Next Article in Journal
The Cerebellum Gets Social: Evidence from an Exploratory Study of Cerebellar, Neurodevelopmental, and Psychiatric Disorders
Previous Article in Journal
Role of the Endocannabinoid System in Metabolic Control Processes and in the Pathogenesis of Metabolic Syndrome: An Update
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

mRNA—From COVID-19 Treatment to Cancer Immunotherapy

Independent Researcher, 13505 Berlin, Germany
Biomedicines 2023, 11(2), 308; https://doi.org/10.3390/biomedicines11020308
Submission received: 1 December 2022 / Revised: 5 January 2023 / Accepted: 9 January 2023 / Published: 22 January 2023
(This article belongs to the Special Issue mRNA-Based Cancer Immunotherapy)

Abstract

:
This review provides an overview covering mRNA from its use in the COVID-19 pandemic to cancer immunotherapy, starting from the selection of appropriate antigens, tumor-associated and tumor-specific antigens, neoantigens, the basics of optimizing the mRNA molecule in terms of stability, efficacy, and tolerability, choosing the best formulation and the optimal route of administration, to summarizing current clinical trials of mRNA vaccines in tumor therapy.

1. Introduction

mRNA—messenger ribonucleic acid—is a single-strand copy of a selected part of the genetic sequence of a gene, negatively charged with secondary and tertiary structure formations. It is produced from DNA and—after appropriate modification—leads to the synthesis of a specific protein by a ribosome. It was first described in 1961 [1]. The feasibility of in vivo expression following the direct injection of in vitro-transcribed mRNA was demonstrated in 1990 [2]. The first commercially available mRNA-based vaccines were Comirnaty (BNT162b2, tozinameran) and Spikevax (mRNA-1273, elasomeran) [3,4]. The major hurdles that had to be overcome before the successful in vivo use of mRNA as a vaccine were instability of the molecule, innate immunogenicity, and delivery. The general structure of mRNA is illustrated in Figure 1.
mRNA is composed of nucleotide building blocks consisting of a selection of four different nucleobases, adenine, cystine, guanine, and uracil, coupled to ribose which contains a phosphate group as the linker to the next nucleotide. mRNA transfers the genetic information for the synthesis of a specific protein from the genes via the DNA to the protein production facility: the ribosome. The nucleotides constitute the alphabet of the genetic code. Three consecutive nucleotides, for example, AUG, function as codons. Accordingly, by using four letters of the alphabet, A, C, G, and U, 64 different codons are possible. In general, codons define the amino acid which will be recruited next in the protein synthesis. On top of that, a single codon or groups of codons have specific functions, for example, the codon AUG codes for the amino acid methionine and operates as the start codon for the coding part of the mRNA. Table 1 provides an overview of the functionality of codons.
The task mRNA has to fulfil involves the following steps. It starts with the production of a single-strand copy of the genetic code laid down in the DNA, which is performed by RNA polymerase. This process is called transcription and leads to precursor mRNA (pre-mRNA), which—via splicing procedures—finally becomes mature mRNA. In parallel to transcription, the 5′ cap is added to the molecule and the poly(A) tail is attached at the end of the chain. After that, the mRNA is transported from the nucleus of the cell to the cytosol. Various mechanisms can be utilized for this step involving different proteins, such as CBP20 and CBP80. Subsequently, ribosomes take over, read the code, and start the protein synthesis according to the codons provided in the coding section of the mRNA. The synthesis is terminated as soon as a stop codon is reached. Stop codons are UAG (amber), UAA (ochre), and UGA (opal). Thereafter, the mRNA is degraded by ribonucleases. mRNA activates the innate immune response through various RNA sensors such as toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I), and protein kinase R (PKR) [5,6,7,8].

2. Lessons Learned from COVID-19

The unprecedented speed of the global spread of the COVID-19 pandemic caused by the coronavirus, SARS-CoV2, resulted in an extremely rapid development of mRNA vaccines [9]. In January 2020, the genetic sequence of SARS-CoV-2 was available; in March 2020, the first Phase I trial (NCT04283461) started with an mRNA vaccine (BNT162b2) and a rolling submission procedure was initiated, in which data were provided to the authorities as soon as they were available. In December 2020, BNT162b2 received a temporary emergency use authorization in the UK followed by a conditional marketing authorization (CMA) by the EMA based on data from a global Phase I/II/III study (NCT04368728).
Although SARS viruses are common in humans, vaccines had not been developed since the course of the infection normally was very mild. The SARS outbreak in early 2000 triggered DNA vaccine development [10] but was stopped very soon as the disease vanished on its own. Together with research on MERS-COV, the antigen target rapidly emerged [11]. The spike protein is the major surface protein for most coronaviruses. The virus enters the host cell by endocytosis after binding to angiotensin-converting enzyme 2 [12], followed by the release of the viral DNA. An antibody developed against the spike protein would then inhibit cell entry and thereby neutralize the virus [13]. Furthermore, it could be shown in rhesus monkeys that SARS-CoV-2 infection protects against re-infection [14], mostly via neutralizing antibodies but not via T cell responses [15]. Intramuscularly injected mRNA vaccines mostly induce IgG-type antibodies and less IgA antibodies [16], which provide disease prevention or attenuation but not sterilizing immunity [9]. The preclinical data necessary for the development of a COVID-19 mRNA vaccine were essentially available from the previous SARS and MERS experiments and so saved a lot of development time. In addition to mRNA vaccines, other functional types have been and are in development for the treatment of COVID-19 including DNA and virus vaccines. The latter range from live attenuated virus vaccines via inactivated virus vaccines to recombinant protein vaccines. These types will not be addressed further in this review. For the mRNA-based treatment of COVID-19, various positions of the mRNA molecule had been modified as listed in Table 2.
mRNA capping determines the stability and maturity of the mRNA molecule [41,42]. A eukaryotic translation initiation factor (eIF4E of the eIF4F complex) couples to the 5′ cap and subsequently initiates the translation process [20]. Targets of modification include the 5′ cap [43,44], 5′- and 3′-UTRs, the coding region, and the poly(A) tail [25]. Modification of the 5′- and 3′-UTRs improves the translation and increases the half-life of in vitro transcription mRNA. The 5′-UTR can be rather short containing only three nucleotides [45] or up to several hundreds. The coding sequence is responsible for the target protein that is produced in the ribosomes and determines the rate of mRNA translation and the stability of mRNA in the cytosol [46,47]. The 3′-UTR is vital for the recognition by proteins in the cytosol and also determines the mRNA stability [25,34]. Again, most details are proprietary and have not been disclosed. The addition of a suitable-length poly(A) tail at the 3′ end of mRNA also plays an important role in its successful translation and stability containing ideally more than 90 nucleotides. In some cases, uridine is replaced by pseudouridine, denoted as Ψ . The poly(A) tail can be added to in vitro transcription (IVT) mRNA either through a template vector or by recombinant poly(A) polymerase after the transcription process has occurred [24,25].
mRNA which encodes the protein of interest, in the case of SARS-CoV-2, the spike protein, can be administered as such or as self-replicating or self-amplifying mRNA (saRNA) [48,49]. Antigen expression is proportional to the number of conventional mRNA transcripts successfully delivered during vaccination [50], which might result in the need for relatively high vaccine doses. saRNA vaccines are derived from alphaviruses such as Sindbis and Semliki Forest viruses [51,52]. The viral genome is divided into two open reading frames (ORFs): the first ORF encodes for the RNA-dependent RNA polymerase (replicase), and the second ORF encodes the antigen (spike protein) [53,54]. Unlike saRNA, ordinary mRNA is small due to its simpler structure and is characterized by only one ORF. According to Bidram [41], there are three types of saRNA available: plasmid-based DNA saRNA, virus-like particle delivery saRNA, and IVT saRNA [41,55]. Figure 2 provides an illustration of the saRNA design. Due to self-replication, considerably lower doses of vaccines are needed. Cancer mRNA vaccines are mostly non-replicating [56,57].
Another alternative is trans-amplifying RNA (taRNA) [58]. In this model, the vector cassette encoding the vaccine antigen originates from the saRNA, from which the replication was deleted to form a trans-replicon. Replicase activity is provided in taRNA by a second molecule, either by a standard saRNA or an optimized non-replicating mRNA (nrRNA), which results in a 10- to 100-fold increase in transcription expression.
mRNA vaccines are different from classic viral vaccines by providing, instead of the antigen itself, the genetic information for producing the antigen. Now, it is up to the host to start protein production. This is a huge benefit compared to administering the antigen to the host. No longer are cell lines required with their potential to produce multiple impurities and the consequence to establish tedious purification and quality control procedures. This is the same issue for DNA vaccines. mRNA vaccines have another significant advantage over DNA vaccines. They have one major hurdle less. DNA needs to enter the cell nucleus [59], while for mRNA vaccines, it is sufficient to reach the cytosol of the cell, which itself is already rather cumbersome.
Vaccine mRNA manufacturing is rapid and cheap by using an IVT from a DNA template with T7 RNA polymerase [60,61]. Since mRNA and appropriate delivery systems are self-adjuvant, they result in strong and long-lasting adaptive immune responses. This is not the case for protein or peptide-based vaccines, which need the addition of adjuvants [62]. Another benefit of mRNA vaccines is that mRNA is much less likely integrated into the host DNA genome than is the case for DNA vaccines [63]. The major reason is that for mRNA manufacturing bacterial fermentation with all its sequelae of isolation and purification is not necessary. Following the expression of antigens, the activity of mRNA is short-lived due to its clearance by RNases, thereby lowering the burden to the host homeostasis.
mRNA is a large hydrophilic, negatively charged molecule with secondary and tertiary structure formations. The cell membranes are negatively charged as well. Additionally, ion pumps and ion channels maintain a negative potential (−40 to −80 mV) across the cell membrane, keeping the cytosol negatively charged by controlling the balance of most of the essential metal ions (for example, K+, Na+, Ca2+, and Mg2+). Naked mRNA is therefore not able to pass this barrier [44]. It was, however, hypothesized that uptake of naked mRNA might occur by endocytosis using cells as mediators. For dendritic cells, this pathway has been described by various groups [64,65,66].
Although the mRNA molecule itself is chemically very stable in the dry state, it is rather unstable in the solution, and, moreover, is rapidly degraded by extracellular and intracellular exo- and endo-ribonucleases [23,67,68]. As a consequence, and in order to mask the negative charge, mRNA needs packaging to be able to pass the cell membrane barrier and to protect itself inside the cell cytosol against degradation before it reaches its target, the ribosome. On the other hand, the package needs to be such that the ribosome is still able to detect and then to process the mRNA [69].
Most mRNA vaccines use lipid nanoparticles (LNPs) as the carrier with a particle size between 1 and 100 nm. LNP formulations are composed of an ionizable or cationic lipid, a helper phospholipid, cholesterol or a cholesterol derivative, and a polyethylene glycol (PEG)-modified lipid. The purpose of the ionizable or cationic lipid is an interaction with the negatively charged mRNA. Examples are DLin-MC3-DMA (MC3) [70] or DOPE [71]. The helper phospholipid, for example, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine or DSPC, stabilizes the bilayer structure of the LNP. The pegylated lipid contributes to the stability of the LNP and prevents opsonization followed by uptake in the liver [3,71,72]. However, the PEG group in the LNP formulation is considered to be a possible allergen for anaphylaxis due to approximately 72% of people having some antibodies against PEGs [73]. LNPs are taken up by apolipoprotein or albumin receptor-mediated endocytosis and can electrostatically attach and fuse with the cell membrane using inverted non-bilayer lipid phases [74] or by non-specific pinocytosis [75]. LNPs modified with mannose target DCs through the mannose receptor CD206 [76]. Once inside the cell, LNPs are routed into early endosomes, followed by late endosomes, and finally the lysosomes where the mRNA contents are enzymatically degraded [77,78].
In addition to LNP, other types of formulations such as liposomes [79], ionizable lipids [80], pH-dependent ionizable materials [81], MC3 [70], polymers [82], dendrimers [83], cell-penetrating peptides [84], and other materials have been investigated [44,56,85,86]. Table 3 provides a summary of COVID-19 vaccines with their active ingredients and formulations for vaccines approved in the European Union comprising both mRNA and viral-based vaccines.
The approved SARS-CoV-2 mRNA vaccines use intramuscular (i.m.) administration. During their development, alternative injection routes have been evaluated, for example, intradermal application. A general consensus on what is the best route has not yet been achieved [3]. Although the i.m. administration route induces strong IgG responses that are thought to protect the lower respiratory tract, unlike natural infection it does not drive the secretory IgA responses that are thought to protect the upper respiratory tract [9]. Most vaccines will protect only against infection of the lower respiratory tract and might not induce sterilizing immunity in the upper respiratory tract, which might still enable transmission of the virus. Live attenuated vaccines or viral vectors that can be applied intranasally would probably also lead to a strong mucosal immune response as well as an IgG response. Currently, 68 clinical studies on intranasal administration are listed at www.clintrials.gov. An overview can be found at [87].
Another important issue of mRNA vaccines, which had to be addressed carefully, is manufacturing. A major lesson from the COVID-19 pandemic is that synthesis of the active ingredient is simple because it is cell-free, scalable, and cost effective. Large-scale production of mRNA vaccines consists of a 1- or 2-step in vitro reaction followed by a purification platform with multiple steps that can include DNase digestion, precipitation, chromatography, or tangential flow filtration [88]. A facility dedicated to mRNA production is able to rapidly manufacture vaccines against multiple targets, with minimal adaptation to processes and formulation [61]. Expression may be possible for complex proteins including monoclonal antibodies that are difficult or impossible to generate with conventional expression systems [89]. The manufacturing process comprises the following steps: generation of a plasmid DNA with an RNA polymerase promoter, e.g., T7, and the mRNA sequence of interest, followed by DNA linearization and transcription of the RNA template and by degradation of the DNA. The 5′ cap and the poly(A) tail can be added either during or after transcription. Purification of the mRNA is an important aspect that needs careful attention. For example, various pollutants such as dsRNA in mRNA may activate pattern recognition receptors. HPLC is a suitable method to remove impurities [90,91]. Alternative methods have been described by Shivalingam et al. [92]. Whereas current methods use enzymatic ligation for nucleic acid assembly, their approach is based on the formation of urea and squaramide artificial backbones from minimally modified, commercially available 3′- and 5′-amino oligonucleotides, which provide a one-pot linkage that can be modified on demand for use with stable pre-activated precursor oligonucleotides under reagent-free, mild conditions.
A comparative evaluation of the efficacy in clinical trials of a total of 19 COVID-19 vaccines including mRNA and viral versions has been performed by Fiolet [93]. High efficacy was observed for all the vaccines against SARS-CoV-2. BNT162b2, mRNA-1273, and Sputnik V were superior (>90%) compared to the other vaccines. AZD1222, and CoronaVac were effective in preventing symptomatic COVID-19 and severe infections against the Alpha, Beta, Gamma, or Delta variants. Real-life data revealed effectiveness against the Alpha and Beta variants and reduced efficacy against Delta. A decline was observed for BNT162b2 and AZD1222 after six months, indicating a need for booster vaccinations. In another study on the efficacy of different COVID-19 vaccines, Krammer [9] described the following ranking of neutralizing antibodies elicited by the vaccine candidates: inactivated and AdV5 vaccine candidates < ChAdOx1 nCoV-19 mRNA vaccines < recombinant protein vaccine candidate [9]. Tolerability was excellent. The inflammatory activity of mRNA vaccines can result in local and systemic inflammation and more autoimmune responses. Serious adverse event rates were rare including myocarditis and pericarditis, cytokine release syndrome, and cerebral venous thrombosis [94,95]. Anaphylaxis was found in 2.5–4.7 cases per million doses, and myocarditis in 3.5 cases per million doses [63].
The lessons to learn from the COVID-19 pandemic regarding mRNA vaccines are the following. The pandemic was the strongest driver possible for speeding up the development, production, and distribution of vaccines by providing more than necessary funding options and an immense public pressure to find solutions. Furthermore, mRNA is the ideal candidate for a vaccine since the target antigen is specific for the virus and—so far—has been rather stable regarding evading mutations. If the antigen should slip away, the development of new vaccines is rather straightforward and quick to establish from modifying the mRNA to manufacturing the final product. A still-unresolved issue is the storage temperature, which means freezing, in the case of Comirnaty at −90 °C to −60 °C and for Spikevax at −50 °C to −15 °C during storage and transport, which makes distribution rather cumbersome, in particular in hot areas such as Africa. Freeze-drying of the formulation has been tried as alternative; however, there is a danger of decreasing stability and losing activity [96]. The addition of cryoprotectants, for example, mannitol or sucrose, does preserve the stability and might be a way out of the dilemma [97].

3. mRNA-Based Cancer Immunotherapy

The objective of cancer immunotherapy is to manipulate the immune system to effectively eliminate cancer cells [98,99,100]. Immunotherapy mainly targets immune cells. It activates the body’s immune system by inhibiting negative immune regulatory factors and enhancing the ability of immune cells to recognize tumor cell surface antigens to eliminate tumor cells [101,102,103]. Cellular immune responses are mediated by T cells; in particular, CD8+ T cells can eliminate tumor cells. Humoral immune activity is mediated by antibodies, which induce clearance by phagocytic cells. On the other hand, the suppressive tumor microenvironment characterized by acidity, hypoxia, and an overexpression of enzymes results in a low immunogenicity of tumor cells and subsequent immunosuppression [104] by preventing T cell infiltration into cancers and causing T cell exhaustion [3]. According to Liu [105], immunosuppressive cells in the microenvironment of tumors include myeloid-derived suppressor cells (MDSCs) [106], tumor-associated macrophages [107], T regulatory cells [108], pro-tumor N2 neutrophils, and cancer-associated fibroblasts. Remodeling the microenvironment, promoting immune cell infiltration, as well as inhibiting tumor angiogenesis and tumor metastasis are imperative for immunotherapy to be effective [109,110,111].
The first marketed immunotherapies for cancer were recombinant versions of the cytokine interferon-α (IFNα), which were approved by the FDA in 1986 for hairy cell leukemia [112,113]. Other FDA-approved immunotherapy drugs are monoclonal antibodies functioning as checkpoint inhibitors such as ipilimumab (melanoma), pembrolizumab (various tumors), and nivolumab (various tumors); bispecific antibodies such as blinatumomab (ALL), which is directed against CD19 and CD3; cytokines such as Intron A which is recombinant INF α 2a (hairy cell leukemia, melanoma, follicular lymphoma, and Kaposi sarcoma); engineered T cell therapies, such as tisagenlecleucel (ALL and NHL), which are CD19-specific CAR T cells; oncolytic viruses such as talimogene laherparepvec (melanoma), which is genetically modified HSV type 1 designed to replicate within tumors and produce GM-CSF; and last but not least, the cancer vaccine Sipuleucel-T (prostate cancer), obtained from a strain of Mycobacterium tuberculosis [114]. However, the therapeutic effect was far from satisfying [115]. Potential reasons are the low specificity of TAAs, immune escape of cancer cells, and immune suppression in the tumor microenvironment [116]. An overview of therapies is provided in Figure 3.
Cancer immunotherapeutics may be divided into four categories: use of recombinant viruses, tumor and immune cell (mainly dendritic cells)-constructed immunizations, and peptide and nucleic acid-based (DNA or RNA) vaccines [41,98] as illustrated in Figure 3. The term “vaccine” is often used instead of immunotherapeutics. Vaccines can be prophylactic or therapeutic. In the treatment of cancer, both prophylactic and therapeutic treatments have been used. Examples for prophylaxis are HPV (Gardasil-9) [117] leading to cervical cancer and HBV (HEPLISAV-B) leading to hepatocellular carcinoma. The topic of this review is focused on mRNA vaccines for the treatment of cancer. All the other therapies will not be covered, as they have been addressed in various review articles [63,118,119,120,121].
mRNA represents the minimal genetic vector and contains only the elements directly required for the expression of the encoded protein [56]. mRNA vaccines constitute an excellent platform for immunotherapy for a number of reasons [122,123,124]. A major point is the possibility for the simultaneous injection of more than one antigen, resulting in both immune and cell-mediated immunity, thereby increasing the likelihood of tumor tissue eradication [125,126,127].
Antigen selection is the most difficult task in mRNA-based immunotherapy. In the COVID-19 case, the opposite was the case. The choice was easy and straightforward, at least at the beginning when the selected antigen, the spike protein of SARS-CoV-2, was rather stable and more or less exempt from mutations. This changed as soon as the Delta and Gamma variants appeared. In cancer immunotherapy, choices for antigens are tumor-associated antigens (TAAs) [128]. The problem is, however, that these structures are also shared by healthy tissues—although at lower expression levels [129]—and therefore likewise become a target of the mRNA administration leading to autoimmunity and resistance. The ideal antigen should be highly immunogenic, expressed only in cancer cells and not or only at very reduced levels in normal tissue, and it should be essential for cancer cell survival [130]. Tissue-specific antigens (TSAs), therefore, are preferable but much less available. Examples are PSA and HER2. However, immunotherapies based on these TSAs were only of limited success [131]. More preferable options are neoantigens, which originate from non-synonymous mutations in tumor cells and are absent in normal cells [132,133,134,135]. Neoantigens are presented by major histocompatibility complex (MHC) molecules.
Furthermore, mRNA vaccines are not constrained by the patient’s HLA class. mRNA vaccines are preferable over DNA vaccines for several reasons. DNA has to reach the nucleus of the tumor cells in contrast to mRNA, for which it is sufficient to reach the cytosol. For mRNA, there is no danger of splice mutations [64,136]. Modifications addressing efficacy, half-life, etc. are easier to achieve by either modifying the molecule itself, the route of administration, or the formulation [69,137,138,139,140,141].
The mRNA modifications described in Section 2 to improve stability and efficacy apply to cancer vaccines as well. Since uridine-rich sequences activate Toll-like receptors [142], which suppress RNA recognition [32], uridine should be replaced by N1-methyl-pseudouridine (1mΨ), 5-methyluridine, or 2-thiouridine [33]. Other possible modifications include the replacement of cytidine with 5-methylcytidine (m5C) and of adenosine with N1-methyladenosine or N6-methyladenosine. GC-rich mRNA results in several-fold higher transcription efficacy [143] without decreasing the half-life [31]. Further improvements include synthetic analogs of the cap and cap enzymes, which are vital for the stability and maturity of mRNA [42], regulatory elements in the 5′-UTR and 3′-UTR, and the use of poly(A) tails that screen mRNA [24,25,27,31,144,145,146].
Despite all these efforts, the tumor is trying to avoid the effect of the treatment by various methods. Tumor escape mechanisms down-regulate the tumor cell surface antigens which means reducing the immunogenicity and thereby the efficacy of the mRNA vaccine [147]. Another action is the up-regulation of immune checkpoint expression on cell surfaces, for example, PD-L1, which inhibits T lymphocyte activity and induces immune evasion [148]. Furthermore, immunosuppressive cells, MDSCs, and Tregs can be recruited into the tumor microenvironment and cytokines can be secreted which inhibit the immune response [149].
The route of administration of mRNA-based vaccines plays an important role [150]. In addition to intramuscular injection, subcutaneous, intradermal, intranodal [151], and intratumoral routes have been used [152]. Adjuvants such as protamine, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 2 (IL2) cause strong activation of the innate immune system, which leads to a potent adaptive immune response [153].
mRNA vaccines are not immunogenic, so that multiple administrations are possible [154,155,156]. However, as already mentioned above, there is one caveat. Formulations containing pegylated moieties might still run into the problem of pre-existing antibodies [71,156]. This issue had first been detected in animal models leading to modifications of the formulations [157,158,159] and verification in animals and in patients [160].
According to Barbier [3], the following objectives should be taken into account in the development of an mRNA cancer vaccine. A strong cytotoxic CD8+ T cell response is needed to eradicate cancer cells. The antigens should be selected such that they are able to induce highly tumor-specific immune responses. Potential targets are TAAs, TSAs, and neoantigens.
The major obstacle to this objective is the high variability of antigens across different individuals [161] or even within the same patient. The heterogeneity of tumors—not only between patients but also within the same patient when looking at the time axis—is one of the highest hurdles to treatment success. Most likely, there are not two identical tumors in the whole patient population of a cancer type. This issue is illustrated in Figure 4. A possible way out of this dilemma is the selection of more than one antigen within the mRNA vaccine.
Neoantigens should be the major target whenever possible [135,162]. During carcinogenesis, malignant cells acquire somatic mutations that lead to the production of protein sequences not expressed by normal cells [132,163]. These proteins are called neoantigens. They may be either common across various patients or specific for each patient, which then can be used to develop personalized treatment [164,165,166]. To that end, the tumor is biopsied, and the neoantigens are analyzed by sequencing, encoded in mRNA, and injected into the patient [3,167,168,169]. Common or shared neoantigens include, for example, BRAF and NRAS mutations, which are observed in approx. 50% and 15–25% of melanoma patients, respectively [170,171]. The advantage of this approach is manifold. First, it is an individual treatment, which is a major point taking into consideration the heterogeneity of tumors. Second, mRNA cannot only encode one whole neoantigen but also as many as there are found in the individual patient [3]. Sahin et al. [172] reported that the first injectable mRNA cancer vaccine encoding neoantigens for advanced melanoma patients through intranodal injection achieved potent T cell responses against multiple neoantigens in all patients after vaccination. However, although personalized cancer vaccines based on neoantigens have shown encouraging results, a large number of predicted neoantigens tend to trigger very few actual anti-tumor responses [173]. Table 4 provides a selection of neoantigens.
Dosing of mRNA can be achieved by titrating up or down, depending on the need, weight, and disease state of the patient. The duration of action is intrinsically limited by mRNA degradation, reducing the likelihood of irreversible side effects, and enabling the treatment of acute indications [184,185].
Formulations for use in cancer treatment are similar to those described for COVID-19 administration. However, as opposed to the delivery systems of mRNA vaccines for pathogen infections, therapeutic mRNA vaccines for cancer treatments are required to generate both robust CD8+ and CD4+ T cell responses [186]. The activation of type I interferon (IFN) proved important in developing a cytotoxic T cell response [187]. Early on, naked mRNA dissolved in Ringer’s solution at a concentration of 1.0 mg mL−1 was injected into separate inguinal lymph nodes in thirteen melanoma patients [172]. However, since naked mRNA cannot enter cell membranes freely but only by cell-mediated endocytic pathways [56,64,65,66], preferred formulations not only for the treatment of COVID-19 but also for cancer therapy are LNPs which can substantially affect intracellular delivery efficiency, determine cell specificity of delivery, and modulate immunogenicity [3]. Ionizable lipid components of LNPs play a key role in multiple aspects of mRNA delivery, including particle formation, cellular uptake, and endosomal escape [44,57,188]. Their self-adjuvant activity, resulting in the stimulation of specific parts of the immune system, is an important aspect of mRNA LNPs. Examples are the stimulator of IFN-γ (STING) pathway and the TLR–RIG-I-like receptor (RLR)-independent mediator of innate immune responses [3,189]. Other formulations that have been developed are hybrid lipopolymer shell mRNA nanoparticles or lipoplexes [190]; nanocapsules with flexible polysaccharide shells and hollow cores, termed a sugar-capsule composed of mannan carrying mRNA [191]; and a nanoparticle platform, called mRNA Galsomes [192]. Reviews on formulations are available from various groups [193,194,195,196]. Large-scale production has been established extremely rapidly [88]. Table 5 provides a selection of mRNA vaccines in development.
As can be seen from Table 5, the range of indications is rather broad reaching from head and neck, breast, lung, pancreas, prostate, gastric, and rectal cancer to melanoma. BNT111 encoding the antigens, tyrosinase, NY-ESO-1, MAGE A3, and TPTE, is currently being investigated in melanoma. A Phase I trial (Lipo-MERIT) using liposomal mRNA, injected intravenously, in combination with or without the checkpoint inhibitor PD1, showed in an interim analysis induction of strong CD4 and CD8 T cell immunity against the vaccine antigens [197]. A Phase II trial with BNT111 ± cemiplimab is ongoing. Preliminary data from a Phase I/II trial of liposomal BNT112 encoding the five prostate cancer-specific antigens, kallikrein-2, kallikrein-3, acid phosphatase prostate, HOXB13, and NK3 homeobox 1, as monotherapy or in combination with cemiplimab, in metastatic castration resistant prostate cancer (mCRPC) showed that all five antigens were immunogenic and responses to each antigen were observed in at least two patients [198]. BNT121 was studied in melanoma. Thirteen metastatic patients received repeat administrations in inguinal lymph nodes showing clear immunological responses and some evidence of clinical activity [3]. Data from a Phase I trial in solid tumors with nine i.v. injections of BNT122 (RO7198457) encoding twenty patient-specific antigens have been reported by Braiteh [199]. BNT122 induced the pulsatile release of pro-inflammatory cytokines with each dose, consistent with the innate immune agonist activity of the RNA. Neoantigen-specific T cell responses were observed in peripheral blood in 14/16 patients (87%). Overall, 1 out of 26 patients had a complete response and 11 had a stable disease. mRNA-2416 is encoding the immune checkpoint modulator, OX40L, and is administered intratumorally. In a first trial with 41 patients with different malignancies, the compound did not meet the response criteria for solid tumors. A Phase II study in ovarian cancer in combination with durvalumab (NCT03323398) is ongoing [3]. mRNA-2752 encoding OX40L, IL-23, and IL-36γ was investigated in colorectal cancer (NCT03739931). However, in 17 patients there were no responses [198]. mRNA-4157 is a personalized mRNA encoding up to 34 neoantigens. In a Phase II solid tumor study, either as monotherapy (N = 16) or in combination with pembrolizumab (N = 63), multiple disease-free patients were observed during the study [199]. The safety and immunogenicity of mRNA-4650 encoding-defined neoantigens, mutations in driver genes, and HLA-I–predicted epitopes were determined in patients with metastatic gastrointestinal cancer (NCT03480152). CD8+ and CD4+ neoantigen-specific T cells elicited by the vaccine could be detected. However, since in 3 of 4 patients, no clinical response was observed, Phase II was not initiated [205]. TriMix, a mixture of monocyte-derived dendritic cells electroporated with mRNA encoding CD70, CD40 ligand, and constitutively active TLR4, as well as the tumor-associated antigens tyrosinase, gp100, MAGE-A3, or MAGE-C2 were administered together with ipilimumab in patients with advanced melanoma [206]. Enzyme-linked immunospot assay responses detected after in vitro T cell stimulation were shown in 12/15 patients. Vaccination in combination with ipilimumab resulted in robust CD8+ T cell responses in a meaningful portion of late-stage melanoma patients, and obviously in patients with a clinical response. TriMixDC-MEL, autologous monocyte-derived mRNA co-electroporated dendritic cells with mRNA encoding CD40L, CD70, and caTLR4, was administered i.v. to 21 late-stage melanoma patients. The control group without treatment comprised 20 patients. One year after randomization, 71% of patients in the study arm were alive and free of disease compared to 35% in the control arm [208]. The median time to non-salvageable recurrence was superior in the TriMixDC-MEL arm (median 8 months (range 1–6) vs. not reached. CV9103 (RNActive®) is based on four prostate-specific antigens, PSA, PSMA, PSCA, and STEAP and was administered intradermally in a Phase I trial in prostate cancer patients [210]. In a subsequent Phase II study, antigen-specific T cells were detected in around 80% of prostate carcinoma patients independent of their HLA background. A majority of immune responders, around 58%, reacted against multiple antigens, and the responses were detected against all antigens independent of their cellular localization. Individual patients were showing prolonged stabilization of PSA levels after initial rises. One patient had a greater than 85% drop in his PSA level [211]. CV9104 is a mixture of six mRNAs, each encoding one antigen, PSA, PSCA, PSMA, STEAP1, PAP, or MUC1. No significant difference in OS was found between the vaccine and control arm. There were also no significant differences in the rPFS endpoints and time to symptom progression [212]. CV9201 is an RNActive®-based cancer immunotherapy encoding five NSCLC antigens, NY-ESO-1, MAGE C1, MAGE C2, survivin, and TBG. In Phase IIa, antigen-specific immune responses against ≥1 antigen were detected in 63% of evaluable patients after treatment. The frequency of activated IgD CD38 B cells increased >2-fold. In total, 31% of evaluable patients in Phase IIa had a stable disease and 69% had a progressive disease [213]. MEDI1191, which is encoding IL-12, showed in a Phase I study in combination with durvalumab two partial responses in patients who had received prior immunotherapy and also had PD-L1-negative tumors [217]. The direct injection in 15 melanoma patients of naked mRNA encoding various antigens has been described by Weide et al. [218]. An increase in the anti-tumor humoral immune response was seen in some patients. However, a demonstration of the clinical effectiveness of the direct injection of copy mRNA for anti-tumor immunotherapy was not shown. Another Phase I trial in 30 renal cell cancer patients using naked mRNA coding for MUC1, CEA, Her-2/neu, telomerase, survivin, and MAGE-A1 has been published by Rittig et al. [219]. The induction of CD4+ and CD8+ T cell responses was shown for several TAAs. In summary, the clinical efficacy of mRNA vaccines looks like a mixed bag of different outcomes ranging from no effect to a rather good response, which means that the route to sufficient efficacy still is rather steep but, nevertheless, nourishes high hopes for the future.

4. Conclusions

The tremendous success and unbelievable speed in developing and bringing to the market COVID-19 mRNA vaccines has raised high expectations for duplicating this performance in other indications, in particular in cancer therapy. mRNA vaccines are easy to design and can rapidly be modified if there is a need for change, exhibiting an extremely broad versatility of building blocks, structural elements, and formulations of the synthetic mRNA including the targeting of defined cells, duration of expression, and immunological effects. Dosing can be adjusted according to the individual needs of the patients, and there is the possibility to individualize treatment by adjusting the selection of antigen(s) to the specific tumor type of each patient. However, the hurdles in cancer immunotherapy are much higher than in fighting coronaviruses. In particular, the following issues need to be addressed: neoantigen use, LNP modifications, combination treatments, immune escape of cancer, and the therapeutic vs. prophylactic use of mRNA vaccines.
If available, neoantigen-specific vaccines, which are currently considered a top priority in cancer immunotherapy, result in a potential killing of all cells exhibiting the neoantigen epitope but leave any other cancer cells unharmed (which might then start or continue multiplying). As a consequence, the cancer genotype and phenotype changes, and the tumor will continue growing [220]. Targeting multiple neoantigens within a single vaccine and/or combining the vaccine with non-mRNA tumor therapies should be targeted to reduce immune evasion and effectively eliminate tumors. Similarly, the general mechanisms for the immune escape of cancer, which are much more complex than those for breakthrough infections of pathogens [221], need to be addressed. Combinations of mRNA vaccines with agents that can reverse immunosuppression such as immune agonists or cytokines and compounds that block immune checkpoints have been shown to be more potent than a single administration of vaccine therapy [41,64,214,222,223]. However, not all patients are responsive to these treatments [224]. Nevertheless, this approach, and the combination of one or more non-mRNA agents fighting immune escape with multitarget mRNAs, should be continued even more extensively in the future.
LNPs are state-of-the-art for mRNA vaccines. However, they bear several pitfalls. Since they are particles, they can be taken up by macrophages of the liver or the spleen—irrespective of their administration route—thereby reducing the activity at the intended site of action, the tumor, and/or leading to side effects at off-target accumulation sites. The side effects might include toxicity and/or immunogenicity [150,225] and inflammation exacerbation [226]. The effects depend on various factors including the constituents of the vaccine carrier and the antigen. For example, cationic/ionizable lipids can induce inflammation by activating TLR pathways and lead to cell toxicity. On the other hand, this inflammatory effect is a useful contributor to the adjuvant activity of the LNP. Accordingly, the positive adjuvant activity and the negative inflammatory action need to be carefully balanced by selecting an appropriate cationic/ionizable lipid [227]. Other strategies have been described including the effort to make the particles invisible to macrophages by modifying their surface with, for example, PEG derivatives. The caveat for this solution has been illustrated above: the potential immunogenicity of PEG compounds [160]. Yet another possibility has been published by Dirisala [228], regarding the in situ stealth coating of liver sinusoids using linear or two-armed PEG–conjugated oligo(L-lysine). Another approach could be the reduction in the size of the particles. Small particles are no longer taken up by the liver or the spleen; they keep circulating in the blood, and therefore prolong the activity, as has been described for blood-pool contrast agents [229]. In summary, the mRNA vaccine carrier for cancer immunotherapy, in particular LNPs, needs further consideration to optimize activity and tolerability.
A further issue that needs to be addressed is the observation that vaccine treatment may not successfully reach the tumor site, in particular solid tumors [175]. Probably, cancer vaccine treatments are more suitable for patients with an undisturbed immune system, a relatively small tumor load, and a greater risk of recurrence.
The focus in cancer immunotherapy should also be directed towards prophylactic vaccination, which has already been very successful in HPV and HBV leading to cervical cancer and hepatocellular carcinoma. An extension to other tumor types seems mandatory.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflicts of Interest

Krause is a retired employee of Bayer AG, Berlin, Germany.

Abbreviations

1mΨN1-methyl-pseudouridine
ADAMDEC1ADAM like decysin 1
ADAMTSL4ADAMTS Like 4
ADAMTS18ADAM metallopeptidase with thrombospondin type 1 motif 18
ALC-01592-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide
ALC-0315((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)
ALDH3A2aldehyde dehydrogenase 3 family member A2
ALLacute lymphocytic leukemia
AP2S1AP-2 complex subunit sigma
AUNIPaurora kinase A and ninein interacting protein
BRAFB-RAF, B rapidly accelerated fibrosarcoma
caTLR4constitutively activated TLR4
CARD11caspase recruitment domain family member 11
CEAcarcinoembryonic antigen
CLLchronic lymphocytic leukemia
CMAconditional marketing authorization
COL10A1collagen type X alpha 1 chain
COL6A1collagen type VI alpha 1 chain
COVID-19coronavirus disease 2019
CpG 1018synthetic oligomer cytosine phospho-guanine
CPIcheckpoint inhibitor
CRCcolorectal cancer
CTSLcathepsin L
CYTH4cytohesin 4
DCdendritic cell
DLin-MC3-DMAdilinoleylmethyl-4-dimethylaminobutyrate
DSPC1,2-distearoyl-sn-glycero-3-phosphocholine
dsRNAdouble-stranded RNA
DOPE1,2-dioleoyl-sn-glycerol-3phosphoethanolamine
eIF4Eeukaryotic translation initiation factor 4E
EGFLAMEGF-like, fibronectin type III and laminin G domains
ESCCesophageal squameous cell carcinoma
FAM134Bfamily with sequence similarity 134, member B
FANCIFA complementation group I
FCRL4Fc receptor-like 4
flLAMPfull-length (fl) lysosomal-associated membrane protein (LAMP)
FN1fibronectin 1
GM-CSFgranulocyte-macrophage colony-stimulating factor
Her-2/neuhuman epidermal growth factor receptor 2
HLTFhelicase-like transcription factor
HBVHepatitis B
HOXB13homeobox B13
HPLChigh-pressure liquid chromatography
HPVhuman papillomavirus
IFNinterferon
ITGA10integrin subunit alpha 10
IVTin vitro transcription
KRASKirsten rat sarcoma virus
LASP1LIM And SH3 protein 1
LCP2lymphocyte cytosolic protein 2
LILRB1leukocyte immunoglobulin-like receptor B1
LSP1lymphocyte-specific protein 1
MAGE-A1melanoma-associated antigen family A1
MAGE-A3melanoma antigen family A3
MAGE-C1melanoma antigen family C1
MAGE-C2melanoma antigen family C2
MERSMiddle East respiratory syndrome
MERS-CoVMiddle East respiratory syndrome-related coronavirus
MHCmajor histocompatibility complex
MPZL2Myelin protein zero like 2
mRNAmessenger ribonucleic acid
MDSCmyeloid-derived suppressor cell
MUC1mucin 1
NHLnon-Hodgkin lymphoma
NLRC5NOD(nucleotide-binding oligomerization)-like receptor family CARD domain containing 5
NRASneuroblastoma RAS
NSCLCnon-small cell lung cancer
NY-ESO-1New York esophageal squamous cell carcinoma 1
ORFopen reading frame
OX40LOX40 ligand
P3H4prolyl 3-hydroxylase family member 4
PAPphosphatidate phosphatase
PEGpolyethylene glycol
PEG2000-DMG1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000
PKRprotein kinase R
PLCG1phospholipase C gamma 1
Poly(A)multiple adenosine units
PPEF1protein phosphatase with EF- protein phosphatase with EF-hand domain 1
PPRpentatricopeptide repeat
PSAprostate-specific antigen
PSCAprostate stem cell antigen
PSMAprostate-specific membrane antigen
PSMD8proteasome 26S subunit, non-ATPase 8
PTPRCprotein tyrosine phosphatase receptor type C
RAC3Rac family small GTPase 3
RBDreceptor-binding domain
RIG-Iretinoic acid-inducible gene I
RORCRAR-related orphan receptor C
SAA2serum amyloid A2
saRNAself-amplifying RNA
SARSsevere acute respiratory syndrome
SARS-CoV-2severe acute respiratory syndrome coronavirus 2
SAV1salvador family WW domain containing protein 1
SCLCsmall cell lung cancer
SIGLEC10sialic acid binding Ig like lectin 10
SM-102(heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate)
STEAP1six transmembrane epithelial antigen of the prostate 1
STRA6signaling receptor and transporter of retinol STRA6
taRNAtrans-amplifying RNA
TAAtumor-associated antigen
TPBGtrophoblast glycoprotein
TLRtoll-like receptor
TLR4toll like receptor 4
TLR7toll like receptor 7
TMEM229Btransmembrane protein 229B
TPTEtransmembrane phosphatase with tensin homology
TSAtumor-specific antigen
TTC3tetratricopeptide repeat domain 3
XPO5exportin 5

References

  1. Brenner, S.; Jacob, F.; Meselson, M. An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 1961, 190, 576–581. [Google Scholar] [CrossRef] [PubMed]
  2. Wolff, J.A.; Malone, R.W.; Williams, P.; Chong, W.; Acsadi, G.; Jani, A.; Felgner, P.L. Direct gene transfer into mouse muscle in vivo. Science 1990, 247, 1465–1468. [Google Scholar] [CrossRef]
  3. Barbier, A.J.; Jiang, A.Y.; Zhang, P.; Wooster, R.; Anderson, D.G. The clinical progress of mRNA vaccines and immunotherapies. Nat. Biotechnol. 2022, 40, 840–854. [Google Scholar] [CrossRef]
  4. Corbett, K.S.; Edwards, D.K.; Leist, S.R.; Abiona, O.M.; Boyoglu-Barnum, S.; Gillespie, R.A.; Himansu, S.; Schäfer, A.; Ziwawo, C.T.; DiPiazza, A.T.; et al. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature 2020, 586, 567–571. [Google Scholar] [CrossRef]
  5. Alexopoulou, L.; Holt, A.C.; Medzhitov, R.; Flavell, R.A. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 2001, 413, 732–738. [Google Scholar] [CrossRef]
  6. Heil, F.; Hemmi, H.; Hochrein, H.; Ampenberger, F.; Kirschning, C.; Akira, S.; Lipford, G.; Wagner, H.; Bauer, S. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 2004, 303, 1526–1529. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Rehwinkel, J.; Tan, C.P.; Goubau, D.; Schulz, O.; Pichlmair, A.; Bier, K.; Robb, N.; Vreede, F.; Barclay, W.; Fodor, E.; et al. RIG-I detects viral genomic RNA during negative-strand RNA virus infection. Cell 2010, 140, 397–408. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  8. Nallagatla, S.R.; Hwang, J.; Toroney, R.; Zheng, X.; Cameron, C.E.; Bevilacqua, P.C. 5’-Triphosphate-dependent activation of PKR by RNAs with short stem-loops. Science 2007, 318, 1455–1458. [Google Scholar] [CrossRef] [Green Version]
  9. Krammer, F. SARS-CoV-2 vaccines in development. Nature 2020, 586, 516–527. [Google Scholar] [CrossRef]
  10. Martin, J.E.; Louder, M.K.; Holman, L.A.; Gordon, I.J.; Enama, M.E.; Larkin, B.D.; Andrews, C.A.; Vogel, L.; Koup, R.A.; Roederer, M.; et al. A SARS DNA vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a Phase I clinical trial. Vaccine 2008, 26, 6338–6343. [Google Scholar] [CrossRef]
  11. Yong, C.Y.; Ong, H.K.; Yeap, S.K.; Ho, K.L.; Tan, W.S. Recent advances in the vaccine development against middle east respiratory syndrome-coronavirus. Front. Microbiol. 2019, 10, 1781. [Google Scholar] [CrossRef] [Green Version]
  12. Letko, M.; Marzi, A.; Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol. 2020, 5, 562–569. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Alsoussi, W.B.; Turner, J.S.; Case, J.B.; Zhao, H.; Schmitz, A.J.; Zhou, J.Q.; Chen, R.E.; Lei, T.; Rizk, A.A.; McIntire, K.M.; et al. A potently neutralizing antibody protects mice against SARS-CoV-2 infection. J. Immunol. 2020, 205, 915–922. [Google Scholar] [CrossRef] [PubMed]
  14. Deng, W.; Bao, L.; Liu, J.; Xiao, C.; Liu, J.; Xue, J.; Lv, Q.; Qi, F.; Gao, H.; Yu, P.; et al. Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques. Science 2020, 369, 818–823. [Google Scholar] [CrossRef]
  15. Yu, J.; Tostanoski, L.H.; Peter, L.; Mercado, N.B.; McMahan, K.; Mahrokhian, S.H.; Nkolola, J.P.; Liu, J.; Li, Z.; Chandrashekar, A.; et al. DNA vaccine protection against SARS-CoV-2 in rhesus macaques. Science 2020, 369, 806–811. [Google Scholar] [CrossRef] [PubMed]
  16. Su, F.; Patel, G.B.; Hu, S.; Chen, W. Induction of mucosal immunity through systemic immunization: Phantom or reality? Hum. Vaccines Immunother. 2016, 12, 1070–1079. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Shuman, S. What messenger RNA capping tells us about eukaryotic evolution. Nat. Rev. Mol. Cell Biol. 2002, 3, 619–625. [Google Scholar] [CrossRef]
  18. Cao, J.; He, L.; Lin, G.; Hu, C.; Dong, R.; Zhang, J.; Zhu, H.; Hu, Y.; Wagner, C.R.; He, Q.; et al. Cap-dependent translation initiation factor, eIF4E, is the target for Ouabain-mediated inhibition of HIF-1α. Biochem. Pharmacol. 2014, 89, 20–30. [Google Scholar] [CrossRef] [PubMed]
  19. Vaidyanathan, S.; Azizian, K.T.; Haque, A.A.; Henderson, J.M.; Hendel, A.; Shore, S.; Antony, J.S.; Hogrefe, R.I.; Kormann, M.S.; Porteus, M.H.; et al. Uridine depletion and chemical modification increase Cas9 mRNA activity and reduce immunogenicity without HPLC purification. Mol. Ther.-Nucleic Acids 2018, 12, 530–542. [Google Scholar] [CrossRef]
  20. Pascolo, S. Synthetic messenger RNA-based vaccines: From scorn to hype. Viruses 2021, 13, 270. [Google Scholar] [CrossRef]
  21. Muttach, F.; Muthmann, N.; Rentmeister, A. Synthetic mRNA capping. Beilstein J. Org. Chem. 2017, 13, 2819–2832. [Google Scholar] [CrossRef] [PubMed]
  22. Kocmik, I.; Piecyk, K.; Rudzinska, M.; Niedzwiecka, A.; Darzynkiewicz, E.; Grzela, R.; Jankowska-Anyszka, M. Modified ARCA analogs providing enhanced translational properties of capped mRNAs. Cell Cycle 2018, 17, 1624–1636. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Ross, J. mRNA stability in mammalian cells. Microbiol. Rev. 1995, 59, 423–450. [Google Scholar] [CrossRef] [PubMed]
  24. Gallie, D.R. The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 1991, 5, 2108–2116. [Google Scholar] [CrossRef] [Green Version]
  25. Holtkamp, S.; Kreiter, S.; Selmi, A.; Simon, P.; Koslowski, M.; Huber, C.; Türeci, O.; Sahin, U. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 2006, 108, 4009–4017. [Google Scholar] [CrossRef]
  26. Warren, L.; Manos, P.D.; Ahfeldt, T.; Loh, Y.-H.; Li, H.; Lau, F.; Ebina, W.; Mandal, P.K.; Smith, Z.D.; Meissner, A.; et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 2010, 7, 618–630. [Google Scholar] [CrossRef] [Green Version]
  27. Miao, L.; Zhang, Y.; Huang, L. mRNA vaccine for cancer immunotherapy. Mol. Cancer 2021, 20, 1–23. [Google Scholar] [CrossRef]
  28. Pickering, B.M.; Willis, A.E. The implications of structured 5’ untranslated regions on translation and disease. In Seminars in Cell & Developmental Biology; Academic Press: Cambridge, MA, USA, 2005; Volume 16, pp. 39–47. [Google Scholar] [CrossRef]
  29. Linares-Fernández, S.; Lacroix, C.; Exposito, J.-Y.; Verrier, B. Tailoring mRNA vaccine to balance innate/adaptive immune response. Trends Mol. Med. 2019, 26, 311–323. [Google Scholar] [CrossRef]
  30. Mauger, D.M.; Cabral, B.J.; Presnyak, V.; Su, S.V.; Reid, D.W.; Goodman, B. mRNA structure regulates protein expression through changes in functional half-life. Proc. Natl. Acad. Sci. USA 2019, 116, 24075–24083. [Google Scholar] [CrossRef] [Green Version]
  31. Kudla, G.; Lipinski, L.; Caffin, F.; Helwak, A.; Zylicz, M. High guanine and cytosine content increases mRNA levels in mammalian cells. PLoS Biol. 2006, 4, e180. [Google Scholar] [CrossRef]
  32. Karikó, K.; Buckstein, M.; Ni, H.; Weissman, D. Suppression of RNA recognition by toll-like receptors: The impact of nucleoside modification and the evolutionary origin of RNA. Immunity 2005, 23, 165–175. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Weng, Y.; Li, C.; Yang, T.; Hu, B.; Zhang, M.; Guo, S.; Xiao, H.; Liang, X.-J.; Huang, Y. The challenge and prospect of mRNA therapeutics landscape. Biotechnol. Adv. 2020, 40, 107534. [Google Scholar] [CrossRef] [PubMed]
  34. Orlandini von Niessen, A.G.O.; Poleganov, M.A.; Rechner, C.; Plaschke, A.; Kranz, L.M.; Fesser, S.; Diken, M.; Löwer, M.; Vallazza, B.; Beissert, T.; et al. Improving mRNA-based therapeutic gene delivery by expression-augmenting 3′ UTRs identified by cellular library screening. Mol. Ther. 2018, 27, 824–836. [Google Scholar] [CrossRef] [Green Version]
  35. Schwanhäusser, B.; Busse, D.; Li, N.; Dittmar, G.; Schuchhardt, J.; Wolf, J.; Chen, W.; Selbach, M. Global quantification of mammalian gene expression control. Nature 2011, 473, 337–342. [Google Scholar] [CrossRef] [Green Version]
  36. Tanguay, R.M.; Jorquera, R.; Poudrier, J.; St-Louis, M. Tyrosine and its catabolites: From disease to cancer. Acta Biochim. Pol. 1996, 43, 209–216. [Google Scholar] [CrossRef] [Green Version]
  37. Mangus, D.A.; Evans, M.C.; Jacobson, A. Poly(A)-binding proteins: Multifunctional scaffolds for the post-transcriptional control of gene expression. Genome Biol. 2003, 4, 223. [Google Scholar] [CrossRef] [Green Version]
  38. Roy, B.; Jacobson, A. The intimate relationships of mRNA decay and translation. Trends Genet. 2013, 29, 691–699. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  39. Jalkanen, A.L.; Coleman, S.J.; Wilusz, J. Determinants and implications of mRNA poly(A) tail size—Does this protein make my tail look big? Semin. Cell Dev. Biol. 2014, 34, 24–32. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  40. Lima, S.A.; Chipman, L.B.; Nicholson, A.L.; Chen, Y.-H.; Yee, B.A.; Yeo, E.; Coller, J.; Pasquinelli, A.E. Short poly(A) tails are a conserved feature of highly expressed genes. Nat. Struct. Mol. Biol. 2017, 24, 1057–1063. [Google Scholar] [CrossRef] [Green Version]
  41. Bidram, M.; Zhao, Y.; Shebardina, N.G.; Baldin, A.V.; Bazhin, A.V.; Ganjalikhany, M.R.; Zamyatnin, A.A.; Ganjalikhani-Hakemi, M. mRNA-based cancer vaccines: A therapeutic strategy for the treatment of melanoma patients. Vaccines 2021, 9, 1060. [Google Scholar] [CrossRef]
  42. Ziemniak, M.; Strenkowska, M.; Kowalska, J.; Jemielity, J. Potential therapeutic applications of RNA cap analogs. Future Med. Chem. 2013, 5, 1141–1172. [Google Scholar] [CrossRef] [PubMed]
  43. Kuhn, A.N.; Diken, M.; Kreiter, S.; Selmi, A.; Kowalska, J.; Jemielity, J.; Darzynkiewicz, E.; Huber, C.; Türeci, Ö.; Sahin, U. Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Ther. 2010, 17, 961–971. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Kowalski, P.S.; Rudra, A.; Miao, L.; Anderson, D.G. Delivering the messenger: Advances in technologies for therapeutic mRNA delivery. Mol. Ther. 2019, 27, 710–728. [Google Scholar] [CrossRef] [Green Version]
  45. Elfakess, R.; Dikstein, R. A translation initiation element specific to mRNAs with very short 5′UTR that also regulates transcription. PLoS ONE 2008, 3, e3094. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Karikó, K.; Muramatsu, H.; Keller, J.M.; Weissman, D. Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol. Ther. 2012, 20, 948–953. [Google Scholar] [CrossRef] [Green Version]
  47. Van Gulck, E.R.A.; Ponsaerts, P.; Heyndrickx, L.; Vereecken, K.; Moerman, F.; De Roo, A.; Colebunders, R.; Van den Bosch, G.; Van Bockstaele, D.R.; Van Tendeloo, V.F.I.; et al. Efficient stimulation of HIV-1-specific T cells using dendritic cells electroporated with mRNA encoding autologous HIV-1 Gag and Env proteins. Blood 2006, 107, 1818–1827. [Google Scholar] [CrossRef] [PubMed]
  48. Ballesteros-Briones, M.C.; Silva-Pilipich, N.; Herrador-Cañete, G.; Vanrell, L.; Smerdou, C. A new generation of vaccines based on alphavirus self-amplifying RNA. Curr. Opin. Virol. 2020, 44, 145–153. [Google Scholar] [CrossRef]
  49. Lundstrom, K. Self-amplifying RNA viruses as RNA vaccines. Int. J. Mol. Sci. 2020, 21, 5130. [Google Scholar] [CrossRef]
  50. Bloom, K.; van den Berg, F.; Arbuthnot, P. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 2021, 28, 117–129. [Google Scholar] [CrossRef]
  51. Vogel, A.B.; Lambert, L.; Kinnear, E.; Busse, D.; Erbar, S.; Reuter, K.C.; Wicke, L.; Perkovic, M.; Beissert, T.; Haas, H.; et al. Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses. Mol. Ther. 2018, 26, 446–455. [Google Scholar] [CrossRef] [Green Version]
  52. Ljungberg, K.; Liljeström, P. Self-replicating alphavirus RNA vaccines. Expert Rev. Vaccines 2015, 14, 177–194. [Google Scholar] [CrossRef] [PubMed]
  53. Zhou, X.; Berglund, P.; Rhodes, G.; Parker, S.; Jondal, M.; Liljeström, P. Self-replicating Semliki Forest virus RNA as recombinant vaccine. Vaccine 1994, 12, 1510–1514. [Google Scholar] [CrossRef] [PubMed]
  54. Chakraborty, C.; Sharma, A.R.; Bhattacharya, M.; Lee, S.-S. From COVID-19 to cancer mRNA vaccines: Moving from bench to clinic in the vaccine landscape. Front. Immunol. 2021, 12, 679344. [Google Scholar] [CrossRef] [PubMed]
  55. Deering, R.P.; Kommareddy, S.; Ulmer, J.B.; Brito, L.A.; Geall, A.J. Nucleic acid vaccines: Prospects for non-viral delivery of mRNA vaccines. Expert Opin. Drug Deliv. 2014, 11, 885–899. [Google Scholar] [CrossRef] [PubMed]
  56. Pardi, N.; Hogan, M.J.; Porter, F.W.; Weissman, D. mRNA vaccines—A new era in vaccinology. Nat. Rev. Drug Discov. 2018, 17, 261–279. [Google Scholar] [CrossRef] [Green Version]
  57. Sahin, U.; Karikó, K.; Türeci, Ö. mRNA-based therapeutics—Developing a new class of drugs. Nat. Rev. Drug Discov. 2014, 13, 759–780. [Google Scholar] [CrossRef]
  58. Beissert, T.; Perkovic, M.; Vogel, A.; Erbar, S.; Walzer, K.C.; Hempel, T.; Brill, S.; Haefner, E.; Becker, R.; Türeci, Ö.; et al. A trans-amplifying RNA vaccine strategy for induction of potent protective immunity. Mol. Ther. 2020, 28, 119–128. [Google Scholar] [CrossRef] [PubMed]
  59. Tregoning, J.S.; Kinnear, E. Using plasmids as DNA vaccines for infectious diseases. Microbiol. Spectr. 2014, 2, 651–668. [Google Scholar] [CrossRef]
  60. Xu, S.; Yang, K.; Li, R.; Zhang, L. mRNA vaccine era—Mechanisms, drug platform and clinical prospection. Int. J. Mol. Sci. 2020, 21, 6582. [Google Scholar] [CrossRef] [PubMed]
  61. Jackson, N.A.C.; Kester, K.E.; Casimiro, D.; Gurunathan, S.; DeRosa, F. The promise of mRNA vaccines: A biotech and industrial perspective. NPJ Vaccines 2020, 5, 11. [Google Scholar] [CrossRef] [Green Version]
  62. Blakney, A.K.; Zhu, Y.; McKay, P.F.; Bouton, C.; Yeow, J.; Tang, J.; Hu, K.; Samnuan, K.; Grigsby, C.; Shattock, R.J.; et al. Big is beautiful: Enhanced saRNA delivery and immunogenicity by a higher molecular weight, bioreducible, cationic polymer. ACS Nano 2020, 14, 5711–5727. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Chen, J.; Ye, Z.; Huang, C.; Qiu, M.; Song, D.; Li, Y.; Xu, Q. Lipid nanoparticle-mediated lymph node–targeting delivery of mRNA cancer vaccine elicits robust CD8+ T cell response. Proc. Natl. Acad. Sci. USA 2022, 119, e2207841119. [Google Scholar] [CrossRef] [PubMed]
  64. Wang, Y.; Wang, M.; Wu, H.; Xu, R. Advancing to the era of cancer immunotherapy. Cancer Commun. 2021, 41, 803–829. [Google Scholar] [CrossRef]
  65. Lorenz, C.; Fotin-Mleczek, M.; Roth, G.; Becker, C.; Dam, T.C.; Verdurmen, W.P.R.; Brock, R.; Probst, J.; Schlake, T. Protein expression from exogenous mRNA: Uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway. RNA Biol. 2011, 8, 627–636. [Google Scholar] [CrossRef] [PubMed]
  66. Selmi, A.; Vascotto, F.; Kautz-Neu, K.; Türeci, Ö.; Sahin, U.; von Stebut, E.; Diken, M.; Kreiter, S. Uptake of synthetic naked RNA by skin-resident dendritic cells via macropinocytosis allows antigen expression and induction of T-cell responses in mice. Cancer Immunol. Immunother. 2016, 65, 1075–1083. [Google Scholar] [CrossRef]
  67. Dirisala, A.; Uchida, S.; Tockary, T.A.; Yoshinaga, N.; Li, J.; Osawa, S.; Gorantla, L.; Fukushima, S.; Osada, K.; Kataoka, K. Precise tuning of disulphide crosslinking in mRNA polyplex micelles for optimising extracellular and intracellular nuclease tolerability. J. Drug Target. 2019, 27, 670–680. [Google Scholar] [CrossRef]
  68. Yen, A.; Cheng, Y.; Sylvestre, M.; Gustafson, H.H.; Puri, S.; Pun, S.H. Serum nuclease susceptibility of mRNA cargo in condensed polyplexes. Mol. Pharm. 2018, 15, 2268–2276. [Google Scholar] [CrossRef]
  69. Aldosari, B.; Alfagih, I.; Almurshedi, A. Lipid nanoparticles as delivery systems for RNA-based vaccines. Pharmaceutics 2021, 13, 206. [Google Scholar] [CrossRef]
  70. Jayaraman, M.; Ansell, S.M.; Mui, B.L.; Tam, Y.K.; Chen, J.; Du, X.; Butler, D.; Eltepu, L.; Matsuda, S.; Narayanannair, J.K.; et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem. Int. Ed. 2012, 51, 8529–8533. [Google Scholar] [CrossRef]
  71. Kauffman, K.J.; Dorkin, J.R.; Yang, J.H.; Heartlein, M.W.; DeRosa, F.; Mir, F.F.; Fenton, O.S.; Anderson, D.G. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett. 2015, 15, 7300–7306. [Google Scholar] [CrossRef]
  72. Lokugamage, M.P.; Gan, Z.; Zurla, C.; Levin, J.; Islam, F.Z.; Kalathoor, S.; Sato, M.; Sago, C.D.; Santangelo, P.J.; Dahlman, J.E. Mild innate immune activation overrides efficient nanoparticle-mediated RNA delivery. Adv. Mater. 2019, 32, e1904905. [Google Scholar] [CrossRef]
  73. Lubich, C.; Allacher, P.; de la Rosa, M.; Bauer, A.; Prenninger, T.; Horling, F.M.; Siekmann, J.; Oldenburg, J.; Scheiflinger, F.; Reipert, B.M. The mystery of antibodies against polyethylene glycol (PEG)—What do we know? Pharm. Res. 2016, 33, 2239–2249. [Google Scholar] [CrossRef]
  74. Yanez Arteta, M.; Kjellman, T.; Bartesaghi, S.; Wallin, S.; Wu, X.; Kvist, A.J.; Dabkowska, A.; Székely, N.; Radulescu, A.; Bergenholtz, J.; et al. Successful reprogramming of cellular protein production through mRNA delivered by functionalized lipid nanoparticles. Proc. Natl. Acad. Sci. USA 2018, 115, E3351–E3360. [Google Scholar] [CrossRef] [Green Version]
  75. Miao, L.; Lin, J.; Huang, Y.; Li, L.; Delcassian, D.; Ge, Y.; Shi, Y.; Anderson, D.G. Synergistic lipid compositions for albumin receptor mediated delivery of mRNA to the liver. Nat. Commun. 2020, 11, 2424. [Google Scholar] [CrossRef] [PubMed]
  76. Gooden, M.; Lampen, M.; Jordanova, E.S.; Leffers, N.; Trimbos, J.B.; van der Burg, S.H.; Nijman, H.; van Hall, T. HLA-E expression by gynecological cancers restrains tumor-infiltrating CD8+ T lymphocytes. Proc. Natl. Acad. Sci. USA 2011, 108, 10656–10661. [Google Scholar] [CrossRef] [Green Version]
  77. Ur Rehman, Z.; Hoekstra, D.; Zuhorn, I.S. Mechanism of polyplex- and lipoplex-mediated delivery of nucleic acids: Real-time visualization of transient membrane destabilization without endosomal lysis. ACS Nano 2013, 7, 3767–3777. [Google Scholar] [CrossRef] [PubMed]
  78. Vermeulen, L.M.P.; Brans, T.; Samal, S.K.; Dubruel, P.; Demeester, J.; De Smedt, S.C.; Remaut, K.; Braeckmans, K. Endosomal size and membrane leakiness influence proton sponge-based rupture of endosomal vesicles. ACS Nano 2018, 12, 2332–2345. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Dimitriadis, G.J. Entrapment of ribonucleic acids in liposomes. FEBS Lett. 1978, 86, 289–293. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  80. Li, B.; Zhang, X.; Dong, Y. Nanoscale platforms for messenger RNA delivery. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2019, 11, e1530. [Google Scholar] [CrossRef]
  81. Ma, Z.; Li, J.; He, F.; Wilson, A.; Pitt, B.; Li, S. Cationic lipids enhance siRNA-mediated interferon response in mice. Biochem. Biophys. Res. Commun. 2005, 330, 755–759. [Google Scholar] [CrossRef]
  82. Kowalski, P.S.; Bhattacharya, C.; Afewerki, S.; Langer, R.S. Smart biomaterials: Recent advances and future directions. ACS Biomater. Sci. Eng. 2018, 4, 3809–3817. [Google Scholar] [CrossRef] [PubMed]
  83. Ping, Y.; Wu, D.; Kumar, J.N.; Cheng, W.; Lay, C.L.; Liu, Y. Redox-responsive hyperbranched poly(amido amine)s with tertiary amino cores for gene delivery. Biomacromolecules 2013, 14, 2083–2094. [Google Scholar] [CrossRef] [PubMed]
  84. Bell, G.D.; Yang, Y.; Leung, E.; Krissansen, G.W. mRNA transfection by a Xentry-protamine cell-penetrating peptide is enhanced by TLR antagonist E6446. PLoS ONE 2018, 13, e0201464. [Google Scholar] [CrossRef] [Green Version]
  85. Sedic, M.; Senn, J.J.; Lynn, A.; Laska, M.; Smith, M.; Platz, S.J.; Bolen, J.; Hoge, S.; Bulychev, A.; Jacquinet, E.; et al. Safety Evaluation of lipid nanoparticle–formulated modified mRNA in the sprague-dawley rat and cynomolgus monkey. Vet. Pathol. 2018, 55, 341–354. [Google Scholar] [CrossRef]
  86. Jorritsma, S.; Gowans, E.; Grubor-Bauk, B.; Wijesundara, D. Delivery methods to increase cellular uptake and immunogenicity of DNA vaccines. Vaccine 2016, 34, 5488–5494. [Google Scholar] [CrossRef]
  87. Alu, A.; Chen, L.; Lei, H.; Wei, Y.; Tian, X.; Wei, X. Intranasal COVID-19 vaccines: From bench to bed. EbioMedicine 2022, 76, 103841. [Google Scholar] [CrossRef]
  88. Rosa, S.S.; Prazeres, D.M.F.; Azevedo, A.M.; Marques, M.P.C. mRNA vaccines manufacturing: Challenges and bottlenecks. Vaccine 2021, 39, 2190–2200. [Google Scholar] [CrossRef]
  89. John, S.; Yuzhakov, O.; Woods, A.; Deterling, J.; Hassett, K.; Shaw, C.A.; Ciaramella, G. Multi-antigenic human cytomegalovirus mRNA vaccines that elicit potent humoral and cell-mediated immunity. Vaccine 2018, 36, 1689–1699. [Google Scholar] [CrossRef]
  90. Karikó, K.; Muramatsu, H.; Ludwig, J.; Weissman, D. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res. 2011, 39, e142. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  91. Weissman, D.; Pardi, N.; Muramatsu, H.; Karikó, K. HPLC purification of in vitro transcribed long RNA. Methods Mol Biol. 2013, 969, 43–54. [Google Scholar] [CrossRef] [PubMed]
  92. Shivalingam, A.; Taemaitree, L.; El-Sagheer, A.H.; Brown, T. Squaramides and ureas: A flexible approach to polymerase-compatible nucleic acid assembly. Angew. Chem. Int. Ed. 2020, 59, 11416–11422. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  93. Fiolet, T.; Kherabi, Y.; MacDonald, C.-J.; Ghosn, J.; Peiffer-Smadja, N. Comparing COVID-19 vaccines for their characteristics, efficacy and effectiveness against SARS-CoV-2 and variants of concern: A narrative review. Clin. Microbiol. Infect. 2022, 28, 202–221. [Google Scholar] [CrossRef] [PubMed]
  94. Abu Mouch, S.; Roguin, A.; Hellou, E.; Ishai, A.; Shoshan, U.; Mahamid, L.; Zoabi, M.; Aisman, M.; Goldschmid, N.; Yanay, N.B. Myocarditis following COVID-19 mRNA vaccination. Vaccine 2021, 39, 3790–3793. [Google Scholar] [CrossRef] [PubMed]
  95. Muthukumar, A.; Narasimhan, M.; Li, Q.-Z.; Mahimainathan, L.; Hitto, I.; Fuda, F.; Batra, K.; Jiang, X.; Zhu, C.; Schoggins, J.; et al. In-depth evaluation of a case of presumed myocarditis after the second dose of COVID-19 mRNA vaccine. Circulation 2021, 144, 487–498. [Google Scholar] [CrossRef]
  96. Zhao, P.; Hou, X.; Yan, J.; Du, S.; Xue, Y.; Li, W.; Xiang, G.; Dong, Y. Long-term storage of lipid-like nanoparticles for mRNA delivery. Bioact. Mater. 2020, 5, 358–363. [Google Scholar] [CrossRef]
  97. Crommelin, D.J.; Anchordoquy, T.J.; Volkin, D.B.; Jiskoot, W.; Mastrobattista, E. Addressing the cold reality of mRNA vaccine stability. J. Pharm. Sci. 2021, 110, 997–1001. [Google Scholar] [CrossRef]
  98. Ahmed, T. Immunotherapy for neuroblastoma using mRNA vaccines. Adv. Cancer Biol.—Metastasis 2022, 4, 100033. [Google Scholar] [CrossRef]
  99. Islam, M.A.; Rice, J.; Reesor, E.; Zope, H.; Tao, W.; Lim, M.; Ding, J.; Chen, Y.; Aduluso, D.; Zetter, B.R.; et al. Adjuvant-pulsed mRNA vaccine nanoparticle for immunoprophylactic and therapeutic tumor suppression in mice. Biomaterials 2021, 266, 120431. [Google Scholar] [CrossRef]
  100. Jahanafrooz, Z.; Baradaran, B.; Mosafer, J.; Hashemzaei, M.; Rezaei, T.; Mokhtarzadeh, A.; Hamblin, M.R. Comparison of DNA and mRNA vaccines against cancer. Drug Discov. Today 2020, 25, 552–560. [Google Scholar] [CrossRef]
  101. Heine, A.; Juranek, S.; Brossart, P. Clinical and immunological effects of mRNA vaccines in malignant diseases. Mol. Cancer 2021, 20, 52. [Google Scholar] [CrossRef]
  102. Kramps, T.; Elbers, K. Introduction to RNA vaccines. Methods Mol Biol. 2017, 1499, 1–11. [Google Scholar] [CrossRef] [PubMed]
  103. Oh, S.J.; Lee, J.; Kim, Y.; Song, K.-H.; Cho, E.; Kim, M.; Jung, H.; Kim, T.W. Far beyond cancer immunotherapy: Reversion of multi-malignant phenotypes of immunotherapeutic-resistant cancer by targeting the NANOG signaling axis. Immune Netw. 2020, 20, e7. [Google Scholar] [CrossRef] [PubMed]
  104. Yang, Q.; Guo, N.; Zhou, Y.; Chen, J.; Wei, Q.; Han, M. The role of tumor-associated macrophages (TAMs) in tumor progression and relevant advance in targeted therapy. Acta Pharm. Sin. B 2020, 10, 2156–2170. [Google Scholar] [CrossRef] [PubMed]
  105. Liu, C.; Papukashvili, D.; Dong, Y.; Wang, X.; Hu, X.; Yang, N.; Cai, J.; Xie, F.; Rcheulishvili, N.; Wang, P.G. Identification of tumor antigens and design of mRNA vaccine for colorectal cancer based on the immune subtype. Front. Cell Dev. Biol. 2022, 9, 783527. [Google Scholar] [CrossRef]
  106. Chesney, J.A.; Mitchell, R.A.; Yaddanapudi, K. Myeloid-derived suppressor cells—A new therapeutic target to overcome resistance to cancer immunotherapy. J. Leukoc. Biol. 2017, 102, 727–740. [Google Scholar] [CrossRef] [Green Version]
  107. Zhou, J.; Tang, Z.; Gao, S.; Li, C.; Feng, Y.; Zhou, X. Tumor-associated macrophages: Recent insights and therapies. Front. Oncol. 2020, 10, 188. [Google Scholar] [CrossRef]
  108. Togashi, Y.; Shitara, K.; Nishikawa, H. Regulatory T cells in cancer immunosuppression—Implications for anticancer therapy. Nat. Rev. Clin. Oncol. 2019, 16, 356–371. [Google Scholar] [CrossRef]
  109. Galluzzi, L.; Chan, T.A.; Kroemer, G.; Wolchok, J.D.; López-Soto, A. The hallmarks of successful anticancer immunotherapy. Sci. Transl. Med. 2018, 10, eaat7807. [Google Scholar] [CrossRef]
  110. Cassetta, L.; Kitamura, T. Macrophage targeting: Opening new possibilities for cancer immunotherapy. Immunology 2018, 155, 285–293. [Google Scholar] [CrossRef]
  111. Saeed, M.; Gao, J.; Shi, Y.; Lammers, T.; Yu, H. Engineering nanoparticles to reprogram the tumor immune microenvironment for improved cancer immunotherapy. Theranostics 2019, 9, 7981–8000. [Google Scholar] [CrossRef]
  112. Riley, R.S.; June, C.H.; Langer, R.; Mitchell, M.J. Delivery technologies for cancer immunotherapy. Nat. Rev. Drug Discov. 2019, 18, 175–196. [Google Scholar] [CrossRef] [PubMed]
  113. Thomas, S.; Prendergast, G.C. Cancer vaccines: A brief overview. Methods Mol Biol. 2016, 1403, 755–761. [Google Scholar] [CrossRef]
  114. Cheever, M.A.; Higano, C.S. PROVENGE (Sipuleucel-T) in prostate cancer: The first FDA-approved therapeutic cancer vaccine. Clin. Cancer Res. 2011, 17, 3520–3526. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  115. Handy, C.E.; Antonarakis, E.S. Sipuleucel-T for the treatment of prostate cancer: Novel insights and future directions. Futur. Oncol. 2018, 14, 907–917. [Google Scholar] [CrossRef] [PubMed]
  116. Song, Q.; Zhang, C.-D.; Wu, X.-H. Therapeutic cancer vaccines: From initial findings to prospects. Immunol. Lett. 2018, 196, 11–21. [Google Scholar] [CrossRef]
  117. Tsai, H.-J. Clinical cancer chemoprevention: From the hepatitis B virus (HBV) vaccine to the human papillomavirus (HPV) vaccine. Taiwan. J. Obstet. Gynecol. 2015, 54, 112–115. [Google Scholar] [CrossRef] [Green Version]
  118. Wei, J.; Hui, A.-M. The paradigm shift in treatment from COVID-19 to oncology with mRNA vaccines. Cancer Treat. Rev. 2022, 107, 102405. [Google Scholar] [CrossRef]
  119. Abbott, M.; Ustoyev, Y. Cancer and the immune system: The history and background of immunotherapy. Semin. Oncol. Nurs. 2019, 35, 150923. [Google Scholar] [CrossRef]
  120. Zhang, Y.; Zhang, Z. The history and advances in cancer immunotherapy: Understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications. Cell. Mol. Immunol. 2020, 17, 807–821. [Google Scholar] [CrossRef]
  121. Velcheti, V.; Schalper, K. Basic Overview of current immunotherapy approaches in cancer. Am. Soc. Clin. Oncol. Educ. Book 2016, 35, 298–308. [Google Scholar] [CrossRef]
  122. Tay, B.; Wright, Q.; Ladwa, R.; Perry, C.; Leggatt, G.; Simpson, F.; Wells, J.; Panizza, B.; Frazer, I.; Cruz, J. Evolution of cancer vaccines—Challenges, achievements, and future directions. Vaccines 2021, 9, 535. [Google Scholar] [CrossRef]
  123. Maiorano, B.; Schinzari, G.; Ciardiello, D.; Rodriquenz, M.; Cisternino, A.; Tortora, G.; Maiello, E. Cancer vaccines for genitourinary tumors: Recent progresses and future possibilities. Vaccines 2021, 9, 623. [Google Scholar] [CrossRef] [PubMed]
  124. Donninger, H.; Li, C.; Eaton, J.; Yaddanapudi, K. Cancer vaccines: Promising therapeutics or an unattainable dream. Vaccines 2021, 9, 668. [Google Scholar] [CrossRef] [PubMed]
  125. Janczar, S.; Bulas, M.; Walenciak, J.; Baranska, D.; Ussowicz, M.; Młynarski, W.; Zalewska-Szewczyk, B. Pulmonary exacerbation of undiagnosed toxocariasis in intensively-treated high-risk neuroblastoma patients. Children 2020, 7, 169. [Google Scholar] [CrossRef] [PubMed]
  126. Kim, C.-G.; Sang, Y.-B.; Lee, J.-H.; Chon, H.-J. Combining cancer vaccines with immunotherapy: Establishing a new immunological approach. Int. J. Mol. Sci. 2021, 22, 8035. [Google Scholar] [CrossRef]
  127. Gautam, A.; Beiss, V.; Wang, C.; Wang, L.; Steinmetz, N.F. Plant viral nanoparticle conjugated with anti-PD-1 peptide for ovarian cancer immunotherapy. Int. J. Mol. Sci. 2021, 22, 9733. [Google Scholar] [CrossRef]
  128. Liu, C.-C.; Yang, H.; Zhang, R.; Zhao, J.-J.; Hao, D.-J. Tumour-associated antigens and their anti-cancer applications. Eur. J. Cancer Care 2017, 26, e12446. [Google Scholar] [CrossRef]
  129. Salomon, N.; Vascotto, F.; Selmi, A.; Vormehr, M.; Quinkhardt, J.; Bukur, T.; Schrörs, B.; Löewer, M.; Diken, M.; Türeci, Ö.; et al. A liposomal RNA vaccine inducing neoantigen-specific CD4+ T cells augments the antitumor activity of local radiotherapy in mice. Oncoimmunology 2020, 9, 1771925. [Google Scholar] [CrossRef]
  130. Hollingsworth, R.E.; Jansen, K. Turning the corner on therapeutic cancer vaccines. NPJ Vaccines 2019, 4, 7. [Google Scholar] [CrossRef] [Green Version]
  131. Melero, I.; Gaudernack, G.; Gerritsen, W.; Huber, C.; Parmiani, G.; Scholl, S.; Thatcher, N.; Wagstaff, J.; Zielinski, C.; Faulkner, I.; et al. Therapeutic vaccines for cancer: An overview of clinical trials. Nat. Rev. Clin. Oncol. 2014, 11, 509–524. [Google Scholar] [CrossRef]
  132. Yarchoan, M.; Johnson, B.A., III; Lutz, E.R.; Laheru, D.A.; Jaffee, E.M. Targeting neoantigens to augment antitumour immunity. Nat. Rev. Cancer 2017, 17, 209–222. [Google Scholar] [CrossRef] [PubMed]
  133. Li, L.; Goedegebuure, S.; Gillanders, W. Preclinical and clinical development of neoantigen vaccines. Ann. Oncol. 2017, 28, xii11–xii17. [Google Scholar] [CrossRef]
  134. Srivastava, P.K. Neoepitopes of cancers: Looking back, looking ahead. Cancer Immunol. Res. 2015, 3, 969–977. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  135. Blass, E.; Ott, P.A. Advances in the development of personalized neoantigen-based therapeutic cancer vaccines. Nat. Rev. Clin. Oncol. 2021, 18, 215–229. [Google Scholar] [CrossRef] [PubMed]
  136. Szanto, C.L.; Cornel, A.M.; Vijver, S.V.; Nierkens, S. Monitoring immune responses in neuroblastoma patients during therapy. Cancers 2020, 12, 519. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  137. Park, J.A.; Cheung, N.K.V. Targets and antibody formats for immunotherapy of neuroblastoma. J. Clin. Oncol. 2020, 38, 1836–1848. [Google Scholar] [CrossRef]
  138. Modak, S.; Cheung, N.-K.V. Disialoganglioside directed immunotherapy of neuroblastoma. Cancer Investig. 2007, 25, 67–77. [Google Scholar] [CrossRef]
  139. Karikó, K.; Muramatsu, H.; Welsh, F.A.; Ludwig, J.; Kato, H.; Akira, S.; Weissman, D. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 2008, 16, 1833–1840. [Google Scholar] [CrossRef]
  140. Thess, A.; Grund, S.; Mui, B.L.; Hope, M.J.; Baumhof, P.; Fotin-Mleczek, M.; Schlake, T. Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals. Mol. Ther. 2015, 23, 1456–1464. [Google Scholar] [CrossRef] [Green Version]
  141. Guan, S.; Rosenecker, J. Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems. Gene Ther. 2017, 24, 133–143. [Google Scholar] [CrossRef]
  142. Hornung, V.; Barchet, W.; Schlee, M.; Hartmann, G. RNA recognition via TLR7 and TLR8. In Toll-Like Receptors (TLRs) and innate immunity; Springer: Berlin/Heidelberg, Germany, 2008; pp. 71–86. [Google Scholar] [CrossRef]
  143. Wilusz, C.J.; Wormington, M.; Peltz, S.W. The cap-to-tail guide to mRNA turnover. Nat. Rev. Mol. Cell Biol. 2001, 2, 237–246. [Google Scholar] [CrossRef] [PubMed]
  144. Martin, S.A.; Paoletti, E.; Moss, B. Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions. J. Biol. Chem. 1975, 250, 9322–9329. [Google Scholar] [CrossRef] [PubMed]
  145. Stepinski, J.; Waddell, C.; Stolarski, R.; Darzynkiewicz, E.; Rhoads, R.E. Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3’-O-methyl)GpppG and 7-methyl (3’-deoxy)GpppG. RNA 2001, 7, 1486–1495. [Google Scholar]
  146. Weissman, D.; Karikó, K. mRNA: Fulfilling the promise of gene therapy. Mol. Ther. 2015, 23, 1416–1417. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  147. Fry, T.J.; Shah, N.N.; Orentas, R.J.; Stetler-Stevenson, M.; Yuan, C.M.; Ramakrishna, S.; Wolters, P.; Martin, S.; Delbrook, C.; Yates, B.; et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat. Med. 2018, 24, 20–28. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  148. Marchioni, M.; Nazzani, S.; Preisser, F.; Bandini, M.; Karakiewicz, P.I. Therapeutic strategies for organ-confined and non-organ-confined bladder cancer after radical cystectomy. Expert Rev. Anticancer Ther. 2018, 18, 377–387. [Google Scholar] [CrossRef] [PubMed]
  149. Zhang, Z.; Liu, S.; Zhang, B.; Qiao, L.; Zhang, Y. T cell dysfunction and exhaustion in cancer. Front. Cell Dev. Biol. 2020, 8, 17. [Google Scholar] [CrossRef] [Green Version]
  150. Pardi, N.; Tuyishime, S.; Muramatsu, H.; Kariko, K.; Mui, B.L.; Tam, Y.K.; Madden, T.D.; Hope, M.J.; Weissman, D. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes. J. Control. Release 2015, 217, 345–351. [Google Scholar] [CrossRef] [Green Version]
  151. Senti, G.; Kündig, T.M. Intralymphatic immunotherapy. World Allergy Organ. J. 2015, 8, 9. [Google Scholar] [CrossRef] [Green Version]
  152. Van der Jeught, K.; Joe, P.T.; Bialkowski, L.; Heirman, C.; Daszkiewicz, L.; Liechtenstein, T.; Escors, D.; Thielemans, K.; Breckpot, K. Intratumoral administration of mRNA encoding a fusokine consisting of IFN-β and the ectodomain of the TGF-β receptor II potentiates antitumor immunity. Oncotarget 2014, 5, 10100–10113. [Google Scholar] [CrossRef] [Green Version]
  153. Beyaert, S.; Machiels, J.-P.; Schmitz, S. Vaccine-based immunotherapy for head and neck cancers. Cancers 2021, 13, 6041. [Google Scholar] [CrossRef] [PubMed]
  154. Karan, D.; Holzbeierlein, J.M.; Van Veldhuizen, P.; Thrasher, J.B. Cancer immunotherapy: A paradigm shift for prostate cancer treatment. Nat. Rev. Urol. 2012, 9, 376–385. [Google Scholar] [CrossRef] [PubMed]
  155. Finn, O.J. The dawn of vaccines for cancer prevention. Nat. Rev. Immunol. 2017, 18, 183–194. [Google Scholar] [CrossRef] [PubMed]
  156. Colella, P.; Ronzitti, G.; Mingozzi, F. Emerging issues in AAV-mediated in vivo gene therapy. Mol. Ther.—Methods Clin. Dev. 2018, 8, 87–104. [Google Scholar] [CrossRef] [Green Version]
  157. Sabnis, S.; Kumarasinghe, E.S.; Salerno, T.; Mihai, C.; Ketova, T.; Senn, J.J.; Lynn, A.; Bulychev, A.; McFadyen, I.; Chan, J.; et al. A novel amino lipid series for mRNA delivery: Improved endosomal escape and sustained pharmacology and safety in non-human primates. Mol. Ther. 2018, 26, 1509–1519. [Google Scholar] [CrossRef] [Green Version]
  158. Chahal, J.S.; Khan, O.F.; Cooper, C.L.; McPartlan, J.S.; Tsosie, J.K.; Tilley, L.D.; Sidik, S.M.; Lourido, S.; Langer, R.; Bavari, S.; et al. Dendrimer-RNA nanoparticles generate protective immunity against lethal Ebola, H1N1 influenza, and Toxoplasma gondii challenges with a single dose. Proc. Natl. Acad. Sci. USA 2016, 113, E4133–E4142. [Google Scholar] [CrossRef] [Green Version]
  159. Besin, G.; Milton, J.; Sabnis, S.; Howell, R.; Mihai, C.; Burke, K.; Benenato, K.E.; Stanton, M.; Smith, P.; Senn, J.; et al. Accelerated blood clearance of lipid nanoparticles entails a biphasic humoral response of B-1 followed by B-2 lymphocytes to distinct antigenic moieties. Immunohorizons 2019, 3, 282–293. [Google Scholar] [CrossRef] [Green Version]
  160. Adams, D.; Gonzalez-Duarte, A.; O’Riordan, W.D.; Yang, C.-C.; Ueda, M.; Kristen, A.V.; Tournev, I.; Schmidt, H.H.; Coelho, T.; Berk, J.L.; et al. Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis. N. Engl. J. Med. 2018, 379, 11–21. [Google Scholar] [CrossRef]
  161. Yamamoto, T.N.; Kishton, R.J.; Restifo, N.P. Developing neoantigen-targeted T cell–based treatments for solid tumors. Nat. Med. 2019, 25, 1488–1499. [Google Scholar] [CrossRef]
  162. Aurisicchio, L.; Pallocca, M.; Ciliberto, G.; Palombo, F. The perfect personalized cancer therapy: Cancer vaccines against neoantigens. J. Exp. Clin. Cancer Res. 2018, 37, 86. [Google Scholar] [CrossRef] [Green Version]
  163. Wells, D.K.; van Buuren, M.M.; Dang, K.K.; Hubbard-Lucey, V.M.; Sheehan, K.C.; Campbell, K.M.; Lamb, A.; Ward, J.P.; Sidney, J.; Blazquez, A.B.; et al. Key parameters of tumor epitope immunogenicity revealed through a consortium approach improve neoantigen prediction. Cell 2020, 183, 818–834. [Google Scholar] [CrossRef] [PubMed]
  164. Gubin, M.M.; Artyomov, M.N.; Mardis, E.R.; Schreiber, R.D. Tumor neoantigens: Building a framework for personalized cancer immunotherapy. J. Clin. Investig. 2015, 125, 3413–3421. [Google Scholar] [CrossRef] [PubMed]
  165. Roudko, V.; Greenbaum, B.; Bhardwaj, N. Computational prediction and validation of tumor-associated neoantigens. Front. Immunol. 2020, 11, 27. [Google Scholar] [CrossRef] [PubMed]
  166. Hu, Z.; Leet, D.E.; Allesøe, R.L.; Oliveira, G.; Li, S.; Luoma, A.M.; Liu, J.; Forman, J.; Huang, T.; Iorgulescu, J.B.; et al. Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma. Nat. Med. 2021, 27, 515–525. [Google Scholar] [CrossRef] [PubMed]
  167. Ott, P.A.; Hu, Z.; Keskin, D.B.; Shukla, S.A.; Sun, J.; Bozym, D.J.; Zhang, W.; Luoma, A.; Giobbie-Hurder, A.; Peter, L.; et al. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature 2017, 547, 217–221, Erratum in Nature 2018, 555, 402. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  168. Rubinsteyn, A.; Kodysh, J.; Hodes, I.; Mondet, S.; Aksoy, B.A.; Finnigan, J.P.; Bhardwaj, N.; Hammerbacher, J. Computational pipeline for the PGV-001 neoantigen vaccine trial. Front. Immunol. 2018, 8, 1807. [Google Scholar] [CrossRef] [Green Version]
  169. Esprit, A.; De Mey, W.; Shahi, R.B.; Thielemans, K.; Franceschini, L.; Breckpot, K. Neo-antigen mRNA vaccines. Vaccines 2020, 8, 776. [Google Scholar] [CrossRef]
  170. Cantwell-Dorris, E.R.; O’Leary, J.J.; Sheils, O.M. BRAFV600E: Implications for carcinogenesis and molecular therapy. Mol. Cancer Ther. 2011, 10, 385–394. [Google Scholar] [CrossRef] [Green Version]
  171. Boespflug, A.; Caramel, J.; Dalle, S.; Thomas, L. Treatment of NRAS-mutated advanced or metastatic melanoma: Rationale, current trials and evidence to date. Ther. Adv. Med Oncol. 2017, 9, 481–492. [Google Scholar] [CrossRef] [Green Version]
  172. Sahin, U.; Derhovanessian, E.; Miller, M.; Kloke, B.-P.; Simon, P.; Löwer, M.; Bukur, V.; Tadmor, A.D.; Luxemburger, U.; Schrörs, B.; et al. Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 2017, 547, 222–226. [Google Scholar] [CrossRef]
  173. Nature Biotechnology. The problem with neoantigen prediction. Nat. Biotechnol. 2017, 35, 97. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  174. Wang, G.; Gao, Y.; Chen, Y.; Wang, K.; Zhang, S.; Li, G. Identification of novel tumor antigens and the immune landscapes of bladder cancer patients for mRNA vaccine development. Front. Oncol. 2022, 12, 921711. [Google Scholar] [CrossRef] [PubMed]
  175. Ping, H.; Yu, W.; Gong, X.; Tong, X.; Lin, C.; Chen, Z.; Cai, C.; Guo, K.; Ke, H. Analysis of melanoma tumor antigens and immune subtypes for the development of mRNA vaccine. Investig. New Drugs 2022, 40, 1173–1184. [Google Scholar] [CrossRef] [PubMed]
  176. Nierengarten, M.B. Messenger RNA vaccine advances provide treatment possibilities for cancer. Cancer 2022, 128, 213–214. [Google Scholar] [CrossRef] [PubMed]
  177. Lu, Y.; He, W.; Huang, X.; He, Y.; Gou, X.; Liu, X.; Hu, Z.; Xu, W.; Rahman, K.; Li, S.; et al. Strategies to package recombinant adeno-associated virus expressing the N-terminal gasdermin domain for tumor treatment. Nat. Commun. 2021, 12, 7155. [Google Scholar] [CrossRef]
  178. Wu, C.-C.; Chiang, C.-Y.; Liu, S.-J.; Chen, H.-W. A novel recombinant Fcγ receptor-targeted survivin combines with chemotherapy for efficient cancer treatment. Biomedicines 2021, 9, 806. [Google Scholar] [CrossRef]
  179. Wu, C.; Qin, C.; Long, W.; Wang, X.; Xiao, K.; Liu, Q. Tumor antigens and immune subtypes of glioblastoma: The fundamentals of mRNA vaccine and individualized immunotherapy development. J. Big Data 2022, 9, 92. [Google Scholar] [CrossRef]
  180. Chen, Z.; Wang, X.; Yan, Z.; Zhang, M. Identification of tumor antigens and immune subtypes of glioma for mRNA vaccine development. Cancer Med. 2022, 11, 2711–2726. [Google Scholar] [CrossRef]
  181. Wang, S.; Yang, Y.; Li, L.; Ma, P.; Jiang, Y.; Ge, M.; Yu, Y.; Huang, H.; Fang, Y.; Jiang, N.; et al. Identification of tumor antigens and immune subtypes of malignant mesothelioma for mRNA vaccine development. Vaccines 2022, 10, 1168. [Google Scholar] [CrossRef]
  182. You, W.; Ouyang, J.; Cai, Z.; Chen, Y.; Wu, X. Comprehensive analyses of immune subtypes of stomach adenocarcinoma for mRNA vaccination. Front. Immunol. 2022, 13, 827506. [Google Scholar] [CrossRef]
  183. Zhang, S.; Li, S.; Wei, Y.; Xiong, Y.; Liu, Q.; Hu, Z.; Zeng, Z.; Tang, F.; Ouyang, Y. Identification of potential antigens for developing mRNA vaccine for immunologically cold mesothelioma. Front. Cell Dev. Biol. 2022, 10, 879278. [Google Scholar] [CrossRef]
  184. Maruggi, G.; Zhang, C.; Li, J.; Ulmer, J.B.; Yu, D. mRNA as a transformative technology for vaccine development to control infectious diseases. Mol. Ther. 2019, 27, 757–772. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  185. Schlake, T.; Thess, A.; Fotin-Mleczek, M.; Kallen, K.-J. Developing mRNA-vaccine technologies. RNA Biol. 2012, 9, 1319–1330. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  186. Tanyi, J.L.; Bobisse, S.; Ophir, E.; Tuyaerts, S.; Roberti, A.; Genolet, R.; Baumgartner, P.; Stevenson, B.J.; Iseli, C.; Dangaj, D.; et al. Personalized cancer vaccine effectively mobilizes antitumor T cell immunity in ovarian cancer. Sci. Transl. Med. 2018, 10, eaao5931. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  187. Snell, L.M.; McGaha, T.L.; Brooks, D.G. Type I interferon in chronic virus infection and cancer. Trends Immunol. 2017, 38, 542–557. [Google Scholar] [CrossRef]
  188. Hassett, K.J.; Benenato, K.E.; Jacquinet, E.; Lee, A.; Woods, A.; Yuzhakov, O.; Himansu, S.; Deterling, J.; Geilich, B.M.; Ketova, T.; et al. Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines. Mol. Ther. Nucleic Acids 2019, 15, 1–11. [Google Scholar] [CrossRef] [Green Version]
  189. Miao, L.; Li, L.; Huang, Y.; Delcassian, D.; Chahal, J.; Han, J.; Shi, Y.; Sadtler, K.; Gao, W.; Lin, J.; et al. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat. Biotechnol. 2019, 37, 1174–1185. [Google Scholar] [CrossRef]
  190. Van der Jeught, K.; De Koker, S.; Bialkowski, L.; Heirman, C.; Tjok Joe, P.; Perche, F.; Maenhout, S.; Bevers, S.; Broos, K.; Deswarte, K.; et al. Dendritic cell targeting mRNA Lipopolyplexes combine strong antitumor t-cell immunity with improved inflammatory safety. ACS Nano 2018, 12, 9815–9829. [Google Scholar] [CrossRef]
  191. Son, S.; Nam, J.; Zenkov, I.; Ochyl, L.J.; Xu, Y.; Scheetz, L.; Shi, J.; Farokhzad, O.C.; Moon, J.J. Sugar-nanocapsules imprinted with microbial molecular patterns for mRNA vaccination. Nano Lett. 2020, 20, 1499–1509. [Google Scholar] [CrossRef]
  192. Verbeke, R.; Lentacker, I.; Breckpot, K.; Janssens, J.; Van Calenbergh, S.; De Smedt, S.; Dewitte, H. Broadening the message: A nanovaccine co-loaded with messenger RNA and α-GalCer induces antitumor immunity through conventional and natural killer T cells. ACS Nano 2019, 13, 1655–1669. [Google Scholar] [CrossRef]
  193. Lou, B.; De Koker, S.; Lau, C.Y.J.; Hennink, W.E.; Mastrobattista, E. mRNA polyplexes with post-conjugated GALA peptides efficiently target, transfect, and activate antigen presenting cells. Bioconjugate Chem. 2019, 30, 461–475. [Google Scholar] [CrossRef] [PubMed]
  194. Coolen, A.-L.; Lacroix, C.; Mercier-Gouy, P.; Delaune, E.; Monge, C.; Exposito, J.-Y.; Verrier, B. Poly(lactic acid) nanoparticles and cell-penetrating peptide potentiate mRNA-based vaccine expression in dendritic cells triggering their activation. Biomaterials 2019, 195, 23–37. [Google Scholar] [CrossRef] [PubMed]
  195. Perche, F.; Clemençon, R.; Schulze, K.; Ebensen, T.; Guzmán, C.A.; Pichon, C. Neutral lipopolyplexes for in vivo delivery of conventional and replicative RNA vaccine. Mol. Ther. -Nucleic Acids 2019, 17, 767–775. [Google Scholar] [CrossRef] [PubMed]
  196. Oberli, M.A.; Reichmuth, A.M.; Dorkin, J.R.; Mitchell, M.J.; Fenton, O.S.; Jaklenec, A.; Anderson, D.G.; Langer, R.; Blankschtein, D. Lipid nanoparticle assisted mRNA delivery for potent cancer immunotherapy. Nano Lett. 2016, 17, 1326–1335. [Google Scholar] [CrossRef] [PubMed]
  197. Sahin, U.; Oehm, P.; Derhovanessian, E.; Jabulowsky, R.A.; Vormehr, M.; Gold, M.; Maurus, D.; Schwarck-Kokarakis, D.; Kuhn, A.N.; Omokoko, T.; et al. An RNA vaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature 2020, 585, 107–112. [Google Scholar] [CrossRef]
  198. Linch, M.; Papai, Z.; Takacs, I.; Imedio, E.R.; Kühnle, M.-C.; Derhovanessian, E.; Vogler, I.; Renken, S.; Graham, P.; Sahin, U.; et al. 421 A first-in-human (FIH) phase I/IIa clinical trial assessing a ribonucleic acid lipoplex (RNA-LPX) encoding shared tumor antigens for immunotherapy of prostate cancer; preliminary analysis of PRO-MERIT. J. Immunother. Cancer 2021, 9, A451. [Google Scholar] [CrossRef]
  199. Braiteh, F.; LoRusso, P.; Balmanoukian, A.; Klempner, S.; Camidge, D.R.; Hellmann, M.; Gordon, M.; Bendell, J.; Mueller, L.; Sabado, R.; et al. Abstract CT169: A phase Ia study to evaluate RO7198457, an individualized Neoantigen Specific immunoTherapy (iNeST), in patients with locally advanced or metastatic solid tumors. Cancer Res. 2020, 80, CT169. [Google Scholar] [CrossRef]
  200. Srikrishna, D.; Sachsenmeier, K. We need to bring R0 < 1 to treat cancer too. Genome Med. 2021, 13, 120. [Google Scholar] [CrossRef]
  201. Bauman, J.; Burris, H.; Clarke, J.; Patel, M.; Cho, D.; Gutierrez, M.; Julian, R.; Scott, A.; Cohen, P.; Frederick, J.; et al. 798 Safety, tolerability, and immunogenicity of mRNA-4157 in combination with pembrolizumab in subjects with unresectable solid tumors (KEYNOTE-603): An update. J. Immunother. Cancer 2020, 8. [Google Scholar] [CrossRef]
  202. Wadhwa, A.; Aljabbari, A.; Lokras, A.; Foged, C.; Thakur, A. Opportunities and challenges in the delivery of mRNA-based vaccines. Pharmaceutics 2020, 12, 102. [Google Scholar] [CrossRef] [Green Version]
  203. Burris, H.A.; Patel, M.R.; Cho, D.C.; Clarke, J.M.; Gutierrez, M.; Zaks, T.Z. A phase I multicenter study to assess the safety, tolerability, and immunogenicity of mRNA-4157 alone in patients with resected solid tumors and in combination with pembrolizumab in patients with unresectable solid tumors. JCO 2019, 37 (Suppl. S15), 2523. [Google Scholar] [CrossRef]
  204. Liang, X.; Li, D.; Leng, S.; Zhu, X. RNA-based pharmacotherapy for tumors: From bench to clinic and back. Biomed. Pharmacother. 2020, 125, 109997. [Google Scholar] [CrossRef]
  205. Cafri, G.; Gartner, J.J.; Hopson, K.; Meehan, R.S.; Zaks, T.Z.; Robbins, P.; Rosenberg, S.A. Immunogenicity and tolerability of personalized mRNA vaccine mRNA-4650 encoding defined neoantigens expressed by the autologous cancer. J. Clin. Oncol. 2019, 37, 2643. [Google Scholar] [CrossRef]
  206. De Keersmaecker, B.; Claerhout, S.; Carrasco, J.; Bar, I.; Corthals, J.; Wilgenhof, S.; Neyns, B.; Thielemans, K. TriMix and tumor antigen mRNA electroporated dendritic cell vaccination plus ipilimumab: Link between T-cell activation and clinical responses in advanced melanoma. J. Immunother. Cancer 2019, 8, e000329. [Google Scholar] [CrossRef] [Green Version]
  207. Wilgenhof, S.; Van Nuffel, A.M.T.; Benteyn, D.; Corthals, J.; Aerts, C.; Heirman, C.; Van Riet, I.; Bonehill, A.; Thielemans, K.; Neyns, B. A phase IB study on intravenous synthetic mRNA electroporated dendritic cell immunotherapy in pretreated advanced melanoma patients. Ann. Oncol. 2013, 24, 2686–2693. [Google Scholar] [CrossRef]
  208. Jansen, Y.; Kruse, V.; Corthals, J.; Schats, K.; Van Dam, P.-J.; Seremet, T.; Heirman, C.; Brochez, L.; Kockx, M.; Thielemans, K.; et al. A randomized controlled phase II clinical trial on mRNA electroporated autologous monocyte-derived dendritic cells (TriMixDC-MEL) as adjuvant treatment for stage III/IV melanoma patients who are disease-free following the resection of macrometastases. Cancer Immunol. Immunother. 2020, 69, 2589–2598. [Google Scholar] [CrossRef]
  209. Deng, Z.; Tian, Y.; Song, J.; An, G.; Yang, P. mRNA vaccines: The dawn of a new era of cancer immunotherapy. Front. Immunol. 2022, 13, 887125. [Google Scholar] [CrossRef]
  210. Rausch, S.; Schwentner, C.; Stenzl, A.; Bedke, J. mRNA vaccine CV9103 and CV9104 for the treatment of prostate cancer. Hum. Vaccines Immunother. 2014, 10, 3146–3152. [Google Scholar] [CrossRef] [Green Version]
  211. Kübler, H.; Scheel, B.; Gnad-Vogt, U.; Miller, K.; Schultze-Seemann, W.; Vom Dorp, F.; Parmiani, G.; Hampel, C.; Wedel, S.; Trojan, L.; et al. Self-adjuvanted mRNA vaccination in advanced prostate cancer patients: A first-in-man phase I/IIa study. J. Immunother. Cancer 2015, 3, 26. [Google Scholar] [CrossRef] [Green Version]
  212. Kimura, H.; Matsui, Y.; Nakajima, T.; Iizasa, T.; Ishikawa, A. Phase III randomized controlled trial of adjuvant chemoimmunotherapy in patients with resected primary lung cancer. Ann. Oncol. 2017, 28, v403. [Google Scholar] [CrossRef]
  213. Sebastian, M.; Schröder, A.; Scheel, B.; Hong, H.S.; Muth, A.; von Boehmer, L.; Zippelius, A.; Mayer, F.; Reck, M.; Atanackovic, D.; et al. A phase I/IIa study of the mRNA-based cancer immunotherapy CV9201 in patients with stage IIIB/IV non-small cell lung cancer. Cancer Immunol. Immunother. 2019, 68, 799–812. [Google Scholar] [CrossRef]
  214. Sabari, J.; Ramirez, K.A.; Schwarzenberger, P.; Ricciardi, T.; Macri, M. Phase 1/2 study of mRNA vaccine therapy + durvalumab (durva) ± tremelimumab (treme) in patients with metastatic non-small cell lung cancer (NSCLC). In Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival, New York, NY, USA, 30 September–3 October 2018. [Google Scholar]
  215. Papachristofilou, A.; Hipp, M.M.; Klinkhardt, U.; Früh, M.; Sebastian, M.; Weiss, C.; Pless, M.; Cathomas, R.; Hilbe, W.; Pall, G.; et al. Phase Ib evaluation of a self-adjuvanted protamine formulated mRNA-based active cancer immunotherapy, BI1361849 (CV9202), combined with local radiation treatment in patients with stage IV non-small cell lung cancer. J. Immunother. Cancer 2019, 7, 38. [Google Scholar] [CrossRef]
  216. Liu, L.; Wang, Y.; Miao, L.; Liu, Q.; Musetti, S.; Li, J.; Huang, L. Combination immunotherapy of MUC1 mRNA nano-vaccine and CTLA-4 blockade effectively inhibits growth of triple negative breast cancer. Mol. Ther. 2018, 26, 45–55. [Google Scholar] [CrossRef] [Green Version]
  217. Hamid, O.; Hellman, M.; Carneiro, B. Preliminary safety, antitumor activity and pharmacodynamics results of HIT-IT MEDI1191 (mRNA IL-12) in patients with advanced solid tumors and superficial lesions. In Proceedings of the ESMO Targeted Anticancer Therapies (TAT), Virtual Congress, 1–2 March 2021. [Google Scholar]
  218. Weide, B.; Carralot, J.-P.; Reese, A.; Scheel, B.; Eigentler, T.K.; Hoerr, I.; Rammensee, H.-G.; Garbe, C.; Pascolo, S. Results of the first phase i/ii clinical vaccination trial with direct injection of mrna. J. Immunother. 2008, 31, 180–188. [Google Scholar] [CrossRef]
  219. Rittig, S.M.; Haentschel, M.; Weimer, K.J.; Heine, A.; Muller, M.R.; Brugger, W.; Horger, M.S.; Maksimovic, O.; Stenzl, A.; Hoerr, I.; et al. Intradermal vaccinations with RNA coding for TAA generate CD8+ and CD4+ Immune responses and induce clinical benefit in vaccinated patients. Mol. Ther. 2011, 19, 990–999. [Google Scholar] [CrossRef]
  220. Rosenthal, R.; Cadieux, E.L.; Salgado, R.; Bakir, M.A.; Moore, D.A.; Hiley, C.T.; Lund, T.; Tanić, M.; Reading, J.L.; Joshi, K.; et al. Neoantigen-directed immune escape in lung cancer evolution. Nature 2019, 567, 479–485. [Google Scholar] [CrossRef]
  221. Beatty, G.L.; Gladney, W.L. Immune escape mechanisms as a guide for cancer immunotherapy. Clin. Cancer Res. 2015, 21, 687–692. [Google Scholar] [CrossRef] [Green Version]
  222. Bialkowski, L.; Van Der Jeught, K.; Bevers, S.; Tjok Joe, P.; Renmans, D.; Heirman, C.; Aerts, J.L.; Thielemans, K. Immune checkpoint blockade combined with IL-6 and TGF-β inhibition improves the therapeutic outcome of mRNA-based immunotherapy. Int. J. Cancer 2018, 143, 686–698. [Google Scholar] [CrossRef]
  223. Wang, Y.; Zhang, L.; Xu, Z.; Miao, L.; Huang, L. mRNA vaccine with antigen-specific checkpoint blockade induces an enhanced immune response against established melanoma. Mol. Ther. 2018, 26, 420–434. [Google Scholar] [CrossRef] [Green Version]
  224. Koyama, S.; Akbay, E.A.; Li, Y.Y.; Herter-Sprie, G.S.; Buczkowski, K.A.; Richards, W.G.; Gandhi, L.; Redig, A.J.; Rodig, S.J.; Asahina, H.; et al. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat. Commun. 2016, 7, 10501. [Google Scholar] [CrossRef] [Green Version]
  225. Zhang, N.-N.; Li, X.-F.; Deng, Y.-Q.; Zhao, H.; Huang, Y.-J.; Yang, G.; Huang, W.-J.; Gao, P.; Zhou, C.; Zhang, R.-R.; et al. A thermostable mRNA vaccine against COVID-19. Cell 2020, 182, 1271–1283.e16. [Google Scholar] [CrossRef]
  226. Parhiz, H.; Brenner, J.S.; Patel, P.N.; Papp, T.E.; Shahnawaz, H.; Li, Q.; Shi, R.; Zamora, M.E.; Yadegari, A.; Marcos-Contreras, O.A.; et al. Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE). J. Control. Release 2021, 344, 50–61. [Google Scholar] [CrossRef]
  227. Igyártó, B.Z.; Jacobsen, S.; Ndeupen, S. Future considerations for the mRNA-lipid nanoparticle vaccine platform. Curr. Opin. Virol. 2022, 48, 65–72. [Google Scholar] [CrossRef]
  228. Dirisala, A.; Uchida, S.; Toh, K.; Li, J.; Osawa, S.; Tockary, T.A.; Liu, X.; Abbasi, S.; Hayashi, K.; Mochida, Y.; et al. Transient stealth coating of liver sinusoidal wall by anchoring two-armed PEG for retargeting nanomedicines. Sci. Adv. 2020, 6, eabb8133. [Google Scholar] [CrossRef]
  229. Krause, W. Liver-specific X-ray contrast agents. In Contrast Agents II; Springer: Berlin/Heidelberg, Germany; New York, NY, USA, 2002; Volume 222, pp. 173–200. [Google Scholar]
Figure 1. (A): General structure of mRNA containing the four nucleobases adenine, cystine, guanine, and uracil, linked to ribose. The resulting nucleosides are connected via phosphate groups. The starting (5′ cap) position is formed by 7-methylguanosine with a three-phosphate moiety as a linker to the first nucleotide. At the end of the molecule, multiple adenosine moieties are attached. A codon comprises three nucleotides, for example, AUG, which is the code for methionine. (B): Functional structure of mRNA; details of functions are provided in Table 1. 5′ UTR: 5′ untranslated region. 3′ UTR: 3′ untranslated region. Poly(A)tail: multiple adenosine units.
Figure 1. (A): General structure of mRNA containing the four nucleobases adenine, cystine, guanine, and uracil, linked to ribose. The resulting nucleosides are connected via phosphate groups. The starting (5′ cap) position is formed by 7-methylguanosine with a three-phosphate moiety as a linker to the first nucleotide. At the end of the molecule, multiple adenosine moieties are attached. A codon comprises three nucleotides, for example, AUG, which is the code for methionine. (B): Functional structure of mRNA; details of functions are provided in Table 1. 5′ UTR: 5′ untranslated region. 3′ UTR: 3′ untranslated region. Poly(A)tail: multiple adenosine units.
Biomedicines 11 00308 g001
Figure 2. Structure of saRNA containing an additional replicase section.
Figure 2. Structure of saRNA containing an additional replicase section.
Biomedicines 11 00308 g002
Figure 3. Major types of cancer immunotherapy with their benefits in green and their downsides in red.
Figure 3. Major types of cancer immunotherapy with their benefits in green and their downsides in red.
Biomedicines 11 00308 g003
Figure 4. Development of cancer heterogeneity and formation of neoantigens. Treatment with a drug (A, B, C, D, or E) can result in cure, partial remission, or primary resistance that can progress to metastases and/or to mutation resulting in new cancer entities presenting neoantigens. Primary tumor: Biomedicines 11 00308 i001. Tumor cells expressing neoantigens A Biomedicines 11 00308 i002, B Biomedicines 11 00308 i003, or C Biomedicines 11 00308 i004.
Figure 4. Development of cancer heterogeneity and formation of neoantigens. Treatment with a drug (A, B, C, D, or E) can result in cure, partial remission, or primary resistance that can progress to metastases and/or to mutation resulting in new cancer entities presenting neoantigens. Primary tumor: Biomedicines 11 00308 i001. Tumor cells expressing neoantigens A Biomedicines 11 00308 i002, B Biomedicines 11 00308 i003, or C Biomedicines 11 00308 i004.
Biomedicines 11 00308 g004
Table 1. Building blocks of mRNA and their functions. The Kozak sequence is a group of nucleotides that initiates the protein translation in the ribosome. An example is 5′-GCCGCCRCCAUGG-3′, where red color indicates that these nucleotides are fixed, AUG is the start codon encoding for methionine, R stands for A or G, and the function of GCC is not well defined.
Table 1. Building blocks of mRNA and their functions. The Kozak sequence is a group of nucleotides that initiates the protein translation in the ribosome. An example is 5′-GCCGCCRCCAUGG-3′, where red color indicates that these nucleotides are fixed, AUG is the start codon encoding for methionine, R stands for A or G, and the function of GCC is not well defined.
ComponentNucleotidesFunction
5′ cap7-Methyl-GEssential for ribosome recognition, transcription, protection against ribonucleases
5′ UTRA, C, G, U; from 3 to several hundred nucleotidesContributes to stability, localization, and translation efficiency
Normally proprietary knowledge
StartAUG within a Kozak sequenceCodes for methione and initiates translation
CodingA, C, G, URegulation of splicing
Decoding (reading) by ribosomes and translation into the target protein
StopUAG: amber
UAA: ochre
UGA: opal
Terminates the translation process
3′ UTR Contributes to stability, localization, and translation efficiency, potentially involved in disease susecptibility
Poly(A) tailAProtects against exonucleases, aids in transport from nucleus to cytosol and in translation
Table 2. Possible modifications of the mRNA molecule for use as COVID-19 vaccines with the spike protein of SARS-CoV-2 as target antigen. Most modifications used for approved vaccines are proprietary and therefore not publicly available.
Table 2. Possible modifications of the mRNA molecule for use as COVID-19 vaccines with the spike protein of SARS-CoV-2 as target antigen. Most modifications used for approved vaccines are proprietary and therefore not publicly available.
PositionModificationEffectReference
5′ cap
  • Methylation of the first nucleotide at position 20 (cap1 structure)
  • CleanCap system: 5′ cap + 2′ methylated adenosine followed by guanosine
  • Allows incorporation of cap1 at the 5′ end of any mRNA and does not limit the concentration of any of the four nucleotides
[17,18,19,20]
5′ UTR
  • Apply 5′ cap in multiple versions
  • Incorporate synthetic cap or anti-reverse cap analogues
  • Strong Kozak translation signal
  • Avoid the presence of start codon (AUG) and non-canonical start codons (CUG)
  • Use shorter 5′ UTR
  • Remove highly stable secondary structures
  • Avoid hairpin loops
  • Increases stability and translation resulting in higher efficiency and longer half-life
  • Increases protein synthesis during fibroblast conversion to induced pluripotent stem cells
  • Improves translation
[21,22,23,24,25,26,27,28,29]
Start
Coding
  • Replace a nucleotide with N1-methyl-pseudouridine (N1mΨ)
  • High GC sequence better than a low GC sequence
  • Replace cytidine with 5-methylcytidine (m5C) or uridine with pseudouridine (Ψ) or 1-methylpseudouridine (m1Ψ)
  • To be carefully optimized since it can affect the rate of translation
  • Better base pair stability and mRNA translation
  • 100-fold higher translation
  • Reduces innate immune activation
  • The rate of translation needs to consider the formation of the tertiary structure of the protein
[30,31,32,33]
Stop
3′ UTR
  • See 5′ UTR (1 + 2)
  • Early-on use of alpha globin 3′ UTR
  • Tandem repeats of alpha globin 3′ UTR
  • Optimal length is mandatory
  • Intracellular kinetics heavily depend on 3′ UTR
  • Increase in protein synthesis during fibroblast conversion to induced pluripotent stem cells
  • Critical for mRNA stability
  • Too long: shorter half-life; too short: less efficient translation
See 5′ UTR
[23,25,34,35,36]
Poly(A) tailLength of the poly(A) tail, ideally > 90 A, shorter sequence is more efficientCritical role for translation and stability[20,37,38,39,40]
Table 3. Active ingredients and formulations of mRNA and viral-based COVID-19 vaccines approved in the European Union. Data have been retrieved from the European Public Assessment Report (EPAR). The structural modifications provided in Table 3 do not reflect all changes that have been introduced into the mRNA molecules; major portions are proprietary and have not been disclosed publicly.
Table 3. Active ingredients and formulations of mRNA and viral-based COVID-19 vaccines approved in the European Union. Data have been retrieved from the European Public Assessment Report (EPAR). The structural modifications provided in Table 3 do not reflect all changes that have been introduced into the mRNA molecules; major portions are proprietary and have not been disclosed publicly.
ProductActive IngredientFormulationStorage
Comirnaty, tozinameran, BNT162b2
BioNTech (Mainz, Germany)
Single-stranded, 5′-capped mRNA, encoding the spike antigen [glycoprotein (S)] of SARS-CoV-2 (isolate Wuhan-Hu-1) containing two consecutive proline mutations (P2 S); uridine substituted by N1-methylpseudouridine (1mΨ) Multidose concentrate to be diluted prior to i.m. injection; dispersion of mRNA in LNPs containing ALC-0315 and ALC-0159 (functional lipids), DSPC and cholesterol (structural lipids) in aqueous cryoprotectant buffer.−90 °C to −60 °C
−25 °C to −15 °C (for 2 weeks)
Spikevax, elasomeran/imelasomeran (Omicron BA.1 variant)
mRNA-1273
Moderna, (Camebridge, MA, USA)
Single-stranded, 5′-capped mRNA, encoding for the full-length SARS-CoV-2 spike protein modified with 2 proline substitutions within the heptad repeat 1 domain (S-2P); S protein composed of two subunits (S1 and S2) and stabilized in the pre-fusion conformation by two amino acid mutations, K986P and V987P; open reading frame of 3819 nucleotides; contains 1mΨ instead of uridine; undisclosed modification of the 5′ capMultidose dispersion for injection with mRNA encapsulated in lipid nanoparticles with the following main components: SM-102, cholesterol, DSPC, and PEG2000-DMG−50 °C to −15 °C
Vaxzevria
AZD1222
COVID-19 Vaccine (ChAdOx1-S [recombinant])
AstraZeneca (Cambridge, UK)
Single recombinant, replication-deficient chimpanzee adenovirus (ChAdOx1) vector expressing the S glycoprotein spike protein of SARS-CoV-2 with a tPA leader sequence; no mutations introduced in the expressed SARS-CoV-2 spike protein; non-encapsulated, icosahedral particles (virions of 80 to 100 nm diameter) containing a single copy of the double-stranded DNA genomeLiquid dosage form for i.m. injection2 °C to 8 °C
COVID-19 Vaccine
Ad26.COV2.S
Janssen (Beerse, Belgium)
Recombinant, replication-incompetent adenovirus serotype 26 (Ad26) encoding the SARS-CoV-2 spike (S) proteinLiquid suspension containing 2-hydroxypropyl-β-cyclodextrin for i.m. injection−25 °C to −15 °C
COVID-19 Vaccine (inactivated, adjuvanted) Valneva
Valneva Austria GmbH (Wien, Austria)
Purified, inactivated, and adjuvanted whole virus SARS-CoV-2 (Italian strain (LAZ-INMI1-isl/2020, GISAID Accession number: EPI_ISL_410545)) vaccine grown on Vero cell cultureLiquid suspension for i.m. injection adjuvanted with hydrated aluminium hydroxide and CpG 1018 and recombinant human albumin produced in yeast2 °C to 8 °C
Nuvaxovid
NVX-CoV2373
COVID-19 vaccine (recombinant, adjuvanted)
Novavax CZ (Jevany, Czechia)
Protein product of a recombinant SARS- CoV-2 S-gene (Wuhan-Hu-1) encoding the 1260 amino acid spike protein (the full-length 1273 amino acid protein minus the signal peptide); S gene codon optimized for expression in Spodoptera frugiperda (Sf9) insect cells; five amino acid changes introduced, including three in the S1/S2 furin cleavage site (RRAR to QQAQ) and two in the HR1 domain Aqueous buffered dispersion for i.m. injection, co-formulated with Matrix-M1 adjuvant2 °C to 8 °C
Table 4. Selected neoantigens for use in mRNA vaccinations.
Table 4. Selected neoantigens for use in mRNA vaccinations.
Tumor TypeNeoantigenReference
Bladder cancerAP2S1, P3H4, and RAC3[174]
MelanomaPTPRC, SIGLEC10, CARD11, LILRB1, and ADAMDEC1[175]
Colorectal, NSCLC, and pancreatic cancersKRAS[176]
Esophageal squamous cell carcinoma (ESCC)NLRC5, FCRL4, TMEM229B, and LCP2[177]
Soft tissue sarcomaHLTF, ITGA10, PLCG1, and TTC3[178]
GlioblastomaADAMTSL4, COL6A1, CTSL, CYTH4, EGFLAM, LILRB2, MPZL2, SAA2, and LSP1[179]
GliomaNAT1, FRRS1, GTF2H2C, BRCA2, GRAP, NR5A2, ABCB4, ZNF90, ERCC6L, and ZNF813[180]
Malignant mesotheliomaFAM134B, ALDH3A2, SAV1, RORC, and FN1[181]
Stomach adenocarcinomaADAMTS18, COL10A1, PPEF1, and STRA6[182]
MesotheliomaAUNIP, FANCI, LASP1, PSMD8, and XPO5[183]
Table 5. Selection of mRNAs in development for cancer treatment. FixVac: Non-mutated antigens shared among patients with a specific cancer type, applicable for almost all types of tumor antigens. iNeST: Targeting 20 neoantigens unique to each patient, applicable for almost all types of tumor antigens.
Table 5. Selection of mRNAs in development for cancer treatment. FixVac: Non-mutated antigens shared among patients with a specific cancer type, applicable for almost all types of tumor antigens. iNeST: Targeting 20 neoantigens unique to each patient, applicable for almost all types of tumor antigens.
NamemRNAIndicationsAdmin.Reference
BNT111
FixVac
4 TAAs: tyrosinase, NY-ESO-1, MAGE A3, TPTE Advanced melanoma
Phase I: Lipo-MERIT trial ± checkpoint inhibitor PD1
Phase II: + cemiplimab
Intravenous (i.v.)
liposomal RNA (RNA-LPX)
[197]
NCT02410733
NCT04526899
BNT112
FixVac
5 prostate cancer-specific antigens: kallikrein-2, kallikrein-3, acid phosphatase prostate, HOXB13, NK3 homeobox 1Prostate cancer
Phase I/II + cemiplimab
PRO-MERIT trial
i.v.
RNA-LPX
[198] NCT04382898
BNT113
FixVac
HPV16-E6 and -E7HPV16+ head and neck cancer; AHEAD-MERIT
Phase II + pembrolizumab
HARE-40
Phase I/II
i.v.
RNA-LPX
NCT04534205
NCT03418480
BNT115
W_ova1 Vaccine
FixVac
3 ovarian cancer TAAsOvarian cancer
Phase I + carboplatin/paclitaxel
i.v.NCT04163094
BNT116
FixVac
6 mRNAs each of which encodes for a different TAANSCLC
Phase I/II + cemiplimab
Phase I + cemiplimab or docetaxel, LuCa-MERIT-1
i.v.
Liposomes
NCT05557591
NCT05142189
BNT121
IVAC MUTANOME
Personalized vaccineMetastatic melanoma
Phase I ± RBL001/RBL002
Intranodal[3,172] NCT02035956
BNT122
(RO7198457 autogene cevumeran) iNeST
20 patient-specific antigensMultiple solid tumors
Phase I
i.v.[199] NCT03289962
Melanoma
Phase II + pembrolizumab
i.v.NCT03815058
NSCLC (adjuvant)
Phase II + atezolizumab
i.v.NCT04267237
CRC
Phase II
i.v.NCT04486378
Pancreatic cancer
Phase I + atezolizumab + mFOLFIRINOX
i.v.NCT04161755
BNT114 + BNT122
Personalized
IVAC_W_bre1_uID and IVAC_W_bre1_uID/IVAC_M_uIDTriple Negative Breast Cancer
TNBC-MERIT
i.v.NCT02316457
SAR441000 (BNT131)IL-12sc, IL-15sushi, GM-CSF, IFNαSolid tumors
Phase I ± cemiplimab
IntratumoralNCT03871348
BNT141Encoded antibodiesMultiple solid tumors
Phase I/II ± nab-paclitaxel and gemcitabine
i.v.NCT04683939
BNT142Encoded antibodiesMultiple solid CLDN6+ tumors
Phase I/II
i.v.NCT05262530
BNT151Encoded cytokines: optimized IL-2Multiple solid tumors (optimized IL-2)
Phase I/II
i.v.NCT04455620
BNT152Encoded cytokines: IL-2, IL-7Multiple solid tumors
Phase I/II
i.v.NCT04455620 NCT04710043
BNT153Encoded cytokines: IL-2, IL-7Multiple solid tumors
Phase I
i.v.NCT04710043
mRNA-2416OX40LAdvanced malignancies
Phase I/II ± durvalumab
Intratumoral LNP[3] NCT03323398
mRNA-2752OX40L, IL-23, IL-36γAdvanced malignancies
Phase I/II ± durvalumab
Intratumoral
LNP
[200] NCT03739931
mRNA-4157
(V941)
Up to 34 neoantigens, personalizedHigh-risk melanoma, solid tumors
KEYNOTE-603
Phase I + pembrolizumab
KEYNOTE-942
Phase II + pembrolizumab
i.m.
LNP
[201,202,203,204] NCT03313778 NCT03897881
mRNA-4650
NCI-4650
Up to 20 antigens + up to 15 HLA class I candidate neoantigensGastric or rectal cancer
Phase I
Melanoma
Phase I/II
i.m.[204,205]
NCT03480152
mRNA-5671
Merck V941
4 KRAS mutations (G12D, G13D, G12C, and G12V), personalized CRC, NSCLC, pancreatic adenocarcinoma
Phase I ± pembrolizumab
i.m.[204]
NCT03948763
ECI-0065 TAAs + 3 DC-activating antigensMelanoma
Phase I ± standard anti-PD-1
Intranodal TriMix[204] NCT03394937
TriMix3 mRNA encoding CD70, CD40L, and a constitutively active form of TLR4Breast cancer
Phase I
IntratumoralNCT03788083
TriMixDC-MEL IPIMAGE-A3, MAGE-C2, tyrosinase, and gp100Melanoma
Phase II + ipilimumab
Intratumoral[206] NCT01302496
TriMix-DC Melanoma
Phase I
Phase I/II
i.v. and intradermal[207]
NCT01066390
TriMix-DC + TLR-DC Melanoma
Phase I
Phase I/II
i.v. (mRNA) intranodal (DCs)[207]
NCT01530698
TriMix:
DC + mRNA (CD70, CD40) +
TLR4
Tyrosinase, gp100, MAGE-A3,
or MAGE-C2
Breast cancer
Phase II + ipilimumab
Intratumoral[206,207] NCT01302496
TriMixDC-MEL: Autologous monocyte-derived mRNA co-electroporated DCs + mRNA CD40L, CD70, caTLR4Melanoma
Phase I
i.v.[208]
CV8102TLR7/8, RIG-1Skin cancer
Phase I
Intradermal
Protamine
[209] NCT03291002
TLHepatocellular carcinoma
Phase I + IMA970A + cyclophosphamide
Intradermal
Protamine
NCT03203005
CV9103
RNActive®
4 antigens for prostate cancer: PSA, PSMA, PSCA,
STEAP
Prostate cancer
Phase I/II
Phase I/II
Intradermal[210,211] NCT00906243 NCT00831467
CV9104
Mixture of 6 mRNAs, each encoding 1 antigen
PSA, PSCA, PSMA, STEAP1, PAP, MUC1Prostate cancer
Phase I/II
Phase II
Intradermal or needle-free injection device (Tropis®, London, UK)
Protamine
[210,212] NCT01817738 NCT02140138
CV92015 mRNAs: NY-ESO-1, MAGE C1, MAGE C2, survivin, TBGNSCLC
Phase I/II
Intradermal[213] NCT00923312 NCT03164772
CV9202
BI 1361849
6 mRNAs encoding 6 different antigens: NY-ESO-1, MAGE C1, MAGE C2, TPBG, survivin, MUC1NSCLC
Phase I + local radiation
Phase I/II ± durvalumab and tremelimumab
Intradermal[214,215,216]
NCT01915524 NCT03164772
MEDI1191IL-12Advanced solid tumors, prostate, breast cancer, NSCLC
Phase I + durvalumab
Intratumoral LNP[204,217]
NCT03946800
Naked mRNAMelan-A, MAGE-A1, MAGE-A3, survivin, GP100, and tyrosinaseMelanoma
Phase I/II + GM-CSF
Phase I/II + GM-CSF
Intradermal GM-CSF as adjuvant[218]
NCT00204516 NCT00204607
Naked mRNAMUC1, CEA, Her-2/neu, telomerase, survivin, MAGE-A1Renal cell cancer
Phase I + durvalumab
Intradermal GM-CSF as adjuvant[219]
SW1115C3Cancer TSAs, personalizedSolid tumors
Phase I
Subcutaneous[209] NCT05198752
Tumor mRNA + pp65 flLAMP Glioma, glioblastoma
Phase I
i.v.
RNA-LP (DOTAP liposome)
NCT04573140
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Krause, W. mRNA—From COVID-19 Treatment to Cancer Immunotherapy. Biomedicines 2023, 11, 308. https://doi.org/10.3390/biomedicines11020308

AMA Style

Krause W. mRNA—From COVID-19 Treatment to Cancer Immunotherapy. Biomedicines. 2023; 11(2):308. https://doi.org/10.3390/biomedicines11020308

Chicago/Turabian Style

Krause, Werner. 2023. "mRNA—From COVID-19 Treatment to Cancer Immunotherapy" Biomedicines 11, no. 2: 308. https://doi.org/10.3390/biomedicines11020308

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop