Modified mRNA as a new therapeutic option for pediatric respiratory diseases and hemoglobinopathies

Pediatric respiratory illness is a major cause of mortality and morbidity among infants and young children as they are more susceptible to respiratory diseases [1]. Asthma, tuberculosis, bronchiectasis, and bronchopulmonary dysplasia are chronic respiratory diseases in children. In addition, congenital respiratory disorders such as cystic fibrosis and primary ciliary dyskinesia are observed at lower incidence in children [2, 3]. Next to the lung diseases, hemoglobinopathies are the most common genetic disorders in pediatrics as at least 60,000 severely affected children are born every year [4, 5]. Although treatments are available for both clinical pictures, the major issue appears to be its limited effectiveness, thus providing only a short-term cure [5, 6]. However, recent advancements in science have paved the way to treat such diseases at the molecular level as new therapeutic targets and pathways are uncovered. In this review, we focus on common pediatric respiratory illness and hemoglobinopathies that are potentially amenable to gene therapy.

For two decades, gene therapy has been focused on plasmid DNA and viral DNA, with severe consequences in early key studies [7]. However, gene therapy has greatly evolved and has become a major focus in nucleotide-based gene therapy. Karikó et al. and Kormann et al. demonstrated that chemical modification of messenger RNA (mRNA) resulted in mRNA transcripts being less immunogenic and more efficiently translated in vitro [8] and in vivo [9]. Such chemically modified messenger RNA (mod. mRNA) has many advantages compared to other therapeutic nucleic acids. The most important features comprise a transient protein expression, reduced immunogenicity, superior translation efficiency, and pharmaceutical safety, as mod. mRNA does not integrate into the host genome [8, 10, 11]. Current progress in targeted genome editing mediated by nucleases and encoded by mod. mRNA to express engineered nucleases such as the CRISPR-Cas9 system, zinc-finger nucleases (ZFNs), and TAL effector nucleases (TALENs) will undoubtedly translate basic research findings to novel molecular therapeutics in the treatment of pediatric diseases [12, 13]. The principle and applications of mod. mRNA are illustrated in Fig. 1.

In a landmark publication, Warren et al. implicated chemically modified mRNA as a powerful tool to express proteins of interest in target cells in vitro. These authors showed that repeated administration of mod. mRNA encoding the Yamanaka factors KLF4, c-MYC, OCT4, and SOX2 could reprogram terminally differentiated human fibroblasts into pluripotent myofibroblasts without the need for retroviral vectors [14]. Consequently, the study provided an efficient way to reprogram cell fate without the risk of potential genomic integration. Subsequent studies have extended these initial observations of mod. mRNA-encoded protein expression to in vivo models, demonstrating specific expression in organs such as the heart, skeletal muscle, and lung [9, 15, 16].

To achieve a beneficial therapeutic effect, reduced activation of innate immune receptors and an increased biological stability are key issues that need to be addressed when considering future clinical applications of mod. mRNA. Chemical substitutions such as the replacement of uridine by pseudouridine (Ψ) or cytosine by 5-methyl-cytosine (m5C) on target mRNA ultimately allow them to evade innate immune responses such as Toll-like receptor (TLRs) signaling pathways [8]. A study by Kormann et al. demonstrated that combining chemical modifications, such as the replacement of 25 % of uridine and cytidine with 2-thiouridine (s2U) and m5C reduced recognition of target mRNA through pattern recognition receptors, including TLR3, TLR7, TLR8, and RIG-I in human PBMCs. In addition, the same modifications also facilitated in vivo delivery of the respective mRNA [9]. These findings were verified by a subsequent study and confirmed that chemical modification effects in reduced innate immune recognition [17]. Very recently, it has been shown that the incorporation of N¹-methylpseudouridine (m1Ψ) in mRNA resulted in innate immune evasion and increased translational capacity in vitro and in vivo [18]. Next to chemical modifications, other approaches to enhance stability and translational efficacy of mRNA include the use of phosphorothioate 5′ cap analogs, HPLC purification, and polyadenylation tail by a defined number of adenosines as well as the incorporation of stabilizer proteins [19‐22]. The progress in lipid nanoparticle mediated delivery of mod. mRNA into target cells in vivo by various routes provided an efficient translation [23].

Cystic fibrosis, SP-B deficiency, asthma, β thalassemia, and sickle cell anemia are all genetic diseases that can affect children. With its potential for protein replacement and gene correction, mod. mRNA presents a powerful tool to minimize or even cure these conditions. The next part of the review will focus on the present evidence of positive results using mod. mRNA against genetic disease.

Asthma is the most common chronic inflammatory disorder in childhood and is associated with airway hyper-responsiveness leading to recurrent episodes of wheezing, breathlessness, chest tightness, coughing, and airflow obstruction. However, in children below 5 years of age, clinical symptoms of asthma differ individually in a nonspecific manner. Usually, inhalation of corticosteroids and bronchodilators is the first choice when it comes to the treatment of asthma. However, some patients face the clinical picture of corticosteroid resistance as a consequence to repeated corticosteroid administration. In this case, treatment by means of gene replacement could be an alternative route and possibly benefit these patients. One such target for gene therapy would be to influence Th2 cytokine reactions [29] by inducing regulatory T cells. Mays et al. demonstrated that administration of modified Foxp3 mRNA in a time- and site-specific fashion in murine lungs prevented allergic asthma in vivo, while suppressing Th2 responses [15].

mRNA has tremendous potential for both gene therapy and gene correction approaches. However, as with every new technology, a number of methodological improvements and specific challenges need to be addressed. The chemical modification needs to be adapted to the target cell type and to the transfection reagent used. In addition, the incorporation of chemically modified nucleosides into mRNA increases protein stability but decreases translational efficiency [24]. Thus, it is recommended that mRNA formulations undergo extensive optimization to achieve a therapeutic benefit. Furthermore, in order to achieve permanent cures for genetic diseases as that mentioned above, the nec-mRNA and repair templates must be directed to stem/progenitor cells by targeted cell therapy.

In conclusion, the therapeutic potential of mod. mRNA has been rapidly recognized for the treatment of pediatric diseases. However, further improvements in systemic delivery, consideration of different cell types or different organs, and effective chemical modifications for each such type are pivotal for efficient utilization. Combining efforts to overcome cell turnover together with appropriate animal models will enable the field to make continued progress and to reach the step into the clinic.