Treatment of ciliopathies

Abstract

The present disclosure provides a pharmaceutical composition comprising a polyribonucleotide for use in treating a ciliopathic disease in a subject suffering from the disease, wherein the polyribonucleotide encodes a functional version of a protein whose deficiency is associated with the ciliopathic disease, and wherein the pharmaceutical composition is administered to the subject's respiratory system when the subject exhibits inflammation of the respiratory system. Additionally, the present disclosure relates to a method for analyzing the effect of a polyribonucleotide on ciliogenesis, wherein the polyribonucleotide encodes a protein associated with and / or required for ciliogenesis.

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition comprising a polyribonucleotide for use in the treatment of a ciliopathies in a subject suffering from a ciliopathies, said polyribonucleotide encoding a functional version of a protein associated with said ciliopathies, and administration of said pharmaceutical composition to the respiratory system of said subject is effected when said subject exhibits inflammation in said respiratory system.The present invention further relates to a method for analyzing the effect of a polyribonucleotide on ciliogenesis, said polyribonucleotide encoding a protein associated with and / or required for ciliogenesis. [Background technology]

[0002] Oxygen is crucial for many multicellular organisms, as it is crucial for the energy supply of cells. In mammals, oxygen contained in the air can enter the organism through the respiratory system, which refers to the organs related to breathing. These include, for example, the nose, throat, larynx, trachea, bronchi, and lungs. In the latter, gases from the environment are exchanged with gases contained in the internal blood circulatory system, which transports oxygen from the lungs to cells in different parts of the organism. However, besides the required oxygen, other components such as irritants contained in the air, including pollutants, disease-causing substances and pathogens (e.g., viruses and bacteria), can also enter the organism through the respiratory system. Therefore, defense mechanisms such as cough reflex and sneezing exist to expel irritants from the respiratory system. For expulsion, the irritants are secreted by epithelial cells of the respiratory system and then embedded in a viscous mucus that is transported across the epithelial surface by ciliated cells.

[0003] Ciliated cells are able to transport mucus and irritants across epithelial surfaces by the synchronized beating of hyperkinetic motile cilia. The ciliary body is a membrane-bounded tubular structure that extends from the epithelial surface into the respiratory space in contact with the environment. Within the cell, the axoneme of the ciliary membrane is anchored to the basal body via an anchoring structure. The axoneme is a central bundle of microtubules in which nine outer doublet microtubules surround a central pair of singlet microtubules (i.e., the respiratory cilia). The outer doublet microtubules and the central microtubule pair are connected by radial spokes. Each of the nine outer doublet microtubules consists of an A-tubule and a B-tubule, and the doublets are connected circumferentially by a nexin-dynein regulatory complex. The inner and outer dynein arms are connected to their respective A-tubules. These dynein arms contain mo-proteins that can walk along microtubules, which leads to bending and thus beating of the cilia (e.g., Lodish et al, 2000, Molecular Cell Biology, 4 th edition, New York: WH Freeman, see Section 19.4). Studies of ciliary structure using the model Trypanosoma brucei have shown that, whereas some of these structural parts exhibit rapid turnover, the scaffolding components of the radial spokes, central pair and lateral dynein arms are primarily incorporated into the distal end of the ciliary structure during development (Vincensini et al., Biol Cell, 2018, 110:1-15).

[0004] Synchronized beating of ciliated cells is essential for the transport of fluids across epithelial surfaces, so any disruption of cilia structure can cause severe disorders. Motility defects, including reduced amplitude and / or frequency of beating and / or asynchrony, can result, for example, from a reduction or loss of dynein arms, disorganization of microtubule arrangement, including mislocalization, and changes in the total number of microtubules per axon, as well as combinations thereof. As far as is known, these alterations in functional axonemal elements are caused by underlying genetic defects. These are primarily due to changes in the sequences of genes that code for axonemal components, including those mentioned above. Since genes can be transcribed into polyribonucleotides such as mRNA and then translated into proteins, the DNA sequence of the genes affects the synthesis of the respective proteins, in terms of quantity and functionality. Since ciliopathy, i.e., ciliopathy caused by changes in base sequence, is inherited, and is associated with impaired cilia motility at all developmental stages, including embryonic or neonatal, ciliopathy can have serious consequences for the organism.

[0005] A well-known example of a ciliopathies is primary ciliary dyskinesia (PCD), a progressive disease that is often associated with a decline in lung function. Thus, long-term treatment is required, including, for example, chest percussion and postural drainage, to increase mucus clearance with increasing frequency, and also lung transplantation in severe cases. In addition, PCD patients often suffer from recurrent infections in the lungs and / or ears, from subfertility, hydrocephalus and body laterality, i.e., left-right axis, defects, as well as retinal and / or neurological problems. However, despite the seriousness of most ciliopathies, no standardized and effective strategies for treating ciliopathies like PCD exist so far. Current treatments are extrapolated from, for example, cystic fibrosis, and in most cases are not validated for the specific ciliopathies to be treated, for example, PCD. Therefore, there is a need to have a solution at hand to be able to efficiently treat subjects suffering from ciliopathies like PCD. Summary of the Invention [Problem to be solved by the invention]

[0006] The present application addresses the need to restore ciliary function in subjects suffering from ciliopathies such as PCD by providing the claimed embodiments. [Means for solving the problem]

[0007] In particular, the present invention relates to a pharmaceutical composition comprising a polyribonucleotide for use in the treatment of a ciliopathies in a subject suffering from a ciliopathies, said polyribonucleotide encoding a functional version of a protein whose deletion is associated with said ciliopathies, and administration of said pharmaceutical composition to the respiratory system of said subject is effected when said subject exhibits inflammation of said respiratory system. [Brief description of the drawings]

[0008] [Figure 1] Figure 1: Translation efficiency of CCDC40 mRNA. 2 / 1.4 / 0.3 / 0.2 / 0.05x10^6 HEK293 cells were seeded in 6-well plates. Seedling cells were transfected with different CCDC40 constructs (ETH031T06-T10, 2.5μg / 9.5cm2) for 24 h using Lipofectamine2000. Cell lysis was performed 6, 24, 48, 72 and 144 h after transfection. 50μg of total protein lysates were analyzed by SDS-PAGE and Western blot. CCDC40 was detected using anti-CCDC40 antibody (HPA022974) from Atlas Antibodies (1:2000) and GAPDH served as a loading control. GFP served as a transfection control. [Diagram 2]Figure 2: Translation efficiency of CCDC40 mRNA constructs in BEAS-2B, RPMI2650, and HEK293 cells: 2x10^6 HEK293, 7.5x10^5 BEAS-2B, and 5x10^5 RPMI 2650 cells were seeded in 6-well plates. 24 h after seeding, cells were transfected with different CCDC40 constructs (2.5μg / 9.5cm2) using Lipofectamine2000. Cell lysis was performed 6 h after transfection. Protein lysates were analyzed by SDS-PAGE and Western blot (HEK293: 50μg, RPMI 2650: 20μg, BEAS-2B: 30μg of total lysate) and CCDC40 was detected using anti-CCDC40 antibody (HPA022974) from Atlas Antibodies (1:2000). [Diagram 3] Figure 3: High-speed video microscopy (HSVM) results after LF92 / CCDC40 transfection. Patient-derived ALI cultures were transfected every other day with 3 μg of LF92 / CCDC40. Videos (20 per insert) were taken before transfection and every 24 h after transfection and the CFB (ciliary beating frequency) was calculated using SAVA (Sisson-Ammons video analysis) software. A total of 16 transfections (1 month) were performed. Measurements were performed at 37 °C with 40x magnification. The mean value of the ciliary beating frequency measurements is calculated (total field analysis, WFA). [Figure 4] Figure 4: Mucociliary clearance (MCC) is shown as Z-projection and polar graph. CCDC40 patient ALI cultures were transfected with CCDC40 mRNA / LF92 at a concentration of 3 μg / insert for 18 days by airlift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). Mucociliary clearance (MCC) was measured at 20x magnification using 0.5 μm fluorescent beads. 30 videos of different areas were taken and analyzed with the Polargraph software from Nikon 1 week after the last TF. One exemplary picture is shown. [Diagram 5]Figure 5: Mucociliary clearance (MCC) shown as Z-projection (top left) and polar graph (top right) for tdTomato and polar graphs of healthy (bottom right) controls. Top row: Cells from a patient with a CCDC40 mutation in ALI culture were transfected with tdTomato mRNA / LF111 at a concentration of 3 µg / insert 18 days after airlift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). Mucociliary clearance (MCC) was measured at 30x magnification using 0.5 µm fluorescent beads. 20 videos of different areas were taken and analyzed with Polargraph software from Nikon 1 week after the last TF. One exemplary video was shown. Bottom right: Particle tracking of a healthy WT ALI culture. MCC was measured at 20x magnification using 0.5 µm fluorescent beads. Twenty videos of different areas were taken and analyzed with the Polargraph software from Nikon one week after the last TF. One exemplary image and two analyzed ROIs are shown. [Figure 6] Figure 6: Cells from patients with CCDC40 mutations in ALI cultures were transfected with either tdTomato mRNA / LF111 or CCDC40 mRNA / LF92 at a dose of 3 µg / insert for 18 d at airlift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). As control, WT ALI from healthy nasal brushes were used. ALI membranes treated with CCDC40 mRNA and tdTomato mRNA were sectioned at 20 µm and stained with anti-GAS8 (red), anti-acetylated tubulin (green) and DAPI (blue). Photographs were taken with a confocal microscope. One exemplary image is shown. Scale bar represents 10 µm. [Figure 7]Figure 7: Cells from patients with CCDC40 mutations in ALI cultures were transfected with either tdTomato mRNA / LF111 or CCDC40 mRNA / LF92 at a dose of 3 μg / insert, 18 d after airlift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). As control, ALI from healthy controls were used. ALI membranes treated with CCDC40 mRNA and tdTomato mRNA were sectioned at 20 μm and stained with anti-DNALI1 (red), anti-acetylated tubulin (green) and DAPI (blue). Photographs were taken with a confocal microscope. One exemplary image is shown. Scale bar represents 10 μm. [Figure 8] Figure 8: Cells from patients with CCDC40 mutations in ALI cultures were transfected with either tdTomato mRNA / LF111 or CCDC40 mRNA / LF92 at a dose of 3 μg / insert for 18 d at air lift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). As control, ALI from healthy controls were used. ALI membranes treated with CCDC40 mRNA and tdTomato mRNA were sectioned at 20 μm and stained with anti-CCDC39 (1:200, red), anti-acetylated tubulin (1:10.000, green) and DAPI (blue). Pictures were taken with a confocal microscope. One exemplary image is shown. Magnification 40x. [Figure 9]Figure 9: CCDC39 protein (110 kDa) expression in HEK-293 and BEAS-2B, 6 and 24 hours after transfection. 1.4x10^5 HEK-293 cells and 3.5x10^5 BEAS-2B cells were seeded in 6 well plates and transfected with CCDC39-RNA (ETH047T02, minimal 5'UTR, Lipofectamine MessengerMax; ratio 1:1.5). After 6 and 24 hours, cells were lysed with M-PER and Triton® X-100 buffer. 50 μg of protein lysate was used for Western blot analysis. As controls, lysates of non-transfected (UT) and EGFP-transfected cells were used. High dose = 0.5 μg / cm2, low dose = 0.25 μg / cm2. [Figure 10] Figure 10: CCDC39 protein (110 kDa) expression in untreated ALI cultures. Two inserts were extracted using the axonal extraction protocol. 30, 10 and 5 μL of protein lysate were used for Western blot analysis. Active Area: Insert 39.9 = 71.25%, Insert 41.2 = 59.88% [Figure 11] Figure 11: CCDC39 protein (110 kDa) expression in 16HBE14 after proteasome inhibitor treatment. 6.0x10^5x16HBE14ocells were seeded in 6-well plates and transfected with CCDC39-RNA (Lipofectamine MessengerMax; ratio 1:1.5). After 6 h, cells were lysed with M-PER buffer. 20 μg of protein lysate was used for Western blot analysis. As controls, lysates of non-transfected (UT) and EGFP-transfected cells were used. High dose = 0.5 μg / cm2, low dose = 0.25 μg / cm2. RNA: ETH047T02: minimal 5'UTR, ETH047T03: TISU, ETH047T04: CYBA, ETH047T05: SP30. [Figure 12]Figure 12: CCDC39 protein (110 kDa) expression upon 16HBE14oafter treatment with proteasome inhibitors. 6.0x10^5 16HBE14ocells were seeded in 6-well plates and transfected with CCDC39-RNA (ETH047T03, TISU 5'UTR, Lipofectamine Messenger Max; ratio 1:1.5). After 24 hours, cells were lysed with M-PER buffer. 20 μg of protein lysate was used for Western blot analysis. As controls, lysates of non-transfected (UT) and EGFP-transfected cells were used. High dose=0.5 μg / cm2, low dose=0.25 μg / cm2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The invention is based on the finding that it is indeed possible to restore proper ciliary function by transfecting cells (which exhibit a defect in a specific protein of the protein complex of the cilia, the defect resulting in the loss of proper ciliary function) with polyribonucleotides encoding a functional version of said protein. However, it was also found that in order to obtain this effect, the ciliated cells must be transfected at an early stage of differentiation. This leads to practical problems, since the precursor cells of the epithelial cells responsible for the cilia, i.e. the basal cells, are located deeper down in the epithelium and are not exposed to the surface, and therefore cannot be accessed for transfection via the airway system. According to the invention, the transfection of epithelial cells by administration of polyribonucleotides is carried out when a subject suffering from a ciliopathy exhibits inflammation of the respiratory system. During respiratory inflammation, the airway epithelium exhibits lesions and wounds that make accessible the precursor cells of the ciliated cells, i.e. the basal cells that have not yet started ciliogenesis. Transfecting these cells with such polyribonucleotides expressing a functional version of the respective protein makes it possible to make these cells cells that form functional cilia or at least partially functional cilia, which can lead to a substantial alleviation of the respective symptoms.

[0010] In the context of the present invention, the term "ciliopathies" refers to diseases associated with and / or characterized by defects in ciliated cells. Thus, ciliopathies include disorders of ciliary structures, including ciliary anchoring structures, basal bodies in which ciliary structures are anchored within cells, and / or ciliary function. Examples of ciliopathies include PCD, Bardet-Biedl syndrome, Simpson-Golabi-Behmel syndrome (type 2), Leber congenital amaurosis, nephronophthisis, and craniectodermal dysplasia (Sensenbrenner) (see, for example, Mitchison et al., 2017, Ultrastructural Pathology,41(6):415-427).

[0011] The term "ciliopathies" as used herein refers to ciliopathies caused by genetic defects in the DNA of a subject, such as chromosomal or mitochondrial DNA. Such genetic defects can be caused by mutations, and can include the loss, addition or exchange of sequence parts. Examples are copy number mutations, presence or absence mutations, deletions (full or partial), insertions, missense mutations, nonsense mutations, splice site mutations, or combinations thereof. Such changes in DNA can result in changes in the availability of encoded proteins (e.g., loss or reduced amount of protein), or proteins with altered functions.

[0012] In a preferred embodiment, the term "ciliopathies" refers to diseases associated with motile ciliary dyskinesia (PCD). PCD is a rare disease caused by the malfunction of motile cilia. PCD is heterogeneous at the genetic, functional and ultrastructural levels. Subjects suffering from PCD show recurrent nasal congestion, sinus infections, ear infections, infertility such as situs inversus and ectopic, and / or hydrocephalus. At the molecular level, PCD is most often associated with abnormalities in the structure, function and biogenesis of cilia in the respiratory system. Examples of such abnormalities are the absence or shortening of dynein arms, complexes, radial spokes or nexin links. Such abnormalities, and thus the ciliary motility defects associated with PCD, are caused by mutations in the genes encoding the respective components, in particular, those genes listed in groups "A" and "B" refer to genes with pathogenic mutations estimated to account for at least 1% and less than 1% of PCD cases, respectively (see Zariwala et al., GeneReviews®, 2007, updated 2015, Primary Ciliary Dyskinesia, editors Adam et al., Seattle (WA): University of Washington, Seattle; 1993-2018).

[0013] Thus, the polyribonucleotides comprised in the pharmaceutical compositions according to the invention are preferably polyribonucleotides that can be translated into functional versions of the proteins listed in Table 1.

[0014] More preferably, the polyribonucleotide comprised in the pharmaceutical composition of the present invention is an mRNA that can be translated into a functional version of a protein selected from the group consisting of DNAH5, DNAH11, CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1, CCDC114, RSPH4A, DNAAF1 (LRC50), DNAAF2 (KTU), and LRRC6.

[0015] [Table 1]

[0016] In some embodiments of any of the foregoing or other aspects and embodiments of the disclosure, the polyribonucleotide or modified polyribonucleotide comprises a primary sequence that is at least 85%, at least 90%, at least 92% or at least 95% identical (e.g., at least 95, 96, 97, 98, 99 or 100% identical) to one or more of SEQ ID NOs: 1 or 5-11 (e.g., a sequence set forth in SEQ ID NOs: 1 or 5-11). In some embodiments, the polyribonucleotide is a modified polyribonucleotide having a level and / or type of modification selected from any such level and / or type described herein. In certain embodiments, the percent identity of the polyribonucleotide is measured only with respect to the CCDC40 coding sequence portion of SEQ ID NOs: 1 or 5-11 (e.g., UTRs, other non-coding sequences and GFP or epitope tags are not considered when calculating percent identity). In certain embodiments of any of the foregoing, such a polyribonucleotide (or modified polyribonucleotide) encodes a functional CCDC40 protein.

[0017] In some embodiments of any of the foregoing or other aspects and embodiments of the disclosure, the polyribonucleotide or modified polyribonucleotide comprises a primary sequence that is at least 85%, at least 90%, at least 92%, or at least 95% identical (e.g., at least 95, 96, 97, 98, 99 or 100% identical) to one or more of SEQ ID NOs: 2 or 12-14 (e.g., a sequence set forth in SEQ ID NOs: 2 or 12-14). In some embodiments, the polyribonucleotide is a modified polyribonucleotide having a level and / or type of modification selected from any such level and / or type described herein. In certain embodiments, the percent identity of the polyribonucleotide is measured only with respect to the CCDC39 coding sequence portion of SEQ ID NOs: 2 or 12-14 (e.g., UTRs, other non-coding sequences, and GFP tags or epitope tags are not considered when calculating percent identity). In certain embodiments of any of the foregoing, such a polyribonucleotide (or modified polyribonucleotide) encodes a functional CCDC39 protein.

[0018] In some embodiments of any of the above or other aspects and embodiments of the disclosure, the polyribonucleotide or modified polyribonucleotide comprises a primary sequence that is at least 85%, at least 90%, at least 92%, or at least 95% identical (e.g., at least 95, 96, 97, 98, 99 or 100% identical) to SEQ ID NO:4 (e.g., to the sequence shown in SEQ ID NO:4). In some embodiments, the polyribonucleotide is a modified polyribonucleotide with a level and / or type of modification selected from any such level and / or type described herein. In certain embodiments, the percent identity of the polyribonucleotide is measured only with respect to the MCIDAS coding sequence portion of SEQ ID NO:4 (e.g., UTRs, other non-coding sequences and GFP or epitope tags are not considered when calculating percent identity). In certain embodiments of any of the above, such a polyribonucleotide (or modified polyribonucleotide) encodes a functional MCIDAS protein.

[0019] In certain embodiments, the present disclosure provides a pharmaceutical composition comprising any of the aforementioned polyribonucleotides. Furthermore, any such polyribonucleotide (or pharmaceutical composition) can be used in any of the methods described herein.

[0020] In one embodiment of a pharmaceutical composition comprising a polyribonucleotide for use in the treatment of a ciliopathies, said ciliopathies are associated with a deficiency in coiled-coil domain-containing 40 (CCDC40; see, e.g., NCBI reference sequences NM_017950.4 and NP_060420.2 for human mRNA and protein CCDC40 sequences, respectively) protein, or a deficiency in coiled-coil domain-containing 39 (CCDC39; see, e.g., NCBI reference sequences NM_181426.2 and NP_852091.1 for human mRNA and protein CCDC39 sequences, respectively) protein.

[0021] CCDC40 and CCDC39 build a complex located between the radial spokes and the A-tubule. The defective version of CCDC40 or CCDC39 protein can be caused by mutations in the CCDC40 gene or the CCDC39 gene, such as insertions, deletions, nonsense and splice site mutations. In particular, deletions at position 248 and TGT insertions between positions 2824 and 2825 in the DNA sequence encoding the CCDC40 protein appear to be quite frequent. The defective version of CCDC40 or CCDC39 results in defects in the structure of the axon (e.g., absence or eccentricity of the central pair, abnormal radial spoke and nexin connections, abnormal assembly of the dynein regulatory complex, and / or reduction of the inner dynein arm). Thus, the polyribonucleotide contained in the pharmaceutical composition according to the present invention is preferably an mRNA that can be translated into a functional version of CCDC40 and / or CCDC39. Further information on proteins associated with ciliogenesis, as well as genes and molecular pathways associated with ciliopathies, can be found, for example, in the review article by Reiter and Leroux (Reiter and Leroux, 2017, Nat Rev Mol Cell Biol,18(9):533-547).

[0022] The pharmaceutical composition of the present invention is administered to a subject suffering from a ciliopathies. In the present specification, a subject suffering from a ciliopathies may be referred to as a patient. The patient may exhibit abnormal ciliary structure and / or function, and / or biosynthesis defects, which lead to accumulation of mucus and bacteria in the airways. The diagnosis of a ciliopathies for a given subject may be based, for example, on clinical findings, molecular analysis and / or ciliary ultrastructural analysis of a biopsy of said subject, as also reviewed, for example, in Goutaki et al. (Goutaki et al., 2016, Eur Respir J, 48(4):1081-1095).

[0023] In the context of the present invention, the term "polyribonucleotide" refers to a single-stranded sequence constructed from adenosine, guanosine, cytidine, and / or uridine residues (modified or unmodified, see below). As used herein, the term "protein-encoding polyribonucleotide" refers to a polyribonucleotide that comprises a coding region that encodes a protein, i.e., a polyribonucleotide that can be translated into an amino acid sequence. Thus, in the context of the present invention, the term "protein-encoding polyribonucleotide" preferably refers to mRNA, which should be understood to mean any polyribonucleotide molecule that is suitable for the expression of a protein or that can be translated into a protein when it enters a cell.

[0024] As used herein, the term "protein" encompasses any type of amino acid sequence, i.e., a chain of two or more amino acids each linked via a peptide bond. The term "protein" as used in this context refers to any amino acid sequence of interest. Preferably, the encoded amino acid sequence is at least 5 amino acids long, more preferably at least 10 amino acids, even more preferably at least 50, 100, 200 or 500 amino acids. Thus, the term "protein" encompasses short peptides and polypeptides. There are no limitations regarding the function of the encoded protein, except that defective variants of the protein are associated with ciliopathies. As used herein, the term "associated with" is intended to encompass the terms "causes", "associated with" and / or "enhances".

[0025] As used herein, the term "protein having a defect" refers to a "defective protein" or a "defective version of a protein" and thus to a version of a protein whose function is altered compared to the functional version of said protein. However, this term can also encompass a version of said protein that has a complete or partial lack of synthesis and therefore availability in the cell. In either case, the defect of the protein version results in a version of said protein that is unable to fulfill the natural function of the protein.

[0026] A version of a protein that fulfills its native function is referred to herein as a "functional protein" or "functional version of a protein" and is encoded by the same DNA sequence as the respective defective protein, but without the deletion causing an alteration in the DNA sequence and therefore without a mutation in the DNA sequence.

[0027] Thus, in the context of the present invention, a "functional protein" is preferably a component of the A-tubule, B-tubule, or nexin-dynein regulatory complex, or a radial spoke, an inner dynein arm, and / or an outer dynein arm.

[0028] Thus, the term "polyribonucleotides encoding a functional version of a protein whose deficiency is associated with said ciliopathies" preferably refers to an mRNA encoding a functional protein associated with the structural organization of the ciliary axons, axon anchoring structures or basal bodies and performing its original function. Thus, polyribonucleotides according to the invention preferably refer to an mRNA encoding a functional protein, the presence of which is necessary or beneficial in the cells of a subject suffering from a ciliopathies to neutralize or prevent the manifestation of said ciliopathies associated with the deficiency of said protein encoded by the DNA sequence of the cells or to alleviate the associated symptoms.

[0029] In addition, the polyribonucleotide used according to the present invention may also contain additional functional regions and / or 3' or 5' non-coding regions. The 3' and / or 5' non-coding regions may be sequences naturally adjacent to the encoded protein or artificial sequences that contribute to the stabilization and / or regulation of said polyribonucleotide. Suitable sequences can be identified and investigated by routine experimentation. In addition, said polyribonucleotide may also have additional functional regions, for example, combined with regulatory elements and target sequences of microRNAs to spatially and temporally control the activity of the desired polyribonucleotide, including sequences encoding proteins, for example, with respect to specific cells or cell types and / or developmental stages or specific time frames.

[0030] The polyribonucleotides used according to the present invention may contain partial or complete codon-optimized sequences derived from the natural sequences used. Codon optimization refers in some cases to a technique applied to maximize protein expression by increasing the translation efficiency of each polyribonucleotide, so that there are codons that are preferentially used by some species for a given amino acid. Furthermore, the polyribonucleotides may contain further modifications to adjust and / or extend the duration of action. The polyribonucleotides may also contain m7GpppG caps, internal ribosome entry sites (IRES), and / or polyA tails at the 3' end, and / or further sequences to facilitate translation.

[0031] In some embodiments of the present invention, the polyribonucleotides used according to the present invention may contain unmodified and modified nucleotides. As used herein, the term "unmodified nucleotides" refers to A, C, G and U nucleotides. As used herein, the term "modified nucleotides" refers to any naturally occurring or non-naturally occurring isomers of A, C, G and U nucleotides, as well as any naturally occurring or naturally occurring analogs thereof, such as alternative or modified nucleotides or isomers thereof with chemical modifications or substituted residues. Modified nucleotides can have base modifications and / or sugar modifications. Modified nucleotides can also have phosphate group modifications, for example, with respect to the 5-prime cap of an mRNA molecule. Modified nucleotides also include nucleotides that are post-transcriptionally synthesized by covalent modification of nucleotides. Furthermore, any suitable mixture of unmodified and modified nucleotides is possible. An infinite number of examples of modified nucleotides can be found in the literature (e.g., Cantara et al., Nucleic Acids Res, 2011, 39(Issue suppl_1):D195-D201; Helm and Alfonzo, Chem Biol, 2014, 21(2):174-185; Carell et al., Angew Chem Int Ed Engl, 2012, 51(29):7110-31), where some preferred modified nucleotides are exemplarily mentioned based on their respective nucleoside residues: 1-Methyladenosine, 2-Methylthio-N6-hydroxynorvalylcarbamoyl adenosine, 2-Methyladenosine, 2'-O-ribosylphosphate adenosine, N6-Methyl-N6-threonylcarbamoyl adenosine, N6-Acetyladenosine, N6-Glycinylcarbamoyl adenosine, N6-Isopentenyladenosine, N6-Methyladenosine, N6-Threonylcarbamoyladenosine, N6,N6-dimethyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, N6-hydroxynorvalylcarbamoyl adenosine, 1,2'-O-dimethyladenosine, N6,2'-O-dimethyladenosine, 2'-O-methyladenosine, N6,N6,2'-O-trimethyladenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-methyladenosine, 2-Methylthio-N6-isopentenyladenosine, 2-Methylthio-N6-threonylcarbamoyladenosine, N6-2-Methylthio-N6-threonylcarbamoyladenosine, 2-Methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 7-Methyladenosine, 2-Methylthio-adenosine, 2-Methoxy-adenosine, 2'-Amino-2'-deoxyadenosine, 2'-Azido-2'-deoxyadenosine, 2' -Fluoro-2'-deoxyadenosine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenosine, 7-deaza-8-aza-adenosine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine; 2-thiocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-hydroxycytidine, lysidine, N4-acetyl-2'-O-methylcytidine, 5-formyl-2'-O-methylcytidine, 5,2'-O-dimethylcytidine, 2-O-methylcytidine, N4,2'-O-dimethylcytidine, N4,N4,2'-O-trimethylcytidine, isocytidine, Pseudocytidine, pseudoisocytidine, 2-thio-cytidine, 2'-methyl-2'-deoxycytidine, 2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, 5-iodocytidine, 5-bromocytidine, 2'-azido-2'-deoxycytidine, 2'-amino-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, 5-aza-cytidine, 3-methyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-methyl-1-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine isocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine; 1-methylguanosine, N2,7-dimethylguanosine, N2-methylguanosine, 2'-O-ribosylphosphateguanosine, 7-methylguanosine, hydroxywybutosine, 7-aminomethyl-7-deazaguanosine, 7-cyano-7-deazaguanosine, N2,N2-dimethylguanosine, N2,7,2'-O-trimethylguanosine, N2,2'-O-dimethylguanosine, 1,2'-O-dimethylguanosine, 2'-O-methylguanosine, N2,N2,2'-O-trimethylguanosine, N2,N2J-trimethylguanosine, isoguanosine, 4-demethylguanosine, epoxyqueuosine, unmodified hydroxywybutosine, methylated unmodified hydroxywybutosine, Isowyosin, Peroxywybutosin, Galactosyl-Queuosin, Mannosyl-Queuosin, Queuosine, Archeuosine, Wybutosine, Methylwyosine, Wyosine, 7-Aminocarboxypropyldemethylwyosine, 7-Aminocarboxypropylwyosine, 7-Aminocarboxypropylwyosine methyl ester, 7-Deaza-guanosine, 7-Deaza-8-aza-guanosine, 6-Thio-guanosine, 6-Thio-7-Deaza-guanosine, 6-Thio-7-Deaza-8-aza-guanosine, 7-Methyl-guanosine, 6-Thio-7-Methyl-guanosine, 7-Methylinosine, 6-Methoxy-guanosine uridine, 1-methylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, N1-methylguanosine, 2'-amino-3'-deoxyguanosine, 2'-azido-2'-deoxyguanosine, 2'-fluoro-2'-deoxyguanosine, 2-thiouridine, 3-(3-amino-3-carboxypropyl)uridine, 3-methyluridine, 4-thiouridine, 5-methyl- 2-Thiouridine, 5-Methylaminomethyluridine, 5-Carboxymethyluridine, 5-Carboxymethylaminomethyluridine, 5-Hydroxyuridine, 5-Methyluridine, 5-Taurinomethyluridine, 5-Carbamoylmethyluridine, 5-(Carboxyhydroxymethyl)uridine methyl ester, Dihydrouridine, 5-Methyldihydrouridine, 5-Methylaminomethyl-2-thiouridine, 5-(Carboxyhydroxymethyl)uridine, 5-(Carboxyhydroxymethyl)-2'-O-methyluridine Lysine methyl ester, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thiouridine, 3,2'-O-dimethyluridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carbamoylmethyl-2-thiouridine, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-(isopentenylaminomethyl)-2'-O-methyluridine, 5,2'-O-dimethyluridine, 2'-O-methyluridine, 2'-O-methyl-2-thiouridine, 2-thio-2'-O-methyluridine, uridine 5-oxyacetic acid, 5-methoxycarbonylmethyluridine, Uridine 5-oxyacetic acid methyl ester, 5-methoxyuridine, 5-aminomethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-taurinomethyl-2-thiouridine, pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 1-methylpseudouridine, 3-methylpseudouridine, 2'-O-methylpseudouridine, 5-formyluridine, 5-aminomethyl-2-geranyluridine, 5-taurinomethyluridine, 5-iodouridine, 5-bromouridine, 2'-methyl-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 2'-fluoro-2'-deoxyuridine, inositol 1-methylinosine, 1,2'-O-dimethylinosine, 2'-O-methylinosine, 5-aza-uridine, 2-thio-5-aza-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine pseudouridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 1,2'-O-dimethyl- adenosine, 1,2'-O-dimethylguanosine, 1,2'-O-dimethylinosine, 2,8-dimethyladenosine, 2-methylthiomethylenethio-N6-isopentenyl-adenosine, 2-geranylthiouridine, 2-lysidine, 2-methylthiocyclic N6-threonylcarbamoyl adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, 2-selenouridine, 2-thio-2'-O-methyluridine, 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, 2'-O-methylinosine, 2'-O-methylpseudouridine, 2'-O-methyluridine, 2'-O-methyluridine 5-oxyacetic acid methyl ester, 2'-O-ribosyladenosine phosphate, 2'-O-ribosylaganosine phosphate, 3,2'-O-dimethyluridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 5,2'-O-dimethylcytidine, 5,2'-O-dimethyluridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 55-(isopentenylaminomethyl)-2'-O-methyluridine, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carboxyhydroxymethyluridine, 5-carboxymethyl-2-thiouridine Auridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 5-cyanomethyluridine, 5-formyl-2'-O-methylcytidine, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-demethylwiocin, 7-methylguanosine, 8-methyladenosine, N2,2'-O-dimethylguanosine, N2,7,2'-O-trimethylguanosine, N2,7-dimethylguanosine, N2,N2,2'-O-trimethylguanosine, N2,N2,7-trimethylguanosine, N2,N2,7-trimethylguanosine, N4,2'-O-dimethylcytidine, N4,N4,2'-O-trimethylcytidine, N4,N4-dimethylcytidine, N4-acetyl-2'-O-methylcytidine, N6,2'-O-dimethyladenosine, N6,N6,2'-O-trimethyladenosine, N6-formyl adenosine, N6-hydroxymethyl adenosine, Agmatidine, 2-methylthio cyclic N6-threonylcarbamoyl adenosine, glutamyl-queosine, Guanosine added to any nucleotide, guanylated 5' terminus, hydroxy-N6-threonylcarbamoyladenosine; most preferably, pseudouridine, N1-methyl-pseudouridine, 2'-fluoro-2'-deoxycytidine, 5-iodocytidine, 5-methylcytidine, 2-thiouridine, 5-iodouridine and / or 5-methyl-uridine.

[0032] Furthermore, the term "modified nucleotide" includes nucleotides that contain isotopes such as deuterium. The term "isotope" refers to elements that have the same number of protons but different numbers of neutrons, resulting in different mass numbers. Thus, for example, isotopes of hydrogen are not limited to deuterium, but also include tritium. Furthermore, polyribonucleotides can also contain isotopes of other elements, including, for example, carbon, oxygen, nitrogen, and fluorophores. Modified nucleotides can be deuterated or contain other isotopes of hydrogen or oxygen, carbon, nitrogen, or fluorophores.

[0033] The total number of modified nucleotide types in a polyribonucleotide can be 0, 1, 2, 3, or 4. Thus, in some embodiments, at least one nucleotide of one nucleotide type can be modified nucleotide, for example, at least one U nucleotide. In some embodiments, at least one nucleotide of a total of two nucleotide types can be modified nucleotide, for example, at least one U nucleotide and at least one C nucleotide. In some embodiments, at least one nucleotide of a total of three nucleotide types can be modified nucleotide, for example, at least one G nucleotide, at least one U nucleotide and at least one C nucleotide. In some embodiments, at least one nucleotide of all four nucleotide types can be modified nucleotide. In all of these embodiments, one or more nucleotides per nucleotide type may be modified and the percentage of modified nucleotides per nucleotide type is 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%.

[0034] In some embodiments, the total percentage of modified nucleotides in the purified mRNA molecules is 0%, 2.5%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%.

[0035] Thus, the polyribonucleotide may be characterized in that, for example, 0.5 to 50%, preferably 5 to 50%, of U nucleotides and 5 to 50% of C nucleotides are modified, the modified U nucleotides being preferably 5-iodouridine and the modified C nucleotides being preferably 5-iodocytidine.

[0036] In some embodiments, the polyribonucleotide may be characterized in that 15-25% U nucleotides and 3-15%, preferably 5-15% C nucleotides are modified, wherein the modified U nucleotides are preferably 5-methyluridine and the modified C nucleotides are preferably 5-iodocytidine.

[0037] In some embodiments, the polyribonucleotide can be characterized in that 30-50% U nucleotides and 10-20% C nucleotides are modified, wherein the modified U nucleotides are preferably 5-iodouridine and the modified C nucleotides are preferably 5-iodocytidine.

[0038] In some embodiments, the polyribonucleotide can be characterized in that 30-50% U nucleotides and 5-15% C nucleotides are modified, wherein the modified U nucleotides are preferably 5-iodouridine and the modified C nucleotides are preferably 5-iodocytidine.

[0039] In some embodiments, the polyribonucleotide can be characterized in that 0.5-5% U nucleotides and 25-35% C nucleotides are modified, wherein the modified U nucleotides are preferably 2-thiouridine and the modified C nucleotides are preferably 5-methylcytidine.

[0040] The polyribonucleotide may be characterized in that, for example, 50 to 100%, preferably 100%, of the U nucleotides are modified. The modified U nucleotides are preferably N1-methyl-pseudouridine.

[0041] As mentioned above, according to the present invention, the administration of the pharmaceutical composition to a patient suffering from a ciliopathies is carried out when the subject shows inflammation of the respiratory system. In the context of the present invention, the term "inflammation" refers to a cellular response to injury, including infection, trauma, and hypersensitivity, and "respiratory inflammation" refers to an inflammatory response in the respiratory system, in particular, but not limited to, the nose, pharynx, larynx, trachea, bronchi, and / or lungs. Inflammatory responses in the respiratory system can be caused by irritants such as pathogens, toxins, pollutants, and / or allergens. During inflammation, certain cell types are activated, which can, for example, release cytokines and mediators to modify the activity of other cells. These processes are as follows. For example, as described in Iwasaki et al. (Iwasaki et al., 2017, Nat Rev Immunol, 17(1):7-20).

[0042] Thus, in one embodiment, the subject suffering from a ciliopathic disease to which a polyribonucleotide is administered is subjected to an assay prior to treatment to determine whether the subject suffers from respiratory inflammation, where the subject is positively determined to have respiratory inflammation.

[0043] Preferably, the inflammation of the respiratory system is acute inflammation.Acute inflammation can occur over seconds, minutes, hours, and days, but not over longer periods.Acute inflammation is therefore inflammation that occurs over a time frame of up to 4 weeks, preferably less than 3 weeks.It can be determined by routine clinical examination based on local increased blood flow, local increased permeability of capillaries, and / or increased number of neutrophils, macrophages and / or lymphocytes.More information on markers of airway inflammation in primary ciliary dyskinesia can be found, for example, in Zihlif et al. (Zihlif et al., 2006, Pediatr Pulmonol, 41(6):509-14).

[0044] In general, inflammation can be classified as either acute or chronic. Acute inflammation is the body's response to harmful stimuli and is characterized, for example, by increased migration of granulocytes to affected tissues. The classical signs of inflammation are heat, pain, redness, swelling, and loss of function. Ciliopathy, such as PCD, is a genetic disease that, if untreated, undergoes permanent, lifelong stimulation by loss of function, resulting in permanent, chronic inflammation, etc., but typically does not exhibit the above symptoms (besides loss of function). Further examples of diseases associated with chronic inflammation are, for example, hay fever, periodontal disease, atherosclerosis, and osteoarthritis. Nevertheless, patients suffering from such diseases may further undergo acute inflammation, for example, by receiving additional harmful stimuli, resulting in one or more of the classical signs of inflammation, such as heat, pain, redness, swelling, and further loss of function.

[0045] Thus, in one embodiment, the subject suffering from a ciliopathies to which a polyribonucleotide is administered is subjected to prior treatment to an assay to determine whether the subject suffers from acute inflammation of the respiratory system, and if the subject is positively determined to have acute inflammation of the respiratory system.

[0046] Alternatively or additionally, respiratory inflammation refers to an inflammation exacerbation, preferably an acute inflammation exacerbation. An exacerbation refers to a worsening of a disease or its symptoms. Exacerbations are most often considered in the context of chronic obstructive pulmonary disease (COPD), as they reduce the quality of life of patients, accelerate the decline of lung function, and contribute substantially to disease-related costs.

[0047] For clinical trials, they state that "a respiratory exacerbation can be defined in a clinical trial as either airway symptoms leading to the initiation of systemic antibiotic treatment, regardless of bacterial culture results, or a decline in forced expiratory volume in 1 second (FEV1)% predicted by 10 percentage points or more compared to the mean of FEV1% predicted at screening and randomization, with or without the prescription of antibiotics. The occurrence of an exacerbation can be assessed by patient history, physical examination, and spirometry. At each study visit and during any special contact with the study site due to an exacerbation, participants can be interviewed about symptoms and concomitant medications since their last contact with the study site. Interviews can be supplemented by a weekly patient diary of symptoms and antibiotics. A physical examination to review the participant's general condition, vital signs, ears, heart, and lungs can be performed at every visit" (see, for example, Kobbernagel et al., 2016, BMC Pulmonary Medicine, 16:104).

[0048] Thus, according to a preferred embodiment, the pharmaceutical composition comprises an mRNA for use in treating a ciliopathic disease in a subject suffering from a ciliopathic disease, the mRNA encoding a functional version of a hair-like structural protein the defect of which is associated with said ciliopathic disease, and administration of said pharmaceutical composition to the respiratory system of a subject suffering from said ciliopathic disease is performed when the subject suffering from a ciliopathic disease exhibits acute inflammation, preferably an acute exacerbation, of the respiratory system.

[0049] Thus, in one embodiment, the subject suffering from a ciliopathic disease to which a polyribonucleotide is administered is subjected to an assay prior to treatment to determine whether the subject is suffering from acute inflammation of the respiratory system, preferably an acute exacerbation, wherein the subject is determined to have a positive chronic inflammation of the respiratory system, preferably an acute exacerbation.

[0050] The presence or absence of respiratory inflammation in a subject suffering from a ciliopathic disease can be determined by routine methods, for example by analyzing a blood sample or by determining whether the patient suffers from a running nose, etc.

[0051] In the respiratory system, inflammation is generally caused by infection, particularly viral or bacterial infection. Therefore, the presence or absence of inflammation in a ciliary disease patient can be preferably evaluated by determining the presence or absence of infection, preferably acute infection. Preferably, the infection is a viral and / or bacterial infection.

[0052] Acute infection is characterized by auscultatory findings, purulent cough, infiltrates, hemoptysis, fever, and elevated blood inflammatory parameters (c-reactive protein (CRP) >200mg / ml, erythrocyte sedimentation rate <100mm / h). Chronic infection is further characterized by migrational infiltrates, antibiotic resistance, persistent systemic symptoms, and moderate blood inflammatory markers (CRP 50-100mg / ml, erythrocyte sedimentation rate <50mm / h) (Klinische Pneumonologie, 1. Aufl. 2014 Georg Thieme Verlag KG, ISBN 978-3-13-129751-8; Jaroszewski et al., 2012, Thorac Surg Clin, 22(3):301-24).

[0053] In the context of the present invention, the term "respiratory system" includes organs related to breathing, such as the nose, pharynx, larynx, trachea, bronchi, and lungs. In particular, the respiratory system is also referred to as the respiratory system in some mammals, including humans, and is also referred to herein as the subject. The terms "respiratory system" and "respiratory system" are used interchangeably herein. The respiratory tract can be divided into the upper and lower respiratory tract. The upper respiratory tract includes the nose, including the nasal cavity, nasal turbinates, nasal vestibule, and nasal cavity; the paranasal sinuses; the pharynx, and the part of the larynx above the chord (cord). The lower respiratory tract includes the part of the larynx below the vocal folds, the trachea, the bronchi, and the bronchioles. Here, the lungs are included in the lower respiratory tract and include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0054] In the context of the present invention, the term "pharmaceutical composition" refers to a composition comprising at least a polyribonucleotide according to the present invention for administration to a subject to treat ciliopathies. The polyribonucleotide is preferably included in an effective amount, i.e., an amount sufficient to induce a detectable therapeutic response in the subject to which the pharmaceutical composition is administered. The pharmaceutical composition of the present invention may be in the form of a sterile aqueous or non-aqueous solution, suspension or emulsion or aerosol. Preferably, the pharmaceutical composition is in a form that allows administration to the respiratory system, for example, via inhalation, nebulization, spray or drops (e.g., nasal spray or nasal drops).

[0055] This is advantageous for the patient because administration using spray, drops, nebulizer, or inhalation can be easily performed by the patient, delivery is comfortable, and therefore readily available to the patient without restricting freedom of action.

[0056] In a preferred embodiment, the pharmaceutical composition comprises an mRNA that can be translated into a functional version of a protein selected from the group consisting of DNAH5, DNAH11, CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1, CCDC114, RSPH4A, DNAAF1 (LRRC50), DNAAF2 (KTU), and LRRC6, and is administered by spray, droplets, using a nebulizer, and / or by inhalation to a subject suffering from PCD caused by a deficiency of said protein. More preferably, the protein is CCDC40 and / or CCDC39.

[0057] Pharmaceutical compositions can include pharmaceutically acceptable carriers, i.e., chemical compounds, materials, ingredients, and / or compositions that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Thus, a pharmaceutically acceptable carrier is an inert substance that is formulated with a pharmaceutically active substance to facilitate its handling, taking into account dosage, adsorption, solubility, or pharmacokinetic considerations. Examples of suitable pharmaceutically acceptable carriers are well known in the art and include phosphate buffered saline solutions, buffer solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, and sterile solutions. In particular, aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate.Further examples of pharma- ceutically acceptable carriers include saline, Ringer's and dextrose solutions, citrates, phosphates, and other organic acids; salt-forming counterions, e.g., sodium and potassium; low molecular weight (>10 amino acid residues) polypeptides; proteins, e.g., serum albumin, or gelatin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., histidine, glutamine, lysine, asparagine, arginine, or glycine; carbohydrates, including glucose, mannose, or dextrin; monosaccharides; disaccharides; other sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; chelating agents, e.g., EDTA; non-ionic surfactants. The present invention also includes, but is not limited to, antioxidants, such as polyoxyethylene sorbitan monolaurate, propylene glycol, pluronic or polyethylene glycol, commercially available under the trademark Tween®; antioxidants, including methionine, ascorbic acid and tocopherol; and / or preservatives, such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. Suitable pharma-ceutically acceptable carriers and their formulations are described in more detail in Remington's Pharmaceutical Sciences, 17th ed., 1985, Mack Publishing Co. In addition, preservatives, stabilizers and other additives, such as antibacterial agents, antiaging agents, chelating agents, and inert gases, nanosystems or liposomes, etc., may also be present.

[0058] The pharmaceutical composition of the present invention can be administered to a patient via a wide class of administration forms known to those skilled in the art to be suitable for administration to the respiratory system, such as using sprays, drops, inhalers, nebulizers, etc. The dose and duration of action depend on the function that the polyribonucleotide must fulfill and must be deliberately adjusted in each case. The duration of action is as long as possible, for example, when the polyribonucleotide is used for chronic treatment of diseases caused by defective genes, i.e. altered DNA sequences, as is the case here. The duration can also be adjusted to a given time window.

[0059] In one embodiment, the pharmaceutical composition is administered at least once a week. This is advantageous to ensure efficient and sustained effects of the treatment of said PCD. Preferably, the pharmaceutical composition is administered weekly for at least 2 weeks, more preferably at least 3 weeks, even more preferably at least 4 weeks. Alternatively, the pharmaceutical composition can be administered twice a week for at least 1 week, preferably at least 2 weeks, more preferably at least 3 weeks, even more preferably at least 4 weeks. In one embodiment, if there is a need for further treatment after 4 weeks of initial treatment as described above, the treatment is performed once a week. Instead of weekly administration, administration can be switched to monthly administration for a longer period, for example, for at least 2 months, preferably at least 3 months, more preferably at least 4 months, even more preferably at least 5 months, and most preferably at least 6 months.

[0060] In one preferred embodiment, the pharmaceutical composition is administered to the respiratory system of a subject after the subject has inhaled a suitable solution, preferably a mucolytic agent, such as a solution of hypertonic saline or N-acetylcysteine ​​(NAC), or after irrigating their nasal and / or paranasal sinuses with a suitable solution, preferably a mucolytic agent, such as a solution of hypertonic saline or N-acetylcysteine ​​(NAC), to remove mucus and potentially sloughed airway epithelial cells. Thus, the pharmaceutical composition is preferably administered to the respiratory system of a subject after the subject has inhaled a suitable solution, preferably a mucolytic agent, such as a solution of hypertonic saline or N-acetylcysteine ​​(NAC), and coughed up the mucolytic agent located on the epithelial cells. This is particularly advantageous for exposing epithelial cells before administering the pharmaceutical composition, thus enhancing the transfection efficacy of polyribonucleotides.

[0061] Thus, in one embodiment, the subject suffering from a ciliopathies to which the polyribonucleotides are administered is one who has been exposed prior to treatment by inhaling a suitable solution, preferably a mucolytic agent such as a solution of hypertonic saline or N-acetylcysteine ​​(NAC), or by irrigating their nasal passages and / or sinuses with a suitable solution, preferably a mucolytic agent such as hypertonic saline or N-acetylcysteine ​​(NAC).

[0062] Such a step is intended to physically remove mucus from the subject's respiratory system prior to administration of the polyribonucleotide.

[0063] Moreover, such subjects are preferably subjects who have been subjected to an assay prior to treatment to determine whether the subject is suffering from respiratory inflammation, preferably acute inflammation or exacerbated inflammation, wherein the subject has been positively determined to have respiratory inflammation, preferably acute inflammation or exacerbated inflammation.

[0064] In a preferred embodiment, the concentration of NAC in said solution is 3%-20%, preferably 5%-15%, more preferably 8%-12%, most preferably 10%. The percentages are on a weight / weight basis. Preferably, the solution containing NAC also contains sodium edetate (edetate refers to ethylenediaminetetraacetate) and / or sodium hydroxide in pharma- ceutically acceptable concentrations. Preferably, the solution is an aqueous solution.

[0065] In some embodiments of the pharmaceutical composition comprising polyribonucleotide for use in treating ciliopathies, the pharmaceutical composition further comprises a mucolytic agent such as N-acetylcysteine ​​(NAC) and / or a hypertonic solution comprising sodium chloride. Both NAC and the hypertonic solution comprising sodium chloride, which acts as phlegm, are advantageous for reducing the retention of viscous mucus in subjects suffering from PCD. This can reduce the risk of respiratory infections and thus further stress for patients.

[0066] In a preferred embodiment, the pharmaceutical composition further comprises NAC in the concentrations as described above.

[0067] In a preferred embodiment, the pharmaceutical composition further comprises a hypertonic solution containing sodium chloride at a concentration of 2% to 8%, preferably 3% to 7%, more preferably 4% to 7%.

[0068] In a preferred embodiment, the pharmaceutical composition further comprises a hypertonic solution containing NAC in a concentration of 3% to 20%, preferably 5% to 15%, more preferably 8% to 12%, and most preferably 10%, and / or sodium chloride in a concentration of 2% to 8%, preferably 3% to 7%, and more preferably 4% to 7%.

[0069] As mentioned above, in a preferred embodiment of the pharmaceutical composition according to the invention, said pharmaceutical composition comprises an mRNA that can be translated into a functional version of a protein selected from the group consisting of DNAH5, DNAH11, CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1, CCDC114, RSPH4A, DNAAF1 (LRRC50), DNAAF2 (KTU), and LRRC6, preferably CCDC40 and / or CCDC39. Such a pharmaceutical composition is also referred to below as "first pharmaceutical composition".

[0070] Multiciliate differentiation and DNA synthesis associated cell cycle (MCIDAS) proteins are transcriptional regulatory proteins that are specifically required for multiciliate cell differentiation, including multikine ciliate formation (see, e.g., NCBI reference sequences NM_001190787.1 and NP_001177716.1 for human mRNA and protein MCIDAS sequences, respectively; or the optimized polyribonucleotide sequence shown in SEQ ID NO:4).

[0071] Thus, in some embodiments of a pharmaceutical composition comprising a polyribonucleotide for use in treating a ciliary disease, the pharmaceutical composition further comprises a polyribonucleotide encoding a functional version of a multiciliate differentiation and DNA synthesis associated cell cycle (MCIDAS) protein.

[0072] In some embodiments, the pharmaceutical composition is a first pharmaceutical composition as described above, administered together with a second pharmaceutical composition comprising a polyribonucleotide encoding an MCIDAS protein.

[0073] Both pharmaceutical compositions, i.e. the first pharmaceutical composition described above further comprising a polyribonucleotide encoding an MCIDAS protein, or the first pharmaceutical composition administered together with the second pharmaceutical composition comprising a polyribonucleotide encoding an MCIDAS protein, are advantageous since the administration of the pharmaceutical composition according to the invention is particularly effective in restoring ciliary cell structure and function when administered to the cells before and / or during ciliary formation. Moreover, both are administered to the patient, preferably by using a nasal spray and / or a nebulizer and / or by inhalation, preferably at least once a week and / or for at least 4 weeks.

[0074] In some embodiments, the pharmaceutical composition comprises a polyribonucleotide that can be translated into functional versions of two or more proteins. This can be done, for example, using a polycistronic polyribonucleotide, which is a single polyribonucleotide that codes for two or more different proteins. Designing such polycistronic polyribonucleotides, for example, using 2A peptides, is well described in the literature. As an example, Liu et al. demonstrated the use of 2A peptides to generate polycistronic mRNAs (Liu et al., 2017, Scientific Reports, 7:2193). These 2A peptides can further be combined with a furin cleavage site, for example, to remove additional amino acids (part of the 2A peptide) that are added to the first protein in the sequence as a result of ribosomal skipping (a functional feature of the 2A peptide). As an example, the use of furin cleavage with 2A peptides is described, for example, by Chng et al. (Chng et al., 2015 MAbs, 7(2):403-412). Thus, a single polyribonucleotide can be designed that encodes two or more, preferably two, therapeutic proteins (e.g., CCDC40 and MCIDAS), where the coding regions of the two therapeutic proteins are separated by a 2A peptide (as described in the above references).

[0075] Thus, in some embodiments, the same applies as described for the above embodiment, except that the pharmaceutical composition comprises a polyribonucleotide that can be translated into two or more, preferably two, functional versions of proteins.Thus, in some embodiments, the pharmaceutical composition described in the above embodiment comprises a polycistronic polyribonucleotide that comprises a sequence that can be translated into a functional version of a protein selected from the group consisting of DNAH5, DNAH11, CCDC39, DNAI1, CCDC40, CCDC103, SPAG1, ZMYND10, ARMC4, CCDC151, DNAI2, RSPH1, CCDC114, RSPH4A, DNAAF1 (LRRC50), DNAAF2 (KTU), and LRRC6, preferably CCDC40 and / or CCDC39, and a sequence that can be translated into a functional version of MCIDAS protein.

[0076] In some embodiments, the pharmaceutical composition comprises a polyribonucleotide that can be translated into a functional version of MCIDAS protein and that comprises an optimized polyribonucleotide sequence as shown in SEQ ID NO: 4, and a polyribonucleotide that can be translated into a functional version of CCDC40 and / or CCDC39 protein and that comprises an optimized polyribonucleotide sequence as shown in SEQ ID NO: 1 (or SEQ ID NO: 5-11) and / or SEQ ID NO: 2 (or SEQ ID NO: 12-14). Preferably, it is administered to the patient by using a nasal spray and / or a nebulizer and / or by inhalation, preferably at least once a week and / or for at least four weeks.

[0077] Instead of or in addition to MCIDAS, for example, GemC1, FoxJ1, and / or E2f4VP16 can be used. GemC1 is specifically expressed in ciliated epithelia and is a central regulator of ciliogenesis. It has been reported that ectopic expression of GemC1 is sufficient to induce the early stages of multiciliogenesis in airway epithelial cells ex vivo by upregulating MCIDAS and FoxJ1, two key transcriptional regulators of multiciliogenesis (Arbi M et al., 2016, EMBO reports, 17(3):400-413). Furthermore, in the same study, it was reported that GemC1 can directly transactivate MCIDAS and FoxJ1 upstream regulatory sequences. E2f4VP16 refers to a form of E2f4 that contains the general activation domain from HSV1 VP16 and can positively affect the activation of expression of key genes related to cilia formation (Kim S et al., Scientific Reports, 2018, 8:12369).

[0078] Thus, in some embodiments of a pharmaceutical composition comprising a polyribonucleotide for use in treating a ciliopathic disease, the pharmaceutical composition is the first pharmaceutical composition described above and further comprises at least one of a polyribonucleotide encoding a functional version of an MCIDAS protein, a polyribonucleotide encoding a functional version of a GemC1 protein, a polyribonucleotide encoding a functional version of a FoxJ1 protein, and / or a polyribonucleotide encoding a functional version of an E2f4VP16 protein.

[0079] In some embodiments of the pharmaceutical composition, the first pharmaceutical composition described above can be administered together with a second pharmaceutical composition comprising at least one of the following polyribonucleotides encoding a functional version of the following: a polyribonucleotide encoding a functional version of an MCIDAS protein, a polyribonucleotide encoding a functional version of a GemC1 protein, a polyribonucleotide encoding a functional version of a FoxJ1 protein, and / or an E2f4VP16 protein.

[0080] It is advantageous if both pharmaceutical compositions, i.e. the first pharmaceutical composition as described above further comprising polyribonucleotides encoding MCIDAS, GemC1, FoxJ1 and / or E2f4VP16 proteins, or the first pharmaceutical composition administered together with the second pharmaceutical composition comprising polyribonucleotides encoding MCIDAS, GemC1, FoxJ1 and / or E2f4VP16 proteins, are administered to cells before the cells start ciliary formation or during ciliary formation, since administration of the pharmaceutical composition according to the invention is particularly effective in restoring ciliary cell structure and function. Moreover, both are administered to the patient, preferably by using a nasal spray and / or a nebulizer and / or by inhalation, preferably at least once a week and / or for at least 4 weeks.

[0081] As mentioned above, the same applies as described for the above embodiment, except that in some embodiments, the pharmaceutical composition comprises a polyribonucleotide that can be translated into two or more, preferably two, functional versions of proteins.Thus, in some embodiments, the pharmaceutical composition described in the above embodiment comprises a multicistronic polyribonucleotide.

[0082] The pharmaceutical composition may comprise a compound that facilitates the transfection of cells with polyribonucleotides. Examples of such compounds are those disclosed in WO 2014 / 20723.

[0083] The pharmaceutical composition may further comprise one or more agents or one or more reagents for delivering and / or introducing the RNA into a target cell or tissue. In particular, it is envisaged that the agent / agents support the delivery and / or introduction of the RNA into a cell or tissue. The agent / agents may be administered together with the RNA. The RNA to be delivered / introduced may also be coupled (e.g., covalently bound or complexed) with the agent / agents (or reagents) or may not be coupled (e.g., simply mixed with the agent / agents). The respective agent or reagent is known in the art (e.g., Tavernier, J Control Release 150(3)(2011),238-47) and may, for example, be selected from the group consisting of lipids and liposomes, micelles, polymers and dendrimers, among others. Specific examples of each agent or reagent are GL67, EDMPC, DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP (1,2-dioleyl-3-dimethylammonium propane), DOTMA (1,2-di-0-octadecenyl-3-trimethylammonium propane), XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) and MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)ethyl). )butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)-Nl,N16-diundecyl-4,7,10,13-tetraazahexadecane-l,16-diamide), C12-200, DLin-KC2-DMA, DODAP, l,2-distearyloxy-N,N-di Methyl-3-aminopropane or "DSDMA", l,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", l,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylammonium bromide or "DDAB", N-(l,2-dimyristyloxy) ... 3-Dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane or "CLinDMA", 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethylll-(cis,cis-9',1-2'-octadecadienoxy)propane or "CpLinDMA", N,N-Dimethyl-3,4-Dioleyloxybenzylamine or "DMOBA", l,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", l,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane or "DLincarbDAP", l,2-dilinoleoylcarbamyl-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane or "DLin-K-DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane or "DLin-K-XTC2-DMA", or mixtures thereof (Heyes, J Controlled Release 107 (2005), 276-287; Morrissey, Nat. Biotechnol. 23(8) (2005), 1003-1007; WO 2005 / 121348). A further example is DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, Biochem. Biophys. Res. Comm. 179 (1991), 280; Wolf, et al BioTechniques 23 (1997), 139; U.S. Pat. No. 5,744,335). Further examples are LIPOFECTIN (DOTMA:DOPE) (Invitrogen, Carlsbad, Calif), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen), LIPOFECTAMINE 2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS), and EFFECTENE. Further examples are modified and unmodified polyacrylates, polyalkoxyanoacrylates, polylactides, polylactide-polyglycolide copolymers, polycaprolactone, dextran, albumin, gelatin, alginates, collagen, chitosan, cyclodextrins, polylysin, polyarginine, oligo / polyamines, and polyethyleneimines.

[0084] The agents or reagents may be oligomers, polymers or lipidoids. They may contain oligo(alkyleneamine) moieties, such as, for example, the characteristic oligo(alkyleneamine) moieties as described in PCT / EP2014 / 063756. In particular, the agents or reagents may be oligomers, polymers or lipidoids as described in PCT / EP2014 / 063756. One main feature of these particular agents or reagents is that they contain a common structural entity of formula (I): [ka]

[0085] Such an agent or reagent may be an oligo(alkyleneamine) selected from the following: a) an oligomer or polymer comprising a plurality of groups of formula (II) as side chains and / or end groups: [ka] where the variables a, b, p, m, n and R 2 ~R 6 is independently defined as follows for each group of formula (II) among multiple such groups: a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1, p is 1 or 2; m is 1 or 2; n is 0 or 1 and m+n is ≧2; and R 2 ~R 5 are, independently of each other, hydrogen; the group -CH-CH(OH)-R 7 , -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 , -CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 wherein R 7is selected from a C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; and a poly(ethylene glycol) chain; R 6 is hydrogen, the group -CH-CH(OH)-R 7 , -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 , -CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 wherein R 7 is selected from a C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; -C(NH)-NH2; a poly(ethylene glycol) chain, and a receptor ligand; and wherein one or more of the nitrogen atoms of formula (II) may be protonated to provide a cationic group of formula (II); b) an oligomer or polymer comprising as repeat units a plurality of groups of formula (III): [ka] where the variables a, b, p, m, n and R 2 ~R 5 is defined independently for each group of formula (III) among multiple such groups as follows: a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1, p is 1 or 2; m is 1 or 2; n is 0 or 1 and m+n is ≧2; and R 2 ~R 5 are, independently of each other, hydrogen; the group -CH-CH(OH)-R 7 , -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 or -CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 wherein R 7is selected from a C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; -C(NH)-NH2; and a poly(ethylene glycol) chain; and wherein one or more of the nitrogen atoms of formula (III) may be protonated to provide a cationic group of formula (III); and c) A lipidoid having the structure of formula (IV): [ka] where the variables a, b, p, m, n and R 1 ~R 6 is defined as follows: a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1, p is 1 or 2; m is 1 or 2; n is 0 or 1 and m+n is ≧2; and R 1 ~R 6 are, independently of each other, hydrogen; the group -CH-CH(OH)-R 7 , -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 , -CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 wherein R 7 is a C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for the amino group; -C(NH)-NH2; a polyethylene glycol chain; and a receptor ligand; provided that R 1 ~R 6 At least two of the residues are the groups -CH-CH(OH)-R 7 , -CH(R 7 )-CH2-OH, -CH2-CH2-(C=O)-OR 7 , -CH2-CH2-(C=O)-NH-R 7 or -CH2-R 7 where R 7is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms of formula (IV) may be protonated to provide a cationic lipidoid of formula (IV).

[0086] Preferably, such an agent or reagent may be an oligo(alkyleneamine) (component comprising the same) selected from a) and b): a) an oligomer or polymer comprising a plurality of groups of formula (IIa) as side chains and / or end groups: -NR 2 {CH2-(CH2) a -NR 3 -CH2-(CH2) b -NR 4} m -[CH2-(CH2) a -NR 5 ] n -R 6 (IIa), In the formula, a, b, m, n, and R 2 ~R 6 is defined as above, and one or more of the nitrogen atoms shown in formula (IIa) may be protonated to provide a cationic oligomeric or polymeric structure; and b) an oligomer or polymer comprising a plurality of groups of formula (IIIa) as repeating units: -NR 2 {CH2-(CH2) a -NR 3 -CH2-(CH2) b -NR 4} m -[CH2-(CH2) a -NR 5 ] n - (IIIa), In the formula, a, b, m, n, and R 2 ~R 5 is defined above, and one or more of the nitrogen atoms shown in formula (IIIa) may be protonated to provide a cationic oligomeric or polymeric structure.

[0087] Additionally, such agents or reagents may be a moiety comprising an oligo(alkyleneamine) selected from lipidoids having the structure of formula (IVa): R 1 -NR 2 {CH2-(CH2) a -NR 3 -CH2-(CH2) b -NR 4} m -[CH2-(CH2) a -NR 5 ] n -R 6 (IVa), In the formula, a, b, m, n, and R 6 R to 1 is defined above, and one or more of the nitrogen atoms shown in formula (IVa) may be protonated to provide a cationic lipidoid.

[0088] With respect to such agents or reagents, in formula (II), (IIa), (III), (IIIa), (IV) or (IVa), n may be 1; or m may be 1 and n may be 1.

[0089] Further, for such agents or reagents, in formula (II), (IIa), (III), (IIIa), (IV) or (IVa), a may be 1 and b may be 2; or a may be 2 and b may be 1.

[0090] In some embodiments, the oligomer, polymer, or lipidoid can be a cationic (eg, protonated) oligomer, polymer, or lipidoid.

[0091] A non-limiting example of such an oligomer, polymer or lipidoid used is a cationic lipid prepared by mixing 100 mg of N,N'-bis(2-aminoethyl)-1,3-propanediamine (0.623 mmol) with 575.07 mg of 1,2-epoxydodecane (3.12 mmol) (N-1) equivalents (where N is 2x the amount of primary amines and 1x the amount of secondary amines / oligo(alkyleneamines)) and mixing for 96 hours at 80° C. under constant shaking. Such oligomers, polymers or lipidoids are also referred to as lipidoids "C12-(2-3-2)".

[0092] The agent or reagent used, particularly the polymer, may be a copolymer, particularly a statistical copolymer. Such copolymer may be a copolymer that contains a statistical / random arrangement of alkyleneamine repeat units of alternating length (as opposed to the less preferred polymer that contains a similar arrangement of alkyleneamine repeat units of non-alternating length). The copolymer may be a cationic (e.g., protonated) copolymer. The copolymers used are known in the art and are described, for example, in EP 14 19 9439.2, WO 01 / 00708, EP-A1 1 198 489 and CA-A1 2,377,207.

[0093] In particular, the copolymer may be a statistical copolymer comprising a plurality of repeat units (a) independently selected from repeat units of the following formulae (a1) and (a2): [ka] , and A plurality of repeating units (b) independently selected from repeating units represented by the following formulae (b1) to (b4): [ka] In the formula, the molar ratio of the total repeating units (a) to the total repeating units (b) is within the range of 0.7 / 1.0 to 1.0 / 0.7; and wherein one or more of the nitrogen atoms of repeat units (a) and / or (b) contained in the copolymer may be protonated to provide a cationic copolymer.

[0094] The copolymer may be a statistical copolymer in which any repeat unit (a) and any repeat unit (b) are statistically distributed in the copolymer macromolecule. It is typically obtained from the copolymerization of a mixture of monomers that produce repeat unit (a) with a monomer that produces repeat unit (b) during the polymerization reaction. Preferably, the copolymer is a random copolymer in which any repeat unit (a) and any repeat unit (b) are randomly distributed in the polymer macromolecule.

[0095] Such copolymers may be linear, branched or dendritic copolymers. As will be appreciated by those skilled in the art, the repeating units of formula (a1), (b1) or (b3) have two valencies (i.e., open bonds to adjacent units), resulting in the propagation of the copolymer structure in a linear fashion. Thus, a linear copolymer may contain a repeating unit of formula (a1), and one or more types of repeating units of formula (b1) and (b3), but not of formula (a2), (b2) or (b4). As will be further appreciated, the presence of a repeating unit of formula (a2), (b2) or (b4) with three valencies provides a branch point in the copolymer structure. Thus, a branched copolymer may contain one or more types of repeating units of formula (a2), (b2) and (b4), and may further contain one or more types of repeating units of formula (a1), (b1) and (b3).

[0096] Such copolymers may comprise a plurality of repeat units (a) independently selected from the repeat units of formulae (a1) and (a2) defined above, and a plurality of repeat units (b) independently selected from the repeat units of formulae (b1) to (b4) defined above. Preferred are copolymers comprising a plurality of repeat units (a) independently selected from the repeat units of formulae (a1) and (a2) defined above, and a plurality of repeat units (b) independently selected from the repeat units of formulae (b1) and (b2) defined above.

[0097] Preferably, such copolymers comprise one or more types of repeat units selected from repeat units (a2), (b2) and (b4), optionally further comprising one or more types of repeat units of formulae (a1), (b1) and (b3), in particular copolymers comprising repeat units of formula (a2) and one or more types of repeat units of formulae (b2) and (b4), optionally further comprising one or more types of repeat units of formulae (a1), (b1) and (b3).Thus, in line with the above, more preferred copolymers are branched copolymers comprising repeat units of formula (a2) and repeat units of formula (b2), optionally further comprising one or more types of repeat units of formulae (a1) and (b1).

[0098] In the copolymer, the total number of repeat units (a) and repeat units (b) is typically at least 20, preferably at least 50, and more preferably at least 100. Typically, the total number of repeat units (a) and repeat units (b) is at most 10,000, preferably at most 5,000, and more preferably at most 1,000.

[0099] Furthermore, it is preferred for the copolymer that the repeat units (a) and (b) account for 80 mol% or more, more preferably 90 mol% or more of all repeat units in the copolymer. More preferred is a copolymer in which the repeat units (a) selected from (a1) and (a2) and the repeat units (b) selected from (b1) and (b2) account for 80 mol% or more, more preferably 90 mol% or more of all repeat units in the copolymer. Most preferred is that all of the repeat units in the copolymer are repeat units (a) or (b), and most preferred is that all of the repeat units in the copolymer are repeat units (a) selected from (a1) and (a2) or repeat units (b) selected from (b1) and (b2).

[0100] The weight average molecular weight of the copolymer, for example, as measured by size exclusion chromatography against linear poly(ethylene oxide) standards, will generally be in the range of from 1,000 to 500,000 Da, preferably from 2,500 to 250,000 Da, and more preferably from 5,000 to less than 50,000 Da.

[0101] The end groups of such copolymers typically contain one or more types of groups (c) independently selected from the group of formulas (c1) to (c3) below, preferably from the group of formulas (c1) and (c2) below: [ka] .

[0102] Preferably, the end groups in the copolymer consist of one or more types of groups (c) independently selected from the group of formulae (c1) to (c3), preferably from the group of formulae (c1) and (c2). As will be appreciated by those skilled in the art, the number of end groups will depend on the structure of the copolymer. Linear copolymers have only two ends, while a greater number of end groups will be included in branched, especially dendritic, copolymers. As will be further appreciated, one or more of the nitrogen atoms of the end groups (c) included in the copolymer may also be protonated to provide a cationic copolymer.

[0103] In the copolymer, the molar ratio of the sum of repeat units (a) to the sum of repeat units (b) is in the range of 0.7 / 1.0 to 1.0 / 0.7, preferably in the range of 0.8 / 1.0 to 1.0 / 0.8. This molar ratio can be determined, for example, by NMR. It will therefore be understood that this ratio is usually determined for multiple polymers of the copolymer, and typically represents the overall ratio of the sum of repeat units (a) to the sum of repeat units (b) in the multiple polymers.

[0104] As indicated above, one or more of the nitrogen atoms of the copolymer can be protonated to provide the copolymer in cationic form, typically in oligocationic or polycationic form. It is understood that the primary, secondary, or tertiary amino groups in the repeating units (a) or (b) or the terminal group (c) can act as proton acceptors, particularly in water and aqueous solutions (including physiological fluids). Thus, such copolymers typically have an overall positive charge in aqueous solutions having a pH of less than 7.5. Aqueous solutions as referred to herein are solutions in which the solvent comprises 50% (vol. / vol.) or more, preferably 80 or 90% or more, and most preferably 100% water. Also, when the compositions are in contact with physiological fluids having a pH of less than 7.5, including, for example, blood and lung fluid, they typically contain repeating units (a) and (b) in which the nitrogen atoms are protonated. The pK of the copolymers used in the compositions a The value is auto pK a It can be determined by acid-base titration using a titrator. The net charge at a given pH value can then be calculated, for example, from the Henderson-Hasselbach equation. Any charge may be shared across several base centers and cannot necessarily be attributed to a single point. Typically, in a solution at physiological pH, the copolymers used in the composition include repeat units with amino groups in a protonated state and repeat units with amino groups in an unprotonated state.

[0105] However, as will be appreciated by those skilled in the art, the copolymers as well as compositions may also be provided as a dry salt form that contains the copolymer in cationic form.

[0106] As will be further understood, the counterions (anions) to the positive charges of the protonated amino groups in compositions comprising copolymers and nucleic acids, particularly RNA, preferably single-stranded RNA such as mRNA, are typically provided by anionic moieties contained in the nucleic acid. When positively charged groups are present in excess compared to the anionic moieties in the nucleic acid, the positive charges are typically provided by other anions, particularly Cl. - or HCO3 - This can be balanced by anions typically encountered in physiological fluids such as:

[0107] In accordance with the above, preferred copolymers are random copolymers. At least 80 mol % of all repeating units, more preferably all repeating units, are formed by A plurality of repeating units (a) independently selected from repeating units of the following formulae (a1) and (a2): [ka] , and A plurality of repeating units (b) independently selected from repeating units of the following formulae (b1) and (b2): [ka] , In the formula, the molar ratio of the total of the repeating units (a) to the total of the repeating units (b) is within the range of 0.7 / 1.0 to 1.0 / 0.7, and more preferably within the range of 0.8 / 1.0 to 1.0 / 0.8; wherein the end groups of the copolymer are formed by A group (c) independently selected from the groups of formulae (c1) and (c2): [ka] ; and wherein one or more of the nitrogen atoms of the repeat units (a) and / or (b) and / or the end group (c) contained in the copolymer may be protonated to provide a cationic copolymer. It is further preferred that the copolymer is a branched copolymer comprising units (a2) and (b2), optionally together with units (a1) and / or (b1).

[0108] The copolymers can be conveniently prepared by procedures similar to those known for the preparation of polyalkyleneimines, such as branched or linear polyethyleneimine (PEI). It will be understood that the monomers used to prepare the copolymers must be adjusted accordingly. It has now been found that in the monomer mixtures subjected to polymerization, the monomers can be reacted quantitatively and conveniently so that the proportion of units (a) and (b) in the copolymer can be adjusted by appropriately adjusting the monomer ratio. Polyethyleneimines can be prepared, for example, by ring-opening polymerization of aziridine, while copolymers can be prepared by ring-opening polymerization of monomer mixtures comprising or consisting of aziridine, azetidine, and, where applicable, pyrrolidine, or in a preferred embodiment, aziridine and azetidine. It will be understood that the expression "where applicable" refers to the presence or absence of repeat units (b3) and (b4) or end groups (c3) formed by pyrrolidine. Ring-opening polymerization of unsubstituted cyclic amines usually results in branched copolymers. Linear copolymers can be prepared, for example, via polymerization of appropriate N-substituted aziridines, N-substituted aziridines and N-substituted pyrrolidines, or N-substituted aziridines and N-substituted aziridines, similar to the procedure published in, for example, Katrien F. Weyts, Eric J. Goethals, New synthesis of linear polyethyleneimine, Polymer Bulletin, 1988, 19(1):13-19, followed by, for example, hydrolytic cleavage of the N-substituents attached to the resulting polyalkylenimine chain. Dendrimers can be synthesized, for example, according to the method described in, for example, Yemul et al, Colloid and Polymer Science, 2008, 286(6-7):747-752, Synthesis and characterization of poly(ethylenimine) dendrimers.

[0109] For the preparation of dendrimers (or dendritic copolymers), the synthetic strategies known for the production of polyethyleneimine or polypropyleneamine dendrimers can be applied as well. Polypropylenimine dendrimers can be synthesized from acrylonitrile building blocks using a repeated sequence of Michael addition to primary amines, followed by heterogeneous catalytic hydrogenation (Newkome and Shreiner Poly(amidoamine), polypropylenimine, and related dendrimers and dendrons possessing different 1→2 branching motifs: An overview of the divergent procedures. Polymer 49 (2008) 1-173; De Brabander-Van Den Berg et al. Large-scale production of polypropylenimine dendrimers, Macromolecular Symposia (1994) 77 (1) 51-62). Polyethylenimine dendrimers can be prepared using a repeating Michael addition sequence of vinyl bromide building blocks to primary amines followed by conversion of the alkyl bromide to an amine using the Gabriel amine synthesis method (Yemul & Imae, Synthesis and characterization of poly(ethyleneimine) dendrimers, Colloid Polym Sci (2008) 286:747-752). Thus, one skilled in the art can not only prepare dendrimers having strictly alternating layers of, for example, propyleneimine and ethyleneimine. Similarly, dendrimer generations can be generated having layers comprising or consisting of repeat units of formulae (a2), (b2) and (b4), and preferably random compositions of repeat units (a2) and (b2).

[0110] The ring-opening polymerization of aziridine and azetidine, or aziridine, azetidine and pyrrolidine, can be carried out in solution, for example, in water. The total monomer concentration is not particularly limited, and typical concentrations range from 10% wt / wt to 80% wt / wt, preferably 30% wt / wt to 60% wt / wt. Typically, since the polymerization is initiated by protons, it is preferable to add a mineral acid, such as a Bronsted acid, especially sulfuric acid, to the reaction system. Small amounts of acid, such as 0.001 to 0.01 equivalents, based on the total monomer concentration, are generally sufficient. The reaction proceeds at a convenient rate, for example, at temperatures in the range of 50 to 150°C, especially 90 to 140°C. In these ranges, higher molecular weight copolymers are usually obtained at higher temperatures, and lower molecular weight copolymers at lower temperatures.

[0111] As a general rule, lipidoids are the preferred agents or reagents to be used, particularly compared to oligomers, and more particularly to polymers.

[0112] Further examples of one or more agents or one or more reagents for delivering and / or introducing RNA into a target cell or tissue are liposomal transfection reagents (LTR's) and magnetic particles (MPs), as described elsewhere herein.

[0113] One particular mode for delivering and / or introducing RNA into target cells or target tissues is transfection.Therefore, it can be assumed that the RNA used is transfected into (target) cells or tissues, delivered / administered via transfection, and / or prepared for transfection.The means and methods for transfecting RNA are well known in the art and are described, for example, in Tavernier (supra), Yamamoto (Eur J Pharm Biopharm.71(3)(2009), 484-9) and Kormann (Nat Biotechnol.29(2)(2011), 154-7).A particular mode of transfection is lipofection, magnetofection or magnetopolypofection.

[0114] Thus, the RNA used may be prepared for lipofection, prepared to be transfected by lipofection, delivered / introduced by lipofection, and / or administered by lipofection.

[0115] Thus, the pharmaceutical composition may (further) comprise at least one lipid or liposomal transfection reagent or enhancer (LTR; liposomal transfection reagent). The RNA used may be contained in, complexed with and / or delivered by the LTR. In particular, the RNA used may be contained in and / or delivered by a (respective) lipofection complex comprising the RNA and the LTR. The pharmaceutical composition may (further) comprise a lipofection complex.

[0116] LTRs are known in the art and are sold, for example, by OzBiosciences, Marseille, France. The LTR used may be selected from the group consisting of the above-mentioned agents or reagents for delivering and / or introducing RNA into target cells or target tissues. For example, such an LTR may be a lipid or lipidoid, preferably a cationic lipid or cationic lipid, such as lipidoids as disclosed in PCT / EP2014 / 063756 (e.g., C12-(2-3-2)), lipids as disclosed in EP2285772 (e.g., Dogtor), and lipopolyamines as disclosed in EP1003711 (e.g., DreamFect™ and DreamFect Gold™). A particular LTR may be selected from the group consisting of (i) C12-(2-3-2); (ii) DreamFect™, preferably DreamFect Gold™ (DF™ / DF-Gold™; OzBiosciences, Marseille, France); (iii) Dogtor (OzBiosciences, Marseille, France); and (iv) Lipofectamine, for example Lipofectamine 2000 (Invitrogene, CA, USA).

[0117] As a general rule, Dogger is preferred, DreamFect™ is more preferred, and DF-Gold™ and C12-(2-3-2) are the more preferred LTRs.

[0118] LTRs such as Dogtor are described, for example, in EP 2285772. LTRs such as DF™ and DF-Gold™ are described, for example, in EP 1003711. In principle, preferred LTRs are oligomers, polymers or lipids such as those disclosed in PCT / EP2014 / 063756, certain cationic lipids such as those disclosed in EP 2285772, and certain lipopolyamines such as those disclosed in EP 1003711. Highest preference is given to LTRs such as C12-(2-3-2) and DF-Gold™.

[0119] Non-limiting examples of lipofection complexes are DF-Gold™ / RNA lipoplexes and C12-(2-3-2) / RNA lipoplexes.

[0120] C12-(2-3-2) is a particularly preferred LTR having the structure shown in formula (V) (see Jarzebinska et al., Angew Chem Int Ed Engl., 2016; 55(33):9591-5): [ka]

[0121] C12-(2-3-2) is preferably prepared as described, for example, in WO 2016 / 075154, EP 3013964, and Jarzebinska et al. (Angew Chem Int Ed Engl., 2016; 55(33): 9591-5). The cationic lipid can be prepared by mixing N1-(2-aminoethyl)-N3-(2-((3,4-dimethoxybenzyl)amino)ethyl)propane-1,3-diamine (8.9 g, 1 eq, 28.67 mmol) with 1,2-epoxydodecane (42.27, 8 eq, 229.4 mmol) and mixing at 80°C for 24 hours under constant shaking, followed by purification and removal of the 3,4-dimethoxybenzyl protecting group.

[0122] Different isomers of C12-(2-3-2) can be used, such as racemic, S-isomer, and / or R-isomer. Preferably, C12-(2-3-2) is used as the pure R-isomer and has the structure shown in formula (VI). To obtain the pure R-isomer of C12-(2-3-2), it can be prepared as described above for C12-(2-3-2) using the R-isomer of 1,2-epoxo-decane for synthesis. [ka]

[0123] Thus, in a preferred embodiment, a pharmaceutical composition comprises a polyribonucleotide for use in the treatment of a ciliopathic disease and further comprises a lipidoid having a structure as shown in formula (V), preferably as shown in formula (VI).

[0124] A further particularly preferred LTR is a cationic lipidoid having formula (VII), also referred to herein as "dL_P", which can be synthesized by reaction of N,N'-bis(2-aminoethyl)-1,3-propanediamine with N-dodecylacrylamide using boric acid as a catalyst. For the reaction, the mixture can be stirred at 100°C under microwave irradiation.

[0125] Thus, in a further preferred embodiment, a pharmaceutical composition comprises a polyribonucleotide for use in the treatment of a ciliopathic disease, further comprising a lipidoid, the polyribonucleotide having the structure shown in formula (VII). [ka]

[0126] Additionally, the pharmaceutical compositions include cationic lipids having the R-isomers of formula (V), (VI) and / or (VII), preferably dL_P and / or C12-(2-3-2), more preferably dL_P and / or C12-(2-3-2), in the formulations described below. In particular, the agents and reagents for delivery and / or introduction of the RNA described herein into a target cell or tissue, and the LTR described herein, can be combined with one or more (e.g., two, three, or four) additional lipids (e.g., cholesterol, DPPC, DOPE, and / or PEG lipids (e.g., DMPE-PEG, DMG-PEG2000)). These additional lipids may support the desired function of the drug / reagent and LTR (supporting and / or increasing the delivery and / or introduction of RNA into cells or tissues and improving transfection efficiency, respectively) and may function as the respective "helper lipids"; specific examples of such "helper lipids" are cholesterol, DPPC, DOPE and / or PEG lipids (e.g., DMPE-PEG, DMG-PEG (e.g., DMG-PEG2000)). Additional lipids (e.g., "helper lipids") may also be part of the complexes / particles disclosed herein. The skilled person is readily in a position to prepare the complexes / particles according to the present invention. Examples of additional lipids (e.g., "helper lipids") are also known in the art. The skilled person is readily in a position to select the appropriate additional lipids (e.g., "helper lipids") and the ratio of the drug / reagent / LTR and additional lipids (e.g., "helper lipids"). Such molar ratios are 1-4:1-5, 3-4:4-6, about 4:about 5, about 4:about 5:about 5.3 of drug / reagent / LTR:plus lipid(s) (narrower ranges are preferred). For example, drug / reagent / LTR can be combined with three additional lipids, such as DPPC, cholesterol, and DMG-PEG2000, in molar ratios of 8:5.3:4.4:0.9, respectively, or more specifically, 8:5.29:4.41:0.88, respectively.

[0127] Preferably, dL_P and / or C12-(2-3-2), more preferably the R-isomers of dL_P and / or C12-(2-3-2), are produced as described above and used in a molar ratio of 8:5.29:4.41:0.88 with the helper lipids DPPC and cholesterol and the PEG-lipid DMG-PEG2000 to formulate lipoid particles.

[0128] A composition in which the R-isomer of C12-(2-3-2) (Formula VI) is formulated with the lipids DPPC and cholesterol and the PEG-lipid DMG-PEG2000 in a molar ratio of 8:5.29:4.41:0.88 is also referred to herein as "LF92." A composition in which dL_P (Formula VII) is formulated with the lipids DPPC and cholesterol and the PEG-lipid DMG-PEG2000 in a molar ratio of 8:5.29:4.41:0.88 is also referred to herein as "LF111."

[0129] Also, as exemplarily described, for example, in WO 2016 / 075154 A1, EP 3013964, and Zhang et al. (TERMIS, 2019, Tissue Engineering: Part A, Vol. 25, Numbers 1 and 2), dL_P and / or C12-(2-3-2) can be used as a non-viral vector to generate stable lipoplexes with mRNA molecules based on electrostatic interactions between the positive amino groups of the lipidoid and negative phosphate groups of the mRNA molecules (Anderson, Human Gene Therapy 14, 2003, 191-202). To stabilize the lipoplex structure and reduce leakage, dL_P and / or C12-(2-3-2), more preferably the R-isomer of dL_P and / or C12-(2-3-2), can be supplied with two helper lipids entitled 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (Anderson, Drug Delivery 11, 2004, 33-39; Liang, Journal of Colliod and Interface Science 278, 2004, 53-62). Finally, 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG) 2 kD (DMG-PEG2000) is added to the lipid mixture to provide PEGylated liposomes. It is already well known that PEGylation improves the physicochemical properties of liposomal formulations by increasing water solubility, protecting them from enzymatic degradation, and limiting immunogenic and antigenic responses (Milla, Current Drug Metabolism 13, 2012, 105-119). The final N / P ratio of the total ethanolic lipid mixture is 8 : 5.29 : 4.41 : 0.88, which represents the molar ratio of amino groups of dL_P and / or C12-(2-3-2) / DPPC / cholesterol / DMG-PEG2000 to one phosphate group of the mRNA molecule, respectively.

[0130] Thus, in a preferred embodiment, a pharmaceutical composition comprises a polyribonucleotide for use in the treatment of a ciliopathic disease and further comprises dL_P and / or C12-(2-3-2), preferably the R-isomer of dL_P and / or C12-(2-3-2), formulated with DPPC, cholesterol, and DMG-PEG2000.

[0131] Furthermore, in a particularly preferred embodiment, the pharmaceutical composition further comprises a polyribonucleotide for use in the treatment of ciliary lipids, preferably dL_P and / or C12-(2-3-2), preferably the R-isomer of dL_P and / or C12-(2-3-2), formulated with DPPC, cholesterol, and DMG-PEG2000, with a final N / P ratio of 8:5.29:4.41:0.88 for the molar ratio of amino groups of dL_P and / or C12-(2-3-2) / DPPC / cholesterol / DMG-PEG2000 to one phosphate group of the mRNA molecule, respectively.

[0132] The R-isomer of C12-(2-3-2) blended with DPPC, cholesterol, and DMG-PEG2000 as described above is also referred to as the LF92 blend.

[0133] Thus, in a particularly preferred embodiment of the pharmaceutical composition comprising a polyribonucleotide for use in the treatment of a ciliary disease, said pharmaceutical composition further comprises an LF92 formulation.

[0134] dL_P formulated with DPPC, cholesterol, and DMG-PEG2000 as described above is also referred to as the LF111 formulation.

[0135] Thus, in a particularly preferred embodiment of the pharmaceutical composition comprising a polyribonucleotide for use in the treatment of a ciliary disease, said pharmaceutical composition further comprises an LF111 formulation.

[0136] In a preferred embodiment of the pharmaceutical composition comprising polyribonucleotide for use in treating ciliopathies, said pharmaceutical composition further comprises LF92 and / or LF111 formulation.LF92 and / or LF111 refer to the carrier formulation used for cell transfection.This is advantageous because the use of LF92 and / or LF111 is associated with high transfection efficiency, thus ensuring that polyribonucleotides encoding functional versions of proteins with defects associated with ciliopathies can efficiently enter cells, preferably undifferentiated hairy cells or basal cells, and thus restore hairy cell function in subjects suffering from PCD.

[0137] In a particularly preferred embodiment, the pharmaceutical composition comprises a LF92 and / or LF111 combination, at least one of the following: an mRNA that can be translated into a functional version of the MCIDAS protein, an mRNA that can be translated into a functional version of the GemC1 protein, an mRNA that can be translated into a functional version of the FoxJ1 protein, an mRNA that can be translated into a functional version of the E2f4VP16 protein; and an mRNA that can be translated into a functional version of the CCDC40 and / or CCDC39 protein. Preferably, such compositions are administered to the patient by using a spray, droplets and / or a nebulizer and / or by inhalation, preferably at least once a week and / or for at least four weeks.

[0138] Thus, in some embodiments, the same applies as described for the above embodiment, except that the pharmaceutical composition comprises a polyribonucleotide that can be translated into two or more, preferably two, functional versions of proteins. Thus, in some embodiments, the pharmaceutical composition described in the above embodiment comprises a multicistronic polyribonucleotide.

[0139] The present invention also relates to a method for treating a ciliopathies in a subject suffering from a ciliopathies, comprising the administration of a pharmaceutical composition comprising a polyribonucleotide, the polyribonucleotide encoding a functional version of a protein whose deficiency is associated with said ciliopathies, said pharmaceutical composition being administered to the respiratory system of said subject, which is carried out when the subject shows inflammation of the respiratory system. With regard to possible and preferred embodiments of the pharmaceutical composition, as well as its method and time of administration, the same applies as described herein above in relation to the pharmaceutical composition according to the invention.

[0140] The present invention also relates to a pharmaceutical composition comprising a polyribonucleotide encoding a protein whose deficiency is associated with a ciliopathies, and a hypertonic solution comprising N-acetylcysteine ​​(NAC), sodium chloride, and / or a LF92 and / or LF111 combination.

[0141] With regard to the pharmaceutical composition and its components, the same applies as described above in relation to the pharmaceutical composition comprising a polyribonucleotide for use in the treatment of a ciliopathic disease in a subject suffering from a ciliopathic disease.Furthermore, other characteristics of such a pharmaceutical composition may be as described above.

[0142] Thus, in a preferred embodiment, the pharmaceutical composition may further comprise an mRNA that can be translated into a functional version of MCIDAS.

[0143] The present disclosure also relates to a polyribonucleotide encoding human CCDC40 as set forth in any of SEQ ID NOs: 1 or 5-11.

[0144] In some embodiments of any of the above or other aspects and embodiments of the disclosure, the polyribonucleotide or modified polyribonucleotide encodes human CCDC40 (e.g., functional human CCDC40) and comprises a primary sequence that is at least 85%, at least 90%, at least 92%, or at least 95% identical (e.g., at least 95, 96, 97, 98, 99 or 100% identical) to one or more of SEQ ID NOs: 1 or 5-11 (e.g., a sequence set forth in SEQ ID NOs: 1 or 5-11).

[0145] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' untranslated region (UTR), a CYBA 5'UTR, a Kozak element followed by a codon-optimized sequence encoding a functional version of the human CCDC40 protein, a CYBA 3'UTR, and a poly(A) tail (see, e.g., SEQ ID NO:1). In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' untranslated region (UTR), a Kozak element followed by a codon-optimized sequence encoding a functional version of the human CCDC40 protein, and a poly(A) tail (see, e.g., SEQ ID NO:5).

[0146] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' untranslated region (UTR), additional U nucleotides, a TISU element followed by a codon-optimized sequence encoding a functional version of the human CCDC40 protein, and a poly(A) tail (see, e.g., SEQ ID NO: 6).

[0147] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' untranslated region (UTR), an hAg 5'UTR, a Kozak element followed by a codon-optimized sequence encoding a functional version of the human CCDC40 protein, and a poly(A) tail (see, e.g., SEQ ID NO: 7).

[0148] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' untranslated region (UTR), a human CMV IE9 5'UTR, a codon-optimized sequence encoding a functional version of the human CCDC40 protein, a human growth hormone 3'UTR, and a poly(A) tail (see, e.g., SEQ ID NO:8).

[0149] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' untranslated region (UTR), a Kozak element, an EGFP coding sequence, a G4S spacer followed by a codon-optimized sequence encoding a functional version of the human CCDC40 protein, and a poly(A) tail (see, e.g., SEQ ID NO: 9).

[0150] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' untranslated region (UTR), a codon-optimized sequence encoding a functional version of the human CCDC40 protein, a G4S spacer, an EGFP coding sequence, and a poly(A) tail (see, e.g., SEQ ID NO: 10).

[0151] In a particular embodiment, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' untranslated region (UTR), a CYBA 5'UTR, a Kozak element, followed by an HA tag, a G4S spacer, a codon-optimized sequence encoding a functional version of the human CCDC40 protein, a T2A peptide, a sequence encoding tdTomato, a CYBA 3'UTR, and a poly(A) tail (see, e.g., SEQ ID NO:11). In some embodiments, the polyribonucleotide is a modified polyribonucleotide having a level and / or type of modification selected from any such level and / or type described herein.

[0152] In certain embodiments, the percent identity of a polyribonucleotide is measured only with respect to the CCDC40 coding sequence portion of SEQ ID NO:1 or 5-11 (e.g., UTRs, other non-coding sequences and GFP or epitope tags are not taken into account when calculating percent identity, and the polyribonucleotide may or may not include such regions).

[0153] In certain embodiments of any of the foregoing, such a polyribonucleotide (or modified polyribonucleotide) encodes a functional CCDC40 protein.

[0154] The present disclosure also relates to a polyribonucleotide encoding human CCDC39 as set forth in any one of SEQ ID NOs: 2 or 12-14.

[0155] In some embodiments of any of the above or other aspects and embodiments of the disclosure, the polyribonucleotide or modified polyribonucleotide encodes human CCDC39 and comprises a primary sequence that is at least 85%, at least 90%, at least 92%, or at least 95% identical (e.g., at least 95, 96, 97, 98, 99, or 100% identical) to one or more of SEQ ID NOs: 2 or 12-14 (e.g., a sequence set forth in SEQ ID NOs: 2 or 12-14).

[0156] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5'UTR, a CYBA 5'UTR, a Kozak element followed by a codon-optimized sequence encoding a functional version of the human CCDC39 protein, a CYBA 3'UTR, and a poly(A) tail (see, e.g., SEQ ID NO: 2).

[0157] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' UTR, a Kozak element followed by a codon-optimized sequence encoding a functional version of the human CCDC39 protein, and a poly(A) tail (see, e.g., SEQ ID NO: 12).

[0158] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5'UTR, additional U nucleotides, a TISU element followed by a codon-optimized sequence encoding a functional version of the human CCDC39 protein, and a poly(A) tail (see, e.g., SEQ ID NO: 13).

[0159] In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5'UTR, SP30 (i.e., a random sequence of 30 nucleotides) as the 5'UTR, a Kozak element followed by a codon-optimized sequence encoding a functional version of the human CCDC39 protein, and a poly(A) tail (see, e.g., SEQ ID NO: 14).

[0160] In some embodiments, the polyribonucleotide is a modified polyribonucleotide having a level and / or type of modification selected from any such level and / or type described herein.

[0161] In certain embodiments, the percent identity of a polyribonucleotide is measured only with respect to the CCDC39 coding sequence portion of SEQ ID NO:2 or 12-14 (e.g., UTRs, other non-coding sequences and GFP or epitope tags are not taken into account when calculating percent identity, and the polyribonucleotide may or may not include such regions).

[0162] In certain embodiments of any of the foregoing, such a polyribonucleotide (or modified polyribonucleotide) encodes a functional CCDC39 protein.

[0163] The present disclosure also relates to a polyribonucleotide encoding human MCIDAS as set forth in SEQ ID NO: 4. In some embodiments of any of the above or other aspects and embodiments of the present disclosure, the polyribonucleotide or modified polyribonucleotide encodes human MCIDAS and comprises a primary sequence that is at least 85%, at least 90%, at least 92% or at least 95% identical (e.g., at least 95, 96, 97, 98, 99 or 100% identical) to SEQ ID NO: 4 (e.g., to the sequence set forth in SEQ ID NO: 4). In certain embodiments, the polyribonucleotide comprises at its 5' end a portion of a T7 promoter, an Ethris' minimal 5' UTR, a codon-optimized sequence encoding a functional version of the human MCIDAS protein, an optional 3' UTR that also serves as a reverse primer binding site for PCR-based template production, and a poly(A) tail. In some embodiments, the polyribonucleotide is a modified polyribonucleotide having a level and / or type of modification selected from any such level and / or type described herein. In certain embodiments, the percent identity of a polyribonucleotide is measured only with respect to the MCIDAS coding sequence portion of SEQ ID NO:4 (e.g., UTRs, other non-coding sequences and GFP or epitope tags are not considered when calculating percent identity, and the polyribonucleotide may or may not include such regions). In certain embodiments of any of the foregoing, such a polyribonucleotide (or modified polyribonucleotide) encodes a functional MCIDAS protein.

[0164] For purposes of determining the percentage identity of a first sequence to a second sequence, an analog (e.g., methylcytidine) corresponds to cytidine, etc. In certain embodiments, the term "primary sequence" may be used to refer to a polynucleotide sequence, regardless of the presence or amount of modification, such that a primary sequence identical to CUCUCUA includes that sequence whether or not any or all of the recited nucleotides are modified (e.g., analogs of any or more of C, U, and A may be present and still be considered the same primary sequence).

[0165] In certain embodiments, the percent identity is determined only by reference to the portion of a given listed sequence that corresponds to, for example, the coding sequence of CCDC40 or CCDC39. In other embodiments, the percent identity is determined by reference to both the coding sequence and one or more non-coding sequences. In certain embodiments, the percent identity is determined over the entire length of the listed sequence (e.g., by reference to the entire length of the sequence listed in the sequence listing herein).

[0166] The present invention further relates to a method for analyzing the effect of a polyribonucleotide on ciliary formation, said polyribonucleotide encoding a protein associated with and / or required for ciliary formation, the method comprising the steps of: (a) obtaining a nasal brush from a subject with a ciliopathic disease, the nasal brush containing undifferentiated basal cells and differentiated ciliated cells; (b) culturing the cells obtained from step (a) as a submerged cell culture to obtain undifferentiated basal cells and dedifferentiated ciliated cells; (c) culturing the undifferentiated basal cells and dedifferentiated ciliated cells obtained from step (b) as an air-liquid interface cell culture and performing an airlift; (d) transfecting the cells obtained from step (c) with a polyribonucleotide encoding a protein associated with and / or required for ciliary formation; (e) culturing the transfected cells obtained from step (d) to obtain differentiated ciliated cells; and (f) determining the effect of the polyribonucleotide on ciliary formation using lactate dehydrogenase measurement, NucGreen assay, high-speed video microscopy, ciliary beat frequency measurement, mucociliary clearance assay, and / or immunofluorescence staining.

[0167] To obtain cells of the patient's airway epithelium, nasal brushes are advantageous because they are easy to handle and are quick and painless for the patient. The cells obtained from nasal brushes contain undifferentiated basal cells and differentiated ciliated cells that can be cultured as submerged cell cultures. Submerged cell culture induces dedifferentiation of epithelial cells, resulting in undifferentiated basal cells and dedifferentiated ciliated cells. The dedifferentiated ciliated cells thus obtained as well as the undifferentiated basal cells of the nasal brushes are then cultured as air-liquid interface (ALI) cell cultures.

[0168] The air-liquid interface cell culture of step (c) refers to a cell culture characterized in that the basal surface of the cells is in contact with the liquid culture medium, while the apical surface is exposed to air. The cells can be, for example, seeded on the septum of a cell culture insert, which is initially supplied with culture medium in both the apical and basal compartments. This is also called "air cell culture", and upon reaching confluence, the cells are subjected to an "air lift" step, where medium is supplied only to the basal chamber. This mimics the conditions found, for example, in the respiratory system, and induces cell differentiation, including ciliogenesis, in undifferentiated ciliary body cells. Thus, cells mimicking the epithelium of the respiratory system, including, for example, basal and ciliated cells, can be obtained for further study in vitro.

[0169] The resulting epithelial mimetic cells can then be obtained by transfecting polyribonucleotides encoding proteins related to and / or required for production. Thus, said polyribonucleotides are introduced into cells, which can be done artificially by viral infection or other means, to express the exogenous polyribonucleotides in the cells. The transfected cells are then cultured to obtain differentiated ciliated cells from basal and undifferentiated ciliated cells. Thus, the effect of polyribonucleotides such as mRNA on ciliogenesis can be determined in vitro, preserving the environment of cells that mimic the epithelium of the airway system, as may be found in a subject, for example.

[0170] The influence of polyribonucleotides on ciliary formation can be determined by various methods known to those skilled in the art. These methods include, for example, lactate dehydrogenase measurement, NucGreen assay, high-speed video microscopy, ciliary beat frequency measurement, mucociliary clearance assay, and / or immunofluorescence staining. Thus, lactate dehydrogenase measurement (LDH) can be performed to determine the amount of LDH released in the environment surrounding the cells. Each assay can be a colorimetric assay, where the amount of LDH released is measured, for example, in an enzymatic reaction that converts iodonitrotetrazolium or tetrazolium salts into red formazan. Thus, the assay can be easily read by spectroscopy.

[0171] Alternatively or additionally, NucGreen assay can be performed. NucGreen is a permanent green fluorescent nucleic acid stain when bound to nucleic acid, and can be used, for example, to examine the toxicity-related effects of transfection. Alternatively or additionally, immunofluorescence staining of the protein encoded by the transfected polyribonucleotide can be performed to determine its amount and cellular localization, including co-localization with other proteins. Western blot techniques can also be used to determine the amount of expressed protein. Furthermore, defects in ciliary structures or absence of ciliary bodies can be detected, for example, using specialized microscopy. Screening tests include measuring the rate of nasal nitric oxide production, ciliary motility by high-speed video microscopy of nasal cells, or in vivo tests, including, for example, the saccharin test. A more specific diagnosis requires examination of the cilia, for example, by immunofluorescence and transmission electron microscopy.

[0172] Alternatively or additionally, ciliary beating frequency measurements can be applied to investigate beating frequency, number of beating cilia and ciliated cells, and beating synchrony using high-speed video microscopy. The same applies to mucociliary clearance assays that can be performed to measure mucus clearance rates that are influenced by ciliary function.

[0173] Regarding the polyribonucleotides used in the described methods, they are similar to those described above in connection with the pharmaceutical composition of the invention for use in treating ciliopathies. Furthermore, other characteristics of such polyribonucleotides may also be as described above.

[0174] Furthermore, the polyribonucleotide may contain a sequence encoding a marker. Examples include tdTomato (e.g., contained in SEQ ID NO: 3), green fluorescent protein (GFP), and enhanced GFP (eGFP), as well as the respective detection methods described in the appended examples. This is particularly advantageous for confirming successful transfection of cells with the polyribonucleotide.

[0175] In some embodiments of the method for analyzing the effect of polyribonucleotides on ciliary body formation, the cells are transfected within 0-12 days, preferably within 0-11 days, more preferably within 0-6 days, even more preferably within 0-4 days, and most preferably within 0-48 hours after the airlift is performed in step (c). This is advantageous because the airlift step induces ciliary body formation in basal and undifferentiated ciliary body cells.

[0176] In some embodiments of the method of analyzing the effect of polyribonucleotides on ciliary formation, cells are transfected with a polyribonucleotide, preferably an mRNA, encoding a protein associated with and / or required for ciliary formation using a lipidoid having the structure shown in Formula (V), (VI), and / or (VII).

[0177] In some embodiments of the method for analyzing the effect of polyribonucleotides on ciliary formation, the cells are cultured in steps (b) to (e) using medium G. The composition of medium G is typically as shown in Table 2.

[0178] [Table 2]

[0179] In some embodiments of the method of analyzing the effect of polyribonucleotides on ciliary formation, the cells are cultured in steps (b)-(e) using one of the media shown in Table 3. The media shown in Table 3 are commercially available media that can be used to grow ALI cultures.

[0180] [Table 3]

[0181] In some embodiments of the method for analyzing the effect of polyribonucleotides on ciliary body formation, the nasal brush further comprises fibroblasts, the growth of which is inhibited in steps (b)-(e). This is advantageous because fibroblasts proliferate faster compared to basal and undifferentiated ciliary body cells, and thus may hinder the study of the effect of polyribonucleotides on ciliary body formation as described above.

[0182] Before transferring the cells to the stationary phase flask, the fibroblasts can be separated from the remaining cells for a short incubation time of 1-2 hours. During that time, the fibroblasts can settle and attach to the cell flask. All other cells remain in the medium as suspension cells and can then be easily transferred to the "stationary phase flask".

[0183] The method according to the present invention preferably leads to the identification of polyribonucleotides that have a positive effect on ciliogenesis and can therefore be used in the pharmaceutical composition according to the second aspect to restore the ciliary cell structure in function, and their use in the treatment of ciliopathy in subjects suffering from ciliopathy according to the first aspect of the present invention.Therefore, with regard to the characteristics of polyribonucleotides, the same applies as described above in relation to the pharmaceutical composition of the present invention for use in the treatment of ciliopathy.Furthermore, other characteristics of such polyribonucleotides may also be as described above.

[0184] Figure 1: Translation efficiency of CCDC40 mRNA. 2 / 1.4 / 0.3 / 0.2 / 0.05x10^6 HEK293 cells were seeded in 6-well plates. Using Lipofectamine2000, the seeded cells were transfected with different CCDC40 constructs (ETH031T06-T10, 2.5μg / 9.5cm). 2 ) for 24 h. Cell lysis was performed 6, 24, 48, 72 and 144 h after transfection. 50 μg of total protein lysates were analyzed by SDS-PAGE and Western blot. CCDC40 was detected using anti-CCDC40 antibody (HPA022974) from Atlas Antibodies (1:2000), and GAPDH served as a loading control. GFP served as a transfection control.

[0185] Figure 2: Translation efficiency of CCDC40 mRNA constructs in BEAS-2B, RPMI2650, and HEK293 cells: 2x10^6 HEK293, 7.5x10^5 BEAS-2B, and 5x10^5 RPMI 2650 cells were seeded in 6-well plates. 24 hours after seeding, cells were transfected with different CCDC40 constructs (2.5μg / 9.5cm) using Lipofectamine2000. 2 ). Cell lysis was performed 6 hours after transfection. Protein lysates were analyzed by SDS-PAGE and Western blot (HEK293: 50 μg, RPMI 2650: 20 μg, BEAS-2B: 30 μg total lysate) and CCDC40 was detected using anti-CCDC40 antibody (HPA022974) from Atlas Antibodies (1:2000).

[0186] Figure 3: High-speed video microscopy (HSVM) results after LF92 / CCDC40 transfection. Patient-derived ALI cultures were transfected every other day with 3 μg of LF92 / CCDC40. Videos (20 per insert) were taken before transfection and every 24 h after transfection and the CFB (ciliary beating frequency) was calculated using SAVA (Sisson-Ammons video analysis) software. A total of 16 transfections (1 month) were performed. Measurements were performed at 37 °C with 40x magnification. The mean value of the ciliary beating frequency measurements is calculated (total field analysis, WFA).

[0187] Figure 4: Mucociliary clearance (MCC) is shown as Z-projection and polar graph. CCDC40 patient ALI cultures were transfected with CCDC40 mRNA / LF92 at a concentration of 3 μg / insert for 18 days by airlift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). Mucociliary clearance (MCC) was measured at 20x magnification using 0.5 μm fluorescent beads. 30 videos of different areas were taken and analyzed with the Polargraph software from Nikon 1 week after the last TF. One exemplary picture is shown.

[0188] Figure 5: Mucociliary clearance (MCC) shown as Z-projection (top left) and polar graph (top right) for tdTomato and polar graphs of healthy (bottom right) controls. Top row: Cells from a patient with a CCDC40 mutation in ALI culture were transfected with tdTomato mRNA / LF111 at a concentration of 3 µg / insert 18 days after airlift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). Mucociliary clearance (MCC) was measured at 30x magnification using 0.5 µm fluorescent beads. 20 videos of different areas were taken and analyzed with Polargraph software from Nikon 1 week after the last TF. One exemplary video was shown. Bottom right: Particle tracking of a healthy WT ALI culture. MCC was measured at 20x magnification using 0.5 µm fluorescent beads. Twenty videos of different areas were taken and analyzed with the Polargraph software from Nikon one week after the last TF. One exemplary image and two analyzed ROIs are shown.

[0189] Figure 6: Cells from patients with CCDC40 mutations in ALI cultures were transfected with either tdTomato mRNA / LF111 or CCDC40 mRNA / LF92 at a dose of 3 µg / insert for 18 d at airlift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). As control, WT ALI from healthy nasal brushes were used. ALI membranes treated with CCDC40 mRNA and tdTomato mRNA were sectioned at 20 µm and stained with anti-GAS8 (red), anti-acetylated tubulin (green) and DAPI (blue). Photographs were taken with a confocal microscope. One exemplary image is shown. Scale bar represents 10 µm.

[0190] Figure 7: Cells from patients with CCDC40 mutations in ALI cultures were transfected with either tdTomato mRNA / LF111 or CCDC40 mRNA / LF92 at a dose of 3 μg / insert, 18 d after airlift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). As control, ALI from healthy controls were used. ALI membranes treated with CCDC40 mRNA and tdTomato mRNA were sectioned at 20 μm and stained with anti-DNALI1 (red), anti-acetylated tubulin (green) and DAPI (blue). Photographs were taken with a confocal microscope. One exemplary image is shown. Scale bar represents 10 μm.

[0191] Figure 8: Cells from patients with CCDC40 mutations in ALI cultures were transfected with either tdTomato mRNA / LF111 or CCDC40 mRNA / LF92 at a dose of 3 μg / insert for 18 d at air lift. ALI cultures were maintained in medium G for the duration of the experiment. Transfections (TF) were performed once a week for 4 weeks (=4xTF). As control, ALI from healthy controls were used. ALI membranes treated with CCDC40 mRNA and tdTomato mRNA were sectioned at 20 μm and stained with anti-CCDC39 (1:200, red), anti-acetylated tubulin (1:10.000, green) and DAPI (blue). Pictures were taken with a confocal microscope. One exemplary image is shown. Magnification 40x.

[0192] Figure 9: CCDC39 protein (110 kDa) expression in HEK-293 and BEAS-2B, 6 and 24 hours after transfection. 1.4x10^5 HEK-293 cells and 3.5x10^5 BEAS-2B cells were seeded in 6 well plates and transfected with CCDC39-RNA (ETH047T02, minimal 5'UTR, Lipofectamine MessengerMax; ratio 1:1.5). After 6 and 24 hours, cells were lysed with M-PER and Triton® X-100 buffer. 50 μg of protein lysate was used for Western blot analysis. As controls, lysates of non-transfected (UT) and EGFP-transfected cells were used. High dose = 0.5 μg / cm 2 , low dose=0.25μg / cm 2 .

[0193] Figure 10: CCDC39 protein (110 kDa) expression in untreated ALI cultures. Two inserts were extracted using the axonal extraction protocol. 30, 10 and 5 μL of protein lysate were used for Western blot analysis. Active Area: Insert 39.9 = 71.25%, Insert 41.2 = 59.88%

[0194] Figure 11: CCDC39 protein (110 kDa) expression in 16HBE14 after proteasome inhibitor treatment. 6.0x10^5x16HBE14ocells were seeded in 6-well plates and transfected with CCDC39-RNA (Lipofectamine MessengerMax; ratio 1:1.5). After 6 h, cells were lysed with M-PER buffer. 20 μg of protein lysate was used for Western blot analysis. As controls, lysates of untransfected (UT) and EGFP-transfected cells were used. High dose = 0.5 μg / cm 2 , low dose=0.25μg / cm 2RNA: ETH047T02: minimal 5'UTR, ETH047T03: TISU, ETH047T04: CYBA, ETH047T05: SP30.

[0195] Figure 12: CCDC39 protein (110 kDa) expression upon 16HBE14oafter treatment with proteasome inhibitors. 6.0x10^5 16HBE14ocells were seeded in 6-well plates and transfected with CCDC39-RNA (ETH047T03, TISU 5'UTR, Lipofectamine Messenger Max; ratio 1:1.5). After 24 hours, cells were lysed with M-PER buffer. 20 μg of protein lysate was used for Western blot analysis. As controls, lysates of non-transfected (UT) and EGFP-transfected cells were used. High dose = 0.5 μg / cm 2 , low dose=0.25μg / cm 2 .

[0196] Other aspects and advantages of the present invention are described in the following examples, which are given for purposes of illustration and not by way of limitation. Each publication, patent, patent application, or other document cited in this application is incorporated herein by reference in its entirety. EXAMPLES

[0197] Methods and materials for use in the present disclosure are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0198] I. Materials and Methods Materials, equipment, software, and test systems used The materials are listed in Table 4.

[0199] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0200] The devices are listed in Table 5.

[0201] [Table 5]

[0202] The software is shown in Table 6.

[0203] [Table 6]

[0204] The test systems are listed in Table 7.

[0205] [Table 7]

[0206] (I. Underwater cell culture) 1.Culture The cell lines were cultivated in a cell incubator at 37° C. and under a humidified atmosphere with 5% CO2 content. All reagents and solutions were heated to 37° C. in a water bath before use.

[0207] Confluent cells (approximately 90%) were first cultured in 20 mL of DPBS (w / o Mg 2+ / Ca 2+) to remove dead cells. To detach the cells, 2 mL of trypsin / EDTA solution (0.05%) was added per flask. The cells were then incubated at 37°C until cell detachment occurred. Trypsin was stopped with 8 mL of the respective medium. Since LHC-9 medium was almost serum-free, at least an equal volume of trypsin inhibitor was added to the detached BEAS-2B cells to inactivate the trypsinization. Afterwards, the cell solution had to be centrifuged at 1100 revolutions per minute (rpm) for 5 minutes to remove the trypsin inhibitor again. The pellet was then disintegrated in the medium. For passaging, the cells were split into different ratios according to the following specifications:

[0208] HEK 293 (DSMZ number: ACC305) The established cell line was derived from a primary human fetal kidney transformed with adenovirus type 5 (AD 5). It was cultured in Dulbecco's Modified Eagle's Medium (DMEM)-GlutaMAX™ supplement with 10% heat-inactivated (hi) fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). The cell line was split twice a week.

[0209] RPMI 2650 (DSMZ number: ACC207) This cell line originated from a pleural effusion of a 52-year-old man with undifferentiated squamous cell carcinoma of the nasal septum. It was selected for its epithelioid ciliated morphology and similarity to bronchiolar epithelium. It was cultured in Dulbecco's Modified Eagle Medium (DMEM)-10% heat-inactivated (hi) fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S) + GlutaMAX™ supplement with 1x non-essential amino acid solution (NEAA). The cell line was split 1-2 times per week.

[0210] BEAS-2B (ATCC number: CRL9609) BEAS-2B cells were derived from normal bronchial epithelium obtained from autopsy of a non-cancerous individual. The cells were then infected with a replication-deficient SV40 / adenovirus 12 hybrid and cloned. The culture medium used was LHC-9 modified w / o additives. They were also split once or twice a week, depending on confluence.

[0211] Flasks used for BEAS-2B cells were coated with a mixture of 0.01 mg / mL fibronectin, 0.03 mg / mL collagen, and 0.01 mg / mL bovine serum albumin in LHC-9. 2 The flasks were then incubated at 37°C for at least 6 hours. 2+ / Ca 2+ The cells were washed three times with Dulbecco's phosphate buffered saline (PBS) free.

[0212] 2. In vitro transcription To generate templates for in vitro transcription, the circular plasmid was linearized by restriction digestion with BstBI and further purified by chloroform-ethanol precipitation.

[0213] mRNA was produced using a standard in vitro transcription mix (with the indicated modified triphosphate nucleotides) containing T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor. Co-transcriptional capping was achieved by addition of ARCA cap analog. For in vitro transcription of chemically modified RNA, 7.5% of cytidine-5'-triphosphate was replaced with 5-iodocytidine-5'-triphosphate and 30% of uridine-5'-triphosphate was replaced with 5-iodouridine-5'-triphosphate (Jena Biosciences) (see, e.g., SEQ ID NO: 1). In another chemically modified RNA production setup, 3% of cytidine-5'-triphosphate was replaced with 5-iodocytidine-5'-triphosphate and 15% of uridine-5'-triphosphate was replaced with 5-methyl-uridine-5'-triphosphate (Jena Biosciences) (see, e.g., SEQ ID NO: 2). The mRNA was then purified by ammonium acetate precipitation, followed by a washing step with 70% ethanol.

[0214] Dephosphorylation of remaining uncapped mRNA was performed using a Quick Dephosphorylation Kit, followed by purification by ammonium acetate precipitation, followed by a washing step using 70% ethanol, and ultrafiltration using a filter with a 100 MWCO cutoff.

[0215] The mRNA was further polyadenylated using poly(A) polymerase. Again, the mRNA was purified by ammonium acetate precipitation followed by a washing step using 70% ethanol and ultrafiltration using a filter with a 100 MWCO cutoff. The poly(A) length was determined to be 100-250 nucleotides by capillary gel electrophoresis.

[0216] In particular, the CCDC40 mRNA constructs investigated in this experiment contained a 5'ARCA cap and PPA and were: Ethris' minimal UTR (ETH03106; T06; SEQ ID NO: 5), with TISU 5'UTR but no 3'UTR (ETH03107; T07; SEQ ID NO: 6), with hAg 5'UTR but no 3'UTR (ETH03108; T08; SEQ ID NO: 7), with CYBA 5' and 3'UTR (ETH0310T0; T09; SEQ ID NO: 1), and with 5'UTR from human CMV IE9 and 3'UTR from human growth hormone (ETH031T0; T10; SEQ ID NO: 8).

[0217] 3. Transfection Cells were seeded in 6-well plates 24 h prior to transfection to provide at least 90% confluency. Cell numbers varied depending on readout time point and cell type (compare Table 8).

[0218] [Table 8]

[0219] Transfections were performed with different CCDC40 mRNA constructs as well as mRNA encoding enhanced green fluorescent protein (eGFP) as a transfection control. Transfections were performed with Lipofectamine® 2000 and culture medium was replaced prior to transfection to provide optimal conditions for the cells. Lipoplexes required an mRNA / WFI mix as well as a Lipofectamine® 2000 / medium mix. 125 μL of each mix were used with a 1:2 volume ratio of mRNA to Lipofectamine® 2000, as the mRNAs presented a stock concentration of 1 μg / μl. Different mRNA concentrations were used and the mixes were adjusted accordingly (see Table 9 for an exemplary pipetting scheme).

[0220] [Table 9]

[0221] After both mixtures were prepared, the mRNA / WFI mixture was added to the Lipofectamine® 2000 / medium mixture and the solution was incubated for >5 min before adding it dropwise to the seeded cells. An additional medium change was performed 4 h after transfection to obtain better cell viability.

[0222] 4. CCDC40 Western Blot a. Sampling for Western Blot For cell lysis, remove the culture medium from the transfection and resuspend the cells in DPBS (with Mg 2+ / Ca 2+ The cells were washed with 100 μL of lysis buffer (10x Triton® X-100 lysis buffer (250 mM Tris-HCl, 1% Triton® X-100, pH: 7.8) adjusted to 1x in WFI and supplemented with the protease inhibitor cOmplete, after which the samples were frozen at -20°C).

[0223] Total protein concentration was determined using the Pierce™ BCA Protein Assay Kit (BCA Assay). The assay was performed according to the manufacturer's protocol. Briefly, a 1:50 dilution of BCA Working Reagent (WR) was prepared. 5 μL of each sample was transferred to a 96-well plate (flat bottom, clear) and 200 μL of WR was added per well. The final step was a 30-minute incubation at 37°C. Results were measured on a Tecan Infinite® 200 PRO plate reader using Magellan™ data analysis software.

[0224] b. Preparation of NET-gelatin

[0225] [Table 10]

[0226] c. SDS-PAGE and Western Blot To perform sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the lysates were mixed with 5 μL of Volt LDS sample buffer (4x) and 2 μL of Volt sample reducing agent (10x) and then heated to 70°C in a Thermomixer® C for 10 min and 350 rpm. 7 μL of markers (Precision Plus protein™ Dual Color Standards) were loaded into the first pocket of each gel to allow for accurate protein size estimation thereafter. The gel tank was then connected to a power supply (Power PAC 300) at 1 A for 30 min. The cassette was subsequently opened with a spatula and the gel was transferred to the blotting station.

[0227] A Transfer System (Transfer-Blot® Turbo™ Transfer System, 30 min, 25 V, 1 A) and a suitable membrane (Transfer-Blot Turbo Transfer Pack Mini 0.2 μm PVDF) were used for blotting according to the manufacturer's instructions. Any air bubbles that may interfere with protein transfer were eliminated by scrolling a roller on the "sandwich". To block free binding sites, the membrane was placed in NET-gelatin and shaken for 60 min on a plate shaker. The gel was cut at the 75 kD band to simultaneously detect CCDC40 and glyceraldehyde-3-phospha-dehydrogenase (GAPDH). Antibodies were diluted in NET-gelatin (anti-CCDC40 1:2000 in 6 mL, GAPDH 1:10000 in 10 mL of NET-gelatin) and the membrane was incubated overnight at 4° C. on a plate shaker. GAPDH serves as a loading control. A list of antibodies used is shown in Table 11. The next day, the membrane was washed three times for 10 min with NET-gelatin to remove nonspecifically bound or residual antibodies. The secondary antibody goat anti-rabbit IgG-HRP was diluted 1:10,000 in NET-gelatin. After 60 min of incubation on a plate shaker at room temperature (RT), the membrane was washed three times for 10 min with NET-gelatin. The horseradish peroxidase-HRP-conjugated secondary antibody could be detected via a molecular imaging unit (ChemiDoc™ XRS system). Therefore, 5 mL of Luminata Crescendo Western HRP Substrate was added onto the gel and incubated for 5 min. Then, the colorimetric photograph was merged with the last photograph that was not yet overexposed and illustrated with Image Lab™ software. Furthermore, the band intensity was measured afterwards using Image Lab™.

[0228] [Table 11]

[0229] (II. ALI culture) 1. Sampling of human respiratory epithelial cells (hRECs) a. Nose brushing RPMI medium was heated to room temperature and the brush was moistened with sterile isotonic saline. A Celletta brush cell collector with a protective tip was applied. After asking the patient to clean his nose, he had to hold his head while sitting in a chair with his head against the wall. The brush was quickly scraped several times on the inside and upper side of the inferior nasal meatus using rotary and linear movements. Once the brush was removed, it was immediately placed in a collection tube (15 mL cornicle) containing 5 ml of pre-warmed RPMI medium. The brush was vigorously shaken in the tube at least 40 times to separate the cells from the brush.

[0230] b. Stationary culture The tubes were spun at 900 rpm for 5 min at room temperature. After discarding the supernatant, the pellet was dissolved in UG medium [DMEM / Ham's F-12 1:1, 5 mL of sterile 100x anibiotics / mycotics and 10 mL of sterile Ultroser G]. 5 mL of medium was used for T25 and 25 mL for T75 flasks.

[0231] After transferring the cells to cell culture flasks, the cells were incubated overnight at 37 °C in a humidified incubator containing 5% CO2. The cells should not attach after 20-24 h. UG medium was changed three times a week (Monday, Wednesday, and Friday).

[0232] 2. Generation and transfection of ALI cultures a. Coating of transwell inserts (0.33 cm 2 ) The following plates were used for ALI culture: Corning Transwell with a clear polyester membrane (0.4 μm pores, 6.5 mm diameter inserts, 24-well plates). Rat tail collagen was diluted 1:5 in acetic acid and 250 μL was loaded onto the apical side of each insert. After incubating the plates overnight at room temperature, the remaining liquid was aspirated with a cryopipette and then the plates were allowed to dry with an open lid for at least 5 min (sterile). Finally, the inserts were filled with DPBS (w / o Ca) and diluted with 100 μL of DPBS (w / o Ca) for 24 h. 2+ / Mg 2+ ) and then dried overnight at room temperature (sterile). Inserts were stored at 4°C for up to one month.

[0233] b. Cell proliferation of hREC as a submerged culture (static culture) Cells were fed with 15 mL of pre-warmed UG medium every 2-3 days (Monday, Wednesday, and Friday). Care had to be taken not to add new medium directly onto the collagen layer to prevent damage to the collagen layer and detachment of the cells. Cells were transferred to ALI filters (after approximately 3 weeks of submerged culture) as soon as the cells were 80% confluent or the collagen layer had started to detach.

[0234] c. Preparation of medium G Different ALI media were tested, but the self-made medium G is preferred.

[0235] This medium was prepared for one week and needed to be stored at 4° C. The batch was frozen in aliquots based on the upcoming experiment size. Table 2 shows the composition of Medium G.

[0236] d. Seeding and airlift of hREC (ALI culture) 1. Aspirate the UG medium and replace with washing medium [DMEM / Ham's F-12 1:1 (1x sterile antibiotic / antimycotic)] or DPBS (w / o Ca 2+ / Mg 2+ ) once. 2. For homemade rat tail collagen coated flasks: Add 9 mL (1x) collagenase to dissolve the collagen layer and incubate at 37°C for 45-60 minutes. b. Transfer the cells to a 15 mL Kolnik tube and centrifuge at 1000 rpm for 5 minutes at room temperature. c. Wash the cells with washing buffer (1000 rpm, room temperature, 5 min). d. Add 2 mL of trypsin-EDTA (1x), gently pipette up and down to detach the cells, and incubate the cells at 37 °C with shaking until the cell clusters break up (approximately 5 min). For collagen-coated flasks purchased from Gibco: a. Add 3 ml of Trypsin-EDTA (1x) to the cell culture flask until the cells detach (approximately 5 min). 3. Stop trypsin by adding 0.5 mL of FBS and 5 mL of wash medium 4. Transfer the cells to a 15 ml falcon and wash the cells twice with washing medium (1000 rpm, room temperature, 5 min) 5. Resuspend the pellet in an appropriate amount of ALI medium (1-2 mL, depending on pellet size) and count the cells using a cell counter 6. Add 250 µL of cell suspension (400,000 cells / mL) to each collagen transwell coated basolaterally with 500 µL of ALI medium 7. Incubate the cells overnight at 37°C in a humidified 5% CO2 incubator. Days 3-5: ALI-expansion 8. Check inserts consistently under a microscope 9. Feed cells with ALI medium daily until 80–100% confluence (3–4 days) ALI differentiation 10. Airlift cells when they reach 80-100% confluency a. Aspirate the ALI medium from both the apical and basolateral sides. b. Add 500 μL of fresh ALI medium to the basolateral compartment c. Incubate the cells at 37°C in a humidified 5% CO2 incubator. 11. Wash the cells with pre-warmed washing medium (apical) once a week and feed the cells with ALI medium every 2-3 days (or Monday-Wednesday-Friday).

[0237] e. Transfection of hRECs The formulation (LF111-mRNA) was administered at 3 μg / insert (=9 μg / cm 2 The inserts were diluted in 2% sucrose to a final concentration of 100 µL each. To remove the mucus, the inserts were flushed apically with 200 µL of DPBS (w / o Ca 2+ / Mg 2+ ) and rinse the apical side with 200 µL of 37 °C (epithelial ALI) or 200 µL of DPBS (w / o Ca 2+ / Mg 2+ ) for 20 min and then incubated at room temperature for 5 min before transfection. Then, DPBS (w / o Ca) was added from the apical surface. 2+ / Mg 2+ ) was removed by gentle suction without damaging the epithelium. Immediately after mucus washing, the cells were washed again with WFI (100 μL) and diluted with a small amount of DPBS (w / o Ca 2+ / Mg 2+ ) was removed. 25 μL of the formulation was added to the apical side of the insert to allow cell uptake. After 6 h of incubation, removal of the carrier was performed and the cells were resuspended in 200 μL of DPBS (w / o Ca 2+ / Mg 2+ ) and the cultures were maintained as ALI without fluid on the apical side.

[0238] 3. Readout assay Different readout assays are used. LDH measurements and NucGreen assays were used to investigate toxicity-related effects of transfection. Ciliary beating frequency measurements (CBF) were necessary to investigate the manner of ciliary beating in terms of beating frequency and percentage of ciliary area. If ciliary beating leads to directional flow, it can be studied using a mucociliary clearance (MCC) assay. Immunofluorescence (IF) staining for CCDC40, its heterodimer partner CCDC39 and the ciliary marker acetylated α-tubulin was utilized to detect whether CCDC40 protein or its partner CCDC39 could be detected in cilia after treatment. In addition, proteins of the dynein regulator complex (DRC) such as GAS8 and DNALI1 were stained. DRC is anchored by CCDC40 / CCDC39 and is only present if CCDC40 / CCDC39 is correctly integrated into the ciliary axon.

[0239] a. LDH measurement LDH concentrations are measured using the Pierce™ Lactate Dehydrogenase Cytotoxicity Assay Kit. The procedure is performed as described in the manufacturer's protocol. ALI cultures are incubated at 37°C and 5% CO2 for 30 min in 100 μL of DPBS (Mg 2+ / Ca 2+ The plates were incubated on top with 50 μL of LDH reaction mixture and 50 μL of incubation medium using a 96-well format, 50 μL of stop solution was added, and incubated at room temperature for 30 min protected from light. The absorbance of formazan was measured at 490 nm and 680 nm using a microplate reader (TriStar2 Multimode Reader LB 942). The value at 680 nm was subtracted from 490 nm for background normalization. Duplicate measurements were performed for each insert.

[0240] b. Ciliary body frequency (CBF) measurement Ciliary body beat frequency (CBF) analysis of hRECs was performed by high-speed video microscopy and Sisson-Ammons video analysis. Videos (125 fps, 640 × 480 pixel resolution) were recorded with a Megaplus camera model ES 310 Turbo attached to an inverted phase-contrast microscope equipped with an ELWD 40x S Plan Fluor objective under physiological conditions, with the temperature maintained at 36 °C by a Minitube SC300 heating unit.

[0241] c. Mucociliary clearance assay (MCC, fluorescent beads) ALI medium and DPBS (w / o Ca 2+ / Mg 2+ ) was pre-warmed to room temperature and the microscope plate was heated to 37 °C. The ALI inserts were transferred to a new plate and filled with pre-warmed ALI medium (substitute DPBS w / o Ca 2+ / Mg 2+ The tip was washed twice with DPBS (w / o Ca). After adding 100 μL of ALI medium to the tip compartment, 20 videos (magnification 40x, Ph2) per insert were recorded to obtain a comprehensive impression of the insert (SAVA software). Red fluorescent particles with a particle size of 0.5 μm were inoculated into DPBS (w / o Ca). 2+ / Mg 2+ ) and vortexed for at least 1 min, then 10 µL of the particle dilution was added to 100 µL of ALI medium, vortexed, and 110 µL was added to the apical compartment. While handling the beads, it was important to vortex before each step to prevent aggregation. Furthermore, working in dim light was necessary to avoid bleaching.

[0242] The flow of fluorescent particles was recorded with NIS-Elements Basic Research software. The movement was recorded over 20-30 s with 20x magnification (Ph1) while exciting the particles with a 488 nm laser. The exposure time had to be adjusted to the number of recorded frames per second (fps). For 7.5 fps, the exposure time was set to 100 ms, from 15 fps to 50 ms. The exact observation area was analyzed with SAVA (20x magnification, Ph1) to visualize the ciliary movement and the 3D structure of the cell layer.

[0243] Nikon NIS Elemets AR Software, NIS PLUG-IN<ADVANCED 2D TRACKING> <ar>(see NIS Elements AR Tracking Modul above).

[0244] d. IF of ALI cultures Embedding of ALI cultures Prior to membrane preparation, the ALI inserts were resuspended in DPBS (w / o Mg 2+ / Ca 2+ Mucus was removed by washing twice with 100 μL of 1000 mM NaCl (200 μL, 1 min, room temperature). The following membrane preparation is performed according to the following protocol (see below).

[0245] After cutting out the transwell filter membrane using a scalpel or syringe, place the membrane in 3 ml of DPBS (Ca 2+ / Mg 2+ The membrane was cut into equal pieces (4–6) and cryomolds were prepared using Shandon Cryomatrix™ freeze embedding medium (medium size). The membrane pieces were placed in the cryomatrix and positioned parallel to each other before the cryomold was incubated on dry ice until the cryomatrix hardened. The cryomolds were stored at -80 °C for at least 2 h before cutting in a cryostat.

[0246] Cleavage of the ALI membrane ALI membranes embedded in Shandon Cryomatrix™ were cut using a cryostat Microm HM560. Therefore, the cryostat was pre-cooled (2-3 h before cutting) to -20 °C (table at -21 °C and chamber at -25 °C). The ALI membranes with cryomolls were transferred from -80 °C to -20 °C for at least 1 h. Sectioning of the membranes was performed at 20 µm, depending on the handling conditions of the membrane. Membrane slices were transferred to Superfrost Ultra Plus slides, dried at room temperature for at least 1 h, and stored at -80 °C overnight or at -80 °C until use.

[0247] Staining procedure For antibodies applied, see Table 12. Day 1: 1. 1xDPBS (Ca 2+ / Mg 2+ ) for 5 minutes at room temperature 2. Fixation: Fix the tissue in 4% PFA / 1xDPBS (Ca) at room temperature. 2+ / Mg 2+ ) 15 minutes 3. Wash 3x DPBS (Ca 2+ / Mg 2+ )(2x fast, 1x5 minutes) 4. Transparency: a. 0.5% Triton® X-100 for 5 minutes or b. 0.2% Triton® X-100 for 10 minutes (varies depending on the antibody used) 5. Prepare 3x DPBS (Ca) at room temperature 2+ / Mg 2+ ) Wash (2x Quick, 1x 5 min) 6. Blocking: 5% normal goat serum + DPBS (Ca 2+ / Mg 2+ ) with 2% BSA, or DPBS (Ca 2+ / Mg 2+ ) with 5% skim milk → leave at room temperature for 2 hours 7. 200 μl of primary antibody at 4°C overnight Day 2: 8. DPBS (Ca 2+ / Mg 2+ ) for 45 minutes (3x15 minutes) 9. Secondary antibody: 200 μL for 1 h at room temperature 10. Soak the cells in DPBS (Ca) for 10 min at room temperature in the dark. 2+ / Mg 2+ ) Hoechst33342 1:1000 11. DPBS (Ca 2+ / Mg 2+ ) for 6x5 min. 12. Mounting coverslips with Dako Antifade

[0248] [Table 12]

[0249] e. Airbrushing of ALI cultures Differentiated ALI cultures were generated using an airbrush model. The protocol was established after Crespin et al. 2011 (Approaches to Study Differentiation and Repair of Human Airway Epithelial Cells).

[0250] Cells were incubated in pre-warmed DPBS (with Mg 2+ / Ca 2+ The airbrush was then briefly washed at the tip with a compressor at 1kg / cm2 by rotating the airbrush and the small wheel. 2 The airbrush may need to be run for some time to stay at a constant value. Next, fill the small reservoir above the airbrush with DPBS (Mg 2+ / Ca 2+ ) and the insert was placed into a new plate without basal medium. The airbrush was started by pushing to the first pressure point and waiting until the compressor pressure remained constant. Then, the airbrush was placed vertically on top of the insert and the lever of the airbrush was pressed very slightly past the first pressure point (DPBS(Mg 2+ / Ca 2+ ) from the reservoir for 2 seconds. The tip of the insert is then filled with pre-warmed DPBS (Mg 2+ / Ca 2+ ) and the inserts were placed in fresh medium applicable for ALI differentiation. The wound was checked by microscopic observation. If it was too small (less than 1 / 3 of the insert area) or the cells in the wound were only slightly detached, the procedure was repeated. The cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2 and cultivated as ALI.

[0251] (III. Labeled CCDC40 mRNA) The mRNAs under study are shown in Table 13.

[0252] [Table 13]

[0253] a. Cell culture HEK-293 cells are cultured in MEM GlutaMAX™, 10% FBS, 100 U / ml P / S (=culture medium) at 37° C., 5% CO2.

[0254] Before seeding, cells are washed with DPBS and detached using trypsin. After trypsinization, trypsin is inactivated using culture medium containing FBS. Therefore, an equal or greater volume of culture medium is used. After centrifugation at 1100×g for 5 min and resuspension in normal growth medium, cells are counted using a Countess Cell Counting Device.

[0255] b. In vitro transcription To generate templates for in vitro transcription, the circular plasmid was linearized by restriction digestion with BstBI and further purified by chloroform-ethanol precipitation. mRNA was produced using a standard in vitro transcription mix containing T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor. Co-transcriptional capping was achieved by the addition of ARCA cap analog. For in vitro transcription of chemically modified RNA, 7.5% of cytidine-5'-triphosphate was replaced by 5-iodocytidine-5'-triphosphate and 30% of uridine-5'-triphosphate was replaced by 5-iodouridine-5'-triphosphate. In another SNIM® RNA production setup, 3% of cytidine-5'-triphosphate was replaced by 5-iodocytidine-5'-triphosphate and 15% of uridine-5'-triphosphate was replaced by 5-methyl-uridine-5'-triphosphate. Residual template DNA was digested with DNase I. The mRNA was then purified by ammonium acetate precipitation, followed by a washing step with 70% ethanol.

[0256] Dephosphorylation of remaining uncapped mRNA was performed using a Quick Dephosphorylation Kit, followed by purification by ammonium acetate precipitation, followed by a washing step using 70% ethanol, and ultrafiltration using a filter with a 100 MWCO cutoff.

[0257] The mRNA was further polyadenylated using poly(A) polymerase. Again, the mRNA was purified by ammonium acetate precipitation followed by a washing step using 70% ethanol and ultrafiltration using a filter with a 100 MWCO cutoff. The poly(A) length was determined to be 100-250 nucleotides by capillary gel electrophoresis.

[0258] c. Seeding, transfection and readout Cells were seeded in 96-well black microplates (25,000 cells per well) 24 hours prior to treatment. 24 hours after seeding, fresh medium was added to each well (100 μL). mRNA was transfected using Lipofectamine® MessengerMAX™ at an RNA to Lipofectamine ratio of 1:1.5 (w / v). 250 ng / 96 well (758 ng / cm 2 ) eGFP-CCDC40 (ETH031T28 or ETH031T30) or HA-CCDC40-T2A-tdTomato (ETH031T26) mRNA was used. All RNAs had a stock concentration of 1 mg / mL. For lipoplex formation, mRNA was most diluted in water. Lipofectamine® MessengerMAX™ was diluted in SFM (serum-free medium) and mixed by pipetting. After 10 min incubation at RT, the RNA solution was added to the Lipofectamine® MessengerMAX™ solution, mixed and incubated for another 5 min at RT. The mixture was then diluted 2-fold with SFM before the Lipoplex solution was added to the wells. In Table 14, an example for one RNA was calculated.

[0259] [Table 14]

[0260] Six and 24 hours after transfection, the transfection efficiency was examined by fluorescence microscopy and photographed at 10x magnification.

[0261] (IV. CCDC39) The mRNA under study (ETH047T02; SEQ ID NO: 12) encoded hCCDC39 and contained the Ethris minimal UTR. Overall, 15% of the uridine-5'-triphosphates were replaced by 5-methyl-uridine-5'-triphosphate, and a total of 3% of the cytidine-5'-triphosphates were replaced by 5-iodo-cytidine-5'-triphosphate. The antibodies used are listed in Table 15.

[0262] [Table 15]

[0263] 1. CCDC39 transfection and Western blot in submerged cell cultures a. Cell culture BEAS-2B cells were cultured in LHC-9 medium in coated flasks. For details, see I1 above. Experiments were performed in uncoated plates.

[0264] HEK-293 cells were cultured in MEM GlutaMax medium supplemented with heat-inactivated FBS and penicillin / streptomycin (P / S).

[0265] Before seeding, cells were washed with DPBS and detached using trypsin. After trypsinization, trypsin was inactivated using either trypsin inhibitor for BEAS-2B or culture medium containing FBS for HEK-293. Therefore, equal or greater volumes of trypsin inhibitor / culture medium were used. After centrifugation at 1100×g for 5 min and resuspension in normal growth medium, cells were counted using a Countess Cell Counting Device. Considering a seeding volume of 2 ml and a 6-well plate format, 5.0x10 5 Seed BEAS-2B cells and 1.4x10 6 Seeding HEK-293 cells was obtained.

[0266] b. Transfection Cells were seeded at the respective density 24 hours before treatment. 24 hours after seeding, 2 mL of fresh medium was added to each well. mRNA was transfected using Lipofectamine® MessengerMAX™ at an RNA to Lipofectamine ratio of 1:1.5 (w / v). All RNAs had a stock concentration of 1 mg / mL. For lipoplex formation, mRNA was diluted in aqua bidest. Lipofectamine® MessengerMAX™ was diluted in SFM and mixed by pipetting. After 10 minutes of incubation at room temperature, the RNA solution was added to the Lipofectamine® MessengerMAX™ solution, mixed, and incubated for another 5 minutes at room temperature. The lipoplex solution was then added to the wells. In Table 16, the examples for transfection are calculated for one 6-well plate.

[0267] [Table 16]

[0268] c. Cell lysis To obtain optimal cell lysis, two different lysis buffers were compared (Triton® X-100 buffer and the commercial lysis buffer M-PER™), both of which were complemented with the protease inhibitor cOmplete, complete-free, and 40 μL / mL DNase I solution (in a 23:1:1 mixture). Thus, cells were seeded in 6-well plates and treated for 6 and 24 hours. After treatment, cells were harvested. Thus, plates were washed once with 1 mL of DPBS. To remove cells from the plate, another 1 mL of DPBS was added and cells were scraped off the plate and transferred to an Eppendorf tube. To remove DPBS, cells were centrifuged at 6250 × g for 2 min at 4 ° C. Cell lysis was performed by adding 200 μL of the respective buffer. To ensure complete lysis, cells were incubated on ice for 30 min. After lysis, a BCA assay was performed to determine total protein concentration using a BCA protein assay kit according to the manufacturer's instructions with the following modifications: 200 μL of working reagent was added to 5 μL of cell lysate. ● Samples and standards were measured in triplicate. Plates were shaken at 450 rpm for 30 seconds before samples were incubated at 37°C for 30 minutes.

[0269] d. Sample preparation Samples were mixed with 5 μL of Bolt® LDS Sample Buffer and 2 μL of Bolt® Sample Reducing Agent and then heated at 70°C for 10 min before SDS-PAGE. 30 μg of total protein was used for SDS-PAGE using Bolt™ 4-12% SDS-PAGE gels, 10 wells.

[0270] e. SDS Page and Blotting method A Trans-Blot® Turbo™ system transfer was used. SDS-PAGE was performed at 200 V for 40 min. Transfer was performed using the TransBlot® Turbo™ transfer system for 30 min.

[0271] f. Antibodies and blocking methods After transfer, the membrane was blocked for 1 h at room temperature. NET-gelatin was used as a blocking reagent. Antibody HPA035364 from Atlas Antibodies was used for detection of CCDC39, and antibody ab8227 from Abcam was used for detection of actin. Before adding the antibodies, the membrane was cut at approximately 60 kDa. The membrane was incubated with the primary antibody overnight at 4 °C. After washing three times (10 min each) with blocking solution at room temperature, horseradish peroxidase-conjugated secondary antibody was added for 1 h at room temperature (diluted 1:20 000). The membrane was washed again three times with blocking solution at room temperature for 10 min each time.

[0272] g. Chemiluminescence signal development Signals were visualized using a chemiluminescent substrate kit (Luminata Crescendo, Classico or Forte Western HRP Substrate, depending on signal intensity) and the ChemiDocTM MP System.

[0273] 2. Western Blot of Endogenous CCDC39 in Differentiated ALI Cultures a. Cell lysis 250 μL of ALI lysis buffer (see Table 17 for composition details) was added per insert and incubated on ice for 15 min. ALI lysis buffer was pipetted back and forth before, during (every 5 min) and at the end of the incubation. After transferring to an Eppendorf tube and rinsing each insert with 150 μL of ALI lysis buffer, the samples were vortexed thoroughly. At this step, samples can be optionally stored at -20°C until further processing.

[0274] [Table 17]

[0275] b. Isolation of axonemal lysate from whole cell lysate The sample was added to a Lysing Matrix A tube (tube was washed with 70% EthOH to remove garnet). The tube was clamped and frozen in liquid nitrogen for 20 seconds while moving the tube. The tube was quickly inserted into an MP FastPrep-24 (HOM-1), settings: 6.5 m / s, 3 x 1 min. The tube was then condensed on ice for 15 minutes before transferring the liquid to a fresh Eppendorf tube and spinning at 1100 rpm, 4 °C for 20 minutes. The supernatant was transferred to a new Eppendorf tube and the pellet and supernatant were stored at -20 °C until further processing.

[0276] c. Preparation of axonemal extract lysate: High-salt extraction of ciliary axonemal proteins Each pellet was resuspended in 50 μl of MMRB + 0.1% Triton® X-100 (see Table 18 for details of the composition of MMRB + 0.1% Triton® X-100) and incubated on ice for 30 minutes (with occasional gentle vortexing). After spinning at 14,000 rpm for 10 minutes at 4° C., the supernatant was transferred to a new Eppendorf tube and stored at −20° C. until further processing.

[0277] [Table 18]

[0278] [Table 19]

[0279] d. Preparation of lysates for Western blot Samples were thawed on ice and 30 μl of sample was transferred to a new Eppendorf tube. 12 μl of LDS sample buffer and 5 μl of 10× reducing agent were added before samples were heated at 70°C, 350 rpm for 10 min. 5, 10 and 30 μl per well were applied to a Bolt™ 4-12% SDS-PAGE gel, 10 wells.

[0280] e. SDS Page and blotting methods: See IV 1e

[0281] f. Antibodies and blocking methods: See IV 1 f.

[0282] g. Chemiluminescent signal generation: See IV 1g

[0283] (II. Results) CCDC40 expression was determined in HEK293 cells 6, 24, 48, 72 and 144 hours after transfection. 6 Cells were seeded in 6-well plates and transfected with various CCDC40 constructs (2.5 μg / 9.5 cm) using Lipofectamine® 2000. 2 Cells were transfected 24 h after seeding with 1000 ng / ml CCDC40 mRNA constructs. The CCDC40 mRNA constructs used in this experiment were T06-T10 (SEQ ID NO:1, and SEQ ID NO:5-SEQ ID NO:8). Cell lysis was performed 6, 24, 48, 72 and 144 h after transfection and 50 μg of total protein lysates were analyzed by SDS-PAGE and Western blot.

[0284] As can be seen in Figure 1, peak translation efficiency was detectable 6 hours after transfection of CCDC40 mRNA in HEK cells.

[0285] In this case, 2x10 6 HEK293, 7.5x10 5 BEAS-2B and 5x10 5 RPMI 2650 cells were seeded in 6-well plates and protein analysis was performed using SDS-PAGE and Western blot (HEK293: 50 μg, RPMI 2650: 20 μg, BEAS-2B: 30 μg total analysis), and CCDC40 was detected using Anti-CCDC40 Antibody (HPA022974) from Atlas Antibody (1:2000).

[0286] Here it can be observed that T09 (CYBA; SEQ ID NO: 1) and T10 (human CMV IE9 and 3'UTR from human growth hormone SEQ ID NO: 8) UTRs resulted in the highest translation efficiency 6 hours after transfection in BEAS-2B, RPMI2650 and HEK293 cells (Figure 2).

[0287] Two experiments were then performed to investigate the restoration of ciliary motility in patient-derived ALI cultures. In particular, the first experiment used non-differentiated ALI cultures (data not shown) (ciliated cells had a deletion of exons 1 and 2, resulting in very short-positioned, less motile cilia), whereas the second experiment used differentiated ALI cultures. In both cases, ALI cultures were transfected with 3 μg LF92 / CCDC40 (ETH031T09; SEQ ID NO: 1) every other day for one month, for a total of 16 transfections. As readouts, high-speed video microscopy (HSVM) was performed every 24 h and immunofluorescence immunocytochemistry (IF-ICC). Videos (20 per insert) were taken before transfection and every 24 h after transfection, and the CFB (ciliary beat frequency) was calculated using SAVA software. A total of 16 transfections (1 month) were performed. Measurements were performed at 37 °C using 40x magnification. Calculate the mean value of ciliary beating frequency (CBF).

[0288] Repeated transfection of LF92 / CCDC40 was well tolerated in patient-derived fully differentiated ALI cultures: CCDC40 protein could be detected in the naturally occurring subcellular region, i.e., cilia, of airway epithelial cells after 22 days by IF-ICC, and ciliary motility increased after LF92 / CCDC40 transfection to approximately 50% of normal ciliary beat frequency from day 18, as seen in Figure 3 for experiments with differentiated ALI cultures.

[0289] Undifferentiated ALI cultures were obtained using medium G, as described in the Materials and Methods section, and transfections (TF) were started 18 days after airlift. Transfections (TF) of CCDC40 patient ALI cultures with CCDC40 mRNA / LF92 were performed once a week for 4 weeks (=4x TF). Mucociliary clearance (MCC) was measured at 20x magnification using 0.5 μm fluorescent beads. 30 videos of different areas are taken and analyzed with Polargraph software from Nikon 1 week after the last TF.

[0290] Importantly, we could detect restoration of cilia beating in CCDC40 patient ALI after 4 weeks of CCDC40 mRNA / LF92 transfection. Moreover, we synchronized cilia beating and allowed directional particle transport as seen in Figure 4. For comparison, particle transport in the case of the control, i.e., 4-fold higher than that of tdTomato mRNA / LF111 transfection, and in the case of the healthy control, is shown in Figure 5.

[0291] Thus, during ciliary cell differentiation, successful CCDC40 mRNA / LF92TF transfection of patient ALI cultures with LF92 resulted in successful ciliary growth, successful ciliary beating, successful directional particle transport, and successful protein detection (CCDC40 as part of the DRC complex + CCDC39 as a binding partner of GAS8 / DNALI1) as shown by comparison with tdTomato control. Moreover, tdTomato control insertion did not show any of the above effects.

[0292] Figure 6 shows successful incorporation of GAS8 protein into axonemes after repeated patient ALI treatment with CCDC40 mRNA, as well as healthy controls not present in control (tdTomato mRNA) treated patient ALIs. Figure 7 shows successful incorporation of DNALI-1 protein into axonemes after repeated patient ALI treatment with CCDC40 mRNA, as well as healthy controls not present in control (tdTomato mRNA) treated patient ALIs. Figure 8 shows successful incorporation of CCDC39 protein into axonemes after repeated patient ALI treatment with CCDC40 mRNA, as well as healthy controls not present in control (tdTomato mRNA) treated patient ALIs.

[0293] In summary, ciliary motility could not be restored to significant levels using differentiated ALI cultures, but was partially restored using differentiated ALI cultures.

[0294] Regarding the CCDC39 experiment, CCDC39 protein could be detected by Western blot analysis 6 hours and 24 hours after transfection in HEK-293 and BEAS-2B cells, as seen in FIG. 9. As shown in FIG. 11, CCDC39 (ETH047T03; SEQ ID NO: 13) expression could be detected after 6 hours in 16HBE14o using proteasome inhibitor. Similar experiments were performed with ETH047T02 (SEQ ID NO: 12), ETH047T04 (SEQ ID NO: 2), and ETH047T05 (SEQ ID NO: 14) with comparable results. As shown in FIG. 12, CCDC39 (ETH047T03; SEQ ID NO: 13) expression could also be detected after 24 hours in 16HBE14o using proteasome inhibitor.

[0295] Exemplary sequences described in this application are provided below. In some embodiments, the disclosure provides a polyribonucleotide comprising a polyribonucleotide sequence, such as, for example, a UTR sequence as set forth in SEQ ID NO: 1 or 2, or a sequence at least 95%, 96%, 97%, 98%, or 99% identical to such a sequence, or an mRNA corresponding to or encoded by any of the foregoing. In certain embodiments of any of the foregoing, the polynucleotide or polyribonucleotide is modified (e.g., comprises nucleotide analogs as described herein).

[0296] SEQ ID NO:1 TIFF0007607340000037.tif159160TIFF0007607340000038.tif234163TIFF0007607340000039.tif21157

[0297] SEQ ID NO:2 TIFF0007607340000040.tif202157TIFF0007607340000041.tif141160

[0298] SEQ ID NO:3 TIFF0007607340000042.tif81160TIFF0007607340000043.tif128164

[0299] SEQ ID NO:4 TIFF0007607340000044.tif95164TIFF0007607340000045.tif82161

[0300] SEQ ID NO:5 TIFF0007607340000046.tif145161TIFF0007607340000047.tif228158TIFF0007607340000048.tif22158

[0301] SEQ ID NO:6 TIFF0007607340000049.tif200158TIFF0007607340000050.tif185159

[0302] SEQ ID NO:7 TIFF0007607340000051.tif33159TIFF0007607340000052.tif225157TIFF0007607340000053.tif127167

[0303] SEQ ID NO:8 TIFF0007607340000054.tif97166TIFF0007607340000055.tif225162TIFF0007607340000056.tif100159

[0304] SEQ ID NO:9 TIFF0007607340000057.tif121159TIFF0007607340000058.tif228157TIFF0007607340000059.tif117160

[0305] SEQ ID NO:10 TIFF0007607340000060.tif107160TIFF0007607340000061.tif227156TIFF0007607340000062.tif136161

[0306] SEQ ID NO:11 TIFF0007607340000063.tif90161TIFF0007607340000064.tif230156TIFF0007607340000065.tif228162

[0307] SEQ ID NO:12 TIFF0007607340000066.tif203166TIFF0007607340000067.tif132160

[0308] SEQ ID NO:13 TIFF0007607340000068.tif94160TIFF0007607340000069.tif230156 [ka]

[0309] SEQ ID NO:14 TIFF0007607340000071.tif198161TIFF0007607340000072.tif145163< / ar>

Claims

1. 1. A pharmaceutical composition comprising a polyribonucleotide for use in treating a ciliopathies in a subject suffering from a ciliopathies, said polyribonucleotide encoding a functional version of a protein whose deletion is associated with said ciliopathies, and wherein administration of said pharmaceutical composition to the respiratory system of said subject occurs when said subject exhibits inflammation of said respiratory system; the polyribonucleotide is an mRNA that can be translated into a functional version of the coiled-coil domain-containing 39 (CCDC39) protein and / or the coiled-coil domain-containing 40 (CCDC40) protein; The polyribonucleotide may be: a) a functional version of the human CCDC40 protein, wherein the polyribonucleotide encodes a functional version of the human CCDC40 protein that is at least 90% identical to one or more of SEQ ID NOs: 1 or 5-11, and wherein the protein encoded by the polyribonucleotide is capable of restoring ciliary motility in air-liquid interface (ALI) cell cultures derived from a ciliopathy patient; or b) a functional version of the human CCDC39 protein, wherein the polyribonucleotide encodes a functional version of the human CCDC39 protein that is at least 90% identical to one or more of SEQ ID NOs: 2 or 12-14, and wherein the protein encoded by the polyribonucleotide is capable of restoring ciliary motility in air-liquid interface (ALI) cell cultures derived from a ciliopathy patient. Code The ciliopathies are associated with a deficiency of the CCDC40 protein and / or a deficiency of the CCDC39 protein; Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein the ciliopathies is primary ciliary dysfunction (PCD).

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the presence or absence of inflammation in the respiratory system of a subject suffering from a ciliopathic disease is determined by analyzing a blood sample and / or by analyzing the amount of exhaled nitric oxide.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein administration of the pharmaceutical composition comprises administration using a nasal spray and / or a nebulizer and / or administration by inhalation.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is administered at least once per week and / or for at least 4 weeks.

6. The pharmaceutical composition according to any one of claims 1 to 5, further comprising N-acetylcysteine ​​(NAC) and / or a hypertonic solution containing sodium chloride.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition further comprises a polyribonucleotide encoding a multicilitiate differentiation and DNA synthesis associated cell cycle (MCIDAS) protein, and / or the pharmaceutical composition is a first pharmaceutical composition administered together with a second pharmaceutical composition comprising a polyribonucleotide encoding a MCIDAS protein.

8. The pharmaceutical composition comprises a compound represented by formula (IV): 【Chemistry 1】 [wherein the variables a, b, p, m, n and R １ ~R ６ is defined as follows: a is 1 and b is an integer from 2 to 4; or a is an integer from 2 to 4 and b is 1, p is 1 or 2; m is 1 or 2; n is 0 or 1 and m+n is ≧2; and R １ ~R ６ are each independently hydrogen; a group -CH ２ -CH(OH)-R ７ , -CH(R ７ )-CH ２ -OH, -CH ２ -CH ２ -(C=O)-OR ７ , -CH ２ -CH ２ -(C=O)-NH-R ７ or -CH ２ -R ７ wherein R ７ is a C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; a protecting group for an amino group; -C(NH)-NH ２ a polyethylene glycol chain; and a receptor ligand; where R １ ~R ６ At least two of the residues are —CH ２ -CH(OH)-R ７ , -CH(R ７ )-CH ２ -OH, -CH ２ -CH ２ -(C=O)-OR ７ , -CH ２ -CH ２ -(C=O)-NH-R ７ or -CH ２ -R ７ Wherein R ７ is selected from C3-C18 alkyl or C3-C18 alkenyl having one C-C double bond; and wherein one or more of the nitrogen atoms of formula (IV) may be protonated to provide a cationic lipidoid of formula (IV). and further comprising a lipidoid having the structure shown in The lipidoid has the formula (V): 【Chemistry 2】 having the structure shown in The pharmaceutical composition according to any one of claims 1 to 7.

9. a polyribonucleotide encoding a functional version of a protein whose deficiency is associated with a ciliopathies, and N-acetylcysteine ​​(NAC), a hypertonic solution comprising sodium chloride, and / or a formulation selected from the following: a) a compound represented by formula (VI) 【Chemistry 3】 a formulation of the R-isomer of C12-(2-3-2) of the formula with DPPC, cholesterol, and the PEG lipid DMG-PEG2000; or b) Formula (VII) 【Chemistry 4】 A formulation of the R-isomer of C12-(2-3-2) of the compound of formulating with lipids of DPPC, cholesterol, and the PEG lipid DMG-PEG2000.

1. A pharmaceutical composition for use in the treatment of a ciliopathic disease in a subject suffering from a ciliopathic disease, comprising: said polyribonucleotide is an mRNA that can be translated into a functional version of a CCDC39 protein and / or a CCDC40 protein, The polyribonucleotide may be: a) a functional version of the human CCDC40 protein, wherein the polyribonucleotide encodes a functional version of the human CCDC40 protein that is at least 90% identical to one or more of SEQ ID NOs: 1 or 5-11, and wherein the protein encoded by the polyribonucleotide is capable of restoring ciliary motility in air-liquid interface (ALI) cell cultures derived from a ciliopathy patient; or b) a functional version of the human CCDC39 protein, wherein the polyribonucleotide encodes a functional version of the human CCDC39 protein that is at least 90% identical to one or more of SEQ ID NOs: 2 or 12-14, and wherein the protein encoded by the polyribonucleotide is capable of restoring ciliary motility in air-liquid interface (ALI) cell cultures derived from a ciliopathy patient. Code The ciliopathies are associated with a deficiency of the CCDC40 protein and / or a deficiency of the CCDC39 protein; Pharmaceutical compositions.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the pharmaceutical composition is administered to the respiratory system of a subject after the subject has inhaled a suitable solution or has irrigated their nasal passages and / or paranasal sinuses with a suitable solution to remove mucus and potentially sloughed airway epithelial cells.

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