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Challenges and prospects of mRNA therapy.
In the pharmaceutical industry, mRNA-based treatment methods are more accurate in practical applications and can be used for individualized treatment.
Compared with recombinant proteins, therapeutic proteins produced in patients will not encounter complex production problems, so more With development potential, it may cause a major revolution in the industry.
Compared with current therapies, the production of mRNA is much more economical, faster and more flexible, because it can be easily produced by in vitro transcription, and the process has nothing to do with the mRNA sequence.
In addition, based on sequence and/or individual conditions, mRNA vaccines allow people to develop personalized therapies.
As the potential of mRNA drugs from the early stage of development to clinical development is highlighted, the technical obstacles it faces are also obvious.
Stability, immunogenicity, transformation efficiency and drug delivery, all these key issues need to be solved urgently.
Recent research results indicate that due to the development of high-end technology, these obstacles are gradually being overcome.
In this review, we described the structural features and modification techniques of mRNA, summarized the latest progress in the development of mRNA delivery systems, and reviewed its preclinical and clinical applications.
The challenges and prospects of developing mRNA into a new class of drugs in the future provide our views.
Messenger RNA (mRNA) was first discovered by researchers in the 1960s and has now become a basic subject and applied research field that has attracted much attention.
The understanding of mRNA ranges from being an intermediate between DNA and protein to thinking that it is a multifunctional molecule that regulates gene functions in all living organs.
Based on these changes, many different types of mRNA-based therapies have emerged.
In 1990, Wolff et al. reported for the first time that intramuscular injection of mRNA into mouse skeletal muscle produced the expression of the encoded protein.
Since then, mRNA-based therapy has been widely used, including tumor immunotherapy, infectious disease vaccines, protein replacement and cell genetic engineering.
In 2001, ex vivo mRNA transfection of dendritic cells entered clinical trials for the first time. In the past two decades, hundreds of mRNA-based clinical trials have been carried out.
However, in the first few decades when mRNA was discovered, people did not recognize it as a new class of drugs. Issues such as instability and immunogenicity hinder its development, and the attention paid to gene therapy is not as good as DNA.
In recent years, through the introduction of modified nucleosides into mRNA sequences and the development of various RNA packaging and delivery systems, these key issues have basically been solved.
Many evidences show that mRNA can not only mediate better transfection efficiency and longer protein expression time, but also has greater advantages over DNA.
These advantages include:
(1) mRNA can function without entering the nucleus. When reaching the cytoplasm, mRNA initiates protein translation. In contrast, DNA needs to enter the nucleus first and then be transcribed into mRNA. This process makes DNA less efficient than mRNA because its function depends on the destruction of the nuclear envelope during cell division.
(2) Compared with DNA and viral vectors, mRNA does not insert into the genome, but only expresses the encoded protein transiently. Therefore, due to its low insertion risk, it provides an excellent safe choice for researchers and pharmaceutical companies.
(3) mRNA is easily synthesized through the in vitro transcription (IVT) process. This process is relatively inexpensive and can be quickly applied to various therapies. Moreover, mRNA can theoretically express any protein, so it can be used to treat almost all diseases.
Therefore, from the perspective of the pharmaceutical industry, mRNA is a very potential drug candidate that can meet the needs of gene therapy, cancer therapy, and vaccines.
In this review, we summarized the latest progress in solving a series of key issues related to mRNA therapy, including avoiding immunogenicity, increasing stability, and improving translation and delivery efficiency.
In the treatment of infectious diseases, tumors, and genetic diseases, we have also made an overview of the product line of mRNA therapy candidate drugs and the progress of pre-development and clinical research.
Finally, we also discussed the mRNA industry and its related biopharmaceutical companies.
2 mRNA structural elements
Generally, natural mRNA has a single-stranded structure consisting of 7′-methylguanosine residues bound at the 5′-end (5′-cap), and a polyadenylation tail at the 3’end.
The open reading frame (ORF) encoding the protein is identified by a start codon and a stop codon, and the untranslated region (UTR) is located between the cap/tail and the ORF.
Plasmid DNA, PCR products or synthetic double-stranded oligonucleotides can be used as transcription templates for in vitro mRNA synthesis.
The transcription process is mediated by T7, T3 or SP6 phage RNA polymerase to synthesize complementary RNA strands through ribonucleoside triphosphates.
During this process or after transcription, the 7-methylguanosine cap is enzymatically capped at the 5’end of the mRNA. Studies have shown that 5’caps play a crucial role in mRNA maturation, splicing, translation, and nonsense-mediated degradation.
The 3’polyadenylic acid tail is very important for mRNA stability and translation process.
However, the 3′-UTR region contains alpha and beta globulin sequences, which can also enhance mRNA stability and translation efficiency. Both the 3′- and 5′-UTR regions can inhibit mRNA degradation and uncapping.
3 Improve mRNA stability and translation level
One of the main challenges of naked mRNA-based therapy is the short half-life, which is caused by the rapid degradation of a large number of extracellular RNases.
The half-life of in vitro transcribed mRNA (IVT mRNA) and its protein product is a key factor affecting pharmacokinetics (PK) and pharmacodynamics (PD). In order to optimize the efficiency of mRNA, various chemical modifications to the mRNA structure were explored, including modifications to the 5′-cap, polyadenylic acid (A) tail, 5′- and 3′-UTR, and coding regions. To modify the 5′-cap of mRNA, several cap analogs were designed (Figure 1).
The mRNA cap is composed of 7-methylguanine (m7G), which is connected to the RNA nucleotides after the initial transcription via a 5′, 5′-triphosphate bridge during transcription. It not only participates in the translation process by binding to translation initiation factor 4E (EIF4E), but also regulates mRNA degradation by binding to DCP1/DCP2.
The most reported cap analog is part of the anti-reverse cap analog (ARCA) m7G modified in ribose.
ARCA capped mRNA prevents the cap from being mistakenly combined during mRNA synthesis, thus exhibiting excellent translation efficiency.
Recently, another cap analog has been developed called the S analog, and the triphosphate bridge contains a monothiophosphate substitution (O-to-S).
It is reported that ARCA (β-S-ARCAs) substituted with S at the β position of the triphosphate bridge has two advantages: high affinity of the cap to EIF4E and low sensitivity to the detachment complex DCP1/DCP2.
Experiments have shown that β-S-ARCAs enhances the expression of mRNA encoding antigens in vivo and in vitro, and is used in ongoing clinical trials of mRNA vaccines against melanoma (Kuhn et al., 2010).
Recently, Jacek Jemielity et al. synthesized a new class of cap analogs, called 2S analogs, which combined dithiodiphosphate modifications, ARCA, and extended polyphosphate chains.
They found that this 2S analog increased its translation level in human immature dendritic cells and was superior to previously published phosphate-modified cap analogs for clinical trials.
The poly(A) tail modifies the 3’end of mature mRNA in eukaryotes.
It is formed by transcribing its DNA template or using poly(A) polymerase after transcription.
The latter is limited, because the length of the poly(A) tail may be different in different mRNA batches, which makes the repeatability of the poly(A) with a certain length in different batches become very difficult.
The poly(A) tail is transcribed in vitro by using a DNA template, which is very popular in manufacturing. As we all know, the poly(A) tail plays a key role in regulating mRNA stability and translation efficiency.
Studies have found that in many different types of cells, longer tails increase protein expression. Mockey et al. found that as the poly(A) tail continues to increase to 100 nucleotides and combined with 5’ARCA cap analogs, the protein translation level in dendritic cells continues to increase.
According to reports by Holtkamp et al., compared with the traditional 64-nucleotide poly(A) tail, 120 protein expression levels are higher.
However, some experts believe that the poly (A) tail is not as long as possible.
They believe that proper adjustment of poly(A) tail length is very important for maintaining specific biological behaviors in cells, but whether the tail needs to be shorter or longer seems to be transcription-specific.
The 5′- and 3′-UTR in mRNA contain specific regulatory sequence elements that can regulate the translation and stability of mRNA.
The half-life of mRNA can be optimized by introducing stability elements in the UTR. For example, the 3′-UTR of α- and β-globin mRNA plays a key role in its half-life of more than 1 day.
To increase stability and translation efficiency, many IVT mRNAs incorporate the 3′-UTR of α- and β-globin mRNA. The stability can be further improved by inserting two β-globin 3′-UTRs in a head-to-tail direction. In addition to the widely used globulin UTR, various UTRs, such as human shock protein 70, 5′-UTR of internal ribosome entry sites (IRESs) and 3′-UTR of eukaryotic elongation factor 1α (EEF1A1), etc. Has been included in the research of therapeutic mRNA.
For the protein coding region of mRNA, codon optimization results in the controllability of translation of the sequence into the desired protein.
Synonymous codon substitution may have a significant impact on protein expression, folding and cell function.
Because the same amino acid can be translated from a set of different codons, there are many options to rewrite the mRNA code to produce the exact same protein.
Recently, Moderna researchers have observed that mRNA secondary structure can regulate protein expression by changing the half-life of mRNA translation, and modified nucleotides can stabilize mRNA spatial structure to achieve high protein expression levels.
Computer assistance is also used to design mRNA sequences that produce more or less desired proteins.
So far, this technology has been successfully used in mRNA-based therapies, such as the expression of non-viral proteins and the development of infectious disease vaccines.
In general, 5′-cap, 3′-poly(A) tail, 5′- and 3′-UTR and coding regions can all be used as modification targets. In order to obtain the best mRNA treatment efficiency, it is necessary to make an optimized combination for specific applications.
4 Solve the immunogenicity of mRNA
An important issue with IVT mRNA is its immunogenicity, because foreign RNA will be regarded as a signal of viral infection.
Non-immune cells recognize RNA through retinoic acid-induced gene I (RIG-I) receptors, and then trigger the innate immune response. Immune cells can be activated by IVT mRNA and induce inflammation through Toll-like receptors.
U-rich RNA sequences are known to be effective activators of Toll-like receptors. Therefore, the immunogenicity problem can be solved by reducing the content of U.
So far, several strategies for chemical modification of nucleotides can be selected to reduce immunogenicity without affecting its translation properties.
For example, replace natural adenosine with N1-methyladenosine (m1A) or N6-methyladenosine (m6A). Replace natural cytosine with 5-methylcytidine (m5C); replace natural uridine with 5-methyluridine (m5U), 2-thiouridine (s2U), 5-methoxy Uridine (5moU), pseudouridine (ψ) or N1-methyl pseudorhododendron (m1ψ) (Figure 2).
Among them, m5C and ψ have attracted much attention, because both in vivo and in vitro have shown that they reduce the immunogenicity of mRNA and also improve translation efficiency.
At the same time, researchers found that increasing the length of the poly(A) tail and reducing the U content or hiding it in the sequence would produce low immunogenicity.
In addition to modifying nucleotides and adding poly(A) tails, optimizing GC-rich codons and minimizing U content is another effective method to eliminate RNA immunogenicity.
CureVac and Acuitas Therapeutics have developed a sequence engineering method that does not require any chemical modification of mRNA.
They designed the EPO mRNA sequence by selecting GC-rich codons for each amino acid, and used lipid nanoparticles (LNP) to administer systemically to pigs.
The results indicate that the expression of EPO protein leads to a positive physiological response without detectable immunogenicity.
However, it should be noted that the more GC content is not the better, because excessive GC content is not conducive to protein expression.
After in vitro transcription, the production of mRNA requires a series of purification processes including concentration, precipitation, extraction and chromatography.
Sophisticated technology, such as anion exchange chromatography, size exclusion chromatography, high performance liquid chromatography (HPLC) and affinity chromatography to remove double-stranded RNA and transcript fragments.
These purification procedures are necessary to eliminate immunogenicity.
It is reported that the mRNA with ψ modification after HPLC purification has no immunogenicity and the protein translation efficiency is significantly improved. In a representative example, Pardi et al. modified the light chain and heavy chain mRNA encoding the broadly neutralizing HIV-1 antibody VRC01 by m1ψ modification and HPLC purification, and wrapped them in LNPs. It was found that after systemic administration, mRNA-LNPs quickly turned into functional antibodies in mice. A single injection of mRNA-LNP completely protected mice from HIV-1 infection.
5 mRNA delivery
Safe and effective delivery is one of the biggest challenges in developing mRNA therapies, which is more challenging than delivering small oligonucleotides.
The size of mRNA (300–5,000 kDa, 1–15 kb, Figure 3a) is significantly larger than siRNA and miRNA analogs (13-15 kDa), antisense oligonucleotides (4-10 kDa) and anti-miR (4-10 kDa).
N-acetylgalactosamine (GalNAc)-oligonucleotide conjugates exhibit excellent liver targeting efficiency and safety in vivo, but are not effective for mRNA delivery.
Due to their size, charge, and degradability, naked mRNA cannot easily pass through cell membranes and effectively penetrate into the cytoplasm.
Studies have shown that naked mRNA is taken up by cells and accumulated in the endosome through the scavenger receptor-mediated endocytic pathway.
The uptake of mRNA in most cells is inefficient, with the exception of immature dendritic cells, which can take up and efficiently accumulate mRNA through the macropinocytosis pathway.
However, the wide application of therapeutic mRNA requires more efficient and safer delivery methods, which are critical to the realization of promising translational therapies, such as vaccination, protein replacement therapy, and genome editing.
Therefore, suitable mRNA preparations such as liposomes, polysomes, lipid complexes and polymers are necessary for effective delivery of mRNA into most types of cells (Figure 3b).
Usually, the nanoparticles loaded with mRNA are taken up by endocytosis, and then the mRNA is released from the endosome. The lysosome will initiate translation and produce any type of protein.
Including secreted, transmembrane, intracellular and mitochondrial proteins (Figure 3c).
In recent years, various materials, such as lipids, lipids, polymers, peptides, proteins, and extracellular vesicles, have been designed and explored for the delivery of mRNA in vivo and in vitro.
Most of these materials are inspired by siRNA and plasmid DNA delivery technology.
The chemical structure of representative lipids, lipids and polymer-based materials are shown in Figure 4.
See Table 1 for detailed information about its composition and ratio, mRNA load, route of administration, indications, applicants and references.
Lipids and lipid-derived materials are the main components of the delivery system (Figure 4). By using lipids or lipid-like substances (lipidoids), various vesicles, liposomes, lipid nanoparticles (LNP), lipid emulsions, lipid implants, etc. can be designed, for example, N- [1-(2,3-dioleoyloxy]propyl] -N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyloxy-3-trimethyl chloride Ammonium propane (DOTAP), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), these are used as classic cationic lipids to deliver DNA, siRNA and mRNA.
Recently, BioNTech used DOTMA, DOTAP, DOPE and cholesterol deliver mRNA to dendritic cells, macrophages, lung endothelial cells, and chimeric antigen receptor T (CAR-T) cells. This technology is undergoing clinical trials. It has been applied previously.
A series of lipid and lipid-like delivery of siRNA are explored and further researched to deliver mRNA in vivo. These materials include DlinDMA, Dlin-MC3-DMA, C12-200, cKK-E12, 5A2-SC8, 7C1 and 1,3 ,5-triazinane-2,4,6-trione (TNT) derivatives, etc. (Figure 4).
Based on these key lipids or lipids, through the adjustment of key lipids and auxiliary lipids, PEG-lipid and cholesterol Molar ratio, changing the auxiliary lipid or PEG-lipid, adding other ingredients (such as protamine), or using the same formula or optimized formula of siRNA can achieve effective mRNA delivery and protein expression. Its DLin-MC3-DMA is The FDA-approved material is also used in the first FDA-approved siRNA therapeutic Onpattro (patisiran), EC.
Several other cKK-E12-derived lipidoids, including OF-02, OF-DegLin and OF-C4-Deg -Lin, has been shown to deliver mRNA to the liver and/or spleen and effectively express protein by systemic (intravenous) administration.
Other lipids and lipids, including I-DD3/A-DD3/B-DD3, lipid 5 and H, TT3, LP01, C14-113, ZA3-Ep10, MPA-A/MPA-B, C12-( 2-3-2), 306Oi10, ssPalm/ssPalmO-Paz4-C2 and ATX-100 (Arcturus’s representative lipid) (Figure 4), have been designed and studied to deliver mRNA intravenously or locally to the target In tissues and cells, Intellia Therapeutics, Ethris and Arcturus Therapeutics have developed mRNA delivery materials for R&D and clinical research: DOTMA, Lipid 5, LP01, C12-(2-3-2) and ATX-100.
In a recent study, an mRNA vaccine encapsulated with DOTMA-lipid complexes bioengineered T cells, which encodes a single-chain variable fragment (scFv) that specifically binds to CLDN6 and targets the surface antigen of carcinoembryonic cells.
Functionalized CAR-T cells show excellent anti-tumor effects in refractory mouse models. Moderna’s recently developed DOTMALNP targeted mRNA treatment of inherited arginase deficiency metabolic liver disease, an autosomal recessive metabolic disease caused by (ARG1) gene mutations.
By using the LNP-INT01 delivery system, the Intellia Therapeutics team obtained clinically relevant levels of in vivo genome editing of the mouse thyroxine gene, which contains the biodegradable ionizable lipid LP01.
In addition, we have developed some lipid or lipid-derived materials and achieved effective gene or siRNA delivery.
Therefore, we will continue to develop and research lipid-based mRNA delivery systems and obtain excellent liver-targeted mRNA delivery systems.
The principle of lipid design for mRNA delivery remains to be further elucidated. However, several aspects have been proven to be key factors determining delivery efficiency and safety.
First, the component ratio and the selected phospholipid have an important effect on delivery efficiency.
Although phospholipids are not necessary for siRNA delivery in some cases, there are relatively few ionizable cationic lipids, and more zwitterionic phospholipids facilitate mRNA delivery.
The delivery efficiency of mRNA-LNP containing zwitterionic lipid DOPE is higher than that of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), which is often used for siRNA delivery.
Presumably, mRNA is larger and more flexible than siRNA.
A small amount of ionizable cationic lipids is sufficient to load and concentrate mRNA to form stable nanoparticles, which can be effectively released when entering cells.
However, a large amount of ionizable cationic lipids may bind tightly to mRNA and cannot be released in cells, which may result in low delivery efficiency.
Due to the difference between the molecular interactions surrounding RNA in the water sac of LNP, the performance of DOPE and DSPC are also different.
Second, biodegradability is important for both efficacy and safety. As mentioned earlier, Dlin-MC3-DMA is an FDA approved excipient.
However, in order to further increase the therapeutic index of siRNA-LNP, the biodegradable version of L319, developed by adding ester bonds to the hydrophobic dialkyl chain, enables siRNA-LNP to be quickly eliminated in the liver.
Designed ATX lipid (Arcturus), which contains an ionizable amino head group and a biodegradable lipid backbone (Figure 4), which degrades and removes a large amount of lipids in the liver faster than Dlin-MC3-DMA.
Therefore it is tolerated in non-human primates (NHP).
Lipid 5, a biodegradable ionizable lipid, uses a primary ester on one of the hydrophobic tails to enhance liver clearance.
In addition, OF-Deg-Lin is a biodegradable ester derived from OF-02, which can deliver mRNA to the spleen, while the non-biodegradable OF-02 accumulates in the liver and promotes mRNA expression (Figure 4).
Third, lipid saturation significantly affects the delivery of mRNA in cells.
According to reports, as the degree of saturation increases from 2 double bonds to 0, the temperature of the lamellar (Lα) to reverse hexagonal (HII) phase transition increases, indicating a decrease in fusion.
DLin-DMA has the lowest phase transition temperature, is the easiest lipid to fuse, and shows the most effective siRNA delivery efficiency.
Therefore, unsaturated lipids, especially cis-double bond lipids, are designed to facilitate mRNA delivery. For example, OF-02, designed based on cKK-E12, increases mRNA expression in vivo by introducing unsaturated fatty chains.
It is speculated that, similar to the unsaturated lipid tail of linoleic acid, the fluidity of the cell membrane can be improved by establishing structural defects, which will promote its entry into the cell and escape from the endosome. These two key factors determine the ultimate efficiency.
Polymers or their derivatives constitute another large family of mRNA delivery vehicles. Linear or branched polyethyleneimine (PEI) is a widely used cationic polymer for nucleic acid delivery in vivo and in vitro.
It is also used to package influenza virus hemagglutinin and nucleocapsid mRNA (saRNA) that encodes itself to protect people from viral infections.
In addition to the classic gene delivery polymer PEI, many different polymers have been synthesized to evaluate their mRNA delivery capabilities.
TarN3C10, DD90-C12-122, A1, C1 (PBAE-PCL), aminopolyester (APE) and hDD90-118 developed by Daniel G.
Anderson and colleagues, these are used alone or in combination with other lipids ( For example, DSPC, cholesterol, 14/0 PEG2000 PE or 18:1 PEG2000 PE) deliver mRNA to hepatocytes or enter the lung epithelium by intravenous injection or inhalation, and achieve ideal titer and safety results.
The polymer of N5 (PEH) and polyamine (2-2-2-2) forms nanocomplexes with mRNA and ribonucleoprotein through electrostatic interaction, and causes high levels in different cells by enhancing the stability of mRNA and protein synthesis Transfection of mRNA.
In addition to the above-mentioned materials, there is another biodegradable polymer designed by Robert M. Waymouth and colleagues, called the Charge Modified Releasable Transporter (CART) (Figure 4, CART D13 / A117).
These materials, especially oligomers (carbonate-b-α-amino ester), use an unprecedented mRNA delivery mechanism.
Oligo (α-amino ester) polycations, they can non-covalently complex, protect and deliver mRNA, and then through degradation, charge-neutral intramolecular rearrangement to change physical properties, resulting in the release of functional mRNA and Efficient protein translation in the cell.
TriManlip, a trimannosyl diether lipid, combined with Lip1 (O, O-diol-N- [3N-(N-methylimidazolium) propylene] phosphoramidate), Lip2 (O, O -Diol-N-histamine phosphoramidate) and PEG-HpK (PEGylated histidine-ylated polylysine) to form lipoprotein complexes to effectively deliver mRNA to dendritic cells and achieve cancer treatment .
In addition, it has been reported that CP2k, aPACE, PEI10k-LinA15-PEG3.0, PEG-PAsp(TEP)-Chol, cRGD-PEG-P(Lys-MP), PEG [Glu(DET)] 2 etc. can be passed intravenously Or subcutaneous administration delivers mRNA to lung, liver or tumor (Figure 4).
In the past, we designed and evaluated a large number of polymers for nucleic acid delivery, and studied the molecular structure of the polymer, the form and degree of polymerization, hydrophobic core, hydrophilic chain, PEG fragments, targeted partial modification and other nucleic acid (siRNA) The impact of delivery performance.
Various polymers have been further synthesized and are being evaluated for their effect on mRNA delivery. As a representative example, cRGD-poly(ethylene glycol) (PEG)-polylysine (PLys) (thiol) and poly(N-isopropylacrylamide) (PNIPAM)-PLys (thiol) ) The hybrid polymer composed of successfully delivered mRNA to tumor tissues and mediated gene expression.
Compared with lipids, polymers are less used in nucleic acid therapy, largely due to their molecular complexity and uncontrollable manufacturing.
In this case, simple and effective polymers are most likely to be used clinically. At the same time, biodegradation and biological response are also key factors to be considered in mRNA delivery.
The former is beneficial to reduce toxicity and enhance the therapeutic index, while the latter may promote cellular uptake and escape of bioreactive endosomes. PBAE, CART and APE are representative biodegradable polymers that exhibit effective mRNA delivery efficiency in animals.
In addition, targeting moieties can be introduced into the polymer to enhance its tissue targeting performance, including TriMan-Lip and cRGD-PEG-P (Lys-MP) (Figure 4).
Another commonly used material for mRNA transfection is protamine, a small protein rich in arginine.
Protamine can be tightly combined with mRNA to form nanoparticles, which can protect mice, ferrets, and pigs against influenza viruses.
The protamine/mRNA complex has also made progress, and a large number of clinical trials are underway.
However, mRNA pharmaceutical companies are gradually inclined to replace protamine with LNP because the former has better mRNA protection and delivery efficiency.
In addition, some other protein or peptide-derived materials, such as OM-PBAE, RALA and extracellular vesicles, virus-like particles, chitosan-alginate gel scaffolds, fluorinated peptide crystals, DNA-modified gold nanoparticles and Polycation functionalized zirconium (Zr)-based metal organic framework (MOF) has been proven to be used for in vivo/external mRNA transport.
Several polymer or lipid-based commercial transfection reagents, such as in vivo jetPEI™, Lipofectamine™, MegaFectin™, Stemfect™ and TranslT™, are also capable of concentrating and loading mRNA, protecting its cargo from degradation and transporting in and out of the body. cell.
Compared with other delivery systems, nano-sized preparations have many advantages, such as easy manufacturing, small batch-to-batch variation, good biocompatibility and scalability.
In addition, some liposomes and polysomes can be functionalized by coupling chemical groups with specific cell or tissue ligands.
Recently, these nanoparticles or nanostructures have been widely used in mRNA-based cancer immunotherapy, antiviral vaccines and the expression of functional proteins in specific tissues.
In addition to the delivery system, choosing an appropriate injection method for a specific tissue or disease is also important to ensure the successful delivery of mRNA.
Electroporation and microinjection are commonly used for mRNA transfection in vitro.
Some preclinical and clinical studies are used to evaluate the treatment of cancer immunotherapy with electroporation for IVT mRNA or patient-derived mRNA (Table 2).
Intravenous injection of naked mRNA can activate the innate immune system, which indicates that the technology can be applied to require an immune response and a relatively small amount of encoded protein.
It may be the kind of other clinical applications that need to replace large amounts of protein therapy.
However, when the combined delivery vehicle forms a formulation, mRNA can be administered through a variety of routes, such as intravenous, subcutaneous, intradermal, intramuscular, intratumor, intranasal, intraperitoneal, intratracheal and retroorbital injection.
So far, dozens of pre-clinical and clinical developments have been carried out to study different mRNA administration routes in the fields of infectious diseases, cancer and protein deficiency diseases.
6 Clinical and preclinical research progress of mRNA
6.1 mRNA vaccine in tumor immunotherapy
According to ongoing clinical trial information (see website http://www.clinicaltrials.gov), mRNA therapy is mainly used in the following areas: cancer immunotherapy, specifically for mRNA-based dendritic cell (DC) vaccines. In inducing a potential immune response, DCs play a key role. They can guide cytotoxic T lymphocytes and natural killer cells to form a powerful anti-tumor weapon that can attack tumor cells. For mRNA-based DC vaccines, IVT mRNA and mRNA derived from autologous tumor stem cells are used to load the DC with tumor-specific antigens.
The mRNA can be used to engineer DCs ex vivo or in situ. For the ex vivo method, the precursor DCs isolated from the patient are activated to form mature DCs, which are loaded with mRNA encoding the antigen and injected into the patient again.
There are several methods that can be applied to DC antigen loading, including nuclear transfection, liposome transfection, ultrasound, and electroporation. Among them, electroporation is a commonly used technique.
The most commonly used for DC differentiation is granulocyte-macrophage colony stimulating factor (GM-CSF) combined with IL-4. GM-CSF can effectively stimulate the immune system.
It can recruit immune effector cells at the injection site and promote antigen presentation. DC vaccine, GM-CSF adjuvant and mRNA have been used in several clinical trials (NCT03396575, NCT00204516, NCT00204607, NCT00626483, etc.).
The maturation status of dendritic cells is crucial for vaccination. Dendritic cells express high levels of costimulatory surface markers, resulting in better therapeutic effects.
However, there are also reports to the contrary that mRNA uptake and subsequent antigen expression only occur in immature dendritic cells. In addition to the mature state, the ability of DCs to produce IL-12p70, an important cytokine that drives differentiation to TH1, has been shown to affect the clinical response of DC vaccines.
The production of IL-12p70 can be achieved by stimulating DCs with TLR ligands or pro-inflammatory cytokines.
For in situ transfection of DC, the common practice is to inject the mRNA encoding the antigen directly into the lymph node or co-deliver it with TriMix.
Both methods are undergoing clinical trials. For example, the phase I clinical trial (NCT01684241) for advanced melanoma injected into the nodule has been completed.
TriMix is a mixture of three mRNAs, encoding immunomodulators CD40L, CD70, truncated constitutively expressing activated TLR4.
According to reports, this so-called TriMix platform exhibits a higher stimulation ability than other classic stimulatory cytokine complexes, as well as enhanced effector T cell expansion.
The latter includes a complex composed of IL-1β, TNF-α, IL-6 and prostaglandin E2 to stimulate DC.
Although naked mRNA can activate TLR and induce DC activation, this process is not enough to fully activate the DC’s antigen presentation ability.
The use of stimulants (such as TriMix) is an effective way to deliver mRNA encoding the antigen. In 2010, the TriMix-DC vaccine (NCT01066390) for the treatment of advanced melanoma was implemented for the first time.
Afterwards, the TriMix-DC vaccine combined with the checkpoint inhibitor ipilimumab has achieved encouraging results (NCT01302496).
Boczkowski et al. reported for the first time that DC induced a potent antigen presentation ability and inhibited tumor growth in mice after stimulation with mRNA encoding tumor antigens.
Since then, the availability of tumor-associated antigens has increased, such as carcinoembryonic antigen (CEA), human telomerase reverse transcriptase (hTERT), prostate cancer-associated antigen (PSA), Wilm’stumor-1 (WT1), gp100, MUC1, Tyrosinase and survivin, etc., the number of preclinical and clinical studies of mRNA is flourishing just like the ready-made anticancer vaccines (Table 2).
For example, Antwerp University Hospital initiated a number of clinical trials to study the role of autologous DC loaded with mRNA encoding WT1 antigen in cancer treatment (NCT02649829, NCT02649582, NCT01291420, NCT01686334, NCT01686334).
The patient-derived hTERT and survivin mRNA are loaded into DC, and curative patients who have been treated with a vaccine to cure prostate cancer are in clinical phase I/II (NCT01197625).
The route of administration of DC vaccine has an important influence on the distribution of DC. Several routes of administration of DC have been in clinical trials, such as intravenous, subcutaneous, intradermal, intranodal and intratumoral administration, and intradermal administration is most commonly used.
This is because there are multiple types of immune cells in different layers of the skin, including Langerhans cells, T cells, skin DCs, and plasma cell-like DCs.
Some studies have shown that intravenous DC vaccine has a poor effect on lymph node migration. Other studies have shown that compared with intradermal injection of mRNA, lymphatic internode injection of mRNA is more effective in inducing antigen-specific T cell responses, because the resident DC of the lymph node can quickly and effectively phagocytose mRNA.
However, more and more evidence proves that the debate on which route of DC injection is superior is important. Combining different routes of administration may be a good choice for inducing systemic immune response.
In addition to DC, mRNA is also transfected into other immune cells to produce cancer vaccines, such as Langerhans cells (LC), cytotoxic T lymphocytes, and natural killer (NK) cells. LC is a type of DC in the skin. Many studies have shown that LC is very significant in inducing cytotoxic lymphocyte (CTL) response.
Clinical trials using LC-based cancer vaccines for the treatment of melanoma or myeloma are ongoing (NCT01995708, NCT01456104).
In addition, T cells and NK cells can be transfected with mRNA encoding chimeric antigen receptors (CAR) to promote antigen binding and cell activation, thereby identifying and killing tumor cells that express these antigens on the cell surface. According to reports, the CAR strategy is effective in several animal tumor models and has entered clinical trials (NCT01355965, NCT03415100).
Through the ongoing clinical trials of mRNA vaccines for tumor immunotherapy, we found that the application of DC vaccines in combination with other anti-tumor therapies is increasing, such as chemotherapy, siRNA, cytokines and antibodies (NCT00672542, NCT02649829, NCT03396575) ).
According to Anguille et al., the combination therapy is based on three main mechanisms: enhancing immune response, maintaining tumor-related immunosuppression, reducing tumor burden and increasing tumor cell immune sensitivity.
Reasonable use of DC vaccine combined with other therapies can improve the overall curative effect and cure rate.
6.2 mRNA vaccine can prevent infectious diseases
Vaccination of infectious diseases is a broad application area of mRNA therapy. Some mRNA-based vaccines against infectious diseases are being studied to treat diseases such as influenza, rabies, HIV, and Zika virus infection. Among these mRNA technology companies, Moderna, Inc. has the most product lines for developing infectious disease vaccines, mainly based on its LNP platform.
Their H10N8 influenza vaccine, a modified mRNA vaccine formulated with LNP that encodes the viral antigen protein hemagglutinin (HA), has been undergoing a phase I clinical study (NCT03076385) in healthy volunteers.
The data shows that a 100 μg intramuscular injection dose can induce a high level of immunogenicity, and is safe and well tolerated.
Human cytomegalovirus (CMV) is the main cause of neonatal infection. It can cause serious complications such as deafness, microcephaly, vision loss and mental deficits.
There is currently no approved vaccine for CMV infection. The latest published data show that Moderna’s mRNA vaccine encoding CMV glycoprotein gB and pentamer complex (PC) produced effective and durable neutralizing antibody titers in immunized mice and NHPs.
The vaccine is currently undergoing phase I clinical trials (NCT03382405).
Another modern vaccine formulated by LNP, which encodes viral antigen proteins (Zika virus prM and E), Zika mRNA vaccine, is currently in phase I/II clinical studies for healthy volunteers (NCT03014089).
Children born to mothers infected with Zika virus have microcephaly, a serious disease characterized by abnormally small heads and severe neurological disabilities.
There are currently no more treatment options or no approved vaccine against Zika virus infection or congenital Zika syndrome.
Studies in mice have shown that the Zika mRNA vaccine can prevent intrauterine infection of mouse Zika virus and protect the fetus from Zika-related congenital damage.
This is the first study of a vaccine to prevent Zika virus during pregnancy. So far, some mRNA infectious disease vaccines are undergoing clinical trials, mainly rabies, HIV, CMV, influenza and Zika vaccines (Table 2). More preclinical studies are underway, and the prospects for success are bright.
6.3 Protein replacement therapy
One of the most common applications of mRNA is the introduction of therapeutic antibodies and functional proteins, which are misexpressed or lost function due to gene mutations. Although dozens of these concepts have been proposed, mRNA molecules were not initially promising.
In recent years, advanced technologies such as chemical modification of nucleosides, refined purification processes and new delivery strategies have largely overcome these shortcomings.
mRNA-mediated transcription replacement therapy can be applied to produce functional cystic fibrosis transmembrane transduction regulator (CFTR) protein, which is defective in CF patients.
In in vitro studies, wild-type CFTR mRNA was transfected into primary cultured human nasal epithelial (HNE) cells and human bronchial epithelial cells, and its expression level increased almost twice, and a large amount of CFTR was located in the cell membrane.
Translate Bio’s CF mRNA treatment is currently undergoing a phase I clinical study, making it the first company to conduct mRNA-mediated treatment of rare genetic diseases in humans (NCT03375047). Moderna is currently exploring the use of pulmonary mRNA delivery to treat CF.
Currently, most mRNA-based gene therapies are in preclinical research. In 2016, AstraZeneca and Moderna initiated a phase I clinical trial of AZD8601 (mRNA-based investigative therapy, encoding vascular endothelial growth factor A (VEGF-A)). Before, scientists injected mRNA encoding VEGFA directly targeting mice Heart. They found that this mRNA can produce enough protein to improve the survival and health of animals after a heart attack.
On May 1, 2018, a randomized, double-blind phase II clinical trial of AZD8601 was conducted with moderately impaired contractile function Of coronary heart disease patients undergoing arterial bypass grafting to study the safety and tolerability after epicardial injection (CABG) (NCT03370887). This is the fastest-growing project of Moderna so far.
The protein translated by mRNA can be transformed into therapeutic protein through a series of processes, including folding, post-translational modification, aggregation into secretory granules, and transport to the outside of the cell. In these processes, a variety of factors may affect the ultimate physiological role of proteins.
For example, signal peptides play an important role in directing protein secretion and are widely used to improve protein expression in cells.
Ideally, extracellular mRNA should be transfected into cells that naturally secrete the encoded protein, otherwise the signal sequence needs to be optimized. Another major factor that should be considered is the cell or tissue specific delivery of IVT mRNA.
Different cells have different post-translational modifications. For example, glycosylation, proteolysis, cofactor-dependent folding and clearance of misfolded proteins are cell-dependent in heterologous tissues.
In addition, compared with long-term gene therapy (such as plasmid DNA transfection), mRNA delivery results in a shorter duration of protein expression.
Sometimes, this is considered a limitation of mRNA therapy. However, in the case of repeated administration, by transiently expressing the therapeutic protein, many pathological defects can be treated. In addition, an understanding of mRNA pharmacokinetics and pharmacodynamic characteristics is necessary to guide its dosage.
6.4 Regulating cell fate and differentiation
Another promising direction of mRNA-based therapy is the use of mRNA to program and remodel cells. Induced pluripotent stem cells (iPSCs) provide a potential source of cells for the establishment of disease models, regenerative medicine and tissue bioengineering.
DNA and RNA-based technologies have been successfully applied to transfect somatic cells to produce iPS cells. The use of IVT mRNA shows faster gene expression and can produce clinically relevant iPSCs without integration.
So far, the application of RNA-based technology to cell reprogramming and lineage switching is still in the laboratory research stage. Many attempts have been made to focus on the main considerations of mRNA, such as cytotoxicity, immunogenicity and transfection efficiency.
Warren et al. reprogrammed them into pluripotent cells by transfecting chemically modified mRNAs encoding KLF4, c-MYC, OCT4, and SOX2 factors into somatic cells. Yoshioka et al. reported another method based on Venezuelan equine encephalitis reprogramming factor (VEE-RF) RNA.
The VEE-RF RNA replicon expresses four reprogramming factors. IPS cells can be effectively produced by transfecting VEE-RF RNA into human fibroblasts in a single time. These mRNA-based iPS cell reprogramming technologies may have a great opportunity to be transferred to clinical applications.
IVT mRNA can also be used to guide iPSC differentiation into terminally differentiated cells.
Warren et al. also proved this. Repeated transfer of MYOD1 mRNA into iPSC can transform it into terminally differentiated myoblasts.
In addition, researchers and biotechnology companies have also noticed the use of therapeutic mRNA to program unwanted, diseased cells to synthesize endotoxins, thereby inducing cell suicide.
Moderna has designed an mRNA encoding a toxin protein that contains two different miRNA binding sites (miRts) in its 3’UTR.
After being wrapped by LNP and injecting mRNA into the tumor, a large number of toxic proteins are expressed in hepatocellular carcinoma cells (HCC) to induce apoptosis, but the expression of toxic proteins in healthy liver cells is limited.
This is because different miRNAs are recruited into HCC and healthy liver cells through mRNA, and a large number of miRNAs degrade the mRNA in normal liver cells through the mechanism of small interfering RNA.
Other recruited miRNA142 is abundant in hematopoietic cells, resulting in the suppression of protein expression in many antigen-presenting cells.
The miRNA-mediated Trojan horse provides a solution for alleviating off-target expression and immune response in mRNA therapy.
6.5 Using mRNA recombination technology for gene editing
The use of engineered nucleases to precisely knock in or knock out defective genes makes gene editing technology promising in the treatment of genetic diseases.
These nucleases include zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and RNA-guided clusters regularly spaced short palindrome repeats (CRISPR/CRISPR-associated protein (Cas) endonucleases.
So far, they have DNA plasmid vector-mediated editing was successfully carried out. However, the sustained expression of plasmid-mediated nuclease is a double-edged sword.
The disadvantage is that the sustained expression of nuclease increases the loss of gene editing.
Target opportunity. In this regard, ZFNs, TALENs, and Cas endonucleases encoded by mRNA seem to be a good alternative because their expression is transient.
In recent years, mRNA encoding gene editing tools have been widely used in the construction of human primary cells and transgenic animals.
CRISPR/Cas9 is one of the most common and simple systems used to generate modified genomes of carrying animals.
For example, through microinjection or electroporation, Cas9 mRNA and guide RNA (gRNA) were transferred into fertilized eggs, which successfully produced large-scale mutant mice.
Other Cas nucleases, such as Cpf1, have also been used to generate knockout mice. In a proof-of-concept study, Cas9 mRNA (≈4500nt) and sgRNA (≈100nt) were injected intravenously into mice through a new zwitterionic amino lipid (ZAL) delivery system to induce targeted DNA editing.
This non-viral RNA delivery system provides a powerful tool for in vivo gene editing.
The transfer of ZFN mRNA and adeno-associated virus (AAV) serotype 6 vector into human hematopoietic stem cells, progenitor cells (HSPC) and T cells by electroporation can also effectively achieve genome editing of these cells.
However, in the transition to clinical medicine, the safety and effectiveness of gene editing technology should be paid attention to.
Because a CRISPR/Cas9-AAV6 gene editing study on hematopoietic stem cells and progenitor cells found that Cas9 mRNA activates transcriptional changes, causing viral responses and overall transcription down-regulation.
7 Thoughts on the mRNA drug industry
Based on the promise of mRNA as a powerful therapy for the treatment of genetic diseases, cancer, infectious diseases and other diseases, more and more well-funded biotechnology companies have been established on the market to set foot in this field, such as Moderna, CureVac, BioNTech, Argos Therapeutics, RaNA, Translate Bio, Ethris, Arcturus, Acuitas, etc.
These companies have achieved some breakthroughs in technology, but the key issues of mRNA delivery, off-target, and immunogenicity have not been completely resolved.
There is still a long way to go before the mRNA can be turned into a marketed drug.
For protein replacement therapy, the dose needs to be carefully considered, because the amount of protein produced by the same dose of mRNA may vary from person to person.
It is reasonable to screen candidate mRNAs that encode effective proteins, and these proteins should have a wide therapeutic window at low doses.
Therefore, the first choice for cancer immunotherapy and infectious disease vaccines is mRNA. For cancer immunotherapy, the choice of mRNA antigen can be a big problem.
For example, mRNA-4157 (Moderna) is composed of 20 mRNAs and is obtained by sequencing and screening genes derived from patient tumors and blood.
The technology to screen neoantigens and predict their potential to generate sufficient immune responses is still under development. Important new antigens may be missed by R&D personnel, but the selected ones are inefficient or off-target antigens, which will lead to safety problems.
Mutant clones in tumor tissues may vary greatly, so it is difficult to determine how much antigen is needed to generate a sufficient antigen immune response.
Another challenge is to deliver mRNA into the cell and promote its release from the endosome.
The mRNA length spans hundreds to thousands of nucleotides and is larger than other kinds of RNA drugs (such as siRNA). Therefore, a new delivery system needs to be designed.
The greater long-term challenge will be the tissue selectivity of mRNA. Commonly used LNP tends to be enriched in the liver, so that mRNA can be used for liver targeted therapy.
However, delivery of mRNA to other organs requires an appropriate route of administration.
For example, AstraZeneca’s clinical trial for heart attacks was to administer VEGF mRNA through epicardial injection or an intelligent delivery system.
Another noteworthy issue is the transparency of mRNA companies, including advanced technology and patent disclosure.
These companies continue to raise funds from private investors, but largely have reservations about their scientific details.
Research data may only be seen by investors, and outsiders can only guess.
8 Summary and outlook
In the past two decades, there has been very little involvement in the field of mRNA in the development of human drugs.
Now this field has attracted billions of dollars of investment.
Compared with traditional protein drugs, mRNA production cycle is shorter, lower cost and easier to control pollution.
NA vaccines also avoid several problems associated with DNA vaccines.
In addition, the two most interesting issues of mRNA are immunogenicity and stability, which are controlled to a certain extent after chemical modification of selected nucleotides.
As other RNA drugs (ASO and RNAi) are approved, the field of mRNA research will become more popular once more positive data is released.
There may be many problems with mRNA in the short term, but in the long term, this is definitely worth exploring.
Challenges and prospects of mRNA therapy
(source:internet, reference only)