Oligonucleotide Therapies for the Lung: Ready to Return to the Clinic?
Authors: Manish Kumar, Sterghios A. Moschos
Journal: Molecular Therapy (2017)

The most promising targets for treating lung diseases are often intracellular molecules recalcitrant to conventional pharmacotherapy. In this respect, antisense oligonucleotides (ASOs) would be valuable tools to treat airways disease as the plurality of their potential mechanisms of action is well-established, their efficiency has been extensively validated in vitro, and their potential clinical value has been demonstrated with numerous regulatory approvals. Following the two eye-injectable formulations fomivirsen and pegaptanib, the recent successes of the systemically active ASOs mipomersen, defibrotide, and eteplirsen have been underscored by the remarkable clinical outcomes of the intrathecally dosed RNA splicing modulator nusinersen, a 2nd generation 2′-methoxy ethyl ASO indicated for spinal muscular atrophy.1Christie-Brown V. Mitchell J. Talbot K. The SMA Trust: the role of a disease-focused research charity in developing treatments for SMA.Gene Ther. 2017; 24: 544-546Crossref PubMed Scopus (7) Google Scholar For lung disease, it is also clear that relatively facile topical administration, i.e., inhaled or intranasal dosing, results in efficient and uniform distribution across the lumen of large and small airways, accessing both epithelial and sub-epithelial compartments. Despite high confidence in the treatment rationale and mechanisms of action, it appears that oligonucleotides delivered topically to the lung either rapidly access circulation via epithelial transcytosis or are removed by alveolar macrophages, exerting minimal, if any, action in the cytosol of cells relevant to lung disease. Moreover, the use of cell-penetrating peptides, liposomes, or nanoparticle delivery systems has, so far, not been shown to eliminate circulatory clearance; may activate immune responses; or drive macrophage phenotypic changes that, together or in isolation, may present risks to patients.2Moschos S.A. Usher L. Lindsay M.A. Clinical potential of oligonucleotide-based therapeutics in the respiratory system.Pharmacol. Ther. 2017; 169: 83-103Crossref PubMed Scopus (22) Google Scholar Importantly, the recently published study by Crosby et al.3Crosby J.R. Zhao C. Jiang C. Bai D. Katz M. Greenlee S. Kawabe H. McCaleb M. Rotin D. Guo S. Monia B.P. Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.J. Cyst. Fibros. 2017; (Published online May 20, 2017)https://doi.org/10.1016/j.jcf.2017.05.003Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar signifies a new addition to the evidence that airways disease, and cystic fibrosis (CF) patients in particular, may stand to benefit greatly from splicing modulator or RNase H-active ASO therapeutics when 3rd generation, so-called bi-cyclic or bridged (2′-4′ constrained) nucleoside chemistry is used. Specifically, the authors use constrained ethyl (cEt) as opposed to the simpler and more widely available constrained methyl, or "locked" nucleic acid (LNA) chemistry.4Pallan P.S. Allerson C.R. Berdeja A. Seth P.P. Swayze E.E. Prakash T.P. Egli M. Structure and nuclease resistance of 2′,4′-constrained 2′-O-methoxyethyl (cMOE) and 2′-O-ethyl (cEt) modified DNAs.Chem. Commun. (Camb.). 2012; 48: 8195-8197Crossref PubMed Scopus (57) Google Scholar Representing continued efforts by Ionis Pharmaceuticals to exploit the affinity and stability advantages of bridged nucleic acids and their long-standing interest in the airways disease space,2Moschos S.A. Usher L. Lindsay M.A. Clinical potential of oligonucleotide-based therapeutics in the respiratory system.Pharmacol. Ther. 2017; 169: 83-103Crossref PubMed Scopus (22) Google Scholar Crosby et al.3Crosby J.R. Zhao C. Jiang C. Bai D. Katz M. Greenlee S. Kawabe H. McCaleb M. Rotin D. Guo S. Monia B.P. Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.J. Cyst. Fibros. 2017; (Published online May 20, 2017)https://doi.org/10.1016/j.jcf.2017.05.003Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar report on the potentially therapeutic effect of an inhaled 16-mer RNase H-active ASO against Scnn1a, a subunit of the amiloride-sensitive luminal sodium uptake channel (ENaC) for the treatment of CF. CF is an autosomal recessive disorder characterized by mucous over-secretion in epitheliated tissue, such as the respiratory, intestinal, and pancreatic duct lumens. In the airways, additional physiological changes include reduction of airway surface liquid, mucus dehydration, and mucus accumulation. The resulting mucous plugs cause inflammation and promote infection and tissue damage, resulting in 80% mortality and a median life expectancy of 37 years of age.5Collawn J.F. Lazrak A. Bebok Z. Matalon S. The CFTR and ENaC debate: how important is ENaC in CF lung disease?.Am. J. Physiol. Lung Cell. Mol. Physiol. 2012; 302: L1141-L1146Crossref PubMed Scopus (64) Google Scholar The majority of patients suffer loss-of-function mutations in the CF transmembrane conductance regulator (CFTR) gene, but the precise site of its expression across the respiratory tree remains unclear due to the very low expression levels of the transcript. However, as CFTR encodes for a luminal chloride secretion channel, extensive research6Hobbs C.A. Da Tan C. Tarran R. Does epithelial sodium channel hyperactivity contribute to cystic fibrosis lung disease?.J. Physiol. 2013; 591: 4377-4387Crossref PubMed Scopus (74) Google Scholar points to solute imbalance in the airway lumen as the mechanism that underlies disease. Elimination of ENaC hyperactivity in CF, which is thought to contribute to luminal solute imbalance and airway surface liquid volumes, has been long proposed as a promising treatment approach if persistent, rather than transient, ENaC activity downregulation can be achieved.6Hobbs C.A. Da Tan C. Tarran R. Does epithelial sodium channel hyperactivity contribute to cystic fibrosis lung disease?.J. Physiol. 2013; 591: 4377-4387Crossref PubMed Scopus (74) Google Scholar This is supported by complementary data, including knockout of Nedd4L, a suppressor of ENaC that induces a CF-like phenotype in mice and is lethal by 3 weeks of age. Thus, using two different mouse models of CF, an adult mouse βENaC overexpression model and a Nedd4L knock-out neonate mouse model, the authors showed that ENaC-specific ASOs resulted in ∼50% (neonate) to ∼85% (adult) reduction of ENaC mRNA levels. Physiologically, the treatment also reduced mucus metaplasia, inflammation, and airway hyper-responsiveness (AHR) in an experimental model of CF and reduced mucous hypersecretion and inflammation in neonate airways. Taken together, these results indeed suggest that treating CF by oligonucleotide therapies might be within clinical reach. These results follow the recent publication of relevant 3rd party patents (US20150211010) and data7Igreja S. Clarke L.A. Botelho H.M. Marques L. Amaral M.D. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.Hum. Mutat. 2016; 37: 209-215Crossref PubMed Scopus (51) Google Scholar on the in vitro utility of exon skipping antisense for known CFTR mutations and join the continued debate around whether ENaC is indeed a suitable target for CF treatment.6Hobbs C.A. Da Tan C. Tarran R. Does epithelial sodium channel hyperactivity contribute to cystic fibrosis lung disease?.J. Physiol. 2013; 591: 4377-4387Crossref PubMed Scopus (74) Google Scholar On the other hand, despite similarly exciting preclinical disease biomarker data with viral infection, asthma, and emphysema models, all clinical efforts to date in respiratory disease oligonucleotide therapeutics have been poor.2Moschos S.A. Usher L. Lindsay M.A. Clinical potential of oligonucleotide-based therapeutics in the respiratory system.Pharmacol. Ther. 2017; 169: 83-103Crossref PubMed Scopus (22) Google Scholar Thus, studies with three different 2nd generation ASO compounds developed originally by Ionis (previously known as ISIS), AIR645 against interleukin-13 (IL-13)/IL-4 (Altair Therapeutics, Inc.), ASM8 dual ASO formulation against CCR3 (Topigen/Pharmaxis Pharma), as well as the siRNA compound ALN-RSV01 against RSV1 (Alnylam) have failed in phase II clinical trials.2Moschos S.A. Usher L. Lindsay M.A. Clinical potential of oligonucleotide-based therapeutics in the respiratory system.Pharmacol. Ther. 2017; 169: 83-103Crossref PubMed Scopus (22) Google Scholar Across these studies, despite high confidence in the treatment rationale and drug mechanism of action, it appeared that oligonucleotides delivered topically to the airways either rapidly entered circulation or were removed by alveolar macrophages, perhaps too efficiently, offering limited (<50%) benefit at the tissue level and inducing a foamy macrophage phenotype.8Forbes B. O'Lone R. Allen P.P. Cahn A. Clarke C. Collinge M. Dailey L.A. Donnelly L.E. Dybowski J. Hassall D. et al.Challenges for inhaled drug discovery and development: Induced alveolar macrophage responses.Adv. Drug Deliv. Rev. 2014; 71: 15-33Crossref PubMed Scopus (60) Google Scholar, 9Moschos S.A. Frick M. Taylor B. Turnpenny P. Graves H. Spink K.G. Brady K. Lamb D. Collins D. Rockel T.D. et al.Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.Mol. Ther. 2011; 19: 2163-2168Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 10Alton E.W. Boushey H.A. Garn H. Green F.H. Hodges M. Martin R.J. Murdoch R.D. Renz H. Shrewsbury S.B. Seguin R. et al.Clinical expert panel on monitoring potential lung toxicity of inhaled oligonucleotides: consensus points and recommendations.Nucleic Acid Ther. 2012; 22: 246-254Crossref PubMed Scopus (24) Google Scholar Notably, in 1998, Ionis published on the rapid systemic access of 30-mer 2nd generation ASOs delivered topically to the lung,11Nicklin P.L. Bayley D. Giddings J. Craig S.J. Cummins L.L. Hastewell J.G. Phillips J.A. Pulmonary bioavailability of a phosphorothioate oligonucleotide (CGP 64128A): comparison with other delivery routes.Pharm. Res. 1998; 15: 583-591Crossref PubMed Scopus (60) Google Scholar and evidence of systemic access and even urinary elimination at the lower limit of assay detection for this compound class was reported even in man.12Zamora M.R. Budev M. Rolfe M. Gottlieb J. Humar A. Devincenzo J. Vaishnaw A. Cehelsky J. Albert G. Nochur S. et al.RNA interference therapy in lung transplant patients infected with respiratory syncytial virus.Am. J. Respir. Crit. Care Med. 2011; 183: 531-538Crossref PubMed Scopus (174) Google Scholar More recently, 16-mer 3rd generation LNA ASOs, chemically comparable to the 3rd generation cEt chemistry4Pallan P.S. Allerson C.R. Berdeja A. Seth P.P. Swayze E.E. Prakash T.P. Egli M. Structure and nuclease resistance of 2′,4′-constrained 2′-O-methoxyethyl (cMOE) and 2′-O-ethyl (cEt) modified DNAs.Chem. Commun. (Camb.). 2012; 48: 8195-8197Crossref PubMed Scopus (57) Google Scholar employed by Crosby et al.,3Crosby J.R. Zhao C. Jiang C. Bai D. Katz M. Greenlee S. Kawabe H. McCaleb M. Rotin D. Guo S. Monia B.P. Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.J. Cyst. Fibros. 2017; (Published online May 20, 2017)https://doi.org/10.1016/j.jcf.2017.05.003Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar were also shown to rapidly reach circulation and end up in the urine as early as 15 min after intratracheal dosing in mice. Crucially, no on-target effect was detected in lung cells after in vivo dosing using a high precision, high power bioanalytical approach involving comparisons of fixed numbers of pneumonocytes obtained by tissue disruption and cell sorting by cell type: epithelial cells, macrophage cells, or mixed cell populations representative of the entire lung cell type diversity.6Hobbs C.A. Da Tan C. Tarran R. Does epithelial sodium channel hyperactivity contribute to cystic fibrosis lung disease?.J. Physiol. 2013; 591: 4377-4387Crossref PubMed Scopus (74) Google Scholar As 16-mer oligonucleotides have a lower molecular weight than 30-mer oligonucleotides, the observation of circulatory access was not entirely unexpected. Indeed, many biopharmaceutical peptides and proteins are very efficient at crossing the pulmonary epithelia into circulation.13Morales J.O. Fathe K.R. Brunaugh A. Ferrati S. Li S. Montenegro-Nicolini M. Mousavikhamene Z. McConville J.T. Prausnitz M.R. Smyth H.D.C. Challenges and Future Prospects for the Delivery of Biologics: Oral Mucosal, Pulmonary, and Transdermal Routes.AAPS J. 2017; 19: 652-668Crossref PubMed Scopus (92) Google Scholar However, because imaging showed ASO association with cells across the entire lung, lack of any appreciable effect was surprising. Furthermore, building on the predicate work with 30-mer 2nd generation phosphorothioate ASOs, the 2011 study demonstrated that the phosphorothioate backbone drove alveolar macrophage loading; in addition, after exit from the airways, it allowed for retention in circulation, followed by biological activity in the liver and kidney.9Moschos S.A. Frick M. Taylor B. Turnpenny P. Graves H. Spink K.G. Brady K. Lamb D. Collins D. Rockel T.D. et al.Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense.Mol. Ther. 2011; 19: 2163-2168Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar More importantly, from a patient safety perspective, a separate clinical expert panel on the safety of inhaled drugs recommended studies of the foamy macrophage phenotype induced by oligonucleotides upon topical dosing to the lung to rule out long-term immune risks.10Alton E.W. Boushey H.A. Garn H. Green F.H. Hodges M. Martin R.J. Murdoch R.D. Renz H. Shrewsbury S.B. Seguin R. et al.Clinical expert panel on monitoring potential lung toxicity of inhaled oligonucleotides: consensus points and recommendations.Nucleic Acid Ther. 2012; 22: 246-254Crossref PubMed Scopus (24) Google Scholar This matter is acutely relevant to airways disease when such phenotypes have been linked with deterioration of respiratory status, at least in asthma.14Zasłona Z. Przybranowski S. Wilke C. van Rooijen N. Teitz-Tennenbaum S. Osterholzer J.J. Wilkinson J.E. Moore B.B. Peters-Golden M. Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma.J. Immunol. 2014; 193: 4245-4253Crossref PubMed Scopus (129) Google Scholar Despite these reports, Crosby et al.3Crosby J.R. Zhao C. Jiang C. Bai D. Katz M. Greenlee S. Kawabe H. McCaleb M. Rotin D. Guo S. Monia B.P. Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.J. Cyst. Fibros. 2017; (Published online May 20, 2017)https://doi.org/10.1016/j.jcf.2017.05.003Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar claim lack of systemic access or activity in the kidney following inhaled administration of their 3rd generation ENaC ASOs. Interestingly, ENaC is expressed only in the collecting ducts and distal convoluted tubules of the kidney—sites acknowledged by the authors as refractory to ASO treatment. Thus, with no effort undertaken to quantify the ASO localization after inhalation in the kidney, these results perhaps are more relevant to showing which kidney cells are not accessible by 3rd generation ASOs, rather than confirming lack of access to this tissue. The authors also pursue a conceptually smart approach in anticipating the efficacy of their ASO chemistry in the lung. Thus, they replicate the Nedd4L knockout effect on inducing CF-like phenotype by administering a Nedd4L ASO to wild-type mice. Unfortunately, looking at the pro- and anti-inflammatory effects of the CF-inducing Nedd4L ASO and the CF symptom-alleviating ENaC ASO, respectively, it is difficult to disentangle the extent of the on-target mechanism of action for both of these oligonucleotides. While molecular evidence of ENaC mRNA cleavage is taken for granted by the authors, at the same time, it is well established that minimal motifs may activate immune responses where others may indeed unexpectedly suppress them.15Hartmann G. Nucleic Acid Immunity.Adv. Immunol. 2017; 133: 121-169Crossref PubMed Scopus (133) Google Scholar Yet, despite the hard lessons in the field, no immune profiling appears to have been attempted by Crosby et al.3Crosby J.R. Zhao C. Jiang C. Bai D. Katz M. Greenlee S. Kawabe H. McCaleb M. Rotin D. Guo S. Monia B.P. Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.J. Cyst. Fibros. 2017; (Published online May 20, 2017)https://doi.org/10.1016/j.jcf.2017.05.003Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar for either of these ASOs. Thus, Opko Health abandoned the first phase III siRNA candidate bevasiranib due to lack of evidence that the trial would meet its primary outcome. A close analogue to bevasiranib targeting the same VEGF sequence was independently shown16Kleinman M.E. Yamada K. Takeda A. Chandrasekaran V. Nozaki M. Baffi J.Z. Albuquerque R.J. Yamasaki S. Itaya M. Pan Y. et al.Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.Nature. 2008; 452: 591-597Crossref PubMed Scopus (803) Google Scholar to activate TLR3 to the point of inhibiting choroidal neovascularisation. These data have raised questions around the TLR3 agonism potential of bevasiranib and other clinical siRNA candidates. The authors of this study also showed that many other siRNAs were TLR3 agonists and that a sequence-irrelevant, luciferase-targeting, alternately 2'-O-methylated siRNA also inhibited choroidal neovascularization when injected intraperitoneally in mice. At the same time, while bevasiranib was not linked to adverse events in the clinic, it is presently unclear how weak TLR agonism by siRNA or other oligonucleotides manifests clinically or how this affects biomarker responses in preclinical models of respiratory disease. However, these events support the rationale of applying careful, appropriate exploratory controls when inflammatory intervention is attempted before concluding fully on-target mechanisms of action. Thus, with the explicit absence of an on-target mechanism of action in the lung cells and evidence ruling out unexpected immunomodulatory effects, it remains to be proven whether the fascinating CF biomarker changes reported are indeed on account of the expected mechanism of action. While these pre-clinical results are as convincing as those reported in previous studies evaluating RNase H-active antisense for asthma, the outcomes in Crosby et al.3Crosby J.R. Zhao C. Jiang C. Bai D. Katz M. Greenlee S. Kawabe H. McCaleb M. Rotin D. Guo S. Monia B.P. Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.J. Cyst. Fibros. 2017; (Published online May 20, 2017)https://doi.org/10.1016/j.jcf.2017.05.003Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar could be due to agonist/antagonist effects in inducing and inhibiting, respectively, severe inflammation. After all, the TLR4 agonist lipopolysaccharide (LPS) is a well-established tool for the induction of mucous hypersecretion as well as the host of associated osmoregulatory gene expression changes reported in the proposed Nedd4L ASO CF model.17Zhao J. Wei J. Dong S. Bowser R.K. Zhang L. Jacko A.M. Zhao Y. Destabilization of Lysophosphatidic Acid Receptor 1 Reduces Cytokine Release and Protects Against Lung Injury.EBioMedicine. 2016; 10: 195-203Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Thus, as with previous efforts, interpretation of the reported data as a mechanistically-relevant, on target, and effective treatment of CF might be premature. At the peak of excitement over the potential of RNAi and subsequently 3rd generation antisense for treating lung diseases, many sought to drug the undruggable targetome with this therapeutic class. Large pharma, which actively engaged in evaluating these modalities in airways disease, eventually concluded that intracellular delivery in the airways remained an outstanding issue, as much as local immunomodulation was a risk. Both of these issues remain unaddressed in Crosby et al.3Crosby J.R. Zhao C. Jiang C. Bai D. Katz M. Greenlee S. Kawabe H. McCaleb M. Rotin D. Guo S. Monia B.P. Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.J. Cyst. Fibros. 2017; (Published online May 20, 2017)https://doi.org/10.1016/j.jcf.2017.05.003Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar and the few other reports since 2011. Notably, emerging alternative approaches, such as in vitro transcribed RNA therapy tested in the airways, have proactively explored these aspects,18Johler S.M. Rejman J. Guan S. Rosenecker J. Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration.PLoS ONE. 2015; 10: e0137504Crossref PubMed Scopus (27) Google Scholar with CureVac being a positive example of exploratory safety practice. Such efforts are commendable and set a new standard in qualifying nucleic acid therapy data; the practice is aligned to calls from large pharma to pursue exploratory safety studies that confirm the proposed mechanisms of action of therapeutics. Widespread adoption is likely to reduce assumptions around the translation of drug function across the drug development pipeline.19Cook D. Brown D. Alexander R. March R. Morgan P. Satterthwaite G. Pangalos M.N. Lessons learned from the fate of AstraZeneca's drug pipeline: a five-dimensional framework.Nat. Rev. Drug Discov. 2014; 13: 419-431Crossref PubMed Scopus (856) Google Scholar Irrespective of historical data on what is and is not immunomodulatory in oligonucleotides versus the intricacies of nucleoside chemistry, a clinically proven and pharmaceutically relevant solution to achieving intracellular delivery, as recognized by Ionis for hepatic indications, involves the use of receptor-specific ligands. Thus, conjugation of N-acetyl-glucosamine (GalNAc) to oligonucleotide drugs has been shown to target the hepatocyte-specific asialglycoprotein receptor (ASGPR) and reduce drug doses by up to 60-fold in phase II clinical trials.20Prakash T.P. Graham M.J. Yu J. Carty R. Low A. Chappell A. Schmidt K. Zhao C. Aghajan M. Murray H.F. et al.Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice.Nucleic Acids Res. 2014; 42: 8796-8807Crossref PubMed Scopus (379) Google Scholar Unfortunately, a prototype GalNAc-ASGPR pair suitable for oligonucleotide delivery into airways cells remains undiscovered but appears necessary. We believe that with rational, tractable, and systematic design and validation of oligonucleotide-conjugated ligands to well-studied respiratory cell receptors, antisense and other oligonucleotide therapeutics may yet still offer clinically and commercially attractive solutions for the treatment of airways disease. Success is likely to largely depend on focusing treatment on cell types with functions relevant to the disease being treated, considering carefully the need for the phosphorothioate backbone, which drives alveolar macrophage and systemic tissue loading, and producing evidence of exclusively on-target molecular pharmacology in a statistically robust and appropriate manner. Thus, we urge the community to capitalize on the GalNAc paradigm and, in seeking to extend the clinical potential of oligonucleotide drugs, intensify the exploration of targeted delivery, both into airways cells and disease-relevant tissues beyond.