https://orcid.org/0000-0003-2549-2569
Some of the major challenges facing the translation of miRNA-based therapeutics designed to treat the pulmonary manifestations of cystic fibrosis (CF) from the bench to the clinic include; optimizing their in vivo stability and organ specific delivery; testing their functionality in appropriate human CF lung models and assessing their usefulness as adjunct therapies to be used with existing approved systemically administered CFTR modulators.
Although great strides have been made in recent years in developing a cure for CF, there is still some way to go to complete this goal because not all CF sufferers can benefit from the current CFTR potentiator and corrector drugs. Adjunct gene therapy strategies designed to enhance or inhibit the expression of specific proteins that contribute to CF lung disease pathophysiology have therapeutic potential. Nevertheless, a number of substantial challenges remain to be overcome in order to transfer such new molecular therapies for the treatment of the manifestations of CF lung disease successfully from bench to bedside. This is particularly true for miRNA-based therapeutics. miRNAs are naturally occurring endogenous negative regulators of gene expression that function in a species, tissue and cell specific manner. Their artificial augmentation or inhibition via various nucleic acid-based strategies has been widely demonstrated in a variety of contexts both in vitro and ex vivo. Currently, to our knowledge, there are no miRNA-based or miRNA-targeting medicines in clinical trials for CF, although human trials of miRNA-based drugs for other diseases are ongoing. For example, a Phase I open label dose-escalation study of TargomiR (an intravenous miR-16 delivery therapy for the treatment of solid tumors in malignant pleural mesothelioma patients) has demonstrated its tolerability and early signs of anti-tumor activity [Citation1]. miRNA inhibition therapies for lung disease have yet to progress to first-in-human studies.
Regarding CF, there are various experimental medicines in development that may have therapeutic use in the near future. These include both delivery (miRNA mimic) and inhibition (antagomiR, peptide nucleic acid) strategies designed to decrease or increase the expression of particular genes, respectively [Citation2–6]. Interestingly, because a single miRNA can often regulate multiple targets within a pathway or network these two approaches can have more potent effects than individual siRNA or gene replacement approaches, albeit with the risk of unpredictable and mostly unwanted off-target effects on additional unrelated miRNA targets. Catalytic knockdown of miRNAs using artificial, sequence-specific ribonucleases called miRNases [Citation7] has not been reported in the CF field to date, however, because these agents tend to display potent, irreversible and persistent suppression of their targets, which are desirable attributes for a high cost, potentially personalized, therapy, development of such drugs is certainly worth exploring. Another more precise miRNA inhibition approach involves the use of target site blockers, which can selectively and specifically inhibit miRNA-mediated knockdown of a single target without off-target effects [Citation3,Citation8].
After the selection of the type of miRNA-based therapeutic (i.e. mimic, inhibitor, target site blocker, etc.), the next challenge is represented by the natural barriers that the human body, and in particular the CF lung, offer toward exogenous nucleic acids. Those delivered systemically can undergo degradation by RNases in the bloodstream and in the endocytic compartments of the cells [Citation9]. In order to limit this effect, chemical modifications can be added to the backbone of miRNA therapeutics such as methylation or addition of locked-nucleic acid bases, which increases their stability and binding strength. At the same time, delivery strategies are under development to create vehicles to encapsulate RNA-based therapeutics for protection against nucleases. We recommend that these two converging approaches could be used in designing a miRNA-based therapeutic strategy aimed to rescue CFTR expression and function in the lung. CFTR is known to be regulated by a set of lead miRNAs that are overexpressed in the lungs of people with cystic fibrosis [Citation4,Citation5]. Therefore, a strategy that can specifically interfere with miRNA-mediated inhibition of CFTR expression in CF bronchial epithelial cells should be able to increase CFTR expression and function in the lung. By encapsulating CFTR-specific target site blockers within nanoparticles and administering these in vitro to polarized CF bronchial epithelial cell lines and primary CF bronchial epithelial cells grown at an air-liquid interface in order to mimic the CF bronchial epithelium in vivo, their ability to enhance CFTR expression and function can be determined. Furthermore, coupling these nanoparticles with a nebulizer that can generate stable and respirable aerosols, would yield a drug-device combination that could be translated to the clinic.
Important within this field is the choice of appropriate model systems in which to test the experimental therapies. Any model system derived from a species other than a human has inherent, insurmountable problems. Although miRNAs can be conserved across species, the 3′ untranslated region (3′UTR) of messenger RNAs are rarely identical between species. Consequently, 3′UTR miRNA recognition elements are almost never 100% conserved between species, although they may be present elsewhere within the 3′UTR in the same or different quantities in another species other than human. Therefore, we believe it is best to concentrate studies on human CF models because the 3′UTRs of most murine genes, for example, ATF6, KC, TGF-β, etc., and likely those of pig and ferret, are not regulated by the same miRNAs as their human counterparts [Citation10–12]. Escalating from the simplest and widely used model, in other words, human CF bronchial epithelial cell lines grown in monolayers, there are now many more specialized human CF model systems available that mimic the CF lung and in which miRNA-based medicines can be tested. These range from primary bronchial epithelial cells, air-liquid interface cultures, nasal biopsies and nasospheres, gut organoids, induced pluripotent stem cell derived lung organoids and even a CF lung on a chip. Each of these can be used to test certain aspects of new therapies and ideally, a combination of methods would be used in the development of any new therapy. Further streamlining and standardization of such human CF models will assist the more rapid translation of miRNA therapeutics from the laboratory to the clinic.
In our opinion, miRNA therapeutics should be developed to be organ-specific, rather than to be administered via the oral route and therefore could be used as adjunct therapies with existing approved systemically administered CFTR modulators. Local delivery of nucleic acid-based therapies to the CF lung is a challenge due to its highly inflamed status and the presence of a thick mucus layer that could impair effective drug delivery to bronchial epithelial cells [Citation13], however, novel strategies are under development to successfully overcome these issues [Citation14–16]. Part of the reason for adopting this organ-specific ‘local’ approach is that it should thereby decrease the dose required and potential side effects of CFTR modulators. Indeed, notwithstanding the success of systemic CFTR modulator therapies since the approval of ivacaftor monotherapy in 2012 and lumacaftor or tezacaftor co-therapy with ivacaftor in 2015 and 2018, and most recently trikaftor triple therapy, these treatments are not without some shortcomings. One problem recognized throughout the CF medical community is that these treatments are neither effective in all Phe508del heterozygous individuals with a second minimal function CFTR mutation, nor in people with CF with rare CFTR mutations. Unwanted side effects have also been reported in some cases. Therefore, internationally within the CF R&D field, there is an urgency to develop alternative adjunct therapies for co-administration with existing CFTR modulator therapies [Citation17]. It is also well recognized that combinations of mechanistically different CFTR modulators are required to obtain meaningful clinical benefit [Citation18,Citation19].
In conclusion, promising adjunct miRNA-based CF therapies that show functionality in human CF models of lung disease would ideally be modified for stability, encapsulated within nanoparticles to enhance their delivery and be administered directly to the lung using a suitable device. Conceptually, their use should lower the dose of oral CFTR modulator co-therapy necessary to restore CFTR function in other organs, as well as potentially decrease their unwanted known side effects. As such, effective CFTR-targeting target site blockers, which function in a CFTR mutation independent manner, could in theory be administered as an adjunct treatment regardless of an individual’s CFTR genotype.
Financial & competing interests disclosure
Funding for research in this group is gratefully acknowledged from Cystic Fibrosis Foundation Therapeutics (GREENE15XXO) and the NIH (R01HL144539). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Additional information
Funding
References
- Van Zandwijk N , PavlakisN , KaoSCet al. Safety and activity of microRNA-loaded minicells in patients with recurrent malignant pleural mesothelioma: a first-in-man, Phase I, open-label, dose-escalation study. Lancet Oncol.18(10), 1386–1396 (2017).
- Viart V , BergougnouxA , BoniniJet al. Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis. Eur. Respir. J.45(1), 116–128 (2015).
- Sonneville F , RuffinM , CorauxCet al. microRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology. Nat. Commun.8(1), 710 (2017).
- Oglesby IK , ChotirmallSH , McElvaneyNG , GreeneCM. Regulation of cystic fibrosis transmembrane conductance regulator by microRNA-145, -223, and -494 is altered in ΔF508 cystic fibrosis airway epithelium. J. Immunol.190(7), 3354–3362 (2013).
- Lutful Kabir F , AmbalavananN , LiuGet al. microRNA-145 antagonism reverses TGF-β inhibition of F508del CFTR correction in airway epithelia. Am. J. Respir. Crit. Care Med.197(5), 632–643 (2018).
- Amato F , TomaiuoloR , NiciFet al. Exploitation of a very small peptide nucleic acid as a new inhibitor of miR-509-3p involved in the regulation of cystic fibrosis disease-gene expression. Biomed. Res. Int.2014, 610718 (2014).
- Patutina OA , MiroshnichenkoSK , MironovaNLet al. Catalytic knockdown of miR-21 by artificial ribonuclease: biological performance in tumor model. Front. Pharmacol.10, 879 (2019).
- Fernández Fernández E , Santos-CarballalB , de SantiCet al. Biopolymer-based nanoparticles for cystic fibrosis lung gene therapy studies. Materials11(1), pii: E122 (2018).
- Rupaimoole R , SlackFJ. microRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat. Rev. Drug Discov.16(3), 203–222 (2017).
- Oglesby IK , AgrawalR , MallMA , McElvaneyNG , GreeneCM. miRNA-221 is elevated in cystic fibrosis airway epithelial cells and regulates expression of ATF6. Mol. Cell Pediatr.2(1), 1 (2015).
- Oglesby IK , VenckenSF , AgrawalRet al. miR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production. Eur. Respir. J.46(5), 1350–1360 (2015).
- Mitash N , MuF , DonovanJEet al. Transforming growth factor-β1 selectively recruits microRNAs to the RNA-induced silencing complex and degrades CFTR mRNA under permissive conditions in human bronchial epithelial cells. Int. J. Mol. Sci.20(19), pii: E4933 (2019).
- Gill DR , HydeSC. Delivery of genes into the CF airway. Thorax69(10), 962–964 (2014).
- Craparo EF , PorsioB , SardoC , GiammonaG , CavallaroG. Pegylated polyaspartamide-polylactide-based nanoparticles penetrating cystic fibrosis artificial mucus. Biomacromolecules17(3), 767–777 (2016).
- Nafee N , ForierK , BraeckmansK , SchneiderM. Mucus-penetrating solid lipid nanoparticles for the treatment of cystic fibrosis: proof of concept, challenges and pitfalls. Eur. J. Pharm. Biopharm.124, 125–137 (2018).
- Osman G , RodriguezJ , ChanSYet al. PEGylated enhanced cell penetrating peptide nanoparticles for lung gene therapy. J. Control Release285, 35–45 (2018).
- Bell SC , BarryPJ , DeBoeck Ket al. CFTR activity is enhanced by the novel corrector GLPG2222, given with and without ivacaftor in two randomized trials. J. Cyst. Fibros.18(5), 700–707 (2019).
- de Wilde G , GeesM , MuschSet al. Identification of GLPG/ABBV-2737, a novel class of corrector, which exerts functional synergy with other CFTR modulators. Front. Pharmacol.10, 514 (2019).
- Holguin F . Triple CFTR modulator therapy for cystic fibrosis. N. Engl. J. Med.379(17), 1671–1672 (2018).