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European Biopharmaceutical Review

mRNA: Get the Message

Messenger ribonucleic acids (mRNAs) are highly versatile, non-toxic molecules that have become the focus of significant research in the field of molecular medicine. mRNAs are considered to be a safe and efficient alternative to protein-, recombinant virus- or DNA-based therapies in the vaccination and gene therapy fields. Due to their great flexibility with respect to production and application, mRNAs are currently being investigated as cancer vaccines, prophylactic vaccines for infectious diseases and, most recently, as a source of therapeutic gene products and protein replacement therapies.

Nucleic Acid-Based Therapeutics
Advances in molecular medicine have given researchers a better understanding of disease causes and mechanisms on a molecular level. It was recognised early on that proteins could principally be expressed by direct injection of the corresponding nucleic acid into target organs (1). Thus, mRNA and DNA have been extensively investigated for potential use as gene therapeutics and tools for genetic immunisation to elicit a specific immune response.

Although mRNA has many advantages over DNA, initial efforts focused on DNA-based strategies because virtually ubiquitous ribonucleases (RNases) hydrolyse mRNA instantly, leading to its rapid degradation. This made the clinical use of mRNA nearly impossible for a long time.

The development of DNA vaccines for therapeutic and prophylactic purposes has not been overly successful when compared to more conventional approaches, and many strategies have been developed and evaluated for enhancing the potency of DNA-based vaccines (2). However, recent setbacks for DNA vaccines, as well as technological advances in the generation and stabilisation of mRNA for medical use, have shifted the attention to mRNA as the basis for a new class of vaccines and drugs (3).

mRNA Applications
mRNA is recognised as a completely novel class of therapeutic with a wide range of potential applications. The physiological role of mRNA is to transfer genetic information from the nucleus to the cytoplasm, where this information is translated into the corresponding protein. Therefore, mRNA-based therapeutics need to cross only one membrane – the plasma – whereas DNA-based therapeutics require nuclear localisation, which includes the crossing of a second membrane before the DNA molecule is transcribed into mRNA.

Additionally, mRNA is non-replicative and its half-life does not usually exceed a few days, making it a transient genetic carrier. On the contrary, DNA-based therapeutics can contain virus-derived promoter elements and have the potential to integrate into the host genome as the result of homologous recombination or a random event. This may lead to inactivation or activation of genes, which can affect regulatory elements, drive oncogenesis or induce pathogenic anti-DNA antibodies. Although this is a rare event, the favourable safety aspect of mRNA is especially important for prophylactic vaccines. Hundreds of millions of doses could be administered to healthy individuals, and any rare safety event could become a serious safety problem.

The versatile activities of mRNA give it the potential to become a treatment or prevention option for any disease, including genetic disorders, infections, cancer and degenerative diseases. RNA also provides enormous flexibility with respect to production. Any protein with a known sequence can be encoded and expressed at large scale under Good Manufacturing Practice regulations. Importantly, different mRNA products can be manufactured by using the same established production process without any adjustment. This is because the physico-chemical characteristics of mRNA molecules are basically unaffected by the sequence of the molecule, saving time and reducing cost, especially when compared with other vaccine platforms (4).

Cancer Immunotherapy
Immunotherapy has always been considered an attractive and potentially efficient treatment for cancer, and during the last decade many strategies have been investigated to develop safe and efficient approaches. Active cancer immunotherapies aim to elicit a specific immune response against tumourspecific antigens or tumour-associated antigens through the activation of the adaptive and innate immune system. Additionally, a long-lasting immune response must include balanced humoral and antigen-specific cytotoxic T lymphocyte-mediated immune responses.

The activation of antigen-presenting cells (APCs) is the centerpiece for effective cancer immunotherapy. Thus, dendritic cells (DCs) – the most potent APCs – are critical for initiating effective immune responses. DCs express or process the captured antigen for its presentation or cross-presentation on major histocompatibility complex molecules. Importantly, the capture and presentation of antigens must occur in the presence of an immunogenic maturation stimulus – for example via toll-like receptors (TLRs) – in order to stimulate both humoral (antibodymediated) responses and cellular (antigen-specific T-cell) responses. DCs that have not received an immunogenic maturation signal will induce tolerance by the production of regulatory T-cells.

Foreign Acids
The human immune system recognises bacterial and viral DNA and viral RNA as 'foreign' nucleic acids, stimulating the mammalian innate immune system – the non-specific immune system – through the activation of TLRs. In detail, the common viral intermediate, double-stranded RNA (dsRNA), activates TLR3, whereas synthetic single-stranded RNA (ssRNA) and virus-related RNA activate human TLR7 and TLR8 (5).

Several mRNA-based vaccination strategies have been evaluated, such as mRNA encapsulated in liposomes, the injection of self-replicative mRNA based on Semliki Forest virus vectors (replicons) – immunisation experiments against lethal challenges with influenza – the use of a gene gun and the ex vivo transfection of dendritic cells with mRNA. Only the latter approach was tested in clinical trials as an anti-tumour immunotherapy (6).

A clinical trial investigated an autologous, mRNA-electroporated dendritic cell vaccine in patients with advanced melanoma. The investigators observed one partial response and five stable diseases in 17 out of the 27 patients treated. It was concluded that therapeutic vaccination with autologous TriMix-DC was feasible, safe and immunogenic, and can be combined with sequential IFN-α-2b (7).

Naked mRNA Molecules
Another approach, which is currently being investigated in a Phase 2b study in prostate cancer and in a Phase 1/2 study in patients with non-small cell lung cancer (NSCLC), is based on the observation that direct injection of naked, sequence-modified mRNA results in protein expression and induction of specific cytotoxic T lymphocytes and antibodies (8). This study laid down the foundation for a novel protocol for vaccination – intradermal injection of non-encapsulated mRNA-based vaccines.

Importantly, naked mRNA molecules are only weak inducers of the maturation process of antigen-presenting cells, as shown in in vitro experiments with mouse dendritic cells. The complexation of mRNA with protamine – a small arginine-rich nuclear protein – induces the maturation of DCs and, therefore, greatly enhances immunogenicity (9).

Cancer Therapy

Today, mRNA-based cancer immunotherapy is being developed as a novel platform approach that also shows promise in combination with other cancer therapies. The two-component mRNA vaccines are composed of free and protaminecomplexed mRNA. The core is the uncomplexed, sequence-modified mRNA, which encodes the desired tumour antigen. The sequencemodification greatly enhances the stability and the translation levels without changing the amino acid sequence of the corresponding protein. The minimal mRNA vector is produced in a cell-free system by using naturally occurring nucleotides – the building blocks of RNA (see Figure 1) (7).

These self-adjuvanted RNA vaccines are administered intradermally without any additional adjuvant, inducing balanced adaptive immune responses and providing humoral and T-cellmediated immunity. Importantly, this two-component mRNA-based tumour vaccine supports both antigen expression and immune stimulation, mediated by toll-like receptor 7 (TLR7) (see Figure 2, page 18) (10).

Immune Responses
The first clinical trials with the two-component mRNA-based immunotherapy were conducted in patients with castrate-resistant, nonmetastatic or mildly symptomatic metastatic prostate carcinoma and NSCLC patients with stage 3b/4 disease. In both trials, the administration of the vaccines was shown to be safe and able to induce a balanced immune response. A high level of antigen-specific T-cells were observed in around 80 per cent of prostate carcinoma patients independent of their human leukocyte antigens-background, and 58 per cent of the immunological responders reacted against multiple antigens. Immune responses were detected against all antigens independent of their cellular localisation, and the appearance of antigen-unspecific B-cells increased in 74 per cent of prostate cancer patients (11). In NSCLC patients, an antigen-specific humoral and cellular immune response was observed in roughly two-thirds of the treated patients. A placebo-controlled randomised Phase 2b study in patients with castrate-resistant prostate carcinoma is currently being enrolled in Europe.

Prophylactic Vaccines

Prophylactic vaccines are designed to prevent or ameliorate the effects of a future infection by bacterial or viralpathogen, and are typically comprised of dead, inactivated organisms and purified products derived from the pathogen or synthetic protein or peptide vaccines. Despite improvements, many vaccines remain sub-optimal due to a range of factors, including the genetic variety of viruses and the production of a specific vaccine in a pandemic outbreak. Nucleic acid vaccines have the potential to address these needs, generating effective prophylactic vaccines, but despite decades of research, there is still no commercial product available for human use (12).

Although plasmid DNA (pDNA) vaccines have proven to be a flexible platform, and are shown to be effective in small animal models, so far they have generally lacked potency in human clinical trials.

The safety profile and ease-of-use widens the use of the two-component, self-adjuvanted RNA-based vaccines for prophylactic vaccines, and can be produced easily and quickly to match any sequence provided. In vivo data has shown that immune responses similar or superior to those triggered by commercially available vaccines were achieved in animal models for influenza. The RNA-based vaccines elicited B and T-cell-dependent protection and targeted multiple antigens, including the highly conserved viral nucleoprotein (13).

Optimised Tests
An optimised mRNA vaccine for prophylactic vaccination against influenza A H1N1, H3N2 and H5N1 viruses was then designed. It tested these mRNA-based vaccines by intradermal injection in various animal models and induced a balanced, long-lived and protective immunity to influenza A virus infections in very young and very old mice, while remaining protective upon thermal stress. The vaccinations with the conserved and intracellular antigen could further improve the protection. Additionally, immunisation with a combination of conserved antigens is feasible and may form the basis for optimised and more broadly protective vaccines (13).

Another research group evaluated the use of a synthetic lipid nanoparticle formulation of self-amplifying RNA (LNP/RNA) to increase the efficiency of antigen production and immunogenicity in vivo, without the need for a viral delivery system. A mouse immunogenicity study of a LNP/RNA vaccine encoding the HIV glycoprotein140 surface elicited functional immune responses (14).

mRNA-based vaccines offer the flexibility to encode virtually any protein as an antigen in a very short timespan. Thus, heath stable novel vaccines could be quickly made. This is a fact of great importance for pandemic scenarios in infectious diseases.

In 1990, JA Wolff and his colleagues achieved gene expression for a variety of proteins after direct injection of the corresponding mRNA into the muscles of mice (15). The innate immune system detects RNA lacking nucleoside modifications, which are found on mammalian mRNA, but not on bacterial mRNA. The incorporation of modified nucleosides – such as m5C, m6A, m5U, s2U or pseudouridine – suppresses the immunostimulatory activity of RNA (4). Another study injected mice with highperformance liquid chromatographypurified pseudouridine containing mRNA-encoding erythropoietin (EPO), complexed with TransIT-mRNA. It measured elevated levels of functional EPO that caused a significant increase of both reticulocyte counts and hematocrits, demonstrating the great potential for clinical applications of therapeutic mRNA (16).

Additionally, synthetically modified mRNA was used as a safe strategy – without genomic integration – for efficiently reprogramming cells to pluripotency, as well as for the differentiation of RNA-induced pluripotent stem cells into terminally differentiated myogenic cells (17). mRNA as a novel therapeutic class has almost limitless opportunities, in addition to its broad applicability for the treatment of human disease. It has now begun to transform modern molecular medicine.

1. Tang DC et al, Genetic immunization is a simple method for eliciting an immune response, Nature, 1992
2. Vergati M et al, Strategies for cancer vaccine development, J Biomed Biotechnol, 2010
3. Vical Phase 3 trial of Allovectin® fails to meet efficacy endpoints, News release, 2013
4. Schlake T et al, Developing mRNAvaccine technologies, RNA Biol, 2012
5. Karikó K et al, Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA, Immunity, 2005
6. Pascolo S, Messenger RNA-based vaccines, Expert Opin Biol Ther, 2004
7. Wilgenhof S et al, Therapeutic vaccination with an autologous mRNA electroporated dendritic cell vaccine in patients with advanced melanoma, J Immunother, 2011
8. Hoerr I et al, In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies, Eur J Immunol, 2000
9. Scheel B et al, Immunostimulating capacities of stabilized RNA molecules, Eur J Immunol, 2004
10. Fotin-Mleczek M et al, Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity, J Immunother, 2011
11. Kübler H et al, Final analysis of a phase I/IIa study with CV9103, an intradermally administered prostate cancer, ASCO, 2011
12. BIO Ventures for Global Health, What are DNA vaccines? Visit: /itemid/5.aspx
13. Petsch B et al, Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection, Nat Biotechnol, 2012
14. Geall AJ et al, Nonviral delivery of self-amplifying RNA, Proc Natl Acad Sci, 2012
15. Wolff JA et al, Direct gene transfer into mouse muscle in vivo, Science, 1990
16. Karikó K et al, Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin, Mol Ther, 2012
17. Warren L et al, Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA, Cell Stem Cell, 2010

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About the author

Ingmar Hoerr is cofounder and Chief Executive Officer of CureVac, an integrated biopharmaceutical company in the field of mRNAbased therapeutics and prophylactic vaccines. CureVac has secured about €145 million in equity financing and currently employs over 110 people. Ingmar received his PhD in Biology from Tübingen University in 1999, and an MBA from Danube University in Krems, Austria in 2001.

Ingmar Hoerr
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