RNA-based treatment methods and vaccine production technology trends

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RNA-based treatment methods and vaccine production technology trends

RNA-based treatment methods and vaccine production technology trends.  In recent years, the growth rate of new drugs, new targets, and new molecules in the monoclonal antibody drug market is the highest (19%).

DNA and RNA therapies are not far behind, with a year-on-year increase of 12%. Industry analysis data shows that in 2020, the RNA-based therapy market will reach 1.2 billion US dollars.

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This article reviews the current situation of the RNA therapy and vaccine market, and compares the difference among small interfering RNA (siRNA), RNA interference (RNA interference, RNAi), microRNA (MicroRNA, miRNA) and messenger RNA (messenger RNA, mRNA).  In addition, it also outlines the general production process of these platforms, the challenges encountered in process development and production, and the strategies to overcome these challenges.

Market overview

RNA-based therapies are aimed at the treatment of diabetes, cancer, tuberculosis and some cardiovascular diseases. There is currently a large amount of money invested in this relatively new therapy and vaccine, and the market will reach 1.2 billion US dollars in 2020.

As shown in Figure 1 below, it shows the RNA R&D pipeline (for reference only) in the previous years (2015). There are more than 700 nucleic acid-based therapies (DNA and RNA) under development, and more than 60% of nucleic acid-based therapies are in the preclinical development stage. Interestingly, 35% of such R&D projects focus on oncology.

Figure 1: Expansion of R&D projects.

Many companies (about 160) and many academic institutions (about 65) are developing RNA-based therapies. Table 1 below shows the companies and institutions that are conducting research. Among them, two companies have already sold RNA-based therapies: NeXstar and Ionis Pharmaceuticals. There are 12 mRNA vaccines under development, 7 of which are being developed by Curevac (Germany). Based on current prospects, the RNA therapy market seems to be more promising than the DNA therapy market.

Table 1: Biopharmaceutical companies developing RNA-based therapies and vaccines. siRNA: small interfering RNA, miRNA: microRNA, mRNA: messenger RNA.

From the perspective of partners, Ionis Pharmaceuticals has reached a global collaboration with Janssen Biotech, Inc. to discover and develop antisense drugs for the treatment of gastrointestinal autoimmune diseases. Merck & Co. (MSD) has made a bet 100 million US dollars to support Moderna’s mRNA technology. Moderna also previously announced cooperation with Alexion, AstraZeneca and the Defense Advanced Research Projects Agency (DARPA) for a total of $450 million. Moderna has raised $625 million in equity financing.

RNA interference (RNAi) and RNA reverse transcription technology seem to dominate the market. RNAi is a gene silencing technology in which RNA molecules inhibit gene expression by targeting and destroying specific mRNA molecules. RNA reverse transcription technology involves the synthesis of RNA strands that bind to splice sites on specific mRNA or mRNA precursor molecules to prevent translation. The main challenges associated with the commercialization of these RNA-based therapies are toxicity and drug delivery.

RNA-based therapy

With the advent of RNA-based therapies and their potential in the treatment of various chronic diseases, it is important to pay attention to the technologies used to develop RNA mechanisms and drug pathways. We will discuss some aspects.

RNAi

RNAi technology uses the DNA sequence of the gene itself to “silence” or turn off the gene (Figure 2 below). The process is initiated by double-stranded RNA (dsRNA), which is expressed as RNA (shRNA) or microRNA (miRNA) transcripts. Using this silencing mechanism, RNAi is usually used to better understand gene function and then can be used To produce other targeted therapies. Small interfering RNA (siRNA) and miRNA are the core elements of therapies based on RNAi technology.

Figure 2: In-depth understanding of the RNAi pathway. RNA (shRNA) is a type of double-stranded RNA (dsRNA). The dsRNA is cleaved or degraded by enzymes into oligonucleotide fragments called small interfering RNA (siRNA), and then enters the cell to form an RNA-induced silencing complex (RISC). The siRNA strands then separate or unwind to form an activated RISC complex, which can then target messenger RNA (mRNA), bind to it and cut it.

siRNA

RNAi uses the “dicer” enzyme to cut dsRNA into 21 oligonucleotide fragments called siRNA. These siRNAs can then bind to a specific family of proteins called Argonaute proteins, of which there are two types: Ago and Piwi. Ago protein binds to siRNA or miRNA, and Piwi protein binds to Piwi-interacting RNA (piRNA) to silence mobile genetic elements. The siRNA, miRNA, or piRNA complex that binds to the Argonaute protein is called the RNA-induced silencing complex (RISC). Once bound to the Argonaute protein, one strand of the dsRNA will be removed, and the remaining strand will bind and guide the degradation of the complementary RNA target sequence, resulting in the loss of protein expression.

It is reported that synthetic siRNA can knock out targets in many diseases in vivo, including hepatitis B, human papilloma virus, ovarian cancer, bone cancer, hypercholesterolemia and liver cirrhosis. Only a few siRNA molecules per cell can produce effective gene silencing. siRNAs are most commonly delivered into cells using microinjection or transfection agents. Many companies now provide siRNA delivery reagents to simplify this process.

miRNA

miRNAs do not code for proteins because they belong to a specific category of non-coding RNA. miRNAs are 19-25 nucleotides in length and are encoded by introns (that is, parts of the gene sequence that are not expressed in proteins). In vertebrates, miRNA acts as the guide strand of the RISC complex to reach its mRNA target. About 30% of the genes in the human genome are regulated by miRNAs.

Although siRNA silencing requires an exact match between the target and the small interfering RNA, miRNA is non-specific and can work through imperfect base pairing. In addition, miRNA triggers translation inhibition (that is, prevents RNA from synthesizing protein from amino acids), while siRNA triggers mRNA degradation.

mRNA

The mRNA encoding the protein is an important part of the central law of life (DNA-mRNA-protein). mRNA is transcribed from a DNA template. mRNA carries the genetic code from DNA to the ribosome, where the mRNA is translated into protein. The significant increase in mRNA-based therapies is due in large part to the fact that mRNA has many advantages over DNA in terms of gene expression and transfer. Although RNAi and reverse transcription RNA technology are mainly used for gene silencing, mRNA technology is usually used for vaccines or gene therapy. In both cases, after being injected into the human body, the mRNA is translated into protein, which can eventually replace the missing protein (therapeutic) or induce an immune response (preventive method). The production of synthetic mRNA for therapeutic use is relatively simple.

RNA-based vaccine

Conceptually, an mRNA-based vaccine is a simple method of inducing an immune response by delivering coding genetic elements as translation-ready molecules. After direct vaccination with mRNA molecules, dendritic cells (antigen-presenting cells) ingest, process, and encode the target antigen, thereby inducing an immune response. Generally, mRNA vaccines are produced by in vitro synthesis through an enzymatic process. This synthesis process can be strictly controlled, resulting in a high-quality and predictable product profile. The mRNA can be easily customized to provide specific immunogenicity characteristics and pharmacokinetics. The stability and antigenic properties of mRNA can be controlled by changing codons or modifying bases.

Ongoing clinical trials have shown that mRNA can be delivered as naked mRNA; immobilized on particles or in liposomal nanoparticles; or transfected in dendritic cells in vitro, resulting in a distinguishable immune response and protective efficacy. mRNA can also be used as an adjuvant, and mRNA has also been explored to stimulate the innate immune system through toll-like receptors. Based on the relatively simple production of RNA vaccines, they can be developed, manufactured and administered in a short time, so they are suitable for pandemic situations. The thermal stability of mRNA vaccines also helps reduce costs because they do not require cold chain delivery.

Manufacturing RNA-based biopharmaceuticals

As more and more experimental RNA drugs enter the clinic and enter large-scale trials, the demand for efficient and cost-effective manufacturing strategies will grow. RNA-based biopharmaceuticals are inherently sensitive to endonucleases, so production and purification require special treatment. The degradation of the product during the manufacturing process increases the heterogeneity and chemical instability of the product. Therefore, the manufacturing and purification methods used in RNA-based therapies are different from DNA and other proteins.

mRNA purification (post-chemical synthesis) includes concentration precipitation, extraction and chromatographic methods (including high performance liquid chromatography). The purpose of the upstream concentration and ultrafiltration step is to concentrate (if the titer is low) and change the buffer to the pH and conductivity required for the first chromatographic step. The purpose of the final concentration and ultrafiltration step is to desalinate and achieve the necessary final concentration before sterile filtration. The 5kD membrane cut-off value is usually used for concentration and ultrafiltration in the mRNA process. Since siRNA is smaller than mRNA, the membrane cutoff value of 1kD is used to fully retain the siRNA product.

Chromatographic purification steps

Since the breakthrough discovery of catalytic RNA in the early 1980s and RNA interference in the late 1990s, more than 50 RNA or RNA-derived therapies have entered clinical trials. In RNA purification, despite the use of different technologies, such as arginine affinity, ion-pair reversed phase, or membrane anion exchange, traditional ion exchange (IEX) media—especially anion exchange (AEX)—are still the best. Commonly used techniques.

Sm and Sm-like proteins can form heteromeric complexes or bind to various RNAs, and have been shown to contain oligo (U) specific RNA binding sequences (Sm domains). FractogelTMAE (MilliporeSigma) is a strong anion exchange resin for purification of small nuclear ribonucleoprotein (snRNP). The snRNP molecule was eluted with Tris/HCl and 300mM NaCl. Ribonucleoprotein and uncoupled RNA are separated from free protein, and the sample is immediately used for negative staining electron microscopy.

Both AEX and reverse phase (RP) technologies are widely used in RNA purification processes. Quaternary amine (Q) and dimethylaminoethyl (DMAE) are AEX’s choices. A study proved that some AEX resins can be used for RNA purification to optimize experimental conditions to achieve high dynamic binding capacity. In this study, of the 18 AEX media screened, only four resins-Q Sepharose FF (GE Healthcare), POROS 50HQ (Applied Biosystems), Q CeramicHyperD F (Pall) and Fractogel DEAE (MilliporeSigma) showed baseline separation RNA and plasmid DNA. After optimizing the loading and elution conditions, Fractogel DEAE has a wider operating range, higher dynamic binding capacity, and the breakthrough RNA is completely separated from the eluted plasmid. High recovery rate, stability, and reproducibility also meet the requirements of large-scale manufacturing. These binding and elution conditions can be used as a starting point for optimal experimental conditions in RNA purification.

In general, many biochromatographic resins are suitable for RNA purification similar to other biomolecule separations. In many cases, Fractogel resin exhibits excellent capacity and efficiency, mainly due to the “tentacles” structure, in which functional groups are located at the ends of the long arms grafted to the surface of the beads, thereby avoiding steric hindrance caused by large biomolecules .

Formulation and delivery

The most challenging aspect of RNA-based therapy is its delivery to target cells. Several methods have been explored and tested in clinical trials. The next article introduces some of the most promising methods.

Polymer bonding/chemical modification

Natural RNA and RNA-based therapies are easily degraded by many ribonucleases found in cells. Chemical modification is a method of hardening RNA to resist such enzymatic attacks. Modifications to the molecule can also increase its target affinity, reduce its undesirable immunogenicity, and increase its overall efficacy. The hardening strategy involves modifying the backbone, sugar or base of the RNA molecule.

The combination of RNA therapeutic agents is a strategy that is increasingly being used to improve delivery and uptake. Alnylam Pharmaceuticals has adopted a method combining the galactosamino sugar derivative N-acetylgalactosamine (GalNac) to improve the delivery of siRNA therapy to the liver. GalNac-coupled siRNA is absorbed by the asialoglycoprotein receptor in the liver, and its efficacy is increased by five times compared with the parent molecule.

Arrowhead Research is developing a competitive conjugate strategy. Arrowhead’s delivery technology, called Dynamic Polyconjugate (DPC), is a type of siRNA that binds to the endosomal dissolving polymer backbone through disulfide bonds. The endosome lysing polymer can release siRNA from the endosome quickly and effectively. Arrowhead’s latest strategy involves linking cholesterol to siRNA and GalNac to endosomal solubilizing polymers to ensure that they are all delivered to liver cells. Combined injection therapy has been shown to increase the efficacy of siRNA-cholesterol by 500 times.

The main and most studied strategy for the delivery of RNA-based therapies is lipid-based delivery systems. A successful platform is the use of stable nucleic acid lipid particles (SNALP), which are lipid particles formed from fusion lipids, cationic lipids, and PEG-lipid mixtures. The SNALP delivery system was developed and supported by Tekmira Pharma; the company now refers to it as LNP technology. According to Tekmira, LNP “encloses siRNA (including mRNA) efficiently in uniform lipid nanoparticles, which can effectively deliver RNAi therapeutics to disease sites in many preclinical models.”

Another promising lipid delivery technology is the proprietary Smarticles delivery platform developed by Novosom and now owned by Marina Biotech. Similar to SNALP, Smarticles technology can change its surface charge to promote stability and endosome release. Smarticles can encapsulate single-stranded and double-stranded nucleic acid therapy. Smarticles are composed of cations, anions and neutral lipids. Negatively charged Smarticles avoid the common toxic effects of positively charged lipids at physiological pH, but are converted to positive charges in the acidic environment of the endosome to promote their release. Other interesting technologies involve PLGA nanoparticles.

Summary

RNA-based therapy is a relatively new type of therapy, which has broad prospects in the treatment and prevention of refractory chronic diseases and rare diseases. RNAi works by interfering with the transcription process, thereby inhibiting protein translation.

Although this treatment is highly selective and targeted, special care needs to be taken in the production of these therapies and vaccines because they are sensitive to ubiquitous RNase-induced degradation. The large-scale manufacturing of new therapies requires RNase-free biological processing components and chemicals.

Technology and tool suppliers need to consider providing such products to achieve large-scale production of RNA-based therapies. Before such products can be commercialized, challenges related to potential toxicity and drug delivery need to be addressed.

However, new technologies are emerging to overcome some of these challenges, and the future of RNA-based therapies is very promising.

(source:internet, reference only)

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