Patent Application
20230416738
The term interstitial lung disease (ILD) encompasses a large and heterogeneous group of over 200 pulmonary disorders, most of which are classified as rare. The major abnormality in ILDs is the disruption of the distal lung parenchyma resulting in impaired gas exchange and restrictive ventilatory defects. It is generally agreed that some form of injury of the alveolar epithelial cells initiates an inflammatory response coupled with repair mechanisms. The injury-repair process is reflected pathologically as inflammation, fibrosis or a combination of both. Irrespective of the underlying pathophysiology, the resulting alteration of the interstitial space leads to clinical symptoms such as dyspnoea and cough, and results in restrictive ventilatory and gas exchange deficits on pulmonary function testing (Schwartz M I et al., 2011). There is no universally accepted single classification of ILDs. They can generally be categorized based on their etiology (idiopathic or ILDs with known association or cause), clinical course (acute, subacute or chronic ILDs), and based on the main pathological features (inflammatory or fibrotic ILDs). Fibrotic ILDs can be subdivided into 3 groups based on their longitudinal disease behavior (Wells A U, 2004):
While IPF is the best-known and prototypical form of a progressive fibrosing ILD (PF-ILD), there is a group of patients with different clinical ILD diagnoses other than IPF who develop a progressive fibrosing phenotype during the course of their disease. These patients demonstrate a number of similarities to patients with IPF, with their disease being defined by increasing extent of pulmonary fibrosis on imaging, declining lung function, worsening respiratory symptoms and quality of life despite management considered appropriate in individual ILDs, and, ultimately, early mortality (Flaherty K R et al., 2017; Wells A U et al., 2018; Cottin V et al., 2019; Kolb M et al., 2019). Similar to IPF, a decline in FVC is predictive of mortality in patients with these other fibrosing ILDs (Jegal Y et al., 2005; Solomon J J et al., 2016; Gimenez A, et al., 2017; Goh N S et al., 2017; Volkmann E R et al. 2019). There is a high unmet medical need, as no approved disease-modifying pharmacological therapies for patients with progressive fibrosing ILDs exist, except for patients with IPF. Along with their clinical similarities, progressive fibrosing ILDs share pathophysiological mechanisms that represent a common fibrotic response to tissue injury (see
According to the scientific literature, ILDs that can be complicated by progressive fibrosis include, but are not limited to, idiopathic non-specific interstitial pneumonia (iNSIP) (Kim M Y et al., 2012), unclassifiable idiopathic interstitial pneumonia (IIP) (Guler S A et al., 2018), hypersensitivity pneumonitis (HP) (Sadeleer L J et al., 2019), autoimmune ILDs such as rheumatoid arthritis-associated ILD (RA-ILD) (Doyle T J & Dellaripa P F 2017) and SSc-ILD Guler S A et al, 2018), sarcoidosis (Walsh S L et al., 2014), and occupation associated lung disease (Khalil N et al., 2007). The etiology of progressive fibrosing ILDs like IPF is still unknown; however various irritants including smoking, occupational hazards, viral and bacterial infections as well as radiotherapy and chemotherapeutic agents (like e.g. Bleomycin) have been described as potential risk factors for the development of IPF. Due to changes in IPF diagnostic criteria over the past years, the prevalence of IPF varies considerably in the literature. According to recent data, the prevalence of IPF ranges from 14.0 to 63.0 cases per 100,000 while the incidence lies between 6.8 and 17.4 new annual cases per 100,000 (Ley B et al., 2013). IPF is usually diagnosed in elderly people with an average age of disease onset of 66 (Hopkins R B et al., 2016). After initial diagnosis IPF progresses rapidly with a mortality rate of approximately 60 percent within 3 to 5 years. In contrast to IPF, a variable portion of the patients with CTD (including e.g. rheumatoid arthritis (RA), Sjögren's syndrome and systemic sclerosis (SSc)) or sarcoidosis display a progressive fibrosing phenotype, with about 10-20% of RA patients, 9-24% of Sjögren's syndrome, >70% of SSc (Mathai S C and Danoff S K, 2016) and 20-25% of sarcoidosis patients (Spagnolo P et al., 2018) developing pulmonary fibrosis.
There are two main histopathological characteristics observed in PF-ILDs, namely non-specific interstitial pneumonia (NSIP) and usual interstitial pneumonitis (UIP). The histopathological hallmarks of IPF are UIP and progressive interstitial fibrosis caused by excessive extracellular matrix deposition. UIP is characterized by a heterogeneous appearance with areas of subpleural and paraseptal fibrosis alternating with areas of less affected or normal lung parenchyma. Areas of active fibrosis, so-called fibroblastic foci, are characterized by fibroblast accumulation and excessive collagen deposition. Fibroblastic foci are frequently located between the vascular endothelium and the alveolar epithelium, thereby causing disruption of lung architecture and formation of characteristic “honeycomb”-like structures. Clinical manifestations of IPF are dramatically compromised oxygen diffusion, progressive decline of lung function, cough and severe impairments in quality of life.
UIP is also one of the main histopathological hallmarks in RA-ILD and late-stage sarcoidosis; however, other CTDs, such as SSc or Sjögren's, are mainly characterized by non-specific interstitial pneumonia (NSIP).
NSIP is characterized by less spatial heterogeneity, i.e. pathological anomalies are rather uniformly spread across the lung. In the cellular NSIP subtype, histopathology is characterized by inflammatory cells, whereas in the more common fibrotic subtype, additional areas of pronounced fibrosis are evident. However, pathological manifestations can be diverse, thereby complicating correct diagnosis and differentiation from other types of fibrosis, such as UIP/IPF.
Due to the unknown disease cause of IPF, the knowledge regarding pathological mechanisms on the cellular and molecular level is still limited. However, recent advances in translational research using experimental disease models (in vitro and in vivo) for functional studies as well as tissue samples from IPF patients for genomics/proteomics analyses enabled valuable insights into key disease mechanisms. According to our current understanding, IPF is initiated through repeated alveolar epithelial cell (AEC) micro-injuries, which finally result in an uncontrolled and persistent wound healing response. In more detail, AEC damage induces an aberrant activation of neighboring epithelial cells, thereby leading to the recruitment of immune cells and stem or progenitor cells to the sites of injury. By secreting various cytokines, chemokines and growth factors, infiltrating cells produce a pro-inflammatory environment, which finally results in the expansion and activation of fibroblasts. Under physiological conditions these so-called myofibroblasts produce extracellular matrix (ECM) components to stabilize and repair damaged tissue. Moreover, myofibroblasts contribute to tissue contraction and wound closure in later stages of the wound healing process via their inherent contractile function. In contrast to physiological wound healing, inflammation and ECM production are not self-limiting in IPF. As a consequence this leads to a continuous deposition of ECM, which finally results in progressive lung stiffening and the destruction of lung architecture. Indeed, ECM biomarkers can be used to determine the onset of the treatment of PF-ILD, see WO2017/207643. On the molecular level the pathogenesis of IPF is orchestrated by a multitude of pro-fibrotic mediators and signaling pathways. Besides TGFβ, which plays a central role in IPF due to its potent pro-fibrotic effects, tyrosine kinase signaling and elevation of various corresponding growth factors like e.g. platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) contribute to the pathogenesis of IPF.
In recent years several drugs have been clinically tested for the treatment of IPF. However, so far only two drugs, Pirfenidone (Esbriet®; Roche/Genentech) and Nintedanib (Ofev®; Boehringer Ingelheim), showed convincing therapeutic efficacy by slowing down disease progression as demonstrated by reduced rates of lung function decline. Despite these encouraging results, the medical need in IPF is still high and additional therapies with improved efficacy and ideally disease modifying potential are urgently needed. Nintedanib is also approved for the treatment of systemic sclerosis associated ILD as well as for chronic fibrosing interstitial lung disease with progressive phenotype other than IPF. In general, the current management of ILDs is centred on the suppression of inflammation with corticosteroid or immunomodulatory therapy. The latter is based on anecdotal reports and uncontrolled treatment responses in small case series with the use of azathioprine, cyclosporine, cyclophosphamide, mycophenolate mofetil, rituximab, and tacrolimus. Some ILDs, e.g. some cases of CTD-ILDs can be stabilized by immunomodulation (Tashkin D P et al., 2006; Fischer A et al. 2013; Morisset J et al., 2017; Adegunsoye A et al., 2017), others are progressive despite (pharmacological and/or non-pharmacological) treatment considered appropriate in individual ILDs (Wells A U 2004), again demonstrating a remaining high demand for innovative therapeutic approaches.
Pulmonary hypertension (PH) is one of the most frequent complications in ILDs, which could be an independent driver of early mortality (Galiè N et al., 2015). PH is defined as a disease with elevated right ventricular systolic pressure (RVSP), right ventricular pressure overload and right atrial and ventricular dilatation (Smith et al. (2013), Am J Med Sci, 346(3):221-225).
Chronic, fibrotic silicosis belongs to the family of ILDs. It is caused by a chronic, recurrent inhalation to crystalline silica, damaging the epithelial cells in the alveolar space and activates macrophages to produce an inflammatory response. Both factors, lead to an activation of resident fibroblasts and the associated massive deposition of extra cellular matrix in these lung areas.
Due to the plethora of pathways involved in the pathogenesis of IPF and other fibrosing ILDs, multi-target therapies aiming to simultaneously modulate various disease mechanisms are likely to be most effective. However, respective approaches are difficult to implement by classical pharmacological strategies using small molecule compounds (NCEs) or biologicals (NBEs) like e.g. monoclonal antibodies, since both modalities are typically designed to specifically inhibit or activate a single drug target or a small set of closely related molecules. To enable multi-targeted therapies for PF-ILDs, microRNAs (miRNAs) represent a novel and highly attractive target class based on their ability to control and fine-tune entire signaling pathways or cellular mechanisms under physiological and pathophysiological conditions by regulating mRNA expression levels of a specific set of target genes. miRNAs are small non-coding RNAs, which are transcribed as pre-cursor molecules (pri-miRNAs). Inside the nucleus pri-miRNAs undergo a first maturation step to produce so called pre-miRNAs, which are characterized by a smaller hairpin structure. Following nuclear export, pre-miRNAs undergo a second processing step mediated by the Dicer enzyme, thereby generating two single strands of fully maturated miRNAs of approximately 22 nucleotides in length. To exert their gene regulatory function, mature miRNAs are incorporated into the RNA Induced Silencing Complex (RISC) to enable binding to miRNA binding sites positioned within the 3′-UTR of target mRNAs. Upon binding, miRNAs induce destabilization and cleavage of target mRNAs and/or modulate gene expression by inhibition of protein translation of respective mRNAs. To date more than 2000 miRNAs have been discovered in humans, which potentially regulate up to 30% of the transcriptome (Hammond S M, 2015).
The present invention discloses the identification of miRNAs involved in the pathogenesis of fibrosing lung disease and methods for the treatment of lung diseases such as PF-ILD by functional modulation of respective miRNAs in ILD patients, preferably in PF-ILD patients, in particular IPF patients, using viral vectors, in particular an Adeno-associated virus (AAV). The present invention focusses on the treatment of humans though mammals of any kind, especially companion animal mammals, such as horses, dogs and cats are also within the realm of the invention.
Treatment of patients with moderate (Child Pugh B) and severe (Child Pugh C) hepatic impairment with Ofev is not recommended (see EPAR). Esbriet must not be used by patients already taking fluvoxamine (a medicine used to treat depression and obsessive compulsive disorder) or patients with severe liver or kidney problems (see EPAR). Thus, there is still a high medical need for PF-ILD patients, and in particular for IPF patients that have severe liver and kidney problems. It is an object of this invention to provide treatment alternatives. An alternative object of the invention is to provide treatment alternatives that might be eligible even for the patient group that cannot benefit from the existing therapies.
While Esbriet and Ofev have shown convincing efficacy in clinical trials, also side effects are associated that potentially limit the options for a combined therapy of both drugs (see both EPARs). Thus, there is still a high medical need for ILD treatments, such as PF-ILD and in particular IPF treatments, with less side effects or at least with side effects different from those seen with Ofev or Esbriet, so that combined therapy with either Esbriet or Ofev may be viable option to increase the overall treatment efficacy. It is an alternative object of the invention to provide treatment alternatives with a different risk/benefit profile compared to the established treatment options, e.g. with lesser side effects or with different side effects compared to the established treatment options. While Esbriet and Ofev are intended for oral, i.e. systemic use, there is still a need for a treatment option that can be administered by local administration or both via local and systemic routes. It is an alternative object of the invention to provide a treatment option that can be administered by local administration or both via local and systemic routes.
It is a further alternative object of the invention to provide
The present invention relates in one aspect to therapeutic agents, i.e. viral vectors or miRNA mimetics, for the treatment of ILD in general, and PF-ILD and IPF in particular.
The viral vectors according to the invention stop or slow one or more aspects of the tissue transformation seen in ILD, preferably in PF-ILD and more preferably IPF, such as the ECM deposits, by modulating miRNA function and thus stop or slow the decline in forced vital capacity seen in these diseases (see WO2017/207643 and references). The viral vectors according to the invention may be administered to the patient via local (intranasal, intratracheal, inhalative) or systemic (intravenous) routes. Especially AAV vectors can target the lung quite efficiently, have a low antigenic potential and are thus particularly suitable also for systemic administration.
From a therapeutic perspective, miRNA function can be modulated by delivering miRNA mimetics to increase effects of endogenous miRNAs, which are downregulated under fibrotic conditions, or by delivering molecules to block miRNAs or to reduce their availability by so-called anti-miRs or miRNA sponges, thus inhibiting functionality of endogenous miRNAs, which are upregulated under pathological conditions.
Moreover, miRNAs described in the present invention, which are upregulated, might also exert protective functions as part of a natural anti-fibrotic response. However, this effect is apparently not sufficient to resolve the pathology on its own. Therefore, in specific cases, the delivery of a miRNA mimetic for a sequence which is already elevated under fibrotic conditions can potentially further enhance its anti-fibrotic effect, thereby offering an additional model for therapeutic interventions.
Based on the fact that miRNAs orchestrate the simultaneous regulation of multiple target genes, viral vector mediated modulation of miRNA function represents an attractive strategy to enable multi-targeted therapies by affecting different disease pathways. The lung-fibrosis associated miRNAs described in the present invention distinguish from previously identified miRNAs by modulating different sets of target genes, thereby offering potential for improved therapeutic efficacy.
In the present invention a set of miRNAs associated with lung fibrosis has been identified by in-depth characterization and computational analysis of two disease-relevant animal models, in particular, Bleomycin-induced lung injury, characterized by a patchy, acute inflammation-driven fibrotic phenotype and AAV-TGFβ1 induced fibrosis that is reminiscent of the more homogenous NSIP pattern. Longitudinal transcriptional profiles of miRNAs and mRNAs as well as functional data have been generated to enable the identification of disease-associated miRNAs. Additionally, high confidence miRNA-mRNA regulatory relationships have been built based on sequence and expression anti-correlation, allowing for characterization of miRNAs in the context of the disease models based on their target sets. To further substantiate these findings, synthetic RNA oligonucleotide mimetics of selected miRNA candidates (mir-29a-3p, mir-10a-5p, mir-181a-5p, mir-181b-5p, mir-212-5p) were generated and used for transient transfection experiments in cellular fibrosis models in primary human lung fibroblasts, primary human bronchial airway epithelial cells and A549 cells. By investigating the effect of transiently transfected miRNAs on major aspects of TGFβ-induced fibrotic remodeling (inflammation, proliferation, fibroblast to myofibroblast transition (FMT), epithelial to mesenchymal transition (EMT)) the predicted anti-fibrotic effects of the selected miRNAs could be confirmed. Finally, to translate these findings into clinical applications, novel therapeutic approaches for fibrosing lung diseases to enable modulation of PF-ILD associated miRNAs by using viral gene delivery based on Adeno-associated virus (AAV) vectors are described.
The miRNA mimetics according to the invention stop or slow one or more aspects of the tissue transformation seen in ILDSs like PF-ILD and IPF, such as the ECM deposits, by modulating miRNA function and thus stop or slow the decline in forced vital capacity seen in these diseases (see WO2017/207643 and references). Compared to viral vectors according to the invention, they have a different profile of side effects, such as a potentially lower antigenicity, thereby potentially allowing multiple treatments without immunosuppressive combined treatment.
By conducting a longitudinal in depth analysis of two disease-relevant animal models, namely the Bleomycin- and the AAV-TGFβ1-induced lung fibrosis model in mice, a novel set of 28 miRNAs has been identified. To select the most relevant miRNAs, the inventors developed a hit selection strategy based on systematic correlation analyses between gene expression profiling data and key functional disease parameters. Under consideration of the chronic nature of PF-ILDs the inventors describe expression of miRNAs, anti-miRs or miRNA sponges by viral vectors especially those based on Adeno-associated virus (AAV) as a novel therapeutic concept to enable long lasting expression of therapeutic nucleic acids for functional modulation of fibrosis-associated miRNAs.
The closest human homologs of the mouse sequences that are highly similar (albeit not identical) are shown in
The shown sequences are also compiled in a sequence listing. In case of contradictions between the sequence listing and
The invention relates to a viral vector comprising: a capsid and a packaged nucleic acid, wherein the packaged nucleic acid codes for two or more miRNAs, wherein the two or more miRNAs comprise the miRNA of Seq ID No. 92 and the miRNA of Seq ID No. 15 or a fragment of the latter having the sequence of Seq ID No. 99. The invention also relates to a viral vector comprising: a capsid and a packaged nucleic acid, wherein the packaged nucleic acid codes for two or more miRNAs, wherein the two or more miRNAs comprise the miRNA of Seq ID No. 92 and the miRNA of Seq ID No. 17 or a fragment of the latter having the sequence of Seq ID No. 100. In a particularly preferred embodiment, the invention relates to a viral vector comprising: a capsid and a packaged nucleic acid, wherein the packaged nucleic acid codes for two or more miRNAs, wherein said miRNAs comprise the miRNA of Seq ID No. 92 and the miRNA of Seq ID No. 15 or a fragment thereof having the sequence of Seq ID No. 99 and the miRNA of Seq ID No. 17 or a fragment thereof having the sequence of Seq ID No. 100. The invention therefore refers to the use of selected miRNAs that have been found effective when being used in combination with each other. The miRNAs include the miRNA of mir-29a-3p (Seq ID no. 92) either in combination with the miRNA of mir-212-5p (Seq ID no. 15) or the miRNA of mir-181a-5p (Seq ID no. 17). In addition, it has been found herein that the miRNA mir-29a-3p can also be combined with fragments of the miRNAs mir-212-5p and mir-181a-5p that lack the terminal nucleotide at the 3′ end of the molecule. These fragments of the miRNAs mir-212-5p and mir181a-5p are set forth herein as Seq ID No. 99 and Seq ID No. 100, respectively. The RNA molecules of Seq ID No. 99 and Seq ID No. 100 are considered as self-contained miRNAs in the context of the present invention. Since the deletion at the 3′-terminus in Seq ID No. 99 and Seq ID No. 100 compared to the authentic mRNA of Seq ID No. 15 and 17, respectively, is remote from the seed region and the region of nucleotides at 13-16 of the miRNA, the specifity of the miRNA according to Seq ID No. 99 and Seq ID No. 100 is acceptable (Grimson et al., 2007). It was shown by Chen, T. et al. that miR-212-5p increase could reduce RVSP and pulmonary vessel wall remodeling in a mouse model of pulmonary hypertension (Chen, T. et al., 2018, Chen, T. et al., 2019). For silicosis context see Jiang, R. et al., 2019 and Yang, X. et al. 2018.
The invention therefore relates to a viral vector comprising: a capsid and a packaged nucleic acid, wherein the nucleic acid augments either (i) the miRNA of Seq ID No. 92 or (ii) miRNA downregulated in a Bleomycin-induced lung fibrosis model or in an AAV-TGFβ1-induced lung fibrosis model, wherein the miRNA comprises miRNA of Seq ID 15 or a fragment thereof having the sequence of Seq ID No. 99 or the miRNA of Seq ID No. 17 or a fragment thereof having the sequence of Seq ID No. 100, or (iii) both (i) and (ii). In one embodiment, the miRNA(s) that are downregulated in a Bleomycin-induced lung fibrosis model or in an AAV-TGF01-induced lung fibrosis model and which are augmented by the packaged nucleic acid further comprise the miRNA of Seq ID No. 19. In another embodiment, the one or more miRNAs which are augmented by the packaged nucleic acid comprise the miRNA of Seq ID No. 92 and the miRNA of Seq ID No. 15 or a fragment thereof having the sequence of Seq ID No. 99 and the miRNA of Seq ID No. 19. In another embodiment, the one or more miRNAs which are augmented by the packaged nucleic acid comprise the miRNA of Seq ID No. 92 and the miRNA of Seq ID No. 17 or a fragment thereof having the sequence of Seq ID No. 100 and the miRNA of Seq ID No. 19.
Augmentation in this context means that the level of the respective miRNA in the transduced cell is increased as a result of the transduction of the target cell, which is preferably a lung cell.
The invention further relates to a viral vector comprising: a capsid and a packaged nucleic acid, wherein the nucleic acid augments either (i) the miRNA of Seq ID No. 92 or (ii) miRNA downregulated in a Bleomycin-induced lung fibrosis model or in an AAV-TGFβ1-induced lung fibrosis model, wherein the miRNA comprises the miRNA of Seq ID 15 or a fragment thereof having the sequence of Seq ID No. 99 or the miRNA of Seq ID No. 17 or a fragment thereof having the sequence of Seq ID No. 100, or (iii) both (i) and (ii) and wherein the nucleic acid further inhibits miRNA selected form the group consisting of miRNAs of Seq ID No 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 16 or the closest human homolog of respective sequences in case of miRNAs with partial sequence conservation.
Inhibition in this context means that the function of the respective miRNA in the transduced cell is reduced or abolished by complementary binding as a result of the transduction of the target cell.
In one embodiment, the invention relates to a viral vector comprising: a capsid and a packaged nucleic acid that codes for one or more miRNA that are downregulated in a Bleomycin-induced lung fibrosis model or in an AAV-TGFβ1-induced lung fibrosis model:
It is understood that the nucleic acid usually comprises coding and non-coding regions and that the encoded miRNA up- or downregulated in a Bleomycin-induced lung fibrosis model or in an AAV-TGFβ1-induced lung fibrosis model results from transcription and subsequent maturation steps in target cell transduced by the viral vector.
It is understood that the nucleic acid usually comprises coding and non-coding regions and that the encoded RNA inhibiting the function of one or more miRNA that is upregulated in a Bleomycin-induced lung fibrosis model or in an AAV-TGFβ1-induced lung fibrosis model results from transcription and potentially, but not necessarily, subsequent maturation steps in target cell transduced by the viral vector.
Viral vectors according to the present invention are selected so that they have the potential to transduce lung cells. Non-limiting examples of viral vectors that transduce lung cells include, but are not limited to lentivirus vectors, adenovirus vectors, adeno-associated virus vectors (AAV vectors), and paramyxovirus vectors. Among these, the AAV vectors are particularly preferred, especially those with an AAV-2, AAV-5 or AAV-6.2 serotype. AAV vectors having a recombinant capsid protein comprising Seq ID No. 29, 30 or 31 are particularly preferred (see WO 2015/018860). In one embodiment, the AAV vector is of the AAV-6.2 serotype and comprises a capsid protein of the sequence of Seq ID No. 82.
The sequence coding for the miRNA thereby augmenting its function and the sequence coding for an RNA that inhibits the function of one or more miRNA may or may not be within the same transgene.
In one embodiment, the invention relates to a viral vector comprising: a capsid and a packaged nucleic acid comprising one or more transgene expression cassettes comprising:
Accordingly, the transgene that codes for a miRNA thereby augmenting its level and the transgene that codes for an RNA that inhibits the function of one or more miRNA are contained in different expression cassettes.
In one embodiment, the invention relates to a viral vector comprising: a capsid and a packaged nucleic acid comprising one or more transgene expression cassettes comprising a transgene that codes
Accordingly, one transgene codes for both a miRNA thereby augmenting its function and for a RNA that inhibits the function of one or more miRNA.
In another embodiment of the invention a viral vector is provided, wherein the miRNA is selected from the group consisting of miRNAs of Seq ID No. 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 92, 99 and 100 or the closest human homolog of respective sequences in case of miRNAs with partial sequence conservation. In this group, the conserved miRNA, namely 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 92, 99, 100 or their closest human homolog are most preferred. The closest human homolog of the respective sequences is shown in
In a further embodiment of the invention a viral vector is provided, wherein the nucleic acid has an even number of transgene expression cassettes and optionally the transgene expression cassettes comprising (or consisting of) a promotor, a transgene and a polyadenylation signal, wherein promotors or the polyadenylation signals are positioned opposed to each other.
The viral vector is a recombinant AAV vector in one embodiment of the invention and has either the AAV-2 serotype, AAV-5 serotype or the AAV-6.2 serotype in other embodiments of the invention.
In a different embodiment of the invention a viral vector is provided, wherein the capsid comprises a first protein that comprises the sequence of Seq ID No. 29 or 30 (see WO 2015/018860).
For all embodiments (i) to (iii): For the determination of the identity between a first protein and a reference protein, any amino acid that has no identical counterpart in the alignment between the two proteins counts as mismatch (including overhangs with no counterpart). For the determination of identity, the alignment is used which gives the highest identity score.
The packaged nucleic acid may be single or double-stranded. An alternative especially for AAV vectors is to use self-complementary design, in which the vector genome is packaged as a double-stranded nucleic acid. Although the onset of expression is more rapid, the packaging capacity of the vector will be reduced to approximately 2.3 kb, see Naso et al. 2017, with references.
A further aspect of the invention is one of the described viral vectors for use in the treatment of a lung disease, preferably an ILD. The diseases that can be treated according to the present invention are preferably selected from the group consisting of PF-ILD, IPF, connective tissue disease (CTD)-associated ILD, rheumatoid arthritis ILD, chronic fibrosing hypersensitivity pneumonitis (HP), idiopathic non-specific interstitial pneumonia (iNSIP), unclassifiable idiopathic interstitial pneumonia (IIP), environmental/occupational lung disease, pulmonary hypertension (PH), fibrotic silicosis, systemic sclerosis ILD and sarcoidosis, and fibrosarcoma.
Delivery Strategies for Recombinant AAV Therapeutics are also referred in e.g. Naso et al, 2017.
A double stranded plasmid vector comprising said AAV vector genome is a further embodiment of the invention.
A further embodiment of the invention relates to this miRNA inhibitor for use as a medicinal product.
The present invention also contemplates the use of miRNA mimetics for the prevention and/or treatment of a of a lung disease, preferably an ILD. The lung diseases that can be treated with the miRNA mimetics of the invention are set out above and include fibroproliferative disorder such as ILD, PF-ILD, and IPF. The miRNA mimetics of the present invention typically and preferably consist of a contiguous nucleotide sequence of a total of 21, 22 or 23 contiguous nucleotides in length. The length of the miRNA mimetics (i.e. the length of the “oligomer of nucleotides” in case of a single-strand mimetic or the length of the “oligomer of nucleotides” (i.e. the sense strand) in case of a double-strand mimetic that contains said oligomer besides other oligonucleotides bound to said oligomer) typically and preferably matches the length of the respective miRNA they mimic. In case of miRNA mimetics of a miRNA that has 23 nt, such as miR-181a-5p or miRNA-212-5p, the length of the miRNA mimetics (i.e. the oligomer in case of a single-strand mimetic or the sense strand of the double-strand mimetic) is either 23 nt (preferred) or 22 nt with the proviso that one nucleotide at the 3′-terminus is missing. Since the deletion at the 3-terminus compared to the authentic mRNA (see e.g. Seq ID No. 99 and 100) is remote from the seed region and the region of nucleotides at 13-16 of the miRNA, the specificity of the corresponding miRNA mimetics is acceptable (Grimson et al., 2007).
A further embodiment of the invention therefore is a combination of miRNA mimetics for use in a method of prevention and/or treatment of a fibroproliferative disorder, such as ILD, PF-ILD, or IPF wherein the combination comprises (i) a mimetic of the miRNA having the sequence of Seq ID No. 92, and (ii) a mimetic of the miRNA having the sequence of Seq ID No. 15 and/or a mimetic of the miRNA having the sequence of Seq ID No. 17. The combination of miRNA mimetics may further comprise one or more mimetic of anmiRNA which has a sequence selected from the group consisting of Seq ID No. 18, 19, 21, 22, 23, 24, 25, 26, 27, 37, 38 and 39, preferably selected from the group consisting of Seq ID Nos. 18 and 19. In one embodiment, a miRNA mimetic is provided for use in a method of prevention and/or treatment of a fibroproliferative disorder, such as ILD, PF-ILD or IPF, wherein miRNA has the sequence of Seq ID No. 92, and wherein the method further comprises the administration of a mimetic of an miRNA that has the sequence of Seq ID No. 15. In another embodiment, a miRNA mimetic is provided for use in a method of prevention and/or treatment of a fibroproliferative disorder, such as ILD, PF-ILD or IPF, wherein miRNA has the sequence of Seq ID No. 92, and wherein the method further comprises the administration of a mimetic of a miRNA that has the sequence of Seq ID No. 17. The prevention and/or treatment preferably further comprises the administration of a mimetic for a miRNA having the sequence of Seq ID No. 18 or of a mimetic for a miRNA having the sequence of Seq ID No. 19.
Likewise, in a further embodiment a miRNA mimetic is provided for use in a method of prevention and/or treatment of a fibroproliferative disorder, such as ILD, PF-ILD or IPF, wherein the miRNA has the Seq ID No. 92. The prevention and/or treatment further comprises the administration of a mimetic for a miRNA having the sequence of Seq ID No. 15 or of a mimetic for a miRNA having the sequence of Seq ID No. 17. Even more preferably,
A further embodiment of the invention is (i) a miRNA mimetic of a miRNA having the sequence of Seq ID No. 92 and (ii) a miRNA mimetic of a miRNA having the sequence of Seq ID No. 15 or a miRNA mimetic of a miRNA having the sequence of Seq ID No. 17, for the treatment of a fibroproliferative disorder such as ILD, PF-ILD or IPF and a pharmaceutical composition comprising these miRNA mimetics and a pharmaceutical-acceptable carrier or diluent.
A further embodiment of the invention is a pharmaceutical composition comprising a miRNA mimetic of a miRNA having the sequence of Seq ID No. 92 and a miRNA mimetic of a miRNA having the sequence of Seq ID No. 15, and a pharmaceutical-acceptable carrier or diluent. Another embodiment of the invention is a pharmaceutical composition comprising a miRNA mimetic of a miRNA having the sequence of Seq ID No. 92 and a miRNA mimetic of a miRNA having the sequence of Seq ID No. 17, and a pharmaceutical-acceptable carrier or diluent. Preferably, the miRNA mimetics in the composition are packed in lipid nanoparticles (LNPs). The LNPs may preferably have a mean particle size of the LNPs is between 30 and 200 nm. The pharmaceutical composition may further comprise 25 to 65 mol % of ionizable lipids.
In any of the above embodiments, the mimetic of the miRNA having the sequence of Seq ID No. 92 preferably is (in case of a single-single stranded mimetic) or contains (in case of a double-stranded mimetic) an oligomer that has the sequence of Seq ID No. 92. Similarly, the mimetic of the miRNA having the sequence of Seq ID No. 15 preferably is or contains an oligomer that has the sequence of Seq ID No. 15 or an oligomer that has the sequence of Seq ID No. 99. The mimetic of the miRNA having the sequence of Seq ID No. 17 preferably is or contains an oligomer that has the sequence of Seq ID No. 17 or an oligomer that has the sequence of Seq ID No. 100.
The invention also provides an miRNA mimetic of miRNA m29a-3p for use in the treatment of a fibroproliferative disorder, such as ILD, PF-ILD or IPF, wherein the miRNA mimetic is (less preferred) or contains (preferred) an oligomer of nucleotides that consists of the sequence selected form the group consisting of Seq ID No. 92, with the following proviso:
In one embodiment, the prevention and/or treatment further comprises the administration of a mimetic of a miRNA having the sequence of Seq ID No. 15. Preferably, the mimetic of the miRNA having the sequence of Seq ID No. 15 is or contains an oligomer of nucleotides that consists of the sequence of Seq ID No. 15 (preferred) or Seq ID No. 99 (less preferred) with the following proviso:
In another embodiment, the prevention and/or treatment further comprises the administration of a mimetic of a miRNA having the sequence of Seq ID No. 17. Preferably, the mimetic of the miRNA having the sequence of Seq ID No. 17 is (less preferred) or contains (preferred) an oligomer of nucleotides that consists of the sequence of Seq ID No. 17 (preferred) or Seq ID No. 100 (less preferred) with the following proviso:
In case, the miRNA mimetics are not delivered being packed in lipid based nano particles (LNPs), it is preferred that the oligomer mentioned in the proviso is lipid conjugated to facilitate drug delivery.
In yet another embodiment, the prevention and/or treatment further comprises the administration of a mimetic of a miRNA having the sequence of Seq ID No. 19. Preferably, the mimetic of the miRNA having the sequence of Seq ID No. 19 is (less preferred) or contains (preferred) an oligomer of nucleotides that consists of the sequence of Seq ID No. 19 with the following proviso:
Further embodiments of the invention are miRNA mimetics of miRNA 29a-3p (Seq ID No. 92), in combination with mimetics of the miRNA 212-5p (Seq ID No. 15) or miRNA 181a5p (Seq ID No. 17) for use in the treatment of a fibroproliferative disorder, such as ILD, PF-ILD or IPF, and wherein the miRNA mimetics are oligomers of nucleotides that consist of the sequence of Seq ID No. 92, Seq ID No. 15 or 99, and Seq ID No. 17 or 100, respectively, with the following proviso:
Further embodiments of the invention are miRNA mimetics of miRNA 29a-3p (Seq ID No. 92) in combination with miRNA 212-5p (Seq ID No. 15 or 99) or miRNA 181a-5p (Seq ID No. 17 or 100) for use in the treatment of a fibroproliferative disorder, such as ILD, PF-ILD or IPF, and wherein the miRNA mimetic is or contains an oligomer of nucleotides that consists of the sequence of Seq ID No. 92, Seq ID No. 15 or 99, and Seq ID No. 17 or 100.
These embodiments are preferred in case, the miRNA mimetics are delivered being packed in lipid based nanoparticles (LNPs). If LNP particles are used for delivery, the dose might be between 0.01 and 5 mg/kg of the mass of miRNA mimetics per kg of subject to be treated, preferably 0.03 and 3 mg/kg, more preferably 0.1 and 0.4 mg/kg, most preferably mg/kg. The administration is of the LNP particles preferably systemic, more preferably intravenous.
In case of a double-strand miRNA mimetic, the miRNA mimetic contains an oligomer of nucleotides (sense strand) that is bound to one or more oligonucleotides that are fully or partially complimentary to the sense strand of said miRNA mimetic, said sense strand of miRNA mimetic may or may not form with these one or more oligonucleotides overhang(s) with single stranded regions.
Double-strand miRNA mimetics are preferred.
A further embodiment of the invention relates to a pharmaceutical composition as defined herein above wherein the composition is an inhalation composition.
A further embodiment of the invention relates to a pharmaceutical composition as defined herein above wherein the composition is intended for systemic, preferably intravenous administration.
A further embodiment of the invention is a method of treating or preventing of a fibroproliferative disorder, such as ILD, PF-ILD or IPF, in a subject in need thereof comprising administering to the subject a pharmaceutical composition as defined above.
For example, the use of a miRNA inhibitor or a miRNA mimetic can be effected by the aerosol route for inhibiting fibrogenesis in the pathological respiratory epithelium in subjects suffering from pulmonary fibrosis and thus restoring the integrity of the pathological tissue so as to restore full functionality.
Further embodiments of the invention are described hereafter from 1 to 55:
The viral vector is preferably administered as in an amount corresponding to a dose of virus in the range of 1.0×1010 to 1.0×1014 vg/kg (virus genomes per kg body weight), although a range of 1.0×1011 to 1.0×1012 vg/kg is more preferred, and a range of 5.0×1011 to 5.0×1012 vg/kg is still more preferred, and a range of 1.0×1012 to 5.0×1011 is still more preferred. A virus dose of approximately 2.5×1012 vg/kg is most preferred. The amount of the viral vector to be administered, such as the AAV vector according to the invention, for example, can be adjusted according to the strength of the expression of one or more transgenes.
A further aspect of the invention is the use of viral vectors, miRNA inhibitors and miRNA mimetics according to the invention for combined therapy with either Nintedanib or Pirfenidone.
An expression cassette comprises a transgene and usually a promotor and a polyadenylation signal. The promotor is operably linked to the transgene. A suitable promoter may be selectively or constitutively active in a lung cell, such as an epithelial alveolar cell. Specific non-limiting examples of suitable promoters include constitutively active promoters such as the cytomegalovirus immediate early gene promoter, the Rous sarcoma virus long terminal repeat promoter, the human elongation factor 1a promoter, and the human ubiquitin c promoter. Specific non-limiting examples of lung-specific promoters include the surfactant protein C gene promoter, the surfactant protein B gene promoter, and the Clara cell 10 kD (“CC 10”) promoter.
A transgene, depending on the embodiment of the invention, either codes for (i) one or more miRNA e.g. a miRNA having the sequence of Seq ID No. 92 or one or more miRNA that are downregulated in a Bleomycin-induced lung fibrosis model or in an AAV-TGFβ1-induced lung fibrosis model, or (ii) for an RNA that inhibits the function of one or more miRNA that is upregulated in a Bleomycin-induced lung fibrosis model and in an AAV-TGFβ1-induced lung fibrosis model, or for both alternatives (i) and (ii). The transgene may also contain an open reading frame that encodes for a protein for transduction reporting (such as eGFP, see
An RNA that inhibits the function of one or more miRNA reduces or abolishes the function of its target miRNA by complementary binding. Two different vector design strategies can be applied, as described in
The term miRNA inhibitor according to the present invention refers to oligomers consisting of a contiguous sequence of 7 to at least 22 nucleotides in length.
The term nucleotide, as used herein, refers to a glycoside comprising a sugar moiety (usually ribose or desoxyribose), a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate internucleotide linkage group. It covers both naturally occurring nucleotides and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as nucleotide analogues herein. Non-naturally occurring nucleotides include nucleotides which have sugar moieties, such as bicyclic nucleotides or 2′ modified nucleotides or 2′ modified nucleotides such as 2′ substituted nucleotides.
Nucleotides with chemical modifications leading to non-naturally occurring nucleotides comprise the following modifications:
(i) Nucleotides which have Non-Natural Sugar Moieties,
Examples are bicyclic nucleotides or 2′ modified nucleotides or 2′ modified nucleotides such as 2′ substituted nucleotides.
(ii) Nucleotides with Phosphorothioate (PS) and Phosphodithioate (PS2) Modifications
To improve serum stability and increase blood concentrations as well as improve nuclease resistance of the miRNAs, a sulfur in one or more nucleotides of the miRNA inhibitor or mimic could exchange an oxygen of the nucleotide phosphate group, which is defined as a phosphorothioate (PS). For some sequences, this could be combined or complemented by a second introduction of a sulfur group to an existing PS, which is defined as a Phosphodithioate PS2. PS2 modifications on distinct positions of the sense strand, like on nucleotide 19+20 or 3+12 (counting from the 5′ end), could further increase serum stability and therefore the pharmacokinetic characteristics of the miRNA inhibitor/miRNA mimetic (ACS Chem. Biol. 2012, 7, 1214-1220).
(iii) Nucleotides with Boranophosphat Modifications
For some miRNA oligonucleotides, it could be beneficial to exchange one oxygen of the ribose phosphate group against a BH3 group. Boranophosphat modifications on one or more nucleotides could increase serum stability, in case the seed region of miRNA oligonucleotides are not modified by other chemical modifications. Boranophosphat modifications could also increase serum stability of miRNA oligonucleotides (Nucleic Acids Research, Vol. 32 No. 20, 5991-6000).
(iv) Nucleotides with 2′O-Methyl Modification
Besides or in addition to phosphate modifications, methylation of the oxygen, bound to the carbon C2 in the ribose ring, could be further options for oligonucleotide modifications. 2′O-methyl ribose modification of the sense strand could increase thermal stability and the resistance to enzymatic digestions.
(v) Nucleotides with 2′OH with Fluorine Modification
It may could also beneficial to modify miRNA oligonucleotides with 2′ OH fluorine modification to enhance serum stability of the oligonucleotide and improve the binding affinity of the miRNA oligonucleotide to its target. 2′ OH fluorine modification, exchanges the hydroxyl group of the carbon C2 in the ribose ring against a fluorine atom. Fluorine modifications could be applied on both strands, sense and anti-sense.
“Nucleotide analogues” are variants of natural oligonucleotides by virtue of modifications in the sugar and/or base moieties. Preferably, without being limited by this explanation, the analogues will have a functional effect on the way in which the oligomer works to bind to its target; for example by producing increased binding affinity to the target and/or increased resistance to nucleases and/or increased ease of transport into the cell. Specific examples of nucleoside analogues are described by Freier and Altman (Nucl. Acid Res, 25: 4429-4443, 1997) and Uhlmann (Curr. Opinion in Drug Development, 3: 293-213, 2000). Incorporation of affinity-enhancing analogues in the oligomer, including Locked Nucleic Acid (LNA™), can allow the size of the specifically binding oligomer to be reduced and may also reduce the upper limit to the size of the oligomer before non-specific or aberrant binding takes place. The term “LNA™” refers to a bicyclic nucleoside analogue, known as “Locked Nucleic Acid” (Rajwanshi et al., Angew Chem. Int. Ed. Engl., 39(9): 1656-1659, 2000). It may refer to an LNA™ monomer, or, when used in the context of an “LNA™ oligonucleotide” to an oligonucleotide containing one or more such bicylic analogues.
Preferably, a miRNA inhibitor of the invention refers to antisense oligonucleotides with sequence complementary to Certain upregulated miRNA (miRNAs selected from the group consisting of the miRNAs of Seq ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 34, 35 and 36.). These oligomers may comprise or consist of a contiguous nucleotide sequence of a total of 7 to at least 22 contiguous nucleotides in length, up to 70% nucleotide analogues (LNA™). The shortest oligomer (7 nucleotides) will likely correspond to an anti-sense oligonucleotide with perfect sequence complementarity matching to the first 7 nucleotides located at the 5′ end of mature to Certain up regulated miRNA, and comprising the 7 nucleotide sequence at position 2-8 from 5′ end called the “seed” sequence) involved in miRNA target specificity (Lewis et al., Cell. 2005 Jan. 14; 120(1):15-20).
A Certain upregulated miRNA Target Site Blocker refers to antisense oligonucleotides with sequence complementary to Certain upregulated miRNA binding site located on a specific mRNA. These oligomers may be designed according to the teaching of US 20090137504. These oligomers may comprise or consist of a contiguous nucleotide sequence of a total of 8 to 23 contiguous nucleotides in length. These sequences may span from 20 nucleotides in the 5′ or the 3′ direction from the sequence corresponding to the reverse complement of Certain upregulated miRNA “seed” sequence.
The term miRNA mimetic of the invention is a single-stranded or double-stranded oligomer of nucleotides capable of specifically increasing the activity of certain miRNA wherein the term certain miRNA means a miRNA that has a sequence selected from the group consisting of Seq ID No. 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 37, 38, 39, and 92 preferably of Seq ID No. 92, 15, 17, 19, 18, and 20, most preferred 15, 17 and 19, even more preferred Seq ID No. 15. The term miRNA mimetic encompasses salts, including pharmaceutical acceptable salts. The miRNA mimetic of a miRNA elevates the concentration of functional equivalents of said miRNA in the cell thereby increasing the overall activity of said miRNA.
These miRNA mimetics of the present invention typically and preferably consist of a contiguous nucleotide sequence of a total of 21, 22 or 23 contiguous nucleotides in length. The length of the miRNA mimetics (i.e. the oligonucleotide in case of a single-strand mimetic or the sense strand in case of a double strand mimetic) typically matches the length of the respective miRNA they mimic (preferred).
In case of miRNA mimetics of a miRNA that has 23 nt, such as miR-181a-5p or miRNA-212-5p, the length of the miRNA mimetics (i.e. the oligonucleotide in case of a single strand or the sense strand of the double strand mimetic) the is either 23 nt (preferred) or 22 nt with the proviso that one nucleotide at the 3′-terminus is missing. Since the deletion at the 3′-terminus compared to the authentic mRNA (see e.g. Seq ID NO. 100, 99) is remote from the seed region and the region of nucleotides at 13-16 of the miRNA, the specificity of the corresponding miRNA mimetics is acceptable (Grimson et al., 2007).
miRNA mimetics of miRNA 29a-3p, 212-5p, miRNA 181a-5p, miRNA 181b-5p, and miRNA 10a-5p, respectively are intended for use in the treatment of a fibroproliferative disorder, such as ILD, PF-ILD or IPF, and wherein the miRNA mimetic is or contains an oligomer of nucleotides that consists of the sequence of Seq ID No. 92, of Seq ID No. 15 or 99, of Seq ID No. 17 or 100, Seq ID No. 18, and Seq ID No. 19, respectively with proviso (a), (b) and (c), (a) and (c), (a) and (d), or (c) and (d),
Lipid conjugated oligomers are well known in the art, see Osborne et al. NUCLEIC ACID THERAPEUTICS Volume 28, Number 3, 2018 with references.
Oligomer consisting of the sequence of Seq ID No. x means that the oligomer comprises the sequence of Seq ID No. x and has as many covalently attached nucleotide building blocks (optionally with chemical modifications) or nucleotide analogues as indicated in the Seq ID No. x.
The miRNA mimetic may be a single-strand miRNA mimetic or a double-strand miRNA mimetic. A single-stand mimetic is an oligonucleotide with no other oligonucleotide molecule bound thereto with full or partial base-pairing. Double-strand miRNA mimetics are defined as miRNA mimetics that are bound to one or more oligonucleotides that are fully or partially complimentary to the miRNA mimetic and that may or may not form with these oligonucleotides overhangs with single stranded regions. The triple RNA strand design referred to under Example 1.11 is an example for double-stranded miRNA mimetics. A further example is disclosed in Vinnikov et al. (2014), p. 10661, 1st col, last paragraph. It is preferred that the miRNA mimetic has at least 80%, more preferably at least 90%, even more preferably more than 95% of the biologic effect of the same amount of the natural miRNA as determined by one or more experiments as described under Example 1.11.
miRNA mimetics or miRNA inhibitors can also be delivered as naturally- and non-naturally occurring nucleotides, packed in lipid nano particles (LNPs). For RNA as cargo molecules, the most effective LNPs contain ionizable lipids with pKa values typically below pH 7 and are composed of up to four components, i.e. ionizable lipids, structural lipids, cholesterol, and polyethyleneglycol (PEG) lipids.
Ionizable lipids include but are not limited to 1,2-dilinoleoyl-3-dimethylamine (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLinMC3-DMA) (Naseri N, Valizadeh H, Zakeri-Milani P. Solid lipid nanoparticles and nanostructured lipid carriers: structure, preparation, and application. Adv Pharm Bull. 2015; 5(3):305-313), ATX-lipids (Ramaswamy S, Tonnu N, Tachikawa K, et al. Systemic delivery of factor IX messenger RNA for protein replacement therapy. Proc Natl Acad Sci USA. 2017; 114(10):E1941-50.), or YSK12-C4-lipids (Sato Y, Hashiba K, Sasaki K, et al. Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo. J Control Release. 2019; 295:140-152.)
Structural lipids include but are not limited to dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), distearoyl-sn-glycero-3-phosphatidylocholine (DSPC), dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE), dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), hydrogenated soybean phosphatidylcholine (HSPC), etc. Cholesterol includes but is not limited to cholesterol and 3-(N—(N0,N0-dimethylaminoethane)-carbamoyl) cholesterol, sterols, steroids, etc.
PEG-lipids include but are not limited to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-mPEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DMPE-mPEG2000), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DPPE-mPEG2000), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DOPE-mPEG2000) and variations of those PEG-lipids with respect to the PEG length, e.g. PEG500, PEG1000, PEG5000, etc.
The LNP formulations can contain distinct proportions of the single LNP components, distinct particle size, and a distinct ratio of positively-chargeable polymer amine (N=nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups (N/P ratio). The preferred formulations comprise or contain 25 to 65 mol % of ionizable lipids, preferably 40 mol %, 5 to 30 mol % of structural lipids, preferably 15 mol %, 15 to 50 mol % cholesterol, preferably 40 mol %, and 1 to 5 mol % of PEG-lipids, preferably 2 mol %. The mean particle size of LNPs can vary between 30 and 200 nm and N/P ratios can vary between 2 to 4, whereas the most preferred nanoparticle size is 100 nm with a N/P ratio of 3.
The most preferred LNP formulation will have the following composition: 40 mol % ionizable lipid consisting of DLinMC3-DMA or ATX lipids, or YSK12-C4-lipids, 15 mol % DSPC, 40 mol % cholesterol, 2 mol % DSPE-mPEG2000 with a particle size of 100 nm and a N/P ratio of 3.
The most preferred miRNA modality for LNP delivery of miRNA mimetics is a double-strand miRNA mimetic, consisting of a complementary passenger sense strand to an anti-sense strand. The passenger strand will protect the anti-sense strand from endonucleases. Like described by Vinnikov et al., both strands have LNA modified overhangs on the 3′ site, consisting of two nucleotides with LNA modification (Vinnikov et al, 2014). LNA stands for locked nucleic acid and is defined by two sugar moieties containing a methylene bridge between the 2-oxygen and the 4-carbon of the ribofuranose ring a two nucleotide LNA-modified overhang on the 3′ site. Additionally the first nucleotide on the 5′ site of the sense strand is also LNA modified to facilitate strand discrimination in the RISC complex. LNA-moiety restricts the flexibility of the monomer and locks it in a rigid bicyclic N-type conformation conferring exceptional tolerance against nucleases and extremely low cellular toxicity. Moreover, these minimal modifications provide a compromise between stability and functionality both for in vitro and in vivo applications (Elmén et al., 2005; Mook et al., 2007 as cited in Vinikov et al). LNA modifications will lead to greater melting temperatures (Tm values) for hybridization with complimentary sequences. Each LNA modified nucleotide can increase Tm up to 8° C. of a formed nucleotide pair (DOI: 21). The length of the sense- and anti-sense strand typically comprises 20-22, 20-23, 20-24 or 20-25 nucleotides.
Another alternative is to design the microRNA mimetics in the triple RNA strand design described under point 1.11 (Functional characterization of miRNAs in cellular assays).
If LNP particles are used for delivery, the dose might be between 0.01 and 5 mg/kg of the mass of miRNA mimetics per kg of subject to be treated, preferably 0.03 and 3 mg/kg, more preferably 0.1 and 0.4 mg/kg, most preferably 0.3 mg/kg. The administration of the LNP particles is preferably systemic, more preferably intravenous.
1. Materials and Methods
1.1 AAV Production, Purification and Quantification
HEK-293h cells were cultivated in DMEM+GlutaMAX media supplemented with 10% fetal calf serum. Three days before transfection, the cells were seeded in 15 cm tissue culture plates to reach 70-80% confluency on the day of transfection. For transfection, 0.5 total DNA per cm 2 of culture area were mixed with 1/10 culture volume of 300 mM CaCl2 as well as all plasmids required for AAV production in an equimolar ratio. The plasmid constructs were as follows: One plasmid encoding the AAV6.2 cap gene (Strobel B et al., 2015); a plasmid harboring an AAV2 ITR-flanked expression cassette containing a CMV promoter driving expression of a codon-usage optimized murine Tgfb1 gene and a hGh poly(A) signal, whereby the Tgfb1 sequence contains C223S and C225S mutations that increase the fraction of active protein (Brunner A M et al., 1989); a pHelper plasmid (AAV Helper-free system, Agilent). For GFP and stuffer control vector production, the Tgfb1 plasmid was exchanged for an eGFP plasmid, harboring an AAV2 ITR-flanked CMV-eGFP-SV40pA cassette and AAV-stuffer control plasmid, containing an AAV2 ITR-flanked non-coding region derived from the 3′-UTR of the E6-AP ubiquitin-protein ligase UBE3A followed by a SV40 poly(A) signal, respectively.
The plasmid CaCl2 mix was then added dropwise to an equal volume of 2×HBS buffer (50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4), incubated for 2 min at room temperature and added to the cells. After 5-6 h of incubation, the culture medium was replaced by fresh medium. The transfected cells were grown at 37° C. for a total of 72 h. Cells were detached by addition of EDTA to a final concentration of 6.25 mM and pelleted by centrifugation at room temperature and 1000× g for 10 min. The cells were then resuspended in “lysis buffer” (50 mM Tris, 150 mM NaCl, 2 mM MgCl2, pH 8.5). AAV vectors were purified essentially as previously described (Strobel B et al., 2015): For iodixanol gradient based purification, cells harvested from up to 40 plates were dissolved in 8 mL lysis buffer. Cells were then lysed by three freeze/thaw cycles using liquid nitrogen and a 37° C. water bath. For each initially transfected plate, 100 units Benzonase nuclease (Merck) were added to the mix and incubated for 1 h at 37° C. After pelleting cell debris for 15 min at 2500× g, the supernatant was transferred to a 39 mL Beckman Coulter Quick-Seal tube and an iodixanol (OptiPrep, Sigma Aldrich) step gradient was prepared by layering 8 mL of 15%, 6 mL of %, 8 mL of 40% and 5 mL of 58% iodixanol solution diluted in PBS-MK (lx PBS, 1 mM MgCl2, 2.5 mM KCl) below the cell lysate. NaCl had previously been added to the 15% phase at 1 M final concentration. 1.5 μL of 0.5% phenol red had been added per mL to the 15% and 25% iodixanol solutions and 0.5 μL had been added to the 58% phase to facilitate easier distinguishing of the phase boundaries within the gradient. After centrifugation in a 70Ti rotor for 2 h at 63000 rpm and 18° C., the tube was punctured at the bottom. The first five milliliters (corresponding to the 58% phase) were then discarded, and the following 3.5 mL, containing AAV vector particles, were collected. PBS was added to the AAV fraction to reach a total volume of 15 mL and ultrafiltered/concentrated using Merck Millipore Amicon Ultra-15 centrifugal filter units with a MWCO of 100 kDa. After concentration to ˜1 mL, the retentate was filled up to 15 mL and concentrated again. This process was repeated three times in total. Glycerol was added to the preparation at a final concentration of 10%. After sterile filtration using the Merck Millipore Ultrafree-CL filter tubes, the AAV product was aliquoted and stored at −80° C.
1.2 Mouse Models and Functional Readouts
For reporter gene studies, 9-12 week old female C57Bl/6 or Balb/c mice, purchased from Charles River Laboratories, either received 2.9×1010 vector genomes (vg) of AAV5-CMV-fLuc or 3×1011 vg of AAV6.2-CMV-GFP, respectively, by intratracheal administration under light anesthesia (3-4% isoflurane). Alternatively, C57Bl/6 mice received 3×1011 vg of AAV2-L1-CMV-GFP by intravenous (i.v.) administration. Two to three weeks after AAV administration (see figure descriptions), reporter readouts were performed. For luciferase imaging, mice received 30 mg/kg luciferin as a substrate via intraperitoneal administration prior to image acquisition. In the case of GFP reporters, either histological fresh-frozen lung sections were prepared and analyzed for direct GFP fluorescence by fluorescence microscopy or formalin-fixed paraffin embedded slices were prepared for GFP IHC analysis (see detailed description further below).
For the fibrosis models, male 9-12 week old C57Bl/6 mice purchased from Charles River Laboratories received intratracheal administration of either 2.5×1011 (vg) of AAV-TGFβ1 or AAV-stuffer, 1 mg/kg Bleomycin or physiological NaCl solution in a volume of 50 μL, which was carried out under light anesthesia. Fibrosis was assessed at day 3, 7, 14, 21 and 28 after AAV/Bleomycin administration. Briefly, to assess lung function, mice were anesthetized by intraperitoneal (i.p.) administration of pentobarbital/xylazine hydrochloride, cannulated intratracheally and treated with pancuronium bromide by intravenous (i.v.) administration. Lung function measurement (i.e. lung compliance, forced vital capacity (FVC)) was then conducted using the Scireq flexiVent FX system. Mice were then euthanized by a pentobarbital overdose, the lung was dissected and weighed prior to flushing with 2×700 μL PBS to obtain BAL fluid for differential BAL immune cell and protein analyses (data not shown). The left lung of each mouse was processed for histological assessment by a histopathologist, whereas the right lung was used for total RNA extraction, as detailed below.
For the miR-212-5p AAV pharmacokinetic study we used male, C57BL/6JRj mice, 10-12 weeks old from Janvier Labs. Mice were intratrachealy (i.t.) instilled with stuffer negative control AAV (1×1011 vg) or three rising dosages (9×109 vg, 10×1010 vg and 1×1011 vg) of miR-212-5p-AAV. i.t. instillation was carried out under light anesthesia with short exposure to isoflurane. Mice were euthanized on day 7, day 14 and day 28 after AAV instillation. Lungs were snap frozen in liquid nitrogen and processed to frozen lung powder for total RNA isolation (using miRNAeasy kit from Qiagen).
1.3 Histology
For the preparation of histological lung samples, the left lung lobe was mounted to a separation funnel filled with 4% paraformaldehyde (PFA) and inflated under 20 cm water pressure for 20 minutes. The filled lobe was then sealed by ligature of the trachea and immersed in 4% PFA for at least 24 h. Subsequently, PFA-fixed lungs were embedded in paraffin. Using a microtome, 3 μm lung sections were prepared, dried, deparaffinized using xylene and rehydrated in a descending ethanol series (100-70%). Masson's trichrome staining was performed using the Varistain Gemini ES Automated Slide Stainer according to established protocols. For GFP-IHC, enzymatic antigen retrieval was performed and antibodies were diluted at indicated ratios in Bond primary antibody diluent (Leica Biosystems). Slides were stained with the 1:1000 diluted polyclonal Abcam rabbit anti-GFP antibody ab290 and appropriate isotype control antibodies, respectively. Slides that had only received antigen retrieval served as an additional negative control. Finally, sections were mounted with Merck Millipore Aquatex medium.
1.4 RNA Preparation
For total lung RNA preparation, the right lung was flash frozen in liquid nitrogen immediately after dissection. Frozen lungs were homogenized in 2 mL precooled Qiagen RLT buffer+1% β-mercaptoethanol using the Peqlab Precellys 24 Dual Homogenizer and 7 mL-ceramic bead tubes. 150 μL homogenate were then mixed with 550 μL QIAzol Lysis Reagent (Qiagen). After addition of 140 μL chloroform (Sigma-Aldrich), the mixture was shaken vigorously for 15 sec and centrifuged for 5 min at 12,000×g and 4° C. 350 μL of the upper aqueous RNA-containing phase were then further purified using the Qiagen miRNeasy 96 Kit according to the manufacturer's instructions. After purification, RNA concentration was determined using a Synergy HT multimode microplate reader and the Take3 module (BioTek Instruments). RNA quality was assessed using the Agilent 2100 Bioanalyzer.
1.5 RNA Sequencing
cDNA libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit. Briefly, 200 ng of total RNA were subjected to polyA enrichment using oligo-dT-attached magnetic beads. PolyA-containing mRNAs were then fragmented into pieces of approximately 150-160 bp. Following reverse transcription with random primers, the second cDNA strand was synthesized by DNA polymerase I. After an end repair process and the addition of a single adenine base, phospho-thymidine-coupled indexing adapters were coupled to each cDNA, which facilitate sample binding to the sequencing flow cell and further allows for sample identification after multiplexed sequencing. Following purification and PCR enrichment of the cDNAs, the library was diluted to 2 nM and clustered on the flow cell at 9.6 pM, using the Illumina TruSeq SR Cluster Kit v3-cBot-HS and the cBot instrument. Sequencing of 52 bp single reads and seven bases index reads was performed on an Illumina HiSeq 2000 using the Illumina TruSeq SBS Kit v3-HS. Approximately 20 million reads were sequenced per sample.
For miRNA, the Illumina TruSeq Small RNA Library Preparation Kit was used to prepare the cDNA library: As a result of miRNA processing by Dicer, miRNAs contain a free 5′-phosphate and 3′-hydroxal group, which were used to ligate specific adapters prior to first and second strand cDNA synthesis. By PCR, the cDNAs were then amplified and indexed. Using magnetic Agencourt AMPure XP bead-purification (Beckman Coulter), small RNAs were enriched. The samples were finally clustered at 9.6 pM and sequenced, while being spiked into mRNA sequencing samples.
1.6 Computational Processing and Data Analysis (mRNA-Seq and miRNA-Seq Data Processing)
mRNA-Seq reads were mapped to the mouse reference genome GRCm38.p6 and Ensembl mouse gene annotation version 86 (http://oct2016.archive.ensembl.org) using the STAR aligner v. 2.5.2a (Dobin et al., 2013). Raw sequence read quality was assessed using FastQC v0.11.2, alignment quality metrics were checked using RNASeQC v1.18 (De Luca D. S. et al., 2012). Subsequently, duplicated reads in the RNA-Seq samples were marked using bamUtil v1.0.11 and subsequently duplication rates assessed using the dupRadar Bioconductor package v1.4 (Sayols-Puig, S. et al., 2016). Read count vectors were generated using the feature counts package (Liao Y. et al., 2014). After aggregation to count matrices data were normalized using trimmed mean of M-values (TMM) and voom transformed to generate log(counts per million) (CPM) (Ritchie M. E., 2015). Descriptive analyses such as PCA and hierarchical clustering were carried out to identify possible outliers. Differential expression between treatment and respective controls at each time points were carried out using limma with a significance threshold of p adj≤0.05 and abs(log2 FC)≥0.5. Two samples out of 124 in total were excluded for not passing QC criteria. miRNA-Seq reads were trimmed using the Kraken package v.12-274 (Davis M. P. A. et al., 2013) and subsequently mapped to the mouse reference genome GRCm38.p6 and the miRbase v. 2l mouse miRNA (http://mirbase.org) using the STAR aligner v. 2.5.2a. Raw sequence read quality was assessed using FastQC v0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), trimming size and biotype distribution assessed using inhouse scripts. After aggregation to count matrices data were normalized using trimmed mean of M-values (TMM) and voom transformed to generate log(counts per million) (CPM). Descriptive analyses such as PCA and hierarchical clustering were carried out to identify possible outliers. Differential expression between treatment and respective controls at each time points were carried out using limma with a significance threshold of p adj≤0.05 and abs(log2 FC)≥0.5.
1.7 Integrated Data Analysis (Correlation of Functional Parameters and Expression)
Spearman's rho between the measured values for lung function and lung weight vs. the voom transformed log(CPM) of each miRNA and mRNA across all samples of both models and all time points.
1.8 Determination of Putative miRNA-mRNA Target Pairs
To determine mRNA targets of miRNAs, a stepwise approach has been carried out. First lowly expressed miRNAs and mRNAs were removed from the expression matrix. Subsequently the Spearman's rho was calculated between voom transformed log(CPM) of each miRNA vs. each mRNA across all samples of both models and all time points, using the corAndPvalue function from WGCNA v. 1.60 (Langfelder & Horvath, 2008) The set of correlation based putative miRNA-mRNA pairs is defined as all combinations with a correlation≤−0.6. To add sequence based prediction of putative miRNA-mRNA pairs, all combinations with predictions in at least two out of five most cited miRNA target prediction algorithms (DIANA, Miranda, PicTar, TargetScan, and miRDB) available in the Bioconductor package miRNAtap v. 1.10.0/miRNAtap.db v. 0.99.10 (Pajak & Simpson, 2016) were taken as sequence based pairs. The final set of miRNA-mRNA pairs is the intersection of anticorrelation based and sequence based interaction pairs, reducing the number of predictions significantly to a high-confidence subset.
1.9 Mouse-Human Conservation of miRNA Sequences
For all murine and human miRNAs from miRBase 21 seed regions (position 2 to 7) were extracted. For all combinations of murine and human miRNAs global alignments between the seed regions and the mature were calculated using the pairwise Alignment function from the Bioconductor Biostrings package (v2.46.0). We applied the Needleman-Wunsch algorithm using an RNA substitution matrix with a match score of 1 and a mismatch score of 0. We assigned two categories to the miRNA candidates—“conserved” for miRNAs with an alignment score of 6 in the seed region for mouse-human pairs of miRNAs with the same name, “non-conserved” for miRNAs with an alignment score<6 in the seed region for mouse-human pairs of miRNAs with the same name. In addition, miRNAs with an alignment score for the alignment of the respective mature sequences above 20 is assigned to the category “mature high similarity”.
1.10 Characterization of miRNAs Based on Gene Set Enrichment of Target Gene Sets
The functional characterization of miRNAs is carried out using the enrichment function on the predicted mRNA targets from the MetabaseR package v. 4.2.3 and the gene set categories “pathway maps”, “pathway map folders”, “process networks”, “metabolic networks”, “toxicity networks”, “disease genes”, “toxic pathologies”, “GO processes”, “GO molecular functions”, “GO localizations”. The enrichment function performs a hypergeometric test on the overlap of the query gene set and the reference sets from Metabase. The data retrieval for the characterization of miRNA target sets was carried out on Metabase on Mar. 12, 2018.
1.11 Functional Characterization of miRNAs in Cellular Assays
miRNAs were characterized regarding their impact on the cellular production of the pro-inflammatory cytokine IL-6 and the pro-fibrotic processes fibroblast proliferation, fibroblasts-to-myofibroblasts transition (FMT), collagen expression and epithelial-to-mesenchymal transition (EMT). Unless stated differently in the Figures or Figure Legends, A549, NHBEC (normal human bronchial epithelial cells) or NHLF (normal human lung fibroblast) cells were transiently transfected with miRNA mimetic at a concentration of 2 nM for single miRNAs or 2+2 nM for miRNA combinations.
All miRNA mimetics used in the experiments shown in the Figures were purchased from Qiagen in the three stranded miRCURY LNA miRNA Mimic format. The design of miRCURY LNA miRNA Mimics includes three RNA strands, rather than the two RNA strands that characterize traditional miRNA mimics. The miRNA (guide) strand is an unmodified RNA strand with a sequence corresponding exactly to the annotation in miRBase. However, the passenger strand is divided into two LNA-enhanced RNA strands (https://www.qiagen.com/de/products/discovery-and-translational-research/functional-an-dcell-analysis/mirna-functional-analysis/mircury-lna-mirna-mimics/mircury-lna-mirna-mimics/#orderinginformation). When designed correctly, these triple RNA strand mimics are as potent as traditional double-strand RNA mimics. The great advantage is that the segmented nature of the passenger strand ensures that only the miRNA strand is loaded into the RNA-induced silencing complex (RISC) with no resulting miRNA activity from the two complementary passenger strands. Phenotypic changes observed with miRCURY LNA miRNA mimics can therefore be safely ascribed to the miRNA simulated by the mimic (see figure miRNA target identification with biotinylated mimics).
The distinct triple RNA strand design is enabled by incorporation of high-affinity LNA nucleotides into the two passenger strands. The sequence, length and LNA spiking pattern of the two passenger strands have been optimized using a sophisticated and empirically derived design algorithm. Bramsen, J. B., et al. (2007) Improved silencing properties using small internally segmented interfering RNAs. Nucleic Acids Research 35:5886-5897. PMID: 17726057. Griffiths-Jones, S. (2004) The miRNA Registry. Nucleic Acids Research Database Issue 32:D109-111. 3. miRBase: www.mirbase.org. Kahn, A. A., et al. (2009) Transfection of small RNAs globally perturbs gene regulation by endogenous miRNAs. Nature Biotechnology 27(6):549-555. doi: 10.1038/nbt.1543.
The miRNA mimetics for miR-29a-3p, miR-181a-5p and miR-212-5p and the corresponding control were used in
All other miRNA mimetics used for the other Figures were designed analogously. Thus, for miR-181b-5p a sequence of Seq ID No. 19 was used, and for miR-10a-5p a sequence according Seq ID No. 18 was used.
For the latter condition, 4 nM miRNA controls were used. Twenty-four hours later, TGFβ1 was added to the cells at 5 ng/mL concentration and cells were incubated for 24 h (IL-6, proliferation assays and collagen mRNA expression) or 72 h (collagen protein expression, FMT and EMT assays). For the measurement of gene expression, total RNA was extracted from the cells using the Qiagen RNeasy Plus 96 Kit and reversely transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). IL-6 gene expression was detected by a Taqman qPCR assay (Hs00174131_m1). IL-6 protein was quantified in the cell supernatant using the MSD V-PLEX Proinflammatory Panel 1 Human kit. To assess cell proliferation, cells were grown in presence of TGFβ1 for 24 h and assayed using a WST-1 proliferation assay kit (Sigma/Roche). FMT was assessed by growing NHLF cells as described above, followed by fixation and fluorescent immuno-staining of Collagen 1a1. Images were taken using an IN Cell Analyzer 2000 high-content cellular imaging system and collagen was quantified and normalized to cell number (identified by DAPI-stained nuclei). EMT assessment relied on the same principle, however, using NHBEC cells and immuno-staining of E-cadherin.
Immunoblots were done according to standard methods using novex gels and according buffers from ThermoFisher and electrophoresis devices from BioRad. All primary antibodies were ordered from Cell Signaling Technology.
All cellular assays were performed with either primary lung epithelial cells or primary lung fibroblasts derived from human patient material. Thus, by the heterogeneity of each individual patient donor, e.g. its genetics, environment, cause of disease/surgery, cell isolation, etc., the derived cell also underlie a certain heterogeneity. Thus, it can happen that there are slight assay-to assay variabilities, which explain a certain standard deviation and different assay windows between equal assay formats. Nevertheless, we used primary cells because they are primary patient material and therefore more relevant for the human disease.
2. Results
AAV-TGFβ1 and Bleomycin administration induce fibrosing lung pathology in mice. Following administration of either AAV-TGFβ1, Bleomycin or appropriate controls (NaCl, AAV-stuffer), longitudinal fibrosis development was measured over a time period of 4 weeks, as illustrated in
Transcriptional characterization of chronological disease manifestation. In order to dissect the molecular pathways and overall changes in gene expression underlying disease development and progression in the two models of pulmonary fibrosis, RNA was prepared from lung homogenates of each animal and applied to next generation sequencing (NGS) analysis. The number of differentially expressed mRNAs and miRNAs is depicted in
Identification of miRNAs associated with clinically relevant disease phenotypes. To identify candidate miRNAs likely to be directly associated with disease development, a staggered selection strategy using multiple filter criteria was set up (
miRNA target prediction (
Functionality of miRNAs in mir-E backbone (
miRNA expression in primary human lung fibroblasts (
Functional characterization of miRNAs in cellular assays (
Because we also wanted to assess other miRNA combinations, beyond miR-10a+miR-181a-5p+miR-181b-5p, we repeated the former EMT assay, depicted in
In addition to airway epithelial cells, fibroblasts are considered as a highly relevant cell type for fibrotic processes. By acting as the main source for excessive production of collagen and other extracellular matrix components, fibroblasts directly contribute to lung stiffening associated with impaired lung function and finally loss of structural lung integrity. To further investigate the function of candidate miRNAs during fibroblast activation, transient transfection experiments were carried out in primary human lung fibroblasts under unstimulated and TGFβ-stimulated (pro-fibrotic) conditions. As functional readouts IL6 expression, collagen expression and fibroblast proliferation were assessed in absence or presence of miRNAs. As shown in
In accordance to collagen 1 deposition in primary lung fibroblasts, miR-181a-5p and miR-212-5p profoundly inhibit intracellular collagen 1 synthesis, especially when they were dosed in combination (
To characterize the performance of a viral construct, we transduced naive mice with increasing dosages of an AAV 6.2 construct, containing a miR-212-5p expression cassete (see Seq ID NO: 61, derived from plasmid according to Seq ID No: 91, see
In summary, the functional characterization in human airway epithelial cells and human lung fibroblasts demonstrates anti-inflammatory, anti-proliferative and anti-fibrotic effects for selected miRNA candidates. The most pronounced effects across all assay formats were observed for miR-181a-5p, mir-181b-5p and mir-212-5p, whereas mir-10a-5p and mir212-3p showed similar profiles although at weaker efficiency compared to the aforementioned miRNAs. In the FMT assay we observed positive effects by miR-10a-5p, miR-181a-5p, miR-181b-5p and miR-212-5p, whereas a triple combination of mir-10a-5p, mir-181a-5p and mir-181b-5p showed an improved inhibitory effects in the FMT assay, indicating an additive or synergistic effect for this combination. Overall we observed a very potent anti-fibrotic effect of miR-181a-5p on lung epithelial cells and a very potent anti-fibrotic effect of miR-212-5p on fibroblasts, which suggests that the combination of these two miRNAs are very potent anti-fibrotic combination affecting the two most important cell types in pulmonary fibrosis. Therefore, combinations of miRNA candidates, and especially mimetics of miR-181a-5p and miR-212-5p or its respective mimetics, provide a preferred option for the development of therapeutic approaches with superior efficiency profiles compared to single miRNAs. In addition, we were able to validate the published collagen inhibitory effects of single miR-29a-3p under fibrotic conditions. By specific combination of miR-29a-3p with miR-212-5p in a dual combination or with miR-212-5p and miR-181a-5p in a triple combination, we could show that this leads to an even more pronounced anti-fibrotic effect compared to the single miRNAs or the dual combination of miR-212-5p with miR-181a-5p. By using lower doses of the involved miRNAs in the combinations, compared to their single use, we are able to keep their anti-fibrotic effects and will likely lead to reduced unwanted/unspecific effects in transduced cells. Furthermore, the specific combination of mirR-29a-3p with either miR-212-5p or with both miR-212-5p and miR-181a-5p would allow to potentially address pulmonary hypertension (PH) in ILD, PF-ILD or IPF patients that either already have a PH co-morbidity or would otherwise develop one. It was shown by Chen, T. et al. that miR-212-5p increase could reduce RVSP and pulmonary vessel wall remodeling in a mouse model of pulmonary hypertension. Besides (super-) additive or synergistical advantages the triple combination also combines anti-fibrotic effects on two key cell types in the pathogenesis of lung fibrosis: epithelial cells and fibroblasts. By combining miR-181a-5p, which has a very pronounced anti-fibrotic effect on the transformation of lung epithelial cells, and miR-212-5p and miR29a-3p, which possess massive anti-fibrotic effects on fibroblast activation and inhibition of ECM deposition, the triple combination of these three miRNAs increases the biological therapeutic spectrum against the single miRNAs.
Therapeutic applications of miRNAs. To translate the discovery of novel lung-fibrosis associated miRNAs into therapeutic applications, approaches based on vector-mediated expression offer an attractive opportunity for chronic diseases like pulmonary fibrosis by enabling long-lasting expression of miRNAs or miRNA-targeting sequences. As illustrated in
For the delivery of the aforementioned expression constructs to the lung non-viral as well as viral gene therapy vectors can be applied. However, compared to currently available non-viral delivery systems like e.g. liposomes, viral vectors demonstrate superior properties with regard to efficacy and tissue/cell-type selectivity, as demonstrated in various publications over the past years. Moreover, viral vectors offer great potential for engineering approaches to further improve potency, selectivity and safety properties. In recent years, viral vectors based on Adeno-associated virus (AAV) have emerged as one of the most favorable vector systems for in vivo gene therapy based on their excellent pre-clinical and clinical safety profile combined with highly efficient and stable gene delivery to various target organs and cell-types including fully differentiated and non-dividing cells. Since the discovery of the prototypic AAV serotype AAV2 in 1965 (Atchison et al.), various additional serotypes have been isolated from humans, non-human primates and from phylogenetically distinct species such as pigs, birds and others. To date more than 100 natural AAV isolates have been described, which interestingly differ with regard to tissue tropism. By applying capsid engineering approaches the repertoire of available AAV vectors for gene therapy approaches has been further expanded in recent years. Based on a landmark paper by Limberis et al. (2009), in which a systematic comparison of 27 AAV capsid variants and natural serotypes regarding lung transduction is described, AAV5, AAV6 and AAV6.2 were identified as highly suitable capsids for lung delivery following local routes of administration (e.g. intransal or intratracheal instillation). In addition, an engineered AAV capsid variant based on AAV2 (AAV2-L1) has been described recently as a novel vector enabling specific gene delivery to the lung after systemic vector administration (Körbelin et al., 2016). As described in
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/081019 | 11/4/2020 | WO |
