiated
the clinical use of directly injected ivt mRNAs through academic
(University of Tübingen, Germany) and industrial (CureVac AG,
Germany) co-developments. As a key milestone, we implemented
the first large scale GMP (good manufacturing practice) produc-
tion of an ivt mRNA (CureVac AG, Germany) worldwide[3] and
led the first clinical studies in which patients with cancer were
injected with ivt mRNAs.[3–6] This pioneering work has revealed
for the first time that an ivt mRNA can be produced at a large scale
in injectable quality, and that its direct administration in humans is
safe and leads to the expected pharmacodynamics (expression of
the encoded protein[6] and development of an immune response[7]).
The most recently developed ivt mRNA-based approaches were
reported to induce very strong immune responses in patients with
cancer.[8,9] Key to this notably high efficacy are the formulation
of the ivt mRNA, the site of injection, as well as the structure of
ivt mRNA.[10] The present review will focus on this latter aspect.
2. Structure of the ivt mRNA
2.1 The 5' Cap
Eukaryotic mRNAs contain a 5' Cap called Cap 0 that con-
sists of a methyl-7 guanine nucleotide connected to the RNA via
a 5' to 5' triphosphate linkage. If the first base of the mRNA is
methylated at the 2' position, the Cap is called Cap 1. It is the
optimal structure to stabilise the mRNA and recruit the eukaryotic
initiation factor 4E (eIF4E) that binds to eIF4G and facilitates the
subsequent assembly of the eIF4F complex. Alternatives to the
5' Cap for the recruitment of the translation machinery are Cap-
independent viral sequences, such as the 5' untranslated region of
the tobacco etch virus,[11] or internal ribosomal entry site (IRES)
sequences.[12] However, such sequences at the 5' end of an ivt mR-
NA are not sufficient to achieve high levels of protein expression
after transfection of the RNA in cells. Thus, the production of a
5' capped ivt mRNA is recommended. Two methods for capping
have been reported: (i) co-transcriptional capping, where a Cap
analogue (or an anti-reverse Cap analogue ‘ARCA’ eventually
optimised[13] or a Cap dinucleotide ‘CleanCap®’ from Trilink) is
present in the transcription reaction, and (ii) post-transcriptional
capping, where the transcribed mRNA is capped using enzymes
such as the vaccinia virus capping enzyme. In all cases, a Cap
392 CHIMIA 2019, 73, No. 5 NCCR RNA & DiseAse
able for codon optimisation that incorporate several features (e.g.
predicted secondary RNA structure and avoiding consecutive rep-
etition of identical codons). Thus, we advise codon optimisation
for the production of mRNAs encoding non-mammalian proteins
(optionally using two independent companies/software packag-
es and comparing the functionality of the resulting ivt mRNAs),
while, a priori, the wild-type coding sequence should be used to
produce mRNAs coding mammalian proteins (a subsequent test
of codon-optimised forms can be envisioned).
2.4 The 3'UTR and Poly-A Tail
In ivt mRNAs, the most frequently used 3'UTR is the globin
3'UTR.[14,17–20] An optimised version of this 3'UTR is a tandem
repeat of the sequence,[21] which contains then approximately 300
bases. Because the presence of repeated sequences may hamper
the synthesis of the synthetic DNA that is used as a matrix for tran-
scription, Orlandini von Niessen et al. recently reported the iden-
tification of new stabilising 3'UTRs.[22] The researchers started
from a library of long-lived mRNAs (derived from dendritic cells
treated with actinomycin D for a few hours to block transcription)
and cloned fragments of those reverse transcribed molecules at
the 3' end of an ORF encoding destabilised eGFP. This library
was then transcribed into mRNAs, transfected into dendritic cells,
RNA was extracted at different time points, and reverse transcrip-
tion followed by PCR amplification was performed. Using an
iterative process of transfection/RNA extraction/amplification/
transcription, optimal 3'UTR sequences were identified. The best
sequences corresponded to UTRs from mRNAs with long half-
lives (this result validated the approach). The combination of two
of those sequences in the 3'UTR, one from mitochondrially en-
coded 12S rRNA ‘mtRNR1’ and one from the amino-terminal
enhancer of split mRNA ‘AES’, produced an ivt mRNA with the
highest expression (the combined sequences produced a UTR of
less than 300 bases). This mRNA’s expression in vitro (several cell
types) and in vivo (mice) was greater than two-fold higher than
the expression of the usual ivt mRNA with a tandem repeat of the
globin 3'UTR. The poly-A tail can be encoded by the DNA matrix
directly such that the ivt mRNA contains a 3' stretch of at least
90 A bases. Importantly, extra C, G or U bases at the 3' end of the
poly-A sequence should be avoided.[21] If the DNA template does
not encode this 3' poly-A sequence, an incubation of the ivt mR-
NA in a reaction with a poly-A polymerase post-transcriptionally
generates the poly-A tail.
2.5 Purification Methods
The RNA polymerases that are usually used to produce ivt
mRNAs (T3, T7, and SP6) are very processive and specific for their
promoters. However, ivt mRNAs visualised on a gel[3] or with an
Agilent analysis may show some aberrant shorter or longer tran-
scripts. Those transcripts might be derived from cryptic promoters
or cryptic terminators of transcription (e.g. highly structured RNA
regions). Therefore, we have implemented a dedicated HPLC-
based purification method for large-scale GMP production.[6] This
method allows identifying and isolating the mRNA with the cor-
rect size, hence removing potential by-products that are longer or
shorter. In addition, the RNA polymerase may generate hairpins
at the 3' end by synthesizing a complementary RNA sequence. A
newly available T7 RNA polymerase that is optimised to generate
RNA at 50 °C (Hi-T7™ RNA Polymerase from NEB Biolabs)
was reported to reduce dsRNA by-product formation. We also
noticed that this Hi-T7™ can produce full-length mRNA from
hard-to-transcribe DNA templates (e.g. containing a GC rich 5'
region, Fig. 2). Meanwhile, for research-grade ivt mRNAs, usual-
ly the classical production using wild type polymerases and sim-
ple purification of ivt mRNAs using selective precipitation (LiCl
that precipitates only long RNA molecules, not oligonucleotides,
DNA or proteins) or columns (silica membranes) is sufficient. A
0 or Cap 1 can be generated (the latter requires the utilisation
of a Cap 2'-O-methyltransferase in addition to the vaccinia virus
capping enzyme in the context of enzymatic capping). Based on
our experience, the most functional ivt mRNA was produced by
co-transcriptional capping using the CleanCap® Reagent AG (3'
OMe) from Trilink (m7(3'OMeG)(5')ppp(5')(2'OMeA)pG). It has
the dual advantages of being an anti-reverse Cap 1 and of not
competing with GTP to initiate transcription. Thus, the GTP con-
centration in the transcription reaction can be maintained at high
levels without reducing the percentage of capped mRNA (using
usual Cap mononucleotide analogues, the GTP concentration
must be reduced to favour initiation by Cap and not by G). The
method’s disadvantage is that the DNA template must contain a
modified T7 promoter, where the C that is used to base pair with
the first nucleotide of the ivt mRNA (usually a G) is a T (allowing
incorporation of the A of the CleanCap® Reagent AG). Thus, new
dedicated DNA constructs must be produced to use the CleanCap®
Reagent AG.
2.2 The 5'UTR
Located between the 5' Cap and the first ATG codon (Fig.
1), the 5'UTR sequence must provide a good Kozak consensus
sequence that favours initiation of the translation process at the
expected ATG. An 80 base sequence from the globin 5'UTR is
frequently used.[14] We recently reported the superior efficacy of
a short sequence of 40 nucleotides corresponding to an aptamer
that binds eIF4G.[15] The rationale was that in some cell types, the
availability of eIF4E is a limiting factor for translation; therefore,
the direct recruitment of eIF4G to the 5'UTR would likely pro-
mote translation. We now typically produce ivt mRNAs with this
5' aptamer sequence, as follows: CapAG - G A C T C A C T A T T
T G T T T T C G C G C C C A G T T G C A A A A A G TG TC G
C C A C C A T G (italicized bases represent the eIF4G aptamer
and underlined bases represent the start codon).
2.3 Coding Sequence
Depending on the species, codon usage can vary and reflects
the relative abundance of tRNAs; efficient translation relies on
optimal codons that correspond to abundant tRNA species.[16]
Rare codons in an open reading frame hinder the production of
full-length proteins. These codons may also decrease the stability
of the mRNA, as slow-moving ribosomes may trigger mRNA de-
cay. Therefore, ivt mRNAs are typically produced from synthetic
DNA templates that have been ‘codon optimised’. However, the
natural sequence of a gene may have evolved to favour proper
protein production, particularly folding, by controlling the speed
of translation; rare codons are used to slow translation between
structural domains allowing time for each domain to fold proper-
ly. Therefore, the replacement of suboptimal codons with optimal
codons may increase protein misfolding. Indeed, we observed
that some codon-optimised ivt mRNAs encoding secreted pro-
teins were less functional than ivt mRNAs with the WT sequences
(data not shown). Notably, multiple software packages are avail-
AAAAAAAAAA
AUGSTOP
40S
β
β
CAP
Apt
Figure 1
Fig. 1. Schematic representation of optimised ivt mRNA. A 5' Cap (op-
timally a methyl-7 guanine nucleotide connected via a 5' to 5' triphos-
phate linkage to a 2'-O-methyl modied residue: Cap1) is followed by a
5'UTR (optimally an aptamer that binds eIF4G: ‘Apt’) and an open read-
ing frame (optimally a codon optimised sequence depicted in yellow) be-
tween a start (AUG) and a stop codon. The 3' end of the mRNA can con-
tain a stabilising 3'UTR (for example a tandem repeat of the beta-globin
3'UTR ‘β’) and a poly-A tail (ideally of more than 90 residues).
NCCR RNA & DiseAse CHIMIA 2019, 73, No. 5 393
genicity (for a completely 1-methyl-pseudoUridine -substituted
mRNA, Fig. 3B). Thus, LiCl does not exert any deleterious effects
on ivt mRNAs that impair their subsequent applications, and it is
a simple, inexpensive and scalable method for purifying mRNAs
from in vitro transcription reactions.
3. Conclusions
The production of ivt mRNAs for research and clinical in-
terventions is easy and inexpensive. However, because this tech-
nology has many facets, including the structure of the ivt mRNA
and its formulation, substantial expertise is helpful for producing
optimal ivt mRNAs. To this end, the implementation of a central-
ised ivt mRNA platform, which we have used at the University of
Zurich (https://www.cancer.uzh.ch/en/Research/mRNA-Platform.
html), represents a significant asset for researchers and clinicians
aiming to use ivt mRNAs for research or clinical interventions.
Investigator-driven clinical trials have been initiated based on
findings from academic studies, in particular for vaccinations
that we have pioneered[3–5,19,20,23,24] and for modified T-cells.[25,26]
Meanwhile, several dedicated biotechnology companies have
initiated human studies, including BioNTech AG, CureVac AG,
eTheRNA, Moderna Inc. Ongoing company-sponsored clinical
trials include vaccinations (e.g. vaccines against solid cancers and
viruses, such as rabies, HPV16 and HIV), and non-immunogenic
protein expression (e.g. ivt mRNAs encoding cytokines that are
injected into the tumour). Furthermore, the versatility of the ivt
mRNA format allows the design of individualized therapeutics;
mRNA vaccines encoding mutated peptides corresponding to pa-
tient-specific cancer mutations have been designed, produced and
administered.[9] In addition, ivt mRNAs can be combined with
comparison of both purification methods (Fig. 3) revealed that
they are equivalent and present similar efficacy in terms of the
amount of recovered RNA, OD260/OD280 ratio (ca. 2), of the
observed transfection efficiencies (Fig. 3A) and reduced immuno-
Luciferaseexpression
Luciferaseactivity
Pseudo LiCl
Ps
eudo MinElute
No RNA
0
1000000
2.0
1006
3.0
1006
PBMCs
HEK
CT26
Figure 3
AB
Immunostimulation
pg/ml
Pseudo LiCl
Ps
eudo MinElute
U LiCl
No RNA
Protamine
-10000
0
10000
20000
30000
Interferon-alpha
TNF-alpha
Fig. 3. Effects of the purication method on the functionality of ivt mRNAs. A completely 1-methyl-pseudoUridine-substituted synthetic mRNA
encoding luciferase was puried either with LiCl precipitation (2.5 M LiCl nal concentration at –20 °C for one hour, followed by centrifugation and
washing of the pellet with 75% ethanol), ‘Pseudo LiCl’ or MinElute columns (‘Pseudo MinElute’) from Qiagen. A, The ivt mRNA was formulated with
MessengerMax (ThermoFisher) according to the manufacturer’s recommendations and added to 100 µL of cells (in each well: 200 ng of mRNA per
1 million fresh human peripheral blood mononuclear cells (PBMCs'') or 10 ng of mRNA per 100,000 human HEK cells or mouse CT26 tumour cells)
in RPMI containing 10% foetal bovine serum and antibiotics. Cells that were not treated with the RNA were used as a negative control (‘No RNA’).
Twenty hours later, the luciferase activity in each well was recorded following the addition of 25 µL of BrightGlo (Promega) and measurement on a
GloMax instrument (Promega). The results in A represent the average luciferase activity ± the standard deviation (from triplicates). They show that
the two purication methods provide mRNAs with comparable expression capacities. B, The ivt mRNA was formulated with protamine to gener-
ate 130 nm nanoparticles[29,30] and 10 µg of the preparation (5 µg mRNA plus 5 µg Protamine) were added to 200 µL of cells (1 million fresh human
peripheral blood mononuclear cells) in RPMI containing 10% foetal bovine serum and antibiotics. Twenty hours later, the interferon-alpha and TNF-
alpha levels in the supernatants were measured using ELISAs (from Mabtech and Bioscience, respectively). A luciferase mRNA generated with the
four canonical bases ACGU and precipitated with LiCl (‘U LiCl’) was used as a positive control. As negative controls, cells without added RNA (‘No
RNA’) or treated with only 5 µg Protamine (‘Protamine’) were used. The results in B represent the average cytokine concentrations ± standard devia-
tions (from triplicates). They show that the two purications method give 1-methyl-pseudoUridine substituted RNA with very reduced immunogenic
capacities even at the high concentration used here in the cell culture (25 µg/mL nal).
2000
1500
1000
500
T7 Hi-T7
Figure 2
Fig. 2. Transcription of difcult DNA matrices. Transcriptions were
performed using three PCR matrices with a GC content of 78% in the
rst 161 bases. The produced mRNAs were analysed on a denaturing
Formaldehyde MOPS agarose gel. The expected size was over ca.
2500 bases. The canonical T7 RNA polymerase (‘T7’) at 37 °C produces
abundant shorter transcripts while the Hi-T7™ RNA Polymerase (‘Hi-T7’)
at 50 °C generates mostly full-length transcripts.
394 CHIMIA 2019, 73, No. 5 NCCR RNA & DiseAse
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“block a protein or pathway”.[27] Meanwhile, drugs have been
used to enhance the efficacy of ivt mRNA-based therapies.
[28]
Thus, ivt mRNAs are a highly versatile tool for research and clin-
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apies in the future.
Conict of Interest
The authors have no conicts of interest to disclose.
Acknowledgements
This work was supported by the University Research Priority
Project (URPP) ‘Translational Cancer Research’ and by the Monique
Dormonville de la Cour Stiftung.
Received: March 1, 2019
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12152.2001.
... The Cap 1 structure is achieved by the methylation of the mRNA first nucleotide at the ribose 2 0 -O position. Both caps can be added during in vitro mRNA transcription using a synthetic cap analogue [27] or the proprietary Cap dinucleotide CleanCap Ò [28]. Another capping approach uses a posttranscription enzymatic reaction based on the vaccinia capping system [29]. ...
... These regions strongly affect translation efficiency as the sequences used are involved in the translation machinery recognition, recruitment, and mRNA trafficking. Strategies to modulate the innate immune response, such as the introduction of unnatural nucleosides (NTPs), and to improve translation efficiency, by using codon optimisation, are also commonly used in mRNA production [27,28]. ...
... Nevertheless, a cost analysis should be performed to compare the costs of the one-step and two-step production options [93]. Alternatively, co-transcriptional capping can be performed using CleanCap Ò Reagent AG [28]. Although this method does not compete with GTP and delivers a Cap 1 construct, it requires the use of templates with a modified T7 promoter. ...
Article
Vaccines are one of the most important tools in public health and play an important role in infectious diseases control. Owing to its precision, safe profile and flexible manufacturing, mRNA vaccines are reaching the stoplight as a new alternative to conventional vaccines. In fact, mRNA vaccines were the technology of choice for many companies to combat the Covid-19 pandemic, and it was the first technology to be approved in both United States and in Europe Union as a prophylactic treatment. Additionally, mRNA vaccines are being studied in the clinic to treat a number of diseases including cancer, HIV, influenza and even genetic disorders. The increased demand for mRNA vaccines requires a technology platform and cost-effective manufacturing process with a well-defined product characterisation. Large scale production of mRNA vaccines consists in a 1 or 2-step in vitro reaction followed by a purification platform with multiple steps that can include Dnase digestion, precipitation, chromatography or tangential flow filtration. In this review we describe the current state-of-art of mRNA vaccines, focusing on the challenges and bottlenecks of manufacturing that need to be addressed to turn this new vaccination technology into an effective, fast and cost-effective response to emerging health crises.
... The Cap 1 structure is generated by the methylation of the mRNA first nucleotide at the ribose 2 ′ -O position. Both caps can be added during in vitro mRNA transcription using a synthetic cap analogue or the proprietary Cap dinucleotide CleanCap® (Sahin, Kariko and Tureci, 2014;Tusup et al., 2019). The Cap modification improves the translation initiation by recruiting translation initiation factors, protects the synthetic mRNA against exonuclease degradation and avoids an innate immunity overactivation response ( Fig. 3) (Linares-Fernandez, Lacroix, Exposito and Verrier, 2020). ...
... These regulatory regions strongly affect the translational efficiency by recognition and recruitment of translational machinery and mRNA trafficking. Other critical modifications such as incorporation of unnatural nucleosides (NTPs) for modulating the innate immune response and codon optimization for improving translational efficiency are also commonly used in mRNA production ( Fig. 3) (Sahin et al., 2014;Tusup et al., 2019). Purification of the mRNA from double-stranded contaminants as well as the incorporation of chemically modified nucleosides like pseudouridine,1-methyl-pseudouridine, and 5 methyl cytosine (5 mC) are crucial for the potency of the vaccine and is essential for efficient translation and reduced immune activation (Kariko et al., 2008;Maruggi et al., 2019) (Fig. 3). ...
Article
The functional and structural versatility of Ribonucleic acids (RNAs) makes them ideal candidates for overcoming the limitations imposed by small molecule-based drugs. Hence, RNA-based biopharmaceuticals such as messenger RNA (mRNA) vaccines, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNA mimics, anti-miRNA oligonucleotides (AMOs), aptamers, riboswitches, and CRISPR-Cas9 are emerging as vital tools for the treatment and prophylaxis of many infectious diseases. Some of the major challenges to overcome in the area of RNA-based therapeutics have been the instability of single-stranded RNAs, delivery to the diseased cell, and immunogenicity. However, recent advancements in the delivery systems of in vitro transcribed mRNA and chemical modifications for protection against nucleases and reducing the toxicity of RNA have facilitated the entry of several exogenous RNAs into clinical trials. In this review, we provide an overview of RNA-based vaccines and therapeutics, their production, delivery, current advancements, and future translational potential in treating infectious diseases.
... These results reinforce the potential of Bayesian optimization to be applied on the optimization of (bio)chemical reactions for industrial applications. (Tusup et al., 2019). Two β-globin tandem repeats are used as a 3′-UTR, followed by a 120 bp poly-A, segmented with a 6 bp spacer (Trepotec et al., 2019). ...
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mRNA vaccines are a new alternative to conventional vaccines with a prominent role in infectious disease control. These vaccines are produced in in vitro transcription (IVT) reactions, catalyzed by RNA polymerase in cascade reactions. To ensure an efficient and cost‐effective manufacturing process, essential for a large‐scale production and effective vaccine supply chain, the IVT reaction needs to be optimised. IVT is a complex reaction that contains a large number of variables that can affect its outcome. Traditional optimisation methods rely on classic Design of Experiments (DoE) methods, which are time‐consuming and can present human bias or based on simplified assumptions. In this contribution, we propose the use of Machine Learning approaches to perform a data‐driven optimization of an mRNA IVT reaction. A Bayesian optimization method and model interpretability techniques were used to automate experiment design, providing a feedback loop. IVT reaction conditions were found under 60 optimization runs that produced 12 g.L−1 in solely two hours. The results obtained outperform published industry standards and data reported in literature in terms of both achievable reaction yield and reduction of production time. Furthermore, this shows the potential of Bayesian optimization as a cost‐effective optimisation tool within (bio)chemical applications. This article is protected by copyright. All rights reserved.
... Cloning vectors of plasmid origin come with incorporated promoters facilitating in-vitro transcription, upstream of multiple cloning sites (MCS). Furthermore, poly A tail sequence, 5 and 3 UTR, and linearization restriction sites are also included at the pDNA template [56]. Currently, the in vitro transcription method using a highly processive bacteriophage RNA single-subunit polymerase (e.g., T7, T3, and SP6) has been widely established, as it is considered a cost-effective and easily scalable mRNA manufacturing process [38,57]. ...
Article
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In the quest for a formidable weapon against the SARS-CoV-2 pandemic, mRNA therapeutics have stolen the spotlight. mRNA vaccines are a prime example of the benefits of mRNA approaches towards a broad array of clinical entities and druggable targets. Amongst these benefits is the rapid cycle “from design to production” of an mRNA product compared to their peptide counterparts, the mutability of the production line should another target be chosen, the side-stepping of safety issues posed by DNA therapeutics being permanently integrated into the transfected cell’s genome and the controlled precision over the translated peptides. Furthermore, mRNA applications are versatile: apart from vaccines it can be used as a replacement therapy, even to create chimeric antigen receptor T-cells or reprogram somatic cells. Still, the sudden global demand for mRNA has highlighted the shortcomings in its industrial production as well as its formulation, efficacy and applicability. Continuous, smart mRNA manufacturing 4.0 technologies have been recently proposed to address such challenges. In this work, we examine the lab and upscaled production of mRNA therapeutics, the mRNA modifications proposed that increase its efficacy and lower its immunogenicity, the vectors available for delivery and the stability considerations concerning long-term storage.
... In co-transcriptional capping, the mRNA is capped by the polymerase through the addition of a cap analogue. Post-transcriptional capping is performed as a second enzymatic reaction, using the vaccinia capping enzyme [22][23][24]. ...
Article
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The global coronavirus pandemic continues to restrict public life worldwide. An effective means of limiting the pandemic is vaccination. Messenger ribonucleic acid (mRNA) vaccines currently available on the market have proven to be a well-tolerated and effective class of vaccine against coronavirus type 2 (CoV2). Accordingly, demand is presently outstripping mRNA vaccine production. One way to increase productivity is to switch from the currently performed batch to continuous in vitro transcription, which has proven to be a crucial material-consuming step. In this article, a physico-chemical model of in vitro mRNA transcription in a tubular reactor is presented and compared to classical batch and continuous in vitro transcription in a stirred tank. The three models are validated based on a distinct and quantitative validation workflow. Statistically significant parameters are identified as part of the parameter determination concept. Monte Carlo simulations showed that the model is precise, with a deviation of less than 1%. The advantages of continuous production are pointed out compared to batchwise in vitro transcription by optimization of the space–time yield. Improvements of a factor of 56 (0.011 µM/min) in the case of the continuously stirred tank reactor (CSTR) and 68 (0.013 µM/min) in the case of the plug flow reactor (PFR) were found.
... These are of great interest for anticancer and antiviral therapies. On the other hand, larger particles activate monocytes and production of TNF-α [270]. ...
Article
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Macromolecular biomolecules are currently dethroning classical small molecule therapeutics because of their improved targeting and delivery properties. Protamine-a small polycationic peptides represent a promising candidate. In nature, it binds and protects DNA against degradation during spermatogenesis due to electrostatic interactions between the negatively charged DNA-phosphate backbone and the positively charged protamine. Researchers are mimicking this technique to develop innovative nanopharmaceutical drug delivery systems, incorporating protamine as a carrier for biologically active components such as DNA or RNA. The first part of this review highlights ongoing investigations in the field of protamine-associated nanotechnology, discussing the self-assembling manufacturing process and nanoparticle engineering. Immune-modulating properties of protamine are those that lead to the second key part, which is protamine in novel vaccine technologies. Protamine-based RNA delivery systems in vaccines (some belong to the new class of mRNA-vaccines) against infectious disease and their use in cancer treatment are reviewed, and we provide an update on the current state of latest developments with protamine as pharmaceutical excipient for vaccines.
... These are of great interest for anticancer and antiviral therapies. On the other hand, larger particles activate monocytes and production of TNF-α [270]. ...
Preprint
In our modern days, macromolecular biomolecules are dethroning classical small molecule therapeutics because of improved targeting and delivery properties. Protamine – a small polycationic peptide represents such a promising candidate. In nature, it binds and protects DNA against degradation during spermatogenesis due to electrostatic interaction between the negatively charged DNA-Phosphate backbone and the positively charged protamine. Researchers are mimicking this technique in order to develop innovative nanopharmaceutical drug delivery systems, incorporating protamine as carrier for biologically active components such as DNA or RNA. The first key part of this review highlights ongoing investigation in the field of protamine-associated nanotechnology, discussing the self-assembling manufacturing process and nanoparticle engineering. Immune-modulating properties of protamine are referred which lead to the second key part protamine in novel vaccine technologies. Protamine-based RNA delivery systems in vaccines (some of them belong to the new class of mRNA-vaccines) against infectious disease and their use in cancer treatment are reviewed and an update on the current state of latest developments with protamine as pharmaceutical excipient for vaccines is given.
... The 5 0 end consisted of a CleanCap TM (Trilink) followed by an eIF4G aptamer as the 5 0 untranslated region (Tusup and Pascolo, 2018) and by a codon-optimized firefly luciferase open reading frame. The 3 0 end consisted of a tandem repeat of the mouse beta-globin 3 0 UTR and a poly-A tail (Tusup et al., 2019). The transcription was made in the presence of the four canonical bases (alanine(A), cysteine (C), glycine (G) , and uracil (U)) to obtain immunostimulatory RNA and in the presence of pseudo-uridine instead of uridine to obtain immuno-silent mRNA used for luciferase expression assays. ...
Article
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Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can be used for the stabilization and delivery of nucleic acids (antisense, small interfering RNA (siRNA), immunostimulatory nucleic acids, plasmid DNA, or messenger RNA) and is therefore included in several compositions that are in clinical development. Notably, when mixed with RNA, protamine spontaneously generates particles in the size range of 20–1000 nm depending on the formulation conditions (concentration of the reagents, ratio, and presence of salts). These particles are being used for vaccination and immuno-stimulation. Several grades of protamine are available, and we compared them in the context of complex formation with messenger RNA (mRNA). We found that the different available protamine preparations largely vary in their composition and capacity to transfect mRNA. Our data point to the source of protamine as an important parameter for the production of therapeutic protamine-based complexes.
Article
Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.
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mRNA therapeutics have become the focus of molecular medicine research. Various mRNA applications have reached major milestones at high speed in the immuno-oncology field. This can be attributed to the knowledge that mRNA is one of nature’s core building blocks carrying important information and can be considered as a powerful vector for delivery of therapeutic proteins to the patient. For a long time, the major focus in the use of in vitro transcribed mRNA was on development of cancer vaccines, using mRNA encoding tumor antigens to modify dendritic cells ex vivo. However, the versatility of mRNA and its many advantages have paved the path beyond this application. In addition, due to smart design of both the structural properties of the mRNA molecule as well as pharmaceutical formulations that improve its in vivo stability and selective targeting, the therapeutic potential of mRNA can be considered as endless. As a consequence, many novel immunotherapeutic strategies focus on the use of mRNA beyond its use as the source of tumor antigens. This review aims to summarize the state-of-the-art on these applications and to provide a rationale for their clinical application.
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Background: in vitro-transcribed messenger RNA (ivt mRNA) is a safe genetic vector that can be used for vaccination and gene therapy. We investigated the impact of the 3’ untranslated region (UTR) and of modified nucleotides on the functionality of ivt mRNA in vitro as well as in vivo. We confirmed that a 3’ UTR consisting of a tandem repeat of beta-globin 3’ UTR enhances the expression of ivt mRNA in non-transformed cells in vitro and in liver in vivo. In addition, we compared ivt mRNA made with the four canonical bases (“ACGU”), with uridine replaced by N1-methyl pseudouridine (“ACGPseudo”) or with both uridine and cytidine replaced by N1-methyl pseudouridine and 5-methylcytidine, respectively (“A5mCGPseudo”). We report that the “ACGU” and “ACGPseudo” ivt mRNA have superior functionality in tumour cells. However, in immune cells and in vivo, the “ACGPseudo” composition allows better expression of the synthetic mRNA. The additional substitution of cytidine with 5-methylcytidine in “A5mCGPseudo” ivt mRNA is deleterious for expression. The “ACGU” ivt mRNA induces cytokines in immune cells, while both “ACGPseudo” and “A5mCGPseudo” do not. We conclude that an ivt mRNA containing A, C, G and N1-methyl pseudouridine residues and having a 3’ tandem repeat of the beta-globin UTR is the optimal design of this vector for gene therapy.
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Nanoparticles of defined size can be easily obtained by simply mixing Protamine, a pharmaceutical drug that is used to neutralize heparin after surgery, and RNA in the form of oligonucleotides or messenger RNA. Depending on the concentrations of the two reagents and their salt contents, homogenous nanoparticles with a mean diameter of 50 to more than 1000 nm can spontaneously be generated. RNA is a danger signal because it is an agonist of for example TLR-3, -7, and -8; therefore, Protamine–RNA nanoparticles are immunostimulating. We and others have shown in vitro that nanoparticle size and interferon-alpha production by human peripheral blood mononuclear cells (PBMCs) are inversely correlated. Conversely, nanoparticle size and TNF-alpha production by PBMCs are positively correlated (Rettig et al., Blood 115:4533–4541, 2010). Particles of less than 450 nm are most frequently used for research and clinical applications because they are very stable, remain polydispersed and induce interferon-alpha proteins, which are a natural antiviral and anticancer protein family with 12 members in humans. Herein, we describe a method to generate 130 nm nanoparticles as well as some of their physical and biological characteristics.
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Poly ribonucleic acid (RNA) is the only polymer capable to recapitulate all processes of life: containment of genetic information, enzymatic activities and capacity to create defined 3D structures. Since it has a remarkable chemical stability (at neutral or acidic pH) and can be modified to enhance/reduce particular features (e.g., stability in biological RNase containing milieus or recognition by immune sensors), it is a particularly versatile and ideal active pharmaceutical ingredient. However, the utilization of RNA as a gene vehicle (messenger RNA, mRNA) for therapy has only recently been exploited. Within this scope, mRNA-based vaccines designed to trigger anti-cancer, anti-virus or anti-allergy immune responses have been developed. Modifications of mRNA vectors and implementation of adequate formulations have allowed to turn this natural superlative biological molecule into a safe active pharmaceutical ingredient that can virtually address any medical need including vaccination or immunotherapy. This is the newest great message delivered by this messenger.
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The aim of this phase I/II nonrandomized trial was to assess feasibility, safety as well as immunological and clinical responses of a mRNA-based vaccination in patients with stage IV renal cell cancer using granulocyte-macrophage colony stimulating factor (GM-CSF) as adjuvant. Intradermal injections of in vitro transcribed naked mRNA, which was generated using plasmids coding for the tumor-associated antigens mucin 1(MUC1), carcinoembryonic (CEA), human epidermal growth factor receptor 2 (Her-2/neu), telomerase, survivin, and melanoma-associated antigen 1 (MAGE-A1) were performed in 30 enrolled patients. In the first 14 patients (cohort A) vaccinations were administered on days 0, 14, 28, and 42 (20 µg/antigen) while in the consecutive 16 patients (cohort B) an intensified protocol consisting of injections at days 0-3, 7-10, 28, and 42 (50 µg/antigen) was used. In both cohorts, after this induction period, vaccinations were repeated monthly until tumor progression analyzed by Response Evaluation Criteria In Solid Tumors criteria (RECIST). Vaccinations were well tolerated with no severe side effects and induced clinical responses [six stable diseases (SD) and one partial response in cohort A and nine SD in cohort B]. In cohort A, 35.7% survived 4 years (median survival 24 months) compared to 31.25% in cohort B (median survival 29 months). Induction of CD4(+) and CD8(+) T cell responses was shown for several tumor-associated antigens (TAA) using interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) and Cr-release assays.
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In mice, injection of messenger RNA (mRNA) coding for tumor-associated antigens can induce antitumor immune responses and therefore offers a broadly applicable immunotherapy approach. We injected intradermally protamine-stabilized mRNAs coding for Melan-A, Tyrosinase, gp100, Mage-A1, Mage-A3, and Survivin in 21 metastatic melanoma patients. In 10 patients keyhole limpet hemocyanin (KLH) was added to the vaccine. Granulocyte macrophage colony-stimulating factor was applied as an adjuvant. Endpoints were toxicity and immune responses. No adverse events more than grade II have been observed. During treatment the frequency of Foxp3+/CD4+ regulatory T cells was significantly decreased upon mRNA vaccination in peripheral blood of the patients in the KLH arm, whereas myeloid suppressor cells (CD11b+HLA-DR lo monocytes) were reduced in the patients not receiving KLH. A reproducible increase of vaccine-directed T cells was observed in 2 of 4 immunologically evaluable patients. One of 7 patients with measurable disease showed a complete response. In conclusion, we show here that direct injection of protamine-protected mRNA is feasible and safe. The significant influence of the treatment on the frequency of immunosuppressive cells, the increase of vaccine-directed T cells upon treatment in a subset of patients together with the demonstration of a complete clinical response encourage further clinical investigation of the protamine-mRNA vaccine.
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RNA is the only molecule known to recapitulate all biochemical functions of life: definition, control and transmission of genetic information, creation of defined three-dimensional structures, enzymatic activities and storage of energy. Because of its versatility and thanks to several recent scientific breakthroughs, RNA became the focus of intense research in molecular medicine at the beginning of the millennium. In particular, mRNA can be seen as a safe and efficient alternative to protein-, recombinant virus- or DNA-based therapies in the field of vaccination. This review summarises the most remarkable advances in this area and presents the advantages and limits of the five different mRNA-based vaccination methods. The paper will present the official, industrial and financial aspects of mRNA-based vaccination that are paving the way for therapeutic and prophylactic drugs with mRNA as the active component.
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Dicistronic reporter plasmids, such as the dual luciferase-containing pR-F plasmid, have been widely used to assay cellular and viral 5' untranslated regions (UTRs) for IRES activity. We found that the pR-F dicistronic reporter containing the 5' UTRs from HIF-1alpha, VEGF, c-myc, XIAP, VEGFR-1, or Egr-1 UTRs all produce the downstream luciferase predominantly as a result of cryptic promoter activity that is activated by the SV40 enhancer elements in the plasmid. RNA transfection experiments using dicistronic or uncapped RNAs, which avoid the complication of cryptic promoter activity, indicate that the HIF-1alpha, VEGF, c-myc, and XIAP UTRs do have some IRES activity, although the activity was much less than that of the viral EMCV IRES. The translation of transfected monocistronic RNAs containing these cellular UTRs was greatly enhanced by the presence of a 5' cap, raising questions as to the strength or mechanism of IRES-mediated translation in these assays.
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Both DNA and mRNA can be used as vehicles for gene therapy. Because the immune system is naturally activated by foreign nucleic acids thanks to the presence of Toll-like Receptors (TLR) in endosomes (TLR3, 7, and 8 detect exogenous RNA, while TLR9 can detect exogenous DNA), the delivery of foreign nucleic acids usually induces an immune response directed against the encoded protein. Many preclinical and clinical studies were performed using DNA-based experimental vaccines. However, no such products are yet approved for the human population. Meanwhile, the naturally transient and cytosolically active mRNA molecules are seen as a possibly safer and more potent alternative to DNA for gene vaccination. Optimized mRNA (improved for codon usage, stability, antigen-processing characteristics of the encoded protein, etc.) were demonstrated to be potent gene vaccination vehicles when delivered naked, in liposomes, coated on particles or transfected in dendritic cells in vitro. Human clinical trials indicate that the delivery of mRNA naked or transfected in dendritic cells induces the expected antigen-specific immune response. Follow-up efficacy studies are on the way. Meanwhile, mRNA can be produced in large amounts and GMP quality, allowing the further development of mRNA-based therapies. This chapter describes the structure of mRNA, its possible optimizations for immunization purposes, the different methods of delivery used in preclinical studies, and finally the results of clinical trial where mRNA is the active pharmaceutical ingredient of new innovative vaccines.
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Vaccination against tumor antigens has been shown to be a safe and efficacious prophylactic and therapeutic antitumor treatment in many animal models. Clinical studies in humans indicate that specific immunotherapy can also result in clinical benefits. The active pharmaceutical ingredient in such vaccines can be DNA, RNA, protein, or peptide and can be administered naked, encapsulated, or after delivery in vitro into cells that are then adoptively transferred. One of the easiest, most versatile and theoretically safest technologies relies on the direct injection of naked messenger RNA (mRNA) that code for tumor antigens. We and others have shown in mice that intradermal application of naked mRNA results in protein expression and the development of an immune response. We used this protocol to vaccinate 15 melanoma patients. For each patient a growing metastasis was removed, total RNA was extracted, reverse-transcribed, amplified, and cloned. Libraries of cDNA were transcribed to produce unlimited amounts of copy mRNA. Autologous preparations were applied intradermally in combination with granulocyte macrophage colony-stimulating factor as adjuvant. We demonstrate here that such treatment is feasible and safe (phase 1 criteria). Furthermore, an increase in antitumor humoral immune response was seen in some patients. However, a demonstration of clinical effectiveness of direct injection of copy mRNA for antitumor immunotherapy was not shown in this study and must be evaluated in subsequent trials.
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T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of β2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.