Messenger RNA-based vaccines

Review Vaccines & Antibodies

Messenger RNA-based vaccines Steve Pascolo †CureVac

1. Introduction 2. All in one 3. The more we know… 4. RNA for therapeutics 5. Messenger RNA for vaccination 6. Strength and memory of the immune response 7. The legislation: safety issues 8. Manufacturing of messenger RNA 9. The costs

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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.

10. Expert opinion on the development of

Keywords: gene therapy, messenger RNA, vaccination

messenger RNA-based vaccines

Expert Opin. Biol. Ther. (2004) 4(8):1285-1294

1. Introduction

mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments (in gene therapy or vaccination) make this molecule one of the most promising active components of future therapeutic or prophylactic methods. It can address viral, fungal, bacterial, autoimmune or tumour diseases, as well as allergies. This review summarises the different methods and presents the industrial tools (Good Manufacturing Practice [GMP] production) and legal considerations that have been developed over the last 15 years for the development of mRNA-based vaccinations. 2.

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All in one

RNA is the only molecule that can alone fulfil all biochemical activities necessary for life. It may have been the single organic component of a replicating complex that can be considered as a basic form of life at the time of ‘RNA world’, 4.2 billion years ago. Such RNA-alone ‘living’ structures may have evolved into organisms composed of DNA, lipid, protein and RNA (reviewed by Joyce [1]). In this post-RNA world, which may have started 3.6 billion years ago, RNA remained the central player in all biochemical processes. In every organism, RNA is the component (RNA-based viruses) or the mediator (DNA-based viruses and all cellular organisms) of genetic information and directs its replication (RNA primers are used for the initiation of DNA replication). In addition, RNA is the omnipresent constituent of the translation machinery (ribosomes, tRNA and mRNA). It can also control the entire gene expression by regulating mRNA half-life: ribozymes and siRNA can specifically destroy targeted mRNA. RNA is also the principal source of energy in the form of phosphate bonds in ATP or GTP, which are the RNA building blocks.

2004 © Ashley Publications Ltd ISSN 1471-2598

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Messenger RNA-based vaccines

3. The

more we know…

Over the last 15 years, two new important roles of RNA came to light. First was the discovery that senescence is linked to telomere length and that this parameter is controlled by a ribonucleic protein complex known as the telomerase [2]. In this complex, RNA molecules are used as templates to generate chromosomal DNA [3]. Thus, some RNA molecules are central in dictating the lifespan of cells. Second was the finding that small double-stranded RNAs, known as small interfering RNAs (siRNAs), can specifically block protein expression through selective mRNA cleavage [4]. siRNA appears to be more efficient in gene silencing than their previously characterised analogues that are antisense RNA and catalytic ribozymes. 4.

RNA for therapeutics

The utilisation of RNA for therapeutic strategies began with the discovery that double-stranded RNA is immunostimulating [5]. In the 1970s, it was shown to be capable of blocking the growth of transplanted tumours in mice [6,7] and gave promising results in Phase I/II human clinical trials when injected intravenously to cancer patients [8] or HIV-infected patients [9]. Further clinical studies will indicate whether or not doublestranded RNA can be used as a nonspecific treatment or a complementary treatment in the context of chronic diseases where immune stimulation may be therapeutic. Instead of synthetic double-stranded RNA, some RNA viruses, such as Newcastle disease virus, may also be used in association with an antigenic formulation (an autologous tumour lysate for example) in order to achieve specific T cell priming [10]. Recently, a new type of immunostimulating RNA has emerged: the stabilised RNA [11]. These stimulate immune cells through Toll-like receptor (TLR)7 (in mice) or -8 (in humans) [12,13]. Doublestranded RNA and stabilised RNA have different immunostimulation profiles: low cytokine secretion, but high increased expression of costimulation molecules, such as CD86 by antigen-presenting cells (APCs), is stimulated with doublestranded RNA, wheras the reverse is true for stabilised RNA or CpG DNA [11]. Consequently, in future studies, stabilised RNA may show a different potency in the field of immunotherapies compared with double-stranded RNA or CpG DNA. Aside from immunostimulating RNA, some other RNA molecules, such as haptamer, antisense, ribozymes or the newly discovered siRNA, may be components of very effective antitumour, antiviral, antiallergy or antiautoimmunity treatments. The mode of action of these molecules and the results obtained in human clinical trials with ribozymes were recently reviewed [14]. The following will focus on the utilisation of coding mRNA in immune interventions. 5.

Messenger RNA for vaccination

DNA- and mRNA-based vaccination methods were published at approximately the same time (1992 by Tang et al. [15] 1286

and 1993 by Martinon et al. [16], respectively). Both methods are genetic vaccinations in which the foreign nucleic acid is being translated into proteins by host cells. Complete or defective ribosomal products (DRiPs) [17,18] can be degraded into peptides by the proteasome in the cytosol. The peptides are translocated into the endoplasmic reticulum where they are loaded on nascent major histocompatibility complex (MHC) class I molecules. The presentation of these complexes at the surface of specialised APCs (which either directly took up the nucleic acid, or phagocytosed and translocated into the cytosol proteins from dying cells that had themselves taken up and translated the nucleic acid) primes CD8+ cytotoxic T cells. In the endosomes of APCs, proteins encoded by the foreign nucleic acids can also be chopped by resident acidic proteases and generate peptides that are bound on MHC class II molecules. The MHC class II–peptide complexes expressed at the surface of APCs will stimulate CD4+ T helper (Th) cells. The foreign proteins that end up in the extracellular matrix (after secretion of the protein by, or death of, the cells that took up and translated the exogenous nucleic acid) can activate specific B cells. For reviews on MHC antigen processing see Bryant et al. [19] and Rock et al. [20]. Thus, both DNA- and mRNA-based vaccines can stimulate all effectors of the adaptive immune response: B lymphocytes, cytotoxic T cells and Th cells. The 10 years following the publications of Tang and Martinon were vastly dominated by DNA-based vaccination strategies. In particular, plasmids, which are inexpensive to purify on a small scale in laboratories, were seen as the efficient and easyto-produce vaccination method of the future (compared with proteins, which are more or less soluble, difficult to purify and expensive to store). In the late nineties, all of these assumptions were challenged: DNA vaccinations present potential risks such as integration into the host genome or induction of pathogenic anti-DNA antibodies [21,22]; large-scale GMP production of high quality plasmid DNA (in the absence of mammalianderived products, antibiotic and antibiotic resistance genes; free of contaminating genomic DNA; homogenous with a stable and defined structure; etc.) appeared to be much more difficult than expected [23]; DNA-based vaccines seem not to be as efficient in primates as in rodents [24]. Consequently, more interest was raised in the utilisation of mRNA as the active component of a nucleic acid-based vaccine. Some of the most relevant research articles published in this field are listed in Table 1. Five methods of vaccination using mRNA have been described (chronologically listed): naked mRNA directly injected in the tissues (first report in 1990 [25]), mRNA entrapped in liposomes (first report in 1993 [16]), replicative RNA (first report in 1994 [26]), mRNA loaded on gold particles and sprayed in the dermis by gene gun (first report in 1996 [27]) and mRNA-transfected in vitro in APCs (first report in 1996 [28]). The first attempt to use mRNA for in vivo protein expression was reported by Wolff et al. in 1990 [25]. The authors made the curious observation that injection into mouse muscle of DNA or mRNA coding for a protein marker was followed

Expert Opin. Biol. Ther. (2004) 4(8)

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Table 1. List of selected articles important in the context of mRNA-based vaccines. Method of mRNA vaccination

Authors [ref.]

Results

Methods

Naked

Wolff et al. [25]

Injection of naked mRNA results in antigen expression

Intramuscular injection of mRNA encoding CAT, β-galactosidase or luciferase

Conry et al. [29]

Injection of naked mRNA results in antigen expression and induction of a protective antitumour response

Intramuscular injection of globin UTR-stabilised mRNA encoding CEA

Hoerr et al. [31]

Injection of naked or protamine-stabilised mRNA triggers an antibody and a CTL response

Ear pinna injection of UTR-stabilised mRNA (defined antigen and library)

Granstein et al. [32]

Injection of naked mRNA extracted from a tumour cell line protects against tumour challenge

Intradermal injection of total RNA extracted from cells

Martinon et al. [16]

Injection of mRNA entrapped in liposomes triggers an anti-influenza CTL response

Subcutaneous or intavenous injection of a mRNA encoding flu nucleoprotein

Zhou et al. [33]

Injection of mRNA encapsulated in liposomes triggers antibodies and CTLs that can protect against tumour challenge

Intra-spleen injection of gp100-coding mRNA

Zhou et al. [26]

Injection of Semliki Forest-based Intramuscular injection of replicative mRNA self-replicative mRNA triggers an encoding flu nucleoprotein anti-influenza cellular and humoral immunity

Ying et al. [34]

Injection of Semliki Forest-based self-replicative mRNA triggers anti-β-galactosidase CTLs and antibodies

Intramuscular injection of replicative mRNA encoding β-galactosidase

Gene gun

Qiu et al. [41]

Bombardment of mouse skin with gold particles coated with mRNA coding for an antigen triggers an antibody response

Gene gun application; RNA coding for human alpha-1 antitrypsin

Transfected in vitro in APCs

Boczkowski et al.

Pulsing of mouse DCs with OVA-coding mRNA or total mRNA from tumour cells prime, in vitro and in vivo, a CTL response that protects against tumour challenge

Defined mRNA; total tumour RNA or mRNA; in vitro amplified mRNA library

Nair et al. [59]

Vaccination with DCs transfected with mRNA encoding proteins expressed during neoangiogenesis plus mRNA coding for tumour antigens delivers synergistic cancer treatment

Defined mRNA encoding VEGFR-2, Tie2 or VEGF plus total tumour RNA or mRNA encoding TERT or TRP-2

Coughlin et al. [60]

RNA-transfected CD40-activated B cells instead of DCs

Flu M1, MART1 or total tumour RNA

Ponsaert et al. [61]

RNA-transfected monocytes instead of DCs

Flu M1 encoding mRNA

Liposomes

Replicative

[28]

A non-exhaustive list of the most relevant articles documenting the efficiency of the five different mRNA-based vaccination strategies is shown. The ‘results’ column indicates the read-out, and the ‘method’ column indicates the antigen used and the site of injection. CAT: Chloramphenicol acetyl transferase; CEA: Carcinoembryonic antigen; CTL: Cytotoxic T lymphocyte; DC: Dendritic cell; MART: Melanoma antigen recognised by T cells; OVA: Ovalbumin; TERT: Telomerase reverse transcriptase; TRP: Tyrosine-related protein; UTR: Untranslated region; VEGF: Vascular endothelial growth factor; VEGFR: VEGF receptor.

by the detection of the reporter molecule. Five years later, in 1995, Conry et al. [29] showed that such an approach can result in the triggering of protective antitumour immunity. In these experiments, mRNA coding for carcinoembryonic antigen (CEA) was repeatedly injected into mouse muscles. Following such a vaccination protocol, the animals developed an antibody response to CEA after a challenge with a CEA-expressing tumour cell line (control mice that did not receive mRNA injections did not develop antibodies to CEA after tumour challenge). In 2000, 10 years after the report by Wolff et al.,

two new articles confirmed that direct injection of mRNA induces an immune response: Hoerr et al. documented antibody and cellular immune responses against β-galactosidase after application in the ear pinna [30] of naked or stabilised (associated to the cationic peptide protamine) mRNA coding for this antigen [31]; Granstein et al. showed that intradermal injection of total RNA extracted from a cell line protects the mice against a challenge with the same tumour cells [32]. Recently, the author’s laboratory has shown that the immune response triggered against the antigen encoded by the injected


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mRNA is a Th2 type response (ear pinna as an injection site). It can be reverted to a Th1 type of immunity when granulocyte-macrophage colony-stimulating factor is used as an adjuvant (Carralot et al., submitted). These new findings may stimulate the further study and development of mRNA-based vaccines where naked or stabilised mRNA is directly injected into patients or healthy people to be immunised. In spite of the Wolff report from 1990, Martinon et al. assumed that mRNA should preferably be protected and delivered to the cytosol through encapsulation in liposomes [16]. Their report published in 1993 is the first where mRNA is successfully used as a tool for vaccination. A flu nucleoprotein-encoding mRNA–liposome formulation injected subcutaneously or intravenously (intraperitoneal was not efficient, intramuscular was not tested) triggered an anti-influenza response in mice. These results were confirmed using other liposome formulations in two reports that were published by Zhou et al. [33] and Hoerr et al. [31]. In the former, an antitumour immune response documented by antibody, cytotoxic T lymphocyte (CTL) assays and tumour challenge, was triggered by the intra-spleen injection of encapsulated mRNA coding gp100; in the latter, an immune response against β-galactosidase was detected in mice that received an intravenous or subcutaneous β-galactosidase-encoding mRNA–liposome formulation. These promising methods do not appear to have been investigated further, as no follow-up articles were published and no human clinical trials were initiated. The toxicity of most liposomes may prevent the use of such formulations in humans. Some new non-toxic particle formulations may be tested and may turn liposome-encapsulated mRNA into a safe, easy and efficient vaccination strategy. A third mRNA-based vaccination method relies on the active self-replication of the injected ribonucleic acid. Such replicative mRNAs are derived from RNA-viruses that code for an RNA replicase. They were shown in several reports to be very efficient in priming an immune response against an antigen encoded by a gene artificially associated to a RNA replicase gene through gene engineering methods. Zhou et al. [26] disclosed this original method in 1994. They used Semliki Forest virus (an alpha virus) as a vector. The virus was modified to contain a transgene coding for the influenza nucleoprotein. Whether encapsulated or injected naked into mouse muscles, the self-replicative mRNA triggers an immune response against the flu nucleoprotein. Several subsequent reports confirmed that the application of replicative RNA triggers a strong immune response: direct injection [34,35], delivery by adoptive transfer of in vitro transfected cells [36] or delivery by gene gun [37]. In this system, not only may the amount of produced antigen be responsible for the strength of the triggered immune response, but also the intrinsic capacity of such vectors to nonspecifically stimulate the immune system. Immunostimulation may be due to the double-stranded RNA intermediates that are formed during RNA replication. They activate APCs through TLR3 [38] and 1288

induce apoptosis. However, the immunostimulating capacities of some RNA viruses may also correlate to other components of these infectious agents (Fournier et al. showed that an RNA virus, such as Newcastle disease virus, is a potent activator of the immune system not only through the production of double-stranded RNA intermediates [39]). Although they proved to be efficient, the safety issues addressed by the utilisation of recombinant replicative mRNA coding for pathogen- or tumour-derived antigens may prevent the further development and application of such vaccine vectors. Indeed, it cannot be excluded that replicative mRNA recombines with other RNA viruses and generates new pathogens. A needle-less method of mRNA vaccination was also developed based on the gene gun technology. The precipitation of mRNA on gold particles to be used as a vaccine has several advantages. First, the amount of nucleic acid needed to prepare a vaccine dose is very low (less than a nanogram [37]). Second, such dried preparations can be stored at room temperature for extended periods of time without being degraded [40]. Third, the delivery of the vaccine by gene gun is painless and insures that the vaccine is delivered to a large surface of skin, that is, to many dendritic cells (DCs) resident in the skin, such as Langerhans cells or dermal DCs. Initially used in the context of a DNA-based vaccine, the method was quickly adapted for mRNA-based vaccination. In 1996, Qiu et al. [41] published the first report demonstrating that mRNA vaccination with gene gun is efficient. Mice bombarded with beads coated with mRNA encoding human alpha-1 antitrypsin generated antibodies against the human protein. Vassilev et al. [40] developed and tested in cattle and sheep an antibovine viral diarrhoea virus vaccine using mRNA-coated gold particles. Preference of DNA over mRNA for laboratory use, the cost of gold particles and gene gun, as well as the inconvenience of mouse bombardment (mice must be shaved before treatment), probably hindered the development of ballistic delivery of mRNA for vaccination. However, this method would probably deserve more interest, as it may be the most powerful, reliable and safe method for the vaccination of large populations with mRNA in the context of prophylactic vaccination (for example, anti-HIV or antimalaria vaccines). The most recently published mRNA-based vaccination strategy, the adaptive transfer of in vitro mRNA-transfected DCs [28], is actually the only method that was tested in humans (Phase I/II clinical trials) [42-44]; it is also by far the most popular method (> 40 original research articles dealing with its use, study and improvement have been published since the original description in 1996). Many reports have documented the efficiency of this technology for the induction of antigen-specific T cells in vitro or in vivo (in mice). Moreover, three articles disclosed the results of RNA-loaded DCs used in human clinical trials as an antitumour immunotherapy approach [42-44]. They demonstrate feasibility, lack of toxicity and promising efficacy based on clinical and immunological


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read-outs. Summarising all of these studies in vitro, in mice and in humans, it can be concluded that mRNA-transfected DCs can prime CD4 and CD8 T cells directed against epitopes derived from the mRNA-encoded protein. Defined mRNA (coding for a chosen protein) or total tumour-derived RNA can be introduced in the DCs. The method can be used to induce immunity against many different types of tumours [45-51], fungi [52] or viruses [53-57]. The company Merix Bioscience [101], which was founded under the impulsion of Dr E Gilboa, undertook the industrial development of mRNA-transfected DCs as a vaccination strategy. The most innovative development of this technology may be: • the use of mRNA coding for chimeric antigens (here the encoded protein is targeted to the endosomes, facilitating MHC class II epitope presentation [58]) • to combine several mRNAs coding for tumour antigen and antigen expressed locally during neoangiogenesis [59] • to replace or supplement DCs with other potent APC types (such as activated B cells [60] or monocytes [61]). Some researchers claim that mRNA transfection does not need to be efficient, and others, on the contrary, believe that the better the transfection, the better the potency of the DCs to specifically stimulate T cells. Supporting the former hypothesis, it has been shown that passive pulsing of DCs with mRNA allows the production of MHC-associated epitopes, even if the antigen (the full-length protein encoded by the mRNA) cannot be detected [28]. No antigen was detected, but CTL priming was described when the immune response triggered against the polio virus (RNA virus that cannot infect mouse cells) was studied in wildtype mice [62]. In addition, Grunebach et al. did not see any difference in T cell stimulation when using efficient or less efficient mRNA transfection methods [63]. These results may be explained by the ‘DRiPs hypothesis’ from J Yewdell, which postulates that the MHC-associated epitopes come mainly from defective ribosomic products (e.g., unfinished or misfolded protein) that are very quickly catabolised into peptides [17,18]. Thus, low level de novo translation is enough to generate MHC class I epitopes. On the contrary, the obvious theoretical assumption that the more antigen produced, the more MHC epitopes will be presented at the cell surface was documented when mRNA-transfected DCs were used to trigger T cell responses [64]. Finally, it is reasonable for human clinical trials to use the most efficient and less toxic transfection method to reliably deliver mRNA in as many DCs as possible. Electroporation is probably the best solution. Although vaccination with mRNA-transfected DCs is efficient, at present it is an expensive method, as the in vitro derivation of DCs in GMP conditions is a costly process. Such a technology can be viewed as a therapeutic vaccine approach, but, as opposed to all of the other mRNA-based vaccination strategies listed above, could not be considered as a large-scale prophylactic vaccination method.

Strength and memory of the immune response 6.

One concern of mRNA-based vaccination strategies is that the mRNA-encoded antigen will be transiently present in the body. Although this feature supports safety, it may go against efficacy, especially when non-replicative mRNA is being used. Freigang et al. demonstrated that de novo translation of mRNA in DCs triggers a cytotoxic T cell response that disappears relatively quickly [62]. A sustained T cell response may require the persistence of the antigen or of the genetic information encoding it [65]. Therefore, in a prophylactic setting, the mRNA-based vaccine may have to be applied frequently. Nevertheless, in a therapeutic setting, where a persistent virus or tumour disease assures the availability of large amounts of antigens, sustaining the CTL response could be achieved after the vaccine mRNA has been degraded. Here the vaccine is expected to trigger an immune response that cannot be primed (or is prevented) by the pathogen itself, but would efficiently recognise virus-infected or tumour cells. In this context, the therapeutic vaccine should lower virus or tumour load, but would probably never result in their total elimination from the organism. Thus, CTL responses induced by a vaccine may be sustained by traces of the pathogen in vaccinated patients. In addition, through epitope spreading [66], the strength of mRNA-based vaccination may be enhanced: efficient antidisease CTLs triggered by the therapeutic vaccination would attack the pathogen, releasing its information (genes and proteins), which can be taken up by local APCs. These may be matured in situ by efficient CD4 T cells that were primed during vaccination. This cascade of events would boost and widen the immune response. In this scenario, the amount of vaccine-derived antigen and its persistence is not a concern, but the efficiency of T cell priming is critical. 7.

The legislation: safety issues

Although located mainly in the cytosol and not in the nucleus, mature mRNAs belong to the biochemical family of nucleic acids. mRNA, similarly to DNA, may be considered as a gene and, consequently, its use as a vaccine may be viewed as ‘gene therapy’. The very strict regulations associated with gene therapy are primarily based on the fact that DNA plasmids or DNA-modified cells may trigger permanent and dangerous changes in the genetic information of treated people. mRNA does not have such drawbacks. It is, however, unclear from the legal authorities what type of regulations should be applied on mRNA-based vaccination. Nevertheless, in February 1997, the FDA approved the first trial based on the utilisation of RNA-transfected DCs to develop immunity in cancer patients. The Recombinant DNA Advisory Committee of the National Institute of Health voted to continue approval in June 1997. This allowed the first mRNA-based vaccination in humans. The results from three trials made in the US are now published [42-44].


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The promising results obtained will trigger the initiation of new trials worldwide. An mRNA-based vaccine trial (again, mRNA-transfected DCs) was launched recently in Germany. The Kommission Somatische Gentherapie, the legal organ in charge of gene therapy issues in Germany, decided that the utilisation of RNA-transfected DCs is not a gene therapy approach. In these trials, the DCs are prepared under GMP conditions, but the active component of the vaccine, mRNA, is not. Increased interest in mRNA-based vaccination as a safe and efficient replacement for DNA-based vaccines is expected to result in a larger number of human trials, regardless of which method of mRNA delivery listed above would be used (direct injection of naked mRNA, liposome encapsulation, replicative mRNA, gene gun application or adoptive transfer of in vitro transfected autologous cells). The authorities will have to develop clear rules to guide the use of mRNA-based approaches in human clinical trials. 8.

Manufacturing of messenger RNA

Apart from mRNA-transfected DCs, all of the mRNA-based vaccination strategies were tested in animal models only. The extension to human therapy using these very promising and safe approaches requires a large amount of mRNA produced according to GMP guidelines. Production of mRNAs, which are usually > 500 bases and up to 12 kilobases, is always achieved through utilisation of enzymes: a plasmid DNA template is linearised using a restriction enzyme that cuts after the gene to be transcribed, an RNA polymerase capable of recognising a promoter ahead of the gene of interest will produce the mRNA and, finally, a treatment with DNase will destroy the DNA template. The RNA can be recovered and cleaned by several methods, including precipitation or chromatography. Because the neosynthesised mRNA is released from its template DNA, this matrix is used several times: an average of 30 µg of capped RNA is obtained from 1 µg of DNA. This means that each DNA template can be transcribed > 100 times. The RNA polymerases are very processive and generate very long mRNA. One of the advantages of this technology is its high production efficiency. For example, in order to obtain a batch of 30 mg of mRNA, 1 mg of plasmid DNA (easily obtained using a 1 litre fermentation, standard DNA purification methods and laboratory tools) is enough. The production of 30 mg of DNA would, on the contrary, require an ∼ 30 litre fermentation and the lysis of a large bacterial pellet. This is often associated with the damage of bacterial DNA from shearing forces used to disperse the pellet. Thus, plasmid DNA becomes contaminated with bacterial DNA fragments. Moreover, different forms of plasmid DNA (supercoiled, relaxed circle and linear) will be contained in the final product and limit the reproduction of the quality between batches. When producing mRNA, the quality of the DNA, in particular the amount of contaminating genomic DNA, is of no concern, as a DNase will destroy all 1290

DNA molecules anyway (plasmid and contaminating bacterial genomic DNA) after transcription. Surprisingly, it may be easier to produce, in a standard high quality, large amounts of GMP mRNA than large amounts of GMP plasmid DNA. So far, only two companies in the world can provide mRNA produced under GMP conditions: Ambion [102] in the US (Austin, Texas) and CureVac [103] in Europe (Tübingen, Germany). In both facilities, the production of mRNA is made with only recombinant or chemical products: no reagents from animal origins are used. Thus, the contamination of the mRNA with viruses, retroviruses or prion proteins is excluded. At CureVac, the mRNA purification is refined by a chromatography step that separates mRNA according to size (see Figure 1). This prevents contamination of the mRNA of interest with smaller or larger by-products that may result from abortive transcription (in the former case) or from traces of nonlinearised DNA template (in the latter). Besides, it has been experienced that some genes contain cryptic transcription termination signals or cryptic promoters recognised by RNA polymerases and, thus, generate shorter or alternative (eventually antisense) mRNA products. For these reasons, the chromatographic purification of mRNA according to its size is a useful step to achieve the best quality in a GMP production. In these conditions, the complete process of GMP production of mRNA takes only ∼ 1 month (from the entry of the template plasmid to the release of the qualified mRNA batch). As opposed to the production of proteins, different mRNA molecules are produced and purified in the same way, regardless of their sequence. Such standardisation makes mRNA-based vaccine production a fast and easy step towards clinical trials, whatever disease is targeted. 9. The

costs

The main obstacle in the development of mRNA-based vaccines compared with genetic vaccinations, such as plasmid DNA-based vaccines, or recombinant virus-based vaccines is probably the cost of mRNA production at the laboratory level. Most scientists assume that mRNA-based therapies would be too expensive to be realistic. This assumption is wrong. First, as demonstrated above, it is actually easy to produce large amounts of high quality mRNA. Second, > 90% of the costs of GMP production are due to the facilities (set-up and maintenance), the documentation, the expertise of qualified personnel, quality controls and certification (series of tests is being made by specialised companies; each test is costly and must be performed regardless of the pharmaceutical product: peptide, DNA, RNA, proteins, viruses, bacteria, etc.). The cost of the chemicals required for production barely affects the price of GMP production. Discrepancies between the costs of laboratory-made immunotherapeutics in inbred mice and the cost of similar therapies in humans are best exemplified with DC-based vaccines. Although it is relatively cheap and easy to test vaccines based on in vitro-derived autologous DCs in laboratories, this method is very expensive


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1

A

2

3

B 200 UV260 absorption

Length in bases 6000 4000 3000 2000 1500 1000 750 500

150

12

4

100

50 3

5

6

-10 Time

Figure 1. Production of mRNA. A shows a picture of an agarose/formaldehyde electrophoresis gel. In line 1, 2 µg of the precipitated mRNA product of a standard transcription reaction was run, where not many contaminants (longer or shorter than the expected 1450 bases product transcript) can be seen. On the contrary, line 2 shows 2 µg of the transcription product obtained from another DNA template, which gave several other mRNA fragments. The expected product is 700 bases. The longer mRNA may originate from transcription of nonlinearised contaminating DNA template or from cryptic promoters in the gene. These would generate antisense mRNA. They may, of course, interfere with the therapeutic activity of the mRNA. Such a transcription product was purified by chromatography after precipitation. The UV absorption profile during elution is shown in B. Peak 1 and 2 are contaminating small fragments (DNA and/or RNA short fragments that still precipitate). Peak 3 is an mRNA smaller than the expected product, which is contained in peak 4. Peaks 5 and 6 are longer mRNA transcripts that can also be seen on the gel (line 2). When peak 4 is collected and analysed on a formaldehyde/agarose gel (A, line 3: 2 µg of RNA from peak 4), it is shown to be a very pure single mRNA product. UV: Ultraviolet.

and strategically difficult to establish under GMP conditions. Consequently, it must be stressed that, contrary to the belief at present, mRNA-based immunotherapy is not more costly than other strategies, such as protein-, peptide-, DNA-, cell- or recombinant pathogen-based strategies, and is actually less expensive than strategies requiring autologous in vitro produced cells.

Expert opinion on the development of messenger RNA-based vaccines 10.

From a ‘DNA world’ that dominated the development of nucleic acid-based therapies in the nineties, the author believes that we are turning towards an ‘RNA world’, where the newly discovered siRNA demonstrates clear promise, and mRNA-based technologies described in this review are now considered a realistic, safe and efficient alternative to vaccination using plasmid DNA, recombinant pathogens or transgenic cells. Besides the development of human clinical trials, more fundamental research is needed to understand the mechanisms underlying mRNA-based vaccination, especially in the context of ‘passive pulsing’ of DCs in vitro or application of naked mRNA (replicative or not) in vivo. In both cases, mRNA spontaneously enters the cytosol, where it can be translated. It is surprising that mRNA reaches the cytosol before it is degraded by the ubiquitous RNases. One hypothesis explaining this phenomenon would be that some cells

express a receptor that can specifically recognise and translocate exogenous mRNA. Such a receptor may have evolved and been conserved for communication among neighbouring cells or for triggering an immune response against pathogens that cannot infect APCs (cross-priming). It was recently demonstrated that the immune system uses the genetic information of a pathogen, rather than its protein content, in order to trigger a cytotoxic T cell response [62]. Thus, a strong CD8 T cell response may depend mainly on de novo protein synthesis from mRNA and not on an abundant delivery of protein antigens. This would explain the efficiency of mRNA-based vaccination strategies. Whatever the mechanisms, all kinds of RNA-based therapies, including mRNA-based vaccines, will certainly be more and more utilised, tested and developed thanks to: • the availability of unlimited amounts of mRNA that can be produced under GMP conditions • the results from the first human clinical trials • the introduction of official regulations As described above, the preclinical development of these strategies at the laboratory scale is more costly for mRNA-based therapies than for DNA- or recombinant virus-based therapies, but this does not apply to clinical trial situations where GMP products are required. Because RNA is easier to purify and store than proteins, and is safer and less toxic than recombinant DNA, it is forecast that mRNA will be the


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active component of new, efficient and widely applicable therapeutics active against a broad spectrum of pathologies, such as tumour diseases, infections with pathogens, autoimmune disorders and allergies.

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•

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Affiliation Steve Pascolo CureVac GmbH, Paul Ehrlich Strasse 15, 72076 Tübingen, Germany Tel: +49 7071 9205312; Fax: +49 7071 9205311; E-mail: [email protected] Website: http://www.curevac.de