Panoramic interpretation of mRNA nanomedicines: latest progress, challenges, clinical translation and future directions

mRNA is an emerging class of therapeutic drugs that can be used to prevent and treat a wide range of diseases
.
The success of two highly effective mRNA vaccines produced by Moderna and Pfizer-BioNTech in preventing coronavirus pneumonia highlights the great potential
of mRNA technology to transform life science and medical research.

Challenges in mRNA stability, immunogenicity, in vivo delivery, and ability to cross multiple biological barriers have been largely addressed
in recent years through advances in mRNA engineering and delivery.

On November 10, 2022, Wei Tao of Harvard Medical School, Robert Langer of MIT, Yihai Cao of Karolinska Institutet and others published a review paper
in Nature Medicine entitled: The landscape of mRNA nanomedicine.

This review provides an overview of mRNA nanomedicines, discusses the technical challenges of mRNA-based therapeutics, and links
them to biological mechanisms and clinical outcomes.
This review presents the latest advances and innovations in the evolving field of mRNA nanomedicine, as well as ongoing clinical translations and future directions
to improve clinical efficacy.

Cao Yihai (left), Robert Langer (middle), Tao Wei (right)

Cao Yihai is a tenured professor at Karolinska Institutet in Sweden and a foreign academician of the Chinese Academy of
Engineering.
Recognized as a leading scientist in angiogenesis in the world, he is also the first Asian in
Sweden to be promoted to a tenured professor of medicine.

Robert Langer is one of MIT's most honored professors and one of the world's top and most prolific biomedical engineering and nanoscientists
.

Wei Tao, a fellow at Harvard Medical School, is the first person in Harvard Medical School's history to serve as an assistant professor as a chair professor
.
His main research interests are the design and synthesis of functional biomaterials, revealing nano-biological interaction mechanisms, and exploring their different biomedical applications, including RNA therapy, cancer treatment, wound healing, cardiovascular diseases, bone regeneration, diabetes treatment, etc
.

Messenger RNA (mRNA) is a transient carrier that transfers the genetic information of life from DNA to the ribosome, where it is translated into proteins that perform life functions
.

By delivering mRNA expressing infectious diseases, cancer antigens, gene-editing components, or disease-related therapeutic proteins, a variety of clinical applications
including vaccines, gene editing, and protein therapeutics can be realized.

Back in 1976, Robert Langer published a paper in Nature that first demonstrated that nucleic acids can be packed and delivered by tiny particles composed of polymers, but this was initially ridiculed
by the scientific community.
Two years later, research began to show that mRNA could be delivered through liposomes and expressed proteins
in human and cell number cells.
Since then, mRNA has shown therapeutic efficacy in various preclinical studies, laying the foundation
for mRNA as a drug and vaccine.

However, the path to successfully applying mRNA to drugs is not simple
.
Initially, the instability of mRNA, its immunogenicity, and high production costs greatly limited the enthusiasm
of companies and the scientific community to invest resources.
Encouragingly, these issues have been addressed with the rapid development of mRNA engineering techniques, including chemical modifications, sequence optimization, and production purification, thanks to decades of
research by numerous researchers.

Key advances in the development of mRNA therapeutics

1.
In 1961, mRNA was discovered;

2.
In 1976, nucleic acids were delivered in vivo by polymer particles for the first time;

3.
In 1995, mRNA-based cancer vaccines were evaluated in mice for the first time;

4.
In 2005, Kariko and Weissman et al.
first reported that nucleoside modification greatly reduced the immune response caused by mRNA;

5.
From 2008 to 2012, Kariko and Weissman et al.
further confirmed that nucleoside modification can enhance the translation ability and stability of mRNA;

6.
In 2017, BioNTech conducted the first clinical trial of mRNA-based personalized cancer vaccine;

7.
In 2020, the mRNA new crown vaccine of Moderna and Pfizer-BioNTech obtained FDA emergency use authorization;

8.
In 2021, it was found that the lipid nanoparticles (LNP) used as delivery vehicles in mRNA new crown vaccines also have vaccine adjuvant activity
.

These advances have laid the foundation for therapeutic applications of mRNA, but clinical translation needs to be expressed
in vivo target cells or tissues.
Although these technological advances have enabled preclinical and clinical studies of many mRNA drugs over the past 20 years, regulatory agencies have not approved any mRNA nanomedicines (including mRNA vaccines or mRNA therapeutics) until the advent
of two mRNA coronavirus vaccines.
Their successful development has stimulated new and strong research interest in mRNA engineering and delivery technologies, providing unprecedented promise
for the clinical translation of various mRNA-based therapies.

Challenges in the clinical application of mRNA

Clinical use of mRNA for treatment requires adequate mRNA translation in the cells concerned without causing an unwanted immune response
.
However, achieving this goal requires overcoming several barriers
to extracellular and intracellular mRNA synthesis and delivery.
Synthesis of therapeutic mRNA
by in vitro transcription (IVT) in cell-free systems using plasmids or linear DNA templates generated by PCR and RNA polymerase.
The mRNA
is then purified using conventional laboratory-scale nucleic acid purification methods.
However, these methods often fail to remove impurities such as double-stranded RNA and RNA fragments, which reduces the efficacy in clinical applications and causes adverse reactions
.
When administered locally or systemically, these mRNAs are also rapidly degraded by extracellular space-rich nucleases, phagocytosed by macrophages, or cleared by renal filtration
.

At the same time, mRNA is a large, negatively charged single-stranded polynucleotide that has difficulty passing through negatively charged cell membranes
.
With mRNA injected directly, only 0.
01% of the mRNA enters the target cell, and most of the mRNA is trapped in the endosomes of the target cell and subsequently degraded
.
Eventually, only a few mRNAs escape from endosomes and reach ribosomes for protein translation
.

Immune stimulation caused by exogenous mRNA is another major barrier
to clinical translation.
Exogenous mRNAs can be sensed by pattern recognition receptors (PRRs), in endocytosis, mRNAs can be detected by Toll-like receptors (TLRs), and mRNAs escaping from endosomes can be sensed
by cytosolic PRRs.
These stimuli eventually lead to the production
of type I interferons (IFNs) and other pro-inflammatory cytokines.
These secreted IFNs bind to receptors on stimulated cells and neighboring cells, activating the JAK-STAT pathway, triggering the transcription of more than 300 interferon-induced genes
.
Among them, interferon-induced protein kinase R (PKR) inhibits the activity of translational initiation factor 2, resulting in translation inhibition of mRNA, and 2'-5'-oligoadenosine synthase and RNA-specific adenosine deaminase can reduce the stability
of mRNA.

Challenges in the clinical application of mRNA

All of these challenges greatly limit the clinical application of mRNA, and advances in mRNA-related technologies are needed to address these challenges
before the therapeutic potential of mRNA can be fully realized.

Design of mRNA and its delivery tools

The rapid development of mRNA engineering and mRNA delivery of non-viral vectors has provided various solutions
for the clinical application of mRNA.
For example, issues such as the translational ability, stability, and immune stimulation of mRNA can be addressed
by introducing innovative mRNA designs.
In addition, mRNA delivery vectors can solve some of the delivery challenges
.
Properly designed mRNA delivery vectors need to protect mRNA from nuclease degradation, help cross various biological barriers, and efficiently deliver mRNA into the cytoplasm, so as to achieve robust protein expression
.

Design of mRNA

In vitro transcription (IVT) mRNAs are structurally similar to the naturally mature mRNAs of eukaryotes and consist of five main domains – a 5' end cap, a 5' end untranslated region, an open reading frame encoding the protein of interest, a 3' end untranslated region, and a Poly(A) tail
.

Optimization of untranslated regions (UTRs) facilitates the translation and stability
of mRNA.
UTR sequences from highly expressed genes, such as those of the human β-globin gene, are widely used in mRNA synthesis because mRNAs containing these UTRs typically have high levels of translation and stability
.
Identifying new UTR sequences through high-throughput screening or deep learning can improve mRNA expression, and a reasonable combination of 5' UTR and 3' UTR can maximize
translation efficiency.
In addition, Poly(A) tails with a length of 100-150 nucleotides can improve the stability of mRNA and efficiently initiate translation
by forming complexes with Poly(A)-binding proteins.

One of the most effective strategies for eliminating immunostimulation of in vitro transcription (IVT) mRNA is nucleoside modification, where incorporation of modified nucleosides, such as pseudouracil (ψ), 5-methylcytidine, N6-methyladenosine, 5-methyluracil, 2-thiuridine, prevents recognition of human toll-like receptors (TLRs) to reduce cytokine production
compared to unmodified mRNA.

Mechanistically, the increased translation and stability of mRNA incorporated with pseudouracil (ψ) is attributed to decreased
activity of protein kinase R (PKR) and 2'-5'-oligopolyadenylate synthetase.
Simultaneous substitution of some nucleosides with 2-thiuridine and 5-methylcytidine significantly inhibits the activation
of TLRs and RIG-1 in vitro and in vivo.
Compared with ψ, N1-methylpseudouridine has lower cytotoxicity and immunostimulating capacity
.
Notably, both the mRNA vaccines developed by Moderna and Pfizer-BioNTech use nucleoside-modified mRNA to avoid unexpected immune responses
.

Another immunostimulating strategy to reduce the production of mRNA is to enhance mRNA purification
.
In vitro transcription (IVT) mRNAs purified by high performance liquid chromatography do not contain double-stranded RNA (dsRNA) byproducts, are 10-1000 times more protein expressed in primary cells than unpurified mRNA, and do not induce IFN or inflammatory cytokine production
.
While HPLC purification is widely used for the production of mRNA, a simple, rapid, and economical cellulose-based purification method provides another option
for producing high-purity in vitro transcription (IVT) mRNA.

The 5' end cap design provides another way to
reduce unwanted immune responses caused by mRNA.
The naturally mature mRNA formed by post-transcriptional modification of eukaryotes has a cap structure (Cap-0), that is, an m7GPPPN structure
, at the 5' end.
Cap-0 spatially inhibits the degradation of mRNA by nucleases and initiates translation by binding to eukaryotic translation
initiation factors.
Compared to Cap-0, two other 5' end caps (Cap-1 and Cap-2) are widely used
in mRNA synthesis due to their lower immunostimulating potential.
At present, mRNAs with Cap-1 cap structures can be manufactured in more aspects, with minimal immune stimulation and satisfactory translation efficiency
.
In addition, the computational experimental platform can simultaneously enhance the stability and translation ability of mRNA, and translation and immunostimulation can also be regulated
by chemical-enzymatic modifications.

mRNA delivery tools

Rapid clinical translation of mRNA-based vaccines or therapeutics has benefited from the development of delivery vectors to protect and deliver highly volatile mRNA molecules
.
At present, the main mRNA delivery systems are lipid-based nanoparticles, polymer-based nanoparticles, and lipid-polymer hybrid nanoparticles
.

Lipid-based nanoparticles

Lipid-based nanoparticles are the most deeply studied and clinically advanced mRNA delivery vehicles, the most widely used of which are cationic, ionizable lipid nanoparticles (LNP), usually lipids modified by cations or ionizable lipids, cholesterol, helper lipids, and polyethylene glycol
.

Lipid-based nanoparticles

Cationic lipids, such as DOTMA or DOTAP, carry quaternary ammonium groups that maintain a positive charge
in a pH-independent manner.
This cationic environment allows negatively charged mRNA to efficiently coagulate, making the cationic lipid-based system the most widely used mRNA delivery system
in early clinical research.
However, its potential cytotoxicity and relatively short blood circulation time hinder its clinical translation
.
To address these issues, lipid polyethylene glycols and various new ionizable lipids
are employed.
Unlike cationic lipids, which have a permanent positive charge, ionizable lipids remain neutral at physiological pH but protonated at acidic
pH.
Ionizable lipids remain neutral in body fluids to reduce toxicity and increase the cycling half-life of ionizable LNPs to some extent
.
In addition, protonation of ionizable lipids at acidic pH not only facilitates polycondensation and encapsulation of mRNA in acidic buffers, but also facilitates mRNA escape
from acidic endosomes.
The introduction of lipid peg greatly improves the cycling half-life of ionizable lipid-based nanoparticles, reduces the aggregation of nanoparticles, and reduces adverse interactions
with serum proteins.

The development of ionizable lipid-based nanoparticles for mRNA delivery has benefited in large part from decades of research
into ionizable lipid-based nanoparticles for siRNA delivery 。 For example, Patisiran, the first siRNA drug approved by the US FDA, uses DLin-MC3-DMA (MC3), and by optimizing these formulation parameters, such as the ratio of RNA to total lipids and the ratio of aqueous solutions to organic solvents, MC3-based LNPs have been used to develop various mRNA therapies, including vaccines against Zika virus, HIV and Lyme disease, as well as the treatment
of cystic fibrosis and lymphedema.

One limitation of MC3 is its poor degradability, which can lead to toxicity problems
when repeated dosing is required.
To address this challenge and further improve the efficacy of MC3, Moderna has developed a biodegradable lipid called lipid 5 that has more branching tails
than MC3.
In animal models, lipid-5-based LNP-delivered mRNA, repeated systemic and topical administration, alleviated acute intermittent porphyria and achieved durable anti-cancer immunity without any significant toxicity
.

In addition to MC3, several other ionizable lipids originally developed for siRNA delivery, such as cKK-E12 and C12-200, have also been redeveloped for delivery of mRNA into the liver for gene editing and protein replacement
.
However, due to the selective accumulation of these LNPs in the liver, therapeutic mRNA is difficult to transport to extrahepatic tissues through these LNPs, a phenomenon that may be determined by the ionizing properties of these lipids
.

Through the in-depth study of a new generation of ionizable lipids, the lipid H/SM-102 and lipid ALC-0315 were finally produced, the former for Moderna's mRNA new crown vaccine, and the latter for Pfizer-BioNTech's mRNA new crown vaccine, which also greatly promoted the development of
new crown mRNA vaccine.

The good safety profile of these vaccines may be attributed to the biodegradability
of lipids.
In addition, Intellia utilizes another LNP, based on the biodegradable ionizable lipid LP01, which carries Cas9 mRNA and gRNA, to achieve robust and durable in vivo gene editing
in animal models.
Compared with non-biodegradable ionizable lipids, LP01 has less liver bioaccumulation and less
safety risk.

Ionizable lipids undoubtedly play a key role in the activity of LNPs, but other components are also important
.
Accessory lipids improve the efficacy of LNP by regulating the fluidity of LNP and promoting the escape of LNP from endosomes, while cholesterol plays an important role
in the stability of LNP.

Lipid-polymer composite nanoparticles

Lipid-polymer hybrid nanoparticles, which typically include ionizable/cationic lipids, hydrophobic polymers, and pegylated lipids
.
These nanoparticles have been used to effectively restore the tumor suppressor PTEN and inhibit tumor growth
in multiple mouse models of prostate cancer.
In this carrier, the hydrophobic polymer polylactic acid-glycolic acid (PLGA) replaces the helper lipids and cholesterol
in LNP.

Replacing PLGA with the redox reaction polymer PDSA forms a redox reaction platform for systemic delivery of tumor suppressor p53 mRNA, enabling potent tumor suppression
in preclinical models.
The addition of pegylated lipid PEG-DMPE to enhance efficacy
.
In addition, local delivery or organ-specific delivery
can be easily achieved by simply changing the end or functionalization of the lipid polyethylene glycol covering the surface of the polymer core.
For example, by using DSPE-PEG-NH2 or DSPE-PEG-SH, adherent mRNA nanoparticles can be generated for delivery of mRNA within the bladder to upregulate proteins needed in mouse bladder tissue in situ
.
Other lipid-polymer hybrid nanoparticles have also recently been developed for efficient mRNA delivery and tested
in preclinical models.

Lipid-polymer composite nanoparticles

Polymer nanoparticles

Polymer nanoparticles consist of cationic polymers simplely, and early research focused on nucleic acid delivery using polyethyleneimine or polyL-lysine, but their significant toxicity limited applications
.
To solve this problem, a series of biodegradable poly β-amino esters (PBAE)
were synthesized.
For example, PBAE-based nanoparticles have been used to deliver functional mRNA in vivo to circulating T cells and a variety of tissues, in addition, a hyperbranched PBAE (hPBAE) that delivers mRNA directly to the lungs
by inhalation 。 Another promising mRNA-delivered polymer nanoparticle is the charge-variable releaseable transporter (CART), which, unlike traditional cationic polymers, can release mRNA in the cytoplasm through a unique mechanism, the initial positive charge of oligomeric α-amino esters can efficiently compress and encapsulate mRNA and deliver it to the cell, through which CART undergoes degradable, charge-neutralizing intramolecular rearrangements, resulting in rapid release
of functional mRNA 。 This property leads to the delivery of mRNA into lymphocytes in vivo, which can support
therapeutic strategies for a variety of diseases.

Recent promising innovations

The successful development of mRNA vaccines has led to innovation
in mRNA engineering and delivery.
These new technologies may yield more stable and robustly expressed next-generation mRNAs, which could lead to applications in protein therapy and gene editing, which typically require higher expression levels and longer expression times
than mRNA vaccines.
In addition, innovations in mRNA delivery can greatly improve the efficiency
of in vivo delivery of mRNA in a variety of applications.
These innovations will further facilitate the clinical translation
of different mRNA therapeutics.

Innovation in mRNA engineering

Self-amplifying mRNA

Self-amplifying mRNA contains an alphavirus-based replicon that amplifies the expression of the coding protein and therefore requires much lower doses
than traditional mRNA vaccines/therapeutics in most applications.
At the same time, the additional replicon gene also makes the self-amplified mRNA larger than the traditional mRNA, so the formulation of the traditional mRNA needs to be further optimized to accommodate the larger size of the self-amplifying mRNA
.

While nucleoside modifications are widely used in currently approved or under study mRNA vaccines/therapies, self-amplifying mRNAs cannot contain these modifications because they interfere with the autoamplification process
.
The self-amplifying mRNA-based coronavirus vaccine has demonstrated the ability to induce high altitude and antibody titers in animals and is currently being tested
in clinical trials.
Self-amplifying mRNA can be used at lower doses (1-10 μg)
than the 30-100 μg injection dose of traditional mRNA vaccines.

Comparison of traditional mRNA vaccines with self-amplifying mRNA vaccines

Circular RNA

The stability
of mRNA can be significantly improved by nucleoside modification, coding region and noncoding region optimization.
In addition to this, stability can be improved by circularizing mRNA
.

Circular RNA (circRNA) is a non-coding RNA with a wide range of biological functions in cells and organisms, circular RNA is a single-stranded, closed circular structure
formed by splicing precursor mRNA.
Recent studies have shown that the protein-coding function of circular RNAs holds great promise
in protein translation.
Compared with linear mRNA, the unique closed circular structure makes circular RNA more stable and less easily degraded
by nucleases.

Mechanism of circular RNA formation

In 2018, Professor Daniel Anderson of the Massachusetts Institute of Technology published a groundbreaking paper in the journal Nature Communications on the use of circular RNAs constructed from self-splicing introns to achieve robust and stable protein expression in eukaryotic cells, which pioneered new applications of exogenous circular RNAs in eukaryotic cells to express proteins and demonstrated that circular RNAs are an effective alternative
to linear mRNA 。 Based on this research, he founded ORNA, the first circular RNA therapy company, leading a new boom
in circular RNA therapy development.
Details: Orna, the world's first circular RNA therapy company, challenges and prospects

In addition to being more stable, circular RNAs induce much fewer adverse immune responses than unmodified linear mRNAs because they do not activate RNA sensors
in vivo.

The paper published in Cell by Professor Wei Wensheng's team of Peking University showed that the new crown vaccine based on circular RNA produced a higher level of neutralizing antibodies than the linear mRNA vaccine, showing that it has a strong protective effect
against the new crown virus and mutant strains in mice and rhesus monkeys.
Details: Cell: Wei Wensheng's team develops circular RNA vaccine, which is effective against Delta and Omicron

Recently, Professor Zhang Yuanhao's team of Stanford University published a paper in the journal Nature Biotechnology, successfully increasing the protein yield expressed by circular RNA translation by hundreds of times through multiple optimization designs of circular RNA structure, which can achieve efficient and durable protein production
in vivo.
Details: Zhang Yuanhao's team increased the production of cyclic RNA proteins in vivo by hundreds of times

In addition, there are companies that are exploring other variants of circular RNAs, such as optimized ribosome entry sites (IRES).

Innovation in mRNA delivery

Novel mRNA delivery system

Lipid nanoparticles (LNPs) are currently the most advanced and widely used mRNA delivery system, but many other non-LNP systems also have great potential
for mRNA delivery.

In August 2021, Zhang Feng's team published a paper in Science to develop a new RNA delivery platform - SEND (Selective Endogenous eNcapsidation for Cellular Delivery), the core of SEND is retrovirus-like protein PEG10, which can bind to its own mRNA and form a spherical protective sac
around it 。 Details: Zhang Feng's Science paper a year ago successfully transformed a new company, raised $200 million, and created a new way of mRNA delivery

The research team used the SEND system to deliver the CRISPR-Cas9 gene-editing system to mouse and human cells and successfully edited the target gene.

This will provide a completely new delivery vector for gene therapy, and the SEND system uses components in humans to self-assemble into virus-like particles, which elicit less immune response and is safer
than other delivery vectors.

PEG10 nanoparticles

Zhang said that SEND technology can complement existing viral delivery vectors and lipid nanoparticles (LNPs) to expand the toolbox
of gene delivery and editing therapies to cells.

The escape of mRNA from endosomes is a major challenge for mRNA delivery, which can be improved using ionizable lipids, while another strategy is to deliver mRNA directly into the cytoplasm
.
Entos Pharmaceuticals has developed a Fusogenix protein-lipid carrier platform
using low-toxicity-neutral lipids and a patented fusion-associated small transmembrane protein.
Unique fusion-associated small transmembrane proteins facilitate rapid fusion of protein-lipid carriers and cell membranes, allowing their loaded cargo, such as mRNA, to be delivered directly into the
cytoplasm.

Fusogenix protein-lipid vector

Similarly, pH- and redox-reaction agglomerates formed by phase-separated peptides enable direct cytoplasmic delivery of mRNA and release of redox activation, bypassing the classical endocytic pathway
.

In addition to these new platforms, innovation will continue to produce more powerful LNPs with multiple capabilities, including enhanced delivery capabilities
.
The LNPs containing heterocyclic lipids identified by the combination library can not only effectively deliver antigen mRNA to mouse tumors, but also promote antigen-presenting cell maturation by stimulating the interferon gene pathway, and synergistically improve the anti-tumor efficacy
.
The efficacy of LNPs can be enhanced by the introduction of unsaturated lipids or alkynes, while modification of LNPs with thiol or bisphosphate groups can direct mRNA delivery to mucus or bone
.
In addition, a single-component ionizable amphiphilic Janus dendritic molecule is capable of efficiently delivering mRNA to different organs, offering hope
for simplifying the current four-component LNPs system.

Biofilm-based mRNA delivery vectors

Biofilm vectors are another novel biocompatible platform
for mRNA delivery.
Different types of biofilm-based systems, including cell membrane vesicles, bacterial-derived outer membrane vesicles, and extracellular vesicles (e.
g.
, exosomes), have been used for in vitro and in vivo delivery
of therapeutic mRNA.

Exosomes, as nanoscale extracellular vesicles, have been extensively studied as drug delivery vehicles
.
For example, Codiak BioSciences has launched a human trial of an engineered exosome therapy, exoSTING, for the treatment
of solid tumors.
There are also preclinical studies showing that exosome-based mRNA vaccines induce mice to produce powerful IgG and IgA, which are stronger
than liposome-based vaccines.
Biocompatible exosomes could be a promising platform
for mRNA delivery.
A major challenge in the current use of mRNA-LNP in protein replacement therapy is the potential toxicity
that may result from repeated dosing in the short term.
The immunogenicity and toxicity of biological vesicles are lower than most existing platforms, which makes them particularly suitable for repeated administration
of mRNA in clinical trials.

Organ- or cell-specific mRNA delivery

After intravenous injection, most nanoparticles preferentially accumulate in the liver, so the ability to target mRNA delivery to extrahepatic tissues will greatly broaden the use of
mRNA technology.

In April 2020, Professor Daniel Siegwart, Cheng Qiang (currently a researcher at the School of Future Technology of Peking University), and Wei Tuo (now a researcher at the Institute of Zoology, Chinese Academy of Sciences) of Texas Southwest Medical Center published papers in Nature Nanobitechnology [4], developing a novel selective organ-targeted nanoparticle platform - SORT
.

The SORT delivery system enables selective delivery of mRNA to the lungs, spleen, or liver of mice by adding cationic lipids, anionic lipids, or the fifth component of ionizable lipids (SORT) to the widely used four-component LNP system, and validates the gene editing effect
of delivering CRISPR-Cas9 gene-edited mRNA components.
They have since published a paper in PNAS revealing the delivery mechanism of this system: the binding of specific proteins of different tissues/cells to the surface of the nanoparticles so that the nanoparticles can selectively accumulate
in different tissues.
Details: PNAS: Mechanisms to Crack the Organ-Selective mRNA Delivery System

SORT system

It is important to note that while the development and optimization of new nanoparticle carrier formulations makes sense for organ-specific delivery via intravenous mRNA, in some cases changing the route of administration may be a more practical solution
.
For example, mRNA-nanoparticles can be specifically targeted to the bladder by intravesical administration; mRNA is specifically targeted to the gastrointestinal site by oral administration via robotic capsules, and mRNA-nanoparticles are specifically targeted to the lungs
by inhalation administration.

In addition to selectively targeting organs, selective delivery of mRNA to specific cell types can lead to more precise and effective treatments
.
One strategy for cell type-specific mRNA delivery is to develop LNPs or polymer nanoparticles
optimized for specific target cell types.
For example, mRNA is delivered to T cells through optimized LNP for use in cancer immunotherapy
.
Another strategy is to use cell-specific ligands, such as conjugating anti-Ly6c-targeting ligands to LNPs and therapeutic mRNA-specific targeting to Ly6c-positive inflammatory leukocytes
delivered to mice with inflammatory bowel disease.
Antigen or CD4 antibodies are conjugated to LNPs, respectively, and mRNA-specific targeting is delivered to antigen-specific CD8+ T cells or CD4+ T cells
.

With more research, these organ- or cell-specific mRNA delivery platforms will expand the types
of diseases that can be prevented or treated through mRNA therapy.

Inhalable, intranasal, or oral delivery of mRNA

Inhalable delivery allows rapid and selective accumulation of mRNA drugs in the lungs, offering great promise
for treating lung-related diseases since the beginning of the COVID-19 pandemic.
Hyperbranched PBAE (hPBAE) can deliver mRNA directly to the lungs
by inhalation.
The hPBAE platform also enables efficient delivery of Cas13a mRNA to the lungs of mice and hamsters, which can promote the degradation of influenza virus RNA, reduce SARS-CoV-2 replication and infection symptoms
.

Intranasal administration is another non-invasive mode of administration that can cause mucosal immunity to respiratory pathogens and is a promising method
of new crown vaccine administration.
Studies in mice have shown that intranasal mRNA vaccines induce a lower immune response than intramuscular injections, possibly because LNP is not designed for intranasal administration, and optimizing LNP formulations for respiratory cells is expected to alleviate this problem
.

Intramuscular injection is the main route of administration of the currently approved new crown vaccine, but this requires medical staff to inject, which may not be conducive to the promotion
of the vaccine.
Oral administration offers a promising and attractive alternative
to COVID vaccination due to its non-invasive, patient-friendly and rapidly scalable nature.
Encouragingly, an orally administered adenovirus 5 vaccine has successfully reduced virus transmission and disease severity in hamsters and has begun phase I clinical trials
.
Although oral administration is a more challenging pathway for fragile mRNA, studies in pigs confirm the feasibility and effectiveness of using digestible robotic capsules to deliver mRNA, offering great promise
for the development of oral mRNA vaccines.
Details: Goodbye needle injection: The oral capsule mRNA vaccine is coming, inspired by the turtle

Recently, BioNTech and Matinas BioPharma announced an exclusive research collaboration to develop oral mRNA vaccines
utilizing a novel lipid nanocrystal platform.
This lipid nanocrystal is a stable crystalline nanoparticle containing multiple layers formed by the interaction of calcium and anionic phospholipids, in which active drug molecules such as mRNA can be loaded into the
layer.

Translational and clinical research of mRNA nanomedicines

Abnormal expression of proteins is characteristic of
many diseases.
With the rapid development of mRNA technology, it is easy to precisely regulate the expression level
of a specific protein by delivering mRNA encoding the protein of interest (up-regulated expression) or mRNA encoding gene-editing components (down-regulated expression) into the cell.
This makes mRNA nanomedicine a promising multifunctional tool
for treating a variety of diseases.

Currently, a range of mRNA nanomedicines, including vaccines and protein therapies or gene-editing therapies, are undergoing intensive clinical trials
.

vaccine

Completed and ongoing mRNA vaccine clinical trials

Two effective mRNA vaccines have been developed and widely used at an unprecedented speed, allowing us to see the great potential
of mRNA technology.
But in fact, the mRNA vaccine is not the first mRNA nanomedicine to enter the clinic, and many companies have carried out clinical trials of other mRNA nanomedicines before the start of the new crown epidemic, but progress has been slow
.
The success of the mRNA vaccine has strongly stimulated the enthusiasm of investors and researchers for mRNA nanomedicines, thereby greatly accelerating innovation and clinical development
.

The first unmodified mRNA coronavirus vaccine (CVnCoV) developed by CureVac showed 48% protection in Phase 2/3 clinical trials, which is not satisfactory, but their second-generation mRNA vaccine, CV2CoV, has shown improved efficacy in preclinical studies and is currently in Phase 1 clinical trials
.

ArcGIS has developed a self-replicating mRNA new crown vaccine, a single dose of 2 μg to protect mice from new crown virus infection, phase III clinical trials have shown that the vaccine is 95% effective in preventing severe new crown disease, 55% effective in preventing symptomatic new crown, it should be noted that the viruses in this clinical trial are mainly Delta and Omicron mutant strains
.
Details: Phase 3 clinical results of the first self-replicating mRNA new crown vaccine announced, with a protection rate of 55% and a protection rate of 95% for severe disease

In addition to the new crown vaccine, the development of many mRNA vaccines against other infectious diseases has also made good progress
in recent years.
For example, Moderna's mRNA-1647 vaccine (CMV vaccine) and mRNA-1345 vaccine (respiratory syncytial virus vaccine) are undergoing Phase III clinical trials
.
Recently, Moderna's mRNA-1010 (seasonal influenza quadrivalent vaccine) entered Phase III clinical trials, making it Moderna's fourth mRNA vaccine
to reach Phase III.

In addition to infectious diseases, cancer is the direction of intensive research in clinical trials of mRNA vaccines, and the mRNA-4157 vaccine for advanced melanoma jointly developed by Moderna and Merck is undergoing phase II clinical trials
.
BioNTech also launched the BNT111 vaccine against melanoma, which induced a durable objective response
in melanoma patients treated with checkpoint inhibitors in Phase 1 clinical trials.

Protein therapy and gene editing

Completed and ongoing mRNA nanomedicine clinical trials for protein replacement and gene editing

mRNA-based protein replacement therapy and gene editing therapy have multiple clinical applications
.
CAR-T cell therapy has shown great efficacy in hematological tumors, but its application to solid tumors is challenging, in part because solid tumors lack available targets
.
In addition, standard CAR-T cell therapies require T cells from cancer patients to be engineered in vitro, which is expensive and time-consuming
.

To address these challenges, BioNTech identified several solid tumor neoantigens and developed CAR-T cell therapy (BNT211) for solid tumors, in which mRNA liposomes encoding CAR-T target antigens are injected into patients and functional CAR-T cells
are generated in vivo.
Details: BioNTech's CAR-T+mRNA vaccine for the treatment of solid tumors has been granted priority drug qualification by the European Food and Drug Administration

In addition to tumors, mRNA-based CAR-T cell therapies have shown potential to treat heart damage, creating fibrotic-resistant CAR-T cells
in situ in mice by using modified mRNA.
Details: Injecting mRNA to generate CAR-T in vivo, Xinrui Company raised $165 million to promote CAR-T in vivo to the clinic

Gene editing is another important application of mRNA nanomedicines, which can downregulate the expression levels
of specific proteins through mRNA.
In primates, a single-dose LNP-delivered mRNA treatment encoding adenine base editor (ABE) developed by Verve almost completely suppressed PCSK9 in the liver, while blood PCSK9 and LDL cholesterol levels were reduced by 89 percent and 61 percent, respectively, surprisingly lasting more than 8 months
.
Details: The first in vivo base editing therapy was approved for clinical use, and one injection can permanently prevent heart disease

Intellia developed a biodegradable LP01 ionizable lipid-based delivery system for Cas9 mRNA and sgRNA delivery, achieving an inhibitory efficiency
of more than 97% serum transthyroxine after a single dose of therapy.
These data led to a human clinical trial of its LNP-based gene-editing therapy (NTLA-2001), which reduced serum transthyretin levels by 87%
after a single treatment at a dose of 0.
3 mg/kg.
Details: One injection, long-term effectiveness, the latest clinical data of in vivo CRISPR therapy delivered by LNP developed by the Nobel Prize team

These encouraging data will strongly drive the clinical translation
of more mRNA-based gene editing therapies.

Current challenges for mRNA nanomedicines

Despite the success of the vaccine, mRNA nanomedicines in development still face some challenges
.
Further innovation and progress are needed to overcome these challenges and accelerate the clinical translation
of more mRNA nanomedicines.

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MC3 is a potent ionizable lipid that is undergoing some clinical trials
.
However, some preclinical studies have shown that MC3-based LNPs have an immunostimulating effect, inducing higher expression of pro-inflammatory cytokines in mice than other LNPs
.
In addition, intravenous injection of MC3-based human erythropoietin (hEPO) mRNA-LNP to rats and monkeys produced a slight toxicological effect (at a dose of 0.
3 mg/kg), which improved
when the dose was reduced to 0.
03 mg/kg.
Off-target effects of mRNA-LNP vaccines or therapies can cause mRNA to translate in other undesirable cells or organs, leading to potential side effects
.
Continuous optimization of organ- or cell-specific mRNA delivery systems will help address this issue
.

There have been sporadic reports that RNA from the new coronavirus or mRNA from the new crown vaccine may be integrated into the host cell genome through the LINE-1-mediated reverse transcription mechanism, but these studies have been questioned by others, and more detailed studies are needed to test this conclusion
.
If more studies in the future demonstrate that exogenous mRNA sequences can be integrated into the genome of host cells, we will need to specifically design existing mRNAs to inhibit their reverse transcription
.

Clinical trials have shown that the two approved mRNA vaccines have a good safety profile, with most local and systemic adverse events being mild to moderate
.
But as with most other types of vaccines, rare allergic reactions have been observed, and one possible cause of these allergic reactions is the pegylated lipids in the LNP component, as antibodies against pegylated lipids are pre-existing in more than 40% of people
.
It is worth noting that the current dose of pegylated lipids used in mRNA new crown vaccines is much lower than other clinical preparations or therapies
using pegylated lipids that cause rare allergic reactions.
There are also studies that show that a person with pegylated lipid antibodies did not develop allergies
after receiving mRNA new crown vaccination.
Therefore, there may be other underlying factors associated with
these rare adverse events.

It is now important to recognize exactly which mRNA vaccine components cause these adverse events, particularly in the context of treating protein deficiencies and chronic diseases, requiring high doses or repeated dosing, which may further increase the risk of
these adverse events.
A better understanding of these mechanisms can guide us to optimize formulations to reduce or substitute undesirable ingredients, thereby reducing the risk of
adverse events.

Adjuvant activity

Depending on the actual application, adjuvant activity can be an advantage or a disadvantage
.
With the improvement of translation efficiency and stability, foreign mRNA that does not cause an immune response is being widely used in clinical trials
.
The mRNA in mRNA-LNP formulations is not usually used as an adjuvant itself, while LNP is known to have adjuvant activity
.
Mice injected intramuscularly with LNP and 10 μg of recombinant hemagglutinin immunogen produced a higher number of antigen-specific follicular helper T cells and germinal center B cells than mice injected with immunogen alone, which showed adjuvant activity
of LNP.
LNP in mRNA vaccines can be used as an adjuvant to induce powerful follicular helper T cell and humoral responses
.
Notably, LNP showed stronger adjuvant activity
than AddaVax, a widely used adjuvant.
Although neither LNP-based mRNA vaccine contains any adjuvant components, the strong cellular and humoral immune response to the virus caused by these vaccines may be due in part to the adjuvant activity
of the LNP component itself.
In addition to being used as an adjuvant for infectious disease vaccines, LNP also enhances the anti-tumor effect
of mRNA cancer vaccines by activating TLR4 signaling.

Although more and more studies confirm the adjuvant activity of LNP, how to manipulate this adjuvant activity remains a challenge
.
It is necessary to study the mechanism of action of LNP adjuvant activity and the structure-activity relationship
of lipid components.

Future direction

mRNA nanomedicines have shown great power
in preventing COVID and reducing the risk of hospitalization and death.
Encouraged by this success, more and more mRNA-based vaccines and therapies are expected to be clinically transformed
.
However, several key goals
must be met before the potential of mRNA nanomedicines can be fully realized.

There is growing evidence that specific biological pathways may interfere with mRNA delivery or translation
.
Therefore, understanding how these biological pathways affect mRNA delivery and translation in vivo can further improve the efficacy of mRNA drugs, while also carefully considering the toxicity and immune response
that mRNA and its vectors may cause.
Ultimately, the rapid and effective implementation of mRNA nanomedicines depends largely on their stability and logistical needs, which will impact real-world implementation and rollout
.

Therefore, innovations to improve the stability of mRNA nanomedicines will be critical
.
In January 2022, the more stable (long-term stable at 2-8°C) mRNA developed by Abogen Biosciences released the results of the Phase 1 clinical trial, details: The results of the first domestic mRNA vaccine Phase 1 clinical trial were announced
.
The next-generation coronavirus vaccine mRNA-1283 developed by Moderna can also remain stable
at 2-5°C.

New engineering advances will facilitate the myriad of real-world applications
of mRNA nanomedicines.
For example, novel PLGA microparticles could be a promising mRNA delivery platform that can program drug release and even enable vaccine self-enhancement
in vivo.
Details: Robert Langer's team develops a new vaccine delivery platform that delivers in batches in one injection to achieve self-enhancement
in the body.
In addition, the microneedle patch as a carrier for seasonal influenza and new crown vaccines has been shown to be safe and immunogenic, and may become a convenient and minimally invasive mRNA delivery platform
.

The unprecedented success of mRNA vaccines demonstrates the great potential of mRNA nanomedicines, and we expect continued innovation to lead to new, highly effective mRNA-based therapeutics, including vaccines for other non-COVID-19 infectious diseases, cancer immunotherapy, protein replacement therapy, gene editing therapy, and many other types or diseases
.