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.

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mRNA-based therapeutics — developing a new class of drugs

Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting β polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) α and β] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology.

Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

The human SLC29 family of proteins contains four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterised family member, hENT1. They belong to the widely-distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporters and are distantly related to a lysosomal membrane protein, CLN3, mutations in which cause neuronal ceroid lipofuscinosis. A predicted topology of 11 transmembrane helices with a cytoplasmic N-terminus and an extracellular C-terminus has been experimentally confirmed for hENT1. The best-characterised members of the family, hENT1 and hENT2, possess similar broad substrate specificities for purine and pyrimidine nucleosides, but hENT2 in addition efficiently transports nucleobases. The ENT3 and ENT4 isoforms have more recently also been shown to be genuine nucleoside transporters. All four isoforms are widely distributed in mammalian tissues, although their relative abundance varies: ENT2 is particularly abundant in skeletal muscle. In polarised cells ENT1 and ENT2 are found in the basolateral membrane and, in tandem with concentrative transporters of the SLC28 family, may play a role in transepithelial nucleoside transport. The transporters play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis, and are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. In addition, by regulating the concentration of adenosine available to cell surface receptors, they influence many physiological processes ranging from cardiovascular activity to neurotransmission.

The equilibrative nucleoside transporter family, SLC29.

Drug transport in the central nervous system is highly regulated not only by the blood-brain and the blood-cerebrospinal fluid barriers but also in brain parenchyma. The novel localization of drug transporters in brain parenchyma cells, such as microglia and astrocytes, suggest a reconsideration of the present conceptualization of brain barriers as it relates to drug transport. That is, the cellular membranes of parenchyma cells act as a second "barrier" to drug permeability and express transporters whose properties appear similar to those localized at the conventional brain barriers. This review will focus on the molecular characteristics, localization, and substrate specificities of several classes of well known membrane drug transporters (i.e., the organic cation, organic anion, nucleoside, P-glycoprotein, and multidrug resistance proteins) in the brain. Comparisons to similar transporters localized within the peripheral system and clinical implications of the functional expression of specific drug transport families will be discussed when appropriate. Nutrient and neurotransmitter transporters, whose characteristics have been reviewed extensively elsewhere, will not be considered in this review.

Drug Transporters in the Central Nervous System: Brain Barriers and Brain Parenchyma Considerations

Nucleoside analogues (NA) are essential components of AML induction therapy (cytosine arabinoside), effective treatments of lymphoproliferative disorders (fludarabine, cladribine) and are also used in the treatment of some solid tumors (gemcitabine). These important compounds share some general common characteristics, namely in terms of requiring transport by specific membrane transporters, metabolism and interaction with intracellular targets. However, these compounds differ in regard to the types of transporters that most efficiently transport a given compound, and their preferential interaction with certain targets which may explain why some compounds are more effective against rapidly proliferating tumors and others on neoplasia with a more protracted evolution. In this review, we analyze the available data concerning mechanisms of action of and resistance to NA, with particular emphasis on recent advances in the characterization of nucleoside transporters and on the potential role of activating or inactivating enzymes in the induction of clinical resistance to these compounds. We performed an extensive search of published in vitro and clinical data in which the levels of expression of nucleoside-activating or inactivating enzymes have been correlated with tumor response or patient outcome. Strategies aiming to increase the intracellular concentrations of active compounds are presented.

Nucleoside analogues: mechanisms of drug resistance and reversal strategies

In most mammalian cells nucleoside uptake occurs primarily via broad-specificity, es (e, equilibrative; 5, sensitive to NBMPR inhibition) transporters that are potently inhibited by nitrobenzylthioinosine (NBMPR). These transporters are essential for nucleotide synthesis by salvage pathways in hemopoietic and other cells that lack de novo pathways and are the route of cellular uptake for many cytotoxic nucleosides used in cancer and viral chemotherapy. They play an important role in adenosine-mediated regulation of many physiological processes, including neurotransmission and platelet aggregation, and are a target for coronary vasodilator drugs. We have previously reported the purification of the prototypic es transporter from human erythrocytes and have shown that this glycoprotein of apparent M, 55,000 is immunologically related to nucleoside transporters from several other species and tissues, including human placenta. Here we report the isolation of a human placental cDNA encoding a 456-residue glycoprotein with functional characteristics typical of an es-type transporter. It is predicted to possess 11 membrane-spanning regions and is homologous to several proteins of unknown function in yeast, nematodes, plants and mammals. Because of its central role in the uptake both of adenosine and of chemotherapeutic nucleosides, study of this protein should not only provide insights into the physiological roles of nucleoside transport but also open the way to improved therapies.

Nick Beaumont

Papers

Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs