Translation of RNA Medicines from Design to Clinic

During development of pharmaceutically-applicable mRNA therapeutics, synthesis, optimization, and formulation must be taken into consideration.

RNA therapeutics comprise a rapidly expanding category of drugs that will speed clinical solutions, actualize personalized medicine, and make the term “undruggable” obsolete, according to a March 2021 Frontiers in Bioengineering and Biotechnology review. Different classes of RNA-based therapeutics include antisense oligonucleotides, aptamers, small interfering RNAs, microRNAs, and messenger RNA (mRNA).1 The widespread use of mRNA vaccines for COVID-19 marked 2020 as a breakout year for mRNA technology platforms.

mRNA therapeutics are applicable to cancer immunotherapies, infectious diseases, and other indications that require protein replacement therapy or antibodies. After cellular uptake, the therapeutic molecule translates genetic information into protein, an antigen, antibody, or other therapeutic protein. During development of the therapeutics several aspects must be taken into consideration–synthesis, optimization, and formulation--to deliver the cargo to the target site.

Although currently best known for their work with Pfizer on the BNT162b2 mRNA vaccine for SARS-CoV-2, according to Heinrich Haas, PhD, Vice President RNA Formulation and Drug Delivery, BioNTech, one of the company’s key areas of development is cancer immunotherapies.

To achieve this particular type of vaccination an mRNA that codes for a tumor-associated antigen (TAA) is transferred into dendritic cells, which have a key function in tumor therapy. Inside the cells the mRNA is translated and stimulates a cascade of immune responses specific to the TAA in addition to a systemic immune response.

R&D Formulation Development

The coding region of mRNA contains the information required to synthesize a protein. Other important elements include the 5’ and 3’ UTR regions, a 5’ cap and a 3’ polyA tail. Synergistically, the elements make a pharmaceutically-applicable molecule and need to be optimized individually to facilitate increased intercellular half-life, translation, and MHC presentation.

“The good thing is that mRNA can be manufactured in a cell-free in vitro transcription (IVT) enzymatic reaction,” said Haas. A DNA plasmid containing the DNA template is linearized, and RNA polymerase, nucleotide triphosphates (NTPs), modified UTP substrates, inorganic pyrophosphatase, ribonuclease inhibitors, and the CAP structure are added. Once the mRNA is transcribed the DNA is digested.

Then, importantly, the RNA needs to be formulated for delivery. “Here the challenges are a bit higher than for small molecules as many functions need to be fulfilled by the formulation,” said Haas. The formulation has to control serum interactions and protect the RNA from degradation in circulation, control biodistribution and deliver the RNA to the target site, enable and improve cellular uptake and translation at the target site, and, finally, ensure protein expression and release into circulation.

Most lipid nanoparticle formulations consist of four components: the ionizable lipid (e.g. DODMA, Dlin-MC3-DMA), cholesterol, helper lipid (DSPC, DOPE) and grafted lipid (PEG-lipid). The ionizable lipid is positively charged at low pH and neutral at higher pH. Thee ionizable lipids bind the RNA in the positively charged state through electrostatic interactions and their pKa values are tailored to favor release from the endosome following cellular uptake.

To manufacture the nanoparticles, the RNA dissolved in buffer, and lipids dissolved in ethanol are rapidly mixed at precise ratios, to induce self-assembly into particles with the desired properties. Further process steps, comprising buffer adjustment, addition of stabilizers, concentration adjustment and sterile filtration may be involved to obtain the end product. After determining various physico-chemically characteristics for quality control, the biological activity is measured in cell or animal models. The correlation between particle characteristics and biology is used as a basis for system optimization.

In lipid-based delivery systems some general features are considered to be related to activity. One is the capacity to undergo transitions between phase states, e.g., hexagonal, inverse hexagonal, or lamellar. Lipids, where the transition between lamellar and hexagonal phases is facilitated are considered to be particularly helpful for uptake or release across bilayer membranes. Another important aspect is the pKa value of ionizable lipids, which is decisive for pH dependent changes of the particle characteristics in circulation and during endosomal processing after uptake.

Two examples of lipid-based delivery systems are lipoplexes (LPX) and lipid nanoparticles (LNPs). Lipoplexes are made by mixing preformed cationic liposomes with RNA to form a lamellar-like lipoplex stack. In contrast, LNPs are formed by mixing lipids (an ionizable lipid, a helper lipid, and a grafted lipid) in ethanolic solution with RNA in an acidic buffer to complex the RNA and form the LNP in one step. Permanently cationic lipoplexes are thought to be more cytotoxic than LNPs, which are largely uncharged at physiological pH, which reduces serum interactions and thus reduces one main cause of potential toxicity.

Alnylam’s Onpattro® for the treatment of polyneuropathy caused by hereditary ATTR (hATTR) amyloidosis and the Moderna mRNA-1273 and Pfizer/BioNTech BNT162b2 SARS-CoV-2 vaccines all use ionizable cationic lipids formed by precipitation in ethanol.

The two systems differ in their internal structure, which can be demonstrated by small angle x-ray scattering (SAXS) where the sample is irradiated with an x-ray beam and the scattered light collected. Scattering profiles are displayed as a function of the angle or the momentum transfer. “From this profile you can determine if a Bragg peak is present along with its height and area to get quantitative information on the internal organization of your particle to assist system specification and optimization,” explained Haas.

The LPX structure is characterized by a narrow and well-defined Bragg peak whereas the LNP peak is broader, indicating a lower degree of internal organization.

A further difference between LNPs and LPXs is, that typically in the former ionizable lipids are used, while lipoplexes typically comprise permanently charged cationic lipids. “This is something that might deserve a closer evaluation,” cautioned Haas. “For example, homologous lipids may be considered, which are identical in structure except for one nitrogen atom in the head group which can be permanently charged or ionizable. If they are ionizable the charge can be switched by adjusting the pH. This impacts packing.”

“SAXS provides accurate information on the pH dependent structural changes inside nanoparticles comprising either permanently charged or ionizable lipid. This allows to determine a structural equivalent to pKa values with high resolution and purpose,” said Haas. “By variation of mixing ratios between lipids and RNA these values can be accurately fine-tuned. This can be helpful along with chemical synthesis of a lipid library. You can tailor the library based on composition and other aspects of the lipoplex system, which allows easier and more accurate adjustment of parameters to optimize endosomal processing.”

There is also another straightforward approach to modulate particle characteristics and activity, added Haas. “If you mix RNA and liposomes in different ratios, you can find conditions where you obtain colloidally-stable particles either with an excess of negative or positive charge,” he said. Animal experiment using luciferase as a reporter gene demonstrated that lipoplexes formed at an excess of positive charge (liposomes) resulted in high transfection efficacy in the lung, whereas those formed with an excess of negative charge (RNA) showed high expression in the spleen. “Just by changing that one physical parameter you can change organ efficacy,” emphasized Haas. Such insight allowed initiation of various clinical studies in the field of cancer immunotherapy.

Thorough characterization of the particles by SAXS and other measurements were here helpful to accurately determine critical quality attributes as a basis for manufacturing of the drug products.

Clinical Product Development

“After you have identified your formulation the development work begins. Now you have to refine all of the parameters and justify them,” said Haas. He recommends review of the FDA Liposome Drug Products guidance that details the requirements for characterization and data generation.

“For liposome manufacturing, when it comes to process development, you start with something adapted for lab-scale experiments,” said Haas. “Since this process may change as it is scaled up into a GMP environment you need to control, detail, and justify all process parameters with more accuracy and care than what was done for the formulation experiments.”

In order to follow up with the requirements in the guidance’s, extensive characterization and thorough understanding of structural and functional coherencies inside the product in development is mandatory. Here, SAXS and other measurements for extended characterization can be useful to generate a sound data base. By performing many measurements for all parameters, the process conditions needed to manufacture and store a pharmaceutical product can be defined.


RNA is labile, highly charged, and complex. As a result, pharmaceutical development becomes a greater challenge than that for small molecules. CMC (chemistry, manufacturing, and controls) aspects are extremely important for successful development of complex nanoparticles that consist of RNA and vehicular molecules. Formulation is of key importance because it may affect the broader qualities.

A thorough understanding of the coherencies inside the delivery systems, such as structure and function as derived from advanced characterization, including SAXS analysis, assists performance of rational formulation development. This also helps define the critical quality attributes required to specify the product that will be important for successful translation of RNA into clinical development.

Listen to Dr. Haas’ full keynote presentation from Precision NanoSystems’ Virtual Symposium: Genetic Medicine from Concept to Clinic.


1. Damase TR, Sukhovershin R, Boada C, Taraballi F, Pettigrew RI., Cooke JP. The Limitless Future of RNA Therapeutics. Frontiers in Bioengineering and Biotechnology 9, 161 (2021).

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