Messenger RNA (mRNA) plays a fundamental and integral role in every living cell: mRNAs are carriers of genetic information and blueprints of proteins. mRNA is a nontoxic molecule that allows transient protein expression in virtually all cell types and has many advantages especially over DNA for gene transfer and expression of target molecules. mRNA doesn’t require nuclear localization or transcription and is therefore a very safe biomolecule.1 Additionally, mRNA provides enormous flexibility with respect to production and application because any protein with a known sequence can be encoded and expressed.
That said, everybody who has worked in the laboratory with mRNA will remember the cumbersome process of isolating and, more importantly, maintaining stable mRNA for further analysis. The human body secretes RNases, enzymes that are responsible for the degradation of single-stranded RNA, in fluids such as tears, saliva, mucus, and perspiration to defend against invading microorganisms by secreting these enzymes. RNases can be found basically everywhere.
Therefore, for many years it was generally accepted that, despite their advantages, mRNAs were too unstable and too difficult to manipulate to be efficiently used as therapeutics. Nevertheless, researchers overcame these limitations and suddenly it seemed (at least in principle) feasible to express therapeutically relevant proteins in vivo. Today, mRNAs are investigated as a novel class of therapeutic in areas such as cancer vaccines, as prophylactic vaccinations for infectious diseases, and as a source of therapeutic gene products and protein replacement therapies.
Cancer Immunotherapy and Prophylactic Vaccines
The ideal cancer vaccine should activate the adaptive and innate immune system and induce a broad, potent, and long lasting immune response that would include balanced humoral as well as T cell-mediated responses. The direct vaccination with mRNA molecules encoding tumor-associated antigens is an elegant approach to let the patient’s body produce its own vaccine.
It is known that the human immune system recognizes bacterial DNA and viral RNA as "foreign" nucleic acids. Foreign DNA and RNA stimulate the mammalian innate immune system, the nonspecific immune system, through activation of Toll-like receptors (TLRs). Double-stranded RNA (dsRNA), a common viral intermediate, activates TLR3, whereas synthetic single-stranded RNA (ssRNA) and virus-related RNA activate human TLR7 and TLR8.2
A study published more than 10 years ago showed that direct injection of naked, unprotected mRNA induces specific cytotoxic T lymphocytes and antibodies suggesting that mRNAs can provide an attractive alternative to peptide and DNA-based vaccines in cancer immunotherapy. These discoveries later laid the foundation for the first mRNA-based immunotherapy.3
However, the use of naked RNA for vaccination in a clinical setting was not feasible. Additionally, naked mRNA molecules were not efficient in inducing maturation of antigen-presenting cells, but mRNA protected against RNase-mediated degradation by association with a cationic peptide or through a phosphorothioate backbone are very potent immunostimulating molecules.4 Conversely, the complexation of the mRNA, required for strong immune-stimulating activity, can inhibit the antigen translation. A two-component mRNA immunotherapeutic that contains free and protamine-complexed mRNA induces balanced adaptive immune responses and provides humoral and T cell mediated immunity; it supports both antigen expression and TLR7-mediated immune stimulation that is entirely HLA independent and is designed to be self-adjuvanting, a property which peptide- and protein-based vaccines lack.5
Early clinical studies in patients with castration-resistant prostate cancer and lung cancer have shown the favorable safety profile of this approach. It is noteworthy that mRNA-based vaccines don’t require any vehicle and are injected intradermally. Data presented earlier have shown that the specific mRNA-based immunotherapies in both trials were doing what they were supposed to do: Antigen-specific T-cells were detected in the majority of patients, independent of their HLA-background. Additionally, the frequency of antigen-unspecific B-cells was also increased in the majority of patients.6
The concept of mRNA-based vaccines is also under investigation for potential use as prophylactic vaccination for infectious diseases, and a recent proof-of-concept study in animal models for influenza was published in Nature Biotechnology.7
Other Therapeutic Approaches, Outlook
mRNA’s capacity to elicit innate immune responses is an important limitation to its in vivo application as a source of therapeutic gene products. Katalin Karikó, Ph.D., and her colleagues have shown that dendritic cells and TLR-expressing cells are potently activated by bacterial and mitochondrial RNA, but not by mammalian total RNA, which is abundant in modified nucleosides.8 The authors concluded that the innate immune system may therefore detect RNA lacking nucleoside modification. Although the incorporation of modified nucleosides both reduced innate immune activation and increased translation of mRNA, the additional removal of double-stranded RNA by high-performance liquid chromatography (HPLC) may avoid the residual induction of type I interferons (IFNs) and proinflammatory cytokines.9
Currently, chemically modified mRNAs that elude the body’s innate immune response are in preclinical development for the in vivo production of both intracellular proteins and secreted proteins.
mRNA has been recognized as a resourceful, nontoxic molecule that may transform the treatment of many human diseases and disorders. Researchers have overcome the hurdles how to handle mRNA, and today, speed, efficiency, and lower cost of the production may open up a new era of mRNA-based therapies.