It is certain that RNA therapy is no longer a no-man's land for medical innovation. Not only have several products been launched and received good market response, but there are also many pipelines in the research and development side to advance to the middle and late clinical stage, and behind the boom are the technological breakthroughs of scientists.
There are three key points in the research and development of RNA drugs:
First, the immunogenicity of RNA drugs: because exogenous RNA will be recognized by the immune system as the signal of virus interference, RNA drugs will more or less stimulate the immune system, and then cause a series of side effects.
Second, the stability of RNA drugs: there are plenty of ribonuclease enzymes in human blood and tissue fluid, usually naked RNA without chemical modification is eliminated by them before it enters the cell. In addition, the efficiency of mRNA translation is closely related to its stability in cells.
Thirdly, the delivery of RNA drugs: RNA molecules are charged and cannot pass through the cell membrane structure freely, so transmembrane transport and endosome escape are the inevitable problems in the design of RNA drugs. In addition, how to achieve targeted delivery is also the focus of current research.
The solutions of the above three concerns are scattered in the three steps of RNA drug preparation, which involve in vitro transcription, chemical modification and delivery vector respectively. Among them, the most difficult or most capable of establishing technical barriers is the drug delivery vector.
The RNA drug delivery system
An excellent delivery system must meet the following basic requirements:
In addition to the above basic requirements, targeted delivery of RNA molecules is the development goal of the next generation vector. There are three main types of delivery systems in the market: nanoparticle delivery system, covalent coupling modification delivery system and exosome delivery system. The most common viral vector delivery system in the field of gene therapy is more used for DNA based therapy.
At present, the most popular nanoparticle delivery system is lipid nanoparticles (LNPS) delivery system. The LNP system used for Moderna's new coronavirus vaccine is derived from Arbutus's technology and has been continuously improved on the basis of it. Through the construction of LNP library and high-throughput screening, Moderna can find the candidate vectors with targeting. Some time ago, gene editing company Beam therapeutics acquired guide therapeutics, which is a LNP screening platform. This platform uses DNA barcodes to label nanoparticles, and finally calculates the distribution of LNP in animal models through gene detection. In addition to finding targeted LNP through screening, some companies choose to introduce ligands on the surface of nanoparticles to achieve ligand mediated targeted delivery. Wei Tao's team at Harvard University has also developed a lipid-polymer hybrid nanoparticle that provides a more flexible surface modification than pure lipid-based nanoparticle with enhanced stability. Sirnaomics completely uses a peptide copolymer composed of histidine and lysine as the nanoparticle carrier of siRNA, so as to bypass the patent barrier of LNP. The LPP/mRNA® nano-delivery platform of Siobacteria utilizes a two-layer structure with a polymer-coated mRNA as the core and a phospholipid-coated outer shell, which has excellent dendritic cell (DC) targeting.
2.Covalent coupling delivery system
The so-called covalent coupling delivery system is similar to the antibody coupled drugs (ADC) in macromolecular drugs, that is, RNA molecules are directly connected with a ligand, and the targeted delivery effect is achieved by the specific binding of the ligand with the receptor on the surface of the target cell.
At present, GalNAc coupling technology is the most widely used technology. GalNAc (N-acetylgalactosamine) can be recognized by asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, so it has good liver targeting. In fact, scientists have used GalNAc to deliver nucleic acid molecules as early as 1995. The quantity of GalNAc, the choice of linker and the connection mode will affect the efficiency of delivery. Academic and industrial circles have done extensive research on this.
At present, companies adopting GalNAc coupling strategy include RNAi enterprises such as Alnylam, Arrowhead and Dicerna, as well as ASO leaders such as Ionis. Of course, GalNAc is not the only coupling ligand, the platform can also achieve targeted delivery of tumor cells by coupling transferrin.
Exosomes are known as "natural nanoparticles". Because of their circulation stability, biocompatibility, low immunogenicity, low toxicity, ability to cross the brain blood barrier and inherent targeting, exosomes have always been a hot topic in the field of drug delivery. Although there is no RNA drug based on exosome delivery system on the market at present, many biotechs and large pharmaceutical companies have actively arranged in this field. Compared with nanoparticle technology and covalent coupling technology, the difficulties in commercialization of exosome technology are now mainly focused on industrial production, among which the core issues involve the separation and purification of exosomes, exosome homogeneity, and the uptake strategy of exosomes into target tissues, etc.
The main purpose of chemical modification is to solve some problems of natural RNA, including immunogenicity, enzyme stability, target affinity, mRNA translation efficiency and so on. According to the position and structure of modification, chemical modification can be divided into base modification, ribose modification and phosphate skeleton modification (Figure 3). The technical details of chemical modification will not be further developed in this paper, but the related patent authorization is more worthy of attention. To take just one example, in the case of base modification (Figure 4), the more successful strategy is to replace cytidine (C) with 5-methylcytidine (m5C) or replace uridine (U) with pseuduridine (ψ), so that the modified mRNA can escape recognition by the immune system. Both of these modifications were first invented by Katalin Kariko and Drew Weissman, biochemists at the University of Pennsylvania, who applied for patents immediately. In order to circumvent this patent, Moderna spent four years to invest a lot of resources, and finally obtained the patent, including patent authorization of a variety of nucleosides such as 1-methylpseudouridine (m1ψ）. Therefore, patent barrier is a common problem for all newcomers, especially for Chinese enterprises which started late.
Synthesis and production of RNA
RNA synthesis is mainly divided into chemical synthesis and biological synthesis. The advantage of chemical synthesis is that it can be produced stably and on a large scale, but the cost is high, and it is generally only suitable for short stranded RNA such as siRNA and ASO. The long stranded mRNA is often the main factor for its synthesis difficulty because of its molecular length and secondary structure. Therefore, when long RNA with more than 40 bases is needed to be synthesized, biosynthesis method is required. At present, it is relatively mature to use RNA transcriptase as template for in vitro transcription production. In addition, when producing mRNAs, it should also be taken into account that mature mRNAs in eukaryotes all have 5 'CAP and 3' Poly (A) tail (Figure 5). The process stability, scale-up, separation and purification, as well as the assembly and quality control of the delivery vector of the biosynthesis method are the key points of RNA drug GMP production.
