Lipid formulations containing nucleic acids and methods for treating cystic fibrosis

Abstract

The present invention provides lipid formulations that contain messenger RNA (mRNA). The mRNA can be used to express CFTR protein in vitro or in vivo. The lipid formulations are typically lipid nanoparticles that contain a mixture of cationic lipids, such as DSPC and DOTAP, cholesterol, and PEGylated lipids. Furthermore, the lipid formulations can be administered by inhalation to treat cystic fibrosis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 275,402, filed November 3, 2021, which is incorporated by reference in its entirety herein.

[0002] The present disclosure relates to lipid formulations, mRNA sequences, compositions and methods for delivery of mRNA to treat cystic fibrosis. More specifically, disclosed herein are lipid formulations for delivering mRNA sequences to express the cystic fibrosis transmembrane conductance regulator (CFTR) protein or a fragment thereof in the lungs of a subject.

[0003] Sequence Listing Reference This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 3, 2022, is entitled "049386-547001WO_ST.26_SL" and is 657,256 bytes in size. [Background technology]

[0004] Cystic fibrosis (CF) is an autosomal inherited disorder caused by mutations in the CFTR gene, which encodes a chloride ion channel that is believed to be involved in regulating several ion channels and transport systems other than chloride ion channels in epithelial cells. The CFTR protein helps maintain salt and water balance on many surfaces in the body, such as the surface of the lungs. When the protein is not expressed or functions properly, chloride becomes trapped inside the cells. When chloride does not move properly, water cannot hydrate the cell surface. The mucus that coats the cells then becomes thick and sticky, causing many of the symptoms associated with cystic fibrosis. When harmful mutations are present in the CFTR gene, the corresponding loss of function of the CFTR gene causes chronic lung disease, abnormal mucus production, and a dramatically shortened life expectancy.

[0005] Currently, CF cannot be cured, but there are several treatments aimed at alleviating the adverse effects of CF, and management of CF has improved significantly over the years. Seventy years ago, infants born with CF were unlikely to survive beyond the age of one year, but today they can live well into adulthood. The current standard of care for CF patients includes proactively treating airway infections and inflammation to maximize organ function and improve the patient's quality of life. However, the best possible outcome with currently available treatments is to slow the decline of organ function.

[0006] Several gene therapy approaches have been proposed as a means of treating CF, but each approach is associated with unwanted effects or significant challenges. For example, despite the successful cloning of the CFTR gene in 1989, attempts to induce CFTR expression in the lung have faced numerous challenges. Some of these past attempts included viral vectors containing CFTR DNA, which, after administration, elicited an immune response and persisted CF symptoms.

[0007] One potential treatment involves the delivery of mRNA encoding the CFTR protein to the lung epithelium of CF patients. However, mRNA-based therapies face several obstacles, including achieving sufficient in vivo half-life of the mRNA, achieving sufficient translation efficiency of the mRNA so that an effective amount of the enzyme is produced, minimizing adverse reactions to the mRNA (e.g., immunogenicity), and effectively delivering the mRNA to the target cell type. Another difficulty in inducing CFTR expression in the lungs of a subject patient relates to the pulmonary environment. Lung-specific challenges have been reported when delivering mRNA using certain lipoplex formulations. For example, a comparison of the in vitro and in vivo performance of lipoplexes bearing mRNA or DNA revealed that although mRNA compositions showed higher expression in cultured cells, only DNA compositions showed measurable expression when administered intranasally to the lungs of mice (Andries et al., Mol. Pharmaceut. 9, 2136-45, 2012).

[0008] Furthermore, CFTR is a large gene compared to model or reporter genes, such as firefly luciferase (FFL), that are commonly used in proof-of-concept studies of mRNA-based therapeutics. Studies of the effect of the length of the coding sequence, comparing wild-type CFTR with FFL, confirmed that the difference in length can affect stability and whether and how much protein is expressed with any given amount of mRNA. Furthermore, it can be difficult to generate large mRNA for therapeutic use. In general, in vitro synthesis of mRNA is preferred over cellular synthesis, since it is free of normal cellular mRNA and other cellular components that constitute undesirable contaminants. However, in vitro synthesis of mRNA with a long coding sequence, such as CFTR mRNA, is substantially more difficult to achieve than in vitro synthesis of mRNA with a relatively short coding sequence, since longer sequences increase the chance of transcription errors and the formation of undesirable by-products.

[0009] Another challenge with mRNA-based therapy is related to the effective, specific and non-toxic delivery of mRNA to target cells.One of the methods that has been successfully adopted to deliver nucleic acid to target cells is to encapsulate nucleic acid in lipid formulations, such as liposomes or lipid nanoparticles.Although lipid formulations have been used with some success, some of the lipids used in these formulations have been found to show poor in vivo degradability, poor efficacy and the possibility of causing adverse reactions.

[0010] In view of the above identified challenges, a need remains for improved agents, formulations, methods of making and delivering CFTR mRNA to induce expression of CFTR in the treatment of CF.

[0011] U.S. Patent Application No. 17 / 246,558, filed April 30, 2021, the entire contents of which are incorporated herein by reference in their entirety, discloses mRNA sequences, compositions and methods for treating cystic fibrosis. Summary of the Invention

[0012] Additional features and advantages of the subject technology will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the subject technology. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology.

[0013] In a general aspect, the present invention discloses a method for producing a medicament for the treatment of a cancer, comprising: a. i. about 20 mol% to about 30 mol% of an ionizable cationic lipid having the structure of ATX-012 (lipid 3) below, [ka] ii. about 20 mol% to about 30 mol% of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); iii. about 7 mol% to about 13 mol% of helper lipid; iv. cholesterol of about 33 mol% to about 44 mol%; v. A lipid formulation comprising about 0.5 mol% to about 3.0 mol% of a PEG-lipid conjugate; b. messenger RNA (mRNA) encoding a peptide having cystic fibrosis transmembrane conductance regulator (CFTR) activity; wherein the mRNA is encapsulated in the lipid formulation.

[0014] In further aspects, the lipid formulation of the composition can be selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymer-based carriers, exosomes, lamellar bodies, micelles, and emulsions. In further aspects, the lipid formulation of the composition can be a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. In further aspects, the lipid formulation of the composition can be a lipid nanoparticle. In more detailed aspects, the lipid nanoparticles can be less than about 200 nm in size (diameter). In further aspects, the lipid nanoparticles can be less than about 150 nm in size (diameter). In further aspects, the lipid nanoparticles can be less than about 100 nm in size (diameter). In further aspects, the lipid nanoparticles can be between about 55 nm and about 90 nm in size (diameter).

[0015] In a further embodiment, the helper lipid of the composition can be a phospholipid. In a further embodiment, the helper lipid of the composition can be selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC) and phosphatidylcholine (PC). In a more detailed embodiment, the helper lipid of the composition can be distearoylphosphatidylcholine (DSPC).

[0016] In further embodiments, the lipid formulation of the composition can include from about 8 mol% to about 12 mol% of the helper lipid.In further embodiments, the lipid formulation of the composition can include from about 9 mol% to about 11 mol% of the helper lipid.

[0017] In a further embodiment, the PEG-lipid conjugate of the composition can be PEG-DMG. In a further embodiment, the PEG-DMG can be PEG2000-DMG.

[0018] In further embodiments, the lipid formulation of the composition can comprise from about 0.75 mol% to about 2.5 mol% of the PEG-lipid conjugate. In further embodiments, the lipid formulation of the composition can comprise from about 1.0 mol% to about 2.0 mol% of the PEG-lipid conjugate. In more detailed embodiments, the lipid formulation of the composition can comprise from about 1.25 mol% to about 1.75 mol% of the PEG-lipid conjugate.

[0019] In further embodiments, the composition can have a total lipid:mRNA weight ratio of about 5:1 to about 25:1. In further embodiments, the composition can have a total lipid:mRNA weight ratio of about 10:1 to about 20:1. In further embodiments, the composition can have a total lipid:mRNA weight ratio of about 12:1 to about 18:1. In more detailed embodiments, the composition can have a total lipid:mRNA weight ratio of about 14:1 to about 17:1.

[0020] In further embodiments, the lipid formulation of the composition may comprise from about 22 mol% to about 28 mol% of the ionizable cationic lipid. In further embodiments, the lipid formulation of the composition may comprise from about 23 mol% to about 27 mol% of the ionizable cationic lipid. In further embodiments, the lipid formulation of the composition may comprise from about 24 mol% to about 26 mol% of the ionizable cationic lipid.

[0021] In further embodiments, the lipid formulation of the composition can include about 22 mol% to about 28 mol% DOTAP. In further embodiments, the lipid formulation of the composition can include about 23 mol% to about 27 mol% DOTAP. In more detailed embodiments, the lipid formulation can include about 24 mol% to about 26 mol% DOTAP.

[0022] In a further embodiment, the lipid formulation of the composition can include about 35 mol% to about 41 mol% cholesterol.In a further embodiment, the lipid formulation of the composition can include about 36 mol% to about 40 mol% cholesterol.

[0023] In further embodiments, the peptide of the composition having CFTR activity can have a sequence at least about 85% identical to the sequence of SEQ ID NO:99. In further embodiments, the peptide having CFTR activity can have a sequence at least about 90% identical to the sequence of SEQ ID NO:99. In further embodiments, the peptide having CFTR activity can have a sequence at least about 95% identical to the sequence of SEQ ID NO:99. In further embodiments, the peptide having CFTR activity can have a sequence at least about 98% identical to the sequence of SEQ ID NO:99. In more particular embodiments, the peptide having CFTR activity can have a sequence at least about 99% identical to the sequence of SEQ ID NO:99. In even more particular embodiments, the peptide having CFTR activity can have a sequence of SEQ ID NO:99.

[0024] In a further aspect, the mRNA of the composition can have a sequence selected from the group consisting of SEQ ID NO: 49, 53, 66, 68, 69, and 72. In one further aspect, the mRNA can comprise SEQ ID NO: 49. In another further aspect, the mRNA can comprise SEQ ID NO: 53. In another further aspect, the mRNA can comprise SEQ ID NO: 66. In another further aspect, the mRNA can comprise SEQ ID NO: 68. In yet another further aspect, the mRNA can comprise SEQ ID NO: 69. In yet another further aspect, the mRNA can comprise SEQ ID NO: 72.

[0025] In a further embodiment, the mRNA of the composition can include a 3' polyA tail of about 50 to about 120 adenosine monomers.

[0026] In a further embodiment, the mRNA of the composition can include a 5' cap. In a further embodiment, the 5' cap has the structure of the following formula (Cap V): 7 GpppAmpG, [ka] In the formula, R 1 , R 2 and R 4 each is OH, n is 1, each L is a phosphate linked by a diester bond, and mRNA is an mRNA of that composition.

[0027] In a further embodiment, the mRNA of the composition comprises, independently, 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fulvenocytidine, 2'-amino-5-methylur ... Oro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N 1 -Hydroxypseudouridine, N 1 -Methylpseudouridine, 2'-O-methyl-N 1 -Methylpseudouridine, N 1 -Ethylpseudouridine, N 1 -Hydroxymethylpseudouridine, aurauridine, N 6 In a further embodiment, the one or more chemically modified nucleotides may include one or more chemically modified nucleotides selected from the group consisting of N-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, and 6-O-methylguanosine. 1 -methylpseudouridine.

[0028] In a further embodiment, the composition may comprise a HEPES or TRIS buffer having a pH of about 7.0 to about 8.5. In a further embodiment, the pH of the HEPES or TRIS buffer is about 7.4 to about 8.2. In another embodiment, the HEPES or TRIS buffer may have a concentration of about 20 mM to about 80 mM. In one particular embodiment, the buffer may have a HEPES concentration of about 35 mM to about 70 mM. In a more detailed embodiment, the buffer may have a HEPES concentration of about 40 mM to about 60 mM. In yet a more detailed embodiment, the buffer may have a HEPES concentration of about 45 mM to about 55 mM. In another particular embodiment, the buffer may have a TRIS concentration of about 20 mM to about 50 mM. In a more detailed embodiment, the buffer may have a TRIS concentration of about 25 mM to about 40 mM. In still more particular embodiments, the buffer can be TRIS at a concentration of about 25 mM to about 35 mM.

[0029] In a further embodiment, the composition can further comprise about 10 mM to about 100 mM NaCl. In a further embodiment, the composition can comprise about 20 mM to about 90 mM NaCl. In a further embodiment, the composition can comprise about 30 mM to about 80 mM NaCl. In a further embodiment, the composition can comprise about 35 mM to about 70 mM NaCl. In a more detailed embodiment, the composition can comprise about 40 mM to about 60 mM NaCl. In a further more detailed embodiment, the composition can comprise about 45 mM to about 55 mM NaCl.

[0030] In a further embodiment, the composition can further comprise one or more cryoprotectants. In a further embodiment, the one or more cryoprotectants of the composition can be selected from the group consisting of sucrose, glycerol, and a combination of sucrose and glycerol. In one embodiment, the cryoprotectant can be sucrose. In another embodiment, the cryoprotectant can be glycerol. In yet another embodiment, the cryoprotectant can be a combination of sucrose and glycerol. In a further embodiment, the composition can comprise a combination of sucrose at a concentration of about 5% (w / v) to about 18% (w / v) and glycerol at a concentration of about 1% (w / v) to about 9% (w / v). In a further embodiment, the composition can comprise a combination of sucrose at a concentration of about 6% (w / v) to about 16% (w / v) and glycerol at a concentration of about 1.5% (w / v) to about 7% (w / v). In further embodiments, the composition can include a combination of sucrose at a concentration of about 7% (w / v) to about 14% (w / v) and glycerol at a concentration of about 1.75% (w / v) to about 6% (w / v). In more specific embodiments, the composition can include a combination of sucrose at a concentration of about 7% (w / v) to about 12% (w / v) and glycerol at a concentration of about 1% (w / v) to about 6% (w / v). In yet more specific embodiments, the composition can include a combination of sucrose at a concentration of about 8% (w / v) to about 11% (w / v) and glycerol at a concentration of about 3% (w / v) to about 6% (w / v).

[0031] In a further embodiment, the helper lipid of the composition can be distearoylphosphatidylcholine (DSPC), the PEG-lipid conjugate of the composition can be PEG2000-DMG, and the mRNA of the composition can comprise SEQ ID NO:53. In a further embodiment, the peptide of the composition having CTFR activity can have a sequence at least about 90% identical to the sequence of SEQ ID NO:99. In a further embodiment, the composition can have a total lipid:mRNA weight ratio of about 15:1. In a further embodiment, the lipid formulation of the composition can be a lipid nanoparticle. In a further embodiment, the lipid nanoparticle can be less than about 100 nm in size. In a further embodiment, the lipid formulation of the composition comprises about 25 mol% ATX-012, about 25 mol% DOTAP, about 10 mol% DSPC, about 38.5 mol% cholesterol, and about 1.5 mol% PEG2000-DMG.

[0032] In another general aspect, the present invention discloses the use of a composition of the present disclosure to make a medicament for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with decreased activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in a subject in need thereof. In a further aspect, the disease can be cystic fibrosis with a cystic fibrosis mutation selected from the group consisting of class 1A, class 1B, class 3, class 4, class 5, and class 6. In one aspect, the cystic fibrosis mutation can be class 1A. In another aspect, the cystic fibrosis mutation can be class 1B. In another aspect, the cystic fibrosis mutation can be class 3. In another aspect, the cystic fibrosis mutation can be class 4. In another aspect, the cystic fibrosis mutation can be class 5. In yet another aspect, the cystic fibrosis mutation is class 6.

[0033] In another general aspect, the present invention discloses a method of ameliorating, preventing, delaying the onset of, or treating a disease or disorder associated with decreased activity of the cystic fibrosis transmembrane conductance regulator (CFTR) in a subject in need thereof, comprising administering to the subject a composition of the present disclosure. In a further aspect, the disease can be cystic fibrosis. In a further aspect, the administration can be intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, nasal, or inhalation. In another further aspect, the administration can be intranasal or inhalation. In a more detailed aspect, the administration can be inhalation. In another aspect, the administration can be once a day, once a week, once every two weeks, or once a month. In a further aspect, the administration can comprise administering an effective dose of about 0.01 to about 10 mg / kg of the mRNA in the composition. In another embodiment, the administration can increase expression of CFTR in the lung epithelium.

[0034] In another general aspect, the invention discloses a method of expressing a CFTR protein in a cell, the method comprising contacting the cell with a composition of the disclosure.

[0035] In another general aspect, the present invention discloses a kit for expressing human CFTR in vivo, comprising a composition of the present disclosure and a device for administering a dose thereof. In a further aspect, the device can be an injection needle, an intravenous needle, or an inhalation device. In a more specific aspect, the device can be an inhalation device.

[0036] Various features of exemplary embodiments of the present disclosure are described below with reference to the drawings, in which the illustrated embodiments are intended to illustrate, but not to limit, the present disclosure. [Brief description of the drawings]

[0037] [Figure 1]FIG. 1 shows the correlation of hCFTR protein expression levels for various hCFTR constructs, as determined by In-Cell Western (ICW) and On-Cell Western (OCW) using a human CFTR antibody for codon-optimized sequences, as described in Example 3. [Diagram 2] 1 shows the expression levels of UTR-optimized hCFTR mRNA sequences measured by ICW using an hCFTR-specific antibody 24 and 48 hours after transfection as described in Example 4. [Diagram 3] FIG. 1 shows C-band levels of CFTR protein (fully mature and fully glycosylated) expressed in vitro by various codon-optimized hCFTR mRNAs as analyzed by Western blot (WB) as shown in Example 5. [Figure 4] Expression levels measured by quantitating C bands using Western blot as described in Example 5 are shown. [Diagram 5] FIG. 1 shows the expression levels of hCFTR-specific bands in cytoplasmic (Cyto) and membrane (Mb) fractions collected from cells transfected with hCFTR mRNA as described in Example 6, analyzed by Western blot (WB) using primary antibodies specific for hCFTR and the plasma membrane (sodium-potassium-ATPase). [Figure 6] FIG. 1 shows confocal immunofluorescence images of CFBE cells transfected with codon-optimized hCFTR mRNA (SEQ ID NO:53) as described in Example 7 and treated with an antibody specific for the hCFTR protein for immunofluorescence. [Figure 7] 1 shows the dose response for protein expression of various hCFTR mRNAs in transfected FRT cells as described in Example 8. [Figure 8]Figure 1 shows the transfection efficiency in FRT cells transfected with mCherry mRNA as described in Example 9. The top panel shows transfected (mCherry) cells and the bottom panel shows non-transfected cells. [Figure 9] As shown in Example 10, measurements of ion channel conductance (Gt values) are shown in FRT cells transfected with various mRNAs and negative controls. [Figure 10] As shown in Example 10, measurements of ion channel conductance (Gt values) are shown in FRT cells transfected with various mRNAs and negative controls. [Figure 11] As shown in Example 10, measurements of ion channel conductance (Gt values) are shown in FRT cells transfected with various mRNAs and negative controls. [Figure 12] As shown in Example 10, measurements of ion channel conductance (Gt values) are shown in FRT cells transfected with various mRNAs and negative controls. [Figure 13] AB show the immunostimulatory levels of IFN-α of mRNA formulated in the indicated lipids for (A) donor 1 and (B) donor 2, as described in Example 11. [Figure 14] AB show the immunostimulatory levels for IL-6 of mRNA formulated in the indicated lipids for (A) donor 1 and (B) donor 2, as described in Example 11. [Figure 15] AB show the immunostimulatory levels of TNF-α from mRNA formulated in the indicated lipids for (A) donor 1 and (B) donor 2, as described in Example 11. [Figure 16]1 shows quantitative PCR (qPCR) measurements of mRNA levels in CF sputum incubated for 24 hours for given hCFTR mRNA-lipid formulations as described in Example 12. [Figure 17] 1 shows luminescence images of luciferase mRNA formulated in lipids administered intratracheally (upper panel) and via nasal nebulization only (lower panel) to wild-type rats as described in Example 13. [Figure 18] Shown are eGFP immunohistochemistry images of PBS controls as compared to animals treated with eGFP mRNA formulated in lipid as described in Example 14. [Figure 19] 1 shows eGFP immunohistochemistry images of animals treated with eGFP mRNA formulated in lipids as described in Example 14. [Figure 20] 1 shows TdTomato (TdT) fluorescence images of lung samples excised from transgenic floxed TdTomato mice following administration of a CRE mRNA-lipid formulation as described in Example 15. [Figure 21] Shown are fluorescent images of lung samples excised from floxed TdTomato mice following administration of a CRE mRNA-lipid formulation and further processed with FoxJ1 and DAPI stains as described in Example 16. The bottom panel shows a high-resolution image of colocalization of TdT and FoxJ1. [Figure 22]A-D show nasal epithelial cell profiling by fluorescence imaging of samples excised from floxed-TdTomato mice following administration of a CRE mRNA-lipid formulation and further processed with FoxJ1 and DAPI stains as described in Example 17. (A) Panoramic image of the nasal septum. (B) High-resolution image of the area indicated by the dotted rectangle in A. (C) High-resolution image of the area indicated by the dotted rectangle in A. (D) Quantitative plot of cell numbers of total TdTomato-expressing cells (TdT+) and cells expressing both TdTomato and FoxJ1 (FoxJ1+ / TdT+). [Diagram 23] FIG. 13 shows the results of fluorescent imaging of lung samples removed from Floxed-TdTomato mice after administration of a given CRE mRNA-lipid formulation and further processed with FoxJ1 and DAPI stains, as described in Example 18. [Figure 24] 1 shows mRNA levels over time as quantified by Quantigene® Assay in CFTR knockout (KO) mice treated intratracheally with various dose levels of lipid-formulated hCFTR mRNA as described in Example 19. [Diagram 25] FIG. 1 shows hCFTR protein levels in membrane (Mb) and cytoplasmic (Cyt) fractions analyzed by WB using an hCFTR-specific antibody in CFTR knockout (KO) mice treated intratracheally with various dose levels of lipid-formulated hCFTR mRNA as described in Example 20. [Figure 26] 2 shows hCFTR mRNA levels quantified by Quantigene® Assay in samples taken from rats 6 or 24 hours after exposure at different exposure lengths as described in Example 21. [Figure 27]1 shows hCFTR mRNA levels quantified by Quantigene® Assay in nasal epithelium samples from CFTR KO mice treated with lipid-formulated hCFTR mRNA as described in Example 22, 6 hours, 40 hours, and 60 hours after the last dose. [Figure 28] 1 shows chloride channel currents measured by nasal potential difference (NPD) in CFTR KO mice treated with lipid-formulated hCFTR mRNA 40 and 60 hours after the last dose as described in Example 22. [Figure 29] 1 shows chloride channel currents measured by intranasal potential difference in CFTR KO mice treated with various hCFTR mRNA-lipid formulations 40 and 60 hours after the last dose, as described in Example 23. [Diagram 30] 2 shows the average droplet size measurements of aerosolized lipid particles as described in Example 24. [Diagram 31] 2 shows the mRNA encapsulation rate as measured by RiboGreen assay, both before and after nebulization, for various lots of mRNA-lipid formulations as described in Example 25. [Diagram 32] 1 shows mRNA recovery as measured by RiboGreen assay in lipid-formulated mRNA both before and after nebulization as described in Example 25. [Diagram 33] 2 shows eGFP fluorescence levels before and after nebulization of lipid-formulated eGFP mRNA used to transfect CFBE cells as described in Example 26. [Diagram 34] FIG. 1 shows eGFP fluorescence levels of lipid-formulated eGFP mRNA used to transfect CFBE cells at different doses before and after nebulization using a vibrating mesh nebulizer as described in Example 27. [Diagram 35]FIG. 14 shows eGFP protein quantification results for three different dose levels of an eGFP mRNA-lipid formulation (LF-1) administered to lung tissue from non-CF subjects and processed for WB and analyzed for eGFP expression 24 hours after incubation, as described in Example 28. [Diagram 36] FIG. 14 shows eGFP protein quantification results for three different dose levels of eGFP mRNA-lipid formulation (LF-2) administered to lung tissue from non-CF subjects and processed for WB and analyzed for eGFP expression 24 hours after incubation, as described in Example 28. [Figure 37] FIG. 14 shows eGFP protein quantification results for three different dose levels of an eGFP mRNA-lipid formulation (LF-3) administered to lung tissue from non-CF subjects and processed for WB and analyzed for eGFP expression 24 hours after incubation, as described in Example 28. [Figure 38] FIG. 14 shows eGFP protein quantification results for three different dose levels of eGFP mRNA-lipid formulation (LF-1) administered to lung tissue from a subject with CF and processed for WB and analyzed for eGFP expression 24 hours after incubation, as described in Example 29. [Figure 39] FIG. 14 shows eGFP protein quantification results for three different dose levels of eGFP mRNA-lipid formulation (LF-2) administered to lung tissue from a subject with CF and processed for WB and analyzed for eGFP expression 24 hours after incubation, as described in Example 29. [Diagram 40] FIG. 14 shows eGFP protein quantification results for three different dose levels of eGFP mRNA-lipid formulation (LF-3) administered to lung tissue from a subject with CF and processed for WB and analyzed for eGFP expression 24 hours after incubation, as described in Example 29. [Diagram 41]1 shows hCFTR expression levels of a given mRNA, a reference mRNA, and a comparative mRNA transfected into CFBE cells at increasing dose levels as described in Example 30. [Diagram 42] A-D show that lipid-formulated mRNA was delivered to ferret lung epithelial cells as described in Example 31. (A) Expression of eGFP shows that CRE mRNA was clearly delivered to epithelial cells in ferrets treated with CRE mRNA-lipid formulation (bright staining around airways). (B) Expression of eGFP shows that CRE mRNA was clearly delivered to epithelial cells in ferrets treated with CRE mRNA-lipid formulation (bright staining around airways). (C) Expression of eGFP shows that CRE mRNA was clearly delivered to epithelial cells in ferrets treated with CRE mRNA-lipid formulation (bright staining around airways). (D) In ​​untreated controls, only TdTomato expression was observed due to the lack of CRE recombination. [Diagram 43] A-D show lipid-formulated mRNA delivered to non-human primate (NHP) lung epithelial cells as described in Example 32. (A) In NHPs treated with lipid-formulated TdTomato mRNA, mRNA was clearly delivered to ciliated cells in the epithelial airways as seen by intense staining of the cells lining the airways. (B) In NHPs treated with lipid-formulated TdTomato mRNA, mRNA was clearly delivered to ciliated cells in the epithelial airways as seen by intense staining of the cells lining the airways. (C) In NHPs treated with lipid-formulated TdTomato mRNA, mRNA was clearly delivered to ciliated cells in the epithelial airways as seen by intense staining of the cells lining the airways. (D) In ​​control PBS-treated NHPs, no TdTomato expression was observed. [Diagram 44]As described in Example 33, lipid-formulated mRNA was shown to be delivered to ciliated epithelial cells of the ferret lung. [Diagram 45] As described in Example 34, results are shown for intranasal administration of LNP-hCFTR mRNA in a class I CFTR knockout (KO) mouse model. [Diagram 46] The effect of a single dose of LNP-hCFTR mRNA compared to multiple doses as described in Example 35 is shown. [Figure 47] As described in Example 36, we demonstrate that LNP-hCFTR was delivered to ferret bronchial epithelial (FBE) cells harboring the CFTR G551D mutation. [Figure 48A] 13 is an immunocytological study showing that LNP-mRNA was delivered to human bronchial epithelial (HBE) cells, as described in Example 37. [Figure 48B] 13 shows that LNP-mRNA was delivered to human bronchial epithelial (HBE) cells, as described in Example 37. Quantitative results of immunocytological studies. [Figure 49A] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 39. Cell viability in CFBE cells. [Figure 49B] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 39. Expression of Tdtomato in CFBE cells. [Figure 49C] 1 shows the results of in vivo delivery of LNP-mRNA as described in Example 39. Immunohistochemistry images of TdTomato in mouse lungs. [Figure 50A] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 41. Cell viability in CFBE cells after transfection. [Figure 50B]1 shows the results of in vitro delivery of LNP-mRNA as described in Example 41. Expression of Tdtomato in CFBE cells after transfection. [Figure 50C] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 41. Cell viability in CFBE cells after transfection. [Figure 50D] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 41. Expression of Tdtomato in CFBE cells after transfection. [Figure 50E] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 41. Cell viability in CFBE cells after transfection. [Figure 50F] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 41. Expression of Tdtomato in CFBE cells after transfection. [Figure 50G] 1 shows the results of in vivo delivery of LNP-mRNA as described in Example 41. tdTomato immunohistochemistry images of mouse lungs. [Figure 51A] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 42. Cell viability in CFBE cells after transfection. [Figure 51B] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 42. Expression of Tdtomato in CFBE cells after transfection. [Figure 51C] 13 shows the results of in vivo delivery of LNP-mRNA as described in Example 42. 14 shows tdTomato immunohistochemistry images of mouse lungs. [Fig. 51D]1 shows the results of in vitro delivery of LNP-mRNA as described in Example 42. Cell viability in CFBE cells after transfection. [Figure 51E] 1 shows the results of in vitro delivery of LNP-mRNA as described in Example 42. Expression of Tdtomato in CFBE cells after transfection. [Fig. 51F] 13 shows the results of in vivo delivery of LNP-mRNA as described in Example 42. 14 shows tdTomato immunohistochemistry images of mouse lungs. [Figure 52A] FIG. 13 shows characterization of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. Cell viability in CFBE cells after transfection. [Figure 52B] 13 shows the characterization of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. Expression of Tdtomato in CFBE cells after transfection. [Figure 52C] FIG. 13 shows characterization of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. Cell viability in CFBE cells after transfection. [Fig. 52D] 13 shows the characterization of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. Expression of Tdtomato in CFBE cells after transfection. [Figure 52E] FIG. 13 shows the characterization of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. Particle size assessment of various concentrations after long-term storage at −70° C. or −20° C. [Fig. 52F] FIG. 13 shows the characterization of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. Particle size assessment of various concentrations after long-term storage at −70° C. or −20° C. [Fig. 52G]FIG. 13 shows the characteristics of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. FIG. 14 shows particle size evaluation results for formulations with various storage buffers as shown in Table 32. [Fig. 52H] FIG. 13 shows the characteristics of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. FIG. 14 shows particle size evaluation results for formulations with various storage buffers as shown in Table 32. [Fig. 52I] FIG. 13 shows the characteristics of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. FIG. 14 shows particle size evaluation results for formulations with various storage buffers as shown in Table 32. [Fig. 52J] 13 shows the characteristics of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. Table 32 shows the evaluation of mRNA purity of formulations upon storage at RT. [Figure 52K] FIG. 13 shows the characteristics of lipid nanoparticle formulations prepared with various buffer components as described in Example 44. FIG. 14 shows the pH evaluation results of the formulations stored at RT for the formulations shown in Table 32. [Figure 53A] FIG. 1 shows parameters of lipid nanoparticle formulations after storage under various conditions as shown in Example 45. pH after storage at room temperature. [Figure 53B] FIG. 1 shows parameters of lipid nanoparticle formulations after storage under various conditions as shown in Example 45. Particle size after storage at −20° C. [Figure 53C] FIG. 1 shows parameters of lipid nanoparticle formulations after storage under various conditions as shown in Example 45. mRNA purity after storage at room temperature. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] It will be understood that from the present disclosure, various configurations of the subject technology will be readily apparent to those skilled in the art, and various configurations of the subject technology have been shown and described by way of example. As will be understood, the subject technology is capable of other and different configurations, and its several details are capable of modification in various other respects, all without departing from the scope of the subject technology. Accordingly, the summary, drawings, and detailed description are to be regarded as illustrative in nature, and not as limiting.

[0039] The detailed description set forth below is intended as a description of various configurations of the subject technology, and is not intended to show the only configurations in which the subject technology may be practiced. The accompanying drawings are incorporated in this specification and form a part of the detailed description. The detailed description includes specific details to provide a thorough understanding of the subject technology. However, it will be apparent to those skilled in the art that the subject technology may be practiced without these specific details. In some cases, well-known structures and components are shown in block diagram form to avoid obscuring the concepts of the subject technology. For ease of understanding, similar components are labeled with the same element numbers.

[0040] In some embodiments, an mRNA is provided that encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein, the mRNA comprising an open reading frame (ORF) that has about 80% sequence identity to one of SEQ ID NOs: 100-105. In some embodiments, the ORF has about 85% sequence identity to one of SEQ ID NOs: 100-105. In some embodiments, the ORF has about 90% sequence identity to one of SEQ ID NOs: 100-105. In some embodiments, the ORF has about 95% sequence identity to one of SEQ ID NOs: 100-105. In some embodiments, the ORF has about 96% sequence identity to one of SEQ ID NOs: 100-105. In some embodiments, the ORF has about 97% sequence identity to one of SEQ ID NOs: 100-105. In some embodiments, the ORF has about 98% sequence identity to one of SEQ ID NOs: 100-105. In some embodiments, the ORF has about 99% sequence identity to one of SEQ ID NOs: 100-105. In some embodiments, the ORF has a sequence selected from the group consisting of SEQ ID NOs: 100-105. In some embodiments, the ORF has a sequence of SEQ ID NO: 100. In some embodiments, the ORF has a sequence of SEQ ID NO: 101. In some embodiments, the ORF has a sequence of SEQ ID NO: 102. In some embodiments, the ORF has a sequence of SEQ ID NO: 103. In some embodiments, the ORF has a sequence of SEQ ID NO: 104. In some embodiments, the ORF has a sequence of SEQ ID NO: 105.

[0041] In some embodiments, the mRNA further comprises a 5' untranslated region (5'UTR). In some embodiments, the 5'UTR comprises a sequence selected from SEQ ID NOs: 106-125. In some embodiments, the 5'UTR comprises SEQ ID NO: 106.

[0042] In some embodiments, the mRNA further comprises a 3' untranslated region (3'UTR). In some embodiments, the 3'UTR comprises a sequence selected from the group consisting of SEQ ID NOs: 126-145. In some embodiments, the 3'UTR comprises SEQ ID NO: 126.

[0043] In some embodiments, the mRNA further comprises a 3' polyadenosine (poly A) tail, hi some embodiments, the 3' poly A tail consists of about 50 to about 120 adenosine monomers.

[0044] In some embodiments, the mRNA further comprises a 5' cap. In some embodiments, the 5' cap has the structure of formula cap IV disclosed herein. 7 GpppGm, where R 1 and R 2 are OH and R 3 OCH 3 wherein each L is a phosphate linked by a diester bond, the mRNA is an mRNA of the disclosure linked at its 5' end, and n is 1. In some embodiments, the 5' cap has the structure of formula cap V disclosed herein. 7 GpppAmpG, where R 1 , R 2 and R 4 each is OH, n is 1, each L is a phosphate linked by a diester bond, and the mRNA is an mRNA of the disclosure linked at its 5' end.

[0045] In some embodiments, the mRNA comprises one or more chemically modified nucleotides, which in some embodiments are each independently 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-Fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N 1 -Hydroxypseudouridine, N 1 -Methylpseudouridine, 2'-O-methyl-N 1 -Methylpseudouridine, N 1 -Ethylpseudouridine, N 1 -Hydroxymethylpseudouridine, aurauridine, N 6 In some embodiments, the one or more chemically modified nucleotides are selected from N-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, and 6-O-methylguanosine. 1 In some embodiments, the one or more chemically modified nucleotides are 5-methyluridine. In some embodiments, the one or more chemically modified nucleotides are 5-methylcytidine and N-methylpseudouridine. 1 In some embodiments, the one or more chemically modified nucleotides are 5-methoxyuridine and N-methylpseudouridine. 1In some embodiments, the one or more chemically modified nucleotides are a combination of 5-methoxyuridine, 5-methylcytidine, and N-methylpseudouridine. 1 -methylpseudouridine. In some embodiments, the one or more chemically modified nucleotides comprise 1-99% of the nucleotide. In some embodiments, the one or more chemically modified nucleotides comprise 50-99% of the nucleotide.

[0046] In some embodiments, the ORF is translatable in a mammalian cell to express a human CFTR protein having CFTR activity. In some embodiments, the ORF is translatable in vivo in a subject to express a human CFTR protein having CFTR activity.

[0047] In some embodiments, the mRNA comprises a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises SEQ ID NO: 49. In some embodiments, the mRNA comprises SEQ ID NO: 53. In some embodiments, the mRNA comprises SEQ ID NO: 66. In some embodiments, the mRNA comprises SEQ ID NO: 68. In some embodiments, the mRNA comprises SEQ ID NO: 69. In some embodiments, the mRNA comprises SEQ ID NO: 72.

[0048] In some embodiments, a pharmaceutical composition is provided comprising an mRNA of the present disclosure and a lipid of formula I, or a pharma- ceutically acceptable salt or solvate thereof, wherein R 5 and R 6 are each independently a straight or branched chain C 1- C 31 Alkyl, C 2- C 31 Alkenyl or C 2- C 31 L is selected from the group consisting of alkynyl and cholesteryl; 5 and L 6 are each independently linear C 1- C 20Alkyl and C 2- C 20 alkenyl; X 5 is -C(O)O- or -OC(O)-, and X 6 is -C(O)O- or -OC(O)-, and X 7 is S or O, and L 7 is absent or is lower alkyl, R 4 is a linear or branched chain C 1- C 6 is alkyl, R 7 and R 8 are each independently hydrogen and straight or branched chain C 1- C 6 is selected from the group consisting of alkyl.

[0049] In some embodiments, a pharmaceutical composition is provided comprising an mRNA of the present disclosure and a lipid selected from the ionizable cationic lipids specifically disclosed herein, or a pharma- ceutically acceptable salt thereof.

[0050] In some embodiments, a combination of an mRNA of the disclosure and an ionizable cationic lipid having the structure of ATX-012 below: [ka] or a pharma- ceutically acceptable salt or solvate thereof.

[0051] In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier, hi some embodiments, the carrier comprises a transfection reagent, a nanoparticle, or a liposome.

[0052] In some embodiments, the pharmaceutical composition comprises a lipid formulation. In some embodiments, the lipid formulation is selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymer-based carriers, exosomes, lamellar bodies, micelles, and emulsions. In some embodiments, the lipid formulation is a liposome. In some embodiments, the liposome is selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. In some embodiments, the lipid formulation is encapsulated with mRNA. In some embodiments, the lipid formulation is at least about 50% encapsulated with mRNA.

[0053] In some embodiments, the pharmaceutical composition comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle is loaded with mRNA. In some embodiments, the lipid nanoparticle is at least about 50% loaded with mRNA. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a helper lipid, cholesterol, and a PEG-lipid conjugate.

[0054] In some embodiments, the lipid nanoparticles are less than about 200 nm in size. In some embodiments, the lipid nanoparticles are less than about 150 nm in size. In some embodiments, the lipid nanoparticles are less than about 100 nm in size. In some embodiments, the lipid nanoparticles are less than about 90 nm in size. In some embodiments, the lipid nanoparticles are at least about 50 nM in size. In some embodiments, the lipid nanoparticles are in the range of about 50 to about 90 nm in size. In some embodiments, the lipid nanoparticles are in the range of about 55 to about 90 nm in size. In some embodiments, the lipid nanoparticles have an average particle size of about 50 to about 85 nm. In some embodiments, the lipid nanoparticles are in the range of about 55 to about 85 nm in size.

[0055] In some embodiments, the pharmaceutical composition comprises a lipid formulation, the lipid formulation comprising a cationic lipid, a helper lipid, cholesterol, and a polyethylene glycol (PEG)-lipid conjugate.

[0056] In some embodiments, the lipid formulation comprises an ionizable cationic lipid. In some embodiments, the lipid formulation comprises about 20 mol% to about 30 mol% of the ionizable cationic lipid. In some embodiments, the lipid formulation comprises about 22 mol% to about 28 mol% of the ionizable cationic lipid. In some embodiments, the lipid formulation comprises about 23 mol% to about 27 mol% of the ionizable cationic lipid. In some embodiments, the lipid formulation comprises about 24 mol% to about 26 mol% of the ionizable cationic lipid. In some embodiments, the lipid formulation comprises about 25 mol% of the ionizable cationic lipid. In some embodiments, the ionizable cationic lipid is ATX-012.

[0057] In some embodiments, the helper lipid is a phospholipid. In some embodiments, the helper lipid is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate) (DOTMA) and phosphatidylcholine (PC), or any combination of the above. In some embodiments, the helper lipid is selected from the group consisting of DOPE, DMPC, DSPC, DMPG, DPPC and PC. In some embodiments, the helper lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the helper lipid is a combination of DOTAP and DSPC.

[0058] In some embodiments, the pharmaceutical composition comprises a lipid formulation, the lipid formulation comprising about 20 mol% to about 30 mol% DOTAP. In some embodiments, the lipid formulation comprises about 22 mol% to about 28 mol% DOTAP. In some embodiments, the lipid formulation comprises about 23 mol% to about 27 mol% DOTAP. In some embodiments, the lipid formulation comprises about 24 mol% to about 26 mol% DOTAP. In some embodiments, the lipid formulation comprises about 25 mol% DOTAP.

[0059] In some further embodiments, the lipid formulation comprising DOTAP further comprises about 7 mol% to about 13 mol% of a second helper lipid. In some embodiments, the lipid formulation comprising DOTAP further comprises about 8 mol% to about 12 mol% of a second helper lipid. In some embodiments, the lipid formulation comprising DOTAP further comprises about 9 mol% to about 11 mol% of a second helper lipid. In some embodiments, the lipid formulation comprising DOTAP further comprises about 10 mol% of a second helper lipid. In some embodiments, the second helper lipid is DSPC. That is, in some embodiments, the lipid formulation comprises about 20 mol% to about 30 mol% of DOTAP and about 7 mol% to 13 mol% of DSPC. In some embodiments, the lipid formulation is encapsulated with mRNA. In some embodiments, the lipid formulation is a lipid nanoparticle formulation.

[0060] In some embodiments, the PEG-lipid conjugate is PEG-dimyristoylglycerol (PEG-DMG). In some embodiments, the PEG-DMG is PEG2000-DMG.

[0061] In some embodiments, the pharmaceutical composition comprises a lipid formulation, the lipid formulation comprising a PEG-lipid conjugate. In some embodiments, the lipid formulation comprises about 0.5 mol% to about 3.0 mol% of the PEG-lipid conjugate. In some embodiments, the lipid formulation comprises about 0.75 mol% to about 2.5 mol% of the PEG-lipid conjugate. In some embodiments, the lipid formulation comprises about 1.0 mol% to about 2.0 mol% of the PEG-lipid conjugate. In some embodiments, the lipid formulation comprises about 1.25 mol% to about 1.75 mol% of the PEG-lipid conjugate. In some embodiments, the lipid formulation comprises about 1.5 mol% of the PEG-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG. In some embodiments, the lipid formulation is encapsulated with mRNA. In some embodiments, the lipid formulation is a lipid nanoparticle formulation.

[0062] In some embodiments, the pharmaceutical composition comprises a lipid formulation, and the lipid formulation comprises cholesterol. In some embodiments, the lipid formulation comprises about 33 mol% to about 44 mol% cholesterol. In some embodiments, the lipid formulation comprises about 35 mol% to about 41 mol% cholesterol. In some embodiments, the lipid formulation comprises about 36 mol% to about 40 mol% cholesterol. In some embodiments, the lipid formulation comprises about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, or about 40 mol% cholesterol. In some embodiments, the lipid formulation comprises about 37 mol%, about 37.5 mol%, about 38 mol%, about 38.5 mol%, or about 39 mol% cholesterol. In some embodiments, the lipid formulation comprises about 38.5 mol% cholesterol. In some embodiments, the lipid formulation is encapsulated with mRNA. In some embodiments, the lipid formulation is a lipid nanoparticle formulation.

[0063] In some embodiments, the lipid nanoparticles comprise about 20 mol% to 40 mol% cationic lipid, about 25 mol% to 35 mol% helper lipid, about 25 mol% to 42 mol% cholesterol, and about 0.5 mol% to 3 mol% PEG2000-DMG.

[0064] In some embodiments, the lipid nanoparticles comprise about 20 mol% to 30 mol% cationic lipid, about 30 mol% to 40 mol% helper lipid, about 34 mol% to 42 mol% cholesterol, and about 1 mol% to 2 mol% PEG2000-DMG.

[0065] In some embodiments, the lipid nanoparticles comprise about 22 mol% to 28 mol% cationic lipid, about 31 mol% to 39 mol% helper lipid, about 35 mol% to 40 mol% cholesterol, and about 1.25 mol% to 1.75 mol% PEG2000-DMG.

[0066] In some embodiments, the lipid formulation comprises about 20 mol% to about 30 mol% of an ionizable cationic lipid, about 20 mol% to about 30 mol% of DOTAP, about 7 mol% to about 13 mol% of a second helper lipid, about 33 mol% to about 44 mol% of cholesterol, and about 0.5 mol% to about 3.0 mol% of a PEG-lipid conjugate. In some further embodiments, the ionizable cationic lipid is ATX-012 or a pharma- ceutically acceptable salt thereof. In some further embodiments, the second helper lipid is DSPC. In some further embodiments, the PEG-lipid conjugate is PEG-DMG. In some further embodiments, the PEG-DMG is PEG2000-DMG. That is, in some further embodiments, the lipid formulation may include lipid nanoparticles, and may include about 20 mol% to about 30 mol% ATX-012, about 20 mol% to about 30 mol% DOTAP, about 7 mol% to about 13 mol% DSPC, about 33 mol% to about 44 mol% cholesterol, and about 0.5 mol% to about 3.0 mol% PEG-DMG. In some embodiments, the lipid formulation may be encapsulated with mRNA. In some embodiments, the lipid formulation is a lipid nanoparticle formulation.

[0067] In some embodiments, the pharmaceutical composition comprises a lipid formulation and an mRNA. In some embodiments, the mRNA encodes a peptide having CFTR activity. In some embodiments, the lipid formulation is encapsulated with an mRNA encoding a peptide having CTFR activity. In some embodiments, the lipid formulation is a lipid nanoparticle formulation. In some embodiments, the mRNA encodes an amino acid sequence at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:99. In some embodiments, the mRNA comprises a sequence selected from the group consisting of SEQ ID NO:49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises SEQ ID NO:49. In some embodiments, the mRNA comprises SEQ ID NO:53. In some embodiments, the mRNA comprises SEQ ID NO:66. In some embodiments, the mRNA comprises SEQ ID NO:68. In some embodiments, the mRNA comprises SEQ ID NO:69. In some embodiments, the mRNA comprises SEQ ID NO:72.

[0068] In some embodiments, the pharmaceutical composition comprises a lipid formulation and an mRNA, wherein the mRNA comprises a 3' poly-A tail, in some embodiments, the 3' poly-A tail consists of about 50 to about 120 adenosine monomers.

[0069] In some embodiments, the pharmaceutical composition comprises a lipid formulation and an mRNA, wherein the mRNA comprises a 5' cap. In some embodiments, the 5' cap is 7 In some embodiments, the m 7 GpppAmpG has the structure of the following formula (Cap V): [ka] In the formula, R 1 , R 2 and R 4each is OH, n is 1, each L is a phosphate linked by a diester bond, and mRNA is an mRNA of that composition.

[0070] In some embodiments, the mRNA of the pharmaceutical composition is selected from the group consisting of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2' -Fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N 1 -Hydroxypseudouridine, N 1 -Methylpseudouridine, 2'-O-methyl-N 1 -Methylpseudouridine, N 1 -Ethylpseudouridine, N 1 -Hydroxymethylpseudouridine, aurauridine, N 6 In some embodiments, the one or more chemically modified nucleotides are selected from the group consisting of N-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, and 6-O-methylguanosine. 1 -Methylpseudouridine.

[0071] In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 5:1 to about 40:1. In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 8:1 to 40:1. In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 10:1 to 30:1. In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 15:1 to 30:1. In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 10:1 to 25:1. In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 5:1 to about 25:1. In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 10:1 to about 20:1. In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 12:1 to about 18:1. In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 14:1 to about 17:1.In some embodiments, the pharmaceutical composition has a total lipid:mRNA weight ratio of about 15:1 to about 16:1.

[0072] In some embodiments, the pharmaceutical composition comprises about 20 w / w% to 60 w / w% of the cationic lipid. In some embodiments, the pharmaceutical composition comprises about 20 w / w% to 50 w / w% of the cationic lipid. In some embodiments, the pharmaceutical composition comprises about 20 w / w% to 40 w / w% of the cationic lipid. In some embodiments, the pharmaceutical composition comprises about 20 w / w% to 30 w / w% of the cationic lipid. In some embodiments, the pharmaceutical composition comprises about 25 w / w% of the cationic lipid.

[0073] In some embodiments, the pharmaceutical composition comprising the lipid formulation and mRNA may further comprise a buffer. In some embodiments, the buffer has a pH of about 7.0 to about 8.5. In some embodiments, the buffer is a HEPES buffer or a TRIS buffer. In some embodiments, the HEPES buffer or the TRIS buffer has a pH of about 7.0 to about 8.5. In some embodiments, the HEPES buffer or the TRIS buffer has a pH of about 7.4 to about 8.2. In some embodiments, the HEPES buffer or the TRIS buffer has a concentration of about 20 mM to about 80 mM. In some embodiments, the buffer is a HEPES buffer. In some embodiments, the buffer is a HEPES buffer with a concentration of about 35 mM to about 70 mM. In some embodiments, the buffer is a HEPES buffer with a concentration of about 40 mM to about 60 mM. In some embodiments, the buffer is a HEPES buffer with a concentration of about 45 mM to about 55 mM. In some embodiments, the buffer is a TRIS buffer. In some embodiments, the buffer is a TRIS buffer at a concentration of about 20 mM to about 50 mM. In some embodiments, the buffer is a TRIS buffer at a concentration of about 25 mM to about 40 mM. In some embodiments, the buffer is a TRIS buffer at a concentration of about 25 mM to about 35 mM.

[0074] In some embodiments, the pharmaceutical composition comprising the lipid formulation and mRNA further comprises sodium chloride (NaCl). In some embodiments, the pharmaceutical composition comprises about 10 mM to about 100 mM NaCl. In some embodiments, the pharmaceutical composition comprises about 20 mM to about 90 mM NaCl. In some embodiments, the pharmaceutical composition comprises about 30 mM to about 80 mM NaCl. In some embodiments, the pharmaceutical composition comprises about 35 mM to about 70 mM NaCl. In some embodiments, the pharmaceutical composition comprises about 40 mM to about 60 mM NaCl. In some embodiments, the pharmaceutical composition comprises about 45 mM to about 55 mM NaCl.

[0075] In some embodiments, the pharmaceutical composition comprising the lipid formulation and the mRNA further comprises one or more cryoprotectants. In some embodiments, the one or more cryoprotectants are selected from the group consisting of sucrose, glycerol, and a combination of sucrose and glycerol. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is glycerol. In some embodiments, the cryoprotectant is a combination of sucrose and glycerol. In some embodiments, the pharmaceutical composition comprises a combination of sucrose at a concentration of about 5% (w / v) to about 18% (w / v) and glycerol at a concentration of about 1% (w / v) to about 9% (w / v). In some embodiments, the pharmaceutical composition comprises a combination of sucrose at a concentration of about 6% (w / v) to about 16% (w / v) and glycerol at a concentration of about 1.5% (w / v) to about 7% (w / v). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 7% (w / v) to about 14% (w / v) in combination with glycerol at a concentration of about 1.75% (w / v) to about 6% (w / v). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 7% (w / v) to about 12% (w / v) in combination with glycerol at a concentration of about 1% (w / v) to about 6% (w / v). In some embodiments, the composition comprises sucrose at a concentration of about 8% (w / v) to about 11% (w / v) in combination with glycerol at a concentration of about 3% (w / v) to about 6% (w / v).

[0076] In some embodiments, the pharmaceutical compositions are provided for use in medical therapy, hi some embodiments, the pharmaceutical compositions are provided for use in the treatment of the human or animal body.

[0077] In some embodiments, a use of the pharmaceutical composition is provided for making a medicament for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with decreased activity of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in a subject in need thereof. In some embodiments, the disease is cystic fibrosis with a cystic fibrosis mutation selected from class 1A, class 1B, class 3, class 4, class 5, and class 6. In some embodiments, the cystic fibrosis mutation is class 1A. In some embodiments, the cystic fibrosis mutation is class 1B. In some embodiments, the cystic fibrosis mutation is class 3. In some embodiments, the cystic fibrosis mutation is class 4. In some embodiments, the cystic fibrosis mutation is class 5. In some embodiments, the cystic fibrosis mutation is class 6.

[0078] In some embodiments, methods are provided for ameliorating, preventing, delaying the onset of, or treating a disease or disorder associated with decreased activity of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in a subject in need thereof, comprising administering to the subject one or more of the mRNA sequences or pharmaceutical compositions described herein. In some embodiments, the disease is cystic fibrosis. In some embodiments, the administration is intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, nasal, or inhaled. In some embodiments, the administration is intranasal or inhaled. In some embodiments, the administration is inhaled. In some embodiments, the administration is once a day, once a week, once every two weeks, or once a month. In some embodiments, the administration comprises an effective dose of 0.01-10 mg / kg. In some embodiments, the administration increases expression of CFTR in the lung epithelium.

[0079] In some embodiments, methods are provided for expressing a CFTR protein in a cell, comprising contacting the cell with one or more of the mRNA sequences or pharmaceutical compositions described herein.

[0080] In some embodiments, a kit for expressing human CFTR in vivo is provided, the kit comprising a 0.1-500 mg dose of an mRNA or pharmaceutical composition described herein and a device for administering the dose. In some embodiments, the device is an injection needle, an intravenous needle, or an inhalation device. In some embodiments, the device is an inhalation device.

[0081] Human CFTR In some embodiments, an mRNA sequence is provided that comprises an mRNA coding sequence that encodes the human CFTR protein. The sequence of the native human CFTR protein is set forth in SEQ ID NO:93.

[0082] In some embodiments, the mRNA encodes a protein substantially identical to the human CFTR protein. In some embodiments, the mRNA encodes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 80% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 85% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 90% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 91% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 92% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 93% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 94% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 95% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 96% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 97% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 98% or more identical to SEQ ID NO:93. In embodiments, the mRNA encodes an amino acid sequence that is at least 99% or more identical to SEQ ID NO:93. In some embodiments, the mRNA encodes a protein having hCFTR activity, the protein having the sequence of SEQ ID NO:93. In some embodiments, an mRNA suitable for the present disclosure encodes a fragment or portion of a human CFTR protein.

[0083] In some embodiments, the disclosure provides an mRNA sequence encoding a homolog or variant of human CFTR. As used herein, a homolog or variant of a human CFTR protein may be a modified human CFTR protein that contains one or more amino acid substitutions, deletions, and / or insertions compared to a wild-type or naturally occurring human CFTR protein, while at the same time substantially maintaining CFTR protein activity. In some embodiments, the mRNA encodes a protein or fragment thereof selected from SEQ ID NOs: 95, 96, 97, and 99. In some embodiments, the mRNA encodes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NOs: 95, 96, 97, and 99. In some embodiments, the mRNA encodes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 95. In some embodiments, the mRNA encodes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 96. In some embodiments, the mRNA encodes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:97. In some embodiments, the mRNA encodes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:99. In some embodiments, the mRNA encodes an amino acid sequence that is at least 80% identical to SEQ ID NO:99. In some embodiments, the mRNA encodes an amino acid sequence that is at least 85% identical to SEQ ID NO:99. In some embodiments, the mRNA encodes an amino acid sequence that is at least 90% identical to SEQ ID NO:99. In some embodiments, the mRNA encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:99.In some embodiments, the mRNA encodes an amino acid sequence that is at least 98% identical to SEQ ID NO: 99. In some embodiments, the mRNA encodes an amino acid sequence that is at least 99% identical to SEQ ID NO: 99. In some embodiments, the mRNA encodes a protein having hCFTR activity, the protein having the sequence of SEQ ID NO: 99.

[0084] In some embodiments, an mRNA suitable for the present disclosure encodes a fragment or portion of a human CFTR protein, where the fragment or portion of the protein still maintains CFTR activity equivalent to or improved over that of the wild-type protein.

[0085] In some embodiments, mRNA suitable for the present disclosure comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 49, 53, 66, 68, 69 or 72. In some embodiments, mRNA provided by the present invention comprises a sequence selected from SEQ ID NO: 49, 53, 66, 68, 69 and 72. In some embodiments, mRNA provided by the present invention comprises SEQ ID NO: 49. In some embodiments, mRNA provided by the present invention comprises SEQ ID NO: 53. In some embodiments, mRNA provided by the present invention comprises SEQ ID NO: 66. In some embodiments, mRNA provided by the present invention comprises SEQ ID NO: 68. In some embodiments, mRNA provided by the present invention comprises SEQ ID NO: 69. In some embodiments, mRNA provided by the present invention comprises SEQ ID NO: 72.

[0086] In some embodiments, an mRNA of the disclosure comprises a coding sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 100, 101, 102, 103, 104 or 105. In some embodiments, an mRNA comprises a coding sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 100, 101, 102, 103, 104 or 105, and further comprises one or more components selected from a 5' cap, a 5' UTR, a translation initiation sequence, a 3' UTR, and a tail region. In some embodiments, the mRNA provided herein comprises a coding sequence selected from SEQ ID NOs: 100, 101, 102, 103, 104, and 105. In some embodiments, the mRNA provided herein comprises a coding sequence selected from SEQ ID NOs: 100, 101, 102, 103, 104, and 105, and further comprises one or more components selected from a 5' cap, a 5' UTR, a translation initiation sequence, a 3' UTR, and a tail region.

[0087] In some embodiments, the mRNAs of the present disclosure provide fusion proteins (e.g., N- or C-terminal fusions) that include a full-length, fragment, or portion of a CFTR protein fused to another sequence, in some embodiments, the N- or C-terminal sequence is a signal sequence or a cell targeting sequence.

[0088] Translatable mRNA sequences and constructs The compositions and methods of the present disclosure include mRNAs that code for active and functional CFTR proteins. The mRNAs can include several features that enhance their in vivo half-life and translation efficiency. In addition, the present disclosure provides DNA scaffolds for generating mRNAs that code for active and functional CFTR proteins via transcription. The DNA scaffolds can be any suitable form of DNA, including plasmid DNA. Polynucleotides contemplated in the present disclosure are further described in detail below.

[0089] The mRNA of the present disclosure, which includes a coding sequence encoding a functional CFTR portion, can be delivered to a patient in need (e.g., a CF patient) and can increase the level of active CFTR in the patient. The mRNA sequence can be used to prevent, treat, ameliorate, or reverse any of the symptoms of cystic fibrosis in the patient. As will be apparent to one of skill in the art armed with the knowledge of this disclosure, the mRNA sequences and constructs of the present disclosure can be used to ameliorate, prevent, or treat any disease or disorder associated with decreased activity (e.g., due to decreased concentration, presence, and / or function) of cystic fibrosis transmembrane conductance regulator (CFTR) in a subject, and / or a disease associated with decreased presence or function of CFTR.

[0090] The disclosed mRNA sequences and constructs can have a long half-life, particularly in the cytoplasm, and can be used to ameliorate, prevent, or treat a disease or disorder associated with decreased activity (e.g., due to decreased concentration, presence, and / or function) of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in a subject.

[0091] The properties of the mRNA sequences and constructs of the present disclosure are manifested according to their molecular structure, and generally, the structure of the molecule in its entirety can provide significant benefits based on these properties.Embodiments of the present disclosure can provide mRNA sequences and constructs that have one or more properties that beneficially increase protein concentration or improve protein activity.The sequences and constructs can further be used in pharmaceutical compositions of the present disclosure to improve, prevent or treat any disease or disorder associated with decreased activity (e.g., due to decreased concentration, presence and / or function) of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in a subject.

[0092] The disclosure herein presents a series of mRNA sequences that demonstrate surprising translatability, resulting in active polypeptides or proteins in vitro, ex vivo and in vivo.

[0093] The mRNA sequences, constructs and compositions may have enhanced translational activity or increased half-life in the cytoplasm, in these embodiments, the mRNA sequences, constructs and compositions may have an increased functional half-life in the cytoplasm of a mammalian cell compared to native mRNA (i.e., mRNA transcribed in vivo from the cell's own genome).

[0094] In a further embodiment, the mRNA sequence can include one or more UNA monomers in the 3' untranslated region of the monomer.

[0095] In a further embodiment, the mRNA sequence can include one or more UNA monomers in the tail region of the monomer.

[0096] In a further embodiment, the mRNA sequence can include one or more UNA monomers in the polyA tail.

[0097] In some embodiments, the mRNA sequence may comprise one or more LNA monomers in the monomeric 3' untranslated region or in the monomeric tail region, for example a polyA tail.

[0098] In another embodiment, the mRNA sequences of the disclosure can be translated at least 2-fold, 3-fold, 5-fold, or 10-fold more efficiently in vivo than a naturally occurring mRNA encoding the same translation product.

[0099] In further embodiments, the mRNA sequence is capable of increasing the levels of a polypeptide or protein in vivo by at least 2-fold, 3-fold, 5-fold or 10-fold compared to the naturally occurring mRNA encoding the same polypeptide or protein.

[0100] In certain embodiments, the mRNA sequence can increase the level of a polypeptide or protein in vivo compared to the native mRNA encoding the same polypeptide or protein, for example, the level of the polypeptide or protein can be increased by 10%, 20%, 30%, 40%, or 50% or more.

[0101] In a further embodiment, the present disclosure provides a method of treating a disease or condition in a subject by administering to the subject a composition comprising an mRNA sequence of the present disclosure.

[0102] The mRNA sequences of the present disclosure may be used to ameliorate, prevent or treat a disease or disorder in a subject, for example a disease or disorder associated with decreased activity (e.g., due to decreased concentration, presence and / or function) of the cystic fibrosis transmembrane conductance regulator (CFTR). In these embodiments, a composition comprising an mRNA sequence of the present disclosure may be administered to modulate or increase the concentration or availability of CFTR in a subject. In one aspect, the protein can be an unmodified native protein in a patient with an abnormal amount (e.g., a patient with a mutant form of CFTR that partially or completely abolishes CFTR activity). In one aspect, the protein can be an unmodified native CFTR protein that can be used to treat a patient with a mutant form of CFTR. In embodiments, the mRNA sequences of the present disclosure may be used to ameliorate, prevent or treat cystic fibrosis.

[0103] In some embodiments, an mRNA sequence may be delivered to a cell or subject and translated to increase CFTR levels in the cell or subject.

[0104] In embodiments, a subject of the present disclosure is a subject having decreased activity (e.g., due to decreased concentration, presence and / or function) of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). In further embodiments, the subject is a human.

[0105] In some embodiments, administering a composition comprising an mRNA sequence of the present disclosure can increase CFTR protein levels in a treated subject. In some embodiments, administering a composition comprising an mRNA sequence of the present disclosure can increase CFTR protein levels by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% compared to baseline CFTR protein levels in a treated subject. In embodiments, administering a composition comprising an mRNA sequence of the present disclosure can increase CFTR levels compared to baseline CFTR levels in a treated subject. In some embodiments, the increase in CFTR levels can be at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 100%, or about 200% or more.

[0106] In embodiments, the CFTR protein is expressed in the lungs of the subject.

[0107] In some embodiments, administration of a composition comprising an mRNA sequence of the disclosure results in expression of non-mutated native human CFTR (i.e., normal or wild-type CFTR, as opposed to abnormal or mutated CFTR) in lung epithelial cells of the treated subject at a protein level of about 10 ng / mg or more, about 20 ng / mg or more, about 50 ng / mg or more, about 100 ng / mg or more, about 150 ng / mg or more, about 200 ng / mg or more, about 250 ng / mg or more, about 300 ng / mg or more, about 350 ng / mg or more, about 400 ng / mg or more, about 450 ng / mg or more, about 500 ng / mg or more, about 600 ng / mg or more, about 700 ng / mg or more, about 800 ng / mg or more, about 900 ng / mg or more, about 1000 ng / mg or more, about 1200 ng / mg or more, or about 1500 ng / mg or more of total protein.

[0108] In some embodiments, the expression of the non-mutated native human CFTR protein is detectable 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, 60 hours, and / or 72 hours after administration of a composition comprising an mRNA sequence of the present disclosure. In some embodiments, the expression of the non-mutated native human CFTR protein is detectable 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and / or 7 days after administration of a composition comprising an mRNA sequence of the present disclosure. In some embodiments, the expression of the non-mutated native human CFTR protein is detectable 1 week, 2 weeks, 3 weeks, and / or 4 weeks after administration. In some embodiments, the expression of the non-mutated native human CFTR protein is detectable after administration of a composition comprising an mRNA sequence of the present disclosure. In some embodiments, the expression of the non-mutated native human CFTR protein is detectable after administration of a composition comprising an mRNA sequence of the present disclosure.

[0109] Design and synthesis of mRNA sequences The mRNA agent of the present disclosure can be obtained by any suitable means.The method of making mRNA is known in the art and will be easily clear to those skilled in the art.The mRNA of the present disclosure can be prepared according to any available method, including but not limited to chemical synthesis, in vitro transcription (IVT), or enzymatic or chemical cleavage of longer precursor.

[0110] In some embodiments, mRNA is generated from a primary complementary DNA (cDNA) construct. The cDNA construct can be generated by the action of a reverse transcriptase (e.g., an RNA-dependent DNA polymerase) on an RNA template. The process of designing and synthesizing a primary cDNA construct described herein generally includes the steps of constructing a gene, generating an mRNA (either with or without modification), and purifying it. In the IVT method, a target polynucleotide sequence encoding a CFTR protein is first selected for incorporation into a vector, and the vector is amplified to generate a cDNA template. Optionally, the target polynucleotide sequence and / or any flanking sequences can be codon-optimized. The cDNA template is then used to generate mRNA by in vitro transcription (IVT). After generation, the mRNA can be subjected to a purification and clean-up process, which are described in more detail below.

[0111] Gene construction steps may include, but are not limited to, gene synthesis, vector amplification, plasmid purification, plasmid linearization and cleanup, and cDNA template synthesis and cleanup. Once the human CFTR protein (e.g., SEQ ID NO: 93 or 99) to be produced is selected, a primary construct is designed. Within the primary construct, the open reading frame (ORF) of a given nucleic acid (DNA or RNA) transcript may be used to construct a first region of linked nucleosides that encodes the polypeptide of interest. The ORF may include a wild-type ORF, an isoform, a variant, or a fragment thereof. As used herein, "open reading frame" or "ORF" is intended to refer to a nucleic acid sequence (DNA or RNA) that can code for a polypeptide of interest. An ORF often begins with an initiation codon, ATG, and ends with a nonsense codon or nonsense signal, or a stop codon or signal.

[0112] The cDNA templates may be transcribed to produce the mRNA sequences described herein using an in vitro transcription (IVT) system. The system typically includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. The NTPs may be selected from, but are not limited to, those described herein, including natural NTPs and non-natural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase, and mutant polymerases, such as, but not limited to, polymerases that can incorporate modified nucleic acids.

[0113] The primary cDNA template or transcribed mRNA sequence may be subjected to a capping and / or tailing reaction. A capping reaction may be performed by methods known in the art to add a 5' cap to the 5' end of the primary construct. Capping methods include, but are not limited to, using Vaccinia Capping enzyme (New England Biolabs, Ipswich, Mass.) or capping at the start of in vitro transcription, for example, by including a capping agent as part of the IVT reaction. (Nuc. Acids Symp. (2009) 53:129). A polyA tailing reaction may be performed by methods known in the art, including, but not limited to, 2'O-methyltransferase, and as described herein. If the primary construct generated from cDNA does not contain polyT, it may be beneficial to perform a polyA tailing reaction before washing the primary construct.

[0114] Codon-optimized cDNA constructs encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein are particularly suitable for generating the mRNA sequences described herein. For example, such cDNA constructs may be used as a base for in vitro transcription of polyribonucleotides encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.

[0115] Examples of DNA ORF sequences are shown in SEQ ID NOs: 1-46, which show sequences that can be used to generate materials for transcription into mRNA of the present disclosure. SEQ ID NO: 1 shows the DNA ORF of a reference hCFTR protein (construct 764), which is commonly used as a reference sequence in the art, and which is slightly modified from wild type and has a point mutation in its coding region to remove the internal cryptic promoter. Preferred DNA ORF sequences include the DNA sequences of SEQ ID NOs: 3, 5, 7, 20, 22, 23, or 26. In some embodiments, the DNA ORF comprises the sequence of SEQ ID NO: 7, which has an optimized coding sequence that encodes the CFTR protein of SEQ ID NO: 93. It will be apparent that T present in the DNA is replaced by U in the RNA, and U present in the RNA is replaced by T in the DNA.

[0116] The disclosure also provides expression vectors comprising a nucleotide sequence encoding a CFTR protein, preferably operably linked to at least one regulatory sequence, which are art-recognized and selected to direct expression of the encoded polypeptide.

[0117] Thus, the term regulatory sequence includes promoters, enhancers and other expression control elements. The design of the expression vector can be determined by such factors as the choice of the host cell to be transformed and / or the type of protein desired to be expressed.

[0118] The present disclosure also provides a polynucleotide (e.g., DNA, RNA, cDNA, mRNA, etc.) encoding human CFTR protein, which may be operably linked to one or more regulatory nucleotide sequences in an expression construct, such as a vector or plasmid. In certain embodiments, such a construct is a DNA construct. The regulatory nucleotide sequence will generally be suitable for the host cell used for expression. For various host cells, a large number of suitable expression vectors and suitable regulatory sequences are known in the art.

[0119] Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplated in the embodiments of the present disclosure.The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of two or more promoters.

[0120] The expression construct may be present in the cell episomally, such as a plasmid, or the expression construct may be inserted into a chromosome. In some embodiments, the expression vector contains a selectable marker gene to allow for the selection of transformed host cells. Selectable marker genes are well known in the art and will vary depending on the host cell used.

[0121] The present disclosure also provides a host cell transfected with mRNA or DNA described herein, encoding a CFTR polypeptide described herein. In some embodiments, the human CFTR polypeptide has the sequence of SEQ ID NO: 99. The host cell may be any prokaryotic or eukaryotic cell. For example, the CFTR polypeptide may be expressed in bacterial cells, such as E. coli, insect cells (e.g., using the baculovirus expression system), yeast or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0122] The present disclosure also provides host cells comprising vectors containing the polynucleotides of SEQ ID NOs:2-46.

[0123] The present disclosure also provides a method for making the human wild-type CFTR protein of SEQ ID NO: 93. For example, a host cell transfected with an expression vector encoding a CFTR protein can be cultured under suitable conditions to express the polypeptide. The polypeptide can be secreted from a mixture of cells and medium containing the polypeptide and isolated. Alternatively, the polypeptide can be retained in the cytoplasm or membrane fraction, and the cells can be harvested and lysed to isolate the protein. A cell culture includes host cells, medium and other by-products. Suitable media for cell culture are well known in the art.

[0124] The expressed CFTR proteins described herein can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification using antibodies specific for particular epitopes of the CFTR polypeptide.

[0125] Codon Optimization A polynucleotide sequence encoding a protein can be altered relative to the wild type of the sequence to select the best combination of codons that code for the amino acids of the protein. For mRNA, all or a portion of the mRNA, such as the coding region or open reading frame (ORF), can be optimized for codons within the region. Codon-optimized sequences can increase the protein expression level of the encoded protein (Gustafsson et al., Codon bias and heterologous protein expression. 2004, Trends Biotechnol 22:346-53), while also providing other benefits. The optimization of codons in a sequence depends on several features of the mRNA construct, including high codon adaptation index (CAI), Low-U method, mRNA secondary structure, cis-regulatory sequences, GC content, and many other similar variables. These variables have been shown to correlate with protein expression levels (Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments. 2006, BMC Bioinformatics 7:285). The High CAI (Codon Adaptation Index) method selects the most frequently used synonymous codon in the entire protein coding sequence. The most frequently used codon for each amino acid has been estimated from 74,218 protein coding genes in the human genome. The Low-U method targets only codons that contain U and can replace them with synonymous codons that have fewer U moieties than the codon. If there are fewer options for the replacement, the more frequently used codon will be selected. The Low-U method leaves the remaining codons in the sequence unchanged. This method can be used in conjunction with the mRNAs of the present disclosure to replace, for example, 5-methoxyuridine or N l -methylpseudouridine may be used to design the coding sequence to be synthesized. Methods for optimizing codons in conjunction with the use of modified nucleotide monomers are described in US2018 / 0327471, the contents of which are incorporated herein by reference.

[0126] In addition, the nucleotide sequence of any of the regions of the mRNA or DNA template may be codon-optimized. Methods of codon optimization are known in the art and may be useful in an attempt to achieve one or more of several goals. These goals include matching codon frequencies in the target and host organisms to ensure proper folding, biasing the GC nucleotide pair content to increase mRNA stability or reduce secondary structures, minimizing tandem repeat codons or stretches of bases that may impair gene assembly or expression, customizing transcriptional and translational control regions, inserting or removing protein trafficking sequences, removing / adding post-translational modification sites (e.g., glycosylation sites) in the encoded protein, adding, removing or shuffling protein domains, inserting or deleting restriction sites, modifying ribosome binding sites and mRNA degradation sites, adjusting the translation rate to ensure proper folding of various domains of the protein, or reducing or eliminating problematic secondary structures in the mRNA. Suitable tools, algorithms and services for codon optimization are known in the art.

[0127] In some embodiments, the nucleotide sequence of any of the regions of the mRNA or DNA templates described herein may be codon-optimized. Preferably, the primary cDNA template may include a reduced abundance or frequency of a particular nucleotide in the template strand. For example, the abundance of a nucleotide in the template may be reduced to a level where the nucleotide is less than 25% in the template. In a further example, the abundance of a nucleotide in the template may be reduced to a level where the nucleotide is less than 20% in the template. In some examples, the abundance of a nucleotide in the template may be reduced to a level where the nucleotide is less than 16% in the template. Preferably, the abundance of a nucleotide in the template may be reduced to a level where the nucleotide is less than 15%, preferably to a level where the nucleotide is less than 12% in the template.

[0128] In some embodiments, the lowering nucleotide is uridine. For example, the present disclosure provides nucleic acids with altered uracil content, where at least one codon in the wild-type sequence is replaced with an alternative codon to produce a uracil-modified sequence. The uracil-modified sequence is (i) an increase or decrease in the total uracil content (i.e., the ratio of uracil to the total nucleotide content in a portion of a nucleic acid, e.g., an open reading frame nucleic acid); (ii) localized increases or decreases in uracil content (i.e., the change in uracil content is restricted to a specific subsequence); (iii) a change in uracil distribution without a change in total uracil content; (iv) a change in uracil clusters (e.g., the number of clusters, the location of clusters, or the distance between clusters); or (v) combinations thereof; The material may have at least one of the following characteristics:

[0129] In some embodiments, the percentage of uracil nucleobases in the nucleic acid sequence is reduced relative to the percentage of uracil nucleobases in wild-type nucleic acid sequence.For example, in wild-type sequence, 30% of nucleobases may be uracil, but the nucleobases in the nucleic acid sequence of the present disclosure are preferably less than 15%, preferably less than 12%, preferably less than 10%.Uracil content can be calculated by dividing the number of uracils in sequence by the total number of nucleotides and multiplying by 100.

[0130] In some embodiments, the percentage of uracil nucleobases in the subsequence of the nucleic acid sequence is reduced relative to the percentage of uracil nucleobases in the corresponding subsequence of the wild-type sequence. For example, the wild-type sequence may have a 5'-end region (e.g., 30 codons) with a local uracil content of 30%, and the nucleic acid sequence of the present disclosure may reduce the uracil content of the same region to preferably 15% or less, preferably 12% or less, preferably 10% or less. These subsequences may be part of the wild-type sequence of the heterologous 5'UTR and 3'UTR sequences of the present disclosure.

[0131] In some embodiments, the codons in the nucleic acid sequences of the present disclosure are reduced or modified in number, size, location or distribution of uracil clusters, which may, for example, adversely affect protein translation. In certain aspects, the smaller the uracil content, the more preferable it is, but some subsequences of the wild-type sequence may have a uracil content, particularly localized uracil content, greater than the wild-type sequence and still maintain beneficial features (e.g., increased expression).

[0132] In some embodiments, uracil modified sequences are less inducible in Toll-like receptor (TLR) responses than wild-type sequences. Several TLRs recognize and respond to nucleic acids. Double-stranded (ds) RNA, a common viral component, has been shown to activate TLR3. Single-stranded (ss) RNA activates TLR7. RNA oligonucleotides, e.g., RNA with phosphorothioate internucleotide linkages, are ligands for human TLR8. DNA containing unmethylated CpG motifs, characteristic of bacterial and viral DNA, activates TLR9.

[0133] As used herein, the term "TLR response" is defined as the recognition of a single-stranded RNA by the TLR7 receptor, preferably including the degradation of the RNA and / or a physiological response resulting from the recognition of the single-stranded RNA by the receptor. Methods for determining and quantifying the binding of an RNA to TLR7 are known in the art. Similarly, methods for determining whether an RNA has elicited a TLR7-mediated physiological response (e.g., cytokine secretion) are also well known in the art. In some embodiments, the TLR response may be mediated by TLR3, TLR8, or TLR9 instead of TLR7. Inhibition of a TLR7-mediated response can be achieved by nucleoside modification. RNA naturally undergoes over 100 different nucleoside modifications. For example, human rRNA has 10 times more pseudouracil ('P) and 25 times more 2'-O-methylated nucleosides than bacterial rRNA. Bacterial mRNA does not contain nucleoside modifications, whereas mammalian RNA has N-methylated nucleosides. 7 -Methylguanosine (m 7 In addition to 5-methylcytidine (m 5 C), N 6 -Methyladenosine (m 6 A), which has modified nucleosides such as inosine and many 2'-O-methylated nucleosides.

[0134] In some embodiments, the uracil content of a polynucleotide disclosed herein, preferably a polynucleotide encoding a CFTR protein of SEQ ID NO: 99, is less than about 50%, less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 44%, less than 43%, less than 42%, less than 41%, less than 40%, less than 39%, less than 38%, less than 37%, less than 36%, less than 35%, less than 38%, less than 39%, less than 38%, less than 37%, less than 38 ... The uracil content of the polynucleotides disclosed herein, preferably those encoding the CFTR protein of SEQ ID NO:99, is about 5% to about 25%. In some embodiments, the uracil content of the polynucleotides disclosed herein, preferably those encoding the CFTR protein of SEQ ID NO:99, is about 5% to about 25%. In some embodiments, the uracil content of the polynucleotides disclosed herein, preferably those encoding the CFTR protein of SEQ ID NO:99, is about 15% to about 25%.

[0135] Natural, modified and chemically modified nucleotides Preferably, the mRNA described herein comprises one or more chemically modified nucleotides. Examples of nucleic acid monomers include non-natural nucleotides, modified nucleotides, and chemically modified nucleotides, including any non-natural nucleotides, modified nucleotides, and chemically modified nucleotides as known in the art. Nucleotides can be artificially modified at either the base or sugar moiety. In nature, most polynucleotides comprise nucleotides that are "unmodified" or "natural" nucleotides, including the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). These bases are typically fixed at their 1' position to ribose or deoxyribose. The use of mRNA polynucleotides comprising chemically modified nucleotides has been shown to improve mRNA expression, expression rate, half-life, and / or concentration of expressed proteins. Additionally, mRNA polynucleotides comprising chemically modified nucleotides continue to be useful in optimizing protein localization to avoid adverse biological responses, such as immune responses and / or degradation pathways.

[0136] Examples of modified nucleotides or chemically modified nucleotides include 5-hydroxycytidine, 5-alkylcytidine, 5-hydroxyalkylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-alkoxycytidine, 5-alkynylcytidine, 5-halocytidine, 2-thiocytidine, N4-alkylcytidine, N 4 -Aminocytidine, N 4 -Acetylcytidine and N 4 ,N 4 -Dialkylcytidines.

[0137] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine, N4 -Methylcytidine, N 4 -Aminocytidine, N 4 -Acetylcytidine and N 4 ,N 4 -Dimethylcytidine.

[0138] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-alkyluridine, 5-hydroxyalkyluridine, 5-carboxyuridine, 5-carboxyalkylester uridine, 5-formyluridine, 5-alkoxyuridine, 5-alkynyluridine, 5-halouridine, 2-thiouridine and 6-alkyluridine.

[0139] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as "5MeOU"), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine and 6-methyluridine.

[0140] Examples of modified or chemically modified nucleotides include 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5-methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'O-methyluridine and 3,2'-O-dimethyluridine.

[0141] Examples of modified or chemically modified nucleotides include N 6-Methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-bromoadenosine, 2-methylthio-N 6 -Methyladenosine, N 6 -Isopentenyl adenosine, 2-methylthio-N 6 -Isopentenyl adenosine, N 6 -(cis-Hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-Hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyl-adenosine, N 6 -Methyl-N 6 -Threonylcarbamoyl-adenosine, 2-methylthio-N 6 -Threonylcarbamoyl-adenosine, N 6 ,N 6 -Dimethyladenosine, N 6 -Hydroxynorvalylcarbamoyl adenosine, 2-methylthio-N 6 -Hydroxynorvalylcarbamoyl-adenosine, N 6 -Acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, alpha-thio-adenosine, 2'-O-methyl-adenosine, N 6 ,2'-O-Dimethyl-adenosine,N 6 ,N 6 ,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine, 2-amino-N 6 -methyl-purine, 1-thio-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine and N 6 -(19-amino-pentaoxanonadecyl)-adenosine.

[0142] Examples of modified or chemically modified nucleotides include N 1 -Alkylguanosine, N 2-Alkylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O6-alkylguanosine, xanthosine, inosine and N l -Alkyl inosines.

[0143] Examples of modified or chemically modified nucleotides include N 1 -Methylguanosine, N 2 -Methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O6-methylguanosine, xanthosine, inosine and N 1 -methylinosine.

[0144] An example of a modified nucleotide or a chemically modified nucleotide is pseudouridine. 1 -Alkylpseudouridine, N 1 -Cycloalkylpseudouridine, N 1 -Hydroxypseudouridine, N 1 -Hydroxyalkylpseudouridine, N 1 -Phenylpseudouridine, N 1 -Phenylalkylpseudouridine, N 1 -Aminoalkylpseudouridines, N 3 -Alkylpseudouridine, N 6 -Alkylpseudouridine, N 6 -Alkoxypseudouridine, N 6 -Hydroxypseudouridine, N 6 -Hydroxyalkylpseudouridine, N 6 -Morpholinopseudouridine, N 6 -Phenylpseudouridine and N 6 Examples of pseudouridines include N 1 -Alkyl-N 6 -Alkylpseudouridine, N 1 -Alkyl-N 6 -Alkoxypseudouridine, N 1 -Alkyl-N6 -Hydroxypseudouridine, N 1 -Alkyl-N 6 -Hydroxyalkylpseudouridine, N 1 -Alkyl-N 6 -Morpholinopseudouridine, N 1 -Alkyl-N 6 -Phenylpseudouridine and N 1 -Alkyl-N 6 In these examples, the alkyl, cycloalkyl, and phenyl substituents may be unsubstituted or further substituted with alkyl, halo, haloalkyl, amino, or nitro substituents.

[0145] Examples of pseudouridines include N 1 -methylpseudouridine (also referred to herein as "N1MPU"), N 1 -Ethylpseudouridine, N 1 -Propylpseudouridine, N 1 -Cyclopropylpseudouridine, N 1 -Phenylpseudouridine, N1-aminomethylpseudouridine, N 3 -Methylpseudouridine, N 1 -Hydroxypseudouridine and N 1 -Hydroxymethylpseudouridine.

[0146] Examples of nucleic acid monomers include modified nucleotides and chemically modified nucleotides, including any modified nucleotides and chemically modified nucleotides known in the art.

[0147] Examples of modified and chemically modified nucleotide monomers include any modified and chemically modified nucleotides known in the art, such as, for example, 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxyribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl-nucleotides, and inverted deoxy base monomer residues.

[0148] Examples of modified nucleotides and chemically modified nucleotide monomers include 3'-terminal stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides and 3'-inverted thymidine.

[0149] Examples of modified nucleotides and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides and 2'-O-methyl nucleotides. In an embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

[0150] Examples of modified nucleotides and chemically modified nucleotide monomers include 2',4'-tethered 2'-O-methoxyethyl (cMOE) and 2'-O-ethyl (cEt) modified DNA.

[0151] Examples of modified and chemically modified nucleotide monomers include 2'-amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides and 2'-O-allyl nucleotides.

[0152] Examples of modified nucleotides and chemically modified nucleotide monomers include N 6 -methyl adenosine nucleotides.

[0153] Examples of modified nucleotides and chemically modified nucleotide monomers include nucleotide monomers having the modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine, 8-bromoguanosine or 7-deazaadenosine.

[0154] Examples of modified nucleotides and chemically modified nucleotide monomers include 2'-O-aminopropyl substituted nucleotides.

[0155] Examples of modified nucleotides and chemically modified nucleotide monomers include those in which the 2'-OH group of the nucleotide is replaced with 2'-R, 2'-OR, 2'-halogen, 2'-SR or 2'-amino, where R can be H, alkyl, alkenyl or alkynyl.

[0156] The above examples of base modifications can be combined with additional modifications of the nucleoside or nucleotide structure, including sugar modifications and linkage modifications.Certain modified or chemically modified nucleotide monomers can be found in nature.

[0157] Preferred nucleotide modifying groups include N 1 -methylpseudouridine and 5-methoxyuridine.

[0158] Examples of modified nucleotides or chemically modified nucleotides include 5-hydroxycytidine, 5-alkylcytidine, 5-hydroxyalkylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-alkoxycytidine, 5-alkynylcytidine, 5-halocytidine, 2-thiocytidine, N 4 -Alkylcytidine, N 4 -Aminocytidine, N 4 -Acetylcytidine and N 4 ,N 4 -Dialkylcytidines.

[0159] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine, N 4 -Methylcytidine, N 4 -Aminocytidine, N 4 -Acetylcytidine and N 4 ,N 4 -Dimethylcytidine.

[0160] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-alkyluridine, 5-hydroxyalkyluridine, 5-carboxyuridine, 5-carboxyalkylester uridine, 5-formyluridine, 5-alkoxyuridine, 5-alkynyluridine, 5-halouridine, 2-thiouridine and 6-alkyluridine.

[0161] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as "5MeOU"), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine and 6-methyluridine.

[0162] Examples of modified or chemically modified nucleotides include 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5-methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'-O-methyluridine, 3'-O-dimethyluridine and 2'-O-dimethyluridine.

[0163] Examples of modified or chemically modified nucleotides include N 6 -Methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-bromoadenosine, 2-methylthio-N 6 -Methyladenosine, N 6 -Isopentenyl adenosine, 2-methylthio-N 6 -Isopentenyl adenosine, N 6 -(cis-Hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-Hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyl-adenosine, N 6 -Methyl-N 6 -Threonylcarbamoyl-adenosine, 2-methylthio-N 6 -Threonylcarbamoyl-adenosine, N 6 ,N 6 -Dimethyladenosine, N 6 -Hydroxynorvalylcarbamoyl adenosine, 2-methylthio-N 6 -Hydroxynorvalylcarbamoyl-adenosine, N 6-Acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenosine, 2-methoxy-adenosine, alpha-thio-adenosine, 2'-O-methyl-adenosine, N 6 ,2'-O-Dimethyl-adenosine,N 6 ,N 6 ,2'-O-trimethyl-adenosine, 2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine, 2-amino-N 6 -methyl-purine, 1-thio-adenosine, 2'-fluoro-ara-adenosine, 2'-fluoro-adenosine, 2'-OH-ara-adenosine and N 6 -(19-amino-pentaoxanonadecyl)-adenosine.

[0164] Examples of modified or chemically modified nucleotides include N 1 -Alkylguanosine, N 2 -Alkylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O 6 -Alkylguanosine, xanthosine, inosine and N 1 -Alkyl inosines.

[0165] Examples of modified or chemically modified nucleotides include N 1 -Methylguanosine, N 2 -Methylguanosine, Thienoguanosine, 7-Deazaguanosine, 8-Oxoguanosine, 8-Bromoguanosine, O 6 -Methylguanosine, xanthosine, inosine and N 1 -methylinosine.

[0166] An example of a modified nucleotide or a chemically modified nucleotide is pseudouridine. 1 -Alkylpseudouridine, N 1 -Cycloalkylpseudouridine, N 1 -Hydroxypseudouridine, N 1 -Hydroxyalkylpseudouridine, N1 -Phenylpseudouridine, N 1 -Phenylalkylpseudouridine, N 1 -Aminoalkylpseudouridines, N 3 -Alkylpseudouridine, N 6 -Alkylpseudouridine, N 6 -Alkoxypseudouridine, N 6 -Hydroxypseudouridine, N 6 -Hydroxyalkylpseudouridine, N 6 -Morpholinopseudouridine, N 6 -Phenylpseudouridine and N 6 Other examples of pseudouridines include N 1 -Alkyl-N 6 -Alkylpseudouridine, N 1 -Alkyl-N 6 -Alkoxypseudouridine, N 1 -Alkyl-N 6 -Hydroxypseudouridine, N 1 -Alkyl-N 6 -Hydroxyalkylpseudouridine, N 1 -Alkyl-N 6 -Morpholinopseudouridine, N 1 -Alkyl-N 6 -Phenylpseudouridine and N 1 -Alkyl-N 6 In these examples, the alkyl, cycloalkyl, and phenyl substituents may be unsubstituted or further substituted with alkyl, halo, haloalkyl, amino, or nitro substituents.

[0167] Examples of pseudouridines include N 1 -methylpseudouridine (also referred to herein as "N1MPU"), N 1 -Ethylpseudouridine, N 1 -Propylpseudouridine, N 1 -Cyclopropylpseudouridine, N 1-Phenylpseudouridine, N 1 -Aminomethylpseudouridine, N 3 -Methylpseudouridine, N 1 -Hydroxypseudouridine and N 1 -Hydroxymethylpseudouridine.

[0168] Examples of nucleic acid monomers include modified nucleotides and chemically modified nucleotides, including any modified nucleotides and chemically modified nucleotides known in the art.

[0169] Examples of modified and chemically modified nucleotide monomers include any modified and chemically modified nucleotides known in the art, such as 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxyribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl-nucleotides, and inverted deoxy base monomer residues.

[0170] Examples of modified nucleotides and chemically modified nucleotide monomers include 3'-terminal stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides and 3'-inverted thymidine.

[0171] Examples of modified nucleotides and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides and 2'-O-methyl nucleotides. In an embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

[0172] Examples of modified nucleotides and chemically modified nucleotide monomers include 2',4'-tethered 2'-O-methoxyethyl (cMOE) and 2'-O-ethyl (cEt) modified DNA.

[0173] Examples of modified and chemically modified nucleotide monomers include 2'-amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides and 2'-O-allyl nucleotides.

[0174] Examples of modified nucleotides and chemically modified nucleotide monomers include N 6 -methyl adenosine nucleotides.

[0175] Examples of modified nucleotides and chemically modified nucleotide monomers include nucleotide monomers having the modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine, 8-bromoguanosine or 7-deazaadenosine.

[0176] Examples of modified nucleotides and chemically modified nucleotide monomers include 2'-O-aminopropyl substituted nucleotides.

[0177] Examples of modified nucleotides and chemically modified nucleotide monomers include those in which the 2'-OH group of the nucleotide is replaced with 2'-R, 2'-OR, 2'-halogen, 2'-SR or 2'-amino, where R can be H, alkyl, alkenyl or alkynyl.

[0178] Some further examples of modified nucleotides are given in Saenger, Principles of Nucleic Acid Structure, Springer-Verlag, 1984.

[0179] Any of the above examples of base modifications can be combined with additional modifications of the nucleoside or nucleotide structure, including sugar modifications and linkage modifications.Certain modified or chemically modified nucleotide monomers can be found in nature.

[0180] Preferred nucleotide modifying groups include N 1 -methylpseudouridine and 5-methoxyuridine.

[0181] 5' Capping Structure The cap structure at the 5' end of mRNA is present in all eukaryotes (and in some viruses) and is important for stabilizing mRNA in vivo. 7 This riboguanosine residue is methylated at position 7. 7 G) is linked to the 5' end of the mRNA molecule via a 5'-5' triphosphate chain. 7 The presence of the Gppp fragment is essential for mRNA maturation because it protects the mRNA from exonucleolytic degradation, facilitates the transport of the mRNA from the nucleus to the cytoplasm, and plays a key role in the formation of the translation initiation complex (Cell 9:645-653, (1976), Nature 266:235 (1977), Federation of Experimental Biologists Society Letter 96:1-11 (1978), Cell 40:223-24 (1985), Prog. Nuc. Acid Res. 35:173-207 (1988), Ann. Rev. Biochem. 68:913-963 (1999), and J Biol. Chem. 274:30337-3040 (1999)).

[0182] Only those mRNAs that have a cap structure are active in cap-dependent translation, and "decapping" an mRNA results in almost complete loss of the template activity of the mRNA for protein synthesis (Nature, 255:33-37 (1975), J. Biol. Chem., vol. 253:5228-5231 (1978) and Proc. Natl. Acad. Sci. USA, 72:1189-1193 (1975)).

[0183] Another element of eukaryotic mRNA is the presence of 2'-O-methylnucleoside residues at transcription position 1 (cap 1) and sometimes at transcription positions 1 and 2 (cap 2). 2'-O-methylation of mRNA increases the efficiency of mRNA translation in vivo (Proc. Natl. Acad. Sci. USA, 77:3952-3956 (1980)) and further improves the nuclease stability of 5'-capped mRNA. mRNAs with cap 1 (and cap 2) are unique marks that allow cells to recognize the true mRNA 5' end and, in some cases, distinguish transcripts derived from infectious genetic elements (Nucleic Acid Research 43:482-492 (2015)).

[0184] Some examples of 5' cap structures and methods of preparing mRNAs containing the structures are provided in WO2015 / 051169A2, WO / 2015 / 061491, US2018 / 0273576, and U.S. Patent Nos. 8,093,367, 8,304,529, and 10,487,105. In some embodiments, the 5' cap is 7 GpppAmpG, which is known in the art. In some embodiments, the 5' cap is 7 GpppG or m 7 GpppGm, which are known in the art. The structural formula of an embodiment of the 5' cap structure is shown below.

[0185] In some embodiments, an mRNA described herein includes a 5' cap having the structure of formula (Cap I): [ka] In the formula, B 1 is a natural or modified nucleobase, and R 1 and R 2 are each independently halogen, OH and OCH 3 wherein each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate, each L is linked by a diester bond, n is 0 or 1, and the mRNA represents an mRNA of the present disclosure linked at its 5' end. 1 , G, m 7 In some embodiments, n is G or A. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, B 1 is A or m 6 A and R 1 OCH 3 where G is guanine and m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 -methyladenine.

[0186] In some embodiments, an mRNA described herein includes a 5' cap having the structure of formula (Cap II): [ka] In the formula, B 1 and B. 2 are each independently a natural or modified nucleobase; R 1 , R 2 and R 3 are each independently halogen, OH and OCH 3wherein each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate, each L is linked by a diester bond, the mRNA represents an mRNA of the present disclosure linked at its 5' end, and n is 0 or 1. In some embodiments, R 1 , R 2 and R 3 At least one of B is OH. 1 , G, m 7 In some embodiments, n is G or A. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, B 1 is A or m 6 A and R 1 OCH 3 where G is guanine and m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 -methyladenine.

[0187] In some embodiments, an mRNA described herein includes a 5' cap having the structure of formula (Cap III): [ka] In the formula, B 1 , B 2 and B. 3 are each independently a natural or modified nucleobase; R 1 , R 2 , R 3 and R 4 are each independently halogen, OH and OCH 3 wherein each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate, each L is linked by a diester bond, the mRNA represents an mRNA of the present disclosure linked at its 5' end, and n is 0 or 1. In some embodiments, R 1 , R 2 , R 3 and R4 At least one of B is OH. 1 , G, m 7 G or A. In some embodiments, B 1 is A or m 6 A and R 1 OCH 3 where G is guanine and m 7 G is 7-methylguanine, A is adenine, m 6 A is N 6 -methyladenine. In some embodiments, n is 1.

[0188] In some embodiments, the mRNA described herein has the structure of formula (Cap IV): 7 It contains a 5' cap analog called GpppG. [ka] In the formula, R 1 , R 2 and R 3 are each independently halogen, OH and OCH 3 wherein each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate, each L is linked by a diester bond, the mRNA represents an mRNA of the present disclosure linked at its 5' end, and n is 0 or 1. In some embodiments, R 1 , R 2 and R 3 In some embodiments, the 5' cap is m 7 GpppG, where R 1 , R 2 and R 3 is OH, n is 1, and each L is phosphate. In some embodiments, n is 1. In some embodiments, the 5' cap is m7GpppGm, where R 1 and R 2 are OH and R 3 OCH3 wherein each L is a phosphate, the mRNA is a CFTR mRNA of the disclosure linked at its 5' end, and n is 1.

[0189] In some embodiments, the mRNA described herein has the structure of formula (Cap V): 7 It contains a 5' cap analog called GpppAmpG. [ka] In the formula, R 1 , R 2 and R 4 are each independently halogen, OH and OCH 3 wherein each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate, each L is linked by a diester bond, the mRNA represents an mRNA of the present disclosure linked at its 5' end, and n is 0 or 1. In some embodiments, R 1 , R 2 and R 4 At least one of is OH. In some embodiments, the compound of formula Cap V is 7 GpppAmpG, where R 1 , R 2 and R 4 is OH, n is 1, and each L is phosphate. In some embodiments, n is 1.

[0190] 3' tail Polyadenylation is the addition of a polyA tail, a chain of adenine nucleotides usually about 100-120 monomers long, to an mRNA. In eukaryotes, polyadenylation is part of the process that generates the mature mRNA for translation and is initiated when transcription of a gene is terminated. First, the 3'-most segment of the newly made pre-mRNA is cleaved by a series of proteins, which then synthesize a polyA tail at the 3' end. The polyA tail is important for the nuclear export, translation, and stability of the mRNA. This tail shortens over time, and when it becomes short enough, the mRNA is degraded by enzymes. However, in some cell types, mRNAs with short polyA tails are stored in the cytosol in preparation for later activation by re-polyadenylation.

[0191] PolyA tails can be added using a variety of methods known in the art, for example, using polyA polymerase to add tails to synthetic RNA or in vitro transcribed RNA. Other methods include using a transcription vector that encodes a polyA tail, or using a ligase (e.g., via splint ligation using T4 RNA ligase and / or T4 DNA ligase), where polyA can be ligated to the 3' end of the RNA. In some embodiments, a combination of any of the above methods is used.

[0192] In some embodiments, the mRNA sequence encoding CFTR includes a tail region, which can function to protect the mRNA from exonuclease degradation. In some embodiments, the tail region can be a polyA tail. The tail region is a 3' polyA and / or 3' polyC region. Preferably, the tail region is a 3' polyA tail. As used herein, a "3' poly-A tail" is a polymer of consecutive adenine nucleotides that can be in the size range of, for example, 10-250 consecutive adenine nucleotides, 60-125 consecutive adenine nucleotides, 90-125 consecutive adenine nucleotides, 95-125 consecutive adenine nucleotides, 95-121 consecutive adenine nucleotides, 100-121 consecutive adenine nucleotides, 110-121 consecutive adenine nucleotides, 112-121 consecutive adenine nucleotides, 114-121 consecutive adenine nucleotides, or 115-121 consecutive adenine nucleotides. Preferably, a 3' poly A tail as described herein comprises 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125 consecutive adenine nucleotides.

[0193] In some embodiments, the mRNA sequence comprises a 3' poly-A tail structure. In some embodiments, the length of the poly-A tail can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. In some embodiments, the 3' poly-A tail comprises about 5 to 300 adenosine nucleotides (e.g., about 30 to 250 adenosine nucleotides, about 60 to 220 adenosine nucleotides, about 80 to 200 adenosine nucleotides, about 90 to about 150 adenosine nucleotides, or about 100 to about 120 adenosine nucleotides). In embodiments, the 3' poly-A tail is about 100 nucleotides in length. In another embodiment, the 3' poly-A tail is about 115 nucleotides in length. In another embodiment, the 3' poly-A tail is about 250 nucleotides in length.

[0194] In some embodiments, the 3' polyA tail comprises one or more UNA monomers. In some embodiments, the 3' polyA tail comprises 2, 3, 4, 5, 10, 15, or 20 or more UNA monomers. In embodiments, the 3' polyA tail comprises two UNA monomers. In further embodiments, the 3' polyA tail comprises two UNA monomers that are found consecutively, i.e., two UNA monomers that are adjacent to each other in the 3' polyA tail. Examples of methods and constructs for the synthesis of UNA-containing polyA tails are described in WO2016 / 070166, the contents of which are incorporated herein by reference.

[0195] In embodiments, the 3' Poly-A tail comprises the sequence Poly-A100 or Poly-A120, which consists of 100 or 120 adenosine nucleotides.

[0196] In some embodiments, the mRNA sequence comprises a 3' poly-C tail structure. In some embodiments, the length of the poly-C tail can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. In some embodiments, the 3' poly-C tail comprises about 5 to 300 cytosine nucleotides (e.g., about 30 to 250 cytosine nucleotides, about 60 to 220 cytosine nucleotides, about 80 to about 200 cytosine nucleotides, about 90 to 150 cytosine nucleotides, or about 100 to about 120 cytosine nucleotides). In embodiments, the 3' poly-C tail is about 100 nucleotides in length. In another embodiment, the 3' poly-C tail is about 115 nucleotides in length. The poly-C tail may be in addition to or in place of a poly-A tail. The poly-C tail may be added to the 5' end of the poly-A tail or to the 3' end of the poly-A tail.

[0197] In some embodiments, the length of the poly-A tail and / or poly-C tail is adjusted to control the stability, i.e., protein transcription, of the modified mRNA of the present disclosure. For example, the length of the poly-A tail can affect the half-life of an mRNA sequence, and thus the time course of polynucleotide expression and / or polypeptide production can be controlled in a target cell by altering the level of resistance of the mRNA to nucleases.

[0198] 5' and 3' untranslated regions (UTRs) In molecular genetics, untranslated region (UTR) refers to either of two portions of an mRNA strand, one at each end of the coding sequence. When the UTR is at the 5' end, it is called 5'UTR (or leader sequence), and when the UTR is at the 3' end, it is called 3'UTR (or trailer sequence). When an mRNA is translated into a protein in vivo, some regions of the mRNA are not usually translated, including the 5'UTR and 3'UTR. In some embodiments, the mRNA described herein further comprises a 5' untranslated region (UTR) sequence. The 5'UTR is upstream from the coding sequence. Within the 5'UTR are sequences recognized by ribosomes, which allow the ribosome to bind and begin translation. In contrast, the 3'UTR is typically found immediately after the translation stop codon of the coding region. The 3'UTR can play an important role in translation termination and post-translational modification. Thus, as understood in the art, the 5'UTR and / or 3'UTR can affect the stability or translation efficiency of the mRNA. The 5'UTR may be derived from an mRNA molecule known in the art to be relatively stable (e.g., histones, tubulin, globin, glyceraldehyde 1-phosphate dehydrogenase (GAPDH), actin, or citric acid cycle enzymes) to improve the stability of the translatable oligomer. In another embodiment, the 5'UTR sequence may include a partial sequence of the cytomegalovirus (CMV) immediate early 1 (IE1) gene.

[0199] In some embodiments, the mRNA sequence may comprise a 5'UTR that is at least about 25, 50, 75, 100, 125, 150, 175, 200, 300, 400, or 500 nucleotides in length. In some embodiments, the 5'UTR comprises about 50-300 nucleotides (e.g., about 75-250 nucleotides, about 100-200 nucleotides, about 120-150 nucleotides, or about 135 nucleotides). In embodiments, the 5'UTR is about 127 nucleotides in length.

[0200] Preferably, the 5'UTR comprises a sequence selected from human IL-6, alanine aminotransferase 1, human apolipoprotein E, human fibrinogen alpha chain, human transthyretin, human haptoglobin, human alpha-1-antichymotrypsin, human antithrombin, human alpha-1-antitrypsin, human albumin, human beta globin, human complement C3, human complement C5, SynK (a thylakoid potassium channel protein from the cyanobacterium Synechocystis sp.), mouse beta globin, mouse albumin and the 5'UTR of tobacco etch virus, or a fragment of any of the above. Preferably, the 5'UTR is derived from tobacco etch virus (TEV). Preferably, the mRNA described herein comprises a 5'UTR sequence derived from a gene expressed by Arabidopsis thaliana. Preferably, the 5'UTR sequence of the gene expressed by Arabidopsis thaliana is AT1G58420. Examples of 5'UTRs and 3'UTRs are described in WO2018 / 222890, the contents of which are incorporated herein by reference. Preferred 5'UTR sequences include sequences selected from SEQ ID NOs: 106-125.

[0201] In some embodiments, the 5'UTR sequence comprises SEQ ID NO: 106 (TEV). In some embodiments, the 5'UTR sequence comprises SEQ ID NO: 107 (AT1G58420).

[0202] In some embodiments, the 3'UTR comprises a sequence selected from the 3'UTR of alanine aminotransferase 1, human apolipoprotein E, human fibrinogen alpha chain, human haptoglobin, human antithrombin, human alpha globin, human beta globin, human complement C3, human growth factor, human hepcidin, MALAT-1, mouse beta globin, mouse albumin, and Xenopus beta globin, or a fragment of any of the above. In some embodiments, the 3'UTR is derived from Xenopus beta globin. Examples of 3'UTR sequences include SEQ ID NOs: 126-145. In some embodiments, the 3'UTR sequence comprises SEQ ID NO: 126 (XBG).

[0203] In certain embodiments, an mRNA sequence encoding CFTR comprises a 5'UTR sequence of SEQ ID NO: 106-125 and a 3'UTR sequence selected from SEQ ID NO: 126-145. In some embodiments, the 5'UTR sequence comprises SEQ ID NO: 106 and the 3'UTR sequence comprises SEQ ID NO: 126.

[0204] Triple stop codon In some embodiments, the translatable oligomer or polymer encoding CFTR may contain a sequence immediately downstream of the coding region (i.e., ORF) that forms a triple stop codon. A triple stop codon is a sequence of three consecutive stop codons. The triple stop codon can ensure that the expression cassette is completely isolated and may be incorporated to increase translation efficiency. In some embodiments, the mRNA may contain a sequence of UAG, UGA, or UAA immediately downstream of the ORF described herein. This triple combination can be either three of the same codons, three different codons, or a different permutation of the three stop codons.

[0205] Translational enhancers and Kozak sequences For translation initiation, proper interaction of the ribosome with the mRNA must be established to accurately position the translation initiation region. However, the ribosome must also dissociate from the translation initiation region and slide toward the downstream sequence during mRNA translation. Translation enhancers upstream from the start sequence of an mRNA increase the biosynthetic yield of the protein. Several studies have investigated the effect of translation enhancers. In some embodiments, the mRNAs described herein contain translation enhancer sequences. These translation enhancer sequences increase the translation efficiency of the mRNAs described herein, thereby increasing the production of the protein encoded by the mRNA. The translation enhancer region may be in the 5'UTR or 3'UTR of the mRNA sequence. Examples of translation enhancer regions include the natural enhancer regions from the TEV 5'UTR and Xenopus beta-globin 3'UTR. Examples of 5'UTR enhancer sequences include, but are not limited to, sequences derived from mRNAs encoding human heat shock proteins (HSPs), including HSP70-P2, HSP70-M1, HSP72-M2, HSP17.9 and HSP70-P1.

[0206] In some embodiments, the mRNA sequence encoding CFTR may include a Kozak sequence. As understood in the art, the Kozak sequence is a short consensus sequence central to the translation initiation site of eukaryotic mRNA, which allows the translation of the mRNA to be efficiently initiated. See, for example, Kozak, Marilyn (1988) Mol. and Cell Biol, 8:2737-2744; Kozak, Marilyn (1991) J. Biol. Chem, 266:19867-19870; Kozak, Marilyn (1990) Proc Natl. Acad. Sci. USA, 87:8301-8305 and Kozak, Marilyn (1989) J. Cell Biol, 108:229-241, and references cited therein. This sequence allows the protein to be correctly translated from the gene message, and mediates the formation of ribosomes and the initiation of translation. The ribosomal translation machinery recognizes the initiation codon AUG associated with a Kozak sequence.

[0207] In some embodiments, the translation start site (e.g., a Kozak sequence) is inserted upstream of the CFTR coding sequence. In some embodiments, the translation start site is inserted downstream of the 5'UTR. In certain embodiments, the translation start site is inserted upstream of the CFTR coding sequence and downstream of the 5'UTR.

[0208] As understood in the art, the length of the Kozak sequence can vary. In general, the longer the leader sequence, the stronger the translation. In some embodiments, the Kozak sequence is immediately downstream of the 5'UTR and immediately upstream of the CFTR coding sequence. In this aspect, Table 1 lists the mRNA constructs exemplified herein. [Table 1-1] [Table 1-2]

[0209] Lipid-Based Formulations Therapeutics based on intracellular delivery of nucleic acids to target cells encounter both intracellular and extracellular barriers. In fact, naked nucleic acid substances cannot be easily administered systemically due to their toxicity, poor stability in serum, rapid renal clearance, low uptake by target cells, uptake by phagocytes, and ability in activating immune responses, all characteristics that hinder their clinical development. When foreign nucleic acid substances (e.g., mRNA) enter the human biological system, they are recognized as foreign pathogens by the reticuloendothelial system (RES) and are removed from the blood circulation before they have a chance to encounter target cells inside or outside the vascular system. It has been reported that the half-life of naked nucleic acids in the bloodstream is around several minutes (Kawabata K, Takakura Y, Hashida MPharm Res. 1995 Jun; 12(6): 825-30). Chemical modification and suitable delivery methods can reduce uptake by RES and protect nucleic acids from degradation by ubiquitous nucleases, thereby improving the stability and efficacy of nucleic acid-based therapeutics.In addition, RNA or DNA is an anionic hydrophilic polymer that is not favorable for uptake by cells that have an anionic surface, similar to RNA or DNA.Therefore, the success of nucleic acid-based therapeutics mainly depends on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells while minimizing toxicity and obtain sufficient levels of expression in vivo.

[0210] Furthermore, once internalized in a target cell, nucleic acid delivery vectors face challenges from intracellular barriers, including uptake into endosomes, degradation by lysosomes, release of nucleic acids from the vector, translocation across the nuclear membrane (for DNA) and release in the cytoplasm (for RNA). Thus, the success of nucleic acid-based therapeutics depends on the ability of the vector to deliver nucleic acids to a target site inside the cell to achieve a desired activity, e.g., gene expression, at a sufficient level.

[0211] Although some gene therapy approaches have been successfully applied to viral delivery vectors (e.g., AAV), lipid-based formulations are increasingly recognized as one of the most promising delivery systems for RNA and other nucleic acid compounds due to their biocompatibility and ease of mass production. One of the most significant advances in lipid-based nucleic acid therapeutics was seen in August 2018, when Patisiran (ALN-TTR02) became the first siRNA drug approved by the U.S. Food and Drug Administration (FDA) and the European Commission (EC). ALN-TTR02 is an siRNA formulation based on the so-called stable nucleic acid lipid particle (SNALP) transfection technology. Despite the success of Patisiran, delivery of nucleic acid drugs, including mRNA, via lipid formulations is still under development.

[0212] Among the several lipid formulation delivery vehicles recognized in the art, delivery vehicles for nucleic acid drugs include, according to various embodiments, polymer-based carriers, such as polyethyleneimine (PEI), lipid nanoparticles, liposomes, nanoliposomes, ceramide-containing nanoliposomes, multivesicular liposomes, proteoliposomes, exosomes, both natural and synthetic, natural, synthetic and semi-synthetic lamellar bodies, nanoparticles, micelles, and emulsions. These lipid formulations may vary in structure and composition, and as can be expected in a fast-evolving field, the art uses several different terms to describe a type of delivery vehicle. At the same time, the intended meaning of lipid formulation terms varies throughout the scientific literature, and this inconsistent use has created confusion about the exact meaning of some of the lipid formulation terms. For the purposes of this disclosure, the following several potential lipid formulations are specifically detailed and defined herein: liposomes, cationic liposomes, and lipid nanoparticles.

[0213] Liposomes Conventional liposomes are vesicles consisting of at least one bilayer and an internal aqueous compartment. The bilayer membrane of liposomes is typically formed by amphiphilic molecules, e.g., synthetic or naturally occurring lipids that contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of liposomes may also be formed by amphiphilic polymers and surfactants (e.g., polymersomes, niosomes, etc.). They generally exist as spherical vesicles, and can range in size from 20 nm to several microns. Liposome formulations can be prepared as colloidal dispersions or lyophilized to reduce stability risks and improve the shelf life of liposome-based drugs. Methods for preparing liposome compositions are known in the art and are within the skill of the art.

[0214] Liposomes with only one bilayer are called unilamellar, and liposomes with two or more bilayers are called multilamellar. The most common types of liposomes are small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast to liposomes, lysosomes, micelles and reverse micelles are composed of a single layer of lipid. Generally, liposomes are considered to have one internal compartment, but some formulations can be multivesicular liposomes (MVLs), which consist of multiple discontinuous aqueous internal compartments separated by several non-concentric lipid bilayers.

[0215] Liposomes have long been recognized as drug delivery vehicles due to their excellent biocompatibility, given that they are essentially analogs of biological membranes and can be prepared from both natural and synthetic phospholipids (Int. J. Nanomedicine. 2014; 9: 1833-1843). Liposomes have an aqueous core surrounded by a hydrophobic membrane, so that when used as a drug delivery vehicle, hydrophilic solutes dissolved in the core cannot easily pass through the bilayer, and hydrophobic compounds will associate with the bilayer. Thus, liposomes can be loaded with hydrophobic and / or hydrophilic molecules. When liposomes are used to carry nucleic acids, such as RNA, the nucleic acid is contained within the aqueous phase liposomal compartment.

[0216] Cationic Liposomes Liposomes can be composed of cationic, anionic and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are liposomes made entirely or partially of positively charged lipids, or more specifically, lipids that contain both cationic groups and lipophilic moieties. In addition to the general properties of liposomes outlined above, the positively charged portion of the cationic lipids used in cationic liposomes provides some advantages and some unique structural features. For example, the lipophilic portion of cationic lipids is hydrophobic, so it will move itself away from the aqueous interior of the liposome and associate with other species that are non-polar and hydrophobic. Conversely, the cationic portion will associate with aqueous media and, more importantly, with polar molecules and species, and can form complexes with them in the aqueous interior of the cationic liposome. For these reasons, cationic liposomes are increasingly being investigated for use in gene therapy, as they favor negatively charged nucleic acids due to electrostatic interactions, resulting in complexes that achieve the biocompatibility, low toxicity, and mass production potential required for in vivo clinical use. Cationic lipids suitable for use in cationic liposomes are listed herein below.

[0217] Lipid Nanoparticles In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNPs) have a structure that includes one monolayer or bilayer of lipids that encapsulates a compound in the solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous or other liquid phase inside, but rather the lipids of the bilayer or monolayer shell directly complex with the compound inside, thereby encapsulating the compound in the solid core. Lipid nanoparticles are typically spherical vesicles with a relatively uniform shape and size distribution. Depending on the size at which lipid particles are considered to be nanoparticles, sources vary, but there is some agreement that lipid nanoparticles can range in diameter from 10 nm to 1000 nm. However, more commonly, lipid nanoparticles are considered to be less than 120 nm or even less than 100 nm.

[0218] In lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to include ionizable cationic lipids that can complex and associate with the negatively charged backbone of the nucleic acid core. Ionizable cationic lipids with apparent pKa values ​​less than about 7 have the advantage that they can complex with the negatively charged nucleic acid backbone and provide the cationic lipid to be loaded onto the lipid nanoparticle at pH values ​​below the pKa of the ionizable lipid (where the cationic lipid is positively charged). Then, at physiological pH values, the lipid nanoparticles can assume a relatively neutral outer surface, which allows the particles to have a significantly extended circulation half-life after iv administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, strong transfection, improved penetration into the tissue to which the drug is delivered, and low levels of cytotoxicity and immunogenicity.

[0219] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely investigated as synthetic materials for the delivery of nucleic acid drugs. In these early efforts, nucleic acids were mixed at physiological pH and then condensed with cationic lipids to form lipid-nucleic acid complexes (known as lipoplexes). However, lipoplexes were found to be unstable and characterized by a wide size distribution (ranging from submicron scale to several microns). Lipoplexes such as Lipofectamine® reagent have proven quite useful in in vitro transfection. However, these first generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (imparted by cationic lipids) result in short plasma clearance, hemolysis and other toxicities, and immune system activation.

[0220] Lipid-mRNA formulation The mRNA as disclosed herein, or a pharma- ceutically acceptable salt thereof, can be incorporated into a lipid formulation (ie, a lipid-based delivery vehicle).

[0221] In the context of the present disclosure, the lipid-based delivery vehicle typically serves to transport the desired mRNA to a target cell or tissue. The lipid-based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid-based delivery vehicle is a liposome, cationic liposome, or lipid nanoparticle comprising the mRNA of the present disclosure. In some embodiments, the lipid-based delivery vehicle comprises a nanoparticle or bilayer of lipid molecules and the mRNA of the present disclosure. In some embodiments, the lipid bilayer preferably further comprises a neutral lipid or polymer. In some embodiments, the lipid formulation preferably comprises a liquid medium. In some embodiments, the formulation preferably further encapsulates a nucleic acid. In some embodiments, the lipid formulation preferably further comprises a nucleic acid and a neutral lipid or polymer. In some embodiments, the lipid formulation preferably encapsulates the nucleic acid.

[0222] Provided herein is a lipid formulation comprising one or more therapeutic mRNA molecules encapsulated within the lipid formulation. In some embodiments, the lipid formulation comprises a liposome. In some embodiments, the lipid formulation comprises a cationic liposome. In some embodiments, the lipid formulation comprises a lipid nanoparticle.

[0223] In some embodiments, the mRNA is fully encapsulated in the lipid portion of the lipid formulation, so that the mRNA in the lipid formulation is resistant to degradation by nucleases in aqueous solution.In another embodiment, the lipid formulation described herein is substantially non-toxic to mammals, such as humans.

[0224] The lipid formulations of the present disclosure typically have a total lipid:RNA ratio (mass / mass ratio) of about 1:1 to about 100:1, about 1:1 to about 50:1, about 2:1 to about 45:1, about 3:1 to about 40:1, about 5:1 to about 38:1, about 6:1 to about 40:1, about 7:1 to about 35:1, about 8:1 to about 30:1, about 10:1 to about 25:1, about 8:1 to about 12:1, about 13:1 to about 17:1, about 18:1 to about 24:1, or about 20:1 to about 30:1. In some preferred embodiments, the total lipid:RNA ratio (mass / mass ratio) is about 10:1 to about 25:1. The ratio may be any value or subvalue within the recited range, including the endpoints.

[0225] The lipid formulations of the present disclosure typically have an average diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, about 35nm, about 40nm, about 45nm, about 50nm, about 55nm, about 60nm, about 65nm, about 70nm, about 75nm, about 80nm, about 85nm, about 90nm, about 95nm, about 100nm, about 105nm, about 110nm, about 115nm, about 120nm, about 125nm, about 130nm, about 135nm, about 140nm, about 145nm or about 150nm, and is substantially non-toxic. The diameter can be any value or part value within the recited range, including the end values. In addition, nucleic acid is resistant to degradation by nuclease in aqueous solution when present in the lipid nanoparticle of the present disclosure.

[0226] In a preferred embodiment, the lipid formulation comprises mRNA, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The lipid formulation can also include cholesterol.

[0227] In the nucleic acid-lipid formulation, the mRNA can be completely encapsulated in the lipid portion of the formulation, thereby protecting the nucleic acid from nuclease degradation.In a preferred embodiment, the lipid formulation comprising mRNA can be completely encapsulated in the lipid portion of the lipid formulation, thereby protecting the nucleic acid from nuclease degradation.In certain cases, the mRNA in the lipid formulation is not substantially degraded even after the particle is exposed to nuclease at 37 ℃ for at least 20 minutes, 30 minutes, 45 minutes or 60 minutes. In certain other cases, the mRNA in the lipid formulation is not substantially degraded after incubating the formulation in serum at 37° C. for at least 30 minutes, 45 minutes, or 60 minutes, or for at least 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, or 36 hours. In another embodiment, the mRNA is complexed with the lipid portion of the formulation.

[0228] For nucleic acids, complete encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that enhances fluorescence when associated with nucleic acid. Encapsulation is determined by adding the dye to the lipid formulation, measuring the resulting fluorescence, and comparing it to that observed when a small amount of a non-ionic detergent is added. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation is determined by the following formula: E=(I 0 -I) / I 0 where I is the fluorescence intensity before the addition of the surfactant, I 0 refers to the fluorescence intensity after addition of surfactant.

[0229] In another embodiment, the present disclosure provides a nucleic acid-lipid composition comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of mRNA-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of mRNA-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of mRNA-lipid nanoparticles.

[0230] In some embodiments, the lipid formulation comprises mRNA that is fully encapsulated within the lipid portion of the formulation, and is present in about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 30% to about 90% of the particle. , about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fractional value or range thereof) of the mRNA is encapsulated. The amount may be any value or subvalue within the recited range, including the endpoints.

[0231] Depending on the intended use of the lipid formulation, the proportions of the components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0232] According to some embodiments, the expressible polynucleotide and mRNA constructs described herein are formulated in a lipid. The lipid formulation is preferably selected from, but not limited to, liposomes, cationic liposomes and lipid nanoparticles. In a preferred embodiment, the lipid formulation is: (a) an mRNA of the present disclosure; (b) a cationic lipid; (c) an aggregation reducing agent (e.g., a polyethylene glycol (PEG) lipid or a PEG-modified lipid); and (d) optionally a non-cationic lipid (such as a neutral lipid); and (e) optionally, a cationic liposome or lipid nanoparticle (LNP) comprising a sterol; It is.

[0233] In some embodiments, the cationic lipid is an ionizable cationic lipid. In one embodiment, the lipid nanoparticle formulation is composed of (i) at least one cationic lipid, (ii) a helper lipid, (iii) a sterol (e.g., cholesterol), and (iv) a PEG lipid in a molar ratio of about 20% to about 40% ionizable cationic lipid, about 25% to about 45% helper lipid, about 25% to about 45% sterol, and about 0.5% to 5% PEG lipid. Examples of cationic lipids (including ionizable cationic lipids), helper lipids (e.g., neutral lipids), sterols, and lipids (e.g., PEG lipids) containing ligands are provided herein below.

[0234] Cationic lipids The lipid formulation preferably comprises a cationic lipid suitable for forming cationic liposomes or lipid nanoparticles. Cationic lipids have been widely studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake. Generally, cationic lipids are amphiphiles that contain a positively charged hydrophilic head group, two (or more) lipophilic tails or steroid moieties, and a link between these two domains. Preferably, the cationic lipid has a net positive charge at around physiological pH. Cationic liposomes have traditionally been the most commonly used non-viral delivery system for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA / small hairpin RNA-shRNA. Cationic lipids, such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate), can form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interactions, resulting in high in vitro transfection efficiency.

[0235] In the lipid formulations disclosed herein, the cationic lipids can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salts), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleo ... (oxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), (N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl) ), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2- Hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriacin-6,9,28 It may be any of 31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), or a combination thereof. Other cationic lipids include N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(1,2-dimyristyloxyprop-3-yl)-N,Cationic lipids include, but are not limited to, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE) and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC). In addition, commercially available preparations of cationic lipids can be used, such as, for example, LIPOFECTIN (containing DOTMA and DOPE, available from GIBCO / BRL) and Lipofectamine (containing DOSPA and DOPE, available from GIBCO / BRL).

[0236] Other suitable cationic lipids are disclosed in International Publication Nos. WO09 / 086558, WO09 / 127060, WO10 / 048536, WO10 / 054406, WO10 / 088537, WO10 / 129709 and WO2011 / 153493, U.S. Patent Application Publication Nos. 2011 / 0256175, 2012 / 0128760 and 2012 / 0027803, U.S. Patent No. 8,158,601, and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are incorporated herein by reference.

[0237] Other suitable cationic lipids include cationic lipids with alternative fatty acid groups and other dialkylamino groups, including dialkylamino groups with different alkyl substituents (e.g., N-ethyl-N-methylamino and N-propyl-N-ethylamino). These lipids are part of a subcategory of cationic lipids called amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. In general, amino lipids with shorter saturated acyl chains are easier to size, especially when the complexes must be sized to less than about 0.3 microns for sterilization purposes. Amino lipids with carbon chain lengths of C 14 ~C 22 Other scaffolds can be used to separate the amino group and the fatty acid or fatty acid alkyl moiety of the amino lipid.

[0238] In some embodiments, the lipid formulation comprises a cationic lipid of formula I according to patent application PCT / EP2017 / 064066, the disclosure of which is hereby incorporated by reference.

[0239] In some embodiments, the amino or cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. Of course, it will be understood that the addition or removal of protons, which correlates with pH, ​​is an equilibrium process, and when referring to charged or neutral lipids, the nature of the predominant species is being described, and not all of the lipids need be present in a charged or neutral form. Lipids having two or more protonatable or deprotonatable groups, or lipids that are zwitterionic, are not excluded from use in the present disclosure. In certain embodiments, the protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11. In some embodiments, the ionizable cationic lipid has a pKa of about 5 to about 7. In some embodiments, the ionizable cationic lipid has a pKa of about 6 to about 7.

[0240] In some embodiments, the lipid formulation comprises an ionizable cationic lipid of formula I: [ka] or a pharma- ceutically acceptable salt or solvate thereof, wherein R 5 and R 6 are each independently a straight or branched chain C 1 -C 31 Alkyl, C 2 -C 31 Alkenyl or C 2 -C 31 L is selected from the group consisting of alkynyl and cholesteryl;5 and L 6 are each independently linear C 1 -C 20 Alkyl and C 2 -C 20 alkenyl; X 5 is -C(O)O- (thereby, -C(O)OR 6 is formed) or -OC(O)- (thereby, -OC(O)-R 6 is formed), and X 6 is -C(O)O- (thereby, -C(O)OR 5 is formed) or -OC(O)- (thereby, -OC(O)-R 5 is formed), and X 7 is S or O, and L 7 is absent or is lower alkyl, R 4 is a linear or branched chain C 1 -C 6 is alkyl, R 7 and R 8 are each independently hydrogen and straight or branched chain C 1 -C 6 is selected from the group consisting of alkyl.

[0241] In some embodiments, X 7 is S.

[0242] In some embodiments, X 5 is -C(O)O-, and therefore -C(O)OR 6 is formed, and X 6 is -C(O)O-, and therefore -C(O)OR 5 is formed.

[0243] In some embodiments, R 7 and R 8 are each independently selected from the group consisting of methyl, ethyl, and isopropyl.

[0244] In some embodiments, L 5and L 6 are each independently 1- C 10 In some embodiments, L 5 is C 1- C 3 is alkyl, and L 6 is C 1- C 5 In some embodiments, L 6 is C 1- C 2 In some embodiments, L 5 and L 6 are linear C 7 In some embodiments, L 5 and L 6 are linear C 9 It is an alkyl.

[0245] In some embodiments, R 5 and R 6 are each independently alkenyl. In some embodiments, R 6 is alkenyl. In some embodiments, R 6 is C 2- C 9 In some embodiments, the alkenyl contains one double bond. In some embodiments, R 5 and R 6 Each is alkyl. In some embodiments, R 5 is branched alkyl. In some embodiments, R 5 and R 6 are each independently 9 Alkyl, C 9 Alkenyl and C 9 In some embodiments, R is selected from the group consisting of alkynyl. 5 and R 6 are each independently 11 Alkyl, C 11 Alkenyl and C 11 In some embodiments, R is selected from the group consisting of alkynyl. 5 and R6 are each independently 7 Alkyl, C 7 Alkenyl and C 7 In some embodiments, R is selected from the group consisting of alkynyl. 5 is -CH((CH 2 ) p CH 3 ) 2 or -CH((CH 2 ) p CH 3 )((CH 2 ) p-1 CH 3 ), where p is 4 to 8. In some embodiments, p is 5 and L 5 is C 1- C 3 In some embodiments, p is 6 and L is alkyl. 5 is C 3 In some embodiments, p is 7. In some embodiments, p is 8 and L is alkyl. 5 is C 1- C 3 In some embodiments, R 5 is -CH((CH 2 ) p CH 3 )((CH 2 ) p-1 CH 3 ) wherein p is 7 or 8.

[0246] In some embodiments, R 4 is ethylene or propylene. In some embodiments, R 4 is n-propylene or isobutylene.

[0247] In some embodiments, L 7 does not exist, and R 4 is ethylene, and X 7 is S and R 7 and R 8 Each is methyl. In some embodiments, L 7 does not exist, and R 4is n-propylene, and X 7 is S and R 7 and R 8 Each is methyl. In some embodiments, L 7 does not exist, and R 4 is ethylene, and X 7 is S and R 7 and R 8 are each ethyl.

[0248] In some embodiments, X 7 is S and X 5 is -C(O)O-, which results in -C(O)OR 6 is formed, and X 6 is -C(O)O-, which results in -C(O)OR 5 is formed, and L 5 and L 6 are each independently linear C 3- C 7 is alkyl, and L 7 does not exist, and R 5 is -CH((CH 2 ) p CH 3 ) 2 and R 6 is C 7- C 12 In some further embodiments, p is 6 and R 6 is C 9 It is alkenyl.

[0249] In some embodiments, the lipid formulation comprises: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The ionizable cationic lipid comprises an ionizable cationic lipid selected from the group consisting of:

[0250] In some embodiments, the lipid formulation can include an ionizable cationic lipid selected from the group consisting of lipid number 1 to lipid number 5 as shown in Table 2. [Table 2-1] [Table 2-2]

[0251] In some preferred embodiments, the lipid formulation comprises an ionizable cationic lipid having the structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0252] Some embodiments may explicitly exclude any one or more of the lipids listed herein.

[0253] Helper lipids and sterols The mRNA-lipid formulations of the present disclosure can include helper lipids, which can be referred to as neutral lipids, neutral helper lipids, non-cationic lipids, non-cationic helper lipids, anionic lipids, anionic helper lipids, or zwitterionic lipids. Lipid formulations, particularly cationic liposomes and lipid nanoparticles, have been shown to have increased cellular uptake when helper lipids are present in the formulation. (Curr. Drug Metab. 2014; 15(9): 882-92). For example, several studies have shown that neutral and zwitterionic lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), di-oleoyl-phosphatidyl-ethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), are more fusogenic (i.e., promote fusion) than cationic lipids and can induce cell membrane fusion and disruption by influencing the polymorphic characteristics of lipid-nucleic acid complexes to promote a lamellar to hexagonal phase transition. (Nanomedicine (Lond). 2014 Jan; 9 (1): 105-20). In addition, the use of helper lipids can help mitigate any adverse effects, such as toxicity and potential immunogenicity, from the use of many common cationic lipids.

[0254] Non-limiting examples of non-cationic lipids suitable for the lipid formulations of the present disclosure include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (DPPC), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (DPPG ...choline (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylcho phospholipids such as palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group of these lipids is preferably C 10 -C 24 The acyl group is derived from a fatty acid having a carbon chain of, for example, lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.

[0255] Further examples of non-cationic lipids include sterols such as cholesterol and its derivatives. One study concluded that cholesterol, as a helper lipid, expands the charge spacing of the lipid layer that interacts with nucleic acid, so that its charge distribution more closely matches that of nucleic acid. (JRSoc.Interface.2012 Mar 7;9(68):548-561). Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5α-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether and 6-ketocholestanol, non-polar analogs such as 5α-cholestane, cholestenone, 5α-cholestanone, 5α-cholestanone and cholesteryl decanoate, and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0256] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivatives.In another embodiment, the helper lipid present in the lipid formulation comprises or consists of one or more phospholipids, for example, lipid formulations that do not contain cholesterol.In yet another embodiment, the helper lipid present in the lipid formulation consists of lipid formulations that do not contain cholesterol or its derivatives, for example, lipid formulations that do not contain phospholipids.

[0257] Other examples of helper lipids include phosphorus-free lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkylaryl sulfate polyethoxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0258] In some embodiments, the helper lipid comprises about 20 mol% to about 50 mol%, about 22 mol% to about 48 mol%, about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol% or about 39 mol% (or any fractional value or range therebetween) of the total lipid present in the lipid formulation.

[0259] In some embodiments, the total helper lipid in the formulation comprises two or more helper lipids, the total amount of helper lipids being about 20 mol% to about 50 mol%, about 22 mol% to about 48 mol%, about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fractional value or range thereof) of the total lipid present in the lipid formulation. In some embodiments, the helper lipid is a combination of DSPC and DOTAP. In some embodiments, the helper lipid is a combination of DSPC and DOTMA.

[0260] The cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 30 mol% to about 40 mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, or about 40 mol% of the total lipid present in the lipid formulation.

[0261] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ±5 mol %.

[0262] A lipid formulation comprising a cationic lipid compound or an ionizable cationic lipid compound may have, on a molar basis, about 20-40% cationic lipid compound, about 25-40% cholesterol, about 25-50% helper lipid, and about 0.5-5% polyethylene glycol (PEG) lipid, the percentages being based on the total lipid present in the formulation. In some embodiments, the composition has about 22-30% cationic lipid compound, about 30-40% cholesterol, about 30-40% helper lipid, and about 0.5-3% PEG lipid, the percentages being based on the total lipid present in the formulation.

[0263] Lipid conjugates The lipid formulations described herein may further comprise lipid conjugates. The conjugated lipids are useful for preventing particle aggregation. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof. In addition, lipid delivery vehicles can be used to target specific targets by attaching ligands (e.g., antibodies, peptides, and sugar chains) to their surface or to the end of the attached PEG chain (Front.Pharmacol.2015 Dec 1;6:286).

[0264] In a preferred embodiment, the lipid conjugate is a PEG-lipid. The inclusion of polyethylene glycol (PEG) as a coating or surface ligand in lipid formulations (a technique called PEGylation) helps protect nanoparticles from the immune system and avoid uptake by the RES (Nanomedicine (Lond). 2011 Jun;6(4):715-28). PEGylation is widely used to stabilize lipid formulations and their payloads by physical, chemical and biological mechanisms. PEG-lipids (e.g. PEG-DSPE), like surfactants, can enter the lipid formulation and form a hydration layer and a steric barrier on the surface. Based on the degree of PEGylation, the surface layer can be generally divided into two types: brush layer and mushroom layer. In PEG-DSPE stabilized formulations, PEG adopts a mushroom-like structure at low levels of PEGylation (usually less than 5 mol%) and transitions to a brush-like structure with increasing PEG-DSPE content above a certain level (J. Nanomaterials. 2011;2011:12). Increasing PEGylation has been shown to significantly increase the circulation half-life of lipid formulations (Annu. Rev. Biomed. Eng. 2011 Aug 15;13():507-30, J. Control Release. 2010 Aug 3;145(3):178-81).

[0265] Suitable examples of PEG lipids include, but are not limited to, PEG conjugated to dialkyloxypropyl (PEG-DAA), PEG conjugated to diacylglycerol (PEG-DAG), PEG conjugated to a phospholipid, such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide, PEG conjugated to cholesterol, or derivatives thereof, and mixtures thereof.

[0266] PEG is a linear water-soluble polymer consisting of repeating ethylene PEG units with two terminal hydroxyl groups. PEG is classified according to its molecular weight, and there are several types of PEG: monomethoxypolyethyleneglycol (MePEG-OH), monomethoxypolyethyleneglycol-succinate (MePEG-S), monomethoxypolyethyleneglycol-succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethyleneglycol-amine (MePEG-NH 2 ), monomethoxypolyethyleneglycol-tresylate (MePEG-TRES), monomethoxypolyethyleneglycol-imidazolyl-carbonyl (MePEG-IM), and compounds containing terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH 2 ) are mentioned.

[0267] The PEG moiety of the PEG-lipid conjugates described herein may have an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain cases, the PEG moiety has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons. The average molecular weight may be any value or subvalue within the recited range, including the endpoints.

[0268] In certain cases, the PEG monomer can be optionally substituted with an alkyl group, an alkoxy group, an acyl group, or an aryl group. The PEG can be directly conjugated to the lipid or can be linked to the lipid via a linker moiety. Any linker moiety suitable for linking the PEG to the lipid can be used, including linker moieties that do not contain esters and linker moieties that contain esters. In a preferred embodiment, the linker moiety is an ester-free linker moiety. Suitable linker moieties that do not contain esters include amide (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-SS-), ether (-O-), succinyl (-(O)CCH 2 CH 2 C(O)-), succinamidyl (-NHC(O)CH 2 CH 2 Linkers that can be used to link the PEG to the lipid include, but are not limited to, linkers that contain both a carbamate linker moiety and an amide linker moiety. In a preferred embodiment, a carbamate linker is used to link the PEG to the lipid.

[0269] In another embodiment, the PEG is linked to the lipid using an ester-containing linker moiety. Suitable ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinoyl, phosphate ester (-O-(O)POH-O-), sulfonate ester, and combinations thereof.

[0270] Phosphatidylethanolamines with various acyl chain groups of different chain length and degree of saturation can be conjugated to PEG to form lipid conjugates. Such phosphatidylethanolamines are commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. 10 ~C 20Phosphatidylethanolamines containing saturated or unsaturated fatty acids ranging from 0 to 100% are preferred. Phosphatidylethanolamines having mono- or di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE) and distearoyl-phosphatidylethanolamine (DSPE).

[0271] In some embodiments, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C 10 ) conjugate, PEG-dilauryloxypropyl (C 12 ) conjugate, PEG-dimyristyloxypropyl (C 14 ) conjugate, PEG-dipalmityloxypropyl (C 16 ) conjugate or PEG-distearyloxypropyl (C 18 ) conjugates. In these embodiments, the PEG preferably has an average molecular weight of about 750 to about 2,000 daltons. In certain embodiments, the terminal hydroxyl group of the PEG is replaced with a methyl group.

[0272] In addition to the above, other hydrophilic polymers can be used instead of PEG. Examples of suitable polymers that can be used instead of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose.

[0273] In some embodiments, the lipid conjugate (e.g., a PEG lipid) comprises about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 0.9 mol% to about 1.6 mol%, about 0.9 mol% to about 1.8 mol%, about 1 mol% to about 1.8 mol%, about 1 mol% to about 1.7 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, or about 1.4 mol% to about 1.6 mol% (or any fractional value or range therein) of the total lipid present in the lipid formulation. In another embodiment, the lipid conjugate (e.g., PEG lipid) comprises about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, or about 5% (or any fractional value or range thereof) of the total lipid present in the lipid formulation. The amount may be any value or subvalue within the recited range, including the endpoints.

[0274] In some preferred embodiments, the PEG lipid is PEG550-PE. In some preferred embodiments, the PEG lipid is PEG750-PE. In some preferred embodiments, the PEG lipid is PEG2000-DMG.

[0275] The percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid formulations of the present disclosure is a target amount, and the actual amount of lipid conjugate present in the formulation may vary, for example, by ±0.5 mol %. It will be apparent to one of skill in the art that the concentration of the lipid conjugate may vary depending on the lipid conjugate used and the rate at which the lipid formulation becomes fusogenic.

[0276] Mechanism of action by which lipid formulations are taken up into cells Lipid formulations for intracellular delivery of nucleic acids, particularly liposomes, cationic liposomes and lipid nanoparticles, are designed to be taken up by cells by penetrating the target cell through the utilization of the endocytosis mechanism of the target cell, where the contents of the lipid delivery vehicle are delivered to the cytosol of the target cell. (Nucleic Acid Therapeutics, 28(3):146-157, 2018). Specifically, in the case of the CFTR mRNA-lipid formulation described herein, the mRNA-lipid formulation enters the lung epithelial cell by receptor-mediated endocytosis. Prior to endocytosis, the surface functionalized ligands of the lipid delivery vehicle, such as PEG lipids, are shed from the surface, which induces internalization into the target cell. During endocytosis, some of the cell's plasma membrane parts surround the vector and incorporate it into a vesicle, which then separates from the plasma membrane, enters the cytosol, and finally follows the endolysosomal pathway. For delivery vehicles that contain ionizable cationic lipids, the acidity of the endosomes increases as they mature, resulting in vehicles with a large positive charge on the surface. The interaction of the delivery vehicle with the endosomal membrane then leads to membrane fusion, delivering the payload to the cytoplasm. For mRNA payloads, the cell's own internal translation process then translates the mRNA into the encoded protein. The encoded protein can then undergo further post-translational processing, including transport to the target organelle or its intracellular location. In the case of the CFTR protein, the CFTR protein is translocated to the cell membrane.

[0277] By controlling the composition and concentration of the lipid conjugate, the rate at which the lipid conjugate is released and exchanged from the lipid formulation, and subsequently the rate at which the lipid formulation becomes fusogenic, can be controlled. In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to vary and / or control the rate at which the lipid formulation becomes fusogenic. Other methods that can be used to control the rate at which the lipid formulation becomes fusogenic will be apparent to those skilled in the art upon reading this disclosure. Also, by controlling the composition and concentration of the lipid conjugate, the particle size of the liposome or lipid can be controlled.

[0278] Preparation of lipid formulations There are many different methods for preparing lipid formulations containing nucleic acids (Curr. Drug Metabol. 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20). Thin film hydration, double emulsion, reverse phase evaporation, microfluidic preparation, asymmetric double centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution, and encapsulation in preformed liposomes are briefly described herein.

[0279] Thin film hydration method In the thin film hydration (TFH) or Bangham method, lipids are dissolved in an organic solvent and then evaporated using a rotary evaporator to form a thin lipid film. After hydration of the layer with an aqueous buffer solution containing the compound to be encapsulated, multilamellar vesicles (MLVs) are formed, and the size of the vesicles can be reduced to produce small or large unilamellar vesicles (LUVs and SUVs) by extruding the starting MLVs through a membrane or by sonication of the MLVs.

[0280] Double Emulsion Lipid formulations can also be prepared by the double emulsion method, which involves dissolving lipids in a water / organic solvent mixture. The organic solution containing aqueous droplets is mixed with an excess of aqueous medium to form a water-in-oil-in-water (W / O / W) double emulsion. After vigorous mechanical shaking, some of the aqueous droplets collapse, forming large unilamellar vesicles (LUVs).

[0281] Reverse Phase Evaporation Reverse phase evaporation (REV) method can also be used to obtain LUVs with encapsulated nucleic acids. In this technique, a two-phase system is formed by dissolving phospholipids in an organic solvent and an aqueous buffer. The resulting suspension is then sonicated for a short period of time until the mixture becomes a clear, one-phase dispersion. The organic solvent is evaporated under reduced pressure to obtain the lipid formulation. This technique has been used to encapsulate a variety of large and small hydrophilic molecules, including nucleic acids.

[0282] Microfluidic preparation method Microfluidic methods offer the possibility to control the lipid hydration process, unlike other bulk techniques. Methods can be classified as continuous-flow and droplet-based microfluidic methods, depending on the way the flow is manipulated. In the microfluidic hydrodynamic focusing (MHF) method, which operates in a continuous-flow regime, lipids are dissolved in isopropyl alcohol and hydrodynamically focused at a microchannel cross-junction between two aqueous buffer streams. By adjusting the flow rate and thus controlling the lipid solution / buffer dilution process, the vesicle size can be controlled. This method can be used to generate oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three inlet ports and one outlet port.

[0283] Asymmetric double centrifugation Asymmetric double centrifugation (DAC) is different from the conventional centrifugation method because it utilizes an additional rotation around its own vertical axis. It effectively homogenizes by creating two overlapping movements, pushing the sample outwards like a normal centrifuge, and then pushing the sample towards the center of the vial with an additional rotation. A viscous vesicular phospholipid gel (VPC) is obtained by mixing lipids with NaCl solution, which is then diluted to obtain a lipid formulation dispersion. The size of the lipid formulation can be adjusted by optimizing the DAC speed, lipid concentration, and homogenization time.

[0284] Ethanol injection method The ethanol injection (EI) method can be used to encapsulate nucleic acids, in which an ethanol solution is rapidly injected with a needle to dissolve lipids in an aqueous medium containing the nucleic acid to be encapsulated. When the phospholipids are dispersed throughout the medium, vesicles spontaneously form.

[0285] Detergent dialysis Nucleic acids can be encapsulated using detergent dialysis. Briefly, lipids and plasmids are solubilized in a detergent solution of appropriate ionic strength, and after the detergent is removed by dialysis, a stabilized lipid formulation is formed. Unencapsulated nucleic acids are then removed by ion exchange chromatography, and empty vesicles are removed by sucrose density gradient centrifugation. This technique is highly sensitive to the cationic lipid content and salt concentration of the dialysis buffer, and the method is also difficult to scale.

[0286] Spontaneous vesicle formation using ethanol dilutions Stable lipid formulations can also be generated by the spontaneous vesicle formation method with ethanol dilution, in which the controlled addition of lipid dissolved in ethanol to a rapidly mixed aqueous buffer containing nucleic acid results in the instantaneous formation of vesicles containing encapsulated nucleic acid upon stepwise or dropwise dilution with ethanol.

[0287] Encapsulation in preformed liposomes Starting from preformed liposomes, the uptake of nucleic acids can be obtained by two different methods: (1) simply mixing cationic liposomes with nucleic acids to obtain electrostatic complexes called "lipoplexes" (which can be successfully used to transfect cell cultures, but which are characterized by low encapsulation efficiency and poor in vivo performance); and (2) slowly adding absolute ethanol to a suspension of cationic vesicles to a concentration of 40% (v / v) to destabilize the liposomes before adding the nucleic acid dropwise to obtain encapsulated vesicles (however, the two main steps that characterize the encapsulation process are very delicate and require the particle size to be reduced).

[0288] CFTR mRNA lipid formulation The present disclosure provides lipid formulations comprising mRNA (CFTR mRNA) encoding an enzyme having cystic fibrosis transmembrane conductance regulator (CFTR) activity. After transfection of one or more target cells with the CFTR mRNA lipid formulation of the present disclosure, the expression of the CFTR enzyme encoded by the mRNA will be stimulated, and the ability of the target cells to express the CFTR enzyme will be enhanced. The CFTR mRNA can be any mRNA suitable for expressing the CFTR enzyme in vivo.

[0289] In a first CFTR mRNA-lipid formulation, the CFTR mRNA-lipid formulation comprises a compound of formula (I) and an mRNA encoding an enzyme having CFTR activity. In some embodiments, the mRNA encodes a CFTR enzyme consisting of a sequence that is 95% identical to SEQ ID NO: 93. In some embodiments, the mRNA encodes a CFTR enzyme consisting of SEQ ID NO: 93. In some embodiments, the mRNA encodes a CFTR enzyme consisting of a sequence that is 95% identical to SEQ ID NO: 99. In some embodiments, the mRNA encodes a CFTR enzyme consisting of SEQ ID NO: 99. Compounds of formula I can be selected based on desirable properties including their lipophilicity, potency, selectivity for a given target cell, in vivo biodegradability, toxicity and immunogenicity profile, and the pKa of ionizable / protonable groups on the compound of formula I.

[0290] In some embodiments of the first CFTR mRNA-lipid formulation, X 7 is S. In some embodiments, X 5 is -C(O)O-, and therefore -C(O)OR 6 is formed, and X 6 is -C(O)O-, and therefore -C(O)OR 5 In some embodiments, R 7 and R 8 are each independently selected from the group consisting of methyl, ethyl, and isopropyl. 5 and L 6 are each independently 1- C 10 In some embodiments, L 5 is C 1- C 3 is alkyl, and L 6 is C 1- C 5 In some embodiments, L 6 is C 1- C 2 In some embodiments, L 5 and L6 are linear C 7 In some embodiments, L 5 and L 6 are linear C 9 In some embodiments, R 5 and R 6 are each independently alkenyl. In some embodiments, R 6 is alkenyl. In some embodiments, R 6 is C 2- C 9 In some embodiments, the alkenyl contains one double bond. In some embodiments, R 5 and R 6 Each is alkyl. In some embodiments, R 5 is a branched alkane. In some embodiments, R 5 and R 6 are each independently 9 Alkyl, C 9 Alkenyl and C 9 In some embodiments, R is selected from the group consisting of alkynyl. 5 and R 6 are each independently 11 Alkyl, C 11 Alkenyl and C 11 In some embodiments, R is selected from the group consisting of alkynyl. 5 and R 6 are each independently 7 Alkyl, C 7 Alkenyl and C 7 In some embodiments, R is selected from the group consisting of alkynyl. 5 is -CH((CH 2 ) p CH 3 ) 2 or -CH((CH 2 ) p CH 3 )((CH 2 ) p-1 CH 3), where p is 4 to 8. In some embodiments, p is 5 and L 5 is C 1- C 3 In some embodiments, p is 6 and L is alkyl. 5 is C 3 In some embodiments, p is 7. In some embodiments, p is 8 and L is alkyl. 5 is C 1- C 3 In some embodiments, R 5 is -CH((CH 2 ) p CH 3 )((CH 2 ) p-1 CH 3 ), and p is 7 or 8. In some embodiments, R 4 is ethylene or propylene. In some embodiments, R 4 is n-propylene or isobutylene. In some embodiments, L 7 does not exist, and R 4 is ethylene, and X 7 is S and R 7 and R 8 Each is methyl. In some embodiments, L 7 does not exist, and R 4 is n-propylene, and X 7 is S and R 7 and R 8 Each is methyl. In some embodiments, L 7 does not exist, and R 4 is ethylene, and X 7 is S and R 7 and R 8 are each ethyl.

[0291] In some embodiments of the first CFTR mRNA-lipid formulation, X 7 is S and X 5 is -C(O)O-, and therefore -C(O)OR 6 is formed, and X 6is -C(O)O-, and therefore -C(O)OR 5 is formed, and L 5 and L 6 are each independently linear C 3- C 7 is alkyl, and L 7 does not exist, and R 5 is -CH((CH 2 ) p CH 3 ) 2 and R 6 is C 7- C 12 In some further embodiments, p is 6 and R 6 is C 9 It is alkenyl.

[0292] Any mRNA encoding an enzyme having CFTR activity is suitable for inclusion in the first CFTR mRNA-lipid formulation of the present disclosure. In some embodiments, a suitable mRNA is wild-type human CFTR mRNA of the sequence of SEQ ID NO: 93. Preferably, the CFTR mRNA has low immunogenicity, high in vivo stability, and high translation efficiency. In some embodiments, the CFTR mRNA is expressible in human lung epithelial cells. In some embodiments, the CFTR mRNA has a coding region that is codon-optimized. In some embodiments, the CFTR mRNA comprises modified uridine nucleotides. In some embodiments, the modified uridine nucleotides are N,N-dimethylformamide, ... 1 In some embodiments, the modified uridine nucleotide is 5-methoxyuridine. In some embodiments, the CFTR mRNA can be any of the CFTR mRNA constructs described herein.

[0293] In some embodiments of the first CFTR mRNA-lipid formulation, the mRNA comprises an open reading frame (ORF or coding region) selected from the sequence including SEQ ID NOs: 100-105. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 100. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 101. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 102. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 103. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 104. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 105. In some embodiments, the mRNA comprises a sequence that is about 85% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 90% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 95% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 96% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 97% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 98% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 99% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 99.5% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence having SEQ ID NO:49.In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 53. In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 66. In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 68. In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 69. In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 72.

[0294] In any of the embodiments of the first CFTR mRNA-lipid formulation, the CFTR mRNA-lipid formulation comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle fully encapsulates the CFTR mRNA.

[0295] In some embodiments, the lipid nanoparticles have an average particle size of less than about 100 nm. In some embodiments, the lipid nanoparticles have an average particle size of about 55 to about 85 nm. In some embodiments, the lipid nanoparticles encapsulate at least about 50% of the mRNA. In some embodiments, the lipid nanoparticles encapsulate at least about 85% of the mRNA. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 90%. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 95%.

[0296] In a second CFTR mRNA-lipid formulation, the CFTR mRNA-lipid formulation comprises an ionizable cationic lipid, [ka] or a pharma- ceutically acceptable salt thereof, and an mRNA encoding an enzyme having CFTR activity.

[0297] Any mRNA encoding an enzyme having CFTR activity is suitable for inclusion in the second CFTR mRNA-lipid formulation of the present disclosure. In some embodiments, a suitable mRNA is a wild-type human CFTR mRNA encoding a protein of SEQ ID NO: 93. In some embodiments, the mRNA encodes a CFTR enzyme consisting of a sequence that is 95% identical to SEQ ID NO: 93. In some embodiments, the mRNA encodes a CFTR enzyme consisting of a sequence that is 95% identical to SEQ ID NO: 99. In some embodiments, the mRNA encodes a CFTR enzyme consisting of SEQ ID NO: 99. Preferably, the CFTR mRNA has low immunogenicity, high in vivo stability, and high translation efficiency. In some embodiments, the CFTR mRNA is expressible in human lung epithelial cells. In some embodiments, the CFTR mRNA has a coding region that is codon-optimized. In some embodiments, the CFTR mRNA comprises modified uridine nucleotides. In some embodiments, the modified uridine nucleotides are N 1 In some embodiments, the modified uridine nucleotide is N-methylpseudouridine or 5-methoxyuridine. In some embodiments, the modified uridine nucleotide is 5-methoxyuridine. In some embodiments, the modified uridine nucleotide is N 1 -methylpseudouridine. In some embodiments, the CFTR mRNA can be any of the CFTR mRNA constructs described herein.

[0298] In some embodiments of the second CFTR mRNA-lipid formulation, the mRNA comprises an open reading frame (ORF or coding region) selected from the sequence including SEQ ID NOs: 100-105. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 100. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 101. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 102. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 103. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 104. In some embodiments, the mRNA comprises an ORF having a sequence of SEQ ID NO: 105. In some embodiments, the mRNA comprises a sequence that is about 85% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 90% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 95% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 96% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 97% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 98% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 99% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence that is about 99.5% identical to a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence selected from SEQ ID NOs: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises a sequence having SEQ ID NO:49.In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 53. In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 66. In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 68. In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 69. In some embodiments, the mRNA comprises a sequence having SEQ ID NO: 72.

[0299] In any of the embodiments of the second CFTR mRNA-lipid formulation, the CFTR mRNA-lipid formulation comprises a lipid nanoparticle. In some embodiments, the lipid nanoparticle fully encapsulates the CFTR mRNA.

[0300] In some embodiments, the lipid nanoparticles have an average particle size of less than about 100 nm. In some embodiments, the lipid nanoparticles have an average particle size of about 55 nm to about 85 nm. In some embodiments, the lipid nanoparticles encapsulate at least about 50% of the mRNA. In some embodiments, the lipid nanoparticles encapsulate at least about 85% of the mRNA. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of greater than about 90%.

[0301] In some embodiments, either the first or second CFTR mRNA-lipid formulation further comprises a helper lipid. In some embodiments, the helper lipid is selected from the group consisting of neutral lipids and anionic lipids. In some embodiments, the helper lipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine (PC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine and dimyristoylphosphatidylglycerol (DMPG). In some embodiments, the non-cationic lipid is distearoylphosphatidylcholine (DSPC).

[0302] In some embodiments, either the first or second CFTR mRNA-lipid formulation further comprises cholesterol.

[0303] In some embodiments, either the first or second CFTR mRNA-lipid formulation further comprises a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG.

[0304] In some embodiments, the lipid portion (meaning the total amount of lipid in the formulation) of either the first or second CFTR mRNA-lipid formulation comprises about 48 mol% to about 66 mol% of the cationic lipid, about 2 mol% to about 12 mol% of DSPC, about 25 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of PEG2000-DMG.

[0305] In some embodiments, the lipid portion of either the first or second CFTR mRNA-lipid formulation comprises about 55 mol% to about 61 mol% of the cationic lipid, about 5 mol% to about 9 mol% DSPC, about 29 mol% to about 38 mol% cholesterol, and about 1 mol% to about 2 mol% PEG2000-DMG.

[0306] In some embodiments, the lipid portion of either the first or second CFTR mRNA-lipid formulation comprises about 56 mol% to about 60 mol% of the cationic lipid, about 6 mol% to about 8 mol% DSPC, about 31 mol% to about 34 mol% cholesterol, and about 1.25 mol% to about 1.75 mol% PEG2000-DMG.

[0307] In some embodiments, either the first or second CFTR mRNA-lipid formulation has a total lipid:mRNA weight ratio of about 50:1 to about 10:1. In some embodiments, either the first or second CFTR mRNA-lipid formulation has a total lipid:mRNA weight ratio of about 40:1 to about 20:1. In some embodiments, either the first or second CFTR mRNA-lipid formulation has a total lipid:mRNA weight ratio of about 35:1 to about 25:1. In some embodiments, either the first or second CFTR mRNA-lipid formulation has a total lipid:mRNA weight ratio of about 28:1 to about 32:1. In some embodiments, either the first or second CFTR mRNA-lipid formulation has a total lipid:mRNA weight ratio of about 29:1 to about 31:1.

[0308] Pharmaceutical composition comprising CFTR mRNA and a lipid formulation comprising the cationic lipid ATX-012 The present disclosure provides a pharmaceutical composition comprising: (a) a lipid formulation comprising an ionizable cationic lipid, where the ionizable cationic lipid is ATX-012; and (b) a messenger RNA (mRNA) encoding a peptide having cystic fibrosis transmembrane conductance regulator (CFTR) activity, where the lipid formulation encapsulates the mRNA.

[0309] In some further embodiments, the lipid formulation of the pharmaceutical composition comprises a first helper lipid that is DOTAP and a second helper lipid that is a PEG-lipid conjugate and cholesterol. In some embodiments, the second helper lipid is DSPC. In some embodiments, the PEG-lipid conjugate is PEG-DMG.

[0310] In some further embodiments, the mRNA of the pharmaceutical composition has a sequence selected from the group consisting of SEQ ID NOs: 49, 53, 66, 68, 69 and 72.

[0311] In some further embodiments, the peptide having cystic fibrosis transmembrane conductance regulator (CFTR) activity has a sequence at least about 90% identical to the sequence of SEQ ID NO: 99. In some further embodiments, the peptide having cystic fibrosis transmembrane conductance regulator (CFTR) activity has a sequence at least about 95% identical to the sequence of SEQ ID NO: 99. In some embodiments, the peptide having CFTR activity has a sequence at least about 98% identical to the sequence of SEQ ID NO: 99. In some embodiments, the peptide having CFTR activity has a sequence at least about 99% identical to the sequence of SEQ ID NO: 99. In some embodiments, the peptide having CFTR activity has a sequence of SEQ ID NO: 99.

[0312] In some particular embodiments, (a) i. about 20 mol % to about 30 mol % of an ionizable cationic lipid having the structure of ATX-012 below, [ka] vi. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) in an amount of about 20 mol% to about 30 mol%; vii. about 7 mol% to about 13 mol% of helper lipids; viii. cholesterol from about 33 mol% to about 44 mol%, and ix. A lipid formulation comprising about 0.5 mol% to about 3.0 mol% of a PEG-lipid conjugate; (b) messenger RNA (mRNA) encoding a peptide having cystic fibrosis transmembrane conductance regulator (CFTR) activity; and wherein the lipid formulation encapsulates the mRNA.

[0313] In some embodiments, the lipid formulation (a) is selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymer-based carriers, exosomes, lamellar bodies, micelles, and emulsions.

[0314] In some embodiments, the lipid formulation (a) is a liposome, hi some embodiments, the liposome is selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes.

[0315] In some embodiments, the lipid formulation (a) is a lipid nanoparticle. In some embodiments, the lipid nanoparticle is less than about 200 nm in size. In some embodiments, the lipid nanoparticle is less than about 150 nm in size. In some embodiments, the lipid nanoparticle is less than about 100 nm in size. In some embodiments, the lipid nanoparticle is about 55 nm to about 90 nm in size. Values ​​and ranges recited herein include any subvalues ​​or subranges within those values ​​and ranges.

[0316] In some embodiments, the helper lipid of the lipid formulation (a) is a phospholipid. In some embodiments, the helper lipid of the lipid formulation (a) is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC) and phosphatidylcholine (PC). In some embodiments, the helper lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the lipid formulation (a) comprises about 8 mol% to about 12 mol% of the helper lipid. In some embodiments, the lipid formulation (a) comprises about 9 mol% to about 11 mol% of the helper lipid. In some embodiments, the lipid formulation (a) comprises about 10 mol% of the helper lipid.

[0317] In some embodiments, the PEG-lipid conjugate of the lipid formulation (a) is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG. In some embodiments, the lipid formulation (a) comprises about 0.75 mol% to about 2.5 mol% of the PEG-lipid conjugate. In some embodiments, the lipid formulation (a) comprises about 1.0 mol% to about 2.0 mol% of the PEG-lipid conjugate. In some embodiments, the lipid formulation (a) comprises about 1.25 mol% to about 1.75 mol% of the PEG-lipid conjugate. In some embodiments, the lipid formulation (a) comprises about 1.5 mol% of the PEG-lipid conjugate.

[0318] In some embodiments, the lipid formulation (a) comprises about 22 mol% to about 28 mol% of the ionizable cationic lipid ATX-012. In some embodiments, the lipid formulation (a) comprises about 23 mol% to about 27 mol% of the ionizable cationic lipid ATX-012. In some embodiments, the lipid formulation (a) comprises about 24 mol% to about 26 mol% of the ionizable cationic lipid ATX-012. In some embodiments, the lipid formulation (a) comprises about 25 mol% of the ionizable cationic lipid ATX-012.

[0319] In some embodiments, the lipid formulation (a) comprises about 22 mol% to about 28 mol% DOTAP. In some embodiments, the lipid formulation (a) comprises about 23 mol% to about 27 mol% DOTAP. In some embodiments, the lipid formulation (a) comprises about 24 mol% to about 26 mol% DOTAP. In some embodiments, the lipid formulation (a) comprises about 25 mol% DOTAP.

[0320] In some embodiments, the lipid formulation (a) comprises about 35 mol% to about 41 mol% cholesterol. In some embodiments, the lipid formulation (a) comprises about 36 mol% to about 40 mol% cholesterol.

[0321] In some embodiments, the pharmaceutical composition has a total lipid:mRNA ratio of about 5:1 to about 25:1 by weight. In some embodiments, the composition has a total lipid:mRNA ratio of about 10:1 to about 20:1 by weight. In some embodiments, the composition has a total lipid:mRNA ratio of about 12:1 to about 18:1 by weight. In some embodiments, the composition has a total lipid:mRNA ratio of about 14:1 to about 17:1 by weight. In some embodiments, the composition has a total lipid:mRNA ratio of about 14:1 to about 16:1 by weight. In some embodiments, the composition has a total lipid:mRNA ratio of about 15:1 by weight.

[0322] In some embodiments, the pharmaceutical composition comprises an mRNA encoding a peptide having CFTR activity, the peptide having a sequence at least about 85% identical to the sequence of SEQ ID NO:99. In some embodiments, the peptide having CFTR activity has a sequence at least about 90% identical to the sequence of SEQ ID NO:99. In some embodiments, the peptide having CFTR activity has a sequence at least about 95% identical to the sequence of SEQ ID NO:99. In some embodiments, the peptide having CFTR activity has a sequence at least about 98% identical to the sequence of SEQ ID NO:99. In some embodiments, the peptide having CFTR activity has a sequence at least about 99% identical to the sequence of SEQ ID NO:99. In some embodiments, the peptide having CFTR activity has a sequence of SEQ ID NO:99.

[0323] In some embodiments, the mRNA of the pharmaceutical composition has a sequence selected from the group consisting of SEQ ID NO: 49, 53, 66, 68, 69, and 72. In some embodiments, the mRNA comprises SEQ ID NO: 49. In some embodiments, the mRNA comprises SEQ ID NO: 53. In some embodiments, the mRNA comprises SEQ ID NO: 66. In some embodiments, the mRNA comprises SEQ ID NO: 68. In some embodiments, the mRNA comprises SEQ ID NO: 69. In some embodiments, the mRNA comprises SEQ ID NO: 72.

[0324] In some embodiments, the mRNA of the pharmaceutical composition comprises a 3' poly A tail, hi some embodiments, the 3' poly A tail consists of about 50 to about 120 adenosine monomers.

[0325] In some embodiments, the mRNA of the pharmaceutical composition comprises a 5' cap. In some embodiments, the 5' cap has the structure of formula (Cap V): 7 GpppAmpG, [ka] In the formula, R 1 , R 2 and R 4 each is OH, n is 1, each L is a phosphate linked by a diester bond, and mRNA is an mRNA of that composition.

[0326] In some embodiments, the mRNA of the pharmaceutical composition is selected from the group consisting of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2' -Fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N 1 -Hydroxypseudouridine, N 1 -Methylpseudouridine, 2'-O-methyl-N 1 -Methylpseudouridine, N 1 -Ethylpseudouridine, N1 -Hydroxymethylpseudouridine, aurauridine, N 6 In some embodiments, the one or more chemically modified nucleotides are selected from the group consisting of N-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, and 6-O-methylguanosine. 1 -Methylpseudouridine.

[0327] In some embodiments, the pharmaceutical composition comprises a buffer. In some embodiments, the buffer is a HEPES buffer or a TRIS buffer. In some embodiments, the pH of the HEPES buffer or the TRIS buffer is about 7.0 to about 8.5. In some embodiments, the pH of the HEPES buffer or the TRIS buffer is about 7.4 to about 8.2. In some embodiments, the concentration of the HEPES buffer or the TRIS buffer is about 20 mM to about 80 mM. In some embodiments, the buffer is HEPES at a concentration of about 35 mM to about 70 mM. In some embodiments, the buffer is HEPES at a concentration of about 40 mM to about 60 mM. In some embodiments, the buffer is HEPES at a concentration of about 45 mM to about 55 mM. In some embodiments, the buffer is TRIS at a concentration of about 20 mM to about 50 mM. In some embodiments, the buffer is TRIS at a concentration of about 25 mM to about 40 mM. In some embodiments, the buffer is TRIS at a concentration of about 25 mM to about 35 mM.

[0328] In some embodiments, the pharmaceutical composition comprises sodium chloride (NaCl). In some embodiments, the NaCl concentration is about 10 mM to about 100 mM NaCl. In some embodiments, the NaCl concentration is about 20 mM to about 90 mM NaCl. In some embodiments, the NaCl concentration is about 30 mM to about 80 mM NaCl. In some embodiments, the NaCl concentration is about 35 mM to about 70 mM NaCl. In some embodiments, the NaCl concentration is about 40 mM to about 60 mM NaCl. In some embodiments, the NaCl concentration is about 45 mM to about 55 mM NaCl.

[0329] In some embodiments, the pharmaceutical composition comprises one or more cryoprotectants. In some embodiments, the one or more cryoprotectants are selected from the group consisting of sucrose, glycerol, and a combination of sucrose and glycerol. In some embodiments, the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is glycerol. In some embodiments, the cryoprotectant is a combination of sucrose and glycerol. In some embodiments, the pharmaceutical composition comprises a combination of sucrose at a concentration of about 5% (w / v) to about 18% (w / v) and glycerol at a concentration of about 1% (w / v) to about 9% (w / v). In some embodiments, the pharmaceutical composition comprises a combination of sucrose at a concentration of about 6% (w / v) to about 16% (w / v) and glycerol at a concentration of about 1.5% (w / v) to about 7% (w / v). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 7% (w / v) to about 14% (w / v) in combination with glycerol at a concentration of about 1.75% (w / v) to about 6% (w / v). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 7% (w / v) to about 12% (w / v) in combination with glycerol at a concentration of about 1% (w / v) to about 6% (w / v). In some embodiments, the pharmaceutical composition comprises sucrose at a concentration of about 8% (w / v) to about 11% (w / v) in combination with glycerol at a concentration of about 3% (w / v) to about 6% (w / v).

[0330] In some more particular embodiments, the pharmaceutical composition comprises: A lipid formulation (a) in which the helper lipid is distearoylphosphatidylcholine (DSPC) and the PEG-lipid conjugate is PEG2000-DMG; mRNA (b) comprising SEQ ID NO: 53; Includes.

[0331] In some further embodiments, the lipid formulation is a lipid nanoparticle that is less than about 100 nm in size.

[0332] In some further embodiments, the total lipid:mRNA weight ratio is within the range of about 10:1 to about 20:1, and the peptide having CFTR activity has a sequence at least about 95% identical to the sequence of SEQ ID NO: 99. In some embodiments, the peptide having CFTR activity has the sequence of SEQ ID NO:99.

[0333] In some further embodiments, the pharmaceutical composition further comprises a HEPES buffer or a TRIS buffer, the pH of which is in the range of about 7.0 to about 8.5.

[0334] In some further embodiments, the pharmaceutical composition comprises NaCl, hi some embodiments, the NaCl concentration of the pharmaceutical composition is about 10 mM to about 100 mM.

[0335] Moreover, in some further embodiments, the pharmaceutical composition comprises one or more cryoprotectants. In some embodiments, the one or more cryoprotectants are selected from the group consisting of sucrose, glycerol, and a combination of sucrose and glycerol. In some embodiments, the cryoprotectant is a combination of sucrose and glycerol.

[0336] In some more particular embodiments, the lipid formulation (a) comprises: About 23 mol% to about 27 mol% of the ionizable cationic lipid ATX-012; DOTAP is about 22 mol% to about 28 mol%. Cholesterol is about 35 mol% to about 41 mol%. PEG-DMG at about 0.75 mol% to about 2.5 mol%; Includes.

[0337] In some further embodiments, the lipid nanoparticles are within the range of about 50 nm to about 90 nm in size.

[0338] The disclosure also provides for the use of a pharmaceutical composition of any one of the above embodiments to make a medicament for ameliorating, preventing, delaying onset, or treating a disease or disorder associated with decreased activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in a subject in need thereof. In some embodiments, the disease is cystic fibrosis with a cystic fibrosis mutation. In some embodiments, the cystic fibrosis mutation is selected from the group consisting of class 1A, class 1B, class 3, class 4, class 5, and class 6. In some embodiments, the cystic fibrosis mutation is class 1A. In some embodiments, the cystic fibrosis mutation is class 1B. In some embodiments, the cystic fibrosis mutation is class 3. In some embodiments, the cystic fibrosis mutation is class 4. In some embodiments, the cystic fibrosis mutation is class 5. In some embodiments, the cystic fibrosis mutation is class 6.

[0339] The present disclosure also provides a method of ameliorating, preventing, delaying onset, or treating a disease or disorder associated with decreased activity of cystic fibrosis transmembrane conductance regulator (CFTR) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of any one of the above embodiments. In some embodiments, the disease or disorder is cystic fibrosis. In some embodiments, the administration is intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, nasal, or inhalation. In some embodiments, the administration is nasal or inhalation. In some embodiments, the administration is inhalation. In some embodiments, the administration is once a day, once a week, once every two weeks, or once a month. In some embodiments, the administration comprises administering an effective dose of about 0.01 to about 10 mg / kg of the mRNA in the pharmaceutical composition. In some embodiments, the administration increases expression of CFTR in the lung epithelium.

[0340] The disclosure also provides a method of expressing a CFTR protein in a cell, comprising contacting the cell with a pharmaceutical composition of any one of the above embodiments.

[0341] The present disclosure also provides a kit for expressing human CFTR in vivo, the kit comprising any one of the pharmaceutical compositions of the above embodiments and an apparatus for administering a dose. In some embodiments, the dose is an effective dose of about 0.01 to about 10 mg / kg of the mRNA in the pharmaceutical composition. In some embodiments, the apparatus comprises an injection needle, an intravenous needle, or an inhalation apparatus. In some embodiments, the apparatus is an inhalation apparatus.

[0342] Pharmaceutical Compositions and Delivery Methods To promote expression of mRNA in vivo, the nucleic acid-lipid formulation delivery vehicles described herein can be combined with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or can be present in a pharmaceutical composition in which the pharmaceutical composition is mixed with a suitable excipient. Methods for formulating and administering drugs can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa. Preferably, the nucleic acid-lipid formulation is a CFTR mRNA-lipid nanoparticle formulation as described herein. Preferably, the mRNA encodes the human CFTR protein of SEQ ID NO: 93 or 99, preferably formulated in a lipid delivery system or lipid carrier, and preferably includes a pharmaceutical acceptable excipient. In some embodiments, the pharmaceutical composition further includes a pharmaceutical acceptable excipient. The pharmaceutical compositions disclosed herein preferably promote expression of CFTR mRNA in vivo.

[0343] The lipid formulations and pharmaceutical compositions of the present disclosure may be administered and dosed according to current medical practice, taking into account the subject's clinical condition, the site and method of administration, the schedule of administration, the subject's age, sex, weight, and other factors that are meaningful to a clinician of ordinary skill in the art. For purposes of the present invention, an "effective amount" may be determined by relevant considerations such as those known to those skilled in the art in experimental clinical research, pharmacology, clinical, and medical fields. In some embodiments, the dosage is effective to at least somewhat stabilize, improve, or eliminate symptoms and other indicators that would be selected by those skilled in the art as appropriate measures of disease progression, reversal, or improvement. For example, an appropriate amount and dosage regimen is one that at least transiently produces a protein (e.g., an enzyme).

[0344] The pharmaceutical compositions described herein can express CFTR protein in lung epithelial cells of a subject. Suitable routes of administration include, for example, intratracheal, inhalation or intranasal. In some embodiments, the administration delivers the mRNA to lung epithelial cells. In some embodiments, the administration is selective for lung epithelial cells over other types of lung and airway cells.

[0345] The pharmaceutical compositions disclosed herein can be formulated with one or more excipients to (1) improve stability, (2) increase transfection into cells, (3) allow for sustained or delayed release (e.g., from a depot formulation of the polynucleotide, primary construct or mRNA), (4) modify its biodistribution (e.g., directing the polynucleotide, primary construct or mRNA to a given tissue or cell type), (5) increase translation of the encoded protein in vivo, and / or (6) modify the release profile of the encoded protein in vivo.

[0346] Preferably, mRNA and its lipid formulations can be administered locally, not in a systemic form.Local delivery can be carried out in various ways, depending on the target tissue.For example, the aerosol containing the composition of the present disclosure can be inhaled (for nasal, tracheal or bronchial delivery).

[0347] The pharmaceutical composition may be administered to any desired tissue. In some embodiments, the CFTR mRNA delivered by the lipid formulation or composition of the present disclosure is expressed in the tissue to which the lipid formulation and / or composition is administered. In some embodiments, the delivered mRNA is expressed in a tissue different from the tissue to which the lipid formulation and / or composition is administered. Examples of tissues to which the delivered mRNA may be delivered and / or expressed include, but are not limited to, the lung, the trachea, and / or the nasal passages.

[0348] The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include combining the active ingredient (i.e., the nucleic acid) with an excipient and / or one or more other accessory ingredients. Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a number of unit doses.

[0349] Pharmaceutical compositions may further comprise pharma- ceutically acceptable excipients, which as used herein include, but are not limited to, any solvents, dispersion media, diluents or other liquid vehicles, dispersing aids, suspending aids, surfactants, isotonicity agents, thickening agents, emulsifying agents, preservatives, and the like, as appropriate for the particular desired dosage form.

[0350] In addition to conventional excipients, such as any solvent, dispersion medium, diluent or other liquid vehicle, dispersion aid, suspension aid, surfactant, tonicity agent, viscosity enhancer, emulsifier, preservative, excipients of the present disclosure can include, but are not limited to, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with primary DNA constructs or mRNA (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0351] Thus, the formulations described herein can include one or more excipients, each in an amount that, in combination, improves the stability of the nucleic acid in the lipid formulation, increases transfection of the nucleic acid (e.g., mRNA) into a cell, increases expression of the encoded protein, and / or modifies the release profile of the encoded protein. Additionally, the mRNA of the present disclosure can be formulated with self-assembling nucleic acid nanoparticles.

[0352] Various excipients for formulation into pharmaceutical compositions and techniques for preparing the compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006, which is incorporated herein by reference in its entirety). The use of conventional excipient vehicles can be envisioned within the scope of the embodiments of the present disclosure, except where any conventional excipient vehicle may be incompatible with a substance or its derivatives, such as by causing any undesirable biological effect or otherwise interacting in a deleterious manner with any of the other component(s) of the pharmaceutical composition.

[0353] The dosage form of the composition of the present disclosure can be a solid, which can be reconstituted with liquid before administration.The solid can be administered as a powder.In some embodiments, the pharmaceutical composition comprises a nucleic acid-lipid formulation that is lyophilized.

[0354] In preferred embodiments, the dosage form of the pharmaceutical compositions described herein can be a liquid suspension of the CFTR mRNA-lipid nanoparticles described herein. In some embodiments, the liquid suspension is a suspension in a buffered solution. In some embodiments, the buffered solution comprises a buffer selected from the group consisting of HEPES, MOPS, TES, and TRIS. In some embodiments, the buffer has a pH of about 7.4. In some preferred embodiments, the buffer is HEPES. In some further embodiments, the buffered solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from a sugar and glycerol, or a combination of a sugar and glycerol. In some embodiments, the sugar is a disaccharide. In some embodiments, the sugar is sucrose. In some preferred embodiments, the buffer comprises HEPES, sucrose, and glycerol at a pH of 7.4. In some embodiments, the suspension is frozen during storage and thawed prior to administration. In some embodiments, the suspension is frozen at a temperature less than about -70°C. In some embodiments, the suspension is diluted with sterile water prior to inhalation administration. In some embodiments, inhaled administration involves diluting the suspension with about 1 volume to about 4 volumes of sterile water. In some embodiments, the lyophilized CFTR-mRNA-lipid nanoparticle formulation can be resuspended in a buffer such as those described herein.

[0355] The compositions and methods of the present disclosure may be administered to a subject by a variety of mucosal administration routes, including intranasal and / or intrapulmonary routes. In some aspects of the present disclosure, the mucosal tissue layer comprises an epithelial cell layer. The epithelial cells can be lung cells, tracheal cells, bronchial cells, alveolar cells, nasal cells, and / or oral cells. The compositions of the present disclosure can be administered using conventional actuators, such as mechanical spray devices, and pressurized actuators, electrically actuated actuators, or other types of actuators.

[0356] The mRNA composition of the present disclosure may be administered as a nasal spray or pulmonary spray in an aqueous solution, and may be dispensed in spray form by various methods known to those skilled in the art. Pulmonary delivery of the composition of the present disclosure is achieved by administering the composition in the form of drops, particles, or spray, which may be, for example, aerosolized, atomized, or nebulized. The composition, spray, or aerosol particles may be in either liquid or solid form, for example, a lyophilized lipid formulation. A preferred system for dispensing liquids as nasal sprays is disclosed in U.S. Pat. No. 4,511,069. Such formulations may be conventionally prepared by dissolving the composition according to the present disclosure in water to make an aqueous solution, and sterilizing the solution. The formulation may be placed in a multi-dose container, for example, the closed dispensing system disclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spray delivery systems are described in TRANSDERMAL SYSTEMIC MEDICATION, YW Chien ed., Elsevier Publishers, New York, 1985, and U.S. Patent No. 4,778,810. Additional aerosol delivery forms may include, for example, compressed air nebulizers, jet nebulizers, ultrasonic nebulizers, and piezoelectric nebulizers, which deliver CFTR mRNA-lipid formulations suspended in a pharmaceutical solvent, such as water, ethanol, or mixtures thereof.

[0357] The nasal and pulmonary spray solutions of the present disclosure typically contain a drug to be delivered, optionally formulated with a surfactant, such as a non-ionic surfactant (e.g., polysorbate 80) and one or more buffers, provided that the inclusion of the surfactant does not disrupt the structure of the lipid formulation. In some embodiments of the present disclosure, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution may be pH 6.8 to 7.2. The pharmaceutical solvent used may also be an aqueous buffer that is weakly acidic, pH 4 to 6. Other components, including preservatives, surfactants, dispersants, or gases, may be added to enhance or maintain chemical stability.

[0358] In some embodiments, the present disclosure provides a pharmaceutical product comprising a solution comprising a composition of the present disclosure and an actuator for a pulmonary, mucosal or intranasal spray or aerosol.

[0359] The dosage form of the composition of the present disclosure can be a liquid in the form of droplets or an emulsion, or in the form of an aerosol.

[0360] The dosage form of the composition of the present disclosure can be a solid, which can be reconstituted with a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0361] To formulate a composition for pulmonary delivery within the scope of the present disclosure, the CFTR mRNA-lipid formulation can be combined with various pharma- ceutically acceptable additives and bases or carriers for dispersing the CFTR mRNA-lipid formulation(s). Examples of additives include pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), isotonicity agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancers (e.g., cyclodextrin and its derivatives), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione). When the composition for mucosal delivery is liquid, the osmolality of the formulation is typically adjusted to a value that does not induce substantially irreversible tissue damage at the mucosa at the site of administration, measured relative to the osmolality of 0.9% (w / v) saline. Generally, the osmolality of the solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and most frequently 3 / 4 to 1.7.

[0362] The CFTR mRNA-lipid formulation may be dispersed in a base or vehicle, which may comprise a hydrophilic compound capable of dispersing the CFTR mRNA-lipid formulation and any desired additives. The base may be selected from a wide range of suitable carriers, including, but not limited to, polycarboxylic acids or their salts, copolymers of carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and non-toxic metal salts thereof. Biodegradable polymers such as polylactic acid, poly(lactic acid-glycolic acid) copolymers, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymers, and mixtures thereof are often selected as bases or carriers. Alternatively or additionally, synthetic fatty acid esters, such as polyglycerol fatty acid esters, sucrose fatty acid esters, etc., can be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and the carriers can be provided with enhanced structural integrity through partial crystallization, ionic bonding, crosslinking, etc. The carriers can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films, for direct application to the nasal mucosa. In this regard, the selected carrier may be used to facilitate absorption of the CFTR mRNA-lipid formulation.

[0363] Alternatively, the compositions of the present disclosure may contain pharma- ceutically acceptable carrier substances, such as pH adjusting agents, pH buffering agents, osmolality adjusting agents and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate and mixtures thereof, as required for appropriate physiological conditions. For solid compositions, conventional non-toxic pharma-ceutically acceptable carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

[0364] In certain embodiments of the present disclosure, the CFTR mRNA-lipid formulation may be administered in a sustained release formulation, for example, in a composition that includes a sustained release polymer. The CFTR mRNA-lipid formulation can be prepared with a carrier that prevents immediate release, for example, a controlled release vehicle, for example, a polymer, a microencapsulated delivery system, or a bioadhesive gel. Sustained delivery of the CFTR mRNA-lipid formulation in various compositions of the present disclosure can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate hydrogel and gelatin.

[0365] It has been shown that nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the nucleic acid composition and by inhalation of an aerosol mist produced by a liquid nebulizer or by use of a dry powder device such as that described in U.S. Pat. No. 5,780,014, incorporated herein by reference.

[0366] In certain embodiments, the compositions of the present disclosure may be formulated to be aerosolized or otherwise delivered as particulate liquid or solid before or at the time of administration to a subject. Such compositions may be administered with the aid of one or more devices suitable for administering such particulate solid or liquid compositions (e.g., aerosolized aqueous solutions or suspensions, etc.) to create particles that are easily respirable or inhalable by the subject. In some embodiments, such devices (e.g., metered dose inhalers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers, propellant-based inhalers or insufflators) prompt the subject to administer a predetermined mass, volume, or dose of the composition (e.g., about 0.5 mg / kg of mRNA per dose). For example, in certain embodiments, the compositions of the present disclosure are administered to a subject using a metered dose inhaler that contains a suspension or solution that includes the composition and a suitable propellant. In certain embodiments, the compositions of the present disclosure may be formulated as particulate powders for inhalation (e.g., respirable dry particles). In certain embodiments, compositions of the present disclosure formulated as respirable particles are appropriately sized so that they are respirable by a subject or can be delivered using an appropriate device (e.g., an average D50 or D90 particle size of less than about 500 μm, less than about 400 μm, less than about 300 μm, less than about 250 μm, less than about 200 μm, less than about 150 μm, less than about 100 μm, less than about 75 μm, less than about 50 μm, less than about 25 μm, less than about 20 μm, less than about 15 μm, less than about 12.5 μm, less than about 10 μm, less than about 5 μm, or less than about 2.5 μm). In yet another embodiment, compositions of the present disclosure are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies).In some embodiments, the compositions of the disclosure provide a single dose of at least 0.05 mg / kg body weight, at least 0.1 mg / kg body weight, at least 0.5 mg / kg body weight, at least 1.0 mg / kg body weight, at least 2.0 mg / kg body weight, at least 3.0 mg / kg body weight, at least 4.0 mg / kg body weight, at least 5.0 mg / kg body weight, at least 6.0 mg / kg body weight, at least 7.0 mg / kg body weight, at least 8.0 mg / kg body weight, at least 9.0 mg / kg body weight, at least 10 mg / kg body weight, at least 15 mg / kg body weight, at least 20 mg / kg body weight, The subject is administered a concentration of at least 25 mg / kg body weight, at least 30 mg / kg body weight, at least 35 mg / kg body weight, at least 40 mg / kg body weight, at least 45 mg / kg body weight, at least 50 mg / kg body weight, at least 55 mg / kg body weight, at least 60 mg / kg body weight, at least 65 mg / kg body weight, at least 70 mg / kg body weight, at least 75 mg / kg body weight, at least 80 mg / kg body weight, at least 85 mg / kg body weight, at least 90 mg / kg body weight, at least 95 mg / kg body weight, or at least 100 mg / kg body weight is administered. In some embodiments, compositions of the disclosure are administered to a subject in one or more doses such that a total amount of at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg of mRNA is administered.The values ​​and ranges recited herein include any subvalues ​​or subranges within those values ​​and ranges.

[0367] In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject once a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject twice a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject three times a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject four times a month.

[0368] According to the present disclosure, a therapeutically effective amount of the compositions provided, when administered periodically, increases the CFTR protein expression level or CFTR protein activity level in a subject compared to the baseline, pre-treatment CFTR protein expression level or CFTR protein activity level. Typically, the CFTR protein expression level or CFTR protein activity level is measured in a biological sample, such as blood, plasma, serum, urine, or solid tissue extract, from the subject. The baseline level can be measured immediately before treatment. In some embodiments, administration of the pharmaceutical compositions described herein increases the CFTR protein expression level or CFTR protein activity level in a biological sample (e.g., plasma / serum or lung epithelial swab) by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% compared to the baseline level before treatment. In some embodiments, administration of provided compositions increases CFTR protein expression levels or CFTR protein activity levels in a biological sample (e.g., plasma / serum or lung epithelial swab) by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% compared to pre-treatment baseline levels for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days.

[0369] Cystic Fibrosis Treatment The compositions of the present disclosure can be used to treat cystic fibrosis. In some embodiments, the present disclosure provides a method for treating cystic fibrosis by administering to a subject in need of treatment an mRNA encoding a CFTR protein, as described herein, or a pharmaceutical composition comprising the mRNA. The mRNA or a pharmaceutical composition comprising the mRNA may be administered directly to the lungs of the subject. Various routes of administration for delivery to the lungs may be used. In some embodiments, the mRNA or a composition comprising the mRNA described herein is administered by inhalation, nebulization, or aerosolization. In various embodiments, administration of the mRNA causes CFTR to be expressed in the lungs of the subject (e.g., in the epithelial cells of the lungs).

[0370] In certain embodiments, the disclosure provides a method of treating cystic fibrosis by administering to the lungs of a subject in need of treatment an mRNA comprising a coding sequence encoding SEQ ID NO: 93. In certain embodiments, the disclosure provides a method of treating cystic fibrosis by administering to the lungs of a subject in need of treatment an mRNA comprising a coding sequence encoding an amino acid sequence at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to SEQ ID NO: 93. In another specific embodiment, the disclosure provides a method of treating cystic fibrosis by administering to the lungs of a subject in need of treatment an mRNA comprising a coding sequence of SEQ ID NO: 100-105. In another embodiment, the disclosure provides a method of treating cystic fibrosis by administering to the lungs of a subject in need of treatment an mRNA comprising a coding sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NOs: 100-105.

[0371] CFTR Mutation Classes The pharmaceutical compositions and methods described herein can be used to treat patients suffering from any of the classes of CF, which classes are described below.

[0372] Class 1A (non-production of mRNA): The first class of mutations prevents the mRNA from even being synthesized. When cells try to produce a protein, an enzyme called RNA polymerase binds to a region of DNA called a promoter. The promoter usually comes just before the section of DNA that codes for a given protein. Mutations in the CFTR promoter prevent RNA polymerase from binding to the DNA, and therefore the gene cannot be transcribed into mRNA. The end result is that no CFTR protein is produced at all. Examples of mutations that prevent CFTR mRNA from being synthesized include Del2,3(21kb) and 1717-1G→A. There are currently no therapies available to correct this type of mutation. However, there are some studies of therapies that do not require the CFTR protein but instead block sodium channels or stimulate other chloride protein channels on the cell surface to balance ion levels.

[0373] Class 1B (non-protein producing): In this class of mutations, CFTR mRNA is produced but is damaged and cannot produce protein. The DNA has a sequence that is carried over to the RNA, signaling the ribosome to stop reading the message and terminate protein production. A mutation can also cause one of these termination sequences to appear prematurely in the mRNA. This results in a truncated CFTR protein being produced that is then degraded by the cell. Gly542X and Trp1282X are types of Class 1B mutations. Read-through compounds can help the ribosome skip the premature termination sequence, allowing the rest of the information in the mRNA to be read and CFTR protein to be produced. Ataluren was one such compound that was being investigated as a possible treatment for this type of mutation, but its development was terminated due to poor results in phase 3 clinical trials.

[0374] Class 2 (non-trafficking): In this class of mutations, the CFTR protein is made but cannot reach the cell membrane. The CFTR protein has 1,480 amino acids, and in some cases, even a single error can cause the protein to misfold. Cells often destroy misfolded proteins, preventing them from being transported to the cell surface. Examples of class 2 mutations include Phe508del, Asn1303Lys, and Ala561Glu. Treatments called CFTR correctors can be used to correct misfolded proteins and help them reach the cell membrane. Some examples of CFTR correctors include lumacaftor / ivacaftor (sold as Orkambi) and tezacaftor / ivacaftor (sold as Simdeco), both manufactured by Vertex Pharmaceuticals.

[0375] Class 3 (Gating Defective): Another type of mutation can produce a CFTR protein that reaches the cell membrane but does not open properly. This is often referred to as "gating defective." Gly551Asp, Ser549Arg, and Gly1349Asp are examples of mutations that cause gating defects. Treatments called CFTR potentiators, such as Kalydeco, can be used to open the channel and / or keep it open for longer.

[0376] Class 4 (reduced conductance): The fourth class of mutations produces a CFTR protein that reaches the cell membrane and responds by signaling the cell to open, but the protein is misshaped so that it can only pass small amounts of chloride ions. This reduction in chloride ion movement is called reduced conductance. Examples of such mutations include Arg117His, Arg334Trp, and Ala455Glu. With these mutations, the CFTR potentiator can still help keep the channel open for longer and allow more chloride ions to pass through.

[0377] Class 5 (reduced protein): In some cases, mutations cause CFTR protein to be produced but not in sufficient quantities. This is often due to a process called alternative splicing, which can sometimes produce the correct protein form but more often produces an incorrect form. The incorrect form never makes it to the cell surface, which reduces the number of CFTR protein channels in the cell membrane. Class 5 mutations include 3272-26A→G, 3849+10kg C→T. Possible treatments for this type of mutation include CFTR correctors, which correct the misformed CFTR protein, CFTR potentiators, which force the functioning CFTR protein to stay open for longer, CFTR amplifiers, which increase the amount of mRNA (i.e., more CFTR protein is produced), or antisense oligonucleotides (which can have many different uses).

[0378] Class 6 (Less Stable Protein): This last type of mutation results in a working CFTR protein, but the protein is not stable enough in shape that it is degraded too quickly once it reaches the cell surface. Class 6 mutations include c.120del123 and rPhe580del. Stabilizers are a type of treatment for this type of mutation. These stabilizers work by inhibiting the enzyme that breaks down CFTR. A treatment called cabosonestat has been studied for this use, but failed to meet its primary goal in a phase 2 clinical trial.

[0379] In some embodiments, the CFTR mRNA-lipid formulation, or a pharmaceutical composition comprising the same, is used to treat a patient with a class 1A mutation. In some embodiments, the CFTR mRNA-lipid formulation, or a pharmaceutical composition comprising the same, is used to treat a patient with a class 1B mutation. In some embodiments, the CFTR mRNA-lipid formulation, or a pharmaceutical composition comprising the same, is used to treat a patient with a class 2 mutation. In some embodiments, the CFTR mRNA-lipid formulation, or a pharmaceutical composition comprising the same, is used to treat a patient with a class 3 mutation. In some embodiments, the CFTR mRNA-lipid formulation, or a pharmaceutical composition comprising the same, is used to treat a patient with a class 4 mutation. In some embodiments, the CFTR mRNA-lipid formulation, or a pharmaceutical composition comprising the same, is used to treat a patient with a class 5 mutation. In some embodiments, the CFTR mRNA-lipid formulation, or a pharmaceutical composition comprising the same, is used to treat a patient with a class 6 mutation.

[0380] combination The CFTR mRNA, formulations thereof, or encoded CFTR protein described herein may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. "In combination with" is not intended to imply that the agents must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of the present disclosure. The composition may be administered simultaneously with, prior to, or after one or more other desired pharmaceutical or medical procedures. In general, each agent will be administered at a dose and / or time schedule established for that agent. Preferably, the therapeutic methods of the present disclosure include delivery of a pharmaceutical, prophylactic, diagnostic, or imaging composition in combination with an agent that may improve its bioavailability, reduce and / or modify its metabolism, inhibit its excretion, and / or modify its biodistribution. As a non-limiting example, the mRNA disclosed herein, and preferably the mRNA sequence comprising SEQ ID NO: 49, 53, 66, 68, 69, 72 or 100-105 encoding the CFTR protein of SEQ ID NO: 99, may be used in combination with pharmaceutical agents for the treatment of CFTR deficiency, including, but not limited to, one or more of Trikafta® (elexacaftor, ivacaftor, tezacaftor (sold by Vertex Pharmaceuticals)), Symdeko® (tezacaftor and ivacaftor (Vertex)), Orkambi® (lumacaftor and ivacaftor (Vertex)), Kalydeco® (ivacaftor (Vertex)), compositions and agents for airway clearance, antibiotics, anti-inflammatories, bronchodilators, mucus thinners, etc., in combination with multiple vitamins, calcium supplements or low protein / high calorie dietary regimens. In general, it is expected that agents used in combination with the disclosed CFTR mRNA and formulations thereof will be used at levels that do not exceed the levels at which the agents are used individually, and in some embodiments, the levels used in combination will be lower than the levels at which the agents are used individually.In one embodiment, these combinations, either alone or together, may be administered according to a split-dosage regimen as known in the art.

[0381] definition At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each individual subcombination of the members of such groups and ranges. For example, "C 1-6 The term "alkyl" specifically refers to methyl, ethyl, C 3 Alkyl, C 4 Alkyl, C 5 Alkyl and C 6 Alkyl is intended to be disclosed individually.

[0382] The phrase "administered in combination" or "co-administration" refers to administering two or more agents to a subject simultaneously or at intervals that allow for overlap of the effects of each agent on the patient. In some embodiments, the agents are administered within about 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute of each other. In some embodiments, the administration of the agents is sufficiently close to each other that a combined effect (e.g., synergistic effect) is obtained.

[0383] As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, generally a genetically engineered animal, or a clone.

[0384] The term "approximately" or "about", when applied to one or more of the values ​​of interest, refers to a value similar to a given reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less of a given reference value in either direction (above or below the reference value) unless otherwise indicated or clearly indicated by context (unless the numerical value exceeds 100% of the possible values).

[0385] The terms "associated with," "conjugated," "linked," "bound," and "tethered," when used in reference to two or more moieties, mean that the moieties are physically associated or linked to one another, either directly or through one or more additional moieties that function as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. The "association" need not be strictly through a direct covalent chemical bond. It may also be indicated that ionic or hydrogen bonds, or hybridization-based linkages are sufficiently stable such that the "associated" entities remain physically associated.

[0386] In the claims, unless specifically stated to the contrary or clearly stated otherwise from the context, articles such as "a," "an," and "the" may mean one or more. A claim or description containing "or" between one or more of the members of a group is deemed to be satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise related to a given product or process, unless specifically stated to the contrary or clearly stated otherwise from the context. The present disclosure includes embodiments in which only one of the members of the group is present in, used in, or otherwise related to a given product or process. The present disclosure includes embodiments in which two or more, or all of the members of the group are present in, used in, or otherwise related to a given product or process.

[0387] The term "acyl," as used herein, refers to a hydrogen or an alkyl group, as defined herein (e.g., a haloalkyl group), attached to the parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl (i.e., a carboxaldehyde group), acetyl, trifluoroacetyl, propionyl, butanoyl, and the like. Examples of unsubstituted acyl groups contain 1 to 7, 1 to 11, or 1 to 21 carbons. In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents, as described herein.

[0388] The term "alkenyl," as used herein, unless otherwise specified, represents a monovalent straight or branched chain radical of 2 to 20 carbons (e.g., 2 to 6 or 2 to 10 carbons) containing one or more carbon-carbon double bonds, and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyl includes both cis and trans isomers. Alkenyl groups may be optionally substituted with one, two, three, or four substituents independently selected from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the examples of alkyl substituents described herein.

[0389] The term "alkoxy" refers to a chemical substituent of the formula -OR, where R is, unless otherwise specified, C 1-20 Alkyl groups (e.g., C 1-6 or C 1-10 Examples of alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituents (e.g., hydroxy or alkoxy) as defined herein.

[0390] The term "alkoxyalkyl" refers to an alkyl group that is substituted with an alkoxy group. Examples of unsubstituted alkoxyalkyl groups contain 2 to 40 carbons (e.g., C 1-6 Alkoxy-C 1-6 Alkyl, C 1-10 Alkoxy-C 1-10 Alkyl or C 1-20 Alkoxy-C 1-20 Like alkyl, it contains 2 to 12 or 2 to 20 carbons. In some embodiments, the alkyl and alkoxy can each be further substituted with 1, 2, 3, or 4 substituents as defined herein in each group.

[0391] The term "alkoxycarbonyl," as used herein, refers to an alkoxy, as defined herein, attached to the parent molecular group through a carbonyl atom (e.g., -C(O)-OR, where R is H or an optionally substituted C 1-6 Alkyl group, C 1-10 Alkyl group or C 1-20 (An alkyl group.) Examples of unsubstituted alkoxycarbonyls include 1 to 21 carbons (e.g., 1 to 11 or 1 to 7 carbons). In some embodiments, the alkoxy group is further substituted with 1, 2, 3, or 4 substituents as described herein.

[0392] The term "alkoxycarbonylalkyl," as used herein, refers to an alkyl group, as defined herein, that is substituted with an alkoxycarbonyl group, as defined herein (e.g., -alkyl-C(O)-OR, where R is an optionally substituted C 1-20 Alkyl group, C 1-10 Alkyl group or C 1-6 Examples of unsubstituted alkoxycarbonylalkyl include those containing 3 to 41 carbons (e.g., C 1-6 Alkoxycarbonyl-C 1-6 Alkyl, C 1-10 Alkoxycarbonyl-C 1-10 Alkyl or C 1-20 Alkoxycarbonyl-C 1-20 and alkyl, which contains 3 to 10, 3 to 13, 3 to 17, 3 to 21, or 3 to 31 carbons. In some embodiments, each alkyl and alkoxy group is further independently substituted with 1, 2, 3, or 4 substituents as described herein (e.g., hydroxy groups).

[0393] The term "alkoxycarbonylalkenyl," as used herein, refers to an alkenyl group, as defined herein, that is substituted with an alkoxycarbonyl group, as defined herein (e.g., -alkenyl-C(O)-OR, where R is an optionally substituted C 1-20 Alkyl group, C 1-10 Alkyl group or C 1-6 Examples of unsubstituted alkoxycarbonylalkenyls include those containing 4 to 41 carbons (e.g., C 1-6 Alkoxycarbonyl-C 2-6 Alkenyl, C 1-10 Alkoxycarbonyl-C 2-10 Alkenyl or C 1-20 Alkoxycarbonyl-C 2-20 and alkenyl, which contains 4 to 10, 4 to 13, 4 to 17, 4 to 21, or 4 to 31 carbons. In some embodiments, each alkyl, alkenyl, and alkoxy group is further independently substituted with 1, 2, 3, or 4 substituents (e.g., hydroxy groups) as described herein.

[0394] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1-20 carbon atoms (wherever alkyl groups appear herein, numerical ranges such as "1-20" refer to the respective integers within the given range, e.g., "1-20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. (up to 20 carbon atoms), although the definition of the present invention also encompasses the occurrence of the term "alkyl" without a numerical range). The alkyl group may be a medium sized alkyl having 1-9 carbon atoms. The alkyl group may also be a lower alkyl having 1-6 carbon atoms. The alkyl group may be a "C 1-4 As merely an example, "C 1-4"Alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethylpropyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0395] The term "lower alkyl" means a group having 1 to 6 carbons in the chain, which can be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl and hexyl.

[0396] The term "alkylsulfinyl," as used herein, represents an alkyl group attached to the parent molecular group via an -S(O)- group. Examples of unsubstituted alkylsulfinyl groups are those containing 1-6, 1-10, or 1-20 carbons. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituents as defined herein.

[0397] The term "alkylsulfinylalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with an alkylsulfinyl group. Examples of unsubstituted alkylsulfinylalkyl groups are those having 2 to 12, 2 to 20, or 2 to 40 carbons. In some embodiments, each alkyl group can be further substituted with 1, 2, 3, or 4 substituents, as defined herein.

[0398] The term "alkynyl," as used herein, refers to a monovalent straight or branched chain radical of 2 to 20 carbon atoms (e.g., 2 to 4, 2 to 6, or 2 to 10 carbons) that contains a carbon-carbon triple bond, and is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groups can be optionally substituted with one, two, three, or four substituents independently selected from aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the examples of alkyl substituents described herein.

[0399] The term "amidine" as used herein means -C(=NH)NH 2 Represents a group.

[0400] The term “amino” as used herein refers to —N(R N1 ) 2 In the formula, each R N1 are independently H, OH, and NO 2 , N(R N2 ) 2 , S.O. 2 OR N2 , S.O. 2 R N2 , SOR N2 , an N-protecting group, an alkyl, an alkenyl, an alkynyl, an alkoxy, an aryl, an alkaryl, a cycloalkyl, an alkylcycloalkyl, a carboxyalkyl (e.g., optionally substituted with an O-protecting group, e.g., an optionally substituted arylalkoxycarbonyl group or any described herein), a sulfoalkyl, an acyl (e.g., acetyl, trifluoroacetyl, or other group described herein), an alkoxycarbonylalkyl (e.g., optionally substituted with an O-protecting group, e.g., an optionally substituted arylalkoxycarbonyl group or any described herein), a heterocyclyl (e.g., a heteroaryl), or an alkylheterocyclyl (e.g., an alkylheteroaryl), and these enumerated R N1Each of the groups may be optionally substituted as defined herein for each group, or may have two R N1 together form a heterocyclyl or N-protecting group, and each R N2 is independently H, alkyl, or aryl. The amino group of the present disclosure includes unsubstituted amino (i.e., -NH 2 ) or substituted amino (i.e. -N(R') 2 In a preferred embodiment, the amino can be -NH 2 or -NHR N1 where R N1 are independently OH, NO 2 , N.H. 2 , N.R. N2 2 , S.O. 2 OR N2 , S.O. 2 R N2 , SOR N2 , alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or other groups described herein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl), or aryl, with each R N2 , H, C 1-20 Alkyl (e.g. C 1-6 Alkyl) or C 1-10 It can be aryl.

[0401] The term "amino acid" as used herein refers to an amino acid having a side chain amino group and an acid group (e.g., -CO 2 H or -SO 3H) and the amino acid is attached to the parent molecular group by a side chain amino or acid group (e.g., side chain). In some embodiments, the amino acid is attached to the parent molecular group by a carbonyl group and the side chain or amino group is attached to the carbonyl group. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclyl, alkylaryl, alkylheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxylnorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine. The amino acid groups are independently selected from the group consisting of (1) C 1-6 Alkoxy, (2) C 1-6 alkylsulfinyl, (3) amino, as defined herein (e.g., unsubstituted amino (i.e., —NH 2 ) or substituted amino (i.e., -N(R N1 ) 2 (In the formula, R N1 is as defined for amino), (4) C 6-10 Aryl-C 1-6 Alkoxy, (5) azido, (6) halo, (7) (C 2-9 heterocyclyl)oxy, (8) hydroxy, (9) nitro, (10) oxo (e.g., carboxaldehyde or acyl), (11) C 1-7 Spirocyclyl, (12) thioalkoxy, (13) thiol, (14) -CO 2 R A’ (In the formula, R A’ (a)C 1-20 Alkyl (e.g. C 1-6 alkyl), (b) C 2-20 Alkenyl (e.g., C 2-6 alkenyl), (c) C 6-10 Aryl, (d) hydrogen, (e) C 1-6 Alkyl-C 6-10Aryl, (f) Amino-C 1-20 Alkyl, (g)-(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 R′ is a polyethylene glycol represented by the formula OR′, where s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R′ is H or C 1-20 alkyl), and (h) -NR N1 (CH 2 ) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1 wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1-6 (15) —C(O)NR B R C’ (In the formula, R B’ and R C’ each independently represents (a) hydrogen, (b) C 1-6 Alkyl, (c) C 6-10 aryl, and (d) C 1-6 Alkyl-C 6-10 aryl), (16)-SO 2 R D’ (In the formula, R D’ (a)C 1-6 Alkyl, (b) C 6-10 Aryl, (c) C 1-6 Alkyl-C 6-10 (17) -SO 2 NR E’ RF’ (In the formula, R E’ and R F’ each independently represents (a) hydrogen, (b) C 1-6 Alkyl, (c) C 6-10 Aryl and (d) C 1-6 Alkyl-C 6-10 aryl), (18)-C(O)R G’ (In the formula, R G’ (a)C 1-20 Alkyl (e.g. C 1-6 alkyl), (b) C 2-20 Alkenyl (e.g., C 2-6 alkenyl), (c) C 6-10 Aryl, (d) hydrogen, (e) C 1-6 Alkyl-C 6-10 Aryl, (f) Amino-C 1-20 Alkyl, (g)-(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 R′ is a polyethylene glycol represented by the formula OR′, where s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R′ is H or C 1-20 alkyl), and (h) -NR N1 (CH 2 ) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1 wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1-6 alkyl), (19) -NR H’ C(O)RI’ (In the formula, R H’ is (a1) hydrogen and (b1) C 1-6 alkyl; R I’ is (a2)C 1-20 Alkyl (e.g. C 1-6 (b2) C 2-20 Alkenyl (e.g., C 2-6 alkenyl), (c2) C 6-10 Aryl, (d2) hydrogen, (e2) C 1-6 Alkyl-C 6-10 Aryl, (f2) Amino-C 1-20 Alkyl, (g2)-(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 R′ is a polyethylene glycol represented by the formula OR′, where s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R′ is H or C 1-20 alkyl), and (h2) -NR N1 (CH 2 ) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1 wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1-6 (20) -NR J’ C(O)OR K’ (In the formula, R J’ is (a1) hydrogen and (b1) C 1-6 alkyl; R K’ is (a2)C 1-20Alkyl (e.g. C 1-6 alkyl), (b2) C 2-20 Alkenyl (e.g., C 2-6 alkenyl), (c2) C 6-10 Aryl, (d2) hydrogen, (e2) C 1-6 Alkyl-C 6-10 Aryl, (f2) Amino-C 1-20 Alkyl, (g2)-(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 R′ is a polyethylene glycol represented by the formula OR′, where s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R′ is H or C 1-20 alkyl), and (h2) -NR N1 (CH) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1 wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1-6 (19) an alkyl group, and (20) an amidine group, optionally substituted with one, two, or three, or in the case of amino acid groups of two or more carbons, four of the substituents selected from the group consisting of (18) an aryl group, (19) an aryl group, (20) an aryl group, and (21) an amidine group, optionally substituted with one, two, or three, or in the case of amino acid groups of two or more carbons, four of the substituents selected from the group consisting of (18) an aryl group, and (22) an aryl group. In some embodiments, each of these groups can be further substituted as described herein.

[0402] The term "aminoalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with an amino group, as defined herein, where the alkyl and amino each may have one, two, three or four substituents, as described herein for the corresponding group (e.g., CO 2 R A’ (In the formula, R A’ (a)C 1-6 Alkyl, (b) C 6-10 aryl, (c) hydrogen, and (d) C 1-6 Alkyl-C 6-10 It may be further substituted with an aryl, for example selected from the group consisting of carboxy and / or an N-protecting group).

[0403] The term "aminoalkenyl," as used herein, refers to an alkenyl group, as defined herein, substituted with an amino group, as defined herein, wherein the alkenyl and amino each may have one, two, three or four substituents, as described herein for the corresponding group (e.g., CO 2 R A’ (In the formula, R A’ (a)C 1-6 Alkyl, (b) C 6-10 aryl, (c) hydrogen, and (d) C 1-6 Alkyl-C 6-10 It may be further substituted with an aryl, for example selected from the group consisting of carboxy and / or an N-protecting group).

[0404] The term "anionic lipid" refers to lipids that are negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0405] The phrase "at least one of" following a list of items, when followed by the term "and" or "or" to separate any of the items, is a phrase that modifies the entire list and not each member (i.e., each item) of the list. The phrase "at least one of" does not require the selection of at least one of each item in the list, but allows for the meaning of at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers, respectively, to A only, B only, or C only, any combination of A, B, and C, and / or at least one of each of A, B, and C.

[0406] Terms such as "include," "having," and the like are used in this specification and in the claims, and such terms are intended to be inclusive terms in the same manner as "comprise" is interpreted when the term "comprise" is used as a transitional phrase in the claims.

[0407] Reference to a singular element is not intended to mean "one and only one" unless specifically stated otherwise, but rather "one or more." Masculine pronouns (e.g., he) include feminine and neuter (e.g., she and it), and feminine and neuter pronouns include masculine and neuter. The term "several" refers to one or more. Underlined and / or italicized headings and subheadings are used for convenience only, do not limit the subject technology, and are not intended to be relevant to the interpretation of the subject technology description. All of the structural and functional equivalents of the various components described throughout this disclosure that are known or will later become known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be included in the subject technology. Furthermore, nothing disclosed herein is intended to be publicly disclosed, whether or not such disclosure is expressly set forth in the above description.

[0408] The term “boranyl” as used herein means —B(R B1 ) 3 In the formula, R B1 is independently selected from the group consisting of H and optionally substituted alkyl. In some embodiments, the boranyl group can be substituted with 1, 2, 3, or 4 substituents as defined herein for alkyl.

[0409] The term "boranophosphate" has its ordinary meaning as understood in the art and includes its protonated, deprotonated and tautomers. For example, boranophosphate refers to the compound: [ka] It can have the following structure.

[0410] The term "biocompatible" means compatible with living cells, tissues, organs or systems and poses little risk of damage, toxicity or rejection by the immune system.

[0411] The term "biodegradable" means capable of being broken down into non-toxic products by the action of living organisms.

[0412] The phrase "biologically active" refers to any characteristic of a substance that has activity in a biological system and / or organism. For example, a substance is considered to be biologically active if, when administered to an organism, it has a biological effect on the organism. In certain embodiments, a polynucleotide of the present disclosure may be considered to be biologically active even if a portion of the polynucleotide is biologically active or mimics an activity that is considered associated with an organism.

[0413] The terms "carbocyclic" and "carbocyclyl" as used herein refer to optionally substituted C 3-12 "Carbocyclic" refers to a monocyclic, bicyclic or tricyclic structure in which the rings (which may be aromatic or non-aromatic) are formed by carbon atoms. Carbocyclic structures include cycloalkyl groups, cycloalkenyl groups and aryl groups.

[0414] The term “carbamoyl” as used herein means —C(O)—N(R N1 ) 2 In the formula, each R N1 The meaning of is found in the definition of "amino" provided herein.

[0415] The term "carbamoylalkyl," as used herein, refers to an alkyl group, as defined herein, that is substituted with a carbamoyl group, as defined herein, which can be further substituted with one, two, three or four substituents, as described herein.

[0416] The term "carbamyl" as used herein means -NR N1 C(=O)OR or -OC(=O)N(R N1 ) 2 In the formula, each R N1 The meaning of is seen in the "amino" definition provided herein, and R is alkyl, cycloalkyl, alkylcycloalkyl, aryl, alkylaryl, heterocyclyl (e.g., heteroaryl) or alkylheterocyclyl (e.g., alkylheteroaryl) as defined herein.

[0417] The term "carbonyl" as used herein refers to a C(O) group, which also may be represented as C=O.

[0418] The term "carboxaldehyde" refers to an acyl group having the structure -C(O)H.

[0419] The term "carboxy" as used herein means -CO 2 It means H.

[0420] The term "cationic lipid" refers to amphipathic lipids and their salts that have a positively charged hydrophilic head group and one, two or more hydrophobic fatty acids or fatty acid alkyl chains and a link between these two domains. Ionizable or protonizable cationic lipids are typically characterized by their pK a It is protonated (i.e., positively charged) at a pH below its pK a At pH levels above 100, the cationic lipids are substantially neutral. Preferred ionizable cationic lipids are those with a pKa below physiological pH (typically about 7.4). The cationic lipids of the present disclosure may also be referred to as titratable cationic lipids. The cationic lipids may be "amino lipids" that have a protonizable tertiary amine (e.g., pH titratable) head group. Some exemplary amino lipids are 18The alkyl chains may each independently comprise 0-3 (e.g., 0, 1, 2, or 3) double bonds and ether, ester, or cathal bonds between the head group and the alkyl chain. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3), and (DLin-MP-DMA) (also known as 1-Bl1).

[0421] The term "comprising" is intended to be open and can, but does not require, the inclusion of additional elements or steps. That is, when the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0422] The term "composition" refers to a product comprising specified ingredients in specified amounts, and any product resulting directly or indirectly from the combination of specified ingredients in specified amounts.

[0423] The term "in combination with" refers to the administration of a lipid-formulated mRNA of the present disclosure and another pharmaceutical agent in the therapeutic methods of the present disclosure, and means that the lipid-formulated mRNA of the present disclosure and the other pharmaceutical agent are administered sequentially or simultaneously in separate dosage forms, or simultaneously in the same dosage form.

[0424] The terms "commercially available chemicals" and "chemicals" used in the examples presented herein can be obtained from common suppliers, such as Acros Organics (Pittsburgh, Pa.), Sigma-Adrich Chemical (Milwaukee, Wis.), Avocado Research (Lancashire, UK), Bionet (Cornwall, UK), Boron Molecular (Research Triangle Park, NC), Combi-Blocks (San Diego, Calif.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, Pa.), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. (Cornwall, UK), Pierce Chemical Co. (Rockford, Ill.), Riedel de Haen (Hannover, Germany), Spectrum Quality Products, Inc. (New Brunswick, NJ), TCI America (Portland, Oreg.), and Wako Chemicals USA, Inc. (Richmond, Va.).

[0425] The phrase "a compound described in the chemical literature" can be identified through reference books and databases of chemical compounds and chemical reactions, as known to those of skill in the art. Suitable reference books and articles detailing the synthesis of reactants useful in the preparation of the compounds disclosed herein or providing references to articles describing the preparation of the compounds disclosed herein include, for example, “Synthetic Organic Chemistry,” John Wiley and Sons, Inc. New York; S.R. Sandler et al., “Organic Functional Group Preparations,” 2nd Ed., Academic Press, New York, 1983; H.O. House, “Modern Synthetic Reactions,” 2nd Ed., W.A. Benjamin, Inc. Menlo Park, Calif., 1972; T.L. Lichrist, “Heterocyclic Chemistry,” 2nd Ed. John Wiley and Sons, New York, 1992; and J. March, “Advanced Organic Chemistry: reactions, Mechanisms and Structure,” 5th Ed., Wiley Interscience, New York, 2001. Certain reactants and similar reactants may be found in the Chemical Abstract Service of the American Chemical Chemicals can also be identified through the Index of Known Chemicals compiled by the American Chemical Society, which is available in most public and university libraries and online databases. (For further information, the American Chemical Society, Washington, DC, can be contacted.) Known but non-catalogued chemicals can be prepared by custom chemical synthesis companies, and many of the standard chemical supply companies (such as those listed above) offer custom synthesis services.

[0426] The term "complementary nucleotide bases" refers to nucleotide base pairs that form hydrogen bonds with each other. Adenine (A) pairs with thymine (T) or, in RNA, with uracil (U), and guanine (G) pairs with cytosine (C). Complementary nucleic acid segments or strands hybridize (i.e., bind by hydrogen bonds) with each other. "Complementary" means that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence, either by the traditional Watson-Crick mode or by another, less traditional, mode of binding.

[0427] The term "cycloalkyl," as used herein, unless otherwise specified, refers to a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group of 3 to 8 carbons and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclic heptyl, and the like. When a cycloalkyl group contains one carbon-carbon double bond, the cycloalkyl group can be referred to as a "cycloalkenyl" group. Exemplary cycloalkenyl groups include cyclopentenyl, cyclohexenyl, and the like. Cycloalkyl groups of the present disclosure include: (1) C 1-7 Acyl (e.g., carboxaldehyde), (2) C 1-20 Alkyl (e.g., C 1-6 Alkyl, C 1-6 Alkoxy-C 1-6 Alkyl, C 1-6 Alkylsulfinyl-C 1-6 Alkyl, Amino-C 1-6 Alkyl, azido-C 1-6 Alkyl, (Carboxaldehyde)-C 1-6 Alkyl, halo-C 1-6 Alkyl (e.g. perfluoroalkyl), hydroxy-C 1-6 Alkyl, nitro-C 1-6 Alkyl or C 1-6 Thioalkoxy-C 1-6 (3) C 12 Alkoxy (e.g. C 1-6 alkoxy, for example perfluoroalkoxy), (4) C 1-6 Alkylsulfinyl, (5)C6-10 Aryl, (6) Amino, (7) C 1-6 Alkyl-C 6-10 Aryl, (8) Azide, (9) C 3-8 Cycloalkyl, (10)C 1-6 Alkyl-C 3-8 Cycloalkyl, (11) halo, (12) C 1-12 Heterocyclyl (e.g., C 1-12 Heteroaryl), (13)(C 1-12 (14) hydroxy, (15) nitro, (16) C 1-20 Thioalkoxy (e.g. C 1-6 Thioalkoxy), (17)-(CH 2 ) q CO 2 R A’ (In the formula, q is an integer of 0 to 4, and R A’ (a)C 1-6 Alkyl, (b) C 6-10 Aryl, (c) hydrogen and (d) C 1-6 Alkyl-C 6-10 aryl), (18)-(CH 2 ) q CONR B’ R C’ (In the formula, q is an integer of 0 to 4, and R B’ and R C’ are independently: (a) hydrogen, (b) C 6-10 Alkyl, (c) C 6-10 Aryl and (d) C 1-6 Alkyl-C 6-10 aryl), (19)-(CH 2 ) q SO 2 R D’ (In the formula, q is an integer of 0 to 4, and R D’ (a)C 6-10 Alkyl, (b) C 6-10 Aryl and (c) C 1-6 Alkyl-C 6-10 aryl), (20)-(CH 2 ) q SO 2 NR E’ RF’ (In the formula, q is an integer of 0 to 4, and R E’ and R F’ each independently represents (a) hydrogen, (b) C 6-10 Alkyl, (c) C 6-10 Aryl and (d) C 1-6 Alkyl-C 1-10 aryl), (21) thiol, (22) C 6-10 Aryloxy, (23)C 3-8 Cycloalkoxy, (24)C 6-10 Aryl-C 1-6 Alkoxy, (25)C 1-6 Alkyl-C 1-12 Heterocyclyl (e.g., C 1-6 Alkyl-C 1-12 Heteroaryl), (26) oxo, (27) C 2-20 Alkenyl, and (28)C 2-20 In some embodiments, each of these groups can be further substituted as described herein. For example, C 1 - Alkaryl or C 1 The alkyl group of the -alkylheterocyclyl can be further substituted with oxo groups to give the corresponding aryloyl and (heterocyclyl)oyl substituents.

[0428] The term "diastereomer," as used herein, means a stereoisomer that is not a mirror image of the other and is not superimposable with one another.

[0429] The term "diacylglycerol" or "DAG" refers to a compound that has two fatty acyl chains, 1 and R 2 and 2-30 carbons, both of which are independently attached to the 1- and 2-positions of glycerol by ester bonds. The acyl groups can be saturated or of various degrees of unsaturation. Suitable acyl groups include lauroyl (C 12 ), myristoyl (C14 ), palmitoyl (C 16 ), stearoyl (C 18 ) and Icosoil (C 20 In a preferred embodiment, R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristoyl (i.e., dimyristoyl), and R 1 and R 2 are both stearoyl (i.e. distearoyl).

[0430] The term "dialkyloxypropyl" or "DAA" includes compounds having two alkyl chains, both of which, R and R, independently, have from 2 to 30 carbons. The alkyl groups can be saturated or have various degrees of unsaturation.

[0431] The term "effective amount" of an agent, as used herein, is an amount sufficient to produce a beneficial or desired result, e.g., a clinical result, and thus "effective amount" depends on the context in which the term is applied. For example, in the context of administering an agent to treat cancer, an effective amount of an agent is, for example, an amount sufficient to achieve cancer treatment as defined herein, as compared to the response obtained in the absence of administration of the agent.

[0432] The term "enantiomer," as used herein, refers to each individual optically active form of a compound of the present disclosure having an optical purity or enantiomeric excess (as determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90%, and more preferably at least 98%.

[0433] "Enzyme with cystic fibrosis transmembrane conductance regulator activity", "enzyme with CFTR activity", "protein with CFTR activity", "protein with cystic fibrosis transmembrane conductance regulator activity", "CFTR enzyme" or "CFTR protein" means a protein or enzyme that conducts chloride ions across epithelial cell membranes, helping to maintain salt and water balance at epithelial surfaces of the body. The CFTR protein is a specific type of protein called an ion channel, which is tubular in shape and allows charged atoms or molecules to move from inside the cell to outside the cell or from outside the cell to inside the cell. In the lungs, the CFTR ion channel moves chloride ions from inside the cell to outside the cell. To leave the cell, the chloride ions move through the middle of the tube formed by the CFTR protein. As the chloride ions leave the cell, they attract a layer of water with them. This layer of water is important because it moves the cilia on the surface of the lung cells back and forth. This back and forth action moves mucus up and out of the airways.

[0434] The term "fully encapsulated" means that the nucleic acid (e.g., mRNA) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or nuclease assay, which would otherwise significantly degrade free RNA.When fully encapsulated, preferably, less than 25% of the nucleic acid in the particle is degraded, more preferably, less than 10%, and most preferably, less than 5% of the nucleic acid in the particle is degraded in a treatment in which 100% of free nucleic acid is normally degraded."Fully encapsulated" also means that the nucleic acid-lipid particle is not rapidly degraded into its component parts when administered in vivo.

[0435] The terms "halo" and "halogen," as used herein, refer to a halogen selected from bromine, chlorine, iodine or fluorine.

[0436] The term "haloalkyl," as used herein, refers to an alkyl group, as defined herein, that is substituted with a halogen group (i.e., F, Cl, Br, or I). Haloalkyl may be substituted with one, two, three, or, in the case of alkyl groups of two or more carbons, four halogens. Haloalkyl groups include perfluoroalkyl (e.g., -CF 3 ), -CHF 2 , -CH 2 F, -CCl 3 , -CH 2 CH 2 Br, -CH 2 CH(CH 2 CH 2 Br)CH 3 And-CHICH 3 In some embodiments, the haloalkyl group can be further substituted with 1, 2, 3, or 4 substituents as described herein for the alkyl group.

[0437] The term "heteroalkyl," as used herein, refers to an alkyl group, as defined herein, in which one or two of its constituent carbon atoms are replaced by nitrogen, oxygen, or sulfur, respectively. In some embodiments, the heteroalkyl group can be further substituted with one, two, three, or four substituents as described herein for the alkyl group.

[0438] The term "hydrocarbon", as used herein, refers to a group consisting solely of carbon and hydrogen atoms.

[0439] As used herein, the term "hydroxy" refers to an -OH group. In some embodiments, the hydroxy group can be substituted with one, two, three, or four substituents (e.g., O-protecting groups) as defined herein for alkyl.

[0440] The term "hydroxyalkenyl," as used herein, refers to an alkenyl group, as defined herein, substituted with one to three hydroxy groups, provided that not more than one hydroxy group may be attached to a carbon atom of the alkyl group, and hydroxyalkenyl is exemplified by dihydroxypropenyl, hydroxyisopentenyl, and the like. In some embodiments, the hydroxyalkenyl group can be substituted with one, two, three, or four substituents (e.g., O-protecting groups) as defined herein for alkyl.

[0441] The term "hydroxyalkyl," as used herein, refers to an alkyl group, as defined herein, substituted with one to three hydroxy groups, provided that not more than one hydroxy group may be attached to a carbon atom of the alkyl group, and hydroxyalkyl is exemplified by hydroxymethyl, dihydroxypropyl, and the like. In some embodiments, the hydroxyalkyl group can be substituted with one, two, three, or four substituents (e.g., O-protecting groups) as defined herein for alkyl.

[0442] The term "hydrate" refers to the solvent molecule being H 2 O.

[0443] The term "isomer," as used herein, refers to any tautomer, stereoisomer, enantiomer, or diastereomer of any of the compounds of the present disclosure. It is recognized that the compounds of the present disclosure can have one or more chiral centers and / or double bonds and can therefore exist as stereoisomers, e.g., double bond isomers (i.e., E / Z geometric isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-), or cis / trans isomers). In accordance with the present disclosure, the chemical structures depicted herein, i.e., the compounds of the present disclosure, can exist in stereoisomerically pure (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) forms, as well as mixtures of enantiomers and stereoisomers, e.g., racemic forms. The term "enantiomeric" includes any of the corresponding stereoisomers, both in the form of a chiral salt, and in the form of a chiral solvent. Enantiomeric and stereoisomeric mixtures of the compounds of the present disclosure can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereoisomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthesis methods.

[0444] The term "nitro" as used herein means -NO 2 Represents a group.

[0445] The term "N / P ratio," as used herein, refers to the ratio of the number of positively charged amine groups (N) of a cationic lipid to the number of negatively charged phosphate groups (P) of the CFTR mRNA that is encapsulated in or targeted for encapsulation by a cationic lipid(s).

[0446] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or modified linkages, including synthetic, natural and non-natural nucleic acids, that have similar binding properties as the reference nucleic acid, and that are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0447] As used herein, the term "oxo" refers to =O.

[0448] The term "stereoisomer," as used herein, refers to any of the various possible isomeric and conformational forms that a compound (e.g., a compound of any formula described herein) may assume, in particular, all possible stereochemical and conformational isomeric forms of the basic molecular structure, all diastereomers, enantiomers and / or conformers. Some compounds of the present disclosure may exist in various tautomeric forms, all of which are included within the scope of the present disclosure.

[0449] The term "sulfonyl" as used herein refers to -S(O) 2 - represents a group.

[0450] The term "compound" is intended to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.

[0451] The term "conserved" refers to nucleotides or amino acids that are found unchanged in the same position of two or more sequences being compared among the nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence. Relatively conserved nucleotides or amino acids are those that are conserved between related sequences to a greater extent than the nucleotides or amino acids found at other positions in the sequences.

[0452] The term "circular" refers to the presence of an unbroken ring. A circular molecule does not necessarily have to be circular, but only connects to form a continuous chain of subunits. A circular molecule, such as the mRNA of the present disclosure, may be a single unit or multimer, or may constitute one or more components of a complex or higher-order structure.

[0453] The term "cytotoxic" refers to killing or having a detrimental, toxic or lethal effect on a cell (e.g., a mammalian cell (e.g., a human cell)), a bacterium, a virus, a fungus, a protozoan, a parasite, a prion, or a combination thereof.

[0454] The term "delivery" refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload.

[0455] The term "delivery agent" refers to any substance that at least partially facilitates the in vivo delivery of a polynucleotide to a target cell.

[0456] The term "digest" means to break down into smaller fragments or components. In reference to polypeptides or proteins, digestion produces peptides.

[0457] The term "distal" means away from the center or from a point or area of ​​interest.

[0458] The phrase "encoding a protein cleavage signal" refers to a nucleotide sequence that encodes a protein cleavage signal.

[0459] The term "engineered" refers to a molecule that is designed to have altered characteristics or properties, whether structurally or chemically, from the starting, wild-type or naturally occurring molecule.

[0460] The term "expression" of a nucleic acid sequence refers to one or more of the following events: (1) the production of an RNA template from a DNA sequence (e.g., by transcription); (2) the processing...

Claims

1. a. i. about 20 mol % to about 30 mol % of an ionizable cationic lipid having the structure ATX-012 below; 【Chemistry 1】 x. about 20 mol % to about 30 mol % of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); xi. about 7 mol% to about 13 mol% of a helper lipid; xii. about 33 mol% to about 44 mol% cholesterol; xiii. A lipid formulation comprising about 0.5 mol% to about 3.0 mol% of a PEG-lipid conjugate; b. messenger RNA (mRNA) encoding a peptide having cystic fibrosis transmembrane conductance regulator (CFTR) activity; A composition comprising: The composition, wherein the mRNA is encapsulated in the lipid formulation.

2. 2. The composition of claim 1, wherein the lipid formulation is selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymer-based carriers, exosomes, lamellar bodies, micelles, and emulsions.

3. 2. The composition of claim 1, wherein the lipid formulation is a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes.

4. The composition of claim 2 , wherein the lipid formulation is a lipid nanoparticle.

5. 5. The composition of claim 4, wherein the lipid nanoparticles have a size of less than about 200 nm, less than about 150 nm, or less than about 100 nm.

6. 5. The composition of claim 4, wherein the lipid nanoparticles have a size of about 55 nm to about 90 nm.

7. The composition of claim 1 , wherein the helper lipid is a phospholipid.

8. 8. The composition of claim 7, wherein the helper lipid is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC) and phosphatidylcholine (PC).

9. The composition of claim 1, wherein the PEG-lipid conjugate is PEG-DMG.

10. The composition of claim 9, wherein the PEG-DMG is PEG2000-DMG.

11. 2. The composition of claim 1, wherein the weight ratio of total lipid to mRNA in the composition is from about 5:1 to about 25:1, from about 10:1 to about 20:1, from about 12:1 to about 18:1, or from about 14:1 to about 17:

1.

12. 2. The composition of claim 1, wherein the lipid formulation comprises (i) about 22 mol% to about 28 mol% of the ionizable cationic lipid, (ii) about 23 mol% to about 27 mol% of the ionizable cationic lipid, or (iii) about 24 mol% to about 26 mol% of the ionizable cationic lipid.

13. 10. The composition of claim 1, wherein the lipid formulation comprises: (i) about 22 mol% to about 28 mol% DOTAP; (ii) about 23 mol% to about 27 mol% DOTAP; or (iii) about 24 mol% to about 26 mol% DOTAP.

14. 10. The composition of claim 1, wherein the lipid formulation comprises from about 8 mol% to about 12 mol% of the helper lipid, or from about 9 mol% to about 11 mol% of the helper lipid.

15. 10. The composition of claim 1, wherein the lipid formulation comprises about 35 mol% to about 41 mol% cholesterol, or about 36 mol% to about 40 mol% cholesterol.

16. 2. The composition of claim 1, wherein the lipid formulation comprises (i) from about 0.75 mol% to about 2.5 mol% of the PEG-lipid conjugate, (ii) from about 1.0 mol% to about 2.0 mol% of the PEG-lipid conjugate, or (iii) from about 1.25 mol% to about 1.75 mol% of the PEG-lipid conjugate.

17. 2. The composition of claim 1, wherein the peptide having CFTR activity has a sequence at least about 85%, 90%, 95%, 98%, or 99% identical to the sequence of SEQ ID NO:

99.

18. 18. The composition of claim 17, wherein the peptide having CFTR activity has the sequence of SEQ ID NO:

99.

19. 2. The composition of claim 1, wherein the mRNA has a sequence selected from the group consisting of SEQ ID NOs: 49, 53, 66, 68, 69 and 72.

20. 2. The composition of claim 1, wherein the mRNA comprises a 3' poly-A tail consisting of about 50 to about 120 adenosine monomers.

21. The composition of claim 1 , wherein the mRNA comprises a 5′ cap.

22. The 5' cap has the structure of the following formula (Cap V): ７ GpppAmpG, 【Chemistry 2】 In the formula, R １ , R ２ and R ４ 22. The composition of claim 21, wherein each is OH, n is 1, each L is a phosphate linked by a diester bond, and the mRNA is the mRNA of the composition.

23. The mRNAs each independently comprise 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylester uridine, 5-formyluridine, 5-methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N １ -hydroxypseudouridine, N １ -methylpseudouridine, 2'-O-methyl-N １ -methylpseudouridine, N １ -Ethylpseudouridine, N １ -hydroxymethylpseudouridine, aurauridine, N ６ 2. The composition of claim 1, comprising one or more chemically modified nucleotides selected from the group consisting of 6-O-methyladenosine, 2-aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 8-oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, and 6-O-methylguanosine.

24. 10. The composition of claim 1, wherein the composition comprises a HEPES or TRIS buffer having a pH of about 7.0 to about 8.5, or about 7.4 to about 8.

2.

25. 25. The composition of claim 24, wherein the HEPES or TRIS buffer is at a concentration of about 20 mM to about 80 mM, about 35 mM to about 70 mM, about 40 mM to about 60 mM, about 45 mM to about 55 mM, about 20 mM to about 50 mM, about 25 mM to about 40 mM, or about 25 mM to about 35 mM.

26. 25. The composition of claim 24, further comprising about 10 mM to about 100 mM, about 20 mM to about 90 mM, about 30 mM to about 80 mM, about 35 mM to about 70 mM, about 40 mM to about 60 mM, or about 45 mM to about 55 mM NaCl.

27. The composition of claim 1 further comprising one or more cryoprotectants.

28. 28. The composition of claim 27, wherein the one or more cryoprotectants are selected from the group consisting of sucrose, glycerol, and a combination of sucrose and glycerol. (i) a combination of sucrose at a concentration of about 5% (w / v) to about 18% (w / v) and glycerol at a concentration of about 1% (w / v) to about 9% (w / v); (ii) a combination of sucrose at a concentration of about 6% (w / v) to about 16% (w / v) and glycerol at a concentration of about 1.5% (w / v) to about 7% (w / v); (iii) a combination of sucrose at a concentration of about 7% (w / v) to about 14% (w / v) and glycerol at a concentration of about 1.75% (w / v) to about 6% (w / v); (iv) a combination of sucrose at a concentration of about 7% (w / v) to about 12% (w / v) and glycerol at a concentration of about 1% (w / v) to about 6% (w / v); or (v) a combination of sucrose at a concentration of about 8% (w / v) to about 11% (w / v) and glycerol at a concentration of about 3% (w / v) to about 6% (w / v); 29. The composition of claim 28, comprising:

30. the helper lipid is distearoylphosphatidylcholine (DSPC); the PEG-lipid conjugate is PEG2000-DMG; The mRNA comprises SEQ ID NO:

53. The composition of claim 1.

31. 31. The composition of claim 30, wherein the lipid formulation is a lipid nanoparticle.

32. 32. The composition of claim 31, wherein the lipid nanoparticles have a size of less than about 100 nm.

33. 33. A medicament for ameliorating, preventing, delaying the onset of, or treating a disease or disorder associated with decreased activity of the cystic fibrosis transmembrane conductance regulator (CFTR) in a subject in need thereof, the medicament comprising the composition of any of claims 1 to 32.

34. A kit for expressing human CFTR in vivo, said kit comprising the composition of any one of claims 1 to 32 and an instrument for administering said composition.

35. 35. The kit of claim 34, wherein the device is an injection needle, an intravenous needle, or an inhalation device.

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