With the first mRNA-based therapeutics and prophylactic vaccines now in clinical testing, this new class of biologics is poised to transform drug discovery and spearhead the transition to an information-guided drug delivery paradigm.

Leveraging the wealth of existing and emerging biological, genetic, biochemical and pathway-related information related to health and disease, a new generation of drugs including enzymes, antibodies and immunostimulatory antigens will be delivered by administering mRNA – in the form of a ‘healthy message’ – thereby enabling the body to produce its own medicine. Any protein can be encoded in an mRNA molecule.

Though not a new concept, mRNA therapies have had to overcome both theoretical and practical hurdles, and the underlying RNA technology has undergone many years of development and refinement before the first molecules achieved proof-of-principle and completed the first human studies demonstrating their safety, tolerability and early evidence of targeted physiologic activity. mRNA drugs can fill critical gaps not met by traditional small molecule drugs, available biological treatments and emerging gene therapies. In addition, they bring advantages related to ease, cost and scalability of manufacturing. These benefits, combined with the ability to create an mRNA for virtually any protein or antibody, and to package multiple molecules into a single therapeutic, have garnered substantial attention from investors and the pharmaceutical industry.

Pioneering RNA medicines
Companies focused on research and clinical development of mRNA therapies are growing in size and number. As the competitive landscape expands, the race to bring the first mRNA product to market intensifies. CureVac, founded in 2000, as a spin-off from University Tübingen, has pioneered the field of mRNA therapies. It has built a drug technology and manufacturing platform based on the scientific research of its co-founder, Ingmar Hoerr, and has established an extensive knowledge base on mRNA therapy, an intellectual property portfolio comprising more than 60 patent families covering broad aspects of the technology and specific indications and expertise in GMP production and scale-up of RNA medicines. In 2008, the company initiated a first-in-human study with an mRNA product and currently has four product candidates in the clinic, with the most advanced being a multi-antigen immunotherapeutic to treat prostate cancer that is in a Phase IIb trial.

Cambridge, MA-based Moderna Therapeutics has attracted substantial investments and formed multiple collaborations to advance its messenger RNA Therapeutics™ platform. Moderna generates mRNA molecules containing modified nucleotides and has product candidates in preclinical development. BioNTech’s (Mainz, Germany) individualised cancer vaccines first entered the clinic in 2013. Ethris, located in Planegg, Germany, has tested its SNIM® RNA technology in preclinical studies and has partnered with Shire in the development of RNA therapies as an alternative to recombinant proteins for treating rare diseases and for regenerative medicine applications.

Information-guided therapeutics
Historically, pharmaceutical companies have chemically synthesised small molecule drugs consisting of complex building blocks through a complicated series of synthetic steps. Time- and cost-intensive screening of large compound collections has been needed to discover a core molecular structure with therapeutic efficacy in a single indication. Too often, however, less than optimal specificity and off-target and sometimes serious side-effects have resulted in costly latestage drug failures.

The 1990s saw the first attempts at DNA-based therapies, with the emergence of antisense oligonucleotide- based drugs containing chemically modified backbones and bases and early versions of gene therapy, intended to deliver the genetic code for a missing or dysfunctional protein. Initial efforts to design and deliver safe and effective gene therapies suffered a series of setbacks, and only recently has the field begun to regain its footing and once again advance therapeutic candidates through the pipeline.

In the 1980s and 1990s, recombinant proteins and antibodies began to fill the pipelines of biopharmaceutical companies, and a new generation of protein- and peptide-based molecules passed regulatory review and came to the market for a variety of indications. During this time, the market share of biopharmaceuticals rapidly increased and their scope of applications broadened. Innovations in the tools, techniques, processes, equipment and upstream and downstream strategies needed to manufacture and scale production of biologicals generally kept pace with advances in product design and development as bottlenecks continually shifted. But even as the biopharmaceuticals market grew, significant challenges related to drug delivery, costs and other factors limited the promise of protein drugs and gene therapies for applications such as enzyme replacement.

Yet RNA did not begin to gain recognition as a potential therapeutic alternative for many years. The concept of mRNA molecules as drugs simply fell through the cracks, mainly due to one misconception: the long-held belief that RNA molecules are inherently unstable and highly susceptible to enzymatic degradation. For enzyme replacement therapy, the dogma was that you needed to deliver either the enzyme or the gene encoding the enzyme. But that view has been changing, with the growing realisation that the most sensible, effective and efficient route to enzyme replacement therapy might just be an mRNA molecule.

It is costly and can take many years to develop a new therapeutic protein or antibody drug on the basis of current state of the art recombinant protein fermentation. In contrast, mRNA molecules can be designed and manipulated quite easily and quickly, and synthesised and mass-produced relatively cost-effectively. Messenger RNA was designed by nature to transmit information – to transfer the instructions for making a protein encoded in a gene to the ribosome, where the codons are read and the appropriate amino acids added on to a growing peptide chain.

In the emerging era of information-guided drug development, designing mRNA therapies is akin to software development. The aim is to compose the necessary medical message on synthetically produced mRNA molecules using specialised technology for engineering, optimising and formulating the mRNA therapy. This technology is now available and ready to be combined with the information about biochemical pathways, gene function and genotype-phenotype relationships needed to guide the design of mRNA pharmaceuticals.

Engineering tomorrow’s RNA drugs
Even in the early years of mRNA technology development, CureVac’s scientific founders rejected the contention that mRNA is unstable per se and were convinced that unmodified, natural mRNA could be sufficiently stable for use as a therapeutic agent. The use of naturally occurring nucleotides would make it easier to design and produce commercial mRNAs, and developing a ‘natural’ product would offer potential advantages related to safety, lower regulatory hurdles and faster time to market. Molecular engineering strategies can be employed to optimise the nucleotide sequence, mRNA formulation and delivery characteristics and protein design. An alternative approach uses chemically modified nucleotides to enhance mRNA stability. In addition to concerns about stability, other factors have also kept mRNA out of the drug discovery spotlight, including the absence of other types of RNA-based therapeutics, comparisons to DNAbased drugs, lack of support for immunotherapeutic approaches for treating cancer and other diseases, and the still-developing information base needed to drive mRNA drug design and optimisation. With the discovery of RNA species such as microRNAs and mechanisms such as RNA interference (RNAi), and the development and testing of small interfering RNA (siRNA)-based therapeutic agents, the research community has been able to learn from the work done on siRNA delivery techniques.

In the area of oncology, in particular, the tremendous amount of attention now focused on immunotherapeutic approaches and the development of therapeutic vaccines designed to activate the immune system to recognise and destroy tumour cells, is driving interest in the immunostimulatory potential of mRNA-based antigen delivery. Cancer immunotherapy represents an exciting opportunity with a large unmet medical need for which mRNA presents a novel solution. First-in-human testing of an mRNA drug evaluated the immunostimulatory potential of a therapeutic vaccine to treat prostate cancer that delivered four different antigens1. Following multiple vaccinations, 79% of patients had antigen-specific immune responses, and 58% of the immune responses were directed against multiple antigens. mRNA-based antigen delivery to elicit an immune reaction has important implications not only for therapeutic vaccine development, but also for producing prophylactic vaccines against infectious disease targets, and as adjuvants to boost immune reactions. Phase I clinical trials of products designed for these applications are under way. These include a first-in-human study of a prophylactic rabies vaccine. Perhaps the most obvious and compelling indications for mRNA-based medicines are therapeutic

protein delivery or enzyme replacement, an application
area more broadly known as molecular
therapy. The aim is to provide cells and tissues the
information they need to produce antibodies or
missing or flawed proteins without stimulating an
immune response or causing unwanted side-effects.
In essence, creating an in vivo protein factory that
enables the body to heal itself.
Recently published data support the ability of
sequence-optimised chemically unmodified mRNA
to achieve sufficient protein expression and avoid
activation of the innate immune system2. In a
model system based on erythropoietin (epo)-driven
production of red blood cells, administration of
engineered epo-mRNA encapsulated in lipid
nanoparticles was associated with substantial
increases in serum epo levels in animals ranging
from mice to pigs to cynomolgus monkeys.
Following a single intravenous dose of epo-mRNA,
reticulocyte counts and hematocrits also rose substantially,
indicating a physiological effect of treatment.
The absence of any remarkable immunostimulatory
reactions validated the potential for multiple
dosing. A comparison of the results obtained
with unmodified mRNA versus mRNA containing
chemically modified nucleosides showed that nonmodified
mRNA gave rise to higher epo yields,
longer lasting protein expression, and significantly
higher numbers of reticulocytes.
Commercialising RNA therapies
Advancing RNA therapies into and through clinical
testing and on to the market will require meeting
all the usual regulatory requirements, including
demonstrating efficacy in large-scale pivotal trials.
However, the challenges that companies commonly
face when developing and scaling up production
of a new class of therapeutics have largely already
been met for unmodified mRNA molecules. The
engineering, production and purification processes
have been refined and optimised.
The composition and structure of these molecules
present unique advantages that translate into
highly flexible, scalable and efficient manufacturing
processes. The mRNA molecule forms off of a
DNA template, mimicking what occurs in nature,
with RNA polymerase carrying out the enzymatic
synthesis. Upstream engineering can optimise the
nucleotide sequence by adapting codon usage and
selecting the most appropriate regulatory
sequences, such as 5’ and 3’ untranslated regions,
and downstream purification selects for molecules
of the desired quality and includes DNase treatment
to remove the DNA template.
Compared to the production of other types of
biologicals, each mRNA product does not need a
dedicated facility. With the use of disposable processing
technology, changing from producing one
mRNA to another only requires switching the
DNA template. This can take no longer than 24
hours. Multiple products can be manufactured in
parallel in one facility.
For prophylactic vaccines, and especially when
used in pandemic settings and in the developing
world, speed and cost of manufacturing are significant
factors. The timeline to design and produce

protein delivery or enzyme replacement, an application area more broadly known as molecular therapy. The aim is to provide cells and tissues the information they need to produce antibodies or missing or flawed proteins without stimulating an immune response or causing unwanted side-effects. In essence, creating an in vivo protein factory that enables the body to heal itself.

Recently published data support the ability of sequence-optimised chemically unmodified mRNA to achieve sufficient protein expression and avoid activation of the innate immune system2. In a model system based on erythropoietin (epo)-driven production of red blood cells, administration of engineered epo-mRNA encapsulated in lipid nanoparticles was associated with substantial increases in serum epo levels in animals ranging from mice to pigs to cynomolgus monkeys. Following a single intravenous dose of epo-mRNA, reticulocyte counts and hematocrits also rose substantially, indicating a physiological effect of treatment. The absence of any remarkable immunostimulatory reactions validated the potential for multiple dosing. A comparison of the results obtained with unmodified mRNA versus mRNA containing chemically modified nucleosides showed that nonmodified mRNA gave rise to higher epo yields, longer lasting protein expression, and significantly higher numbers of reticulocytes.

Commercialising RNA therapies
Advancing RNA therapies into and through clinical testing and on to the market will require meeting all the usual regulatory requirements, including demonstrating efficacy in large-scale pivotal trials. However, the challenges that companies commonly face when developing and scaling up production of a new class of therapeutics have largely already been met for unmodified mRNA molecules. The engineering, production and purification processes have been refined and optimised.

The composition and structure of these molecules present unique advantages that translate into highly flexible, scalable and efficient manufacturing processes. The mRNA molecule forms off of a DNA template, mimicking what occurs in nature, with RNA polymerase carrying out the enzymatic synthesis. Upstream engineering can optimise the nucleotide sequence by adapting codon usage and selecting the most appropriate regulatory sequences, such as 5’ and 3’ untranslated regions, and downstream purification selects for molecules of the desired quality and includes DNase treatment to remove the DNA template.

Compared to the production of other types of biologicals, each mRNA product does not need a dedicated facility. With the use of disposable processing technology, changing from producing one mRNA to another only requires switching the DNA template. This can take no longer than 24 hours. Multiple products can be manufactured in parallel in one facility.

For prophylactic vaccines, and especially when used in pandemic settings and in the developing world, speed and cost of manufacturing are significant factors. The timeline to design and produce an mRNA-based vaccine against an infectious disease target is relatively rapid. A cost of no more than $1/dose is feasible, a target set by the Bill and Melinda Gates Foundation for vaccine developers to quality for research and development funding.

Thermostability and product activity under various storage and transport conditions are also important considerations. Extensive testing of mRNA storage has shown good stability in liquid form both when the product is refrigerated or frozen. In lyophilised form, an mRNA vaccine can remain stable for up to 24 months at ambient temperature, and for shorter periods as storage temperatures rise. Furthermore, these vaccines can withstand some amount of multiple freeze-thaw cycles and retain their activity.

A bright future
With the first mRNA therapies in the clinic and proving their safety, physiologic activity and potential for efficacy in patients, the path forward is bright and will expand to include more products in a range of indications. Leading the way will likely be both therapeutic and prophylactic vaccines intended to activate the immune system, mRNA therapies designed to deliver antibodies, enzymes and hormones, for example, without eliciting an immune reaction, and RNA molecules that act as adjuvants to amplify the response to immunotherapies and preventive vaccines. Each new product will build on knowledge gained from ongoing research and previous trials, improving delivery and efficacy and empowering the body to generate its own medicine.

Acknowledgements
This work was partially supported by grants from the Federal Ministry for Education and Research (BMBF), Germany (KMU-innovativ, grant no 02PK2178).


Dr Ingmar Hoerr is Chief Executive Office and Co-Founder of CureVac. He co-founded CureVac in 2000 together with Florian von der Mülbe and other colleagues in Tübingen. His entrepreneurship was motivated by a surprising discovery during his doctoral research at the University of Tübingen. Experiments conducted for this research showed that the mRNA molecule class is capable of generating a strong specific immune response, contrary to what had previously been believed. From this discovery, Ingmar and Florian founded CureVac, now a global leader in the research and development of mRNA-based drugs.

References
1
Kübler, H, Scheel, B, Gnad- Vogt, U et al. Self-adjuvanted mRNA vaccination in advanced prostate cancer patients: A first-in-man phase I/IIa study. J Immunother Cancer 2015; doi:10.1186/s40425-015-0068-y.

2 Thess, A, Grund, S, Mui, BL et al. Sequence-engineered mRNA without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals. Molec Ther 2015; doi:10.1038/mt.2015.103.