November 27, 2022

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30 Years Development of mRNA Vaccines

30 Years of Development of mRNA Vaccines



 

30 years Development of mRNA vaccines.

 

Abstract:

In the early 1990s , the first use of mRNA vaccine as a therapeutic tool. In the following decades, a better understanding of mRNA pharmacology and new insights in immunology made mRNA- related technologies a new generation of vaccine candidates. 

 

This article summarizes the history and current situation of mRNA vaccines, and evaluates the development of mRNA vaccines from an immunological perspective .

 

In this article, we highlight the challenges in vaccine design, testing and management , the key factors in the design of mRNA- related vaccines, and the new opportunities brought by packaging mRNA in nano-vaccine.

 

Finally, we discussed the mRNA self-adjuvant effect, which, as a key two-sided parameter, determines the safety, effectiveness and strength of the induced immune response.

 

Introduction: The first step in the development of mRNA vaccines

The concept of using mRNA as a new therapeutic drug was born in 1989 , and the San Diego- based Vical company announced its success.

They showed that encapsulating mRNA into lipid nanoparticles can successfully transfer mRNA to various eukaryotic cells.

A few months later, Wolff et al. pointed out that naked mRNA can be injected directly into mouse muscles in their experiments .

 

In fact, this was originally carried out as a control for lipid delivery experiments.

After a few days of intramuscular injection of naked mRNA, the expression of the encoded protein can be detected.

This experiment proved for the first time that genetic information can be used to make proteins in living tissues through in vitro transcription of mRNA .

 

Importantly, this process does not require viral or non-viral vectors, which is far from the hypothesis of mRNA stability in vivo.

This makes mRNA a valuable and safe alternative to plasmid DNA .

Because mRNA can only be translated on the ribosome when it becomes cytosol, which avoids the risk of its integration into the host genome.

 

30 years of development of mRNA vaccines

 

mRNA in addition to therapeutic use of temporary errors substituted protein, the 20 century 90 ‘s proposed early mRNA can present antigen to the antigen presenting cell information.

Martinon showed for the first time that liposomes containing mRNA can encode influenza virus nucleoprotein to induce virus-specific cytotoxic T cell responses.

 

In addition to cellular immunity, Conry showed that humoral immunity was also activated, which indicated that mRNA prophylactic vaccines encoding carcinoembryonic antigens eventually induced anti-tumor antibody responses.

After 30 years of research, mRNA vaccines have ushered in a new situation, and many candidate vaccines have entered clinical trials.

 

In this review, we will list the most important basic content so that we can understand how mRNA vaccines are prepared and delivered.

We will discuss the immune results between different mRNA vaccine platforms, which may help induce more effective and safe immune responses.

 

 

 

Immunity perspectives of mRNA vaccines

 

mRNA is an attractive antigen

A key advantage of using antigen-encoded mRNA is that it provides a simple way to induce MHC I presentation and induce CTL responses.

Similar to viral infection, reverse transcription mRNA forms the instantaneous expression and accumulation of selective antigens in the cytoplasm.

It is then fully processed into polypeptides and enters the MHC I pathway.

 

At this point, a small number of mRNA molecules in the cytoplasm can ensure further antigen presentation to the CTL , and the protein can only rely on the inefficient cross-linking presentation pathway. Interestingly, mRNA antigens can also target the MHC II pathway.

 

This occurs after the secretion and recycling of the mRNA expression protein, or directly through the transfer of the antigen from the cytoplasm to the lysosome.

Such as designing lysosomal targeting sequences in the mRNA structure.

By comparing protein-related vaccines, antigens can be continuously obtained through mRNA , which has a significant effect on the breadth and affinity of antibodies, thereby bringing longer-lasting protection.

 

Customizing a new mRNA structure for a specific disease can be completed simply and quickly, making mRNA an ideal candidate for inducing immunity against infectious diseases.

These diseases that tend to mutate rapidly require flexible and rapid preparation of suitable vaccines to match the virus strains in the circulating system.

 

In this context, preventive mRNA vaccines have been considered safe and effective in dealing with infectious diseases in phase I clinical trials, such as influenza and rabies.

In large and small animal models, mRNA vaccines can induce immune responses against new infections, such as Zika, Ebola, and HIV .

Moreover, by in-situ expression of proteins in cells, mRNA can harvest appropriately folded products and glycosylated antigens, providing solutions for challenging products and limited stability of protein antigens.

 

Modern treatments have prepared an mRNA vaccine encoding five different subunits of the CMV pentamer .

Unlike the mRNA sequence targeting CMV glycoprotein gB , this multi-antigen mRNA vaccine can induce stronger and lasting neutralizing antibodies in immunized mice and non-human primates.

 

mRNA as a danger signal

The binding of mRNA to these hazard sensors leads to downstream signal transmission through specific linker molecules ( ie MyD88 vs. TLR7/8 , TRIF vs. TLR3) , which ultimately produces type I interferon and other pro-inflammatory cytokines ( such as IL-6 and TNF-α ) .

In turn, type I IFNs bind to autocrine or paracrine receptors to activate the Janus kinase signal transduction and transcription activation pathway (JAK-STAT) , which regulates the gene expression of hundreds of proteins involved in antiviral immunity.

Therefore, these signaling pathways coordinately activate and promote different innate and adaptive immune responses, which is called the “self-adjuvant effect” of mRNA .

 

Examples of mRNA vaccine development

Many mRNA vaccine platforms have emerged in recent years . The IVT mRNA basic structure and “mature” eukaryotic mRNA is very similar, the (i) a protein encoded by open reading frame (ORF) composed of, respectively, on both sides of (ii) 5 ‘ and 3’ untranslated regions ( UTRs of ), terminus (iii) 7- methyl guanosine 5 ‘ cap structure and (iv) 3’poly (A) tail.

These structural features in the non-coding mRNA plays an important role in the pharmacology, it can be adjusted by optimizing the individual mRNA stability, translation efficiency and immunogenicity.

 

2004 Nian, Kariko and his colleagues observed that dendritic cells in vitro human exposure to different sources of mRNA , the mammal can tolerate the mRNA , and bacteria, mammalian cells and necrosis of IVT mRNA with a strong inflammatory cells Factor response.

Interestingly, they found that the strong reduction of endogenous mRNA immunomodulatory ability may be due to the presence of modified nucleotides in the mRNA structure, such as methylated nucleosides or pseudouridines.

 

Therefore, it was determined that the naturally occurring post-translational mRNA nucleoside modification protects endogenous mRNA from immunological detection, which allows cells to distinguish pathological or invasive mRNA .

This is the mRNA to provide development of new opportunities : through a combination of modified nucleosides, known as “modified nucleoside mRNA ” of mRNA may reduce the immune stimulatory activity, thereby improving safety.

 

In addition, modified nucleosides enhance the stability and translation ability of designed mRNA vaccines, because they can avoid the direct antiviral pathway induced by type I interferons, and can programmatically degrade and inhibit invading mRNA .

For example, in IVT mRNAIt was found that substituting pseudouridine for uridine can reduce the activity of 2’5 ‘ oligoadenylate synthase, which can regulate RNaseI to cleave mRNA . In addition, the low activity of protein kinase R was also determined . Inhibit the translation process of mRNA .

 

In one treatment, Kormann, et al., Demonstrated a modified nucleoside mRNA encoding erythropoietin (of Epo) , which was added 25% sulfur uridine and 25% of 5- methyl-cytidine, intramuscular Two weeks later, the Epo level of the immunized mice was 5 times that of the control mice .

On the contrary, the unmodified mRNA did not change significantly and only caused a substantial immune response.

 

In addition to adding modified nucleotides, other methods have also been proven to improve the translation ability and stability of mRNA .

One example is the development of sequence design mRNA .

Optimizing mRNA expression by mRNA ORF and UTRs can be increased, for example, by enriching GC components, or by selecting UTRs of natural long-lived mRNA molecules .

Another method is to design a “self-amplified mRNA ” structure. Most of it is derived from alphavirus, and its ORF is composed of an antigen of interest and an ORF encoding viral replicase .

The latter drives the replication of mRNA in the cell , so it can significantly increase the antigen expression ability.

As early as 1995 , Johanning et al. found that intramuscular injection of self-amplified mRNA from Sindbis virus resulted in a 10- fold increase in protein expression compared to non-amplified mRNA .

This protein expression level can be maintained for a longer time ( from 2 Day arrives10 days ) .

 

At the same time, some modifications were made to the mRNA end structure. Antiretroviral cap (ARCA) modified to ensure proper positioning of the hat at the 5 ‘ end direction, which means that the most complete mRNA fragments effective ribosome binding.

Other cap modifications, such as phosphorothioate cap analogs, can further increase its affinity for eukaryotic translation initiation factor 4E (eIF4E) and enhance its resistance to mRNA uncapping complex.

We found that the mRNA polyA extending the time associated with the expression of tail, . 3 ‘the UTR -specific modifications can slow de olefination of polyA degradation tail.

 

In addition, some more exotic methods have been proposed, such as generating circular RNAs that can resist degradation by exonuclease .

In recent years, after artificially synthesized polyamine complexes are pre-assembled with mRNA and eIF4E protein, their expression efficiency is significantly higher than that of mRNA alone .

This may be due to the higher stability of these complexes and higher recruitment of ribosomes effect.

Conversely, by changing its structure, the effectiveness of mRNA to trigger the innate immune response can be further improved, but translation ability will be impaired.

 

CureVac AG found that the phosphorothioate backbone stabilizes mRNA, Or by precipitation with the cationic protein protamine, although the amount of antigen expression is reduced, stronger immune stimulation ability can be obtained.

This has promoted the development of the protamine complex mRNA molecule, making it either alone as an immune adjuvant ( ie RNA adjuvant ) for peptide and protein-based vaccines , or as an antigen-encoded mRNA and protamine mRNA.

The two-component mRNA platform composed of complexes plays a role in improving the immunogenicity ( ie RNA activity ) of the vaccine .

 

Taken together, these findings are used as the principles of mRNA vaccine formulation design.

One strategy is to use mRNA that is fully optimized to harvest a strong adjuvant effect, and the other strategy is to use modified mRNA to obtain high translation capabilities, thereby increasing the bioavailability of the antigen.

Considering the purpose of vaccination, we should pay attention to these modifications not only to facilitate mRNA translation, but also to partially or completely reduce the mutual lease of mRNA molecules and one or more virus-specific PRRs . Because these may weaken the adjuvant effect of mRNA vaccines.

 

After all, both outcomes are back-regulated by type I IFN- induced genes. People usually give priority to the translation ability of mRNA in order to improve the availability of antigen.

However, from an immunological point of view, innate immunity is more sensitive to mRNA , which can cause phenotypic immune responses and cytokine production, at least as important.

 

In fact, these innate immune signals will trigger and guide the choice of effect response, which is crucial to the therapeutic value of vaccines.

Nevertheless, the effectiveness of this mRNA self-adjuvant effect must be weighed against the inherent risks of any adverse reactions, including inflammatory reactions and autoimmune events.

The key to this challenge is to find a good balance between the translation capability of the mRNA vaccine and the adjuvant performance to obtain sufficient but safe immunogenicity, which will be discussed later.

 

 

 

 


mRNA vaccine delivery

 

In vivo approach

The first human experiment to evaluate mRNA delivery used an in vitro method, using mRNA encoding an antigen to transform mononuclear-derived DCs , which were then fed back into patients as a cellular vaccine.

Excellent reviews on mRNA- based DC vaccines can be found everywhere.

In recent years, hot spots have begun to point to direct delivery of mRNA .

Generally speaking, directly targeting mRNA to APCs in vivo has more advantages than obtaining DC vaccines in vitro .

First of all, the steps of isolating and culturing patient-specific DC in vitro can be omitted, thereby reducing manpower and material resources.

Secondly, the delivery of mRNA in vivo is closer to natural viral infection, which is beneficial to improve the vaccine effect.

 

A variety of immune cells and non-immune cells are directly transformed in the natural environment, which can immediately activate innate immunity and sequentially transmit signals to adaptive immunity.

Moreover, key immune events, such as the release of inflammatory cytokines and chemical factors, peak within a few hours after transfection, which cannot be achieved when preparing in vitro DC vaccines.

 

Local delivery of naked, unprotected mRNA vaccines

Only a few years later, the immune cells involved in the ingestion of unmodified mRNA were determined : skin-resident dendritic cells in mice can introduce naked mRNA through macrophagocytosis and trigger a T cell response.

Through RNActive ( RNA active) vaccine technology, it is determined that different types of cells participate in the uptake of mRNA vaccine. Intradermal injection, all vaccine components, including coding mRNA and protamine complex mRNA , are taken up by immune cells and non-immune cells, and the detection frequency is highest in macrophages, dendritic cells and neutrophils.

This is consistent with the increased expression of resident APCs activation markers and the transient production of different cytokines and chemokines, indicating the self-adjuvant effect of the preparation.

In addition to these locally induced effects, two independent studies tested RNActive or coding sequence mRNA (naked mRNA ), respectively, and showed that activated immune cells migrate to lymph nodes (LNs) , and can detect innate immune signals and mRNA- encoded antigens in draining lymph nodes. .

 

In recent years, it has been clarified that the injection route plays an important role in the cell types that mRNA contacts and the immune response induced.

Therefore, injecting mRNA directly into the lymph nodes is the best way to ensure delivery to APC .

Kreiter and colleagues emphasized this point. They pointed out that the unmodified naked mRNA vaccine can induce T- cell immunity through lymph node injection than through skin injection .

They demonstrated that in lymph nodes, naked mRNA is mainly taken up by resident DCs ( and macrophages ) through endocytosis.

In addition, with the increasing awareness of the extensive infiltration of immune cells into various tumor types , research on the application of naked mRNA in tumors has also been promoted .

In fact, in different mouse subcutaneous tumor models, tumor-infiltrating CD8a + cross-expressing DCs are mainly responsible for the uptake of mRNA after injection , resulting in mRNA expression for more than 5 days.

 

It is worth noting that the self-amplified mRNA situation is different. First, it was discovered that self-amplified mRNA cannot be directly transfected into APCs , and the length of self-amplified mRNA is significantly extended (10kb, while non-amplified mRNA is only 2-3kb) .

Nevertheless, this phenomenon of inability to directly transfect APCs did not stop triggering CTLs after intramuscular injection . In order to clarify the underlying mechanism, Lazzaro et al. studied the contribution of ( transfected ) muscle cells and ( untransfected ) professional APCs to CTL priming.

They concluded that APCs can absorb antigens processed by transfected muscle cells, and cross-presenting antigens expressed by muscle cells is the mechanism that induces CTL responses.

Therefore, transfection of somatic cells such as cardiomyocytes may affect the abundance and duration of antigens, and the activation of TLR and cytoplasmic RNA sensors in these cells can lead to local inflammation and dendritic cell infiltration.

 

In order to further improve the innate immune activity, antigen-encoded mRNA is combined with immune activators (proteins) or immunostimulants ( mRNA transcripts) for detection.

Intradermal injection of granulocyte – macrophage colony stimulating factor (GM-CSF) facilitates the infiltration of monocytes and the migration of mature DCs transfected with mRNA to lymph nodes.

The co-delivery of immune adjuvant polyinosinic acid and lipopolysaccharide can make APCs mature quickly and weaken the ability of cells to take up mRNA .

Therefore, these adjuvants can reduce the toxicity in mRNA transport, thereby changing the antigenic bioavailability of T cells.

In addition, VanLint et al . registered RNA immunotherapy by giving a mixture of 4 naked mRNA encoding an antigen and 3 additional immunomodulatory molecules ( CD40 ligand, constitutive activator TLR4 and CD70 , patented TriMix mRNA technology) .

TriMix technology is currently being used to treat melanoma and breast cancerA clinical study of mRNA immunotherapy, in which a mixture of unmodified naked mRNA sequences is injected into lymph nodes or directly injected into accessible tumor lesions.

Recently, a clinical study on dose escalation for HIV-1 infection showed that lymph node infusion of TriMix mRNA vaccine is safe and tolerable, and at the same time, a moderate-intensity HIV- specific T cell response was obtained at high doses ( total mRNA dose is 1.2 mg ) .

 

 

 


Development of mRNA delivery system

 

Nanoparticles open new channels

Despite the success of naked mRNA research, research on nanocarriers has begun. The basic principle behind this is that mRNA nanomedicine can be used as a multifunctional drug to expand the range of options for vaccine delivery.

First, because nano- mRNA can better prevent enzymatic damage, new delivery routes, such as intravenous injection, become possible.

In addition, by making mRNA into nanoparticles, its biodistribution, cell targeting and cellular uptake mechanism can be changed, thereby promoting the delivery of mRNA and vaccines.

 

Although it is now clear that the selection and optimization of nanoparticles is the key to successful mRNA transfection .

However, it is necessary to realize that many studies have shown that transfection experiments in cultured cells may not reliably predict the behavior of mRNA preparations in vivo.

Bhosle et al. further proved the obvious difference between in vitro and in vivo mRNA delivery.

They observed that there are completely different cellular uptake pathways in vitro and in vivo, both naked mRNA and lipid nanoparticle mRNA .

 

In addition, using a high-throughput system, Paunovska et al. were able to test hundreds of lipid nanoparticles (which are used to deliver nucleic acids to macrophages or endothelial cells after intravenous injection) and found that their effects in vitro and in vivo have little correlation.

These studies raise the question : Can a nanoparticle matrix be reasonably designed to give full play to its multifunctional role as an mRNA vaccine enhancer ?

In the following chapters, we will discuss some key considerations when designing mRNA vaccine nanoparticles , Especially for mRNA LNPs , because these have entered ( early ) clinical trials.

For the reader, an extensive summary of nanofragments with different chemical properties including but not limited to protamine, lipids or polymers, and hybrid preparations can be found elsewhere.

 

Design and preparation of lipid nanoparticles for mRNA delivery

The lipid forms tested for mRNA delivery generally consist of cationic / ionized lipids and other “auxiliary” lipids, such as phospholipids, cholesterol, and / or polyethylene glycol (PEG) .

Cationized lipids and negatively charged mRNA molecules form a complex through electrostatic interaction, and can be subdivided into

(i) “permanently charged lipids” based on the pKa of the amino group , such as DOTMA , DOTAP and DC- cholesterol, or

( ii) ” pH -dependent ionized lipids “, Such as D-Lin-MC3-DMA and lipid molecules C12-200 .

These ionizable lipids (pKa <7), originally optimized for siRNA delivery , have a neutral to mild cationic charge at physiological pH .

 

This is more beneficial than permanently charged lipids, the most important of which is that ionizable lipids are related to reducing toxicity and prolonging blood circulation life.

Other lipid components are considered “auxiliary lipids”Because they have unique functional properties, they may affect the structural arrangement of compound mRNA LNPs , thereby improving their stability or promoting mRNA uptake and cytoplasmic entry.

 

One of the key obstacles affecting the overall transfection efficiency is the degradation of endosomal phagocytic mRNA LNPs . Therefore, we strive to modify and optimize mRNA LNPs to promote the transformation of endosomes into cytoplasm.

The mechanism of endosomal mRNA release relies on the protonation (ionization) of LNP and the formation of a lipid mixture by the negative phospholipids of the endosomal membrane.

The formation of ion pairs between these lipids triggers membrane fusion and membrane instability, which in turn enhances the escape of molecules from endosomes.

In addition, this lipid exchange is believed to induce a non-bilayer structure transformation ( ie, the lamellar structure is transformed into an inverted hexagon ) , which will dissociate LNPs and destabilize endosomal membranes.

Research has linked the structural organization of mRNA and lipid components to the ultimate transfection efficiency.

It is pointed out that the physicochemical and structural characteristics of mRNA LNPs will depend on the lipid composition, the ratio of mRNA to total cationic lipids, and the synthesis of LNP .

 

When mRNA is mixed with monolamellar liposomes containing permanently charged lipids to prepare mRNA LNPs , it is assumed that the lipids rearrange to form a multilayer lamellar structure, in which mRNA molecules are interspersed in concentric lipid bilayers.

The addition of saturated phospholipids, such as DPPC and DSPC , increases the phase transition temperature of cationic liposomes and supports the formation of this lipid bilayer structure.

In contrast, unsaturated phospholipids ( DOPE ) help to form a non-lamellar lipid phase ( ie, inverted hexagonal phase ) , which has been found to help endosomes escape. However, the latter is only applicable under in vitro conditions.

In preclinical trials, DOPE is associated with increased disintegration of cationic (DNA) LNPs induced by serum , so the transfection efficiency in vivo is low.

 

Another method for preparing mRNA LNPs is the ethanol dilution method, which is suitable for preparing more advanced LNPs containing ionizable lipids . Here, individual lipids are dissolved in ethanol and then quickly mixed with mRNA in an acidic aqueous solution .

By diluting the ethanol phase, the lipid undergoes a concentration process to form LNPs while effectively encapsulating mRNA .

Then it is dialyzed against a neutralization buffer solution to remove ethanol and neutralize pH .

Kulkarni and his colleagues recently clarified the formation mechanism of these ionized LNP systems ( with different nucleic acid payloads ) .

 

They proposed that in the rapid mixing stage under acidic conditions, ( “empty” ) small liposome structures and ( “loaded” ) particles with greater electron density are formed.

Next, during the dialysis process, the pH is neutralized and these particles tend to fuse, eventually forming the LNP system.

In addition, they proved that the PEG- lipid component exerts a repulsive force on the particle surface to limit further fusion, which determines the final LNP size.

In another study, Yanez ArtetaEt al. pointed out that the structural rearrangement of mRNA LNPs based on D-Lin-MC3-DMA , DSPC , cholesterol and PEG- lipid presents a disordered inverse hexagonal phase, including D-Lin-MC3-DMA , cholesterol, water and mRNA .

 

This core is surrounded by a lipid monolayer and mainly contains DSPC and DMPE-PEG2000 components, as well as a small part of D-Lin-MC3-DMA and cholesterol.

Interestingly, by adjusting the molar ratio of different lipids, the author can prepare mRNA LNPs with different surface properties .

They found that the LNP component that facilitates the separation of D-Lin-MC3-DMA lipid from the inner core part to the outer surface is related to improving the transfection efficiency.

In contrast, the transfection ability of mRNA LNPs rich in DSPC on the surface is weak ( close to DSPC monolayer ) .

In addition, the author believes that the enrichment of cholesterol may be related to the formation of nanocrystals on the surface of LNP .

Although this study has not been studied further, previous reports have linked the emergence of cholesterol nanocrystals with the increase in transfection efficiency.

 

It is worth noting that the benchtop microfluidic mixing device can be used to produce unloaded liposomes or mRNA LNPs through the principle of ethanol dilution , and the scale can be expanded to clinical or industrial applications.

The preparation of LNPs by these microfluidic devices has the advantages of controllability and high throughput, which will inevitably promote the clinical translation of new LNPs gene delivery.

In addition to facilitating the production process, the screening of new lipid libraries also helps to continuously optimize lipid composition.

In addition, this also supports the discovery of new lead ionized lipids, which greatly improves the efficiency and safety of mRNA transfection.

 

In addition to advances in the design and production of LNP , innovation in the mode of action of transfected cells in vivo is the key to further development.

Although the theory of membrane fusion has existed for more than ten years, the mechanism of LNP promoting cytoplasmic delivery of mRNA cannot be limited to this.

Recent studies have shown that the endogenous transport of mRNA LNPs is a dynamic process involving circulation pathways and signal pathways that affect cytoplasmic entry and mRNA translation.

For example, the transporter Niemann-Pick type C-1 , located in the late endolysosome , was found to be responsible for most of the exocytosis of internalization (si)RNA LNPs , thus strongly reducing cell elution.

In contrast, the mechanical targeting of rapamycin complex 1 to the lysosomal membrane is the key to triggering the delivery of mRNA translation.

 

Although the relationship between mRNA entry into cells and intracellular transport mechanisms is rarely studied, they are equally important.

By targeting mRNA LNPs to selective surface receptors expressed by DCs , these particles may enter the intracellular transport pathways, which degrade the mRNA load less.

Therefore, (mRNA) nanovaccines have targeted C -type lectins DEC-205 , CLEC9A and mannose receptors, which are also involved in pathways that facilitate absorption, endocytosis protection, and cross-presentation of dead cell antigens.

 

Injection of mRNA LNPs

Another key parameter is the choice of delivery route for mRNA LNPs , which will determine the effectiveness and safety of mRNA LNPs .

Importantly, depending on the route of administration, specific extracellular barriers may be encountered, leading to the optimization of specific particles.

The skin is a very convenient way. It can cause an immune response through two different mechanisms : either by local transfection of mRNA LNPs and activation of APCs , APC then migrates to the lymphatic vessels, or passively discharged through the lymphatic system, thereby directly The mRNA load is delivered to the lymphatic resident APCs and T cells.

 

In a detailed study of rhesus monkeys, Liang et al. investigated immune cell infiltration, vaccine uptake, immune activation, and mRNA translation by intramuscular or intradermal injection of ionizable LNP adjuvant nucleic acid modified mRNA vaccine .

In both routes of administration, neutrophils, monocytes and different DC subgroups can be seen to quickly accumulate to the injection site ( skin or muscle ) .

Although all these types of cells can internalize mRNA LNPs , only monocytes and myeloid DCs have higher levels of mRNA translation.

 

In addition, they can detect mature, genetically modified APCs in the draining lymph nodes.

Combined with the immune response, their intradermal injection led to early activation of skin DCs and more effective migration to the draining lymph nodes, as well as the long-term availability of antigens at the injection site.

Overall, this resulted in better immunogenicity than intramuscular injection, because intradermal injection resulted in the highest antibody titers and CD4 + T cell activation.

 

According to previous reports, particle size is a key factor in obtaining effective lymphatic transport of nanoparticles.

The report shows that nanoparticles below 200 nanometers can enter lymphocytes, while larger particles are retained at the injection site.

Pegylation accelerates the flow of ( lipid ) nanoparticles into the lymphatic system, while targeting ligands such as mannose and antibodies against selective DC receptors can increase liposome capture into draining lymph nodes.

 

In addition, studies have shown that PEGylation needs to prevent the particles from being completely fixed in the extracellular matrix (ECM) of the skin , which can be explained as ECM components, such as glycosaminoglycans, which can inhibit non-PEGylated lymph at the injection site Distribution and cellular uptake.

 

In order to give an example of optimized mRNA LNPs lymphocyte transport, Wang et al. produced a nanoparticle system composed of mRNA and calcium phosphate pre-concentrated core, stabilized by DOPA , and a lipid shell of DOTAP and DSPE-PEG package.

 

The particle size of these mRNA nanoparticles is relatively small (<50 nm ) , the outer surface is not charged, and the outer surface has a high degree of PEGylation.

Therefore, this system was found to be able to effectively enter the lymphatic tissues, 4 hours after intradermal injection, mRNA can be detected in the draining lymph nodesThe presence of, which can directly deliver ( almost a full dose ) of mRNA to the resident macrophages and dendritic cells.

 

 

For specific cancer immunotherapy, it is very important to trigger an anti-tumor systemic immune response, which makes intravenous injection a very attractive route of administration for mRNA- based cancer vaccines.

There is no doubt that once mRNA LNPs enter the blood, the particle size, surface morphology, structural structure and other characteristics will undergo tremendous changes.

In fact, the interaction between (mrna) nanoparticles and biological fluids causes the adsorption of endogenous proteins and other biomolecules on specific surfaces, leading to the formation of biomolecules or protein corona.

This may reduce the colloidal stability of mRNA LNPs , followed by particle aggregation and premature release of mRNA .

 

Therefore, using advanced microscopy techniques such as fluorescent single particle tracking (fSPT) and fluorescence correlation spectroscopy (FCS) to determine the size, stability and mRNA packaging rate of LNPs in undiluted biological media is a good practice method.

The stability of serum can also be adjusted by changing the composition of auxiliary lipids.

For example, cholesterol is often used to increase the rigidity of LNPs ( ie, reduce the permeability of lipid membranes ) , thus helping to improve the stability and integrity of LNP in serum. In addition, pegylated lipids are widely used to provide “stealth effects.”

 

This reduces the overall protein adsorption and improves the colloidal stability of LNPs , but it also hinders LNPCell uptake and transfection ability. Here, PEG lipids with shorter acyl chains can provide an effective strategy to overcome this ” PEG barrier .”

These PEG lipids gradually diffuse out of LNP , thereby temporarily giving the LNP system stealth performance.

At the same time, PEG lipids exist for a longer period of time to achieve higher transfection efficiency.

 

From another advantageous point of view, the formation of this type of biocorona provides nanoparticles with new ( surface ) properties, which may be used for targeting and / or enhanced transmission.

Importantly, the number and characteristics of individual proteins present in this biomolecular corona will depend on the characteristics of the ( “primitive” ) nanoparticle and its biological background.

Generally speaking, it is known that intravenously injected nanoparticles will react with opsonic blood proteins, such as complement fragments, immunoglobulins, and fibronectin, thereby promoting the uptake of phagocytes and the clearance of nanoparticles.

Recently, Vu et al. clarified how nanoparticles mediate complement action and found that this is quite common in a series of clinically approved liposomes.

 

The formation of biomolecular corona exposes the self-epitope, and naturally occurring antibodies can bind.

Only a few surface-bound immunoglobulin molecules are enough to trigger complement activation.

Several studies have shown that the natural conditioning process may be manipulated by passive targeting of APCs , or the complement cascade may be used as a red flag for vaccination purposes.

Others trigger extensive complement activation through the special diffusion reaction of nanoparticles, and the clinical manifestations are cardiopulmonary discomfort.

However, recent studies have shown that these nanoparticle-mediated fusion reactions are caused by acute lung macrophage poisoning.

 

It should be noted that detailed studies on the role of biomolecular corona formation and the dynamics of LNPs are still very limited.

However, further progress in this field may reveal more systems to deliver specific mRNA LNPs products in terms of biodistribution and cell-specific targeting.

For example, Akinc et al found that, in the selectively adsorbed ionizable ( neutral ) LNP on the E -type carrier lipoprotein, by receptor-mediated endocytosis effects and (si) RNA specific delivery to hepatocytes relevant.

In contrast, cationic mRNA LNPs composed of cationic lipids and cholesterol, or DOPE , were found to target the lungs, where they transfected dendritic cells, macrophages, and endothelial cells that reside in the tissue.

Interestingly, by reducing the ratio of lipid to mRNA , thereby reducing its lipid composition, Kranz et al. found that DOTMA-DOPE mRNA LNPs carry negative charges and specifically target DCs in the spleen .

 

The discovery that these anionic mRNA LNPs specifically target the spleen has been confirmed in patients participating in phase I melanoma clinical trials.

Although the observed site-specific targeting may be due to the surface charge of LNP , the effect of biomolecular corona cannot be ruled out.

Therefore, for these different chargesA detailed comparison of the biomolecular corona of mRNA LNPs helps to further clarify the mechanism behind the changes in organ distribution and cell targeting observed after intravenous injection.

 

As mentioned earlier, antibodies or other targeting proteins can be incorporated into the LNP system to support selective organ treatment of mRNA LNPs , or to promote receptor-mediated absorption of specific ( immune ) cell types.

To cite a few other examples, Parhiz et al. demonstrated that ionized mRNA LNPs bind to antibodies against vascular cell adhesion molecule (PECAM-1) and promote mRNA transfection of lung endothelial cells after systemic delivery .

Perche et al. prepared mannose nanoparticles containing mRNA , which can be used as an active targeting strategy to increase the transfection rate of splenic dendritic cells.

 

For a more detailed summary of potential target receptors and technical details of ligand binding strategies, we also recommend readers to refer to the relevant literature.

It is worth noting that Dan Peer’s laboratory recently proposed a modular targeting platform for the selective delivery of (si) RNA LNPs to different white blood cells in the body.

Here, lipoproteins in the LNP system bind to the Fc region of the target antibody through non-covalent linkage .

 

However, it should be noted that attaching the targeting ligand to the outer layer of the LNP system complex increases the synthesis steps, costs and management barriers.

In addition, the targeting ability of such functionalized nanoparticles can be replaced by the deposition of endogenous proteins and the migration and removal of nanoparticles.

Therefore, use these targeted gene fragments for specificThe potential clinical benefits of the mRNA method should be weighed against the complexity of the cost.

 

mRNA self-adjuvant effect

The effect of type I IFN on the immunogenicity of mRNA vaccines is twofold. Many studies have revealed that the ability of mRNA LNPs to trigger the intensity of CTL response depends on the production of type I IFN .

Two articles showed that intravenous injection of unmodified mRNA LNPs triggers the rapid and systemic production of type I IFN , which can participate in the selective targeting of DCs and macrophages, and the activation of the TLR7 pathway.

Knockout the IFN- [alpha] receptor 1 or TLR7 experiments recipient mice showed that the mRNA LNPs systemic induced I -type IFN for APCs activation and effector cells play a key role.

 

When mice with lung metastasis were injected intravenously with mRNA LNPs and intraperitoneally injected with anti- IFNAR1 antibody, the anti-tumor effect was significantly weakened.

Through comparative experiments between this research group and other laboratories, nucleic acid modification was found.

Compared with unmodified mRNA LNPs, the translation ability of mRNA LNPs is improved, but because the production of type I IFN is greatly reduced, CTL response cannot be successfully induced .

 

Type I IFN has a great effect on inducing effector T cell responses, while other articles have shown that early type I IFN production inhibits the expression of antigen-encoding mRNA , which is quite detrimental to the effect of mRNA vaccines.

De Beuckelaer and colleagues showed that the B16 melanoma model was injected subcutaneously or intracutaneously with DOTAP-DOPE mRNA LNPs , and its effect of inducing CTL anti-tumor immunity was significantly enhanced after type I IFN blockade.

For self-amplified mRNA LNPs vaccines, Pepini et al. reported that when IFN- α / β is missing, the antigen expression is significantly increased, which is related to the increased immunogenicity and IgG antibody response.

In addition, the higher intensity and sustained antigen expression of nucleic acid modified mRNA than unmodified mRNA is related to a more optimized antibody response.

 

Obviously, this raises a basic question, how to deal with the self-adjuvant effect of mRNA vaccines.

Generally speaking, acute type I IFN mediates the multidirectional and pro-inflammatory effects of innate immune cells and adaptive immune cells.

 

In fact, type I IFN signaling induces DC maturation, improves the processing and presentation of antigens, and enhances the migration of DCs to abundant T cell regions ( for example, through CCR7) .

Importantly, type I IFN is directly used as a signal 3 cytokine for T cell activation, promoting clonal expansion, survival, and effective differentiation of CTL and T helper cells.

 

In humoral immunity, type I IFN promotes the production of long-term antibody responses by indirectly enhancing helper T cell responses and direct immune stimulating effects on B cells.

By regulating other pro-inflammatory cytokines, type I IFNIt is also involved in regulating other immune cell types, such as natural killer cell (NK) response.

 

Type I IFN is involved in regulating immune tolerance and can promote the development of self-reactive B cells and T cells, which is a greater threat.

This can lead to serious autoimmune consequences, such as systemic lupus erythematosus and diabetes.

With the first publication of the clinical results of different mRNA vaccines, we have learned more about their efficacy and potential side effects in humans.

Although the number of patients is still limited, Pardi et al. pointed out that some of the observed side effects of clinically tested mRNA vaccines may still be incomplete, including the potential risk of autoimmune diseases.

In a phase I/IIa clinical trial of the RNactive vaccine for non-small cell lung cancer (NSCLC) , after 5 intradermal injections ( dose 400-1600g) , it was found that the common diagnostic index of autoimmunity increased to 20% .

Another study tested the safety and immunogenicity of the RNactive platform as a preventive rabies vaccine.

A week after the second intramuscular injection of the highest dose of 640ug (1/37 patients ) , it caused moderate Bell’s palsy. This is a temporary facial nerve palsy and is related to autoimmunity.

 

The complexity of type I IFN signaling in terms of effectiveness and safety needs further research on how to solve it through the design of mRNA vaccines.

However, there is no doubt that different mRNA vaccines, different mRNA designs, different preparation processes and administration routes will show different mRNA expression kinetics and specificity of natural immune activation cells.

Therefore, changes in these mRNA vaccine parameters will affect type I IFNThe effect of the signal on the outcome of the mRNA vaccine determines whether it is good, bad, or annoying.

Induction of antibody response and CTL response requires a trade-off between translation effect and mRNA vaccine type I IFN activity, which may explain why unmodified mRNA vaccine is more suitable for inducing CTL response, while nucleoside modified mRNA vaccine can detect more Good antibody response.

Nevertheless, it is certain that mRNA vaccines can obtain high-titer and continuously expressed antigens, which will benefit the magnitude and duration of T cell and B cell responses.

 

It is exciting that new imaging tools have been applied to the evaluation of novel mRNA delivery systems that visualize mRNA pharmacology at the single cell and tissue level , including the generation of transgenic reporter mouse strains, such as IFN- β reporter Mouse and Cre recombinase (mRNA) reporter mice, and the development of fluorescent imaging mRNA probes for high-resolution detection of mRNA .

To give a prominent example, the Santan-gelo laboratory combines fluorescently labeled mRNA delivery with proximity-based detection.

Therefore, when mice deliver naked mRNA or LNP- modified mRNA through muscle , they can simultaneously detect cell-specific uptake, transformation, and in situ activation of TLR7 , RIG-I , and MDA5 pathways.

These different tools for real-time tracking of mRNA activity will be very helpful in determining the differences between different mRNA vaccine platforms and the comparison of immune pathways.

Combined with immune readings and thorough safety assessments, this will help develop more effective and safe mRNA vaccines.

 

Other than type I IFN reaction

Considering the two sides of anti- mRNA type I IFN response, we hypothesize that it would be beneficial to dissolve the link between mRNA vaccine translation and type I IFN activity and replace the type I IFN response with another more controllable immune activation .

 

This strategy provides freedom for the optimization of mRNA structure for high translation capacity, for example, the use of nucleoside modified mRNA , while choosing a “smarter” immune adjuvant : one that does not interfere with the mRNA translation process and can provide effective but controllable immune response.

In a proof-of-concept study, we demonstrated that nucleoside-modified mRNA can be co-delivered with the clinically approved TLR4 agonist MPLA to achieve the same functional CTL response as unmodified mRNA , but significantly reduce type I IFN response.

In addition, we have a new optimized mRNA LNP platform that combines a modified nucleoside mRNA and natural killer T (NKT) cell ligand [alpha] – galactosylceramide (mrna galsomes) in vivo delivery, called mRNA Galsomes .

Induce LNP process in type I IFNIn mRNA and naked mRNA mice, through the dual “antigen” signals of traditional T cells and NKT cells , mRNA Galsomes can induce a higher antigen-specific CTLs , NKT and NK cell flow in tumors , and can reduce immunosuppressive bone marrow The presence of cells.

 

In addition to replacing type I IFN responses with more controllable immune stimulation , specific strategies have also been developed within the framework of immunotherapy to avoid type I IFN responses to ensure high protein expression, which is beneficial.

The first alternative method for adaptive immune cell restart is to use mRNA directly encoding different antibody forms , including monoclonal antibodies, antibody fragments or bispecific antibodies to explore passive immunity.

Since the liver is the main target of many preparations after systemic delivery, this organ is used as a biological factory for the production and secretion of mRNA- encoded proteins.

To give two examples, Thran et al. evaluated the sequence design of mRNA encoding LNP formulations for prophylactic or therapeutic antibodies .

 

This can prevent rabies virus infection, botulism and tumors. Stadler et al. designed mRNAs encoding bispecific antibodies against T cell receptor-related CD3 molecules and tumor antigens , called RiboMABs .

This mRNA platform serves as an effective alternative to classic mRNA cancer vaccines, providing a powerful infiltration of T cells that can eliminate mouse tumors .

 

Another method is to directly transfer the mRNACoded immunomodulatory molecules are delivered to the tumor site, such as cytokines and costimulatory molecules.

Breckpot and colleagues demonstrated that intratumoral injection of Trimix the mRNA ( of CD40L, caTLR4 and of CD70 ) containing or encoding IFN-β and TGF-β receptor II fusion factor extracellular domain of the mRNA , the DC reprogramming.

 

Consistent with this, Moderna Therapeutics recently published an article on anti-cancer immunotherapy, using a mixture of IL-23 , IL-36 γ, and OX40(L) ligands encoded by direct intratumoral and LNP- mediated mRNA . 

Through the local expression of these immunomodulatory molecules, this strategy can break DC immune tolerance and activate tumor-specific T cell responses.

 

Moreover, the study of Van Hoecke et al. showed that intratumor electroporation encoding mixed-lineage kinase domain-like protein (MLKL) mRNA has been determined to convert tumors into in situ vaccines.

In fact, the expression of MLKL causes an immunogenic cell death called necrosis, which promotes the release of tumor-specific antigens and induces the formation of anti-tumor response DAMP .

 

Combination of mRNA vaccine and immune checkpoint

For mRNA therapy of cancer or tumor vaccines, activating effector immunity is not enough, and attention should also be paid to several immunosuppressive mechanisms that affect immune activation.

During the activation of T cells, the expression of checkpoint molecules forms a strong negative feedback loop, keeping the response of T cells within the ideal physiological range.

In general, the immune system should be able to quickly respond to out-of-control or potential dangers, but it should also establish a balance to maintain immune homeostasis.

 

Multiple studies have shown that mRNA cancer vaccines up-regulate the expression of effector T cell checkpoint molecular programmed death 1 (PD-1) and its ligand (PD-L1) on tumor cells and APCs .

It is worth noting that the up-regulation of the PD-1/PD-L1 checkpoint pathway is mainly mediated by IFN-γ and can also be induced by type I IFN .

 

Therefore, there is an opportunity for a combination of mRNA vaccine and PD-1 checkpoint inhibitor therapy.

In fact, a large amount of pre-clinical evidence shows that, in combination with checkpoint inhibitors such as anti- PD-1(L) and anti- CTLA-4 antibodies, it can strengthen the T cell response and anti-tumor effect caused by mRNA therapy .

 

This is also being tested in numerous I/IIb clinical trials. These trials are either in progress or in recruiting patients to test the effectiveness of mRNA vaccines combined with checkpoint inhibitors in the treatment of different cancer indications.

In monotherapy, the efficacy and tolerability of checkpoint inhibitors for patients with highly refractory and advanced cancers such as metastatic melanoma and non-small cell lung cancer (NSCLC) are impressive.

 

However, only some patients benefit from these checkpoint therapies.

Therefore, the establishment of effective combination therapies that can improve the response of checkpoint inhibitors is an area that requires intensive research.

Although some mRNA cancer vaccines have failed before obtaining an objective clinical response, the combination of these mRNA platforms and checkpoint inhibitors may be a valuable approach

(i) to ensure the persistence and continued effectiveness of the induced anti-tumor immunity Sex,

(ii) contributes to the T cell inflammatory tumor phenotype, making more patients suitable for checkpoint suppression therapy.

 

 

 

 

 


Summary and outlook

 

From the earliest published literature on the delivery of mRNA in vivo, nucleic acid has become a versatile and promising form of vaccine.

This review pointed out and analyze the determinants of mRNA vaccine for success 2 key factors.

First, ensure that there is a sufficient amount of mRNA for intracellular delivery in the body, especially targeting APCs in vivo .

In recent years, a large number of knowledge gaps in the behavior of mRNA in vivo have been filled.

Including naked mRNA delivery or nanoparticle delivery, as well as immunization through various immune pathways.

 

Nowadays, the use of lipid nanoparticles for local and systemic delivery of mRNA has become a trend.

We discussed the multi-index tasks to be completed in the design of LNPs .

The overall mRNA transfection rate depends on the different requirements for crossing the extracellular and intracellular barriers.

However, preventive measures need to be taken to prevent potential nanoparticle invasion ( immune ) toxicity.

 

If these mRNA LNPs are to be used in the clinic, it is also important to meet the requirements of drugs and registered products, from the large-scale production under GMP conditions, as well as product quality control, stability testing, and pre-clinical stage in related animal models Good safety and effectiveness assessment.

 

It is worth noting that the FDA and EMA approved siRNA therapy (Onpattro TM ) for the first time , using ionizable LNP technology to assist parental siRNA injection, which will be the futureThe use of mRNA vaccines is a pioneer.

 

Another balance that is difficult to find is the balance between the expression of the antigen encoded by the mRNA and adequate immune stimulation.

These two parameters are critical to the development of vaccines, but they are both related to the structural properties of the mRNA molecule itself.

More specifically, although the type I IFN response induced by mRNA may increase the immunogenicity of the mRNA vaccine, it can also interfere with the expression of the antigen encoded by the mRNA and may even cause harmful immune effects.

 

Basic research should pay more attention to how to control the mRNA vaccineType I IFN activity, which may depend on the process of mRNA , route of administration, individual patient, and expected therapeutic application.

Therefore, methods that do not necessarily rely on the self-adjuvant effect of mRNA are emerging, such as replacing type I IFN responses with more controllable adjuvant systems , or using mRNA passive immunization methods.

Whether these new strategies are more beneficial than “traditional” mRNA vaccines is a topic worthy of further discussion.

 

It is an exciting time for different biotech companies to apply mRNA therapy to clinical practice.

In the near future, for the mutation-derived epitopes ( new antigen ) of mRNA cancer vaccine may become the leading clinical cancer vaccines other attractive alternatives, such as those made of synthetic ( long ) vaccine peptides and immune adjuvant composition.

Here, it can be expected that the combination of ( individualized ) mRNA vaccines and checkpoint inhibitors in future clinical trials will have a better chance of success.

 

However, the safety and optimization of this combination therapy will be another research topic.

For mRNA vaccines to prevent infectious diseases , preclinical studies ( in non-human primates ) have shown that mRNA vaccination is feasible, usually well tolerated, and may have advantages over other traditional vaccine methods.

However, in terms of patient response to mRNA vaccines, more extensive clinical experience is needed, including more comparative studies, to select the most suitable mRNA platform and route of administration, and to show clear therapeutic advantages over other vaccine strategies. .

 

In short, it will be a matter of time before we can determine which mRNA vaccine can safely and effectively induce an immune response in the human body. We hope that the mRNA vaccine can become a new generation vaccine.

 

 

30 years Development of mRNA vaccines.

(source:internet, reference only)


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