Patent Application
20150079118
This invention relates to a composition for modifying the activity of microRNA-mediated regulation of mRNA in a cell. In particular, compositions for use in the treatment of asthma and methods of prevention or treatment of asthma.
MicroRNAs, also referred to herein as miRNAs, are a class of small endogenously expressed small regulatory non-coding RNAs of about 18 to 25 nucleotides in length. miRNAs negatively regulate target mRNAs. This negative regulation is mediated by binding to a miRNA binding site which is an imperfect complement to the miRNA in the 3′-untranslated region of the mRNA. These are also referred to herein as 3′-UTRs. The miRNA can alternatively bind in the coding sequence of the mRNA. MicroRNAs play a crucial role in the complex network of gene regulation in eukaryotic cells. As they are the specificity determining subunit in the RNA-induced silencing complex (RISC), miRNAs are capable of inhibiting the translation of genes. Based on early studies in invertebrates, miRNAs are expected to have roles in developmental regulation and cell differentiation in mammals, and roles for miRNAs in cardiogenesis and lymphocyte development have been demonstrated. Several studies suggest a strong connection between miRNA and human cancer. Recent reports implicate roles for mammalian miRNAs in metabolic pathways. Furthermore, miRNAs may contribute to destabilization of the mRNA molecules bound by the miRNA. Despite the growing list of roles for mammalian miRNAs, most of the hundreds of miRNAs identified in mammals have no reported function. Nevertheless, many miRNAs and their respective targets have been found to be evolutionary highly conserved. One single miRNA may regulate a variety of different genes, in some examples said variety consisting of more than 100 genes. Accordingly, it is generally assumed today that between 30 and 60% of all human genes are regulated by miRNAs.
MicroRNAs have also been shown to suppress levels of viral RNA in cells. In mammals, miRNAs can play diverse roles in viral infection through their capacity to regulate both host and viral genes. Recent reports have demonstrated that specific miRNAs change in expression level upon infection and can impact viral production and infectivity. It is clear that miRNAs are an integral component of viral-host interactions, and it is likely that both host and virus contain mechanisms to regulate miRNA expression and/or activity.
Asthma is one of the most common chronic diseases globally, with recurrent exacerbations and resistance to treatment significantly contributing to the morbidity and economic burden of this airway disorder. Up to 85% of exacerbations are caused by respiratory viruses, and asthmatics show increased susceptibility to viral infection. Severe asthma sufferers, even on maximal therapy, experience recurrent exacerbations and thus it is vital that the mechanisms underlying the susceptibility to virally triggered exacerbations are elucidated in order to develop preventive treatment strategies. Bronchial epithelial cells and cells recovered by bronchoalveolar lavage (BAL) in asthmatics appear to have an impaired innate immune response to viruses, producing less interferon (IFN) than cells from healthy subjects. Among the cells recovered in BAL, alveolar macrophages (AM) are key to airway defences, promoting airway tolerance to antigens as well as detecting invading viruses with the consequent secretion of IFN that protects airway epithelium from rhinoviruses. Many aspects of innate immune functions and signalling pathways appear to be controlled by microRNAs (miRNAs). miRNAs are small non-coding RNA molecules that exert post-transcriptional regulation on gene expression by repression of translation or degradation of mRNA.
Therefore, it would be desirable to provide methods and compositions to bring the airway reactivity of asthmatics to viruses and bacteria closer to that of normal subjects.
According to a first aspect of the invention, there is provided a composition for modifying the activity of microRNA-mediated regulation of mRNA in a cell, wherein the composition comprises:
MicroRNAs are small non-coding RNAs that inhibit gene expression by pairing to the 3′ untranslated region (3′UTR) of their target mRNAs facilitating their translational repression or degradation. It has been found that two or more micro RNA molecules may target the 3′untranslated region of the same mRNA. MicroRNA molecules that have a specific target mRNA in common tend to increase their affinity for this target when they are simultaneously manipulated in the cell. Having an increased preference for the common target, these microRNA molecules seem to show less avidity for other targets, thereby reducing the potential for off-target effects of a potential microRNA regulation based therapy. In particular, microRNAs target a region in the mRNA, the 3′UTR (untranslated region) that is in a tight secondary structure (this is difficult to access). MicroRNAs targeting areas that are slightly open are more effective. Thus, more than one microRNA targeting the same mRNA will make it more accessible to the rest (they will co-operate to open this closed region). Not only will this increase their effect on this particular mRNA, but it will favour the mRNA targeted by several microRNAs (because it is open and accessible) over the other targets that are still closed and are difficult to access, which reduces the chance of off target effects. Therefore the advantage of targeting multiple microRNA molecules is that they may eliciting a stronger and/or more controlled response than targeting individual microRNA molecules. It is advantageous to simultaneously or sequentially administer compounds that bind to each of a group of microRNA molecules that all target the same mRNA to provide an enhanced effect on the mRNA.
Advantageously, blocking the binding of microRNA molecules to mRNA can elevate the mRNA levels in the cell (thereby upregulating the expression of the encoded protein), because binding of the microRNA molecules to the mRNA can mark the mRNA for degradation or block their translation into protein.
The cell may be a mammalian cell. The cell may be a human cell. The cell may be an alveolar macrophage. The cell may be human alveolar macrophage.
At least one microRNA molecule may be selected from any of the group comprising miR-150, miR-152 and miR-375. The two or more microRNA molecules may comprise two or more microRNA molecules selected from any of the group comprising miR-150, miR-152 and miR-375. The two or more microRNA molecules may comprise three or more microRNA molecules selected from any of the group comprising miR-150, miR-152 and miR-375. The two or more microRNA molecules may comprise miR-150 and miR-152. The two or more microRNA molecules may comprise miR-150 and miR-375. The two or more microRNA molecules may comprise miR-152 and miR-375. The two or more microRNA molecules may comprise miR 150, miR-152 and miR-375.
The mRNA may encode TLR7. The mRNA may encode RipK1. The microRNA molecules may target the mRNA encoding TLR7 and RipK1.
At least one compound may be arranged to bind to a microRNA molecule to prevent it from engaging with the target mRNA. Two or more compounds may be arranged to bind to the microRNA molecules to prevent them from engaging with the target mRNA. At least one compound may be arranged to bind to the target mRNA in order to block a binding site of a microRNA molecule. Two or more compounds may be arranged to bind to the target mRNA in order to block a binding site of the microRNA molecules.
The compound may be an antagomir, oligonucleotide, or blockmir. At least one compound may be an oligonuleotide. At least one compound may be an antagomir. At least one compound may be a blockmir. A combination of antagomirs and blockmirs may be provided. The two or more compounds may be selected from the group comprising oligonuleotides, antagomirs and blockmirs, or combinations thereof.
The compound may be the Anti-miR™ miRNA inhibitor of miR-150 or mirVana® miRNA inhibitor of miR-150 (available from Life Technologies Corporation Cat. #AM17000 and Cat. #4464084 respectively) The compound may be the Ambion® Anti-mir™ miRNA inhibitor of miR-150 (available from Life Technologies Corporation). The compound may be an antagomir having at least about 80% sequence identity to the Anti-miR™ miRNA inhibitor of miR-150; or mirVana® miRNA inhibitor of miR-150; or Ambion® Anti-mir™ miRNA inhibitor of miR-150. The compound may be an antagomir having at least about 90%, 95%, 98%, or 99% sequence identity to the Anti-miR™ miRNA inhibitor of miR-150 or mirVana® miRNA inhibitor of miR-150; or Ambion® Anti-mir™ miRNA inhibitor of miR-150.
The compound may be the Anti-miR™ miRNA inhibitor of miR-152 or mirVana® miRNA inhibitor of miR-152 (available from Life Technologies Corporation Cat. #AM17000 and Cat. #4464084 respectively). The compound may be the Ambion® Anti-mir™ miRNA inhibitor of miR-152 (available from Life Technologies Corporation). The compound may be an antagomir having at least about 80% sequence identity to the Anti-miR™ miRNA inhibitor of miR-152; or mirVana® miRNA inhibitor of miR-152; or Ambion® Anti-mir™ miRNA inhibitor of miR-152. The compound may be an antagomir having at least about 90%, 95%, 98%, or 99% sequence identity to the Anti-miR™ miRNA inhibitor of miR-152; or mirVana® miRNA inhibitor of miR-152; or Ambion® Anti-mir™ miRNA inhibitor of miR-152.
The compound may be the Anti-miR™ miRNA inhibitor of miR-375 or mirVana® miRNA inhibitor of miR-375 (available from Life Technologies Corporation Cat. #AM17000 and Cat. #4464084 respectively). The compound may be the Ambion® Anti-mir™ miRNA inhibitor of miR-375 (available from Life Technologies Corporation). The compound may be an antagomir having at least about 80% sequence identity to the Anti-miR™ miRNA inhibitor of miR-375; or mirVana® miRNA inhibitor of miR-375; or Ambion® Anti-mir™ miRNA inhibitor of miR-375. The compound may be an antagomir having at least about 90%, 95%, 98%, or 99% sequence identity to the Anti-miR™ miRNA inhibitor of miR-375; or mirVana® miRNA inhibitor of miR-375; or Ambion® Anti-mir™ miRNA inhibitor of miR-375.
At least one compound may be an oligonuleotide comprising a sequence complementary to a sequence selected from any of the group comprising SEQ ID NO. 24, SEQ ID NO. 25, and SEQ ID NO. 26. The sequence may be complementary across between about 5 and 15 nucleotides, or 6 and 10 nucleotides. The sequence may be complementary across between 7 and 9 nucleotides.
The two or more compounds may comprise two or more oligonuleotides comprising a sequence complementary to a sequence selected from any of the group comprising SEQ ID NO. 24, SEQ ID NO. 25, and SEQ ID NO. 26. The two or more compounds may comprise three or more oligonuleotides comprising a sequence complementary to a sequence selected from any of the group comprising SEQ ID NO. 24, SEQ ID NO. 25, and SEQ ID NO. 26. The two or more compounds may comprise an oligonuleotide comprising a sequence complementary to a sequence of SEQ ID NO. 24 and an oligonucleotide comprising a sequence complementary to a sequence of SEQ ID NO. 25. The two or more compounds may comprise an oligonuleotide comprising a sequence complementary to a sequence of SEQ ID NO. 24 and an oligonucleotide comprising a sequence complementary to a sequence of SEQ ID NO. 26. The two or more compounds may comprise an oligonuleotide comprising a sequence complementary to a sequence of SEQ ID NO. 25 and an oligonucleotide comprising a sequence complementary to a sequence of SEQ ID NO. 26. The two or more compounds may comprise an oligonuleotide comprising a sequence complementary to a sequence of SEQ ID NO. 24; an oligonucleotide comprising a sequence complementary to a sequence of SEQ ID NO. 25; and an oligonucleotide comprising a sequence complementary to a sequence of SEQ ID NO. 26.
At least one compound may be an oligonuleotide comprising a sequence complementary to a sequence selected from any of the group comprising SEQ ID NO. 24, or complementary to a sequence having 80%, 90%, 95%, or 99% identity to SEQ ID NO. 24. At least one compound may be an oligonuleotide comprising a sequence complementary to a sequence selected from any of the group comprising SEQ ID NO. 25, or complementary to a sequence having 80%, 90%, 95%, or 99% identity to SEQ ID NO. 25. At least one compound may be an oligonuleotide comprising a sequence complementary to a sequence selected from any of the group comprising SEQ ID NO. 26, or complementary to a sequence having 80%, 90%, 95%, or 99% identity to SEQ ID NO. 26.
The sequence may be complementary across at least 5 nucleotides. The sequence may be complementary across at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, or at least 10 nucleotides.
One or more compounds may be anti-microRNA oligonucleotides that specifically bind to the microRNA molecules. The oligonucleotides may have more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99% or 100% sequence identity to a sequence that is complementary to the microRNA molecule that it specifically binds to. The sequence identity may be defined over the entire region of the anti-microRNA oligonucleotide.
One or more compounds may be oligonucleotides that specifically bind to the microRNA molecule binding site of the target mRNA. The oligonucleotides may have more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99% or 100% sequence identity to a sequence that is complementary to the microRNA molecule binding site of the mRNA that it specifically binds to.
The sequences identities may be determined by BLAST under standard parameters.
One or more compounds may be anti-microRNA oligonucleotides that are long enough to specifically bind to their target microRNA and block or reduce their binding to their target mRNA. The anti-microRNA oligonucleotides may be between 5 and 50 nucleotides in length, between 10 and 40 nucleotides in length, between 15 and 30 nucleotides in length, or between 20 and 25 nucleotides in length. The anti-microRNA oligonucleotides may be between 7 and 9 nucleotides in length.
One or more compounds may be oligonucleotides that are long enough to specifically bind to the microRNA binding site of the mRNA and block or reduce the binding of the microRNA molecules to the target mRNA. The oligonucleotides may be between 5 and 50 nucleotides in length, between 10 and 40 nucleotides in length, between 15 and 30 nucleotides in length, or between 20 and 25 nucleotides in length.
At least one compound may be a small molecule or a peptide. The two or more compounds may be small molecules or peptides.
A small molecule may be a chemical compound. A small molecule may not be a biological molecule. The small molecule may not be any one of a polymeric nucleic acid, a protein, or an antibody. A small molecule may be a low molecular weight <900 Daltons, organic compound that may serve as an enzyme substrate or regulator of biological processes, with a size on the order of 10−9 m. Biopolymers such as nucleic acids, proteins, and polysaccharides (such as starch or cellulose) may not be considered small molecules. The constituent monomers of biopolymers, such as ribo- or deoxyribonucleotides, amino acids, and monosaccharides may be considered to be small molecules. Small oligomers may be considered small molecules, such as dinucleotides, peptides such as the antioxidant glutathione, and disaccharides such as sucrose.
The composition may comprise three or more different compounds wherein each of the three or more different compounds binds to a different microRNA molecule and wherein all of the different microRNA molecules target the same mRNA.
The composition may further comprise four or more, five or more, six or more, or seven or more compounds each capable of blocking the binding of a different microRNA molecule to the same target mRNA, wherein each microRNA molecule is different in structure or sequence to each other.
According to another aspect of the invention, there is provided the composition of the invention herein, for use in the prevention or treatment of asthma in a patient.
According to another aspect of the invention, there is provided a composition for use in the prevention or treatment of asthma in a patient, wherein the composition comprises at least one oligonuleotide capable of blocking a microRNA molecule selected from miR-150, miR-152 and miR-375 from binding to a target mRNA. The composition may comprise at least two oligonuleotides capable of blocking a microRNA molecule selected from miR-150, miR-152 and miR-375 from binding to a target mRNA.
According to another aspect of the invention, there is provided a method of prevention or treatment of asthma, comprising administering a composition of the invention herein.
According to another aspect of the invention, there is provided a method of prevention or treatment of asthma, comprising administering a composition comprising at least one oligonuleotide capable of blocking a microRNA molecule selected from miR-150, miR-152 and miR-375 from binding to a target mRNA. The composition may comprise at least two oligonuleotides capable of blocking a microRNA molecule selected from miR-150, miR-152 and miR-375 from binding to a target mRNA.
According to another aspect of the invention, there is provided a method of prevention or treatment of asthma, comprising administering:
The method may further comprise the administration of a third compound, wherein the third compound is capable of blocking a third microRNA molecule from binding to the same target mRNA, wherein the first and second, and third microRNA molecules are different in sequence or structure relative to each other.
The method may comprise the administration of a plurality of different compounds, wherein the plurality of compounds is capable of blocking the binding of a plurality of different microRNA molecules to the same target mRNA.
The plurality of microRNA molecules may be different in sequence or structure relative to each other.
Administration of the first compound and the second compound may be concurrently, simultaneously, or sequentially. The plurality of different compounds may be administered concurrently, simultaneously, or sequentially, or combinations thereof, relative to each other. The first compound and the second compound may be combined in a single composition. The plurality of compounds may be combined in a single composition, or combined in pairs or groups in multiple compositions.
The asthma may be viral exacerbated asthma. The patient may have, or is at risk of, a respiratory viral infection or viral exacerbated asthma. The patient may have, or is at risk of, a bacterial respiratory infection or bacterial exacerbated asthma. The patient may have, or is at risk of, allergen exacerbated asthma. The patient may have, or is at risk of, pollutant exacerbated asthma.
The administration of the composition may be before or during an asthmatic episode. The administration of the composition may be before, during or after a viral infection or viral exacerbation of the asthma. The administration may be prophylactic. The administration may be provided to a subject during a high risk period for viral exacerbation, for example in autumn and/or spring seasons; during pandemics or epidemics; during local viral outbreaks. The administration may be provided to a subject if a co-habitor, family, colleague or other group member is infected with a virus.
Prophylactic administration advantageously has no effect in the absence of viral exacerbation, yet it enhances the ability of the alveolar macrophage to detect a viral infection, and potentially prevent the infection from spreading or developing. The administration advantageously does not cause the negative secondary effects of other asthma therapies such as interferon treatment.
The composition comprising two or more compounds of two, three, four, five, six or seven compounds that bind to or block the micro RNAs may be administered simultaneously in one formulation. The combination of two three, four, five, six or seven compounds may be administered sequentially.
The composition may comprise pharmaceutically acceptable excipients. The composition may further comprise suitable excipients, carriers and/or diluents.
The composition may be delivered by intravenous or subcutaneous injection. The composition may be delivered by aerosol. An aerosol may be defined as a suspension of small particles or droplets suspended in gas or vapour.
According to another aspect of the invention, there is provided a composition comprising an antogomir or blockmir of miR-150; and/or an antogomir or blockmir of miR-152; and/or an antogomir or blockmir of miR-375.
The composition of the invention may comprise an additional active ingredient. The additional active ingredient may comprise an asthma medicament. The additional active ingredient may comprise an anti-viral medication. The additional active ingredient may comprise an antibiotic. The additional active ingredient may comprise an anti-inflammatory. The additional active ingredient may comprise a bronchodilator. The additional active ingredient may comprise a steroid. Combinations of the above actives may be provided.
The additional active ingredient may comprise a medicament selected from any of the group comprising a short-acting beta2-adrenoceptor agonist (SABA); an anticholinergic medication; an anticholinergic bronchodilator; an adrenergic agonist; a corticosteroid; a long-acting beta-adrenoceptor agonist (LABA); a leukotriene antagonist; and a mast cell stabilizer; or combinations thereof.
The additional active ingredient may comprise a medicament selected from any of the group comprising salbutamol (albuterol); terbutaline; ipratropium bromide; epinephrine; fluticasone propionate; beclometasone; salmeterol; formoterol; montelukast; zafirlukast; cromolyn sodium; cromoglycate; nedocromil; budesonide; ciclesonide; fluticasone; and mometasone; or combinations thereof.
According to another aspect of the invention, there is provided a nebuliser or inhaler comprising at least one compound capable of blocking at least one microRNA molecule selected from miR-150, miR-152 and miR-375 from binding to its mRNA target.
The nebuliser or inhaler may comprise the composition of the invention herein.
The nebuliser or inhaler may comprise an additional active ingredient. The additional active ingredient may comprise an asthma medicament. The additional active ingredient may comprise an anti-viral medication. The additional active ingredient may comprise an antibiotic. The additional active ingredient may comprise an anti-inflammatory. The additional active ingredient may comprise a bronchodilator. The additional active ingredient may comprise a steroid. Combinations of the above actives may be provided.
The additional active ingredient may comprise a medicament selected from any of the group comprising a short-acting beta2-adrenoceptor agonist (SABA); an anticholinergic medication; an anticholinergic bronchodilator; an adrenergic agonist; a corticosteroid; a long-acting beta-adrenoceptor agonist (LABA); a leukotriene antagonist; and a mast cell stabilizer; or combinations thereof.
The additional active ingredient may comprise a medicament selected from any of the group comprising salbutamol (albuterol); terbutaline; ipratropium bromide; epinephrine; fluticasone propionate; beclometasone; salmeterol; formoterol; montelukast; zafirlukast; cromolyn sodium; cromoglycate; nedocromil; budesonide; ciclesonide; fluticasone; and mometasone; or combinations thereof.
According to another aspect of the invention, there is provided a pharmaceutical composition comprising pharmaceutically acceptable excipients and a composition according to the invention herein.
According to another aspect of the invention, there is provided a composition for modifying the activity of microRNA-mediated regulation of mRNA in a cell, wherein the composition comprises:
According to another aspect of the invention, there is provided a composition for use in the prevention or treatment of asthma in a patient, wherein the composition comprises at least one molecule capable of preventing or reducing the expression of a microRNA molecule selected from miR-150, miR-152 and miR-375 in an alveolar macrophage cell. The composition may comprise at least two molecules capable of preventing or reducing the expression of a microRNA molecule selected from miR 150, miR-152 and miR-375 in an alveolar macrophage cell.
The expression may be prevented or reduced by binding of a molecular probe, oligo or other molecule specific for the nucleic acid encoding or controlling the expression of the microRNA molecule. The expression may be prevented or reduced by mutation, removal or knock-out of the nucleic acid encoding or controlling the expression of the microRNA molecule. The mutation, removal or knock-out of the nucleic acid may be provided in the form of gene-therapy. The mutation, removal or knock-out of the nucleic acid may be provided by a nucleic acid vector delivered into the cell, for example by a modified virus.
The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention.
There now follows by way of example only a detailed description of the present invention with reference to the accompanying drawings, in which;
Sequences and accession numbers as provided on miRBase.org.
Asthma exacerbations are predominantly triggered by viral infections, reflecting defective airway innate immunity. Reduced virus-induced production of interferons by airway cells appears to be a critical factor, but the underlying mechanisms are not known. A disregulation in the expression of several microRNAs has been observed in asthma. The implications of this dysregulation were investigated focusing on different microRNAs in different cells types and processes. This approach has facilitated the discovery of synergies or collaborations between multiple microRNAs. It has been found that multiple microRNAs may be used simultaneously as therapeutic targets, eliciting a more controlled response that individual microRNAs. For example, the expression of TLR7 in alveolar macrophages is shown to be reduced because a group of three co-operating microRNAs (miR-150, miR-152 and miR-375) are over-expressed. Targeting these miRNAs restores TLR7 expression and corrects the impaired innate response.
MicroRNAs Predicted to Target TLR7 are Up-Regulated in Macrophages from Asthmatics.
Alveolar macrophages (AM) were purified from bronchoalveolar lavage (BAL) miRNA microarray analyses were performed on samples from both healthy and asthmatic volunteers. This identified 27 miRNAs that were differentially up-regulated in AM from asthmatics (
Quantitative PCR (qPCR) on these four miRNAs confirmed that the expression of three, namely miR-150, miR-152 and miR-375, was significantly increased in severe asthmatic-AM, while the expression of the fourth, miR19b, was similar in severe asthmatic-AM and healthy AMs (
TLR7 Expression is Reduced in Asthmatic Macrophages and Co-Relates with Clinical Status.
According to the in silico prediction above, the increased expression of miRs-150, -152 and -375 would result in reduced levels of TLR7, impairing antiviral response to rhinovirus in severe asthmatics. Therefore, the expression of TLR7 in SA-AM was evaluated and a reduction at both the mRNA and protein level was found as compared to that in healthy AM (
TLR7-Dependent Induction of IFN and IFNα is Impaired in Macrophages from Asthmatics.
It was next investigated whether there was a functional impairment in the response to virus exposure of macrophages from severe asthmatics. The AM was challenged with rhinovirus (RV-16) in vitro and it was found that RV-induction of IFNα and IFNβ mRNA as well as secreted protein was significantly reduced from SA-AMs compared to healthy AMs (
Once it was confirmed that the in silico prediction that over-expression of miRs-150, -152 and -375 in SA-AMs (
Antagomirs Against miRs-150, -152 and -375 Restore IFN Response to Virus in Alveolar Macrophages.
These findings highlight the need for novel therapeutic approaches to restoring the normal innate immune response in order to prevent virally-induced disease exacerbation in asthma. On the basis of these findings, the knock-down of miRs-150, -152 and -375, would restore TLR7 expression and therefore function, thereby ameliorating the defective IFN production by AM in response to viral challenge. Therefore, specifically to test this, AMs were transfected with three antagomirs, anti-miR-150, anti-miR-152 and anti-miR-375. When the transfected cells were then challenged with rhinovirus they showed significantly augmented production of IFNα and IFNβ mRNA and secreted protein compared to mock transfected cells (
Furthermore, when these transfected AM were challenged with imiquimod, a similar, significant increase in the mRNA production of IFNα, IFNβ, M×A and OAS was seen (
For the first time, the specific molecular role of microRNAs in human asthma has been demonstrated. This work not only delivers a cellular and molecular understanding of the role of miRs-150, -152 and -375 in human asthma but provides real therapeutic potential for treatment of severe asthmatics with specific antagomirs to restore their anti-viral innate immunity. Furthermore, the targeting of the three microRNAs studied, which act cooperatively, is likely to limit any off target effects.
In conclusion, bioinformatic predictions and molecular tools with a broad applicability were employed to identify a novel mechanism involved in the deficient innate immune response to virus in asthma. A response that is driven by miRNA-mediated deficiency in the expression of an important PRR in macrophages, namely TLR7. More importantly, it has been shown that by manipulating the expression of miRNAs in AMs, the defective IFN response to virus can be ameliorated, making it an extremely promising and novel treatment for the future prevention of disease exacerbation in asthma.
Study subjects—adults with severe asthma, moderate asthma and healthy subjects were recruited. Severe asthma subjects were recruited from the MRC funded Wessex Severe Asthma Cohort and Difficult Airways clinic held at University Hospitals Southampton Foundation Trust, while subjects in the latter 2 groups were recruited through local advertising. The study was approved by the Southampton and South West Hampshire Local Research Ethics Committee and all subjects gave written informed consent. Baseline spirometry was measured using the Jaeger Masterscreen with Viasys® software. Healthy and moderate asthma subjects underwent methacholine (Stockport Pharmaceuticals) challenge testing to assess airway responsiveness. Skin prick testing was performed using allergen extract from the following: aspergillus fumigatus, alternaria tenius, birch tree pollen, mixed grasses, mixed tree pollen, rape pollen, weed pollen, dermatophagoides pteronyssinus, dermatophagoides farinae, dog fur, cat dander and horse fur, along with positive (histamine) and negative (saline) control (all from Allegopharma, Germany).
Alveolar Macrophages Subjects underwent flexible bronchoscopy in accordance with established guidelines (British Thoracic Society Bronchoscopy Guidelines Committee, 2001). Bronchoalveolar lavage (BAL) was performed by instilling 120 mls (6×20 ml aliquots) pre-warmed (37° C.) normal saline into the right upper lobe. Cells from BAL were washed immediately and resuspended in RPMI 1640 with Glutamax (Invitrogen) containing 10% FCS and antibiotics (penicillin and streptomycin-P/S) at a concentration of 1×106/ml and placed at 37° C. in a humidified 5% CO2 incubator for 2 hours to allow for the AM to adhere. AM were collected in TRI-Reagent (for RNA isolation, Applied Biosystems) and NP-40 protein lysis buffer (Invitrogen, supplemented with PMSF, Sigma-Aldrich, and protease inhibitor cocktail, Sigma-Aldrich).
For the functional studies, AM were cultured in RPMI 1640 with Glutamax containing 10% FCS and antibiotics (P/S). They were then incubated with Imiquimod (5 μg/ml, Invivogen) or rhinovirus-16 at an MOI of 0.6 (or UV-inactivated rhinovirus-16) or polyl:C (10 μg/ml, Invivogen). After 24 hours supernatants were harvested for future evaluation of IFNα and IFNβ protein concentration. Adherent cells (AM) were collected in TRI-Reagent.
For the transfection studies, AM were cultured in RPMI 1640 with Glutamax containing 10% FCS and antibiotics (P/S). They were transfected (transfection occurs by natural phagocytosis or passive diffusion, no transfection reagent used) with a combination of anti-miR-150, anti-miR-152 and anti-miR-375 (50 nM each) or scrambled control (150 nM, Life Technologies). At 48 hours cells were treated with imiquimod, rhinovirus-16 or poly:IC at the concentrations stated above for 24 hours. At this point adherent cells were collected in TRI-Reagent and supernatants harvested.
For transfection studies to evaluate changes TLR7 protein expression, healthy AM were transfected with pre-miR-150, pre-miR-152 and pre-miR-375 (500 nM each, Life Technologies) or scrambled control. At 48 hours macrophages were lysed in NP-40 protein lysis buffer (supplemented with PMSF and protease inhibitor cocktail).
All samples were stored at −80° C. until use.
To study the effects of steroids on miRNAs AM from healthy subjects were treated with dexamethasone (Sigma Aldrich, 1000 nM, 100 nM, 10 nM) and incubated at 37° C. with 5% CO2. Cells were collected at 24 and 48 hours in TRI-Reagent and stored at −80° C. until analysis.
RNA Extraction and qPCR
Total RNA was isolated using TRI-Reagent. For miRNA analysis 5 ng of RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit with specific stem loop primers for each miRNA. All miRNA data was normalized to the internal control RNU44. For mRNA analysis 200 ng of RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit. GAPDH was used as an internal control. qPCR was performed using Taqman® Universal PCR Mastermix on a 7900HT Fast Real-time PCR system (Applied Biosystems) and changes in the expression of miRNA and mRNA were expressed as fold change relative to control. All reagents from Life Technologies.
The Life Technologies Taqman® Low Density Array system was used following manufacturer's instructions. Briefly, 500 ng of total cellular RNA was was reversed transcribed using Megaplex™ RT Primers and the cDNA was then loaded onto the array card for PCR amplification. Arrays were carried out 4 healthy and 4 asthmatic subjects. Results were displayed as a Heat map using MultiExperiment Viewer (MeV: http://www.tm4.org/mev/).
MiRNA targets were predicted using Targetscan 4.2 (http://www.targetscan.org/vert—42/) with default options.
Western blotting and densitometric analysis was performed as previously described (Martinez-Nunez et al., 2009) the following antibodies were used: anti-TLR7 (1:500, Abcam), anti-mouselgG-HRP (Fisher Scientific) and anti-β-actin-peroxidase (1:25,000, Sigma-Aldrich).
The Verikine™ IFNβ enzyme-linked immunoabsorbant assay (range 25-2000 pg/ml) was used which employs a sandwich immunoassay. It was done using manufacturer's instructions and standards, samples and blanks were run in duplicate.
Meso Scale Discovery singleplex kits were also used for evaluation of IFNα and IFNβ concentrations in cell supernatants. It was done using manufacturer's instructions and standards, samples and blanks were run in duplicate.
The 3′UTR of TLR7, containing the predicted miR-150, miR-152 and miR-375 seed sequences was amplified from human genomic DNA and cloned into XbaI and NotI sites of the pRLTK vector (Promega) and called pRLTK_WT—3′UTR_TLR7. Site-directed mutagenesis was performed on this vector to create vectors containing mutations in the seed sequence(s) for miR-150 (pRLTK_MUT—3′UTR_TLR7—150—1, pRLTK_MUT—3′UTR_TLR7—150—2), miR-152 (pRLTK_MUT—3′UTR_TLR7—152) and mir375 (pRLTK_MUT—3′UTR_TLR7—375). For pCDNA3.1—150, pCDNA3.1—152 and pCDNA3.1—375 genomic DNA was amplified by PCR and DNA fragments cut with different pairs of enzymes were cloned into BamHI/XhoI, XhoI/KpnI and XbaI/BamHI pCDNA3.1 multi-cloning sites respectively. In order to generate pCDNA3.1_MIX, these fragments were cloned simultaneously in pCDNA3.1(−) using XbaI/KpnI (order of cloning from promoter: miR-275, miR-150 and miR152).
Primers used to generate the vectors in
Transfections were performed 3 times in duplicate. HeLa cells were co-transfected with the luciferase reporter constructs containing either the WT (wild-type) or MUT (3′UTR mutants) and 800 ng of pCDNA3.1—150 or pCDNA3.1—152 or pCDNA3.1—375. Transfections were carried out using Superfect (Qiagen) and normalization was performed using pGL3 (25 ng/well, Promega). Transfected cells were cultured for 24 hours after which measurements were made using the Dual-Luciferase® Reporter Assay System (Promega).
| Number | Date | Country | Kind |
|---|---|---|---|
| 1316651.7 | Sep 2013 | GB | national |
