Recent Developments in mRNA-Based Protein Supplementation Therapy to Target Lung Diseases

Abstract

Protein supplementation therapy using in vitro-transcribed (IVT) mRNA for genetic diseases contains huge potential as a new class of therapy. From the early ages of synthetic mRNA discovery, a great number of studies showed the versatile use of IVT mRNA as a novel approach to supplement faulty or absent protein and also as a vaccine. Many modifications have been made to produce high expressions of mRNA causing less immunogenicity and more stability. Recent advancements in the in vivo lung delivery of mRNA complexed with various carriers encouraged the whole mRNA community to tackle various genetic lung diseases. This review gives a comprehensive overview of cells associated with various lung diseases and recent advancements in mRNA-based protein replacement therapy. This review also covers a brief summary of developments in mRNA modifications and nanocarriers toward clinical translation.

mRNA has gathered attention as a new class of therapeutics for different diseases. Progress has been made in the field of protein replacement therapy by developing mRNA into a safe and efficient therapy. This article focuses on the delivery of mRNA therapeutics in the lung, and it provides an overview of lung architecture and associated diseases, such as asthma, cystic fibrosis, COPD, and SP-B deficiency.

Main Text

RNA, a fundamental molecule in the eukaryotic and prokaryotic cells and viruses, came recently into focus for therapeutic approaches:1, 2, 3 tRNAs for nonsense mutation correction, RNA aptamers for binding to a specific target molecule, and RNAi and long noncoding RNAs (lncRNAs) for gene regulation.4, 5, 6, 7, 8 mRNA gives rise to a new therapy for diseases associated with functional protein loss by supplementing the protein with a transcript encoding. The first study of in vitro-transcribed (IVT) mRNA in the late 1980s showed that this mRNA can be directly translated into a functional form immediately after transfection in vitro and in vivo.9, 10 This was shortly followed by a therapeutic application of mRNA in a temporary reversal of diabetes insipidus, unraveling its therapeutic potential.¹¹

Therapeutic applications of mRNA are advantageous due to its unique properties. Cytoplasm being mRNA’s functional site requires no transportation across the nuclear membrane. Furthermore, due to the transient nature and biodegradability of mRNA, permanent adverse effects can potentially be avoided.¹² This includes preventing the permanent manipulation of the genome, making mRNA a prevalent molecule for protein supplementation therapy.13, 14, 15 Several aspects of mRNA have to be addressed in order to achieve therapeutic benefits: potential immunogenicity mediated by innate immune system reactions (pattern recognition receptor),16, 17, 18, 19 degradation of single-stranded mRNA by nucleases, and its negative charge that inhibits the passive crossing of the cell membrane. Concerning these obstacles, solutions such as chemical modifications of nucleosides to reduce immunogenicity and usage of nanocarriers to facilitate crossing the cell membrane are emerging. The recent advancements in the field of nanocarriers suggest the possibility to customize particles for target organs.20, 21

Based on our research interest in lungs, this review focuses on protein replacement therapy of lung disease, especially monogenetic diseases, such as cystic fibrosis (CF) and surfactant protein B (SP-B) deficiency, as well as multifactorial diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. To achieve targeted therapy options, lung structure and methods to reach specific lung cell populations are critical. Therefore, this review gives an overview of lung cell populations and diseases associated with them. Furthermore, a comprehensive summary of mRNA transcript improvements by chemical modification of nucleosides or capping and nanocarriers to target lung cell populations is also featured.

Lung Architecture and Disease Pathology

The lung has a very unique architecture to enable efficient transfer of oxygen and carbon dioxide required for oxidative metabolism. Inhaled gases travel through the airway tubes via trachea bronchi and bronchioles to the alveoli enriched with blood vessels, the primary site of gas exchange. Inflation and deflation of the lung is a prerequisite for gas exchange at the alveoli. This process requires multiple components like the extracellular matrix, smooth muscle cells, and cartilage for support and flexible collagen and the elastin fiber network for flexibility during inflation and deflation. Precisely regulated surface fluids, electrolytes, and mechanical activity of secretory and ciliated cells determine the mucociliary clearance, whereas on the other hand the epithelium maintains the barrier function.

The airway epithelium (tracheal and bronchiolar) consists of goblet cells, brush or tuft cells, ciliated cells, basal cells, neuroendocrine or neuroepithelial bodies, club cells, lineage-negative epithelial progenitor (LNEP) cells, and the newly identified ionocytes.22, 23 The ducts of submucosal glands consist of goblet cells, serous cells, and myoepithelial cells. The alveolar epithelium consists primarily of alveolar type I (ATI) cells (∼95%), macrophages, and alveolar type II (ATII) cells, with close association to capillary endothelial cells of pulmonary microcirculation.²⁴ The terminal airway ducts and alveoli are supported by fibroblasts and myofibroblasts producing extensive elastin-collagen networks, which help with inflation and deflation.25, 26, 27 Other important cell populations include stem cells and immune cells, which help in region-specific regeneration and protection against pathogens, respectively. Abnormalities of lung mechanics are observed in various diseases, including CF,28, 29 asthma,30, 31 idiopathic pulmonary fibrosis (IPF),32, 33 COPD,34, 35 and bronchiolitis obliterans.34, 36 These conditions are detrimental in nature and involve multiple factors, such as increased resistance of lung tissue due to fibrosis of the lung; collapsing tubes or thickening of airway walls due to mucus overproduction; and the loss of ciliary function, resulting in airflow obstruction, mucus plugging, chronic infection, and inflammatory damages. In that process, multiple cell populations undergo remodeling, directly contributing to clinical symptoms.

Broncho-Epithelial Cells and Associated Diseases

Goblet cells, present in the broncho-epithelia and submucosal glands, produce mucins (MUC5B and MUC5AC), and they are differentiated from basal and club cells upon various stimuli (toxic substances, pathogens, particles, and neural and innate immune signals). Goblet cells are not only helping to establish the innate immune system but also key players in pulmonary diseases, as described before.³⁷ These cells produce cytokines and chemokines that recruit and educate innate immune cells, including dendritic cells (DCs), innate lymphoid cells, and eosinophils. This contributes to the CD4⁺ T helper cell 2 (Th2)-mediated immune response typical of asthma.38, 39 Hyper-production of mucus and goblet cell metaplasia are characteristics of Th2-mediated and non-mediated inflammation (pathogen mediated), which leads to complications in CF, COPD, and IPF.40, 41 The fluids for hydration are regulated by serous cells that line the acinar region of submucosal glands and are important for the pathology of CF. The mucins are precisely balanced by fluid, and electrolyte transport enables rapid secretion and dispersal of mucus onto the airway surfaces and the movement of the mucus gel up the airway by ciliary activity. In CF, the secretion of chloride and bicarbonate is impaired, disrupting mucociliary clearance due to mutations in CF transmembrane conductance regulator gene (CFTR), which leads to recurrent airway infections, sinusitis, bronchiectasis, and pulmonary tissue remodeling.⁴²

Brush cells, containing distinctive apical microvilli, are found in multiple organs, e.g., pancreas, intestine, nose, and trachea. Brush cells are known to play an important role in activating the innate immune system in the intestine⁴³ and nose (Tas2R receptors),⁴⁴ however, a similar phenomenon in airway trachea requires testing. Recent studies in the trachea have indicated their role as chemosensory for immune surveillance and as respiratory regulators.⁴⁵ These might be responsible in transducing signals regulating wheezing and coughing during episodes of asthma. However, further studies are required to understand their role.⁴⁶

Ciliated cells are characterized by their multiple apical, motile cilia composed of structural proteins and motor proteins (dynein)47, 48, 49 that regulate the coordinated bidirectional beating critical for particle and pathogen clearance.50, 51 Ciliated cells respond to both physical⁵² and chemical⁵³ stimulation. Mucociliary clearance can be affected by ciliary dysfunction, impaired fluid secretion, disruption of epithelial cell lining, or lack of cough. This impairment can initiate an inflammatory response, damaging the airway epithelium. Disruption in ciliated cell function results in recurrent and persistent infections, morbidity, and mortality in chronic pulmonary disorders.⁵⁴ In COPD, direct evidence has been provided of suppressed ciliary beating in nasal epithelium⁵⁵ with normal mucus production. Cigarette smoking has been shown to have a detrimental effect on the number and size of cilia in vitro,⁵⁶ whereas in vivo a slight increase in ciliary beating initially followed by significant loss of cilia over prolonged time was observed.⁵⁷ Primary ciliary dyskinesia (PCD) resulting from ciliary dysfunction is caused by recessive mutations in one of multiple genes involved at different points in cilium structure, assembly, and function, which include DNAI,⁵⁸ DNAH(5,⁵⁹ 11⁶⁰), ARMC4,⁶¹ TXNDC3,⁶² HEATR2,⁶³ HYDIN,⁶⁴ CDC (39,⁶⁵ 40,⁶⁶ 65,⁶⁷ 103,⁶⁸ 114,⁶⁹ 151⁷⁰), DNAAF(1,⁷¹ 2,⁷² 3⁷³), RHSP(4A, 9),⁷⁴ DYX1C1,⁷⁵ LRRC6,⁷⁶ ZMYND10,⁷⁷ CCNO,⁷⁸ and recently identified MCIDAS.⁷⁹

Club cells are columnar, secretory cells that express high levels of cytochrome P450-detoxifying enzyme CYP2F, surfactant proteins (SP-A, SP-B, and SP-D), and innate immune proteins, including defensins, lactoferrin, and secretaglobins (SCGB1A1 and SCGB3A1).⁸⁰ Upon stimulus or injury, these differentiate into alveolar cells,81, 82 goblet cells,38, 83, 84 or ciliated cells.⁸⁵ However, in humans, these are only localized in terminal bronchioles, and they may play a role in the maintenance of distal bronchioles.⁸⁶ Club cells being progenitor cells also plays an important role in the repair mechanism of the airway epithelium, and, therefore, they are connected to damage responses in CF, COPD, and IPF.⁸⁰ Club cells also metabolize chemical toxins (e.g., naphthalene) to toxic compounds that selectively kill club cell subsets. SP-A and SP-D (C-type lectins) are responsible for host defense, enhancing the clearance of various microbial pathogens, whereas the secretory lipids and proteins (SP-B and SP-C) help in minimizing surface tension and collapsing forces caused by inhaled gases when in direct contact with alveolar structures, protecting peripheral saccules from atelectasis during ventilator cycles.⁸⁷

Neuroendocrine cells represent less than 1% of the airway epithelium,⁸⁸ and they are found either isolated or in clusters known as neuroepithelial bodies located at precise airway branch points.⁸⁹ The localization aids in their role as airway (environmental) sensors (acidosis, hypoxia, and hypercarbia),90, 91 and clustering is required for the appropriate innate immune responses.⁹² The response to a stimulus is via the release of stored amines (serotonin) and peptides (calcitonin gene-related peptide [CGRP]).⁹² Hyperplasia is associated with a wide range of congenital and infantile lung disorders. Though the underlying cause is unknown, NKX2-1 mutation has been associated with neuroendocrine hyperplasia of infancy (NEHI).⁹³

Ionocytes (Foxi1⁺) are the rare cell type of the airway epithelium recently characterized, in both mice and humans, by two independent research groups.22, 23 These reside at multiple levels of the airway tree, and they are needed to maintain airway surface physiology, including mucus viscosity.²³ Foxi1 is already known to regulate V-ATPase, which is important for transport and fluid pH in other cell types in skin.94, 95 Knockout in a mouse model reduced Cftr and Ascl3 expression, indicating the role of Foxi1 in CFTR regulation.²³ Montoro et al.²³ also performed pulse-sequence tracing that indicated the basal cell lineage, with an increased expression of CFTR.

Basal cells are progenitor cells96, 97, 98 that are regulated by NOTCH signaling to give rise to ciliated cells (NOTCH⁻), goblet cells (NOTCH⁺), and club cells (NOTCH²⁺).99, 100, 101, 102 Due to their basal proximity, these cells interact with the columnar epithelium; underlying mesenchymal cells; basal membrane; neurons; and also lymphocytes, inflammatory cells, and DCs.¹⁰³ A loss in tight regulation of basal cell differentiation can result in inappropriate cell fate determination, leading to pathological airway remodeling. This includes epithelial hypoplasia (proliferation failure), basal cell hyperplasia (excessive proliferation with no differentiation), goblet cell metaplasia or hyperplasia instead of ciliated cell generation, and squamous metaplasia (suprabasal cells) instead of luminal cells. Pathological airway remodeling occurs frequently in association with CF, COPD, and chronic asthma.¹⁰³ Araya et al.¹⁰⁴ in their study showed that hyper-proliferating basal cells secrete cytokines (interleukin-1β [IL-1β]) that promote airway wall fibrosis via transforming growth factor β (TGF-β) signaling in COPD.

Alveolar Cells and Associated Diseases

Alveolar type I cells (ATI) are squamous cells lining the alveolar compartment involved in gas exchange. These are terminally differentiated cells (lifespan ∼120 days) that form a barrier to sense microbial products and generate inflammatory responses.105, 106 These cells undergo excessive physical and chemical stresses due to their higher exposure, and they require constant regulated repair.107, 108 A defect in repair directly and indirectly contributes toward injurious manifestations of the lung, leading to diseases like acute respiratory distress syndrome (ARDS) and IPF.109, 110 ATII (progenitor of ATI cells) are cuboidal cells and cover about 7% of the total alveolar surface. These produce surfactant lipids (phosphatidylcholine) and surfactant proteins (SP-A to -D). Congenital SP-B deficiency leads to death87, 111 soon after birth; however, targeted disruption of SP-C,¹¹² SP-A,¹¹³ and SP-D114, 115 gene loci does not show detrimental effects. In cases with acute respiratory distress syndrome, a decrease in the expression of SP-B is also observed.116, 117

Stem Cells of the Airways

To maintain the constant dynamic function of the lung, it is very crucial that the respiratory epithelium is equipped with fast and extensive regenerative ability following injury. Airway basal cells and ATII cells have been known for their role in repair of the airway epithelium. Recently, studies have identified distinct niches throughout the lung that can mediate graded and region-specific responses.118, 119 Myoepithelial cells (MECs) and bronchoalveolar stem cells (BASCs) are a couple of the established stem cells in lungs. It was well established that the innervated MECs encircle the submucosal glands and mediate mucus secretion in response to neural inputs, which can activate massive secretory responses after stimulation by irritants and toxins (also reviewed in Boers et al.⁸⁶ and Yei et al.¹²⁰).37, 42, 121 However, recent work by Lynch et al.¹²² and Tata et al.¹²³ have explained the potency of MECs in generating seven cell types of surface airway epithelium and the submucosal gland following airway injury. These can be activated via Sox9 or Lef-1 transcriptional signaling, and they can be exploited for regenerative medicine. BASCs have been identified as stem cells that co-express both club cell and ATII cell markers.¹²⁴ BASCs sorted by flow cytometry and cultured in vitro showed differentiation, self-renewability, and response to injury.124, 125, 126 Lineage-tracing studies have revealed the BASC’s ability to give rise to alveolar epithelial cells in vivo127, 128 and its contribution to homeostasis and repair, along with club and ATII cells.127, 128, 129, 130

Respiratory Diseases Targeted by Protein Replacement Therapy

SP-B Deficiency

SP-B deficiency is a rare genetic disease leading to neonatal lethality, including interstitial lung disease (ILD) and ARDS.131, 132, 133, 134, 135 SP-B is crucial for breathing transition of neonates at birth, and it helps in reducing surface tension of the alveolus. Dipalmitoylphosphatidylcholine (DPPC) is the principal surface tension-reducing component that combines with hydrophobic SP-B or SP-C peptides to form stable surfactant film.¹³⁶ Surfactant supports rapid adsorption and insertion of phospholipids, reduction in surface tension upon compression, and rapid re-spreading during expansion. Changes in surfactant lead to alveolocapillary leakage, alveolar instability, compromised gas exchange, and respiratory failure. Both SP-B and SP-C peptides are processed from their pro-peptide forms to their functional form. Absence of proSP-C processing leads to the accumulation of misprocessed SP-C, consisting of the mature peptide with an N-terminal extension (relative molecular weight [M_r] ∼6,000), and to a significant decrease in mature SP-C peptide in alveolar surfactant. ProSP-C processing is also closely related to SP-B expression.87, 111, 137 The combinatorial effect exacerbates lung function at birth, and in vivo studies showed respiratory failure in selective loss of SP-B in adult.¹³⁸

Natural surfactant replacement, such as Survanta beractant (modified bovine surfactant 8 mg SP-B/mL) by Abbott Laboratories, Curosurf (porcine surfactant 80 mg/mL) by Chiesi, and Infasurf (calf surfactant), are a few of the FDA-approved preventive medications for infants with ARDS or premature babies at the risk of developing RDS. Accompanied by physical measures, corticosteroids, or immunosuppressants and repeated lung lavage, surfactant replacements have shown improvement in the disease condition until lung transplantation.139, 140, 141, 142, 143, 144 The potential risks with animal-derived protein include immunological reactions and transmission of animal-derived diseases, justifying the need for standardized human-like alternatives.145, 146, 147 One alternative involves synthetic mimics that have shown superior surfactant properties.144, 148

Since SP-B deficiency is a monogenetic disorder, it acts as a perfect model for gene therapy. Both DNA- (virus¹²⁰ and plasmid¹⁴⁹) and mRNA-¹⁵⁰ based gene supplementation have been tested in a conditional SP-B-knockout mouse model, which indicated improvements in lung function and SP-B expression and a significant increase in survival. Kormann et al.¹⁵⁰ showed for the first time that intratracheal (i.t.) instillation of modified SP-B mRNA to the lung can restore up to 71% of the wild-type SP-B expression, and the treated conditional SP-B-knockout mouse model survived until the predetermined end of the study of 28 days. Presently, the Rudolph team from Ethris holds a patent for pulmonary delivery of mRNA with polyethylenimine (PEI) (US patent application 20150126589), and their teaming up with AstraZeneca and MedImmune could bring the therapy closer to the reach of patients.¹⁵¹

Asthma

Asthma is a multifactorial disease and can be characterized by airway obstruction, chest tightness, wheezing, cough, and breathlessness, followed by recurrent pneumonia or bronchitis. The initiating event in asthmatic airway diseases revolves around interactions between DCs and T cells. DC and T cell interactions favor the generation of Th2, leading to eosinophilia, mucus hypersecretion, and chronic airway inflammation.152, 153 The overactive Th2 response induces the production of cytokines and chemokines, followed by a cascade of immune-activating events, leading to changes in airway smooth muscle contractility,¹⁵⁴ a characteristic of asthma. Studies by Hellings et al.¹⁵⁵ and Wilson et al.¹⁵⁶ showed that Th17 cells orchestrate airway inflammation by enhancing neutrophil recruitment to the lung. The Th2-mediated immune response can be contained via Th1-type cytokines (by Th1 cells), IL-10, and TGF-β (by T regulatory cells [Tregs]), but the roles of IL-17 and IL-22 (by Th17 cells) are debated.155, 157, 158 Both circulatory and airway fluids of asthmatic patients indicate increased IL-17 levels¹⁵⁵ and decreased airway Tregs,¹⁵⁹ indicating an imbalance in Th2 regulation.

Corticosteroid treatment is found to suppress the Th2 immune response via increased Foxp3⁺ Tregs in asthmatic patients.¹⁶⁰ Similar results were found with exposure to microbes161, 162 influencing Treg expression, modulation of IL-6,¹⁶³ prostanoids,¹⁶⁴ and tumor necrosis factor (TNF) pathway enhancement.¹⁶⁵ Mays et al.¹⁶⁶ has successfully demonstrated the protective role of Foxp3 by delivering chemically modified Foxp3 mRNA into the lung of an asthma mouse model. Site-specific instillation of chemically modified Foxp3 mRNA can modulate both Th2 and Th17 responses in an IL-10-dependent manner.¹⁶⁶ Local administration of Foxp3 mRNA can influence the balance among Treg, Th2, and Th17 responses, and it can reduce side effects in terms of the anti-tumoral and anti-infective167, 168 effects of Tregs in comparison to systemic delivery.¹⁶⁵ Kormann et al.¹⁶⁹ produced a unique insight into Toll-like receptors (TLRs), as polymorphisms in TLRs 1, 6, and 10 have shown protective effects on atopic asthma in humans by forming heterodimers with TLR2. A subsequent study by Zeyer et al.¹⁷⁰ demonstrated that Tlr1/2 and Tlr2/6 mRNA instillation in the lungs of a house dust mite-induced mouse model of asthma reduced airway resistance by 40%.

CF

CF, caused by mutations in the CFTR gene, is the most common life-limiting autosomal-recessive disease in the Caucasian population, and it affects more than 80,000 people worldwide. Around 2,000 mutations have been identified and are categorized into 6 classes, ranging in severity from no production of functional protein to decreased synthesis, stability, or function of CFTR protein. CFTR protein acts as a small conductance ATP and cyclic AMP (cAMP)-dependent chloride channel, found at the apical side of epithelial cell lining of most exocrine glands. In the lung epithelium (ionocytes and ciliated and goblet cells), CFTR ensures the secretion of chloride ions, resulting in more hydration and regulated mucus clearance in the airway. A lack of functional CFTR leads to decreased chloride secretion and increased sodium absorption, resulting in dry and rigid mucus production by goblet cells.¹⁷¹ An increase in inflammatory response is also observed, possibly due to the breakdown of elastin fibers by neutrophil elastase and reduced IL-10.¹⁷² The defective mucus clearance enables further pathogen (Pseudomonas aeruginosa and Staphylococcus aureus) colonization, repetitive inflammatory responses that result in irreparable lung damage, and ultimately cardiac arrest. Defects of the CFTR channel lead to a failure in LPS recognition, endocytosis of pathogens, and changes in airway fluid composition. This inactivates beta defensins, causing detrimental effects on the primary defense in the lung.

A readthrough agent for CFTR class I mutation (Ataluren, PTC Therapeutics) showed potential benefits in vivo by increasing CFTR production and function;¹⁷³ however, it failed phase III clinical trial due to a lack of efficacy.¹⁷⁴ Channel modulators categorized into potentiators, correctors, and amplifiers have been used for CF treatment. Potentiators (ivacaftor, Kalydeco) can help in gating and conduction mutations by increasing the open probability of the CFTR channels. Correctors (tezacaftor and lumacaftor) improve CFTR trafficking by facilitating the formation of correct 3D protein structure. Combinations of potentiator and corrector (Orkambi and Symdeko, Vertex Pharmaceuticals) are commercially available only for patients with F508del mutation, expanding the modulators also for application on class II mutations.175, 176 A triple combination of two correctors (VX-659, tezacaftor) and one potentiator (ivacaftor) has also shown greater potency in pre-clinical trials. PTI-428 (Proteostasis Therapeutics), an amplifier to increase the amount of CFTR protein, is in phase II trial. Eluforsen (QR-010) can bind to defective CFTR RNA, and it has shown increased CFTR function by nasal potential difference test in compound heterozygous or homozygous F508del CF patients.¹⁷⁷

Protein replacement therapy with DNA, protein, or mRNA holds a great potential as a universal therapy for curing the underlying defect of CF. Initial attempts of in vivo protein transfer via phospholipid liposomes into the apical membrane of nasal epithelia of CFTR-knockout mice showed limited membrane incorporation by electron microscopy but improvement of nasal potential difference (NPD) measurement.¹⁷⁸ Similarly, DNA-based vectors (viral and plasmid) were tested by Alton’s group (pGM169/GL67A), reaching phase II clinical trials with modest improvement in FEV₁ after repeated administration but no improvement in patient’s quality of life.179, 180 Haque et al.²⁰ have observed a significant improvement in CFTR protein translation, expression, and function in vitro (CFBE41o- and 16HBE14o-) and in vivo (CFTR-knockout mice) by administering chemically modified human CFTR (hCFTR) mRNAs complexed with chitosan-coated poly(lactic-co-glycolic acid) (PLGA) nanoparticles. The study also showed a substantial improvement in FEV_0.1 up to 89% of the level of a healthy control group. Airway compliance and resistance are also significantly improved by the treatment with chemically modified hCFTR mRNAs. A significant decrease in chloride concentration (around 50%) was also observed, indicating a restoration of CFTR in the duct compartment of submucosal glands and thus leading to improved chloride absorption.²⁰ A separate study from Robinson et al.¹⁸¹ confirmed nasal application of chemically modified CFTR mRNA can recover up to 55% of the net chloride efflux characteristic of healthy mice. Bangel-Ruland et al.¹⁸² demonstrate restoration of cAMP-induced CFTR current following transfection of CFBE41o- cells with wild-type CFTR-mRNA similar to the values seen in 16HBE14o- control cells. Translate Bio is also working on CFTR-encoding mRNA (MRT5005) and has entered phase I/II clinical trial (Table 1). All these studies prove the potential of mRNA as a promising therapeutic in CF patients, irrespective of their CFTR mutation status.

Table 1.

Current Clinical Trials Involving mRNA Delivery

Name	Disease	Genetic/Protein Target	Administration Route	Administration Vehicle	ClinicalTrials.gov Identifier	Phase
Lipo-MERIT	melanoma	tumor-associated antigens	intravenous infusion	mRNA-Lipoplex	NCT02410733	1
TNBC-MERIT	triple-negative breast cancer	tumor-associated antigens	intravenous infusion	mRNA-Lipoplex	NCT02316457	1
IVAC mutanome/warehouse	triple-negative breast cancer	patient-specific antigens	intra-nodal	naked	NCT02035956	1
mRNA-1851	influenza A	Hemagglutinin 7 (H7) protein	intramascular injection	not disclosed	Not disclosed	1
mRNA 1440	influenza A	Hemagglutinin 7 (H7) protein	intramuscular injection	not disclosed	not disclosed	1
CV7201	rabies	rabies virus glycoprotein	intramuscular injection	naked	NCT02241135	1
CV8102	HIV, rabies, RSV	RNA-based adjuvant	intramuscular injection	naked	NCT02238756	1
mRNA MRK-1777	not disclosed	vaccine	intradermal	not disclosed	not disclosed	1
mRNA AZD-8601	cardiovascular diseases	VEGF-A	intramuscular injection	naked	NCT02935712	1
mRNA-1325	Zika	viral antigenic protein	intramuscular injection	lipid nanoparticle	NCT03014089	1/2
CV9103	prostate cancer	tumor-Specific antigen	autologous dendritic cell therapy	naked	NCT01197625 NCT00831467	1/2
MRT5005	cystic fibrosis	CFTR	nebulization to the respiratory tract	lipid nanoparticle	NCT03375047	1/2
AGS-004	HIV	vaccine	autologous dendritic cell therapy	naked	NCT01069809, NCT02707900	1/2
AGS-003-LNG	non-small-cell lung cancer	tumor-specific antigen	autologous dendritic cell therapy	naked	NCT02662634	2
iHIVARNA-01	HIV	HIV-target antigen	intranodal route	naked	NCT02888756	2
AGS-003	renal cell carcinoma	tumor-specific antigen	autologous dendritic cell therapy	naked	NCT01482949 NCT00678119 NCT01582672	2/3

Modified from Kaczmarek et al.³²¹

COPD and COPD-like Symptoms in α1-Antitrypsin Deficiency

COPD is a progressive and largely irreversible smoking-related disease characterized by small airway obstruction, emphysema, and chronic bronchitis. It is mainly attributed to long-term exposure to tobacco, toxic gases, and particles, activating both innate and adaptive immune responses. The innate immune defense includes tight junctions, TLRs of epithelial barrier, macrophages, and alveolar fluid (secreted by ATII cells of lung). A second line of defense includes exudation of plasma and circulating effector cells into damaged tissue, regulated by IL-1, IL-8, and TNF-α. The infiltration of both bronchial and alveolar tissue with macrophages, B and T lymphocytes, and eosinophils has been associated with emphysematous destruction. Both the responses are linked to tissue repair and remodeling that increase mucus content of airway lumen and metaplasia of mucous and squamous cells. This leads to a thickened wall and narrowed lumen of conducting airways. Second, emphysema limits air flow by reducing elastic recoil pressure for exhaling air during forced expiration.¹⁸³

α₁-antitrypsin deficiency (AATD) can cause COPD-associated symptoms like emphysematous destruction along with innate inflammation in lung due to an imbalance in protease and antiprotease homeostasis.¹⁸⁴ In lungs, AAT has the major physiological function of protecting the healthy but fragile alveolar tissue from proteolytic damage of neutrophil elastases.¹⁸⁵ The AATD is largely associated with mutations within SERPINA1, resulting in abnormal protein folding, intracellular retention, and consequently low serum levels.¹⁸⁶ However, multiple other factors also contribute to disease severity, and much research is being done to obtain a comprehensive picture for enabling better diagnosis.187, 188, 189, 190, 191, 192, 193, 194, 195, 196 Danozol was found to significantly improve AAT circulating levels¹⁹⁷ without eliciting side effects. Another approach is to inhibit polymerization of AAT by small molecules,¹⁹⁸ peptides,¹⁹⁹ autophagy-enhancing drugs200, 201 (ClinicalTrials.gov: NCT01379469 Tregretol phase II clinical trial, rapamycin²⁰²), and RNA silencing of mutant AAT in liver hepatocytes.203, 204, 205 Intravenous (i.v.) augmentation of plasma-derived AAT (Bayer Biologicals, ZLB Behring, Baxter Healthcare)206, 207 is an established method to raise circulating levels of AAT in blood and bronchoalveolar lavage fluid (BALF), slowing the progression of lung destruction.²⁰⁸

Various routes of administration and vectors have been tested for gene delivery of AAT in various animal models (rat, mouse, and dog), with varying expression efficiency in terms of time and localization (reviewed in detail²⁰⁹). However, only AAVrh.10hAAT (Adverum Biotechnologies, ClinicalTrials.gov: NCT02168686) has entered the phase I/II clinical trial to assess the safety and therapeutic level expression of M1-type AAT in the serum and alveolar epithelial lining fluid.²¹⁰ Connolly et al.²¹ have shown successful expression of AAT with liposome-encapsulated SerpinA1 mRNA in vitro and in vivo after transfection. In an independent study, Michel et al.²¹¹ observed significant expression of AAT in vitro and ex vivo along with a significant reduction in elastase activity.

Steps toward Pharmacologically Auspicious mRNA

In recent years substantial efforts have been made for engineering mRNA with diverse pharmacokinetic properties (Figure 1). Modifications of structural elements such as 5′ Cap, 5′ and 3′ UTRs, poly(A) tail, and the coding region were the main focus.14, 212, 213, 214, 215

Figure 1.

mRNA IVT, Modifications, and Function and Timeline

Overview of milestones in protein supplementation therapy, in vitro transcription, and mRNA modification. White boxes, important milestones for the development of mRNA therapy;²⁵⁰ blue boxes, evolution of different cap structures;215, 220, 322 red, green, and gray boxes, 5′ UTR, 3′ UTR, and poly(A) tail, respectively, the addition of regulatory elements in the modification of mRNA;14, 312, 313 yellow boxes, nucleoside modifications and sequence optimizations in the development for mRNA therapy.215, 230, 238, 240, 314, 317

5′-Capping for Stability and Immune Evasion

5′-capping is vital for the robust translation of mRNA as the natural 7-methylguanosine (m⁷G/Cap0), and it is connected by a 5′-to-5′ triphosphate bridge to the first nucleotide. Translation is initiated by binding to eukaryotic translation initiation factors (eIF4E and eIF4G), and mRNA deterioration is controlled by binding with Dcp1, Dcp2, and DcpS (mRNA-decaying enzymes).57, 216, 217, 218 In in vitro transcription, m⁷G possesses the risk of constructing uncapped or inactive IVT mRNA, as the m⁷G and GTP compete for incorporation. m⁷GpppG cap was the first step to circumvent the restriction of m⁷G, however, substantial proportions of m⁷GpppG analog were incorporated in the reverse direction and thus yielded substandard translational activity.9, 219 Anti-reverse cap analog (ARCA; m₂^7,3′-OGpppG) can counteract the reverse integration and skip degeneration by Dcp2, and, thus, it results in superior translational efficiency and extended half-life.13, 150, 220 Study on viral capping systems reveals that the 2′ ribose position of the first cap-proximal nucleotide is 2′O-methylated to form a Cap 1 structure (^m7GpppN_2′Om N), and, in ∼50% of transcripts, the second cap-proximal nucleotide is 2′O-methylated to form a Cap 2 (^m7GpppN_2′OmN_2′Om) structure.²²¹ Cap 1 2′O-methylation has been described to reduce recognition by pattern recognition receptors (e.g., interferon [IFN]-induced protein with tetratricopeptide repeats-1 and 5 [IFIT1 and IFIT5] and retinoic acid-inducible gene I [RIG-I]) in comparison to Cap 0.222, 223, 224

Modification in UTRs

The poly(A) tail plays an important role in regulating the stability and translational efficiency (half-life) of mRNA by preventing deadenylation by poly-specific nucleases.²²⁵ Integration of the poly(A) tail during IVT mRNA synthesis can be conducted by encoding the poly(A) stretch in the template or by a two-step enzymatic reaction using recombinant poly(A) polymerase.²²⁶ The ideal length of the poly(A) tail is between 120 and 150 nt, and the 3′ end of the poly(A) tail should not be concealed by additional bases.14, 213 5′ and 3′ UTRs also play a vital role in the stability and expression of IVT mRNA by harboring several sequence elements. For example, mRNAs with adenosine in 5′ UTR can form a complex with Lsm1-7 at both the 5′ and 3′ ends and circularize the transcript to inhibit degradation by exosome and Dcp1/2.²²⁷ Adenine uracil (AU)-rich elements in the 3′ UTR can destabilize the mRNA, and they might provide a mechanism to limit the duration of protein production.²²⁸

Post-transcriptional Modifications

Post-transcriptional chemical modifications of RNA are not uncommon, and over 100 modifications are listed by different studies.²²⁹ In mRNA, only a small subset of these naturally occurring modifications is reported to be essential for reducing innate immune response and improving mRNA expression and stability.215, 230, 231 N6-methyladenosine (m6A) is one of the most frequent modifications in eukaryotic mRNA. Insulin-like growth factor 2 (IGF2) mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) preferentially recognize m⁶A mRNA and guard the modified mRNA against decay.²³² Based on the studies of Kormann and Warren et al.,²³³ the first generation of modified mRNAs containing 5-methylcytidine (m5C) or pseudouridine (Ψ-UTP) reduces innate immune responses and enhances translation. TLR3, TLR7, TLR8, and RIG-I activations were significantly reduced when mRNA contained modified nucleosides such as m5C, m6A, 5-methyluridine (m5U), Ψ-UTP, and 2-thiouridine (s2U).20, 150, 230, 234 RNA-dependent protein kinase (PKR) arbitrated the immune response, and translation inhibition (by phosphorylating the alpha subunit of translation initiation factor 2 [eIF-2α]) can be escaped using Ψ-UTP- or m5C-modified nucleosides.²³⁵ m5C is explicitly recognized by the mRNA export adaptor ALYREF, and it increases mRNA-binding affinity and associated mRNA export.²³⁶ Activation of two important components of the innate immune response against unmodified mRNA, the interferon-induced enzymes 2′-5′-oligoadenylate synthetase (OAS) and RNase L, can be limited by Ψ-UTP.²³⁷ N1-methylpseudouridine (N1-mΨ-UTP) is the most used chemical modification in recent studies, and it showed remarkable expression compared to Ψ-UTP-substituted mRNA, even when delivered by different routes in vivo.²³⁸ N1-mΨ-UTP induces a tight binding to RIG-I but failed to activate RIG-I signaling (Figure 2).234, 239

Figure 2.

Processing of IVT mRNA in a Cell

(A) In vitro-transcribed (IVT) mRNA from linearized DNA or PCR-amplified fragment is used to transfect the cell of interest. Step 1: mRNA protection from RNase degradation and mRNA uptake are facilitated by various carriers. Step 2 of mRNA transport and release inside the cell is still unclear. Different capping modification can increase translation in step 3 and also protect from degradation. In steps 3 and 4, the translated protein from delivered mRNA gets transferred to various parts of the cell system based on post-translational modification. For an immunotherapeutic approach, the translated protein needs to get degraded by proteasome to antigen epitopes and delivered to MHC (major histocompatibility complex) class I located in the endoplasmic reticulum. MHC class I mediates surface presentation of the presented epitope to CD8+ cytotoxic T cells.³²⁰ The T cell further initiates the immune response by relocating the antigen and presenting in to MHC II. (B) IVT mRNA cause inflammatory responses and inhibition of mRNA replication as triphosphorylated mRNA or double-stranded RNA (dsRNA) can be recognized by Toll-like receptors 3, 7, and 8 (endosomal innate immune receptors), which can initiate inflammation associated with type 1 interferon (IFN), interleukin-6 and -12, and tumor necrosis factor (TNF).²³⁰ Cytoplasmic receptors, protein kinase R (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation-associated protein (MDA5), and 2′-5′-oligoadenylate synthase (OAS) can detect triphosphorylated mRNA or dsRNA and stalled translation through eIF2α, RNA degradation by ribonuclease L (RNase L), and inhibition of mRNA replication by IFN.323, 324

Codon Optimization

Codon optimization of mRNA uses the degeneracy of the genetic code to substitute specific nucleosides of a mRNA sequence without altering the resulting amino acid composition. Several recent studies have reported a high expression by codon optimization of unmodified and Ψ-UTP-modified mRNA through enriching guanosine and/or cytosine (GC).231, 240 Cas9 activity has been reported to produce significantly higher insertion or deletion (indel) and to be less immunogenic when uridine depletion has been used with 5-methoxyuridine (5moU) modification compared to unmodified and Ψ-UTP-modified Cas9 mRNA.²¹⁵ Pharmacologically favorable mRNA that has undergone modification and sequence optimization still needs to be transferred in lung using carriers, to circumvent the naturally occurring barriers the lung possesses.

Reaching the Lung

In vivo delivery of mRNA therapeutics remains one of the biggest hurdles for mRNA-based therapies in general. Apart from the fragility of the mRNA molecules and the ubiquitous existence of RNases, there are two main obstacles in the delivery of mRNA in vivo: targeting specific lung cell populations and crossing the cellular membrane. For solving the latter part, nanocarriers as a delivery system have gained increased attention. Therefore, this review focuses on the benefits and obstacles in the use of mRNA-nanocarrier complexes.

In terms of transport of mRNA to a specific tissue, the route of administration plays an important role when discussing the hurdles of organ-specific mRNA therapy. Focusing on the lung, two main routes of administration were investigated in the past: i.v. application of mRNA or mRNA-complexes to reach the lung from the vascular structures and i.t. delivery of mRNA therapeutics via dry powder insufflation or high-pressure liquid suspensions using a microsprayer.150, 166, 241, 242, 243, 244, 245, 246

Intravenous application of RNAs leads to a systemic distribution of the therapeutic throughout the whole organism.²⁴⁷ This can be beneficial for certain pulmonary diseases like CF affecting other organs; on the contrary, only a small percentage of an active agent reaches the desired location.²³⁹ A substantial amount of mRNA is removed from the bloodstream, especially in the liver and spleen.²⁴⁸ This increases the amount of mRNA needed for reaching effective dose levels in the lung.²⁰ Another hurdle emerges in the lung itself. The lung has a capillary system that consists of mostly small and non-fenestrated capillaries.²⁴⁹ This is very efficient for gas exchange at the alveoli, but it does not permit a free exchange of larger molecules out of the blood into the tissue. This phenomenon affects mRNA therapeutics by reaching cells in close proximity, while cells farther away from capillaries are harder to reach by i.v. administration.²⁵⁰ In general, i.v. application of mRNA has the advantage of circumventing some initial innate defense systems and lung barriers while fighting with the problems of systemic application of dispersed distribution and losing the targeted administration toward the lung.

In contrast, i.t. administration of therapeutic substances gives the advantage of local application of mRNA in the lung and airways. Moreover, the alveolar surface area is large and suitable for drug absorption, and the epithelial barrier is thin,²⁴² which could facilitate the delivery of mRNA to lung cells. While endothelial cells and lung stem cells are difficult to access, alveolar cells, epithelial cells, and macrophages can be targeted by i.t. administration.²⁵¹ A potential therapeutic has to be appropriately formulated to be able to reach deep lung surfaces. This includes a particle size of 4–7 μm in diameter²⁵² for targeting the tracheobronchial region and 1–3 μm for targeting the alveolar region, when preparing a powder for insufflation or nebulizing a liquid (Figure 3).²⁵³

Figure 3.

Deposition of Nanoparticles for Delivery in the Lung after Intratracheal or Intravenous Administration

Left: intratracheal instillation requires a particle size of 1–3 μm to reach the alveoli efficiently; particles from 4 to 7 μm are mainly distributed to the upper airways and main bronchioles, and particles smaller than 1 μm are exhaled again.252, 318 Right: inhaled nanoparticles can enter bronchial as well as alveolar epithelium; nanoparticles can enter lymph and blood circulation to be delivered to secondary organs.³¹⁹ Intravenous injection can systemically deliver nanoparticles to a limited part of the alveolar epithelium due to small and non-fenestrated endothelial cells in the capillaries in the lung.²⁵⁰

To reach deep lung structures and alveoli, mRNA therapeutics still has to pass the respiratory mucus. In a non-pathological condition, the thickness of the mucus is between 2 and 5 μm in the bronchi and 10 and 30 μm in the trachea,²⁵⁴ while in CF asthma and COPD the mucus layer is reported to be much thicker.²⁵⁵ The gel-on-brush model of Button et al.²⁵⁶ states that ciliary movement transports all material out of the lung at a rate of 3.6 mm/min,²⁵⁷ while a layer of mucins and glycoproteins form a fine mesh preventing large particles from reaching the periciliary layer and epithelial cells in the lung.²⁵⁶ The mucus layer, lining epithelial cells from the nose to the terminal bronchioles, also affects nanocarriers by sterical obstruction or direct interaction from diffusion to the target cells.²⁵¹ Different independent studies showed a correlation between nanocarrier size and mobility in respiratory mucus. Sanders et al.²⁵⁸ and Dawson et al.²⁵⁹ reported that nanospheres at the size of ∼100 nm were able to pass more or less freely through CF sputum compared to nanospheres larger than 500 nm. Broughton-Head et al.²⁶⁰ detected that CF sputa of three CF patients contained a mesh with a mean size of 300 ± 106 nm, 578 ± 191 nm, and 711 ± 328 nm, respectively, providing evidence for the distinct transport parameters of different-sized nanospheres. Studies by Stern et al.,²⁶¹ Kitson et al., ²⁶² and Ferrari et al.²⁶³ suggested that not only the retention by the mesh structure in the CF sputum but also the direct interaction with free DNA present in CF sputum can reduce the gene transfer of 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC)-cholesterol (Chol)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-based lipoplexes up to 20-fold.

Respiratory mucus is not the only fluid presenting a barrier for the nanocarrier delivery of mRNA. Also, the alveolar fluid is known to inhibit cationic lipid nanocarriers, presumably by disintegration of the lipoplexes by negatively charged lipids in the surfactant.²⁵¹ In contrast, PEI and dendrimer polyamidoamine (PAMAM)-based gene delivery was observed to be resistant to the effects of pulmonary surfactant in vitro and in vivo.264, 265 Moreover, Exosurf (a synthetic surfactant) has been reported to increase the efficiency of PAMAM-pDNA complexes in vitro.²⁶⁵

To overcome the obstacles presented by both respiratory mucus and pulmonary surfactant, various approaches have been tested. Mucolytic agents, which degrade the biopolymer network built up out of DNA, actin, and mucins, are a focus of many research groups.258, 259, 260, 263 Recombinant human DNase (rhDNase) liquifies CF mucus by cleaving DNA chains in the biopolymer, and it has a direct effect on the mobility of nanocarriers of CF sputum. It increased the mobility of 270-nm nanospheres and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/DOPE-based lipoplexes 2.5-fold and 1.4-fold, respectively.258, 266 N-Acetylcysteine (NAC), a clinically used mucolytic agent for asthma, COPD, and CF patients, reduces the disulfate bonds between mucins and lowers the viscosity and elasticity of respiratory mucus.267, 268 NAC-mediated mucus clearance of an ex vivo sheep trachea model increased gene transfer via p-ethyl-dimyristoylphosphadityl choline (EDMPC)-Chol lipoplexes and PEI-based polyplexes 20-fold and 10-fold, respectively. If the nasal epithelium of mice is treated with NAC 30 min before the administration of EDMPC-Chol lipoplexes, the gene expression can be increased up to 100-fold.²⁶³

An alternative strategy is to coat nanocarriers with biocompatible hydrophilic but biologically inert polymers²⁵¹ to shield the nanocarriers from respiratory fluids and surfactant. GL67 (genzyme lipid 67)/DOPE lipoplexes can be coated with polyethylene glycol (PEG), and they have been reported to circumvent the adverse effects of CF mucus during gene transfection in vivo.²⁶⁹ Maisel et al.²⁷⁰ reported that 10–40 kDa PEG-coated nanocarriers can diffuse through the mucus as a mucoinert particle.

Nanotransporters to Target the Lung

The labile nature of mRNA and immunogenicity are the biggest hurdles of mRNA therapeutics. As discussed above, the immunogenicity has been overcome by chemical modifications; however, the instability of mRNA under physiological conditions requires additional action. Electroporation,²⁷¹ gene gun,²⁷² microinjection,²⁷³ and sonoporation²⁷⁴ have been investigated for mRNA delivery; however, these are restricted to ex vivo manipulation and ill suited for systemic delivery. Therefore, suitable mRNA carriers should exhibit the following functions: protection from RNase degradation, evasion of direct renal clearance, avoidance of nonspecific interaction, facilitation of mRNA stability, and sufficient mRNA loading and release.275, 276, 277 The physiochemical properties, such as hydrodynamic diameter, shape, size, surface charge, solubility, flexibility, stability, formulation, and body composition with regard to route of administration, decide the target binding bio-distribution as well as the clearance of the nanocarriers. As many materials used to construct nanoparticles are toxic or potentially toxic, biocompatibility and biodegradability become key factors. Since our focus is delivering mRNA therapeutics to the lung, we cover biomaterials such as lipids, polymers, and combined formulations that are developed for delivery to the lung.

Lipid-based nanoparticles (LNPs) or lipoplexes have gained popularity since the beginning of drug and nucleic acid delivery.9, 231, 240, 278, 279These have the significant advantages of easy synthesis, scalability, low batch variability, and biocompatibility.280, 281, 282 Commercially available lipoplexes, such as RNAiMAX, Stemfect, and Megafectin, have been successfully used in transfecting mRNA in vivo.279, 283, 284, 285 Cationic lipids, such as 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), DOTAP, and zwitterionic DOPE, have been used alone or in combination, as these readily form complexes with mRNA. Variants to reduce toxicity and immunogenicity associated with cationic lipids and with improved efficacy have been developed. Additionally, the ratio of components substantially affects LNP efficacy. These include ionizable lipidic systems that can reduce toxicity by possessing a neutral charge at physiological pH²⁸⁶ and ionizable lipid nanoparticles consisting of ionizable lipid, cholesterol (hydrophobic), helper lipid (DOPE or 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC]), and PEG lipids. DOPE enhances efficacy by promoting membrane fusion (cell and endosomal), and PEG lipids prevent reticuloendothelial clearance and reduce opsonization by serum proteins.

LNP-mediated mRNA delivery has been extensively used in protein replacement therapies, vaccines, and cancer immunotherapies.²⁷⁶ Earlier work by Litzinger et al.²⁸⁷ showed that cationic liposomes of size 2.0 μm are transiently taken up by the lung, followed by rapid distribution to the liver. Similarly, efficient pulmonary endothelial delivery of plasmid was achieved with a lipid vector consisting of DOTAP liposomes, protamine, and oligo deoxynucleotides,²⁸⁸ with minimum cytotoxicity and release of proinflammatory cytokines. The landmark clinical trial of CFTR gene therapy with pGM169¹⁷⁹ and multiple other clinical trials with nasal delivery tested lipid nanoparticle cholest-5-en-3-ol(3β)-,3-[(3-aminopropyl)[4-[(3-aminopropyl)amino] butyl] carbamate] (GL67A) due to its desirable stability during aerosolization,²⁸⁹ gene transfer potency,²⁹⁰ and well-characterized safety parameters.²⁹¹ The Wendel group²⁹² has shown DOPE liposomes as potential transfection agents for AAT mRNA, resulting in prolonged protein production of AAT in vitro with improved stability of mRNA in liposomes for up to 80 days, without the loss of transfection efficacy. Alexion Pharmaceuticals has shown human AAT expression in both mouse liver and lung upon i.v. injection of mRNA-ionizable LNP complex after 24 h.²¹ Both approaches require further testing on knockout mouse models²⁹³ to check its efficiency as a therapy for AATD.

Lipid-enabled and unlocked nucleomonomer agent-modified RNA (LUNAR) technology of Arcturus Therapeutics employs biodegradable ionizable lipids (ATX, Arcturus Therapeutics’s proprietary lipid) that have shown no adverse events, hepatotoxicity, weight loss, or innate or adaptive immune reactions in response to treatment with repeated dosing of up to 4 months.²⁹⁴ Ramaswamy et al.²⁹⁴ observed faster translation (within 6 h) and major deposition of LUNAR-encapsulated mRNA in mouse liver with i.v. injection. Arcturus holds multiple patents on nanoparticles for RNA delivery with the potential of lung epithelial delivery via nebulization. Translate Bio holds a patent on multiple lipid nanoparticles with its collaborator at Massachusetts Institute of Technology (MIT) and Imperial College London, and it is the first one to enter clinical trials with LNPs (ClinicalTrials.gov: NCT03375047) for mRNA-based CF therapy. Valera (by Moderna) has reported efficacy of modified hemagglutinin mRNA-LNP-formulated vaccines against H7N9 and H10N8 influenza virus (presently at clinical phase I), when immunized intradermally or intramuscularly in mice, ferrets, and non-human primates²⁹³ (one must note that localization in lungs is not required for immunization).

Though not as equally advanced as LNPs, polymer-based nanoparticles have shown considerable potential in aiding therapeutics. Cationic polymers (linear or branched) can enable nucleic acid shuttling across membranes by compactly packing them into nanoplexes, and they can help in cellular uptake via endocytosis.²⁹⁵ PEI is the vastly studied polymer for gene or oligonucleotide delivery,296, 297 however, toxicity due to nondegradability, high molecular weight (>20 kDa), and its highly branched formulations has limited its clinical applications.²⁹⁸ The positive charge attributes to interaction with serum proteins (negatively charged), resulting in their aggregation and increase in size that causes toxicity, similar to that of cationic liposomes. Therefore, different groups have tried to modify PEI and achieve higher transfection with lower toxicity, which includes reducing size²⁹⁹ (mRNA release), reducing molecular weight,³⁰⁰ or using additives.³⁰¹

Poly (L-lysine) (PLL), poly(2-(dimethylamino)ethyl methacrylate) (p[DMAEMA]), and PLGA are well known polymers, and diblock³⁰² and triblock polymers³⁰³ have shown encouraging results for nucleic acid transfection. As already discussed, mucus acts as a strong barrier, and mucoadhesive particles can increase the residence time while bulking up the nanoparticle. PEGylated NPs (diblock copolymer composed of PLGA and PEG [PLGA-PEG], namely, PLGA-PEG mucous penetrating particle [MPP]) of >200-nm size are known to penetrate mucus and CF sputum.304, 305 Based on this, Schneider et al.³⁰⁵ and others have shown that MPP (≤300 nm in diameter) exhibits improved particle distribution and lung retention.¹⁵⁰ Chitosan (mucoadhesive) coating20, 306 of PLGA nanoparticles has shown successful delivery of SP-B mRNA and hCFTR mRNA in mouse models, significantly improving survival³⁰⁷ and lung function,³⁰⁸ respectively. It is possible that these enable deeper lung delivery instead of mere epithelial delivery, due to which we could observe the survival of an SP-B-deficient mouse model³⁰⁹ when corrected with zinc-finger nuclease (ZFN) mRNA and donor template. In an attempt to develop nanoparticles for pulmonary delivery, Ethris used mRNA complexed with a polymer scaffold of poly (acrylic acid) of 20 kDa grafted with oligoalkylamines, which showed delivery in cranial parts of pig lung upon nebulization.⁵⁴ A study about the exact localization of developed nanoparticles in different lung cell populations would benefit the scientific society in moving toward disease-specific targeted therapy for different lung diseases.

Further, combinations of lipids and polymers have been tested as nanocarriers. These include self-assembling nano-micelles formed by copolymer consisting of polyamino acid block and PEG with mRNA at core. Commonly used polyamino blocks include poly(Nʹ-(N-(2-aminoethyl)-2-aminoethyl) aspartamide (PAsp[DET]), which has shown protein expression in nasal neurons with mRNA coding for brain-derived neurotrophic factors (BDNFs).³¹⁰ The complex also reduced apoptosis when injected with anti-apoptotic protein B cell lymphoma (Bcl-2) mRNA in a fulminant hepatitis mouse model.³¹¹ The group of Daniel G. Anderson has developed various nanoparticles to be used in therapeutics,14, 312 among which the polymer lipid combination of poly (β-amino esters) (PBAEs) and PEG has shown greater potential in delivering mRNA to lung via i.v. injection,³¹³ aiding successful systemic delivery. Desrosiers et al.³¹⁴ have developed amine-modified polyester-based nanocarriers in combination with triblock copolymers, with specific mRNA delivery to lung. Though degradable and optimized for serum stability and reduced toxicity, these need to be further tested for inflammatory reactions before clinical translation. Recent publication by Patel et al.³¹⁵ on hyperbranched PBAEs has shown ease in nebulization and uniform distribution of mRNA in all 5 lobes of lung, with no measured local or systemic toxicity.

To further increase the specificity of nanocarrier-based delivery, receptor-based technologies have been tested. Arrowhead Pharmaceuticals developed an asialoglycoprotein receptor-targeting nanocarrier to specifically administer an RNAi molecule (targeted RNAi molecule [TRiM]) to reduce the accumulation of AAT protein²³⁰ for AAT-related liver diseases. If combined with pulmonary delivery of AAT mRNA, it can act as a complete therapy for diseases like AATD. A receptor-based method can also be developed for the lung epithelium, but specific markers of lung epithelial cells have to be identified to avoid cross-reactivity with other epithelial cell linings. In another approach for enhancing translation and reducing the degradation of mRNA upon entry, a delivery system has been tested that employs translation initiation factor eIF4E with cationic polyamine. A study showed that these nanoparticles induced mRNA expression in mouse lung upon systemic delivery.³¹⁶ Other nanoparticles developed for lungs include gelatin nanocarriers crosslinked with genipin, monomethoxypoly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly-l-lysine (mPEG-PLGA-PLL) triblock copolymers, MUC-1 aptamer-functionalized hybrid nanoparticles, drug-loaded liposomes, anionic PAMAM dendrimers, and a recently developed virus-inspired polymer for endosomal release (VIPER).215, 230, 238, 240, 250, 252, 317, 318, 319, 320 However, as discussed before, these must be extensively tested for compatibility with mRNA.

Conclusions

This paper focused on respiratory diseases and associated cell populations of the lung. To understand the disease pathology and possible countermeasures, the physiological aspects of various lung cells have to be determined. This includes the awareness of connections of epithelial cells with alveolar cells, serous cells (goblet cells), and also stem and progenitor cells, as lung diseases may originate from a single defect but can affect the whole lung. Recent developments already produced major advancements in therapies such as chemical modulators for CF, but they also produced therapy resistance or were only effective for certain variants of a disease. This makes mRNA-based protein supplementation therapy a viable alternative for diseases such as CF, SP-B deficiency, asthma, IPF, and COPD while offering a treatment independent of the underlying mutational status. Furthermore, stem cells of the lung can also be a target for mRNA-based CRISPR/Cas therapies that hold the potential for permanent cures for monogenetic lung diseases, such as CF, SP-B deficiency, and AATD.

The therapeutic potential of mRNA for protein supplementation therapy was widely unrecognized due to its instability for a long time. Over the last decades, an appreciation of mRNA as the molecule connecting the world of proteins and DNA is renewing the focus of research on mRNA.9, 20, 215 The research focuses on the properties of RNA to increase or modulate stability and evade immune recognition as well as delivery of mRNA specifically to the lung and other organs. The use of naturally occurring nucleoside modifications has diminished the recognition of mRNA by the innate immune system.230, 235 These modifications also help in improving the stability and expression of mRNA. This is further promoted by modifications like 5′-capping and the addition of a poly(A) tail as well as modifications in the 3′ and 5′ UTRs. Sequence optimization increases expression and lowers immunogenicity of mRNA therapeutics. The most optimized RNA still needs to reach the target cell to be expressed into a protein.

The problem of delivery consists of three main parts: (1) how to find a suitable route of administration; (2) as mRNA for protein supplementation therapy usually are of substantial size and negatively charged, they will not cross cellular membranes unfacilitated; and (3) the composition of mRNA and nanocarriers to maintain the optimal stability of mRNA nanocarrier complexes. Looking at protein supplementation therapies for the lungs, i.t. delivery gives the ability to apply mRNA therapeutics locally and concentrated in the lungs. To overcome defense barriers like the respiratory mucus and alveolar fluids as well as the cellular membranes, nanocarriers developed into a favorable type of vehicle for mRNA. To date, research indicates that small nanocarriers (∼100 nm) together with a polymer coating and the potential application of a mucolytic agent can improve the stability of the mRNA nanocarrier complex and mRNA uptake into the target cell. Nonetheless, the mechanism of how a nanocarrier facilitates the cellular uptake of mRNA is still not fully understood.

All in all, the pieces needed for an effective protein supplementation therapy in the lung are available in various preclinical and clinical stages. The next task is to find a formulation to bring all of these components together—route of administration, carrier, mRNA sequence and modification—to form a working therapy for patients. The first clinical trials in the slowly evolving field of protein supplementation therapy show that the concept is viable (ClinicalTrials.gov: NCT02935712 and NCT03375047; see also Table 1).

Author Contributions

I.S., A.K.M.A.H., and B.W. wrote the main parts of the article and produced graphics. P.W. helped in the meticulous preparation of the manuscript. M.S.D.K. drafted the final version of the manuscript. All authors read and approved the final manuscript.

Conflicts of Interest

M.S.D.K. holds a patent on RNA modification (EP2459231B1). M.S.D.K. and A.K.M.A.H. hold a European patent on delivery of hCFTR cmRNA complexed with nanoparticles (17169561.2-1401).