mRNA-based therapeutics — developing a new class of drugs

Nature Reviews Drug Discovery | Review

The therapeutic potential of in vitro-transcribed mRNA (IVT mRNA) extends from prophylactic and therapeutic vaccines to applications such as protein replacement and genome engineering. In this Review, the authors describe the recent developments in the IVT mRNA field, discuss the class-specific challenges with regards to translating IVT mRNA into a biopharmaceutical, and provide an overview of IVT mRNA drugs in development for different indications.

Key points

• Messenger RNA (mRNA) is a pivotal molecule of life, involved in almost all aspects of cell biology.
• As the subject of basic and applied research for more than 5 decades, mRNA has only recently come into the focus as a potentially powerful drug class able to deliver genetic information. 
• Synthetic mRNA can be engineered to resemble mature and processed mRNA molecules as they occur naturally in the cytoplasm of eukaryotic cells and to transiently deliver proteins. 
• Recent advances addressed challenges inherent to this drug class and provided the basis for a broad spectrum of applications 
• Besides cancer immunotherapies and infectious disease vaccines novel approaches such as in vivo delivery of mRNA to replace or supplement proteins, mRNA-based induction of pluripotent stem cells, or mRNA-assisted delivery of designer nucleases for genome engineering rapidly emerged and entered into pharmaceutical development. 
• This Review gives a comprehensive overview of the current state of mRNA drug technologies, their applications and crucial aspects relevant to mRNA based drug discovery and development.

Abstract:

In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.

Figure 1: (Timeline): Key discoveries and advances in the development of mRNA as a drug technology

CAR, chimeric antigen receptor; Cas9, CRISPR-associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeat; DC, dendritic cell; dsRNA, double-stranded RNA; iPSC, induced pluripotent stem cell; RSV, respiratory syncytial virus; ssRNA, single stranded RNA; TALEN, transcription activator-like effector nuclease; TLR, Toll-like receptor. 

Figure 2: Principles of antigen-encoding mRNA pharmacology.

a | A linearized DNA plasmid template with the antigen-coding sequence is used for in vitro transcription. The in vitro transcribed mRNA contains the cap, 5′and 3′ untranslated regions (UTRs), the open reading frame (ORF) and the poly(A) tail, which determine the translational activity and stability of the mRNA molecule after its transfer into cells. b | Step 1: a fraction of exogenous mRNA escapes degradation by ubiquitous RNases and is spontaneously endocytosed by cell-specific mechanisms (for example, macropinocytosis in immature dendritic cells) and enters endosomal pathways. Step 2: release mechanisms of mRNA into the cytoplasm are not fully understood. Step 3: translation of mRNA uses the protein synthesis machinery of host cells. The rate-limiting step of mRNA translation is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the cap structure 222, 223. Binding of the mRNA to ribosomes, the eukaryotic initiation factors eIF4E and eIF4G, and poly(A)-binding protein, results in the formation of circular structures and active translation 224. Step 4: termination of translation by degradation of mRNAs is catalysed by exonucleases 225, 226. The cap is hydrolysed by the scavenger decapping enzymes DCP1, DCP2 and DCPS 32, followed by digestion of the residual mRNA by 5′–3′ exoribonuclease 1 (XRN1). Degradation may be delayed if the mRNA is silenced and resides in cytoplasmic processing bodies 227. Alternatively, endonucleolytic cleavage of mRNA in the exosome may occur 228, 229, 230. The catabolism of abberant mRNA (for example, mRNA with a premature stop codon) is controlled by various other mechanisms 231. Step 5: the translated protein product undergoes post-translational modification, the nature of which depends on the properties of the host cell. The translated protein can then act in the cell in which it has been generated. Step 6: alternatively, the protein product is secreted and may act via autocrine, paracrine or endocrine mechanisms. Step 7: for immunotherapeutic use of mRNA, the protein product needs to be degraded into antigenic peptide epitopes. These peptide epitopes are loaded onto major histocompatibility complex (MHC) molecules, which ensure surface presentation of these antigens to immune effector cells. Cytoplasmic proteins are proteasomally degraded and routed to the endoplasmic reticulum where they are loaded on MHC class I molecules to be presented to CD8+ cytotoxic T lymphocytes. MHC class I molecules are expressed by almost all cells. Step 8: in antigen-presenting cells, to obtain cognate T cell help for a more potent and sustainable immune response, the protein product needs to be routed to MHC class II loading compartments. This can be accomplished by incorporating routing signal-encoding sequences into the mRNA. Moreover, exogenous antigens that are taken up by dendritic cells can also be processed and loaded onto MHC class I molecules by a mechanism that is known as cross-priming 232. Step 9: protein-derived epitopes can then be presented on the cell surface by both MHC class I and MHC class II molecules. 

Figure 3: Tuning mRNA drug dose pharmacokinetics.

a | Key structural elements of in vitro transcribed (IVT) mRNA and strategies for their modifications. b | Depending on which elements (for example, modification of caps, untranslated regions (UTRs) or poly(A) tails) are used alone or in combination, the duration and kinetic profile of expression of the protein product can be modulated and fine-tuned. eIF4E, eukaryotic translation initiation factor 4E; IRES, internal ribosome entry site; ORF, open reading frame. 

Figure 4: Inflammatory responses to synthetic mRNA.

In vitro transcribed (IVT) mRNA is recognized by various endosomal innate immune receptors (Toll-like receptor 3 (TLR3), TLR7 and TLR8) and cytoplasmic innate immune receptors (protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5) and 2′–5′-oligo adenylate synthase (OAS)). Signaling through these different pathways results in inflammation associated with type 1 interferon (IFN), tumour necrosis factor (TNF), interleukin-6 (IL-6), IL-12 and the activation of cascades of transcriptional programmes. Overall, these create a pro-inflammatory microenvironment poised for inducing specific immune responses. Moreover, downstream effects such as slow-down of translation by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, enhanced RNA degradation by ribonuclease L (RNASEL) over expression and inhibition of replication of self-amplifying mRNA are of relevance for the pharmacokinetics and pharmacodynamics of the IVT mRNA. IRF, interferon regulatory factor; ISRE7, interferon-stimulated response element; MAVS, mitochondrial antiviral signaling protein; MDA5, melanoma differentiation-associated protein 5; MYD88, myeloid differentiation primary response protein 88; MX1, myxovirus (influenza) resistance 1; NF-κB, nuclear factor-κB; TRIF, Toll-IL-1 receptor domain-containing adapter protein inducing IFNβ.

Figure 5: Differences in siRNA, pDNA and mRNA technologies in tissues with non-fenestrated or fenestrated capillaries.

All three nucleic acid-based drug modalities are applied as nanosized drug formulations for systemic delivery and reach organs via capillary systems with either non-fenestrated (a) or fenestrated (b) capillaries. The primary pharmacological effect of small interfering RNA (siRNA), namely the deletion of a defined protein function in situ, is restricted to those very cells it has entered. siRNA cannot act in cells that are not directly accessed owing to biological barriers such as non-fenestrated capillaries. In tissues with endothelial fenestration, siRNA may reach a few tissue layers adjacent to capillaries. Plasmid DNA (pDNA) is only incorporated and active in those cells undergoing mitosis at the time of exposure. This impairs its use for tissues with non-fenestrated capillaries and restricts the number of transfectable cells in tissues with endothelial fenestration to those undergoing mitosis at the time of exposure. In contrast to pDNA, mRNA enters and acts in endothelial cells of non-fenestrated tissues, and in fenestrated tissues it reaches both mitotic and non-mitotic cells in cell layers adjacent to the capillaries 233. Non-target cells, such as vascular endothelial cells transfected with mRNA or pDNA, can express pharmacologically active proteins and, via paracrine secretion, can reach target cells that are located behind the mRNA delivery barriers 234 (obviously siRNA cannot rely on such a function). Proteins produced in transfected cells are able to diffuse after secretion into the target tissue and mediate the intended biological effects via paracrine activity on adjacent cell populations. Such paracrine activity may be of particular value in tissues that have non-fenestrated capillaries.

In vitro transcribed (IVT) mRNA is recognized by various endosomal innate immune receptors (Toll-like receptor 3 (TLR3), TLR7 and TLR8) and cytoplasmic innate immune receptors (protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein 5 (MDA5) and 2′–5′-oligoadenylate synthase (OAS)). Signalling through these different pathways results in inflammation associated with type 1 interferon (IFN), tumour necrosis factor (TNF), interleukin-6 (IL-6), IL-12 and the activation of cascades of transcriptional programmes. Overall, these create a pro-inflammatory microenvironment poised for inducing specific immune responses. Moreover, downstream effects such as slow-down of translation by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, enhanced RNA degradation by ribonuclease L (RNASEL) overexpression and inhibition of replication of self-amplifying mRNA are of relevance for the pharmacokinetics and pharmacodynamics of the IVT mRNA. IRF, interferon regulatory factor; ISRE7, interferon-stimulated response element; MAVS, mitochondrial antiviral signalling protein; MDA5, melanoma differentiation-associated protein 5; MYD88, myeloid differentiation primary response protein 88; MX1, myxovirus (influenza) resistance 1; NF-κB, nuclear factor-κB; TRIF, Toll-IL-1 receptor domain-containing adapter protein inducing IFNβ.

Figure 6: Potential therapeutic applications of IVT mRNA.

The therapeutic applications of in vitro transcribed (IVT) mRNA are summarized in detail in Table 1. The solid arrows pointing in the right hand column denote applications that are in the clinic, whereas stippled arrows refer to preclinical applications. Cas9, CRISPR-associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeat; EPO, erythropoietin; FOXP3, forkhead box P3; IL-10, interleukin-10; MSC, mesenchymal stem cell; RSV, respiratory syncytial virus; SPB, surfactant protein B; TALEN, transcription activator-like effector nuclease; VEGFA, vascular endothelial growth factor A; ZNF, zinc finger nuclease.

Title: mRNA-based therapeutics — developing a new class of drugs

Author: Ugur Sahin, Katalin Karikó & Özlem Türeci 

Publication: Nature Reviews Drug Discovery | Review 

Publisher: Nature Publishing Group 

Date:19 September 2014 

Copyright © 2014, Rights Managed by Nature Publishing Group