As a new pharmaceutical technology, mRNA has made breakthroughs in the treatment of infectious diseases and tumors in a short time. At present, one of the major obstacles in clinical mRNA technology is to determine how to deliver mRNA to a specific target cell and protect it from degradation, so the ideal delivery vector must be safe, stable, and organ specific.

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mRNA Technology: How To Achieve Targeted
Delivery to Organs, Cells, Tumors?
As a new pharmaceutical technology, mRNA has made breakthroughs in the
treatment of infectious diseases and tumors in a short time. At present, one of
the major obstacles in clinical mRNA technology is to determine how to
deliver mRNA to a specific target cell and protect it from degradation, so
the ideal delivery vector must be safe, stable, and organ specific.
1. Nucleic Acid Delivery Technology
Since RNA was first discovered, researchers have used a number of methods
to deliver it into cells. The initial technique used is naked RNA, which is easily
degraded by RNase and causes a strong pro-inflammatory response. Later
formulations developed for RNA delivery included carbohydrate polymers,
polyethylenimine (PEI), etc. Due to its positive charge and abundance of
amines, PEI has a good affinity for nucleic acids, resulting in the formation of
positively charged complexes on the surface. In vivo, PEI was successfully
used for aerosol gene delivery to the lungs. Although PEI preparations have
high transfection efficiency in vitro and in vivo, they are also significantly
cytotoxic, partly due to their poor degradation, preventing the wider application
of PEI-based vectors in preclinical and clinical settings.
Polyesters are another group of materials used for RNA delivery, and the
addition of pluripotent F127 reduced the total charge of the nanoparticles and
improved their stability. Manipulating F127 levels can lead to
lung-specific mRNA delivery, which can be used to treat lung diseases.
Natural chitosan is a carbohydrate polymer with biodegradability,
biocompatibility, and cationic charge that allows nucleic acid binding. However,
it also has limitations such as poor water solubility and limited targeting ability.
To date, lipid nanoparticles (LNPs) have been mainly used as RNA therapeutic
carriers in clinical. Lipids are organic, water-insoluble lipid compounds with
hydrophilic heads and hydrophobic tails that enable LNPs to self-assemble
into well-defined structures, such as cell membranes. LNP-RNA systems are
formed through hydrophobic interactions in the aqueous environment and
electrostatic interactions between negatively charged RNAs and cationic or
ionizable lipids. Ionizable lipids are positively charged at low pH (which allows
RNA binding) and become neutral at physiological pH, a change that helps
reduce the toxicity of LNP-RNA complexes in vivo.

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Carriers for Nucleic Acid Delivery: Cargo and Formulation (Adv Drug Deliv Rev.
2020;156:119-132.)
2. Organ-targeted Delivery
Using intramuscular injection of LNP-RNA for systemic immunity, how to
precisely guide LNP-RNA to target organs? Apolipoprotein E (ApoE) in serum
can bind to intravenously injected LNPs, and the liver is the main organ for
clearing ApoE-bound lipoproteins. Therefore, systemically delivered
LNPs-RNA will bind ApoE and preferentially enter the liver. Excessive liver
homing is a significant disadvantage of intravenously administered ionizable
LNPs, which can be overcome by selective organ targeting (SORT) strategies.
The key to organ-specific delivery is to manipulate the internal and/or external
charges of the formulated LNPs.
LNPs charge and organ targeting (Pharmaceutics 2021, 13, 1675)

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In addition to standard LNP components including ionizable cationic lipids,
phospholipids, cholesterol, and PEG, the addition of SORT molecules resulted
in lung, spleen, and liver-specific gene delivery.
Increasing the percentage of permanently positively charged DDAB and EPC
shifted tissue tropism from the liver to the lung.
Increasing the negatively charged 1,2-dienoyl-sn-glycero-3-phosphate (14PA)
SORT molecule, at 10-40%, resulted in spleen-specific delivery. The
appropriate ratio of DODAP and C12-200 as shown in the figure below can
also increase liver targeting.
Nat Nanotechnol . 2020 Apr;15(4):313-320
3. Cell-targeted Delivery
In addition to organ-specific delivery, the researchers also sought to target
specific cell subpopulations in the liver by selectively delivering engineered

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ionizable lipid nanoparticles into liver cells and hepatic sinusoidal endothelial
cells (LSEC). Increased PEG content enhanced hepatocyte targeted delivery,
while addition of mannose increased hepatic sinusoidal endothelial cell (LSEC)
delivery.
Cell-targeted delivery (Sci. Adv. 2021, 7, eabf4398.)
4. Tumor targeted Delivery
Heterogeneity is one of the important markers of high malignancy of cancer, so
efforts to eradicate cancer by targeting a single receptor are likely to fail, and
the loss of this specific targeted receptor will lead to the growth of
drug-resistant cancer cells. Although monoclonal antibodies that bind
specifically to cell surface receptors are widely used, chemical coupling of
multiple different monoclonal antibodies to LNPs is inefficient and requires
careful optimization of each monoclonal antibody.
A recent new LNPs engineering strategy, ASSET, is a membrane-anchored
lipoprotein that is integrated into RNA-loaded lipid nanoparticles and interacts
with the antibody Fc domain. It consists of two functional domains: an
N-terminal signal sequence followed by a short CDQSSS peptide NlpA motif
that undergoes lipidylation in bacteria, and an scFv of a monoclonal antibody
(clone RG7/1.30)9 which binds to the Fc constant region of an IgG2a antibody.
This lipification strategy, originally used to demonstrate proteins anchored to
the inner lining of E. coli, allows the purified recombinant lipified ASSET to be
inserted into any lipid vesicle. An mCherry domain and His tag were fused to
the C-terminal of ASSET for tracking LNPs absorption (for experimental use)
and ASSET purification.

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Nat Nanotechnol . 2018 Mar;13(3):214-219.
After the cells to be targeted are selected, only specific target antibodies or
antibody combinations of corresponding cells need to be injected, and LNPs
can self-assemble into targeted LNPs in vivo by binding with antibody Fc. The
targeting specificity and effectiveness were confirmed by mouse model
experiments (Reference [4]).
5. Commercialization Progress
ReCode Therapeutics in the United States is promoting the commercialization
of SORT LNPs (currently in preclinical stages), and completed its Series B
financing of $80 million on October 21, 2021, led by Pfizer Ventures and
EcoR1 Capital, with participation from Sanofi Ventures, and the funds will be
used for SORT LNPs technology for primary ciliary dyskinesia (PCD) and
cystic fibrosis (CF) clinical research.
6. Conclusion
How to accurately deliver mRNA to the target area is the main strategy to
maximize the effect of mRNA and minimize toxicity. According to the physical
and chemical characteristics of the delivery vector itself, targeted delivery of
lung, liver and spleen has been realized, and even specific cells can be
accurately targeted. The recently developed self-assembled LNPs can
self-assemble into targeted LNPs in vivo with specific targeted antibodies or
groups of targeted antibodies to achieve accurate targeting of specific cancer
tissues or inflammatory tissues. Organ targeted delivery of mRNA will be
the focus of the next generation of mRNA technology development, and
ReCode Therapeutics is ready to enter clinical trials.
mRNA has emerged as a new class of therapeutic agent for the prevention
and treatment of various diseases, such as mRNA vaccines. As a
professional PEG derivatives and pharmaceutical intermediates
supplier, Huateng Pharma is able to provide some PEG products which used

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as ingredients in mRNA vaccines for your mRNA delivery from mg to kg. For
more information, please read the page PEGs for mRNA Delivery.
References:
[1]. Alexandra S Piotrowski-Daspit et al，Polymeric vehicles for nucleic acid delivery，Adv
Drug Deliv Rev . 2020; 156:119-132.
[2]. Qiang Cheng et al，Selective organ targeting (SORT) nanoparticles for tissue-specific
mRNA delivery and CRISPR-Cas gene editing ， Nat Nanotechnol . 2020
Apr;15(4):313-320.
[3[. Kim, M.; Jeong, M.; Hur, S.; Cho, Y.; Park, J.; Jung, H.; Seo, Y.; Woo, H.A.; Nam, K.T.;
Lee, K.; et al. Engineered ionizable lipid nanoparticles for targeted delivery of RNA
therapeutics into different types of cells in the liver. Sci. Adv. 2021, 7, eabf4398.
[4]. Ranit Kedmi et al ， A modular platform for targeted RNAi therapeutics ， Nat
Nanotechnol . 2018 Mar;13(3):214-219.
[5]. Zak, M.M.; Zangi, L. Lipid Nanoparticles for Organ-Specific mRNA Therapeutic
Delivery. Pharmaceutics 2021, 13, 1675