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Understanding mRNA technology as platform for biopharma

The revolutionary potential of mRNA as a platform for biopharma

Synthetic messenger RNA (mRNA) is a new platform technology for biopharma companies with attractive opportunities in therapeutics, gene therapy or vaccine applications.1,2,3  The straightforward adaptation of mRNA to specific and even personalized targets promises to usher in breakthroughs in precision medicine. The in vitro synthesis of mRNA using bacteriophage RNA polymerases like T7 or SP6 4  obviates the need to culture cells and extract proteins from a crude cell mass through elaborate purification steps. This simplified, cell-free manufacturing process is scalable and easily adopted within a short time.5

Advantages of mRNA technology over therapeutic proteins and DNA transfections

  • Therapeutic protein production requires large-scale cell culture and laborious purification protocols specific to each protein.

  • Misfolded or improperly modified proteins can cause immunogenicity and adverse effects.

  • Process complexity of protein production often requires lengthy development cycles and complicates GMP compliance.

  • Transfection of plasmid DNA is less efficient in quiescent cells, and the need for a specific promoter and crossing of the nuclear membrane complicates transfection method.6
  • mRNA is produced in a standardized one-vessel reaction, using the same protocol regardless of coding sequence.7

  • With mRNA technology synthetic mRNA can be engineered to resemble  mRNA molecules as they occur naturally in the cytoplasm of cells and to transiently deliver the proteins of interest directly in the cell.8
  • mRNA technology allows simplified GMP production and reduced development costs.9

  • The mRNA transfection process is simpler and more efficient: with a smaller construct than plasmid DNA, mRNA is directly delivered to and expressed in the cytoplasm and never crosses the nuclear membrane.

mRNA construct design

Today’s knowledge and technology enable the tailored production of mRNA to minimize immunogenicity while tuning expression to the needs and form of a treatment. The commonly used linear and mature mRNA construct usually bears the following structural elements, which are optimized during sequence design:10,11

mrna construct

5’CAP: The cap region at the 5’ end of the sequence is essential for mRNA maturation. It allows ribosome to recognize and efficiently translate the sequence12 and protects the molecule from nuclease digestion.

5´UTR/3´UTR: The untranslated regions (UTRs) upstream and downstream from the mRNA coding region impact translation efficiency, localization and stability. They can be adapted in a way to improve protein expression. 13,14

CDS: The open reading frame or coding sequence (CDS) region contains the gene to be expressed. Codon optimization and modification of nucleotides can contribute to translation efficiency. For example, optimized guanine and cytosine content can have a significant impact on translation.15

Poly(A) tail: The poly(A) tail at the 3’ end of the mRNA is crucial for protein translation 11  and mRNA stability by preventing digestion via 3’ exonuclease. There is also a dependency of translational efficiency and stability on the length of the poly(A) tail.16

Further design features include the addition of modified nucleic acids during transcription. mRNA can cause immunogenicity when delivered to a cell exogenously. However, the incorporation of naturally occurring, chemically modified nucleosides like pseudouridine and 1-methylpseudouridine prevents activation of the innate immune system. Nucleoside-modified mRNA is also translated more efficiently by ribosome than unmodified mRNA.17

Newer developments and applications in biopharma incorporate a growing range of RNA constructs18, including self-amplifying RNAs (saRNAs)19, circular RNAs and RNAs bearing an internal ribosomal entry site or IRES.20  

The first step into the usage of the “novel” saRNA technology was already taken in 1981 when the first cloning of an infective full-length genome of an animal RNA virus was accomplished21. Containing a viral replicon from positive-strand RNA viruses (like picornaviruses or flaviviruses22) synthetic saRNAs are able to self-amplify within host cells23. Synthetic saRNA has therefore the potential to induce higher levels of protein production and immunogenicity relative to the injected dose compared to conventional mRNA constructs24. Synthetic saRNA can be prepared by in vitro transcription25 (as conventional mRNA constructs) but are usually larger (9–12 kb) than non-amplifying mRNAs26. Today the usage of self-amplifying RNA is a promising technology in various therapeutic applications and vaccines27.

Process steps of large-scale mRNA production

Illustration

1. Template DNA design and preparation:

The DNA template for mRNA in vitro transcription (IVT) can be a plasmid construct, a cDNA generated from RNA, an oligonucleotide or a PCR product. The template contains the sequence of interest and a double-stranded promoter region for RNA polymerase (e.g., T7 or SP6) to bind and initiate RNA synthesis. Plasmid constructs are manufactured in-house or purchased from a third party, and the desired DNA sequence is inserted by cloning.

Illustration

2. DNA template linearization:

 

A restriction enzyme like XbaI is used to linearize the template pDNA for IVT. The enzymatic reaction is terminated with a suitable reagent like proteinase K. Linearization increases access of polymerases to the target sequence and ensures that the generated transcripts have a defined length and sequence. Alternatively, the linearized template can also be produced via PCR.

 

mrna in vitro transcription reaction

3. In vitro transcription reaction:

RNA is synthesized in vitro from the linearized plasmid template28 when T7, SP6 or T3 RNA polymerase binds to the respective promoter region adjacent to the transcript sequence. The RNA polymerase uses ribonucleotides (NTPs) to synthesize the RNA. Modified ribonucleotides like pseudouridine and 1-methylpseudouridine are often added to reduce immunogenicity and increase subsequent translation efficiency. 29 Transitioning to large-scale production may require adapting transcription systems to improve the yield of synthetic RNA. For example, ribonuclease inhibitor and inorganic pyrophosphatase significantly improve the quality and yield of products.30

co-or posttranscriptional modification mRNA

4. Co- or posttranscriptional modification:

A mature eukaryotic mRNA contains a 5´cap structure and a 3’ poly(A) tail. They are commonly added during IVT or enzymatically after transcription using capping enzymes and polyA polymerase.31,32,33

mrna purification

5. mRNA purification:

Clinical-grade mRNA for therapeutic applications must be free of impurities from upstream processes. The plasmid DNA backbone is enzymatically digested by DNase I. Enzymes and protein components can be removed using proteinase K. Chromatographic methods are used to remove free nucleotides, residual enzymes, endotoxins, double-stranded RNA or other. Finally, polyadenylated RNAs can be enriched using oligodT column purification.

 

Illustration

6. Final formulation:

Delivery of the mRNA requires cellular uptake followed by endosomal escape of the nucleic acid into the cytosol of targeted cells. At the same time, the mRNA must be protected during delivery from degradation by exosomal RNases. One frequently used delivery system is based on lipid nanoparticles (LNPs).34

Contaminants in mRNA manufacturing


As an easily scalable platform technology for biopharmaceutical manufacturing, mRNA production must be GMP-compliant. This means manufacturing with batch-to-batch consistency and stringent quality control to yield safe products ready for clinical trials and subsequent commercialization. 35,36  Therefore, the raw materials used in mRNA production, including IVT, should be chosen carefully. Inadequate reagents may carry or generate contaminants that lead to problems downstream, impact efficacy and can cause side effects in patients. 


Common contaminants and their effect on mRNA manufacturing
  • Double-stranded RNA contamination:
    RNA polymerases transcribe RNA with high fidelity from a DNA template. However, double-stranded RNA (dsRNA) is generated to varying extents as a by-product during transcription initiation. These by-products can cause downstream issues (immunogenicity) like robust type I interferon production that inhibits the translation and the degradation of cellular mRNA and ribosomal RNA, respectively.37 Contaminating dsRNA is efficiently removed from the mRNA product by chromatographic methods.38

  • DNA contamination:
    DNA transcript carryover E. coli DNA from plasmid preparation decreases the performance and quality of the mRNA product. DNA can be removed through digestion with DNase I and the possibility of residual E. coli DNA carryover can be excluded by testing with the qPCR-based Residual DNA E. coli Kit.

  • RNA instability and RNAse contamination:
    RNases can carryover from the plasmid purification process or be introduced by improper handling and contaminated raw materials. RNases are ubiquitous in the environment. Their presence in production, however, is excluded by working with highly purified raw materials and their activity is blocked with RNAse Inhibitors.39 Cleavage of RNA also happens at a pH exceeding 6 or in the presence of metal ions40 like Mg2+, which are introduced during IVT or are potential contaminants from raw materials. Proper purification and the right choice of raw materials are the key to produce stable and translationally active mRNA.

Bridging the gap between laboratory and industrial scale

Expansion in production scale of mRNA from research and clinical trials to commercial manufacturing entails significant differences in logistics, technical feasibility and requirements. Commercial all-in-one kits often used in the research space are generally not an option in industrial settings.41

For industrial scale and biopharma purposes, the overall protocol and construct design must be optimized, and processes must be adapted to GMP conditions. The narrow product consistency, quality, and safety tolerances of GMP pharmaceutical manufacturing requires high-quality sources of raw materials for each process step, as well as the right purification methods and materials to control immunogenicity and stability.

mRNA as platform technology for a broad range of applications - from vaccines to cellular therapies

mRNA applications
Vaccines
 (e.g. for SARS-CoV2)

Personalized medicine
 (e.g. cancer immunotherapy) 

Cellular therapies

 

mrna cell

mRNA applications

Protein replacement

Gene editing
(e.g. CRISPR CAS)

Rare diseases
(e.g. enzyme replacement)

Regulatory disclaimers are listed on the respective product pages.

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