mRNA: A Novel Avenue to Antibody Therapy?

First attempts to use exogenous mRNA for protein expression in vivo were made more than 25 years ago. However, widespread appreciation of in vitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such in vivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient in vivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.

mRNA as novel technology for passive immunotherapy.

While active immunization elicits a lasting immune response by the body, passive immunotherapy transiently equips the body with exogenously generated immunological effectors in the form of either target-specific antibodies or lymphocytes functionalized with target-specific receptors. In either case, administration or expression of recombinant proteins plays a fundamental role. mRNA prepared by in vitro transcription (IVT) is increasingly appreciated as a drug substance for delivery of recombinant proteins. With its biological role as transient carrier of genetic information translated into protein in the cytoplasm, therapeutic application of mRNA combines several advantages. For example, compared to transfected DNA, mRNA harbors inherent safety features. It is not associated with the risk of inducing genomic changes and potential adverse effects are only temporary due to its transient nature. Compared to the administration of recombinant proteins produced in bioreactors, mRNA allows supplying proteins that are difficult to manufacture and offers extended pharmacokinetics for short-lived proteins. Based on great progress in understanding and manipulating mRNA properties, efficacy data in various models have now demonstrated that IVT mRNA constitutes a potent and flexible platform technology. Starting with an introduction into passive immunotherapy, this review summarizes the current status of IVT mRNA technology and its application to such immunological interventions.

mRNA mediates passive vaccination against infectious agents, toxins, and tumors.

The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA-lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long-lasting serum antibody titers in vivo, thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA-mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization.

A novel, disruptive vaccination technology: self-adjuvanted RNActive(®) vaccines.

Nucleotide based vaccines represent an enticing, novel approach to vaccination. We have developed a novel immunization technology, RNActive(®) vaccines, that have two important characteristics: mRNA molecules are used whose protein expression capacity has been enhanced by 4 to 5 orders of magnitude by modifications of the nucleotide sequence with the naturally occurring nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine) that do not affect the primary amino acid sequence. Second, they are complexed with protamine and thus activate the immune system by involvement of toll-like receptor (TLR) 7. Essentially, this bestows self-adjuvant activity on RNActive(®) vaccines. RNActive(®) vaccines induce strong, balanced immune responses comprising humoral and cellular responses, effector and memory responses as well as activation of important subpopulations of immune cells, such as Th1 and Th2 cells. Pre-germinal center and germinal center B cells were detected in human patients upon vaccination. RNActive(®) vaccines successfully protect against lethal challenges with a variety of different influenza strains in preclinical models. Anti-tumor activity was observed preclinically under therapeutic as well as prophylactic conditions. Initial clinical experiences suggest that the preclinical immunogenicity of RNActive(®) could be successfully translated to humans.

Protein expression from exogenous mRNA: uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway.

Insertional mutagenesis and the inherent risk of malignancy compromise the clinical use of DNA-based therapies. Being a transient copy of genetic material, mRNA is a safe alternative, overcoming this limitation. As a prerequisite for the development of efficient mRNA-based therapies, we investigated the cellular uptake and intracellular fate of mRNA for the first time. To this end we determined cell-type, dose and energy dependence of mRNA internalisation. Moreover, we employed markers for uptake pathways and cellular compartments to analyse the route of mRNA internalisation and its intracellular destination. Finally, we addressed the involvement of receptors and their nature using a competitor-based approach. We found that all cell types tested were amenable to uptake and expression of naked mRNA. Internalisation mainly occurred via caveolae/lipid raft-rich membrane domains and involved scavenger-receptor(s). Following endocytosis, mRNA eventually accumulated in lysosomes, while part of it escaped into the cytosol giving rise to protein synthesis. Taken together, our findings provide unprecedented insights into the internalisation and trafficking of exogenous mRNA, greatly facilitating the development of effective mRNA-based therapies in the future.

Major shifts in genomic activity accompany progression through different stages of the hair cycle.

Hair follicles display a unique pattern of cyclic growth and regression involving cell proliferation, differentiation and migration. The molecular details of these processes are largely unexplored. Global expression analyses on the basis of about 20,000 genes for each morphologically distinguishable stage of the hair cycle revealed unexpected complexities of and major temporal shifts in transcriptional programs involving about 13% of all genes. In particular, hundreds of genes characterise the pattern of genomic activity during regression and resting phases; selected genes can be used to monitor hair growth in mice. We demonstrate that temporal expression patterns predict gene expression domains within the hair follicle. Expression of insulin-like growth factor binding proteins and anti-angiogenic factors is associated with the regression phase of the hair cycle.