Innovative Developments And Emerging Technologies In RNA-based Therapeutics

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Innovative Developments And Emerging
Technologies In RNA-based Therapeutics

The use of synthetic oligonucleotides to alter protein expression by Watson-Crick
hybridization of RNA was first described by Zamecnik and Stephenson in 1978. On this
basis, after years of research on nucleic acid medicinal chemistry, RNA-targeted drugs
(RTD) have become a very active field of drug design and development in the
pharmaceutical industry. However, due to the instability of bare RNA and the existence of
biological barriers to RNA entry into cells, achieving widespread application of RNA
therapeutics in the clinic remains extremely challenging. Despite the difficulties in
developing RNA-based therapies, scientists have explored a variety of techniques to
promote RNA intracellular transport and metabolic stability. Here, we will summarize the
current innovative developments and emerging technologies in RNA-based therapeutics.

Figure 1 Major developments in the field of RNA targeting [1]

Types and Modes of Action of RNA Therapies

1.Antisense Oligonucleotide (ASO)

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Antisense Oligonucleotide (ASO) is a single-stranded oligonucleotide molecule that
binds to target mRNA intracellularly via the Watson-Crick base pairing principle. ASO
mainly regulates the expression of target genes through two mechanisms: ① After binding
to target mRNA, ASO promotes mRNA degradation under the action of Rnase H1 (the
main way) or ribozyme, thus inhibiting protein expression. This mechanism is also known
as enzymatic RNA degradation; ②ASO controls the expression of target proteins by
changing the splicing pattern of RNA without the help of specific enzymes, which is also
known as the steric mechanism.

2. RNA interference (RNAi)

RNAi therapies use the principle of gene silencing and can be further classified as
therapeutic agents using small interfering RNA (siRNA) or microRNA (miRNA).
Double-stranded siRNA molecules recruit RISC to mRNA by the Watson-Crick base
pairing principle to inhibit protein translation. miRNAs are single-stranded noncoding
RNAs that cause mRNA translational repression or degradation in the silencing complex
miRISC they induce by base-pairing with a specific RNA sequence (usually the 3'UTR).

3. CRISPR-based genome editing

The prokaryotic-derived CRISPR-associated protein (Cas) system has been widely used
in mammalian cells and organisms. In this system, Cas protein and sgRNA combine to
form a complex to recognize and cleave target sequences for precise and efficient
genome editing.

4. Aptamer

Aptamer is a structured oligonucleotide sequence that specifically binds and inhibits
protein expression. It is usually obtained by SELEX, an in vitro screening technique. They
are also known as chemical antibodies because they are synthesized in a similar way to
antibodies. Aptamer-based therapies mainly include: ① using Aptamer to disrupt the

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interaction between disease-related targets; ② using cell-specific Aptamer as a carrier to
deliver other therapeutic agents to target cells or target tissues.

5. mRNA drugs

The concept of mRNA-encoded drugs was proposed in 1990 [1], when in vitro
synthesized (IVT) mRNA was directly injected into mouse skeletal muscle, and the protein
encoded by IVT was found to be expressed in skeletal muscle. Preclinical studies of IVT
mRNA have facilitated the clinical development of mRNA vaccines. mRNA vaccines are
delivered to host cells and translated into targeted antigens that activate the body's
immune response. It shows great potential in the treatment of cancer and infectious
diseases.

Figure 2. Mechanisms of action of various RNA therapies

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Chemical modification of RNA drugs

ASO and siRNA are chemically modified mainly at the phosphate backbone, ribose ring,
3'- and 5' -ends to improve their substrate specificity and nuclease resistance, and to
reduce their toxicity and immunogenicity. For example, siRNA 2'-F and 2'-O-Me
modifications can help antagonize RNases and prevent siRNAs from activating innate
immune receptors (TLR, MDA-5, and RIG-I). 5 'cap structure, ORF, flanking 5' 3 '-UTRs,
3' -poly (A) tail were optimized to enhance the translation ability of RNA through sequence
optimization, nucleoside modification or sequence replacement of UTR. 5 'cap structure,
ORF, flanking 5' 3 '-UTRs, 3' -poly (A) tail were optimized to enhance the translation ability
of RNA through sequence optimization, nucleoside modification or sequence replacement
of UTR.

Figure 3 Common chemical modifications of RNA drugs

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Application of nanoparticle drug delivery system

in RNA therapy

Since therapeutic RNAs carry a large number of negative charges and chemical
modifications, it is necessary to develop an effective vector to protect RNA from the
physiological environment [3].

1. Lipid Nanoparticles (LNPs)

Lipid nanoparticles (LNPs) are the most widely used carriers for the delivery of
oligonucleotide drugs. FDA-approved LNPs contain four basic components: cationic or
ionized lipids, cholesterol, helper lipids, and PEG-lipids. Selective organ targeting (SORT)
technology can specifically target liver and extrahepatic tissues (lung and spleen) by
adding SORT molecules to LNPs. This technology enables mRNA delivery and
CRISPR-Cas gene editing in specific tissues [4].

Figure 4 Structure of lipid nanoparticles

2. Polymer nanoparticles

Polyethylenimine (PEI) polymer family is the most widely studied polymer materials for
nucleic acid delivery. They are composed of linear or branched polycations that can form

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nanoscale complexes with miRNA or siRNA. The commercially available linear PEI
derivative jetPEI™ is widely used for DNA, siRNA and mRNA transfection. PBAE
polymers have better biodegradability and lower cytotoxicity. Recently, researchers have
used PBAE-based polymers to deliver Cas13a mRNA into the respiratory tract of mice by
aerosolization for the treatment of SARS-Cov-2 [5] .

Figure 5 Structure of polymer nanoparticles

FDA-Approved RNA Therapies

Fomivirsen is the first ASO drug approved by the FDA for the treatment of
cytomegalovirus retinitis in AIDS patients. Mipomersen and Inotersen are
second-generation ASO drugs that mediate the degradation of ApoB and TTR mRNA,
respectively.

GalNAc-siRNA is a single conjugate formed by carbohydrates and siRNA, which can be
used to treat various diseases only by changing the siRNA sequence. Currently, the FDA
has approved GalNAc-siRNA in combination with Givosiran for the treatment of acute
intermittent hepatic porphyria (AHP) and Lumasiran in the treatment of primary
hyperoxaluria type 1 (PH1).

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Pegaptanib is the first FDA-approved Aptamer drug targeting VEGF for the treatment of
age-related macular degeneration.

During the COVID-19 epidemic, mRNA vaccines have been shown to be effective
against SARS-CoV-2. Pfizer – BioNTech’s vaccine BNT162b2 (Comirnaty®) and
Moderna’s vaccine mRNA-1273 are two FDA-approved SARS-CoV-2 vaccines with >94%
efficacy in Phase III clinical trials.

Conclusion

The emerging applications of RNA drugs in modulating immune responses and the
expression of disease-related proteins have provided new ideas for biomedical research.
While small molecules and antibody drugs target only 0.05% of the human genome, RNA
selectively acts on proteins, transcripts, and genes, thereby broadening the range of drug
targets. Combining RNA chemical modifications with nanocarrier systems can improve
the efficiency of RNA drug delivery. In the future, with the continuous progress of medical
science and technology, RNA-based therapeutic methods are expected to be more
diversified.

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purity PEG derivatives in GMP and non-GMP grades for your research of lipid
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References:
[1]. Zhu Yiran,Zhu Liyuan,Wang Xian et al. RNA-based therapeutics: an overview and
prospectus.[J] .Cell Death Dis, 2022, 13: 644.
[2]. Wolff J A,Malone R W,Williams P et al. Direct gene transfer into mouse muscle in

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vivo.[J] .Science, 1990, 247: 1465-8.
[3]. Paunovska Kalina,Loughrey David,Dahlman James E,Drug delivery systems for RNA
therapeutics.[J] .Nat Rev Genet, 2022, 23: 265-280.
[4]. Dilliard Sean A,Cheng Qiang,Siegwart Daniel J,On the mechanism of tissue-specific
mRNA delivery by selective organ targeting nanoparticles.[J] .Proc Natl Acad Sci U S A,
2021, 118: undefined.
[5]. Blanchard Emmeline L,Vanover Daryll,Bawage Swapnil Subhash et al. Treatment of
influenza and SARS-CoV-2 infections via mRNA-encoded Cas13a in rodents.[J] .Nat
Biotechnol, 2021, 39: 717-726.

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