Comparison of the expression kinetics and immunostimulatory activity

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Comparison of the expression kinetics and immunostimulatory activity of replicating mRNA, non-replicating mRNA and pDNA after intradermal electroporation in pigs Bregje Leyman, Hanne Huysmans, Séan Mc Cafferty, Francis Combes, Eric Cox, Bert Devriendt, and Niek N. Sanders Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00722 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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Molecular Pharmaceutics

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Comparison of the expression kinetics and immunostimulatory activity of replicating mRNA, non-replicating mRNA and pDNA after intradermal electroporation in pigs

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Bregje Leyman1,4, Hanne Huysmans1,4, Séan Mc Cafferty1,2, Francis Combes1,2, Eric Cox3,

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Bert Devriendt3, Niek N. Sanders1,2*

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Ethology, Laboratory for Gene Therapy, Heidestraat 19, 9820 Merelbeke, Belgium

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Ghent University, Faculty of Veterinary Medicine, Department of Nutrition, Genetics and

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Cancer Research Institute (CRIG), Ghent University, Belgium

Ghent University, Faculty of Veterinary Medicine, Department of Virology, Parasitology and Immunology, Salisburylaan 133, 9820 Merelbeke, Belgium *

Corresponding author:

Email address: [email protected]

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Tel: +32 9 264 78 08

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These authors contributed equally to this work

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Abstract

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Synthetic mRNA is becoming increasingly popular as an alternative to pDNA-based gene

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therapy. Currently, multiple synthetic mRNA platforms have been developed. In this study we

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investigated the expression kinetics and the changes in mRNA encoding cytokine and

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chemokine levels following intradermal electroporation in pigs of pDNA, self-replicating

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mRNA, modified- and unmodified mRNA. The self-replicating mRNA tended to induce the

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highest protein expression, followed by pDNA, modified mRNA and unmodified mRNA.

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Interestingly, the self-replicating mRNA was able to maintain its high expression levels

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during at least 12 days. In contrast, the expression of pDNA and the non-replicating mRNAs

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dropped after respectively one and two days. Six days after intradermal electroporation a

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dose-dependent expression was observed for all vectors. Again, also at lower doses, the self-

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replicating mRNA tended to show the highest expression. All the mRNA vectors, including

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the modified mRNA, induced elevated levels of mRNA encoding cytokines and chemokines

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in the porcine skin after intradermal electroporation, while no such response was noticed after

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intradermal electroporation of the pDNA vector.

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Keywords: Pigs, pDNA, mRNA-therapeutics, luciferase expression, innate immune response

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Introduction

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Nucleic acid-encoded drugs based on plasmid DNA (pDNA) and messenger RNA (mRNA)

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form a potential new drug class with multiple applications such as protein replacement

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therapy and vaccination for cancer and infectious diseases1,2. One of the first reports on the

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use of pDNA and mRNA to express proteins in vivo dates from the 1990s, when Wolff et al.

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made the observation that intramuscular injection of mice with non-formulated pDNA or

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mRNA coding for a luciferase reporter protein, resulted in the detection of this protein3.

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Nevertheless, after this report the field of gene therapy has been dominated for many years by

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a focus on pDNA and viral vectors. This conservative attitude was based on the belief that

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mRNA is a very fragile and instable molecule4. However, after more than two decades of

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research the goal of commercialization of pDNA based vaccines for human usage has not

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been reached. The reason for this is not clear but might be a consequence of several

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drawbacks of pDNA. For example, pDNA vectors have a low efficacy in non- or slow

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dividing cells5, they are sensitive to epigenetic silencing6, they often contain an antibiotic

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resistance gene and they result, after e.g. intramuscular injection, in a long-term uncontrolled

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expression7,8. This long-term expression might be an advantage for applications such as

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protein replacement therapy reducing the dosage frequency of the treatment, but may be a

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disadvantage for other applications such as vaccination. Although viral vectors are more

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effective, they are also afflicted with important disadvantages like a complex production

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process and their ability to trigger immune responses against viral epitopes on the vector9-11.

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Additionally, some viral vectors integrate in the genome of the host12. As an alternative to

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pDNA and viral vectors, in vitro transcribed (IVT) mRNA (synthetic mRNA) is becoming

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increasingly popular. In contrast to pDNA and viral vectors, mRNA-based therapeutics are

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expressed very efficiently in dividing and non-dividing cells despite the lower stability as a

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consequence of the ubiquitous RNases. Additionally, they also do not contain antibiotic 3 ACS Paragon Plus Environment


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resistance genes, carry virtually no risk of genomic integration and oncogenic mutagenesis

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and their expression is limited in time1,13-15. Multiple synthetic mRNA platforms, such as

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unmodified mRNA, modified mRNA and self-replicating mRNA, are currently available.

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After intracellular delivery of unmodified synthetic mRNAs, pattern recognition receptors

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(PRRs), such as toll-like receptors (TLRs), NOD-like receptors and RIG-like receptors, sense

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the mRNA and trigger an innate immune response that is characterized by the production of

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cytokines and chemokines (e.g. INF-β, IL-6, IL-12, CCL-5 and CXCL-10)16,17. The

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formulation of synthetic mRNA into particles increases the capacity of synthetic mRNA to

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stimulate the innate immune system because these particles mostly end up in endosomes,

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which contain the RNA sensing TLRs 3, 7 and 818. Notwithstanding that the innate

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immunostimulatory effects of unmodified mRNA may serve as an intrinsic adjuvant, and

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hence might be a major benefit for vaccination, it might hamper the mRNA translation

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process and trigger mRNA degradation19. Therefore, researchers have tried to decrease the

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capacity of synthetic mRNA to stimulate the innate immune system by incorporating

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modified nucleotides such as pseudouridine (Ψ), N(1)-methyl-pseudouridine (m1Ψ), 5-

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methylcytidine (m5C), N6-methyladenosine (m6A), 5-methyluridine (m5U), or 2-thiouridine

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(s2U)16,19,20. Beside these two non-replicating mRNA platforms, also self-replicating mRNA

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is gaining more and more attention. Self-replicating mRNAs are mostly based on positive-

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stranded RNA viruses, like alphaviruses, and contain RNA-dependent RNA polymerase genes

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responsible for amplifying the transgene which replaced the structural viral proteins1,21. Self-

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replicating mRNAs are inherently immunostimulatory, as several single stranded and double

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stranded RNA species are formed during the amplification process, which can activate PRRs

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resulting in secretion of type I IFNs22. Despite the many studies that used mRNA for protein

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replacement therapies and for vaccination against e.g. cancer, allergies, viral- and bacterial

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infections, mRNA-based therapeutics are far from a commercial product1,17,23-28. Moreover,


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most of those studies are based on small animal models (mainly mice) that poorly mimic

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humans. The evaluation of nucleic acid-encoded drugs in large animals (> 10 kg), such as e.g.

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pigs that are physiologically more closely related to humans than mice, is uncommon24,29.

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Additionally, a side-by-side comparison of the expression efficacy and immunogenicity of

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pDNA and the abovementioned different mRNA platforms has not been performed. In this

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study the expression kinetics and the capacity to stimulate the innate immune system of either

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(1) pDNA, (2) self-replicating mRNA, (3) modified- or (4) unmodified mRNA was compared

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in a porcine model. Because of our interest in mRNA vaccination, we focused on intradermal

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electroporation as the skin is extremely immuno-competent and easily accessible.

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Additionally, porcine and human skin show several anatomical, immunological and

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physiological similarities30. The data of this study might be very useful for the further

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development of synthetic mRNA-based therapeutics, and especially the development of

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mRNA-based vaccines.

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Materials and methods

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Mice and pigs

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Seven week old female Balb/c mice (Janvier, France) were housed in individually ventilated

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cages at 25 °C under natural day-night rhythm with ad libitum access to feed and water and

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enriched with mouse houses and nesting material. Belgian landrace pigs of 12 weeks old were

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housed together at 25 °C under natural day-night rhythm with ad libitum access to feed and

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water. Experiments were started after an acclimatization period of at least 1 week. At the start

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of the experiments the pigs weighed about 35-40 kg. All in vivo experiments were approved

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by the ethical committee of the Faculty of Veterinary Medicine, Ghent University (EC

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2013/57, 2015/77 and 2015/156).

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Plasmids



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The pGL4.13 [luc2/SV40] plasmid (pDNA) was purchased from Promega (Wisconsin, USA)

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and contains the luc2 reporter gene and the SV40 early enhancer/promotor and a synthetic β-

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lactamase (Ampr) coding region. This plasmid contains 293 CpG motifs (i.e. 63 CpG per 1000

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base pair (bp); the sequence can be found at GenBank accession number AY738225.1).

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pTK160 (11519 bp.) and pTK305 (4112 bp.) plasmids were used to produce the self-

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replicating or (un)modified mRNAs by in vitro transcription (IVT). pTK305 and pTK160

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contain, besides the luciferase gene, a bacteriophage T7 polymerase promoter, 5′ and 3′

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untranslated regions (UTRs) and consensus recognition sequences for the I-SceI

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endonuclease. pTK160 also contains nonstructural proteins (nSP1-4) of the Venezuelan

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equine encephalitis virus (VEEV), that form the replicase complex. All constructs contain the

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same Fluc2 gene (protein ID = AAV52875.1) which has been codon optimized for

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mammalian expression. Conformity of the luciferase transcripts is essential for proper

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comparison and subsequent selection of the most suited construct for future applications.

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mRNA in vitro transcription

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The unmodified and modified mRNAs were produced by IVT of the I-SceI-linearized

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pTK305 plasmid using a MEGAscript T7 transcription Kit (Invitrogen, Massachusetts, USA)

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with unmodified nucleotides or the m1Ψ modified nucleotides (Tebu-bio, Belgium) replacing

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all the non-modified equivalents. The self-replicating mRNA was produced from the I-SceI-

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linearized pTK160 plasmid using the same kit and unmodified nucleotides. Next, the mRNAs

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were purified and capped using vaccinia virus capping enzymes and 2’-O-Methyltransferase

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(Cellscript, Wisconsin, USA) to create cap1 and were then again purified using the RNeasy®

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Mini kit (Qiagen, Germany). The self-replicating mRNAs have a poly(A) tail of 40

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adenosines. The non-replicating mRNAs also have a poly(A) tail of 40 adenosines after IVT,

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but for these mRNAs the poly(A) tail was extended by poly(A)tailing with the A-plusTM

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Poly(A) polymerase Tailing Kit (Cellscript) to approximately 200 adenosines, followed by 6 ACS Paragon Plus Environment

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purification. All mRNA’s were quantified using a Nanodrop spectrophotometer (Thermo

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Fisher Scientific, Massachusetts, USA) and the purity was determined by formamide agarose

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gel electrophoresis as described previously31. The biological activity of each new batch of

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mRNA was compared with that of previous batches. To this extent 5 µg of mRNA (dissolved

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in 50 µl phosphate buffer saline (Ca2+ and Mg2+ free Dulbecco's Phosphate-Buffered Saline,

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PBS, Ambion, Massachusetts, USA) was intradermally (ID) injected in mice (n = 3).

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Injections were immediately followed by needle electroporation (AgilePulse, BTX Harvard

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Apparatus, Massachusetts, USA) of the injection spot as described previously9 and the

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bioluminescence was measured after 24 h, using an IVIS Lumina II (PerkinElmer, Belgium)

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and the photon flux (photons/s) in the region of interest was calculated using the Living

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IMAGE Software 4.3.1. Bioluminescence of untreated skin was measured and used as

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background. The molecular weight of all final products was calculated by loading the

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sequences of the mRNAs and pDNA into OligoCalc: an online oligonucleotide properties

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calculator of Kibbe WA.

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Optimization of ex vivo quantification of firefly luciferase expression

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In vivo imaging of firefly luciferase expression in pigs is not possible, due to the size of the

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animals. Therefore, we decided to measure the bioluminescence signal ex vivo after excision

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of the injection spots. However, the storage time and temperature of the excised samples may

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affect the bioluminescence signal. Therefore, we first determined the optimal storage

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condition of the biopsies in mice. In more detail, Balb/c mice were ID injected with pDNA

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coding for luciferase (20 µg in 50 µl PBS), followed by electroporation, as described before9.

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Forty-eight hours after the injection 8 mm diameter skin punch biopsies (Miltex, The

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Netherlands) were taken from the injection sites. All mice were humanely euthanized before

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skin biopsies were taken. Half of the biopsies (n = 4) were immediately immersed in 24-well

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plates containing 600 µl ice cold D-luciferin solution (15 mg D-luciferin (Gold 7 ACS Paragon Plus Environment


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Biotechnology, Missouri, USA) per ml PBS). The other half was immediately immersed in

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600 µl warm (37 °C) D-luciferin solution. After 15 min incubation on ice or at 37°C, the

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bioluminescence signal was measured. Additionally, we also followed the evolution of the

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bioluminescence signal in four excised spots during 90 min. This was done in a similar

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manner as described above using an incubation on ice as this condition gave the best results

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(see supplementary Figure 1 for results). These data showed that skin biopsies should be

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immediately transferred to an ice-cold D-luciferin solution which is placed on ice.

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Additionally, the bioluminescence signal should be measured as soon as possible after biopsy

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using a fixed time point (in this study we used 18 min) after biopsy.

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Analysis of pDNA and mRNA expression in pigs: kinetics and dose dependency

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One week after arrival of the pigs, five rectangular (1 cm x 2 cm)-sites were shaved and

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marked using tattoo ink (MS Schippers, Belgium) on the back of each pig, for later

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identification of the injection sites. Subsequently, the expression kinetics of pDNA,

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unmodified mRNA, modified mRNA and self-replicating mRNA was studied after

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intradermal injection of 20 µg of the different vectors (dissolved in 50 µl PBS) in the marked

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spots. Control spots were injected with PBS. Injection spots were immediately electroporated

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using 4 mm gap needle array electrodes (needle length 5 mm) using the AgilePulse-BTX

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Harvard Apparatus-program-protocol (two pulses of 450 V with a pulse duration of 0.050 ms

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and a pulse interval of 0.2 ms; and subsequently after 50 ms eight pulses of 110 V with a

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pulse duration of 10 ms and a pulse interval of 20 ms). One-, two-, six- and twelve days

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following administration, the luciferase expression of the different mRNA vectors and the

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pDNA was determined by taking a skin punch biopsy of the injection spots. Ex vivo

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bioluminescent imaging of the biopsies occurred as optimized in mice. For each construct

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(including control) and time point we had five injection spots. Additionally, we also evaluated

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the dose dependency of the luciferase expression. For that purpose, pigs were again shaved 8 ACS Paragon Plus Environment

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and marked. The marked spots were subsequently ID electroporated with 1, 5 or 20 µg of

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each of the luciferase expression mRNA vectors and pDNA. After 1 and 6 days all animals

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were humanely euthanized, skin biopsies were collected, and ex vivo bioluminescent imaging

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occurred as optimized previously in mice. For each dose and time point we included 4

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injection spots.

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Analysis of the local innate immune response after intradermal electroporation of pDNA

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and mRNA vectors in pigs

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To study the effect on the innate immune system of each of the luciferase expression vectors,

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pigs were ID injected with 20 µg of (1) pDNA, (2) self-replicating mRNA, (3) modified

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mRNA, (4) unmodified mRNA and (5) PBS as a control. All injections were immediately

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followed by electroporation as described above. Twenty-four hours later, pigs were humanely

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sacrificed, and skin biopsies were taken from the injection sites, and immediately frozen in

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RNAlater (Sigma-Aldrich, Germany) and stored at -20 °C until further analysis. RNA was

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isolated from the biopsies following the RNAzol® RT protocol (Sigma-Aldrich). RNA

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concentrations were assessed at 260 nm by a Nanodrop spectrophotometer (Thermo Fisher

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Scientific) and the purity of the RNA samples was checked using an Experion RNA StdSens

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Analysis kit (Bio-rad Laboratories, California, USA). Reverse transcription was carried out

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using the RT² First Strand kit (Qiagen) according to the manufacturer’s instructions. Purity

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and quantity were assessed again after reverse transcription. Prior to gene expression analysis

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using the commercially available RT² profiler PCR Array (PASS-122Z pig antiviral response,

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Qiagen), the cDNA template was mixed with RT² SYBR Green mastermix (Qiagen) and

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diluted with RNase-free water according to the RT² profiler PCR Array Handbook from

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Qiagen. Thermo-cycling was performed in 384-well plates and parameters were set as

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described in the manufacturer’s protocol.

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Statistical analysis 9 ACS Paragon Plus Environment


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Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, California,

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USA). Differences between groups were analysed by means of a non-parametric Kruskal-

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Wallis test and a post-hoc test following Dunn using the Benjamini-Hochberg correction for

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multiple testing. The qPCR results were analyzed using the CFX manager software v3.0 (Bio-

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Rad Laboratories) by a Gene Study with normalized expression (∆∆Cq) based on the mean

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efficiency corrected CT-value and after inter-run calibration. A P-value of p < 0.05 was

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considered significant.

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Results

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The transfection capacity of self-replicating mRNA outperforms pDNA and non-

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replicating mRNA in the porcine skin

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The luciferase expression of the different mRNA vectors was slightly below that of pDNA

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one day after intradermal electroporation in pigs (Figure1). Interestingly, at day 1 and 2 the

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luciferase expression of the different mRNA vectors was similar and not significantly

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different from each other. While the average luciferase expression of modified and

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unmodified mRNA showed a 2.5 and 5-fold decrease at day 6 and 12 respectively, the

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average luciferase signal of the self-replicating mRNA showed, compared to the expression

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on day 1 and 2, a slight increase at day 6 and 12 (Figure 1). The pDNA luciferase expression

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levels tended to outperform the mRNA luciferase expression levels one day after injection,

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however, the average pDNA signal dropped below the signal of all mRNAs on day 2, and was

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inferior to the signal of the self-replicating mRNA until the end of the experiment. After day 2

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the expression of the pDNA stayed constant till day 6 and subsequently showed a further brief

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drop at day 12 (Figure 1). Note that no significant differences have been noticed and only

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trends could be observed. We also calculated the area under the curve as a measure of the

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total amount of luciferase produced during the 12-day follow-up period (Figure 2). Although

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no significant differences could be observed, Figure 2 shows that self-replicating mRNA 10 ACS Paragon Plus Environment

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tends to perform better than pDNA, unmodified as well as modified non-replicating mRNA

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after intradermal electroporation in pigs.

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Figure 1. Quantification of the mean bioluminescence signal after intradermal electroporation

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of the mRNA vectors and pDNA in pigs. Skin biopsies of the injection spots were taken 1

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day, 2 days, 6 days and 12 days after intradermal electroporation of 20 µg of either self-

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replicating mRNA (3.83 x 1012 molecules), unmodified mRNA (1.49 x 1013 molecules),

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modified mRNA (1.48 x 1013 molecules), or pDNA (4.20 x 1012 molecules mole). The value

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between brackets is the copy number, i.e. the number of molecules present in 20 µg of the

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vectors. The mean value of the total flux (p/s) is given along with the standard error of the

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mean (SEM). No significant (p > 0.05) differences were observed. Each data point is the

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mean of five biopsies obtained from five different pigs. The dotted line represents the

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background signal (Bkg).



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Figure 2. The area under the curve (AUC) of the bioluminescence signal versus time graphs

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was calculated, along with the SEM, over a period of 12 days after intradermal

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electroporation of 20 µg of either self-replicating mRNA (3.83 x 1012 molecules), unmodified

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mRNA (1.49 x 1013 molecules), modified mRNA (1.48 x 1013 molecules), or pDNA (4.20 x

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1012 molecules). For these calculations, the bioluminescence signal of the negative controls

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was subtracted from all data points. The value between brackets is the copy number, i.e. the

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number of molecules present in 20 µg of the vectors. Each bar is the mean of five biopsies

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obtained from five different pigs (n=5). No significant differences could be detected.

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Effect of the vector dose on the luciferase expression

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The expression level of different pDNA and mRNA doses (1 µg, 5 µg, 20 µg) were compared

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1 and 6 days after intradermal electroporation (Figure 3). One day after injection, a real dose-

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dependent effect on the luciferase expression was not noticed for the non-replicating mRNAs

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and the pDNA. Nevertheless, the expression of pDNA tends to show an increase when 20 µg

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was used. Surprisingly, one day after administration, the self-replicating mRNA showed the

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highest expression at the lowest dose (1 µg), while the two other doses (5 and 20 µg) showed

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a circa three-fold lower expression (Figure 3). Six days after intradermal electroporation the


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expression levels of all vectors and doses showed a huge drop, except the expression level of

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the self-replicating mRNA, which showed a slight increase when a dose of 20 µg was

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administered (Figure 3). Additionally, a more clear dose-dependent expression was observed

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for all mRNA vectors and pDNA six days after intradermal electroporation but no significant

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differences could be observed.

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Figure 3. Dose-dependent expression after intradermal electroporation in pigs of 1 µg, 5 µg

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and 20 µg of the mRNA vectors or pDNA. The bioluminescence signal in the punch biopsies

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was measured one day and six days after injection. The average value of the total flux (p/s) in

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the skin biopsies from four pigs (1 µg and 5 µg) or five pigs (20 µg) is given along with its

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SEM. The dotted line represents the background signal (Bkg). At each dose and time point

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the expression levels of the different vectors were not significantly different. Note that in the

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x-axis also the copy number of each vector is shown in mole.

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Electroporation of synthetic mRNA but not pDNA induces transcriptional changes

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associated with an innate immune response in the porcine skin 13 ACS Paragon Plus Environment


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Next we analyzed the capacity of the synthetic mRNAs and pDNA to provoke changes in the

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transcriptome of cytokines and chemokines involved in the pig antiviral immune response

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after intradermal electroporation in pigs. Elevated expression of mRNAs coding for these

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cytokines and chemokines might give us information about the immune stimulatory properties

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of the different constructs. Depending on this potential, the constructs can be used for

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different applications. A strong self-adjuvant effect can be advantageous for mRNA based

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vaccines. For protein-replacement therapies, however, stimulation of the innate immune

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system should be kept to a minimum17. Twenty-four hours after intradermal electroporation of

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20 µg of either (1) pDNA, (2) unmodified mRNA, (3) modified mRNA and, (4) self-

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replicating mRNA the innate immune response was characterized by analyzing the expression

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level of 84 cytokines and chemokines in the injections spots (Figure 4). Intradermal

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electroporation of pDNA did, compared to control (i.e. PBS plus electroporation), not

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significantly affect the mRNA expression level of any of the tested genes. In contrast, self-

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replicating mRNA, 1mψ-modified mRNA and unmodified mRNA, induced a significant (p
0.05) than the induced

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upregulation after electroporation of modified mRNA and self-replicating mRNA.

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Figure 4. Gene regulation of 84 genes involved in innate immunity after intradermal

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electroporation of 20 µg of the different vectors in pigs. Twenty-four hours after

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administration of the vectors total RNA was isolated from the excised injection spots and the

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expression of the different innate immune marker genes was measured relative to control (i.e.

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intradermal electroporation of PBS). The heat map represents normalized mean expression

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values depicted as log 2-fold change relative to control. Reported values were obtained from

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three biopsies taken from three different pigs. Values indicated by a star* indicate a

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significant difference (p < 0.05) compared to control (n = 3).

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Figure 5. Innate immune marker genes that were commonly upregulated by all three mRNA

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vectors 24 h after intradermal electroporation in pigs. The graph shows the normalized mean

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expression values of the indicated genes relative to control (i.e. intradermal electroporation of

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PBS). Each bar represents mean ± standard deviation of three biopsies from three different

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pigs. There were no significant differences between the different mRNA vectors.

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Discussion

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In this study, we compared the expression kinetics and intrinsic effect on the innate immunity

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of three mRNA-based platforms and a state-of-the-art pDNA vector in pigs. Messenger RNA

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based therapeutics have the potential to form a new class of drugs with a broad range of

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possible applications. Especially in the field of vaccination, mRNA is very popular because of

333

its safety and its high transfection efficiency in non-dividing cells15. Additionally, mRNA

334

vaccines have a great flexibility with respect to production and application, enabling a fast

335

development of e.g. prophylactic vaccines against emerging diseases. Different mRNA

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platforms have been developed allowing researchers to select the most suited mRNA platform

337

for their application32. Most preclinical studies with mRNA therapeutics have been performed

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in mice. For pDNA therapeutics it is well-known that they have a much lower efficiency in 16 ACS Paragon Plus Environment

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larger animals and humans than in mice, which makes translation of data obtained in mice to

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humans difficult10,11,33,34. In this work, we evaluated different mRNA platforms in pigs. The

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mRNAs were administered via intradermal injection. This route was chosen because of our

342

interest in mRNA vaccination. To prevent carrier related effects on the efficiency and

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immunogenicity of the vectors we used electroporation to increase the intracellular delivery of

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the mRNAs and pDNA.

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In this study we found that the expression after intradermal electroporation of pDNA reaches

346

a maximum after one day and is followed by a sharp drop in expression at day 2. This drop

347

was unexpected as after intradermal electroporation of the same pDNA in mice we observed

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that the pDNA reaches its maximal expression after about two days and stays on a high level

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up to six days after administration (Supplementary Figure 2). This drop in pDNA expression

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in pigs after day 1 might be attributed to epigenetic silencing. It is also surprising that one

351

day after administration pDNA and not the non-replicating mRNAs shows the highest

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expression. Indeed, after delivery of pDNA in the cytosol it has the longest path to fulfil

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before it becomes translated: it has to migrate to the nucleus where it gets transcribed into

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mRNA and subsequently the mRNA must be transported to the cytosol where it is translated

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into proteins.

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The non-replicating mRNAs also reach their maximum one day after injection and

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subsequently show a steady decrease in expression until day six. In mice similar drops in

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expression that start 4 to 24h after carrier mediated delivery of non-replicating mRNAs have

359

been reported35-37. The group of Joachim Rädler determined that the intracellular half-life of

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mRNA after intracellular delivery is about 11h. Taking into account that the half-life of firefly

361

luciferase is only a few hours

362

delivery nicely fits with this degradation kinetics of mRNA. An early drop in expression as

363

seen with the non-replicating mRNAs does occur after administration of the self-replicating

38,39

, the reported luciferase expression kinetics after mRNA



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mRNA. The capacity of the self-replicating mRNA to produce many subgenomic mRNA

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copies in the cell enables this vector to maintain its protein production level during at least 12

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days in the skin of pigs. However, this effect is only obtained when the highest dose (20 µg) is

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used. In mice, only 1 µg of the same self-amplifying mRNA is needed to obtain such

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sustained protein production, which indicates that mice are easier transfected with self-

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replicating mRNA than pigs (Supplementary Figure 3). The self-replicating mRNA needs

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some time before the subgenomic mRNA is abundantly produced in the second cycle of

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replication. This causes a maximum expression around six days after injection as expected

372

taking into account previously reported expression profiles of replicating mRNAs in mice40.

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Furthermore, when interpreting these data one must also take into account that, based on the

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molecular weight, the number of self-replicating mRNA molecules per mass is about 4 times

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lower than the number of non-replicating mRNA molecules and comparable to the number of

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pDNA molecules. Taking into account this difference in copy number and the difference in

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luciferase expression between self-amplifying mRNA, non-replicating mRNA and pDNA at

378

later time points (day 6 and 12) (Figure 1), we can conclude that the self-amplifying mRNA is

379

about 100, 40 and 4 times more effective on day 6 and 50, 30 and 5 times more effective on

380

day 12 than unmodified mRNA, modified mRNA and pDNA, respectively. We also need to

381

take into account that none of the differences between the expression kinetics of the different

382

vectors appeared to be significant. This is the result of the small number of pigs (4 or 5 per

383

experiment) in combination with high variability of the ex vivo bioluminescence signal. The

384

mean values and their standard errors of the individual data points shown in Figures 1, 2, 3

385

and means and the standard deviations of Figure 5 are displayed in Supplementary Table 1.

386

Therefore, we can only draw conclusions about predictive trends in expression kinetics.

387

Next, we compared the capacity of the synthetic mRNAs and pDNA to stimulate the innate

388

immune system by measuring transcriptional changes in innate immune responsive genes one 18 ACS Paragon Plus Environment

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day after intradermal electroporation in pigs. An innate immune alarm might be

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advantageous, as it can serve as a self-adjuvant, for mRNA based vaccines. However, for

391

protein-replacement therapies such innate immune response might diminish the protein

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expression efficiency and promote an immune response against the therapeutic protein36. The

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RT2 profiler PCR array demonstrated that self-amplifying, unmodified and modified mRNA,

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but not pDNA, triggered an innate immune response after intradermal electroporation in pigs.

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This is in accordance with previous studies in large animals and humans, showing poor

396

immunogenicity of DNA vaccines compared to their immunogenicity in small

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animals10,11,33,34. Our results indicate a significantly higher expression of genes involved in

398

signaling downstream of toll-like receptors (IRF7, CCL5/RANTES, CD40), NOD-like

399

Receptors (CASP1, OAS2), RIG-1-Like receptors (DDX58/RIG-1, DHX58, IFIH1, ISG15,

400

IRF7) and type 1 Interferon signaling (MX1) for all tested mRNAs compared to control (i.e.

401

PBS plus electroporation). Signaling through these different pathways results in the activation

402

of e.g. type 1 interferon (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6) and caspase

403

1 activation. This creates a pro-inflammatory microenvironment that can ameliorate the

404

efficiency of mRNA vaccines1. Ishii et al. demonstrated that TANK-binding kinase-1 (TBK1)

405

plays an important role in the innate immune response after intramuscular electroporation of

406

pDNA vaccines in mice41. However, in our study intradermal electroporation of pDNA in pigs

407

causes a downregulation of TBK1. Also others found that stimulation of the IRF3 pathway

408

can result in a temporal decrease in TBK142. It has been described in the past that the

409

incorporation of modified nucleosides such as m1Ψ in mRNA can decrease the activation of

410

the innate immune system43. Therefore, we were surprised that also the m1Ψ modified mRNA

411

induced an upregulation of innate immune responsive genes19. Nevertheless, unmodified

412

mRNA induced the highest upregulation of the innate immune marker genes (Figure 4).

413

Interestingly, self-amplifying mRNA increased the innate immune responsive genes to a



Page 20 of 26

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lesser extent than unmodified mRNA. One would expect the opposite as many uncompleted

415

mRNA strands are present during the self-amplification process. However, our self-

416

amplifying mRNA is based on the genome of a positive-strand RNA virus, which are known

417

to replicate in organelle-like structures, which prevents an exorbitant innate immune

418

response44. The upregulation of genes that signal downstream of toll-like receptors and the

419

weak upregulation of TLR3, 7 and 8 (Figure 4) was remarkable as one may expect that

420

mRNA does not encounter these endosomal TLRs after electroporation16. However, in recent

421

work it has been proposed that electroporation can also induce endocytosis (electro-

422

endocytosis)45. Therefore, it is possible that part of the electro-transferred mRNA uses the

423

classical endosomal trafficking pathway, and is recognized by TLRs residing in endosomes45.

424

In line with this reasoning, we would also expect an upregulation of TLR9 after

425

electroporation of pDNA as TLR9 recognizes unmethylated CpG motifs which are abundantly

426

present in the pGL4.13 plasmid (63 per 1000 bp). However, our data show that

427

electroporation of this pDNA did not cause a significant upregulation of TLR9 mRNA in pigs.

428

It has been reported by Hochrein et al. and Guzylack-Pirou et al. that pigs show a lower

429

response to CpG containing oligodeoxynucleotides compared to mice46,47. This may explain

430

the non-significant upregulation of TLR9 mRNA and the absence of a significant induction of

431

innate immune responsive genes after intradermal electroporation of pDNA in pigs. However,

432

another explanation of the lack of an innate immune response after intradermal

433

electroporation of pDNA in pigs in our study can also be due to the low dose (20µg pDNA

434

per 40kg, i.e. 0.5µg/kg). Indeed, a recent study in non-human primates showed that

435

intradermal electroporation of a HIV pDNA vaccine at a much higher dose of 22µg/kg

436

resulted in a significant, although moderate, induction of IL-1248

437

Finally, the production costs of therapeutic mRNAs will also be of importance for their

438

implementation in future applications. Production of mRNA on research scale is rather 20 ACS Paragon Plus Environment

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439

expensive but the key to reduction of the production cost is upscaling of the production

440

process to an industrial level. The mandatory Good Manufacturing Practice (GMP)

441

regulations require product-specific validation. For mRNA however, the same production

442

process can be used for many different mRNA vaccines, bypassing the need for new

443

validation. This ensures production costs that are competitive with those of protein, peptide,

444

DNA, cell or recombinant pathogen based vaccines14. For vaccination purposes, an adequate

445

immune response is required to induce a memory immune response. Nucleotide modifications

446

have been shown to reduce this immune response by diminishing binding to TLRs, making

447

the modified mRNA maybe less suited for vaccination and more suited for example protein

448

replacement therapy. These modified nucleotides are more expensive compared to the

449

conventional ones. In our modified mRNA, the uridine was completely replaced by N1-

450

methylpseudouridine. Kormann et al., however, showed that a replacement of 25% is

451

sufficient to provoke a decrease of mRNA binding to pattern recognition receptors, what

452

would also lead to reduced production costs17.

453 454

Conclusions

455

Finally, to our knowledge, this is the first study in pigs where the intradermal translational

456

kinetics and the effect on the innate immune system of different synthetic mRNA-platforms

457

and pDNA were compared. In conclusion, our results show that mRNA modifications do not

458

completely prevent recognition by innate immune receptors in pigs, compared to previous

459

studies in mice16. This is probably due to the fact that we used silica columns and not a HPLC

460

based method (to remove double stranded mRNAs) for the purification of our mRNA.

461

Furthermore, synthetic mRNA, and specifically self-replicating mRNA, is a very promising

462

and a cost-effective DNA-alternative for the development of genetic therapeutics for use in

463

farm animals or humans14. In the future, mRNA based therapeutics may replace the current 21 ACS Paragon Plus Environment


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commercial available DNA-based therapeutics in veterinary medicine like Oncept® (a DNA

465

cancer vaccine for dogs, Merial), Apex®-IHN (a DNA vaccine for fish, Vical), Clynav® (a

466

DNA vaccine for fish, Elanco) and LifeTide® SW 5 (a DNA vector for pigs encoding growth

467

hormone releasing hormone, VGX™ Animal Health)49.

468 469

Acknowledgements

470

We are grateful to Prof. dr. Frank Pasmans of the Department of Bacteriology, Pathology and

471

Avian Diseases, Faculty of Veterinary Medicine, UGent, among other things for the use of the

472

CFX384 Bio-rad-cycler. Furthermore, the technical assistance of Sofie De Bruyckere

473

(Department of Bacteriology, Pathology and Avian Diseases, Faculty of Veterinary Medicine,

474

UGent) and Rudy Cooman (Department of Virology, Parasitology and Immunology, Faculty

475

of Veterinary Medicine, UGent) is greatly appreciated. The plasmids pTK160 and pTK305

476

were kind gifts from Tasuku Kitada and Ron Weiss (Massachusetts Institute of Technology,

477

USA). This work was supported by the concerted research action (GOA) fund of Ghent

478

University: Project Code BOF15/GOA/013.

479

Supporting Information

480

Supplementary figure 1: (A) Effect of storage temperature of skin biopsies on the ex vivo

481

bioluminescence signal. (B) Time-dependent evolution of the bioluminescence signal in the

482

skin biopsies.

483

Supplementary Figure 2: Luciferase expression kinetics after intradermal electroporation of

484

10 µg of pDNA in mice.

485

Supplementary Figure 3: Luciferase expression kinetics after intradermal electroporation of 1

486

µg of self-replicating mRNA in mice.


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Supplementary Table 1: Overview of the variance of the data displayed in Figures 1, 2, 3 and

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5. Variance is displayed as standard error of the mean (SEM) of the bioluminescence signal

489

(p/s) for Figures 1-3 and standard deviation of the bioluminescence signal for Figure 5.

490

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Abstract graphics

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For table of contents use only.

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Title: Comparison of the expression kinetics and immunostimulatory activity of replicating mRNA, non-replicating mRNA and pDNA after intradermal electroporation in pigs.

626 627

Authors: Bregje Leyman, Hanne Huysmans, Séan Mc Cafferty, Francis Combes, Eric Cox, Bert Devriendt, Niek N. Sanders

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Comparison of the expression kinetics and immunostimulatory activity

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