Improved compositions for delivery of mRNA

Abstract

The present invention provides, inter alia, improved mRNA-encapsulated lipid nanoparticles that are particularly effective for pulmonary delivery by inhalation administration. The lipid nanoparticles comprise a lipid component consisting of cationic lipids, non-cationic lipids, PEG-modified lipids, and cholesterol or cholesterol analogs, with a lower molar ratio of non-cationic lipids than is typically present in lipid nanoparticles delivered via this route of administration.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 176,549, filed April 19, 2021, and U.S. Provisional Patent Application No. 63 / 251,372, filed October 21, 2021, each of which is incorporated by reference in its entirety. [Background technology]

[0002] Messenger RNA therapy (MRT) is becoming an increasingly important approach for the treatment of various diseases. MRT involves the administration of messenger RNA (mRNA) to a patient in need of therapy for the production of the protein encoded by the mRNA in the patient's body. Lipid nanoparticles are commonly used to encapsulate the mRNA for effective in vivo delivery of the mRNA. Summary of the Invention [Problem to be solved by the invention]

[0003] The success of mRNA therapeutics depends on a well-designed delivery system that should guide the mRNA into the desired compartment of the selected cell. However, humans and other organisms have developed natural barriers that protect the body from different types of pathogens or invaders. Delivery of mRNA therapeutics into the lungs is particularly challenging. For example, inhalation administration of lipid nanoparticles encapsulating mRNA (e.g., mRNA encoding a therapeutic protein) has been found to be effective in inducing expression of the protein encoded by the mRNA in the far reaches of the lungs. However, delivery of large amounts of intact lipid nanoparticles using inhalation administration has proven challenging. In addition, the lungs contain mucus that traps microorganisms and particles that remove them from the lungs via coordinated beating of motile cilia. Thus, there is a need to provide improved lipid nanoparticles for delivery of mRNA to target cells in the lungs by inhalation administration.

[0004] The inhalation dose output and the change in encapsulation efficiency are important design control criteria (DC criteria) for effective pulmonary delivery of lipid nanoparticles encapsulating mRNA. Based on previous research studies, the inventors have identified an inhalation dose output of at least 12 ml / h and a change in encapsulation efficiency (ΔEE%) after inhalation dose of not more than 20% as important for effective pulmonary delivery of intact mRNA into the lungs. [Means for solving the problem]

[0005] The present invention provides lipid nanoparticles encapsulating mRNA that are particularly effective in delivering mRNA to the lungs via inhalation administration. In particular, the lipid nanoparticles described herein can achieve increased inhalation dose output, maintain encapsulation efficiency of mRNA upon inhalation administration, and result in increased expression of proteins that are proteins encoded by the mRNA. Without being bound by theory, these improvements are due to the lower molar ratio of non-cationic lipids present in the lipid nanoparticles of the present invention. Surprisingly, the inventors have found that lipid nanoparticles with reduced total lipid content maintain better encapsulation efficiency and are more effectively nebulized while not degrading in vivo efficacy relative to lipid nanoparticles with higher lipid content. In particular, the inventors have found that the inhalation administration characteristics and in vivo efficacy of mRNA-encapsulated lipid nanoparticles can be improved by adjusting the total lipid:mRNA ratio (mg:mg) to 19:1 or less. This can be achieved by increasing the molar ratio of cationic lipid to more than 40% (molar ratio) and decreasing the molar ratio of non-cationic lipid content. Specifically, increasing the molar ratio of cationic lipid to greater than 40% (e.g., to 50% or 60%) while lowering the overall lipid content through a reduction in the non-cationic lipid content results in lipid nanoparticle formulations with improved in vivo efficacy.

[0006] The lipid nanoparticles of the present invention and compositions comprising the same can be used for the effective treatment or prevention of a number of diseases and disorders, including pulmonary diseases and disorders, e.g., protein deficiency or neoplastic diseases affecting the lungs, and infectious diseases, e.g., via immunization via the lungs, or for the systemic delivery of mRNA therapeutics via the lungs.

[0007] In particular, the present invention relates to, inter alia: (i) mRNA encapsulated within lipid nanoparticles, and (ii) consisting of: a. cationic lipid components; b. non-cationic lipid components; c. a PEG-modified lipid component, and D. Cholesterol components Lipid components consisting of 1. A lipid nanoparticle comprising: (1) the cationic lipid component is greater than 40% (molar ratio); (2) the non-cationic lipid component is less than 25% (by molar ratio); (3) Providing lipid nanoparticles having a total lipid:mRNA ratio (mg:mg) of 19:1 or less.

[0008] In some embodiments, the total lipid:mRNA ratio (mg:mg) is between 11:1 and 19:1.

[0009] In some embodiments, the cationic lipid component is 45%-60% (molar ratio). In particular embodiments, the cationic lipid component is 45%-55% (molar ratio). In particular embodiments, the cationic lipid component is about 50% (molar ratio).

[0010] In some embodiments, the non-cationic lipid component is about 22.5% (molar ratio) or less. In some embodiments, the non-cationic lipid component is less than 18% (molar ratio). In some embodiments, the non-cationic lipid component is 15% (molar ratio) or less. In some embodiments, the non-cationic lipid component is less than 13% (molar ratio).

[0011] In some embodiments, the cholesterol component is cholesterol or a cholesterol analog.

[0012] In a particular embodiment, the molar ratio of the lipid components is: a. Cationic lipids: approximately 47% to 60% b. non-cationic lipids, about 10% to 22.5%; c. Approximately 3% to 5% PEG-modified lipid; d. The remainder is cholesterol or a cholesterol analog.

[0013] In a particular embodiment, the molar ratio of the lipid components is: a. about 50% to 55% cationic lipids, b. about 10-15% non-cationic lipids; c. Approximately 3-5% PEG-modified lipid; d. The remainder is cholesterol or a cholesterol analog.

[0014] In certain embodiments, the molar ratio of the lipid components is: a. approximately 55% cationic lipids; b. about 10% non-cationic lipids; c. about 5% PEG-modified lipid, and d. About 30% cholesterol or cholesterol analogues.

[0015] In another particular embodiment, the molar ratio of the lipid components is: a. approximately 50% cationic lipids, b. about 12.5% ​​non-cationic lipids; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues is about 32.5%.

[0016] In a further particular embodiment, the molar ratio of the lipid components is: a. approximately 50% cationic lipids, b. about 15% non-cationic lipids; c. about 5% PEG-modified lipid, and d. About 30% cholesterol or cholesterol analogues.

[0017] In certain embodiments, the molar ratio of the lipid components is: a. approximately 47% cationic lipids; b. about 22.5% non-cationic lipids; c. about 3% PEG-modified lipid, and d. Cholesterol or cholesterol analogues is about 27.5%.

[0018] In a particular embodiment, the cationic lipid is SY-3-E14-DMAPr. In another particular embodiment, the cationic lipid is TL1-01D-DMAPr.

[0019] For example, in certain embodiments, the lipid nanoparticle is any one of the lipid nanoparticles in Tables A, B, C, D, E, F, G and H.

[0020] In some embodiments, the total lipid:mRNA ratio (mg:mg) is about 18:1 or less. In some embodiments, the total lipid:mRNA ratio (mg:mg) is about 17:1 or less. In some embodiments, the total lipid:mRNA ratio (mg:mg) is about 15:1 or less.

[0021] Typically, lipid nanoparticles according to the present invention can be nebulized at an inhalation throughput of more than about 12 ml / h. In a particular embodiment, lipid nanoparticles can be nebulized at an inhalation throughput of more than about 15 ml / h. In a particular embodiment, lipid nanoparticles can be nebulized at an inhalation throughput of more than about 20 ml / h.

[0022] In some embodiments, the encapsulation efficiency of the lipid nanoparticles after inhalation administration is about 10% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In some embodiments, the lipid nanoparticles have an encapsulation efficiency of at least about 90% before and after inhalation administration.

[0023] The present invention relates to (i) mRNA encapsulated within lipid nanoparticles, and (ii) lipids having the following molar ratio: a) 41% to 70% cationic lipid, b) non-cationic lipids 9% to 18%, c) 2% to 6% PEG-modified lipid, and d) Cholesterol or cholesterol analogues 9% to 48% Lipid components consisting of Also provided are lipid nanoparticles comprising:

[0024] Such lipid nanoparticles can be nebulized, for example, at inhalation dose throughputs of greater than 12 ml / h.

[0025] In some embodiments, the molar ratio of cationic lipids in the lipid nanoparticles according to the present invention is between 45% and 70%. In some embodiments, the molar ratio of cationic lipids is between 45% and 65%. In some embodiments, the molar ratio of cationic lipids is between 50% and 70%. In some embodiments, the molar ratio of cationic lipids is between 50% and 65%. In a particular embodiment, the molar ratio of cationic lipids is between 50% and 60%. In a particular embodiment, the molar ratio of cationic lipids is about 50%. In another particular embodiment, the molar ratio of cationic lipids is about 55%. In yet a further particular embodiment, the molar ratio of cationic lipids is about 60%.

[0026] In some embodiments, the molar ratio of non-cationic lipids in the lipid nanoparticles according to the present invention is 9% to 15%. In particular embodiments, the molar ratio of non-cationic lipids is 10% to 15%. In a particular embodiment, the molar ratio of non-cationic lipids is about 15%. In another particular embodiment, the molar ratio of non-cationic lipids is about 12.5%. In yet a further particular embodiment, the molar ratio of non-cationic lipids is about 10%.

[0027] In some embodiments, the molar ratio of PEG-modified lipid in the lipid nanoparticle according to the present invention is 3%-6%. In a particular embodiment, the molar ratio of PEG-modified lipid is 4%-6%. In a particular embodiment, the molar ratio of PEG-modified lipid is about 5%. In another particular embodiment, the molar ratio of PEG-modified lipid is about 3%.

[0028] In some embodiments, the molar ratio of cholesterol or cholesterol analogues in the lipid nanoparticles according to the present invention is between 10% and 45%. In a particular embodiment, the molar ratio of cholesterol or cholesterol analogues is between 10% and 30%. In one particular embodiment, the molar ratio of cholesterol or cholesterol analogues is between 25% and 30%. In a particular embodiment, the molar ratio of cholesterol or cholesterol analogues is about 25%. In another particular embodiment, the molar ratio of cholesterol or cholesterol analogues is about 30%.

[0029] In one particular embodiment, the molar ratio of lipids in lipid nanoparticles according to the invention is: a. 50%-60% cationic lipid, b. 9%-18% non-cationic lipid, c. 4%-6% PEG-modified lipid, and d. 20-35% cholesterol or cholesterol analog. In another particular embodiment, the molar ratio of lipids in lipid nanoparticles according to the invention is: a. 50%-60% cationic lipid, b. 9%-15% non-cationic lipid, c. 4%-6% PEG-modified lipid, and d. 25-30% cholesterol or cholesterol analog.

[0030] In an exemplary embodiment, the molar ratio of lipids in lipid nanoparticles according to the present invention is: 1) a. about 50% cationic lipid, b. about 15% non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 30% cholesterol or cholesterol analogue. 2) a. about 60% cationic lipid, b. about 10% non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 25% cholesterol or cholesterol analogue. 3) a. about 50% cationic lipid, b. about 10% non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 35% cholesterol or cholesterol analogue. 4) a. about 50% cationic lipid, b. about 12.5% ​​non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 32.5% cholesterol or cholesterol analogue. 5) a. about 50% cationic lipid, b. about 17.5% non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 27.5% cholesterol or cholesterol analogue. 6) a. about 55% cationic lipid, b. about 10% non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 30% cholesterol or cholesterol analogue. 7) a. about 55% cationic lipid, b. about 12.5% ​​non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 27.5% cholesterol or cholesterol analog. In some embodiments, the lipid molar ratio is: a. about 55% cationic lipid, b. about 15% non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 25% cholesterol or cholesterol analog. 8) Ia. about 55% cationic lipid, b. about 17.5% non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 22.5% cholesterol or cholesterol analogue. 9) a. about 60% cationic lipid, b. about 12.5% ​​non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 22.5% cholesterol or cholesterol analogue. 10) a. about 60% cationic lipid, b. about 15% non-cationic lipid, c. about 5% PEG-modified lipid, and d. about 20% cholesterol or cholesterol analogue.

[0031] In some embodiments, the lipid nanoparticles of the present invention are any one of the lipid nanoparticles in Tables A, B, C, D, E, F, G or H.

[0032] In certain embodiments, the lipid nanoparticles of the present invention can be nebulized at an inhalation dose throughput of greater than about 15 ml / h.

[0033] In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency of at least about 90% before and after inhalation administration. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency of at least about 95% before and after inhalation administration. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency of at least about 96% before and after inhalation administration. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency of at least about 97% before and after inhalation administration. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency of at least about 98% before and after inhalation administration. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency of at least about 99% before and after inhalation administration.

[0034] In some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention changes by less than about 20% upon inhalation administration. In some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention changes by less than about 15% upon inhalation administration. In some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention changes by less than about 10% upon inhalation administration.

[0035] In some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 20% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 15% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 10% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In certain embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 5% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In certain embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 3% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is approximately the same as the encapsulation efficiency of the lipid nanoparticles before inhalation administration.

[0036] In some embodiments, the lipid nanoparticles of the present invention are for pulmonary delivery by inhalation administration, hi some embodiments, inhalation administration is performed using a nebulizer that includes vibrating mesh technology (VMT).

[0037] In some embodiments, the cationic lipid in the lipid nanoparticles according to the present invention has the formula (IIA): [ka] (wherein X is O or S; where R' is [ka] and; Here, R 6 teeth, [ka] and;

[0038] wherein m and p are each independently 0, 1, 2, 3, 4, or 5; Here, R 7 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) k R A or -(CH2) k CH(OR 11 )R A Selected from; Here, R 8 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) n R B or -(CH2) n CH(OR 12 )R B Selected from; Here, R 9 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) q R C or -(CH2) q CH(OR 13 )R C Selected from; Here, R 10 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) r R D or -(CH2) r CH(OR 14 )R D Selected from; wherein k, n, q and r are each independently 1, 2, 3, 4 or 5; or (i) R 7 and R 8 or (ii) R 9 and R 10 together form an optionally substituted 5- or 6-membered heterocycloalkyl or heteroaryl, the heterocycloalkyl or heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; Here, R 11 , R 12 , R 13 and R 14 are each independently selected from H, methyl, ethyl, or propyl; Here, R A , R B , R C and R D is optionally substituted (C6-C 20 ) alkyl, optionally substituted (C6-C 20 ) alkenyl, optionally substituted (C6-C 20) alkynyl, optionally substituted (C6-C 20 ) acyl, optionally substituted -OC(O)alkyl, optionally substituted -OC(O)alkenyl, optionally substituted (C1-C6)monoalkylamino, optionally substituted (C1-C6)dialkylamino, optionally substituted (C1-C6)alkoxy, -OH, -NH2; Here, R 7 , R 8 , R 9 , R 10 At least one of the following is R A , R B , R C Or R D Each part contains this R A , R B , R C Or R D is optionally substituted (C6-C 20 ) alkyl, optionally substituted (C6-C 20 ) alkenyl, optionally substituted (C6-C 20 ) alkynyl, optionally substituted (C6-C 20 ) acyl, optionally substituted -OC(O)(C6-C 20 ) alkyl or optionally substituted -OC(O)(C6-C 20 ) alkenyl) or a pharma- ceutically acceptable salt thereof.

[0039] In some embodiments, the cationic lipid in the lipid nanoparticles according to the present invention has formula (IIID): [ka] (wherein X is O or S; where R' is [ka] and Here, R 6 teeth, [ka] and;

[0040] wherein m and p are each independently 0, 1, 2, 3, 4, or 5; Here, R 7 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) k R A or -(CH2) k CH(OR 11 )R A Selected from; R 8 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) n R B or -(CH2) n CH(OR 12 )R B Selected from; Here, R 9 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) q R C or -(CH2) q CH(OR 13 )R C Selected from; Here, R 10 is H, optionally substituted (C1-C6) alkyl, optionally substituted (C2-C6) alkenyl, optionally substituted (C2-C6) alkynyl, optionally substituted (C1-C6) acyl, -(CH2) r R D or -(CH2)r CH(OR 14 )R D Selected from; wherein k, n, q and r are each independently 1, 2, 3, 4 or 5; or (i) R 7 and R 8 or (ii) R 9 and R 10 together form an optionally substituted 5- or 6-membered heterocycloalkyl or heteroaryl, the heterocycloalkyl or heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; Here, R 11 , R 12 , R 13 and R 14 are each independently selected from H, methyl, ethyl, or propyl; R A , R B , R C and R D is optionally substituted (C6-C 20 ) alkyl, optionally substituted (C6-C 20 ) alkenyl, optionally substituted (C6-C 20 ) alkynyl, optionally substituted (C6-C 20 ) acyl, optionally substituted -OC(O)alkyl, optionally substituted -OC(O)alkenyl, optionally substituted (C1-C6)monoalkylamino, optionally substituted (C1-C6)dialkylamino, optionally substituted (C1-C6)alkoxy, -OH, -NH2; Here, R 7 , R 8 , R 9 , R 10 At least one of the following is R A , R B , R C Or R D Each part contains this R A , R B , R C Or R Dis optionally substituted (C6-C 20 ) alkyl, optionally substituted (C6-C 20 ) alkenyl, optionally substituted (C6-C 20 ) alkynyl, optionally substituted (C6-C 20 ) acyl, optionally substituted -OC(O)(C6-C 20 ) alkyl or optionally substituted -OC(O)(C6-C 20 ) alkenyl) or a pharma- ceutically acceptable salt thereof.

[0041] In some embodiments of Formula (IIA) or Formula (IIID), X is O.

[0042] In some embodiments of Formula (IIA) or Formula (IIID), m is 1, 2 or 3.

[0043] In some embodiments of Formula (IIA) or Formula (IIID), p is 1, 2 or 3.

[0044] In some embodiments of Formula (IIA) or Formula (IIID), R' is: [ka] It is.

[0045] In some embodiments of Formula (IIA) or Formula (IIID), i) k, m and n=1; or ii) k, m and n=1 and R 11 and R 12 =H; or iii) k and n=1, m=2; or iv) k and n=1, m=2, and R 11 and R 12 =H; or v) k and n=1, m=3; or vi) k and n=1, m=3, and R 11 and R 12 =H.

[0046] In some embodiments of Formula (IIA) or Formula (IIID), R 6 teeth, [ka] In some embodiments of formula (IIA) or formula (IIID), R 6 teeth, [ka] In some embodiments of formula (IIA) or formula (IIID), R 6 teeth: [ka] In some embodiments of Formula (IIA) or Formula (IIID), R 6 teeth: [ka] is selected from the group consisting of:

[0047] In some embodiments of Formula (IIA) or Formula (IIID), R 6 teeth, [ka] and R' is [ka] where m is 2 and p is 2.

[0048] In some embodiments of Formula (IIA) or Formula (IIID), R 6 teeth, [ka] and R' is [ka] where m is 3 and p is 2.

[0049] In some embodiments of Formula (IIA) or Formula (IIID), R A and R B is optionally substituted (C6-C 20 ) alkyl, optionally substituted (C6-C 20 ) alkenyl, optionally substituted (C6-C 20 In some embodiments of Formula (IIA) or Formula (IIID), R A and R B are identical and optionally substituted (C6-C 20 ) alkyl, optionally substituted (C6-C 20 ) alkenyl, optionally substituted (C6-C 20 In some embodiments of Formula (IIA) or Formula (IIID), R A and R B are each independently an optionally substituted (C 20 In some embodiments of Formula (IIA) or Formula (IIID), R A and R B are identical and optionally substituted (C6-C 20 In some embodiments of Formula (IIA) or Formula (IIID), R A and R B are each independently an optionally substituted (C 20 In some embodiments of formula (IIA) or formula (IIID), R A and R B are identical and optionally substituted (C6-C 20 In some embodiments of formula (IIA) or formula (IIID), R A and R B are each independently an optionally substituted (C 20 In some embodiments of formula (IIA) or formula (IIID), R A and RB are identical and optionally substituted (C6-C 20 In some embodiments of formula (IIA) or formula (IIID), R A and R B are each independently an optionally substituted (C 20 In some embodiments of formula (IIA) or formula (IIID), R A and R B are identical and optionally substituted (C6-C 20 In some embodiments of formula (IIA) or formula (IIID), R A and R B each independently represents an optionally substituted -OC(O)(C 20 In some embodiments of Formula (IIA) or Formula (IIID), R A and R B are identical and optionally substituted -OC(O)(C6-C 20 In some embodiments of Formula (IIA) or Formula (IIID), R A and R B each independently represents an optionally substituted -OC(O)(C 20 In some embodiments of formula (IIA) or formula (IIID), R A and R B are identical and optionally substituted -OC(O)(C6-C 20 ) alkenyl.

[0050] In some embodiments, the cationic lipid has formula (IIIE): [ka] (In the formula, each n is independently 0 or 1; X 1A are independently O or NR 1A and; R 1A is H or C1-C6 alkyl; X 1B is a covalent bond, C(O), CH2CO2 or CH2C(O); X 2A and X 2B one is O and the other is a covalent bond; X 3A and X 3B one is O and the other is a covalent bond; X 4A and X 4B one is O and the other is a covalent bond; R 1 is independently L 1 -B 1 , C6~C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 is alkynyl; R 2 is independently L 2 -B 2 , C6~C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 is alkynyl; R 3 is independently L 3 -B 3 , C6~C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 is alkynyl; R 4 is independently L 4 -B 4 , C6~C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 is alkynyl; L 1 , L 2 , L 3 , and L 4 are each independently C1 to C 30 Alkylene; C2~C 30 Alkenylene; or C2-C 30 alkynylene; B 1 , B 2 , B3 and B. 4 each is independently an ionizable nitrogen-containing group; Cationic lipids contain at least one ionizable nitrogen-containing group. or a pharma- ceutically acceptable salt thereof.

[0051] In some embodiments, the cationic lipid has formula (IIIF): [ka] (In the formula, B 1 is an ionizable nitrogen-containing group; L 1 is C1~C 10 is alkylene; R 2 , R 3 and R 4 Each of the following is independently C6 to C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 alkynyl) or a pharma- ceutically acceptable salt thereof.

[0052] In some embodiments, the cationic lipid has the formula (IIIG): [ka] (In the formula, B 1 is an ionizable nitrogen-containing group; R 2 , R 3 and R 4 Each of the following is independently C6 to C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 alkynyl) or a pharma- ceutically acceptable salt thereof.

[0053] In some embodiments, R in the cationic lipid according to any of formulas IIIE-IIIG is 2 , R 3 and R 4 each independently represents -O(CO)R 5 OR -C(O)OR 5 C6-C substituted by 12 is alkyl, R 5 is unsubstituted C6-C 14 In some embodiments, R in the cationic lipid according to any of formulas IIIE-IIIG is alkyl. 2 , R 3 and R 4 Each of the following is independent:

[0054] [ka] It is.

[0055] In some embodiments, B in the cationic lipid according to any of formulas IIIE-IIIG. 1 teeth, a) NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl; b) [ka] ;or c) [ka] It is.

[0056] In some embodiments, L in the cationic lipid according to any of formulas IIIE-IIIG. 1 is C1-alkylene.

[0057] In some embodiments, the cationic lipid in the lipid nanoparticles according to the present invention is selected from GL-TES-SA-DMP-E18-2, GL-TES-SA-DME-E18-2, TL1-01D-DMA, TL1-04D-DMA, SY-3-E14-DMAPr, TL1-10D-DMA, HEP-E3-E10, HEP-E4-E10, SI-4-E14-DMAPr, TL1-12D-DMA, SY-010 and SY-011. In some embodiments, the cationic lipid is SY-3-E14-DMAPr.

[0058] In some embodiments, the cationic lipid in the lipid nanoparticles according to the present invention is any of the cationic lipids disclosed in PCT / US21 / 25128, which is incorporated herein by reference.

[0059] In some embodiments, the non-cationic lipid in the lipid nanoparticle according to the present invention is selected from DOPE, DLoPE, DMPE, DLPE, DOPC, DEPE, DSPC, DPPC, DMPC, DOPC, DOPS, 16:1PC, and 14:1PC.In some embodiments, the non-cationic lipid is DLoPE, DMPE, DLPE or DOPC.In a particular embodiment, the non-cationic lipid is DOPE.In some embodiments, the non-cationic lipid is DPPC or DSPC.

[0060] In some embodiments, the cholesterol or cholesterol analog in the lipid nanoparticles according to the present invention is cholesterol. In some embodiments, the cholesterol analog in the lipid nanoparticles according to the present invention is selected from β-sitosterol, stigmastanol, campesterol, fucosterol, stigmasterol and dexamethasone. In some embodiments, the cholesterol analog in the lipid nanoparticles according to the present invention is β-sitosterol. In some embodiments, the cholesterol analog in the lipid nanoparticles according to the present invention is stigmastanol.

[0061] In some embodiments, the PEG-modified lipid in the lipid nanoparticle according to the present invention is selected from DMG-PEG2K, 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, and DSPE-PEG2K-COOH. In certain embodiments, the PEG-modified lipid is DMG-PEG2K.

[0062] Exemplary lipid nanoparticles of the present invention include the following lipids: 1) a. a cationic lipid; b. DOPE as a non-cationic lipid, c. DMG-PEG2K as a PEG-modified lipid, and d. cholesterol or a cholesterol analog; or 2) a. cationic lipid, b. DSPC as a non-cationic lipid, c. DMG-PEG2K as a PEG-modified lipid, and d. cholesterol as a cholesterol or cholesterol analogue. It has a lipid component consisting of:

[0063] Exemplary lipid nanoparticles of the present invention include the following lipids: 1) a. SY-3-E14-DMAPr as a cationic lipid, b. DOPE as a non-cationic lipid, c. DMG-PEG2K as a PEG-modified lipid, and d. cholesterol or cholesterol analogue; 2) a. SY-3-E14-DMAPr as a cationic lipid, b. DSPC as a non-cationic lipid, c. DMG-PEG2K as a PEG-modified lipid, and d. cholesterol or a cholesterol analogue. It has a lipid component consisting of:

[0064] In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of less than 20:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of 16 to 19:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 19:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 16:1 (mg:mg).

[0065] In some embodiments, the mRNA encodes a therapeutic protein.

[0066] In some embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes cystic fibrosis transmembrane conductance regulator, ATP-binding cassette subfamily A member 3 protein, axonemal dynein intermediate chain 1 (DNAI1) protein, axonemal dynein heavy chain 5 (DNAH5) protein, alpha-1-antitrypsin protein, forkhead box P3 (FOXP3) protein, or one or more surfactant proteins.

[0067] In some embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention is codon optimized.

[0068] In some embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention comprises at least one non-canonical nucleobase. In some embodiments, the non-standard nucleobase is a nucleoside analog selected from the group consisting of: 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine.

[0069] In some embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes the cystic fibrosis transmembrane conductance regulator (CFTR). In some embodiments, the mRNA encodes the ATP-binding cassette subfamily A member 3 protein. In some embodiments, the mRNA encodes the axonemal dynein intermediate chain 1 (DNAI1) protein. In some embodiments, the mRNA encodes the axonemal dynein heavy chain 5 (DNAH5) protein. In some embodiments, the mRNA encodes the alpha-1-antitrypsin protein, the forkhead box P3 (FOXP3) protein. In some embodiments, the mRNA encodes one or more surfactant proteins. In some embodiments, the mRNA encodes the surfactant A protein, the surfactant B protein, the surfactant C protein, or the surfactant D protein.

[0070] In some embodiments, the lipid nanoparticles of the present invention have a size of less than about 150 nm. In certain embodiments, the lipid nanoparticles of the present invention have a size of less than about 100 nm. In particular embodiments, the lipid nanoparticles of the present invention have a size of 60-150 nm, for example 60-125 nm or 60-100 nm.

[0071] In some embodiments, the lipid nanoparticles have a size of less than about 200 nm. In some embodiments, the lipid nanoparticles have a size of less than about 150 nm. In some embodiments, the lipid nanoparticles have a size of less than about 120 nm. In some embodiments, the lipid nanoparticles have a size of less than about 110 nm. In some embodiments, the lipid nanoparticles have a size of less than about 100 nm. In some embodiments, the lipid nanoparticles have a size of less than about 80 nm. In some embodiments, the lipid nanoparticles have a size of less than about 60 nm.

[0072] In some embodiments, the present invention provides compositions comprising the lipid nanoparticles of the present invention.In typical embodiments, such compositions are formulated for pulmonary delivery by inhalation administration.In particular embodiments, inhalation administration is performed using a nebulizer that includes vibrating mesh technology (VMT).

[0073] In some embodiments, the composition comprising the lipid nanoparticles of the present invention further comprises one or more excipients. In some embodiments, the one or more excipients are selected from a buffer, a salt, a sugar, or a combination thereof. In certain embodiments, the composition of the present invention further comprises a buffer. In certain embodiments, the composition of the present invention further comprises a salt. In certain embodiments, the salt is sodium chloride. In some embodiments, the composition of the present invention further comprises a sugar. In particular embodiments, the sugar is a disaccharide. In certain embodiments, the disaccharide is sucrose or trehalose. In some embodiments, the disaccharide is at a concentration of about 4% w / v, about 6% w / v, about 8% w / v, or about 10% w / v. In some embodiments, the composition of the present invention comprises, for example, TPGS at a concentration of about 0.1% w / v to about 1% w / v in addition to other excipients, such as disaccharides.

[0074] In some embodiments, the mRNA in the compositions of the invention is at a concentration of 0.4 to 0.8 mg / ml. In certain embodiments, the mRNA is at a concentration of about 0.6 mg / ml.

[0075] In certain embodiments, the composition according to the invention comprises: a. mRNA encapsulated in lipid nanoparticles of the present invention at a concentration of about 0.6 mg / ml; b. trehalose at a concentration of about 8% w / v, and c. TPGS at a concentration of about 0.5% w / v Includes.

[0076] In some embodiments, a composition comprising the lipid nanoparticles of the present invention comprises: a. mRNA encapsulated in lipid nanoparticles at a concentration of about 0.6 mg / ml; and B sucrose at a concentration of approximately 8% w / v Includes.

[0077] In certain embodiments, the composition according to the invention comprises: a. mRNA encapsulated in lipid nanoparticles of the present invention; b. a disaccharide, such as trehalose or sucrose, at a concentration of about 3-10% w / v; c. a buffer, optionally a phosphate buffer, and d. Salt, possibly sodium chloride Includes.

[0078] In some embodiments, the compositions of the invention comprise: a. mRNA at a concentration of 0.4 to 0.8 mg / ml; b. trehalose or sucrose at a concentration of about 4% to 6% w / v; c. A buffer solution that is a phosphate buffer solution with a concentration of 1 mM to 10 mM (pH 5 to 5.5); and d. A salt that is sodium chloride at a concentration of at least 75 mM Includes. In some embodiments, the sodium chloride is at a concentration of about 75 mM to about 200 mM.

[0079] In some embodiments, the compositions of the invention comprise: a. mRNA at a concentration of about 0.4 mg / ml; b. a disaccharide that is sucrose at a concentration of about 4% w / v; c. a buffer solution that is a phosphate buffer solution at a concentration of about 2.5 mM (pH 5.5); and d. A salt that is sodium chloride at a concentration of about 150 mM Includes.

[0080] In some embodiments, the compositions of the invention comprise: a. mRNA at a concentration of about 0.4 mg / ml b. trehalose, a disaccharide, at a concentration of about 4% w / v; c. a buffer solution that is a phosphate buffer solution at a concentration of about 10 mM (pH 5); and d. A salt that is sodium chloride at a concentration of about 150 mM Includes.

[0081] The lipid nanoparticles or compositions of the present invention are typically for use in therapy. In such embodiments, the mRNA encodes a therapeutic protein, and the therapy comprises administering the lipid nanoparticles or compositions by inhalation. In some embodiments, the therapy comprises treating or preventing a disease or disorder in a subject. Thus, the present invention provides, inter alia, a method for delivering mRNA encoding a therapeutic protein in vivo, comprising administering the lipid nanoparticles or compositions of the present invention to a subject via pulmonary delivery, wherein the pulmonary delivery is via inhalation, and the composition is nebulized prior to inhalation. The present invention also provides a method for treating or preventing a disease or disorder in a subject, comprising administering the lipid nanoparticles or compositions of the present invention via inhalation.

[0082] In some embodiments, the lipid nanoparticles or compositions of the present invention are provided as dry powder formulations.In some embodiments, the lipid nanoparticles or compositions of the present invention for use in therapy are administered using a nebulizer that includes vibrating mesh technology (VMT).In some embodiments, the lipid nanoparticles or compositions of the present invention are provided in lyophilized form and are reconstituted in aqueous solution before inhalation administration.Thus, the mRNA encapsulated in lipid nanoparticles of the present invention is delivered into the lungs.

[0083] In some embodiments, the therapeutic protein encoded by the mRNA is expressed in the lungs of a healthy subject. In some embodiments, the therapeutic protein is a secreted protein. In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is an antigen.

[0084] In some embodiments, the disease or disorder treated or prevented with the lipid nanoparticles or compositions of the present invention is a pulmonary disease or disorder, e.g., a chronic respiratory disease. In some embodiments, the disease or disorder treated or prevented with the lipid nanoparticles or compositions of the present invention is a protein deficiency, e.g., a protein deficiency affecting the lungs. In some embodiments, the disease or disorder treated or prevented with the lipid nanoparticles or compositions of the present invention is a neoplastic disease, e.g., a tumor. In some embodiments, the disease or disorder treated or prevented with the lipid nanoparticles or compositions of the present invention is an infectious disease.

[0085] In some embodiments, the disease or disorder treated with the lipid nanoparticle or composition of the present invention is a protein deficiency. In these embodiments, the mRNA encodes a defective protein. In some embodiments, the protein deficiency is cystic fibrosis. Thus, in some embodiments, the mRNA encodes the cystic fibrosis transmembrane conductance regulator (CFTR). In some embodiments, the protein deficiency is primary ciliary dyskinesia. Thus, in some embodiments, the mRNA encodes the axonemal dynein intermediate chain 1 (DNAI1) protein. In some embodiments, the protein deficiency is surfactant deficiency. Thus, in some embodiments, the mRNA encodes a surfactant protein. For example, the mRNA can encode surfactant A protein, surfactant B protein, surfactant C protein, or surfactant D protein.

[0086] In some embodiments, the disease or disorder treated with the lipid nanoparticle or composition of the present invention is a chronic respiratory disease.In some embodiments, the chronic respiratory disease is chronic obstructive pulmonary disease (COPD), asthma, pulmonary arterial hypertension or idiopathic pulmonary fibrosis.Thus, in some embodiments, the mRNA encodes a therapeutic protein for treating the symptoms of pulmonary disease or disorder.For example, in some embodiments, the mRNA encodes an antibody against proinflammatory cytokines.

[0087] In some embodiments, the disease treated with the lipid nanoparticles or compositions of the present invention is a neoplastic disease, such as a tumor.Thus, in some embodiments, the mRNA encodes a protein expressed on the surface of a neoplastic cell, such as an antibody that targets the cells that constitute the tumor.

[0088] In some embodiments, the disease or disorder treated with the lipid nanoparticles or compositions of the present invention is an infectious disease. In some embodiments, the infectious disease is caused by a virus. For example, in some embodiments, the mRNA encodes a soluble decoy receptor that binds to a surface protein of the virus. In other embodiments, the mRNA encodes an antibody that targets a surface protein of the virus. In some embodiments, the infectious disease treated with the lipid nanoparticles or compositions of the present invention is caused by a bacterium. Thus, in some embodiments, the mRNA encodes an antibody that targets a surface protein of the bacterium. In some embodiments, the mRNA encodes an antigen derived from the causative agent of the infectious disease.

[0089] In some embodiments, the subject treated with the lipid nanoparticles or compositions of the present invention is a human.

[0090] The figures are for illustrative purposes only and are not limiting. [Brief description of the drawings]

[0091] [Figure 1]FIG. 1 illustrates the effect of decreasing the molar ratio of non-cationic lipid on inhalation throughput. By decreasing the molar ratio of non-cationic lipid from 30% (LNP12) to 15% (LNP3), the inhalation throughput improved from about 10 ml / h to about 15 ml / h. The threshold target is indicated by the dashed horizontal line in the figure. By further decreasing the molar ratio of non-cationic lipid from 15% (LNP3) to 10% (LNP9), the inhalation throughput improved from about 15 ml / h to about 30 ml / h. Thus, the claimed lipid nanoparticles achieve improved inhalation throughput. [Diagram 2] FIG. 2 illustrates the effect of decreasing the molar ratio of non-cationic lipids on the change in encapsulation efficiency of lipid nanoparticles after inhalation administration. This is significant because the decrease in encapsulation efficiency after inhalation administration means that less mRNA is encapsulated by lipid nanoparticles and therefore less mRNA is available to be delivered to the lungs, which leads to, for example, a decrease in protein expression in vivo, since unencapsulated mRNA is rapidly degraded in vivo. As shown in FIG. 2, decreasing the molar ratio of non-cationic lipids from 30% (LNP12) to 15% (LNP3) reduced the loss of encapsulation of mRNA in lipid nanoparticles after inhalation administration (expressed as a percentage change) (compared to encapsulation before inhalation administration) from more than 30% loss to less than 20% loss. Further decreasing the molar ratio of non-cationic lipids from 15% (LNP3) to 10% (LNP9) reduced the loss of encapsulation of mRNA in lipid nanoparticles after even more inhalation administration to less than 5% loss in encapsulation efficiency. Thus, the claimed lipid nanoparticles preserve more mRNA during inhalation administration. [Diagram 3]Figure 1 illustrates the effect of decreasing the molar ratio of non-cationic lipids on protein expression. By decreasing the molar ratio of non-cationic lipids from 30% (LNP12) to 15% (LNP3) and 10% (LNP9), the amount of delivered and thus expressed mRNA was improved. The reference target expression level is indicated by the dashed line. Thus, the claimed lipid nanoparticles achieve improved delivery and expression of mRNA. [Figure 4] 1 illustrates the effect of decreasing the molar ratio of non-cationic lipid on inhalation output for the cholesterol analogs β-sitosterol and stigmastanol. Decreasing the molar ratio of non-cationic lipid from 30% (LNP13 and LNP15) to 15% (LNP14 and LNP16) improved inhalation output. [Diagram 5] FIG. 1 illustrates the effect of decreasing the molar ratio of non-cationic lipids on the loss of encapsulation (expressed as a percentage change) of mRNA in lipid nanoparticles after inhalation administration (compared to encapsulation before inhalation administration) for the cholesterol analogs β-sitosterol and stigmastanol. Decreasing the molar ratio of non-cationic lipids from 30% (LNP13 and LNP15) to 15% (LNP14 and LNP16) reduced the loss in encapsulation or encapsulation efficiency of mRNA in lipid nanoparticles upon inhalation administration. [Figure 6] Figure 1 illustrates the effect of decreasing the molar ratio of non-cationic lipids on protein expression, for cholesterol and cholesterol analogs β-sitosterol and stigmastanol.By decreasing the molar ratio of non-cationic lipids from 30% (LNP12, LNP13 and LNP15) to 15% (LNP3, LNP14 and LNP16), the amount of delivered and thus expressed mRNA is improved.The reference target expression level is indicated by the dashed line. [Figure 7]FIG. 7A illustrates the effect of decreasing the molar ratio of the non-cationic lipid DPPC on inhalation dose output. By decreasing the molar ratio of the non-cationic lipid DPPC from 30% to 15%, the inhalation dose output was improved. FIG. 7B illustrates the effect of decreasing the molar ratio of the non-cationic lipid DPPC on the encapsulation of mRNA in lipid nanoparticles, or the loss in encapsulation efficiency, after inhalation administration, compared to the encapsulation efficiency before inhalation administration. By decreasing the molar ratio of the non-cationic lipid DPPC from 30% to 15%, the loss in the encapsulation efficiency of lipid nanoparticles after inhalation administration was reduced by half or more. FIG. 7C illustrates the effect of decreasing the molar ratio of the non-cationic lipid DPPC on protein expression. By decreasing the molar ratio of the non-cationic lipid DPPC from 30% to 15%, the amount of mRNA delivered and thus expressed was improved. The reference target expression level is indicated by a dashed line. [Figure 8] FIG. 8 illustrates the effect of decreasing the total lipid content of lipid nanoparticles on their inhalation extrusion rate by decreasing the molar ratio of the non-cationic lipid DOPE. The composition of each of the lipid nanoparticles tested is provided in the table above the figure panel. Decreasing the molar concentration of non-cationic lipid from 30% to 25% to 15%, while keeping the molar concentration of cationic lipid constant at 40%, resulted in an increase in inhalation extrusion rate (FIG. 8A). The increase in inhalation extrusion rate was maintained when the cationic lipid content was increased above 40% (molar ratio), while decreasing the non-cationic lipid content to 18% or less (molar ratio) to reach a total lipid:mRNA ratio (mg:mg) of 19:1 or less dramatically increased the encapsulation efficiency after inhalation administration (FIG. 8B). The improvement in inhalation administration characteristics was associated with an increase in in vivo efficacy (FIG. 8C). The line in FIG. 8A indicates the DC criterion (≧12 ml / h) for inhalation extrusion rate. The line in FIG. 8B indicates the DC criteria for encapsulation efficiency following inhalation administration (ΔEE not exceeding 20%). [Figure 9]Figure 9 illustrates the inhalation dosing profile for lipid nanoparticle formulations with reduced total lipid content containing different non-cationic lipids. The composition of each of the lipid nanoparticles tested is provided in the table above the figure panel. The non-cationic lipid was DLPC (12:0 PC) in bars 1-4, DMPC (14:0 PC) in bars 5-8, and DOPC (18:1 PC) in bars 9-13. The inhalation dosing extrusion rate improved significantly for all tested formulations (Figure 9A). The observed changes in encapsulation efficiency were typically around 10% or less (Figure 9B). Although an increase in size was observed, all lipid nanoparticles maintained a size below 150 nm (Figure 9C). [Figure 10] Figure 1 illustrates the in vivo efficacy of lipid nanoparticle formulations with reduced total lipid content containing different non-cationic lipids. The lipid nanoparticle compositions tested contained various concentrations of SY-3-E14-DMAPr as the cationic lipid, and various concentrations of either DOPE, DMPC, DLPC, DPPC or DOPC as the non-cationic lipid. Expression of mRNA encoding firefly luciferase was measured by determining the mean radiance (p / s / cm2 / sr) of the lungs of mice after administration of the test formulation. [Figure 11] Figure 1 illustrates the in vivo efficacy of lipid nanoparticles with reduced total lipid content, including DLPE instead of DOPE. The composition of each of the lipid nanoparticle formulations tested is shown in the table. Expression of mRNA encoding firefly luciferase was measured by determining the mean radiance (p / s / cm2 / sr) of the lungs of mice after administration of the test formulations. [Figure 12]FIG. 1 illustrates the in vivo efficacy of lipid nanoparticle formulations with reduced total lipid content and different molar ratios of cationic lipid. The composition of each lipid nanoparticle formulation tested is shown in the table adjacent to the graph. After pulmonary delivery via inhalation administration of lipid nanoparticles, the lungs of test animals were isolated and homogenized, and mCherry expression levels were determined by ELISA. Increasing the molar ratio of cationic lipid to more than 40% (e.g., to 50% or 60%) while reducing the overall lipid content through the reduction of cationic lipid content resulted in lipid nanoparticle formulations with improved in vivo efficacy. [Figure 13] FIG. 13 illustrates the inhalation administration profile and in vivo efficacy of lipid nanoparticle formulations containing different amounts of DOPE and cationic lipid TL1-01D-DMA. The composition of each lipid nanoparticle formulation tested is shown in the table above the figure panel. Adjusting the molar ratio of both cationic lipid and non-cationic lipid improved inhalation administration extrusion (FIG. 13A) and led to the identification of lipid nanoparticle formulations with high encapsulation efficiency (FIG. 13B). This formulation reduced the total lipid content to a total lipid:mRNA ratio (mg:mg) of less than 19:1 (marked in bold in the bottom row of the table). It also exhibits improved in vivo efficacy against comparative lipid nanoparticle compositions containing ICE or ML-2 as cationic lipid components, and compared to starting compositions with higher total lipid content (FIG. 13C). [Figure 14]FIG. 14 illustrates that reducing the total lipid content results in improved inhalation delivery properties and in vivo efficacy of lipid nanoparticle formulations containing the non-cationic lipid DPPC. The composition of each of the lipid nanoparticle formulations tested is shown in the table above the figure panel. Reducing the total lipid content by lowering the molar ratio of DPPC to 15% while increasing the molar ratio of the cationic lipid (SY-3-E14-DMAPr) to 50% results in increased inhalation delivery extrusion (FIG. 14A) and improved encapsulation efficiency after inhalation delivery (FIG. 14B). In vivo efficacy could also be increased and further improved by adjusting the molar ratio of SY-3-E14-DMAPr to 60% and the molar ratio of DPPC to 10% (FIG. 14C). Surprisingly, none of the lipid nanoparticle formulations containing DPPC had in vivo efficacy comparable to the standard lipid nanoparticle formulation containing 30% DOPE. However, a formulation containing 60% SY-3-E14-DMAPr and 10% DPPC compared favorably with the ML2-based lipid nanoparticles. In vivo efficacy was assessed by determining the mean radiance (p / s / cm2 / sr) of the lungs of mice following administration of a test formulation that induces expression of mRNA encoding firefly luciferase. [Figure 15] FIG. 1 illustrates the effect of different PE and PC lipids as non-cationic lipid components of lipid nanoparticles on in vivo efficacy. The PC lipids DPPC, DSPC, and DOPC, and the PE lipids DLPE, DMPE, DLoPE, and DOPE were evaluated. The composition of the lipid nanoparticle formulations is shown in the table above the graph. Except for DOPC, administration of lipid nanoparticles with PE lipids as the non-cationic lipid resulted in higher mRNA expression levels than lipid nanoparticles with PC lipids as the non-cationic lipid. Expression of mRNA encoding firefly luciferase was measured by determining the mean radiance (p / s / cm2 / sr) of the lungs of mice after administration of the test formulations. [Figure 16]1 is an illustrative bar graph depicting the amount of radiance generated by expressed luciferase protein in mice after administration of mRNA-lipid nanoparticles, each containing a different cationic lipid component. The horizontal line in the graph near 104 p / s / cm2sr represents the historical radiance / expression of encapsulated FFL mRNA in lipid nanoparticles containing a first reference cationic lipid delivered to the lung. The horizontal line in the graph near 106 p / s / cm2sr represents the historical radiance / expression of encapsulated FFL mRNA in lipid nanoparticles containing a second reference cationic lipid delivered to the lung. These thresholds can be used to screen lipids for pulmonary delivery. The lower thresholds are representative of the minimum expression levels typically desired for lipid nanoparticles of the present invention. [Figure 17] Figures 17A, 17C and 17E illustrate the effect of different disaccharides (trehalose, sucrose) and disaccharide concentrations (10%, 8%) on the size (Figures 17B, 17D and 17F) and encapsulation efficiency (Figures 17B, 17D and 17F) of mRNA-encapsulated lipid nanoparticle formulations before and after lyophilization. The composition of each of the lipid nanoparticles tested is provided in the table above the graphs. The dashed lines indicate the size and encapsulation efficiency of the lipid nanoparticles before lyophilization ("pre-lyo"), respectively. The bars indicate the size and encapsulation efficiency after lyophilization and reconstitution of the lipid nanoparticles. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0092] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. Publications and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference.

[0093] Approximately or about: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 5%, 4%, 3%, 2%, or 1% in either direction (e.g., ±2.5% more or less) of the stated reference value, unless otherwise stated or clear from the context (except where such number exceeds 100% of possible values).

[0094] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition can be administered, for example, for experimental, diagnostic, prophylactic, cosmetic and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates and / or humans). In some embodiments, the patient is a human. Human includes prenatal and postnatal forms.

[0095] Pharmaceutically acceptable: The term "pharmacologically acceptable," as used herein, refers to a material that is suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0096] Stable: As used herein, the term "stable" refers to a composition that maintains its physical stability and / or biological activity.In one embodiment, stability is determined based on the percentage of mRNA that is degraded (e.g., fragmented).In another embodiment, stability is determined based on the percentage of lipid nanoparticles that are no longer present in suspension.

[0097] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. In some embodiments, the subject is a patient, which refers to a human who presents to a health care provider for diagnosis or treatment of a disease or disorder. The term "subject" is used interchangeably herein with "individual" or "patient." A subject is afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder. In some embodiments, a subject is healthy and can receive the lipid nanoparticles or compositions of the present invention to prevent a disease or disorder.

[0098] Substantially: As used herein, the term "substantially" refers to a qualitative state of exhibiting the entire or nearly entire extent or degree of a feature or characteristic of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0099] Treating: As used herein, the terms "treat", "treatment" or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be administered to subjects who do not exhibit signs of the disease and / or who exhibit only early signs of the disease, with the aim of reducing the risk of developing pathologies associated with the disease.

[0100] chemical definition Acyl: As used herein, the term “acyl” refers to R Z -(C=O)-, where R Z is, for example, any alkyl, alkenyl, alkynyl, heteroalkyl, or heteroalkylene.

[0101] Aliphatic: As used herein, the term aliphatic refers to any group consisting of C1-C 40 Aliphatic refers to hydrocarbons, including both saturated and unsaturated hydrocarbons. Aliphatic may be linear, branched or cyclic. For example, C1-C 20 Aliphatic: C1-C 20 Alkyl (e.g., linear or branched C1-C 20 Saturated alkyl), C2-C 20 Alkenyl (e.g., linear or branched C4-C 20 Dienyl, linear or branched C6-C 20 trienyl, etc.), and C2-C 20 Alkynyl (e.g., linear or branched C2-C 20 alkynyl). 20 Aliphatic: C3-C 20 Cycloaliphatic (e.g., C3-C 20 Cycloalkyl, C4-C 20 Cycloalkenyl, or C8-C 20In certain embodiments, an aliphatic group can include one or more cycloaliphatic and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and can be optionally substituted with one or more substituents, such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. An aliphatic group is unsubstituted or substituted with one or more substituents as described herein. For example, an aliphatic group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -COH, -COR", -CN, -OH, -OR", -OCOR', -OCOR", -NH, -NHR", -N(R")2, -SR", or -SOR", where each instance of R" is independently selected from C1 to C6. 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted C1-C3 alkyl. In embodiments, the aliphatic is unsubstituted. In embodiments, the aliphatic does not include any heteroatoms. Alkyl: As used herein, the term "alkyl" refers to acyclic linear and branched hydrocarbon groups, e.g., "C1-C 30"Alkyl" refers to an alkyl group having 1 to 30 carbons. The alkyl group may be linear or branched. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl tert-pentyl hexyl, isohexyl, and the like. The term "lower alkyl" refers to a straight chain alkyl group or a branched alkyl group having 1 to 6 carbon atoms. Other alkyl groups will be readily apparent to one of skill in the art given the benefit of this disclosure. The alkyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, an alkyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -COH, -COR", -CN, -OH, -OR", -OCOR', -OCOR", -NH, -NHR", -N(R)2, -SR", or -SOR", where each instance of R" is independently selected from the group consisting of C1 to C6. 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkyl group is substituted with an -OH group, and as such may be referred to herein as a "hydroxyalkyl" group, where the prefix denotes the -OH group and "alkyl" is as described herein.

[0102] As used herein, "alkyl" also refers to a group of linear or branched saturated hydrocarbon groups having 1 to 50 carbon atoms ("C1-C 50 In some embodiments, an alkyl group has 1 to 40 carbon atoms ("C1-C 40 In some embodiments, an alkyl group has 1 to 30 carbon atoms ("C1-C 30 In some embodiments, an alkyl group has 1 to 20 carbon atoms ("C1-C 20 In some embodiments, an alkyl group has 1 to 10 carbon atoms ("C1-C 10In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C1-C9 alkyl"). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C1-C8 alkyl"). In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C1-C7 alkyl"). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C1-C6 alkyl"). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C1-C5 alkyl"). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C1-C4 alkyl"). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C1-C3 alkyl"). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C1-C2 alkyl"). In some embodiments, the alkyl group has 1 carbon atom ("C1 alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups include, without limitation, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"). In certain embodiments, an alkyl group is an unsubstituted C1-C 50 In certain embodiments, the alkyl group is a substituted C1-C 50 It is an alkyl.

[0103] The addition of the suffix "-ene" to the base indicates that the group is a divalent moiety, for example, arylene is a divalent moiety of an aryl, and heteroarylene is a divalent moiety of a heteroaryl.

[0104] Alkylene: The term "alkylene", as used herein, represents a saturated divalent straight or branched chain hydrocarbon group, exemplified by methylene, ethylene, isopropylene, and the like. Similarly, the term "alkenylene", as used herein, represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, and the term "alkynylene", as used herein, represents an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur at any stable point along the chain. In certain embodiments, alkylene, alkenylene, or alkynylene groups can contain one or more cyclic aliphatic and / or one or more heteroatoms, such as oxygen, nitrogen, or sulfur, and can be optionally substituted with one or more substituents, such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester, or amide. For example, an alkylene, alkenylene, or alkynylene can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -COH, -COR", -CN, -OH, -OR", -OCOR", -OCOR", -NH, -NHR", -N(R)2, -SR", or -SOR", where each instance of R" is independently selected from C1 to C6. 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10In certain embodiments, R" is independently an unsubstituted C1-C3 alkyl. In certain embodiments, the alkylene, alkenylene, or alkynylene is unsubstituted. In certain embodiments, the alkylene, alkenylene, or alkynylene does not contain any heteroatoms. Alkenyl: As used herein, "alkenyl" refers to any linear or branched hydrocarbon chain with one or more unsaturated carbon-carbon double bonds that may occur at any stable point along the chain, e.g., "C2-C 30 "Alkenyl" refers to an alkenyl group having 2 to 30 carbons. For example, alkenyl groups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. In embodiments, an alkenyl contains one, two, or three carbon-carbon double bonds. In embodiments, an alkenyl contains a single carbon-carbon double bond. In embodiments, multiple double bonds (e.g., two or three) are conjugated. An alkenyl group can be unsubstituted or substituted with one or more substituents as described herein. For example, an alkenyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -COH, -COR", -CN, -OH, -OR", -OCOR", -OCOR", -NH, -NHR", -N(R)2, -SR", or -SOR", where each instance of R" is independently selected from C1 to C6. 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10In embodiments, R" is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein). In embodiments, the alkenyl group is substituted with an -OH group, and as such may be referred to herein as a "hydroxyalkenyl" group, where the prefix indicates the -OH group and "alkenyl" is as described herein.

[0105] As used herein, "alkenyl" also refers to a group of straight-chain or branched hydrocarbon groups having 2 to 50 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds) ("C2-C 50 In some embodiments, an alkenyl group has 2 to 40 carbon atoms ("C-C 40 In some embodiments, an alkenyl group has 2 to 30 carbon atoms ("C-C 30 In some embodiments, an alkenyl group has 2 to 20 carbon atoms ("C-C 20 In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C-C 10In some embodiments, an alkenyl group has from 2 to 9 carbon atoms ("C2-C9 alkenyl"). In some embodiments, an alkenyl group has from 2 to 8 carbon atoms ("C2-C8 alkenyl"). In some embodiments, an alkenyl group has from 2 to 7 carbon atoms ("C2-C7 alkenyl"). In some embodiments, an alkenyl group has from 2 to 6 carbon atoms ("C2-C6 alkenyl"). In some embodiments, an alkenyl group has from 2 to 5 carbon atoms ("C2-C5 alkenyl"). In some embodiments, an alkenyl group has from 2 to 4 carbon atoms ("C2-C4 alkenyl"). In some embodiments, an alkenyl group has from 2 to 3 carbon atoms ("C2-C3 alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds may be internal (such as 2-butenyl) or terminal (such as 1-butenyl). Examples of C2-C4 alkenyl groups include, without limitation, ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the C2-C4 alkenyl groups previously described, as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted ("unsubstituted alkenyl") or substituted ("substituted alkenyl") with one or more substituents. In certain embodiments, an alkenyl group is an unsubstituted C2-C 50 In certain embodiments, the alkenyl group is a substituted C-C 50 It is alkenyl.

[0106] Alkynyl: As used herein, "alkynyl" refers to any hydrocarbon chain of either linear or branched configuration with one or more carbon-carbon triple bonds occurring at any stable point along the chain, e.g., "C2-C 30 "Alkynyl" refers to an alkynyl group having 2 to 30 carbons. Examples of alkynyl groups include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, and the like. In embodiments, an alkynyl contains one carbon-carbon triple bond. Alkynyl groups can be unsubstituted or substituted with one or more substituents as described herein. For example, an alkynyl group can be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR", -COH, -COR", -CN, -OH, -OR", -OCOR", -OCOR", -NH, -NHR", -N(R)2, -SR", or -SOR", where each instance of R" is independently selected from the group consisting of C1 to C6. 20 Aliphatic (e.g., C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted alkyl (e.g., an unsubstituted C1-C 20 Alkyl, C1-C 15 Alkyl, C1-C 10 In embodiments, R" is independently an unsubstituted C1-C3 alkyl. In embodiments, the alkynyl is unsubstituted. In embodiments, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituents as described herein).

[0107] As used herein, "alkynyl" also refers to a group of straight-chain or branched hydrocarbon groups having 2 to 50 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) and optionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds) ("C2-C 50 Alkynyl groups that have one or more triple bonds and one or more double bonds are also referred to as "ene-ynes." In some embodiments, alkynyl groups have 2 to 40 carbon atoms ("C2-C 40 In some embodiments, an alkynyl group has 2 to 30 carbon atoms ("C-C 30 In some embodiments, an alkynyl group has 2 to 20 carbon atoms ("C-C 20 In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C-C 10In some embodiments, an alkynyl group has from 2 to 9 carbon atoms ("C2-C9 alkynyl"). In some embodiments, an alkynyl group has from 2 to 8 carbon atoms ("C2-C8 alkynyl"). In some embodiments, an alkynyl group has from 2 to 7 carbon atoms ("C2-C7 alkynyl"). In some embodiments, an alkynyl group has from 2 to 6 carbon atoms ("C2-C6 alkynyl"). In some embodiments, an alkynyl group has from 2 to 5 carbon atoms ("C2-C5 alkynyl"). In some embodiments, an alkynyl group has from 2 to 4 carbon atoms ("C2-C4 alkynyl"). In some embodiments, an alkynyl group has from 2 to 3 carbon atoms ("C2-C3 alkynyl"). In some embodiments, an alkynyl group has 2 carbon atoms ("C2 alkynyl"). The one or more carbon-triple bonds may be internal (e.g., 2-butynyl) or terminal (e.g., 1-butynyl). Examples of C2-C4 alkynyl groups include, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-C6 alkenyl groups include the C2-C4 alkynyl groups previously described, as well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted ("unsubstituted alkynyl") or substituted with one or more substituents ("substituted alkynyl"). In certain embodiments, an alkynyl group is an unsubstituted C2-C6 alkenyl group. 50 In certain embodiments, the alkynyl group is a substituted C-C 50 It is alkynyl.

[0108] Aryl: The term "aryl," used alone or as part of a larger moiety as in "aralkyl," refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of 6 to 14 ring members, where the ring system has a single point of attachment to the remainder of the molecule, at least one ring in the system is aromatic, and each ring in the system contains 4 to 7 ring members. In embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl," e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 aryl," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 "Aryl", e.g., anthracen. "Aryl" further includes ring systems in which an aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, where the group or point of attachment is on the aryl ring, and in such instances the number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system. Illustrative aryls include phenyl, naphthyl, and anthracene.

[0109] As used herein, "aryl" also refers to groups of monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring systems (e.g., having 6, 10 or 14 pi electrons shared in the cyclic array) having 6 to 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system ("C6-C 14 In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14"Aryl"; e.g., anthracyl). "Aryl" further includes ring systems in which an aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, where the group or point of attachment is on the aryl ring, and in such instances the number of carbon atoms continues to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, an aryl group is an unsubstituted C6-C 14 In certain embodiments, the aryl group is a substituted C-C 14 It is aryl.

[0110] Arylene: The term "arylene," as used herein, refers to an aryl group that is divalent (i.e., has two points of attachment to the molecule). Exemplary arylenes include phenylene (e.g., unsubstituted or substituted phenylene).

[0111] Carbocyclyl: As used herein, “carbocyclyl” or “carbocyclic” refers to a ring system having 3 to 10 ring carbon atoms (C 10 "C3-C8 carbocyclyl" and "C6 carbocyclyl" refer to groups of non-aromatic cyclic hydrocarbon groups having zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has from 3 to 8 ring carbon atoms ("C3-C8 carbocyclyl"). In some embodiments, a carbocyclyl group has from 3 to 7 ring carbon atoms ("C3-C7 carbocyclyl"). In some embodiments, a carbocyclyl group has from 3 to 6 ring carbon atoms ("C3-C6 carbocyclyl"). In some embodiments, a carbocyclyl group has from 4 to 6 ring carbon atoms ("C4-C6 carbocyclyl"). In some embodiments, a carbocyclyl group has from 5 to 6 ring carbon atoms ("C5-C6 carbocyclyl"). In some embodiments, a carbocyclyl group has from 5 to 10 ring carbon atoms ("C5-C6 carbocyclyl"). 10Illustrative C3-C6 carbocyclyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Illustrative C3-C8 carbocyclyl groups include, without limitation, the C3-C6 carbocyclyl groups previously described, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl (C8), and the like. Illustrative C3~C 10 Carbocyclyl groups include, but are not limited to, the C3-C8 carbocyclyl groups described above, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10As the foregoing examples illustrate, in certain embodiments, a carbocyclyl group can be either monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged, or spiro ring system, such as a bicyclic ring system ("bicyclic carbocyclyl") or a tricyclic ring system ("tricyclic carbocyclyl")) and can be saturated or contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" further includes ring systems in which a carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, where the point of attachment is on the carbocyclyl ring, and in such instances the number of carbons continues to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted ("unsubstituted carbocyclyl") or substituted with one or more substituents ("substituted carbocyclyl"). In certain embodiments, the carbocyclyl group is an unsubstituted C-C 10 In certain embodiments, the carbocyclyl group is a substituted C-C 10 It is a carbocyclyl.

[0112] In some embodiments, a "carbocyclyl" or "carbocyclic" is referred to as a "cycloalkyl", i.e., a monocyclic saturated carbocyclyl group having from 3 to 10 ring carbon atoms ("C 10 In some embodiments, a cycloalkyl group has from 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"). In some embodiments, a cycloalkyl group has from 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"). In some embodiments, a cycloalkyl group has from 4 to 6 ring carbon atoms ("C4-C6 cycloalkyl"). In some embodiments, a cycloalkyl group has from 5 to 6 ring carbon atoms ("C5-C6 cycloalkyl"). In some embodiments, a cycloalkyl group has from 5 to 10 ring carbon atoms ("C5-C6 cycloalkyl"). 10Examples of C5-C6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-C6 cycloalkyl groups include the C5-C6 cycloalkyl groups described above, as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-C8 cycloalkyl groups include the C3-C6 cycloalkyl groups described above, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, a cycloalkyl group is an unsubstituted C3-C6 cycloalkyl group. 10 In certain embodiments, the cycloalkyl group is a substituted C-C 10 It is cycloalkyl.

[0113] Halogen: As used herein, the term "halogen" means fluorine, chlorine, bromine or iodine.

[0114] Heteroalkyl: The term "heteroalkyl" refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having 1 to 14 carbon atoms in addition to 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyl includes tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. Heteroalkyl groups can optionally include monocyclic, bicyclic, or tricyclic rings, where each ring desirably has 3 to 6 members. Examples of heteroalkyl include polyethers such as methoxymethyl and ethoxyethyl.

[0115] Heteroalkylene: The term "heteroalkylene," as used herein, represents a divalent form of a heteroalkyl group, as described herein.

[0116] Heteroaryl: The term "heteroaryl," as used herein, refers to a fully unsaturated heteroatom-containing ring, where at least one ring atom is a heteroatom, such as, but not limited to, nitrogen and oxygen.

[0117] As used herein, "heteroaryl" also refers to a group of 5-14 membered monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring systems (e.g., having 6, 10 or 14 pi electrons shared in the cyclic array) having ring carbon atoms and one or more ring heteroatoms (e.g., 1, 2, 3 or 4 ring heteroatoms) provided in the aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon and phosphorus ("5-14 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon atom or a nitrogen atom, if valence permits. Heteroaryl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, where the point of attachment is on the heteroaryl ring, and in such cases the number of ring members continues to designate the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused with one or more aryl groups, where the point of attachment is on either the aryl or heteroaryl ring, and in such cases the number of ring members continues to designate the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. In polycyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., either the ring with a heteroatom (e.g., 2-indolyl) or the ring without a heteroatom (e.g., 5-indolyl).

[0118] In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-10 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-8 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms provided in the aromatic ring system, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has one or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6 membered heteroaryl has one ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted ("unsubstituted heteroaryl") or substituted ("substituted heteroaryl") with one or more substituents. In certain embodiments, a heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, a heteroaryl group is a substituted 5-14 membered heteroaryl.

[0119] Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Illustrative tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0120] As used herein, "heterocyclyl" or "heterocyclic" refers to a group of 3- to 14-membered non-aromatic ring systems having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("3- to 14-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon atom or at a nitrogen atom, where valence permits. Heterocyclyl groups may be either monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., fused, bridged, or spiro ring systems, such as bicyclic ("bicyclic heterocyclyl") or tricyclic ("tricyclic heterocyclyl") systems), and may be saturated or may contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can contain one or more heteroatoms in one or both rings. "Heterocyclyl" further includes ring systems in which a heterocyclyl ring as defined above is fused to one or more carbocyclyl groups, where the point of attachment is on either the carbocyclyl ring or the heterocyclyl ring, or a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, where the point of attachment is on the heterocyclyl ring, in such instances the number of ring members continues to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl.

[0121] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus (a "5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and one or more (e.g., 1, 2, 3, or 4) ring heteroatoms, where each heteroatom is independently selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus ("5-6 membered heterocyclyl"). In some embodiments, a 5-6 membered heterocyclyl has one or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, a 5-6 membered heterocyclyl has one or two ring heteroatoms selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments, a 5-6 membered heterocyclyl has one ring heteroatom selected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

[0122] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, aziridinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl, and thiocanyl.Illustrative bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepin ... 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo-[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

[0123] Heterocycloalkyl: The term "heterocycloalkyl," as used herein, is a non-aromatic ring in which at least one atom is a heteroatom, such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus, and the remaining atoms are carbon. Heterocycloalkyl groups can be substituted or unsubstituted.

[0124] As understood above, alkyl groups, alkenyl groups, alkynyl groups, acyl groups, carbocyclyl groups, heterocyclyl groups, aryl groups and heteroaryl groups as defined herein are, in certain embodiments, optionally substituted. Optionally substituted refers to groups that may be substituted or unsubstituted (e.g., "substituted" or "unsubstituted" alkyl groups, "substituted" or "unsubstituted" alkenyl groups, "substituted" or "unsubstituted" alkynyl groups, "substituted" or "unsubstituted" heteroalkyl groups, "substituted" or "unsubstituted" heteroalkenyl groups, "substituted" or "unsubstituted" heteroalkynyl groups, "substituted" or "unsubstituted" carbocyclyl groups, "substituted" or "unsubstituted" heterocyclyl groups, "substituted" or "unsubstituted" aryl groups, or "substituted" or "unsubstituted" heteroaryl groups). In general, the term "substituted" means that at least one hydrogen present on a group is replaced with an acceptable substituent, e.g., a substituent that, upon substitution, results in a stable compound, e.g., a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituents are either the same or different at each position. The term "substituted" is intended to include substitution with all acceptable substituents of organic compounds, and any of the substituents described herein will result in the formation of a stable compound. The present invention contemplates any and all such combinations to arrive at a stable compound. For purposes of this invention, heteroatoms such as nitrogen can have hydrogen substituents and / or any suitable substituents as described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0125] Illustrative carbon atom substituents include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb)2、-N(R bb )3+X - 、-N(OR cc )R bb 、-SeH、-SeR aa 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2、-CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3-OSi(R aa )3-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa, -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc ), C1~C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 14 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 aryl, and 5- to 14-membered heteroaryl, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently represents 0, 1, 2, 3, 4, or 5 R dd substituted with a group; Or the two geminal hydrogens on a carbon atom can be =O, =S, or =NN(R bb )2 units, =NNR bb C(=O)R aa base, =NNR bb C(=O)OR aa base, =NNR bb S(=O)2Raa base, =NR bb Group or =NOR cc has been replaced with a radical;

[0126] R aa Each example is independently C1-C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 aryl, and 5- to 14-membered heteroaryl, or two R aa The groups are joined to form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently represents 0, 1, 2, 3, 4, or 5 R dd substituted with a group;

[0127] R bb Each instance of is independently hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, C1~C 50 Alkyl, C2-C 50Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 aryl, and 5- to 14-membered heteroaryl, or two R bb The groups, together with the heteroatom to which they are attached, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently represent 0, 1, 2, 3, 4, or 5 R dd substituted with a group;

[0128] R cc Each example is independently hydrogen, C1-C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 aryl, and 5- to 14-membered heteroaryl, or two R cc The groups, together with the heteroatom to which they are attached, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently represent 0, 1, 2, 3, 4, or 5 R dd substituted with a group;

[0129] R dd Each example is independently a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3+X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee, -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff ) OR ee , -OC(=NR ff )R ee , -OC(=NR ff ) OR ee , -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee , -S(=O)R ee , -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, C1~C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-10 membered heterocyclyl, C6-C 10aryl, and 5- to 10-membered heteroaryl, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg substituted with a group or two geminal R dd The substituents can be joined to form =O or =S;

[0130] R ee Each example is independently C1-C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, C6~C 10 aryl, 3- to 10-membered heterocyclyl, and 3- to 10-membered heteroaryl, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently selected from 0, 1, 2, 3, 4, or 5 R gg substituted with a group;

[0131] R ff Each example is independently hydrogen, C1-C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-10 membered heterocyclyl, C6-C 10 aryl, and 5- to 10-membered heteroaryl, or two R ff The groups, together with the heteroatom to which they are attached, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently represent 0, 1, 2, 3, 4, or 5 R gg substituted with a group;

[0132] R ggEach example is independently a halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC1-C 50 Alkyl, -ON(C1-C 50 Alkyl)2, -N(C1-C 50 Alkyl)2, -N(C1-C 50 Alkyl)3+X - , -NH(C1-C 50 Alkyl)2+X - , -NH2(C1-C 50 Alkyl) + X - , -NH3+X - , -N(OC1-C 50 Alkyl)(C1-C 50 Alkyl), -N(OH)(C1-C 50 alkyl), -NH(OH), -SH, -SC1-C 50 Alkyl, -SS(C1-C 50 Alkyl), -C(=O)(C1-C 50 Alkyl), -CO2H, -CO2(C1-C 50 Alkyl), -OC(=O)(C1-C 50 Alkyl), -OCO2(C1-C 50 Alkyl), -C(=O)NH2, -C(=O)N(C1-C 50 Alkyl)2, -OC(=O)NH(C1-C 50 Alkyl), -NHC(=O)(C1-C 50 Alkyl), -N(C1-C 50 Alkyl)C(=O)(C1~C 50 Alkyl), -NHCO2(C1-C 50 Alkyl), -NHC(=O)N(C1-C 50 Alkyl)2, -NHC(=O)NH(C1-C 50 alkyl), -NHC(=O)NH2, -C(=NH)O(C1-C 50 Alkyl), -OC(=NH)(C1~C 50 alkyl), -OC(=NH)OC1-C 50 Alkyl, -C(=NH)N(C1-C 50 Alkyl)2, -C(=NH)NH(C1-C 50 Alkyl), -C(=NH)NH2, -OC(=NH)N(C1-C50 alkyl)2, -OC(NH)NH(C1-C 50 Alkyl), -OC(NH)NH2, -NHC(NH)N(C1-C 50 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C1-C 50 Alkyl), -SO2N(C1-C 50 Alkyl)2, -SO2NH(C1-C 50 Alkyl), -SO2NH2, -SO2(C1-C 50 Alkyl), -SO2O(C1-C 50 alkyl), -OSO2(C1-C6 alkyl), -SO(C1-C6 alkyl), -Si(C1-C 50 alkyl)3, -OSi(C1-C6 alkyl)3, -C(=S)N(C1-C 50 Alkyl)2, C(=S)NH(C1-C 50 alkyl), C(=S)NH2, -C(=O)S(C1-C6 alkyl), -C(=S)S(C1-C6 alkyl), -SC(=S)S(C1-C6 alkyl), -P(=O)2(C1-C 50 Alkyl), -P(=O)(C1-C 50 Alkyl)2, -OP(=O)(C1-C 50 Alkyl)2, -OP(=O)(OC1~C 50 Alkyl)2, C1-C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, C6~C 10 aryl, 3- to 10-membered heterocyclyl, 5- to 10-membered heteroaryl; or two geminal R gg The substituents can be joined to form =O or =S; where X - is the counter ion.

[0133] As used herein, the term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

[0134] As used herein, a "counterion" is a negatively charged group associated with a positively charged quaternary amine to maintain electronic neutrality. Illustrative counterions include halide ions (e.g., F - , Cl - , Br - , I - ), NO3 - , ClO4 - , O.H. - , H2PO4 - , HSO4 - , sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-l-sulfonic acid-5-sulfonate, ethane-1-sulfonic acid-2-sulfonate, etc.), and carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, etc.).

[0135] Nitrogen atoms may be substituted or unsubstituted, where valence allows, and include primary, secondary, tertiary and quaternary nitrogen atoms. Illustrative nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc ) OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa, -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, C1~C 50 Alkyl, C2-C 50 Alkenyl, C2-C 50 Alkynyl, C3-C 10 Carbocyclyl, 3-14 membered heterocyclyl, C6-C 14 aryl, and 5- to 14-membered heteroaryl; or two R cc The groups, together with the N atom to which they are attached, form a 3- to 14-membered heterocyclyl ring or a 5- to 14-membered heteroaryl ring, where each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl independently represent 0, 1, 2, 3, 4, or 5 R dd is substituted with an R aa , R bb , R cc and R dd is as defined above.

[0136] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group).Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PG M Huts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0137] For example, a nitrogen protecting group such as an amide group (e.g., -C(=O)R aa), include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0138] Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)OR aa), but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2- Trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate ( t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, a alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzoisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chloro Monyl methyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, 2, 2-Dimethoxyacylvinylcarbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propylcarbamate, 1,1-dimethylpropynylcarbamate, di(2-pyridyl)methylcarbamate, 2-furanylmethylcarbamate, 2-iodoethylcarbamate, isobornylcarbamate, isobutylcarbamate, isonicotinylcarbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1 -methylcyclohexyl carbamate, 1-methyl-l-cyclopropylmethyl carbamate, 1-methyl-1(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-l-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0139] Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=O)R aa Examples of the benzenesulfonamides include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethyl ...tb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mtb), 4-methoxybenzenesulfonamide (Mtb), 2,4,6-trimethylbenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenes sulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0140] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethylphenylamine, N-phenylpropanediol ... disilyl azacyclopentane adducts (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-ones, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-ones, 1-substituted 3,5-dinitro-4-pyridones, N-methylamines, N-allylamines, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrroline-3-ylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrroline-3-ylamine), ... -yl)amine, quaternary ammonium salt, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethyl Thiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-Dimethyl-3-oxo-l-cyclohexenyl)amine, N-borane derivatives, N-diphenylborinic acid derivatives, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidates, diphenyl phosphoramidates, benzenesulfenamides, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridine sulfenamide (Npys).

[0141] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group).Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PG M Huts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0142] Illustrative oxygen protecting groups include, but are not limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyeth ... Dimethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]phenyl] nyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloro ethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4,4',4"-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylsexisilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate Acetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyl dithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenyl methyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-Trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl aryl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzyl carbonate, benzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate , (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

[0143] In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PG M Huts, 3rd Edition, John Wiley & Sons, 1999, which is incorporated herein by reference.

[0144] Illustrative sulfur protecting groups include, but are not limited to, alkyl, benzyl, p-methoxybenzyl, 2,4,6-trimethylbenzyl, 2,4,6-trimethoxybenzyl, o-hydroxybenzyl, p-hydroxybenzyl, o-acetoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picolyl, 2-quinolinylmethyl, 2-picolyl N-oxide, 9-anthrylmethyl, 9-fluorenylmethyl, xanthenyl, ferrocenylmethyl, diphenylmethyl, bis(4-methoxyphenyl)methyl, 5-dibenzosuberyl, triphenylmethyl, diphenyl-4-pyridylmethyl, phenyl, 2,4-dinitrophenyl, t-butyl, 1-adamantyl, methoxymethyl (MOM), isobutoxymethyl, benzyloxymethyl, 2-tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidino, acetamidomethyl, trimethylacetamidomethyl, benzamidomethyl, allyloxycarbomethyl, aryloxy ... nylaminomethyl, phenylacetamidomethyl, phthalimidomethyl, acetylmethyl, carboxymethyl, cyanomethyl, (2-nitro-1-phenyl)ethyl, 2-(2,4-dinitrophenyl)ethyl, 2-cyanoethyl, 2-(trimethylsilyl)ethyl, 2,2-bis(carboethoxy)ethyl, (1-m-nitrophenyl-2-benzoyl)ethyl, 2-phenylsulfonylethyl, 2-(4-methylphenylsulfonyl)-2-methylprop-2-yl, Examples of such aryl groups include acetyl, benzoyl, trifluoroacetyl, N-[[(p-biphenylyl)isopropoxy]carbonyl]-N-methyl]-γ-aminothiobutyrate, 2,2,2-trichloroethoxycarbonyl, t-butoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, N-ethyl, N-methoxymethyl, sulfonate, sulfenylthiocarbonate, 3-nitro-2-pyridine sulfenyl sulfide, and oxathiolane.

[0145] Detailed Description The present invention provides lipid nanoparticles encapsulating mRNA that are particularly effective in delivering mRNA to the lungs via inhalation administration. In particular, the lipid nanoparticles described herein achieve increased inhalation dosage output, maintain the encapsulation efficiency of mRNA upon inhalation administration, and result in increased expression of the protein that is the protein encoded by the mRNA.

[0146] In particular, the present invention relates to lipid nanoparticles comprising, inter alia: (i) mRNA encapsulated within lipid nanoparticles, and (ii) consisting of: a. cationic lipid components; b. non-cationic lipid components; c. a PEG-modified lipid component, and D. Cholesterol components Lipid components consisting of Includes: (1) the cationic lipid component is greater than 40% (molar ratio); (2) the non-cationic lipid component is less than 25% (by molar ratio); (3) Providing lipid nanoparticles having a total lipid:mRNA ratio (mg:mg) of 19:1 or less.

[0147] For example, the present invention provides a lipid nanoparticle, comprising: (i) mRNA encapsulated within lipid nanoparticles, and (ii) lipids having the following molar ratio: a) 41% to 70% cationic lipid, b) non-cationic lipids 9% to 18%, c) 2% to 6% PEG-modified lipid, and d) Cholesterol or cholesterol analogues 9% to 48% Lipid components consisting of The present invention provides a lipid nanoparticle comprising:

[0148] The lipid nanoparticles of the present invention and compositions comprising the same can be used for the effective treatment of a number of pulmonary diseases or for the systemic delivery of mRNA therapeutics via the lungs.

[0149] Lipid Nanoparticles The inventors have discovered that lipid nanoparticles encapsulating mRNA with lipid components consisting of cationic lipids, non-cationic lipids, PEG-modified lipids and cholesterol or cholesterol analogues are more effective for pulmonary administration by inhalation administration when a lower molar ratio of non-cationic lipid is used than that typically present in lipid nanoparticles delivered via this administration route.In particular, the inventors have unexpectedly discovered that it is also possible to achieve increased inhalation administration throughput and increased expression of the protein encoded by the mRNA encapsulated in the lipid nanoparticles, while maintaining the encapsulation efficiency of mRNA for inhalation administration when a lower molar ratio of non-cationic lipid is used.

[0150] These observations are independent of the particular non-cationic lipid used.Indeed, the inventors have observed improvements in inhalation administration when cholesterol is replaced with various cholesterol analogues, but the most effective way to increase extrusion and protein expression while maintaining encapsulation efficiency is to reduce the amount of non-cationic lipid present in the formulation.Advantageously, reducing the amount of non-cationic lipid allows the total amount of lipid required for effective delivery and expression of encapsulated mRNA to be reduced.

[0151] Molar ratio As used herein, the molar ratio of lipids in lipid nanoparticles totals 100%. For example, in lipid nanoparticles containing lipid components with the following molar ratios: a) cationic lipid 41%-70%, b) non-cationic lipid 9%-18%, c) PEG-modified lipid 2%-6%, and d) cholesterol or cholesterol analog 9%-48%, if the molar ratio of cationic lipid is 70%, the molar ratio of non-cationic lipid is 9%, the molar ratio of PEG-modified lipid is 2%, and then the molar ratio of cholesterol or cholesterol analog is 19% (70%+9%+2%+19%=100%).

[0152] In some embodiments, when a molar ratio is defined as a range, e.g., 2% to 6% PEG-modified lipid, the limits of the range are the exact values ​​specified, e.g., the lower limit 2% of a molar ratio of 2% to 6% PEG-modified lipid is 2%.

[0153] Cationic lipids As used herein, the term "cationic lipid" refers to an ionizable lipid that has a net positive charge at a pH below physiological pH (e.g., about pH 5.5, about 6.0, or about 6.5).

[0154] Suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those cationic lipids described in International Patent Publication WO2010 / 144740, which is incorporated herein by reference. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharmaceutically acceptable salts thereof.

[0155] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include the ionizable cationic lipids described in International Patent Publication WO2013 / 149140, which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the present invention include ionizable cationic lipids having one of the following formulas: [ka] or a pharma- ceutically acceptable salt thereof, wherein R1 and R2 are each independently hydrogen, an optionally substituted, variably saturated or unsaturated C1-C 20 Alkyl and optionally substituted, variably saturated or unsaturated C6-C 20 acyl; wherein L1 and L2 are each independently selected from the group consisting of hydrogen, optionally substituted C1-C 30 Alkyl, optionally substituted variably unsaturated C1-C 30 Alkenyl, and optionally substituted C1-C 30 alkynyl; where m and o are each independently selected from the group consisting of zero and any positive integer (e.g., when m is 3); and where n is zero or any positive integer (e.g., when n is 1). In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and cationic lipid (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine ("HGT5000") having the compound structure: and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and cationic lipid (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine ("HGT5001") having the compound structure: and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharmaceutically acceptable salts thereof.

[0156] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those cationic lipids described as amino alcohol lipidoids in International Patent Publication WO2010 / 053572, which is incorporated herein by reference. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharma- ceutically acceptable salts thereof.

[0157] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those cationic lipids described in International Patent Publication WO2016 / 118725, which is incorporated herein by reference. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharma- ceutically acceptable salts thereof.

[0158] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those cationic lipids described in International Patent Publication WO2016 / 118724, which is incorporated herein by reference. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharma- ceutically acceptable salts thereof.

[0159] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include cationic lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharma- ceutically acceptable salts thereof.

[0160] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those described in International Patent Publications WO2013 / 063468 and WO2016 / 205691, each of which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein R L Each instance of is independently an optionally substituted C-C 40 In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0161] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those cationic lipids described in International Patent Publication WO2015 / 184256, which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein each X is independently O or S; each Y is independently O or S; each m is independently 0 to 20; each n is independently 1 to 6; and each R A are independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocycle, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen; each R Bare independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocycle, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharma- ceutically acceptable salts thereof.

[0162] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the invention include those cationic lipids described in International Patent Publication WO2016 / 004202, which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the invention include cationic lipids having the compound structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the lipid nanoparticles and compositions of the present invention include a cationic lipid having the compound structure: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the lipid nanoparticles and compositions of the present invention include a cationic lipid having the compound structure: [ka] or a pharma- ceutically acceptable salt thereof.

[0163] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those cationic lipids described in U.S. Provisional Patent Application No. 62 / 758,179, which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein each R 1 and R 2 is independently H or a C1-C6 aliphatic; each m is independently an integer having a value from 1 to 4; each A is independently a covalent bond or arylene; each L 1 is independently an ester, thioester, disulfide, or anhydride group; each L 2 are independent, C2~C 10 is aliphatic; each X 1 is independently H or OH; each R 3 are independent, C6~C 20 In some embodiments, the lipid nanoparticles and compositions of the present invention include a lipid having the following formula: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the lipid nanoparticles and compositions of the present invention include a cationic lipid having the following formula: [ka] or a pharma- ceutically acceptable salt thereof. In some embodiments, the lipid nanoparticles and compositions of the present invention include a cationic lipid having the following formula: [ka] or a pharma- ceutically acceptable salt thereof.

[0164] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the invention include those described in J. McClellan, MCKing, Cell 2010, 141, 210-217, and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference. In certain embodiments, the cationic lipids of the lipid nanoparticles and compositions of the invention include: [ka] and pharma- ceutically acceptable salts thereof.

[0165] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those cationic lipids described in International Patent Publication WO2015 / 199952, which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the present invention include the cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0166] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the invention include those cationic lipids described in International Patent Publication WO2017 / 004143, which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0167] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those cationic lipids described in International Patent Publication WO2017 / 075531, which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the following formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein L 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -OR-NR a C(=O)O-;L 1 or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -OR-NR a C(=O)O- or a direct bond;G 1 and G 2 are each independently unsubstituted C1 to C 12 Alkylene or C1-C 12 Alkenylene; G 3 is C1~C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1~C 12 R is alkyl; 1 and R 2are each independently C6 to C 24 Alkyl or C6-C 24 alkenyl; R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ;R 4 is C1~C 12 R is alkyl; 5 is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0168] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the invention include those cationic lipids described in International Patent Publication WO2017 / 117528, which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] In some embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0169] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include those described in International Patent Publication WO2017 / 049245, which is incorporated herein by reference. In some embodiments, the cationic lipids of the lipid nanoparticles and compositions of the present invention include those having the following formula: [ka] In any one of these four formulas, R4 is -(CH2) n Q and -(CH2) n CHQR are independently selected from; Q is -OR, -OH, -O(CH2) n N(R), -OC(O)R, -CX, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)R, -N(H)S(O)R, -N(R)C(O)N(R), -N(H)C(O)N(R), -N(H)C(O)N(R), -N(R)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(R), -N(H)C(S)N(H)(R) and heterocycle; n is 1, 2 or 3. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0170] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the invention include those cationic lipids described in International Patent Publications WO2017 / 173054 and WO2015 / 095340, each of which is incorporated herein by reference. In certain embodiments, the lipid nanoparticles and compositions of the invention include: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include cationic lipids having the compound structure: [ka] and pharma- ceutically acceptable salts thereof.

[0171] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the present invention include the cleavable cationic lipids described in International Patent Publication WO2012 / 170889, which is incorporated herein by reference. In some embodiments, the lipid nanoparticles and compositions of the present invention include those having the following formula: [ka] wherein R1 is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, optionally substituted alkylamino (e.g., alkylamino, e.g., dimethylamino), and pyridyl; and wherein R2 is selected from the group consisting of the following two formulas: [ka] wherein R and R are each independently an optionally substituted, variably saturated or unsaturated C to C 20 Alkyl and optionally substituted, variably saturated or unsaturated C6-C 20 acyl; where n is zero or any positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more). In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] (HGT4001) and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include the cationic lipid "HGT4001" having the compound structure: [ka] Cationic lipid "HGT4002" (also referred to herein as "Guan-SS-Chol") having the compound structure: and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharma- ceutically acceptable salts thereof. In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] and pharma- ceutically acceptable salts thereof.

[0172] Other suitable cationic lipids for use in the lipid nanoparticles and compositions of the invention include the cleavable cationic lipids described in U.S. Provisional Patent Application No. 62 / 672,194, filed May 16, 2018, and incorporated herein by reference. In certain embodiments, the lipid nanoparticles and compositions of the invention include cationic lipids having any of the general formulas or structures (1a)-(21a) and (1b)-(21b) and (22)-(237) described in U.S. Provisional Patent Application No. 62 / 672,194.

[0173] In certain embodiments, the lipid nanoparticles and compositions of the present invention include a lipid nanoparticle having the formula (I'): [ka] (In the formula: RX are independently -H, -L 1 -R 1 or -L 5A -L 5B -B'; L 1 , L 2 , and L 3 each independently represents a covalent bond, —C(O)—, —C(O)O—, —C(O)S—, or —C(O)NR L - and; Each L 4A and L 5A are independently -C(O)-, -C(O)O- or -C(O)NR L - and; Each L 4B and L 5B are independent, C1~C 20 Alkylene; C2~C 20 Alkenylene; or C2-C 20 alkynylene; Each B and B' is NR 4 R 5 or a 5- to 10-membered nitrogen-containing heteroaryl; Each R 1 , R 2 , and R 3 are independent, C6~C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 is alkynyl; Each R 4 and R 5 are independently hydrogen, C1 to C 10 Alkyl; C2-C 10 Alkenyl; or C2-C 10 is alkynyl; Each R L are independently hydrogen, C1 to C 20 Alkyl, C2-C 20 Alkenyl or C2-C 20 Cationic lipids having a structure according to the formula:

[0174] In certain embodiments, the lipid nanoparticles and compositions of the present invention include: [ka] An example of such a cationic lipid is compound (139), which has the compound structure 62 / 672,194.

[0175] In some embodiments, the lipid nanoparticles and compositions of the present invention include a cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride ("DOTMA"). (Feigner et al. (Proc. Nat'l Acad. Sci. 84, p. 7413 (1987); U.S. Pat. No. 4,897,355, incorporated herein by reference). Other cationic lipids suitable for the lipid nanoparticles and compositions of the present invention include, for example, 5-carboxyspermylglycine dioctadecylamide ("DOGS"); 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium ("DOSPA") (Behr et al., Proc. Nat'l Acad. Sci. 86, p. 6982 (1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"); 1,2-dioleoyl-3-trimethylammonium propane ("DOTAP").

[0176] Additional illustrative cationic lipids suitable for the lipid nanoparticles and compositions of the present invention include: 1,2-distearyloxy-N,N-dimethyl-3-aminopropane ("DSDMA"); 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane ("DODMA"); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane ("DLinDMA"); 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane ("DLenDMA"); N-dioleyl-N,N-dimethylan ammonium chloride ("DODAC"); N,N-distearyl-N,N-dimethylammonium bromide ("DDAB"); N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"); 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane ("CLinDMA"); 2-[5'-(cholest-5-ene-3-beta N,N-dimethyl-3,4-dioleyloxybenzylamine ("DMOBA"); 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane ("DOcarbDAP"); 2,3-dilinoleoyloxy-N,N-dimethylpropylamine ("DLinDAP"); 1,2-N,N'-dilinoleylcarbamyl-3-dimethyl Aminopropane ("DLincarbDAP"); 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane ("DLinCDAP"); 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane ("DLin-K-DMA"); 2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA");(2R)-2-((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ("Octyl-CLinDMA(2R)"); (2S)-2-((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N,fsl-dimethylh3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propane- 1-Amine ("Octyl-CLinDMA(2S)"); 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("DLin-K-XTC2-DMA"); and 2-(2,2-Di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine ("DLin-KC2-DMA") (see WO 2010 / 042877; Semple et al., Nature Biotech. 28:172-176 (2010), incorporated herein by reference). (Heyes, J. et al., J Controlled Release 107:276-287 (2005); Morrissey, DV. et al., Nat. Biotechnol. 23(8):1003-1007 (2005); International Patent Publication WO 2005 / 121348). In some embodiments, the one or more cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety. In some embodiments, cationic lipids suitable for the lipid nanoparticles and compositions of the present invention are selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane ("XTC"); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine ("ALNY-100") and / or 4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide ("NC98-5");

[0177] In some embodiments, the cationic lipid has formula (IIIE): [ka] or a pharma- ceutically acceptable salt thereof, wherein each n is independently 0 or 1; X 1A are independently O or NR 1A and; R 1A is H or C1-C6 alkyl; X 1B is a covalent bond, C(O), CH2CO2 or CH2C(O); X 2A and X 2B one is O and the other is a covalent bond; X 3A and X 3B one is O and the other is a covalent bond; X 4A and X 4B one is O and the other is a covalent bond; R 1 is independently L 1 -B 1 , C6~C 30 Alkyl, C6-C 30 Alkenyl or C6-C 30 is alkynyl; R 2 is independently L 2 -B 2 , C6~C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 is alkynyl; R 3 is independently L 3 -B 3 , C6~C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 is alkynyl; R 4 is independently L 4 -B 4 , C6~C 30Alkyl, C6-C 30 Alkenyl, C6-C 30 is alkynyl; L 1 , L 2 , L 3 , and L 4 are each independently C1 to C 30 Alkylene; C2~C 30 Alkenylene; or C2-C 30 alkynylene; B 1 , B 2 , B 3 and B. 4 each is independently an ionizable nitrogen-containing group; Cationic lipids contain at least one ionizable nitrogen-containing group.

[0178] In some embodiments, the cationic lipid has formula (IIIF): [ka] or a pharma- ceutically acceptable salt thereof, wherein B 1 is an ionizable nitrogen-containing group; L 1 is C1~C 10 is alkylene; R 2 , R 3 and R 4 Each of the following is independently C6 to C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 It is alkynyl.

[0179] In some embodiments, the cationic lipid has the formula (IIIG): [ka] or a pharma- ceutically acceptable salt thereof, wherein B 1 is an ionizable nitrogen-containing group; R 2, R 3 and R 4 Each of the following is independently C6 to C 30 Alkyl, C6-C 30 Alkenyl, C6-C 30 It is alkynyl.

[0180] In some embodiments, R in the cationic lipid according to any of formulas IIIE-IIIG is 2 , R 3 and R 4 each independently represents -O(CO)R 5 OR -C(O)OR 5 C6-C substituted by 12 is alkyl, R 5 is unsubstituted C6-C 14 In some embodiments, R in the cationic lipid according to any of formulas IIIE-IIIG is alkyl. 2 , R 3 and R 4 Each of the following is independent: [ka] It is.

[0181] In some embodiments, B in the cationic lipid according to any of formulas IIIE-IIIG. 1 teeth, d) NH2, guanidine, amidine, mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6-membered nitrogen-containing heteroaryl; e) [ka] ;or f) [ka] It is.

[0182] In some embodiments, L in the cationic lipid according to any of formulas IIIE-IIIG. 1is C1-alkylene.

[0183] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is TL1-04D-DMA: [ka] The compound has the structure:

[0184] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is GL-TES-SA-DME-E18-2: [ka] The compound has the structure:

[0185] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is SY-3-E14-DMAPr: [ka] The compound has the structure:

[0186] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is TL1-01D-DMA: [ka] The compound has the structure:

[0187] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is TL1-10D-DMA: [ka] The compound has the structure:

[0188] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is GL-TES-SA-DMP-E18-2: [ka] The compound has the structure:

[0189] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is HEP-E4-E10: [ka] The compound has the structure:

[0190] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is HEP-E3-E10: [ka] The compound has the structure:

[0191] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is SI-4-E14-DMAPr: [ka] The compound has the structure:

[0192] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is SY-010: [ka] The compound has the structure:

[0193] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is SY-011: [ka] The compound has the structure:

[0194] In some embodiments, a suitable cationic lipid for the lipid nanoparticles and compositions of the present invention is TL1-12D-DMA: [ka] The compound has the structure:

[0195] The molar ratio of cationic lipids in the lipid nanoparticles according to the invention is 41% to 70%. In some embodiments, the molar ratio of cationic lipids is 45% to 70%. In some embodiments, the molar ratio of cationic lipids is 45% to 65%. In some embodiments, the molar ratio of cationic lipids is 50% to 70%. In some embodiments, the molar ratio of cationic lipids is 50% to 65%. In a particular embodiment, the molar ratio of cationic lipids is 50% to 60%. A molar ratio of 50% to 60% of cationic lipids in the lipid nanoparticles according to the invention was found to result in high encapsulation efficiency, which was maintained before and after inhalation administration. Without wishing to be bound by any particular theory, using the smallest possible amount of cationic lipids is particularly advantageous, as it reduces the possibility of toxicity and adverse reactions during treatment with the lipid nanoparticles of the invention. Thus, in one particular embodiment, the molar ratio of cationic lipids is about 50%. In another particular embodiment, the molar ratio of cationic lipids is about 55%. In yet a further particular embodiment, the molar ratio of cationic lipids is about 60%.

[0196] In some embodiments, the cationic lipid in the lipid nanoparticles according to the present invention is any of the cationic lipids disclosed in PCT / US21 / 25128, which is incorporated herein by reference.

[0197] Non-cationic lipids As used herein, the phrase "non-cationic lipid" refers to neutral, zwitterionic or anionic lipid.Specific non-cationic lipids suitable for use in the lipid nanoparticles of the present invention include distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1,2-dilauroyl-sn-glycero-3-phosphorylethanolamine (DLPE), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phospho- L -serine (DOPS), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (14:1PC), 1,2-dipalmiteroyl-sn-glycero-3-phosphocholine (16:1PC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

[0198] Naturally occurring zwitterionic lipids are typically phospholipids, which can be broadly divided into two classes: (i) phospholipids containing ethanolamine substituents in the head group (also called PE lipids); and (ii) phospholipids containing choline substituents in the head group (also called PC lipids). The inventors have found that zwitterionic phospholipids and lipid nanoparticles containing ethanolamine substituents in the head group as the non-cationic lipid generally exhibit higher in vivo efficacy following inhalation administration than phospholipids containing choline substituents in the head group.

[0199] Thus, in some embodiments, the lipid nanoparticles according to the present invention include PE lipid as a non-cationic lipid component.In some embodiments, the lipid nanoparticles according to the present invention include DOPE, DLoPE, DMPE or DLPE as a non-cationic lipid component.In a particular embodiment, the lipid nanoparticles according to the present invention include DOPE as a non-cationic lipid component.In other embodiments, the lipid nanoparticles according to the present invention include DEPE as a non-cationic lipid component.

[0200] In some embodiments, the lipid nanoparticles according to the present invention include DOPC, DPPC or DSPC as a non-cationic lipid component. In a particular embodiment, the lipid nanoparticles according to the present invention include DOPC as a non-cationic lipid component.

[0201] The molar ratio of non-cationic lipids in lipid nanoparticles according to the present invention is 9%-18%. In some embodiments, the molar ratio of non-cationic lipids is 9%-15%. In a particular embodiment, the molar ratio of non-cationic lipids is 10%-15%. The inclusion of non-cationic lipids in lipid nanoparticles at molar ratios falling within this range has been found to provide particularly effective inhalation administration properties, as illustrated in the examples. Furthermore, the reduction in the total amount of non-cationic lipids, and the associated reduction in the overall lipid content of the lipid nanoparticles, did not adversely affect the encapsulation efficiency both before and after inhalation administration. In addition, lipid nanoparticles with a molar ratio of non-cationic lipids of 10%-15% have been found to be particularly potent in inducing in vivo expression of proteins encoded by the mRNAs encapsulated therein. Thus, in a particular embodiment, the molar ratio of non-cationic lipids is about 15%. In another particular embodiment, the molar ratio of non-cationic lipids is about 12.5%. In yet a further specific embodiment, the molar ratio of non-cationic lipids is about 10%.

[0202] PEG modified lipid The lipid nanoparticle of the present invention includes polyethylene glycol (PEG) modified lipid.In one embodiment, PEG modified lipid is PEG modified phospholipid or other derivatized lipid, for example derivatized ceramide (PEG-CER), for example N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide).

[0203] PEG-modified lipids typically include C6-C 20 Polyethylene glycol chains up to 5 kDa in length are covalently attached to lipids having long alkyl chain(s). 14 or C 18) are particularly useful. In some embodiments, the PEG modification (or PEGylated lipid) is PEGylated cholesterol. Lipid nanoparticles according to the present invention typically include PEG-modified lipids, such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K). Alternatively, they include [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or DSPE-PEG2K-COOH.

[0204] The inclusion of such PEG-modified lipids prevents complex aggregation (e.g., during storage and inhalation administration), and their presence can also increase the membrane permeability of the lipid nanoparticles of the present invention once delivered into the lungs.

[0205] In some embodiments, the molar ratio of PEG-modified lipid in lipid nanoparticles according to the invention is 2%-6%, for example 3%-6% or 4%-6%. In a particular embodiment, the molar ratio of PEG-modified lipid is 3%-5%. Lipid nanoparticles with a molar ratio of PEG-modified lipid falling within this range have been found to be particularly effective in delivering mRNA cargo to the underlying epithelium of the lung through the mucus layer. Thus, in a particular embodiment, the molar ratio of PEG-modified lipid is about 5%. In another particular embodiment, the molar ratio of PEG-modified lipid is about 4%. In yet a further particular embodiment, the molar ratio of PEG-modified lipid is about 3%.

[0206] For certain applications, lipid nanoparticles in which the PEG-modified lipid component constitutes about 5% of the total lipid by molar ratio have been found to be particularly suitable.

[0207] Cholesterol and cholesterol analogues Lipid nanoparticles according to the present invention typically include cholesterol as one of the four lipids that constitute its lipid components. In some embodiments, it is advantageous to use cholesterol analogues instead of cholesterol. As used herein, the term "cholesterol analogue" includes compounds that have the same structure as cholesterol, but differ in one or more atoms, functional groups and / or substructures. In some embodiments, cholesterol analogues are functional analogues of cholesterol, e.g., they have the same physical, chemical, biochemical and / or pharmacological properties as cholesterol. Examples of cholesterol analogues include, but are not limited to: β-sitosterol, stigmastanol, campesterol, fucosterol, stigmasterol and dexamethasone.

[0208] Both β-sitosterol and stigmastanol have been found to be particularly suitable for the manufacture of lipid nanoparticles with improved inhalation administration properties.Therefore, in one particular embodiment, the cholesterol analogue used in place of cholesterol in lipid nanoparticles of the present invention is β-sitosterol.In another particular embodiment, the cholesterol analogue used in place of cholesterol in lipid nanoparticles of the present invention is stigmastanol.

[0209] The molar ratio of cholesterol or cholesterol analogues in lipid nanoparticles according to the invention is between 9% and 48%. Cholesterol or cholesterol analogues can be used as "filler lipids" in lipid nanoparticles. In some embodiments, the molar ratio of cholesterol or cholesterol analogues is between 10% and 45%. In a particular embodiment, the molar ratio of cholesterol or cholesterol analogues is between 10% and 30%. More typically, about 25% to 30% (by molar ratio) of the lipid components of lipid nanoparticles according to the invention are cholesterol or cholesterol analogues. Thus, in one particular embodiment, the molar ratio of cholesterol or cholesterol analogues is between 25% and 30%. In a particular embodiment, the molar ratio of cholesterol or cholesterol analogues is about 25%. In another particular embodiment, the molar ratio of cholesterol or cholesterol analogues is about 30%.

[0210] Exemplary Lipid Nanoparticle Formulations The lipid nanoparticles of the present invention can include any of the cationic lipids, non-cationic lipids, cholesterol lipids and PEG-modified lipids described herein.

[0211] Cationic lipids that are particularly suitable for inclusion in such lipid nanoparticles include GL-TES-SA-DME-E18-2, TL1-01D-DMA, SY-3-E14-DMAPr, TL1-10D-DMA, GL-TES-SA-DMP-E18-2, HEP-E4-E10, HEP-E3-E10 and TL1-04D-DMA.These cationic lipids have been found to be particularly suitable for use in lipid nanoparticles administered via pulmonary delivery via inhalation administration.Of these, HEP-E4-E10, HEP-E3-E10, GL-TES-SA-DME-E18-2, GL-TES-SA-DMP-E18-2, TL1-01D-DMA and TL1-04D-DMA have been particularly well-performed.

[0212] Non-cationic lipids particularly suitable for inclusion in such lipid nanoparticles include DOPE, DLoPE, DMPE, DLPE, DOPC, DEPE, DSPC and DPPC.

[0213] PEG-modified lipids particularly suitable for inclusion in such lipid nanoparticles include DMG-PEG2K and DSPE-PEG2K-COOH.

[0214] Cholesterol analogues particularly suitable for inclusion in such lipid nanoparticles include β-sitosterol and stigmastanol.

[0215] Exemplary lipid nanoparticles include one of GL-TES-SA-DME-E18-2, TL1-01D-DMA, SY-3-E14-DMAPr, TL1-10D-DMA, GL-TES-SA-DMP-E18-2, HEP-E4-E10, HEP-E3-E10 and TL1-04D-DMA as a cationic lipid component, DOPE as a non-cationic lipid component, cholesterol as a helper lipid component, and DMG-PEG2K as a PEG-modified lipid component.

[0216] Table A below provides examples of lipid nanoparticles of the invention. In one embodiment, the lipid nanoparticle of the invention is any one of the lipid nanoparticles in Table A. In particular embodiments, the total lipid:mRNA ratio in the lipid nanoparticles of Table A is about 19:1 (mg:mg) or less. In particular embodiments, the total lipid:mRNA is between 11:1 and 19:1.

[0217] [Table 1]

[0218] Table B below provides further examples of lipid nanoparticles of the present invention. In one embodiment, the total lipid:mRNA ratio in the lipid nanoparticles of Table B is about 19:1 (mg:mg) or less. In a particular embodiment, the total lipid:mRNA is between 11:1 and 19:1.

[0219] [Table 2]

[0220] Table C below provides further examples of lipid nanoparticles of the present invention. In one embodiment, the total lipid:mRNA ratio in the lipid nanoparticles of Table C is about 19:1 (mg:mg) or less. In a particular embodiment, the total lipid:mRNA is between 11:1 and 19:1.

[0221] [Table 3]

[0222] Table D below provides further examples of lipid nanoparticles of the present invention. In one embodiment, the total lipid:mRNA ratio in the lipid nanoparticles of Table D is about 19:1 (mg:mg) or less. In a particular embodiment, the total lipid:mRNA is between 11:1 and 19:1.

[0223] [Table 4]

[0224] Table E below provides further examples of lipid nanoparticles of the present invention. In one embodiment, the total lipid:mRNA ratio in the lipid nanoparticles of Table E is about 19:1 (mg:mg) or less. In a particular embodiment, the total lipid:mRNA is between 11:1 and 19:1.

[0225] [Table 5]

[0226] Table F below provides further examples of lipid nanoparticles of the present invention. In one embodiment, the total lipid:mRNA ratio in the lipid nanoparticles of Table F is about 19:1 (mg:mg) or less. In a particular embodiment, the total lipid:mRNA is between 11:1 and 19:1.

[0227] [Table 6]

[0228] Table G below provides further examples of lipid nanoparticles of the present invention. In one embodiment, the total lipid:mRNA ratio in the lipid nanoparticles of Table G is about 19:1 (mg:mg) or less. In a particular embodiment, the total lipid:mRNA is between 11:1 and 19:1.

[0229] [Table 7]

[0230] Table H below provides further examples of lipid nanoparticles of the present invention. In one embodiment, the lipid nanoparticles of Table H have a total lipid:mRNA ratio of about 19:1 (mg:mg) or less. In particular embodiments, the total lipid:mRNA is between 11:1 and 19:1.

[0231] [Table 8]

[0232] Produce lipid nanoparticles Various processes can be used to produce mRNA-encapsulated lipid nanoparticles. Typically, the first step in the production of such suspensions is to provide a lipid solution. The lipid solution contains a mixture of lipids that form lipid nanoparticles. The lipid solution can be mixed with the mRNA solution first without preforming the lipids into lipid nanoparticles for encapsulation of the mRNA (as described in U.S. Patent Application No. 14 / 790,562, entitled "Encapsulation of messenger RNA," filed July 2, 2015, and its U.S. Provisional Patent Application No. 62 / 020,163, filed July 2, 2014, and in International Patent Application WO2016 / 004318, and U.S. Patent Application No. 2016 / 0038432, both of which are incorporated herein by reference in their entirety). Alternatively, a lipid solution is used to produce lipid nanoparticles. The preformed lipid nanoparticles can then be mixed with an mRNA solution to encapsulate the mRNA in the preformed lipid nanoparticles, for example, as described in International Patent Application WO2018 / 089801 and US Patent Application No. 2018 / 0153822, both of which are incorporated herein by reference in their entirety. These exemplary processes result in effective encapsulation of the mRNA in the lipid nanoparticles. Typically, the process can be optimized to achieve an encapsulation efficiency of at least about 90%, for example, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%.

[0233] As used herein, the term "encapsulation" or grammatical equivalents refer to the process of enclosing mRNA molecules in lipid nanoparticles.As used herein, this typically means that all or substantially all of the mRNA is encapsulated in lipid nanoparticles.

[0234] The inventors have found that the encapsulation efficiency of the lipid nanoparticles of the present invention remains relatively unaffected by inhalation administration, for example, when lipid nanoparticles according to the present invention are aerosolized by a vibrating mesh nebulizer. Thus, in some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency before and after inhalation administration of at least about 90%. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency before and after inhalation administration of at least about 95%. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency before and after inhalation administration of at least about 96%. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency before and after inhalation administration of at least about 97%. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency before and after inhalation administration of at least about 98%. In some embodiments, the lipid nanoparticles of the present invention have an encapsulation efficiency before and after inhalation administration of at least about 99%.

[0235] Those skilled in the art will recognize that a small loss in encapsulation efficiency upon inhalation administration of the lipid nanoparticles of the present invention is acceptable, so long as the majority of the lipid nanoparticles in the composition of the present invention (e.g., at least 80% of the lipid nanoparticles) effectively encapsulate the mRNA after being nebulized. Thus, in some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention changes less than about 20% upon inhalation administration. In particular embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention changes less than about 15% upon inhalation administration. In certain embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention changes less than about 10% upon inhalation administration. For example, in some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 20% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In particular embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 15% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In certain embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 10% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In another specific embodiment, the encapsulation efficiency of the lipid nanoparticles after inhalation administration is about 5% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration.In yet another specific embodiment, the encapsulation efficiency of the lipid nanoparticles after inhalation administration is about 3% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration.It is particularly desirable that the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is approximately the same as the encapsulation efficiency of the lipid nanoparticles before inhalation administration.

[0236] Lipid:mRNA ratio A further advantage associated with the lipid nanoparticles of the present invention is that they require less total lipid for effective encapsulation and delivery of mRNA compared to prior art mRNA-lipid nanoparticles commonly used for pulmonary delivery. For example, the lipid nanoparticles of the present invention can be produced using a total lipid:mRNA ratio of less than 20:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of between 11:1 and 19:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of between 16:1 and 19:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 19:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 18:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 17:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 16:1 (mg:mg).

[0237] In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio between 11:1 and 15:1. In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 15:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 14:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 13:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 12:1 (mg:mg). In some embodiments, the lipid nanoparticles of the present invention are produced using a total lipid:mRNA ratio of about 11:1 (mg:mg).

[0238] Lipid:mRNA ratio In some embodiments, the lipid nanoparticles of the present invention have an N / P ratio between 1 and 6. In particular embodiments, the lipid nanoparticles of the present invention have an N / P ratio of about 4. In some embodiments, the lipid nanoparticles of the present invention have an N / P ratio of less than 4. In some embodiments, the lipid nanoparticles of the present invention have an N / P ratio of about 3. In some embodiments, the lipid nanoparticles of the present invention have an N / P ratio of about 2.

[0239] Lipid nanoparticle size The process for producing lipid nanoparticles of the present invention was directed to obtain a composition with particle size fully defined above. In some embodiments, lipid nanoparticles of the present invention have a size of less than about 150 nm. In certain embodiments, lipid nanoparticles of the present invention have a size of less than about 100 nm. In particular embodiments, lipid nanoparticles of the present invention have a size of 60-150 nm, for example, 60-125 nm or 60-100 nm. Lipid nanoparticles within these size ranges have been used to successfully deliver mRNA to the lungs of a subject via inhalation administration.

[0240] In some embodiments, the lipid nanoparticles have a size of less than about 200 nm. In some embodiments, the lipid nanoparticles have a size of less than about 150 nm. In some embodiments, the lipid nanoparticles have a size of less than about 120 nm. In some embodiments, the lipid nanoparticles have a size of less than about 110 nm. In some embodiments, the lipid nanoparticles have a size of less than about 100 nm. In some embodiments, the lipid nanoparticles have a size of less than about 80 nm. In some embodiments, the lipid nanoparticles have a size of less than about 60 nm.

[0241] The size of the lipid nanoparticles of the present invention can be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981), which is incorporated herein by reference. For example, a Malvern Zetasizer can be used to measure particle size in lipid nanoparticle compositions of the present invention.

[0242] Messenger RNA (mRNA) The lipid nanoparticles of the present invention can encapsulate any mRNA. mRNA is typically considered to be a type of RNA that carries information from DNA to ribosomes. Typically, in eukaryotes, mRNA processing involves the addition of a "cap" at the 5' end and a "tail" at the 3' end. A typical cap is the 7-methylguanosine cap, which is a guanosine that is linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond. The presence of the cap is important to provide resistance to nucleases found in most eukaryotic cells. The addition of the tail is typically a polyadenylation event, whereby a polypolyadenylyl portion is added to the 3' end of the mRNA molecule. The presence of this "tail" serves to protect the mRNA from exonuclease degradation. The mRNA is translated by the ribosome into a series of amino acids that make up a protein.

[0243] mRNA encoding a therapeutic protein In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes a therapeutic protein. The term "therapeutic protein" as used herein refers to a protein, polypeptide or peptide. In typical embodiments, the therapeutic protein is an enzyme, a membrane protein, an antibody or an antigen.

[0244] In some embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes cystic fibrosis transmembrane conductance regulator (CFTR), ATP-binding cassette subfamily A member 3 protein, axonemal dynein intermediate chain 1 (DNAI1) protein, axonemal dynein heavy chain 5 (DNAH5) protein, alpha-1-antitrypsin protein, forkhead box P3 (FOXP3) protein, or surfactant proteins, such as surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein.

[0245] In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes the ATP-binding cassette subfamily A member 3 protein. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes the axonemal dynein intermediate chain 1 (DNAI1) protein. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes the axonemal dynein heavy chain 5 (DNAH5) protein. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes the alpha-1-antitrypsin protein. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes the forkhead box P3 (FOXP3) protein. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes more of the surfactant proteins, such as surfactant A protein, surfactant B protein, surfactant C protein, and surfactant D protein.

[0246] In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an antigen. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an antigen associated with the cancer of the subject or identified from the cancer cells of the subject. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an antigen (e.g., tumor neoantigen) determined from the subject's own cancer cells, thus providing a personalized cancer vaccine.

[0247] In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an antibody. In certain embodiments, the antibody is a bispecific antibody. In certain embodiments, the antibody is part of a fusion protein. In certain embodiments, the codon-optimized mRNA encapsulated in such lipid nanoparticles encodes an antibody against OX40. In certain embodiments, the codon-optimized mRNA encapsulated in such lipid nanoparticles encodes an antibody against VEGF. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an antibody against tissue necrosis factor alpha. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an antibody against CD3. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an antibody against CD19.

[0248] In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an immunomodulator. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes interleukin 12. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes interleukin 23. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes interleukin 36 gamma. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes a constitutively active version of one or more stimulator of interferon genes (STING) proteins.

[0249] In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an endonuclease. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes an RNA-guided DNA endonuclease protein, such as a Cas9 protein. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes a meganuclease protein. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes a transcription activator-like effector nuclease protein. In certain embodiments, the mRNA encapsulated in the lipid nanoparticles of the present invention encodes a zinc finger nuclease protein.

[0250] Typically, the mRNA encapsulated in the lipid nanoparticles of the present invention comprises a poly-A tail. In some embodiments, the mRNA comprises a poly-A tail of at least 70 residues in length. In some embodiments, the mRNA comprises a poly-A tail of at least 100 residues in length. In some embodiments, the mRNA comprises a poly-A tail of at least 120 residues in length. In some embodiments, the mRNA comprises a poly-A tail of at least 150 residues in length. In some embodiments, the mRNA comprises a poly-A tail of at least 200 residues in length. In some embodiments, the mRNA comprises a poly-A tail of at least 250 residues in length.

[0251] mRNA synthesis The mRNA can be synthesized according to any of a variety of known methods. Various methods are described in published U.S. Patent Application No. 2018 / 0258423, which can be used to practice the present invention, and are incorporated herein by reference in their entirety. For example, mRNA for use with the present invention can be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that can include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions vary depending on the particular application. In some embodiments, the mRNA can be further purified for use with the present invention. In some embodiments, the in vitro synthesized mRNA can be purified before being formulated and encapsulated to remove undesired impurities, including various enzymes and other reagents used during mRNA synthesis.

[0252] Various methods can be used to purify mRNA for use with the present invention. For example, purification of mRNA can be performed using centrifugation, filtration and / or chromatography methods. In some embodiments, the synthesized mRNA is purified by ethanol precipitation, or filtration, or chromatography, or gel purification, or any other suitable means. In some embodiments, the mRNA is purified by HPLC. In some embodiments, the mRNA is extracted in a standard phenol:chloroform:isoamyl alcohol solution familiar to those skilled in the art.

[0253] In a particular embodiment, mRNA is purified using tangential flow filtration (TFF).Suitable purification methods include those described in published US application No. 2016 / 0040154, published US patent application No. 2015 / 0376220, published US patent application No. 2018 / 0251755, published US patent application No. 2018 / 0251754, US Provisional Patent Application No. 62 / 757,612 filed on November 8, 2018, and US Provisional Patent Application No. 62 / 891,781 filed on August 26, 2019, all of which are incorporated herein by reference and can be used to practice the present invention.

[0254] In some embodiments, mRNA is purified before capping and tailing. In some embodiments, mRNA is purified after capping and tailing. In some embodiments, mRNA is purified both before and after capping and tailing. In some embodiments, mRNA is purified by centrifugation either before or after capping and tailing, or both before and after. In some embodiments, mRNA is purified by filtration either before or after capping and tailing, or both before and after. In some embodiments, mRNA is purified by TFF either before or after capping and tailing, or both before and after. In some embodiments, mRNA is purified by chromatography either before or after capping and tailing, or both before and after.

[0255] A variety of naturally occurring or modified nucleosides can be used to generate mRNA for use with the present invention. In some embodiments, the mRNA can be modified with any of the following nucleosides: natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-amino-2-methyl-1,2-diamino ... -deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N-1-methyl-pseudouridine), 2-thiouridine and 2-thiocytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[0256] In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues include, for example, 5-methyl-cytidine ("5mC"), pseudouridine ("ΨU") and / or 2-thio-uridine ("2sU"). For example, see U.S. Pat. No. 8,278,036 or WO2011012316 for a discussion of such residues and their incorporation into mRNA. An mRNA is an RNA defined as an RNA in which 25% of U residues are 2-thio-uridine and 25% of C residues are 5-methylcytidine. Teachings of the use of the RNA are disclosed in U.S. Patent Publication 20120195936 and International Publication WO2011012316, both of which are incorporated herein by reference in their entirety. The presence of non-standard nucleotide residues can make the mRNA more stable and / or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In further embodiments, the mRNA can include one or more non-standard nucleotide residues selected from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine cytosine, as well as combinations of these and other nucleobase modifications. Some embodiments further include additional modifications to the furanose ring or nucleobase. Additional modifications include, for example, sugar modifications or substitutions (e.g., one or more of 2'-O-alkyl modifications, locked nucleic acids (LNAs)). In some embodiments, the RNA can be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNAs). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modifications include, but are not limited to, 2'-deoxy-2'-fluoro modifications, 2'-O-methyl modifications, 2'-O-methoxyethyl modifications, and 2'-deoxy modifications.In some embodiments, any of these modifications may be present individually or in combination in 0-100% of the nucleotides, e.g., 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, greater than 95% or 100% of the constituent nucleotides.

[0257] Lipid nanoparticles of the present invention can encapsulate various lengths of mRNA.In some embodiments, lipid nanoparticles of the present invention can encapsulate in vitro synthesized mRNA of about 0.5kb, 1kb, 1.5kb, 2kb, 2.5kb, 3kb, 3.5kb, 4kb, 4.5kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, 14kb, 15kb, 20kb, 30kb, 40kb or 50kb or more in length. In some embodiments, the lipid nanoparticles of the present invention can encapsulate in vitro synthesized mRNA ranging in length from about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, about 8-15 kb, or about 8-50 kb.

[0258] Codon-optimized mRNA In some embodiments, the lipid nanoparticles of the present invention are for delivering codon-optimized mRNA encoding therapeutic proteins to subjects for the treatment of diseases.Suitable codon-optimized mRNAs encode either full-length, fragments or portions of proteins that can replace naturally occurring protein activity and / or reduce the intensity, severity and / or frequency of one or more symptoms associated with the disease.

[0259] Generation of optimized nucleotide sequences The present invention provides lipid nanoparticles that can encapsulate mRNAs that contain optimized nucleotide sequences that code for therapeutic proteins. These mRNAs are modified compared to their naturally occurring counterparts to (a) improve the yield of full-length mRNAs during in vitro synthesis, and (b) maximize the expression of encoded polypeptides after delivery of the mRNA to target cells in vivo. Sequence motifs that favor rapid degradation of mRNA in target cells are also removed.

[0260] An exemplary process for generating optimized nucleotide sequences for mRNA encapsulated by lipid nanoparticles of the present invention first generates a list of sequences that are codon-optimized, and then applies three filters to the list. Specifically, it applies a motif screening filter, a guanine-cytosine (GC) content analysis filter, and a codon adaptation index (CAI) analysis filter to generate an updated list of optimized nucleotide sequences. The updated list no longer includes nucleotide sequences that contain features that are predicted to inhibit effective transcription and / or translation of the encoded polypeptide.

[0261] Codon optimization The genetic code has 64 possible codons. Each codon contains a three nucleotide sequence. The frequency of usage of each codon in a protein-coding region of the genome can be calculated by determining the number of instances in which a particular codon appears in the protein-coding region of the genome, and then dividing the value obtained by the total number of codons that code for the same amino acid in the protein-coding region of the genome.

[0262] Codon usage tables often contain empirically derived data regarding how often each codon is used to code for a particular amino acid for the particular biological source from which the table was generated. This information is expressed as a percentage (0-100%) or fraction (0-1) for each codon of how often this codon is used to code for a particular amino acid relative to the total number of times that codon codes for that amino acid.

[0263] Codon usage tables are stored in publicly available databases, such as the Codon Usage Database (Nakamura et al. (2000) Nucleic Acids Research 28(1), 292; available online at https: / / www.kazusa.or.jp / codon / ) and the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs) database (Athey et al. (2017), BMC Bioinformatics 18(1), 391; available online at http: / / hive.biochemistry.gwu.edu / review / codon).

[0264] During the first step of codon optimization, codons are removed from a first codon usage table reflecting the frequency of each codon in a given organism (e.g., mammals or humans) if they are associated with a codon usage frequency below a threshold frequency (e.g., 10%). The codon usage frequencies of the unremoved codons in the first step are normalized to generate a normalized codon usage table. An optimized nucleotide sequence encoding an amino acid sequence of interest is generated by selecting a codon for each amino acid in the amino acid sequence based on the usage frequency of one or more codons associated with the given amino acid in the normalized codon usage table. The probability of selecting a particular codon for a given amino acid is equal to the usage frequency associated with the codon associated with this amino acid in the normalized codon usage table.

[0265] The codon-optimized sequence of the mRNA encapsulated by the lipid nanoparticles of the present invention is generated by a computer-implemented method for generating optimized nucleotide sequences. The method includes: (i) receiving an amino acid sequence, the amino acid sequence encoding a peptide, polypeptide or protein; (ii) receiving a first codon usage table, the first codon usage table comprising a list of amino acids, each amino acid in the table being associated with at least one codon, each codon being associated with a frequency of usage; (iii) removing from the codon usage table any codons associated with a frequency of usage below a threshold frequency; (iv) normalizing the frequency of usage of the codons not removed in step (iii) to generate a normalized codon usage table; and (v) generating an optimized nucleotide sequence encoding the amino acid sequence by selecting a codon for each amino acid in the amino acid sequence based on the frequency of usage of one or more codons associated with the amino acid in the normalized codon usage table. The threshold frequency is in the range of 5% to 30%, in particular 5%, 10%, 15%, 20%, 25% or 30%. In the context of the present invention, the threshold frequency is typically 10%.

[0266] The step of generating a normalized codon usage table includes: (a) distributing the usage frequency of each codon associated with the first amino acid and removed in step (iii) among the remaining codons associated with the first amino acid; and (b) repeating step (a) for each amino acid to generate a normalized codon usage table. In some embodiments, the usage frequency of the removed codon is equally distributed among the remaining codons. In some embodiments, the usage frequency of the removed codon is equally distributed among the remaining codons based proportionally on the usage frequency of each remaining codon. "Distributing" in this context can be defined as considering the combined magnitude of the usage frequency of the removed codons associated with a particular amino acid, and allocating a portion of this combined frequency to each of the remaining codons that code for a particular amino acid.

[0267] The steps of selecting a codon for each amino acid include: (a) identifying, in a normalized codon usage table, one or more codons associated with a first amino acid in the amino acid sequence; (b) selecting a codon associated with the first amino acid, such that the likelihood of selecting a particular codon is equal to the frequency of usage associated with the codon associated with the first amino acid in the normalized codon usage table; and (c) repeating steps (a) and (b) until a codon has been selected for each amino acid in the amino acid sequence.

[0268] The step of generating optimized nucleotide sequences is performed n times by selecting a codon for each amino acid in the amino acid sequence (step (v) in the method above) to generate a list of optimized nucleotide sequences.

[0269] Motif Screening The motif screening filter is applied to the list of optimized nucleotide sequences, and optimized nucleotide sequences that encode any of the known negative cis-regulatory elements and negative repeat elements are removed from the list to generate an updated list.

[0270] For each optimized nucleotide sequence in the list, it is also determined whether it contains a stop signal. Any nucleotide sequence that contains one or more stop signals is removed from the list to generate an updated list. In some embodiments, the stop signal has the following nucleotide sequence: 5'-X1ATCTX2TX3-3', where X1, X2 and X3 are independently selected from A, C, T or G. In some embodiments, the stop signal has one of the following nucleotide sequences: TATCTGTT; and / or TTTTTT; and / or AAGCTT; and / or GAAGAGC; and / or TCTAGA. In a typical embodiment, the stop signal has the following nucleotide sequence: 5'-X1AUCUX2UX3-3', where X1, X2 and X3 are independently selected from A, C, U or G. In certain embodiments, the stop signal has one of the following nucleotide sequences: UAUCUGUU; and / or UUUUU; and / or AAGCUU; and / or GAAGAGC; and / or UCUAGA.

[0271] Guanine-cytosine (GC) content The method further comprises determining the guanine-cytosine (GC) content of each of the optimized nucleotide sequences in the updated list of optimized nucleotide sequences. The GC content of a sequence is the percentage of bases in a nucleotide sequence that are guanine or cytosine. The list of optimized nucleotide sequences is further updated by removing any nucleotide sequence from the list if its GC content is outside a predetermined GC content range.

[0272] Determining the GC content of each of the optimized nucleotide sequences includes, for each nucleotide sequence: determining the GC content of one or more additional portions of the nucleotide sequence, the additional portions not overlapping with each other and with the first portion; updating the list of optimized sequences includes: removing a nucleotide sequence if the GC content of any portion is outside the predetermined GC content range; optionally, determining the GC content of the nucleotide sequences stops if the GC content of any portion is determined to be outside the predetermined GC content range. In some embodiments, the first portion and / or the one or more additional portions of the nucleotide sequence include a predetermined number of nucleotides, optionally the predetermined number of nucleotides is in the range of: 5 to 300 nucleotides or 10 to 200 nucleotides or 15 to 100 nucleotides or 20 to 50 nucleotides. In the context of the present invention, the predetermined number of nucleotides is typically 30 nucleotides. The predetermined GC content range is 15% to 75% or 40% to 60% or 30% to 70%. In the context of the present invention, the desired GC content range is typically between 30% and 70%.

[0273] A suitable GC content filter, in the context of the present invention, analyzes the first 30 nucleotides of an optimized nucleotide sequence, i.e., nucleotides 1 to 30 of the optimized nucleotide sequence. The analysis comprises determining the number of nucleotides in the portion that are either G or C, and determining the GC content of the portion comprises dividing the number of G or C nucleotides in the portion by the total number of nucleotides in the portion. The result of this analysis provides a value describing the proportion of nucleotides in the portion that are G or C, which may be a percentage, e.g., 50%, or a fraction, e.g., 0.5. If the GC content of the first portion is outside a predetermined GC content range, the optimized nucleotide sequence is removed from the list of optimized nucleotide sequences.

[0274] If the GC content of the first portion is within the predetermined GC content range, the GC content filter then analyzes a second portion of the optimized nucleotide sequence, in this example this is the second 30 nucleotides of the optimized nucleotide sequence, i.e. nucleotides 31 to 60. Analysis of this portion may be repeated for each portion and move on to the next optimized nucleotide sequence in the list until either: the optimized nucleotide sequence is removed from the list, a portion is found that has a GC content that is outside the predetermined GC content range, or, if the GC content filter keeps the optimized nucleotide sequence in the list, the entire optimized nucleotide sequence is analyzed and no such portion is found.

[0275] Codon Adaptation Index (CAI) The method further comprises determining the codon adaptation index of each of the optimized nucleotide sequences in the most recently updated list of optimized nucleotide sequences. The codon adaptation index of a sequence is a measure of codon usage bias and is a value between 0 and 1. The most recently updated list of optimized nucleotide sequences is further updated by removing any nucleotide sequence whose codon adaptation index is less than or equal to a predefined codon adaptation index threshold. The codon adaptation index threshold is 0.7 or 0.75 or 0.8 or 0.85 or 0.9. The inventors have found that optimized nucleotide sequences with a codon adaptation index equal to or greater than 0.8 deliver a very high yield of protein. Therefore, in the context of the present invention, the codon adaptation index threshold is typically 0.8.

[0276] The codon adaptation index can be calculated for each optimized nucleotide sequence in any manner apparent to one of skill in the art, e.g., as described in "The codon adaptation index - a measure of directional synonymous codon usage bias, and its potential applications" (Sharp and Li, 1987. Nucleic Acids Research 15(3), pp. 1281-1295); available online at https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC340524 / .

[0277] Implementations of the codon fitness index calculation include the following or similar methods: For each amino acid in a sequence, the weight of each codon in the sequence is calculated as a relative fitness (w i The relative fitness can be expressed by a parameter called the observed codon f for that amino acid. i Frequency of and the most frequent synonymous codon f j The codon adaptation index of a sequence can then be calculated as the geometric mean of the weights associated with each codon over the length of the sequence (measured as codons). The reference sequence set used to calculate the codon adaptation index is the same reference sequence set from which the codon usage table used in the codon optimization method described herein is derived.

[0278] composition The present invention provides compositions comprising the lipid nanoparticles of the present invention. In an exemplary embodiment, such compositions are formulated for pulmonary delivery by inhalation administration. Formulations include the addition of various excipients. These excipients may be useful in maintaining encapsulation efficiency before and after inhalation administration of lipid nanoparticle compositions. Inhalation administration, in the context of the present disclosure, is commonly performed using a nebulizer that includes vibrating mesh technology (VMT).

[0279] In some embodiments, the composition according to the present invention comprises mRNA encapsulated in lipid nanoparticles of the present invention, and the mRNA is present in the composition at a concentration ranging from about 0.5 mg / mL to about 1.0 mg / mL. In some embodiments, the mRNA is present at a concentration of at least 0.5 mg / mL. In some embodiments, the mRNA is present at a concentration of at least 0.6 mg / mL. In some embodiments, the mRNA is present at a concentration of at least 0.7 mg / mL. In some embodiments, the mRNA is present at a concentration of at least 0.8 mg / mL. In some embodiments, the mRNA is present at a concentration of at least 0.9 mg / mL. In some embodiments, the mRNA is present at a concentration of at least 1.0 mg / mL. In typical embodiments, the mRNA is present at a concentration of about 0.6 mg / mL to about 0.8 mg / mL.

[0280] The compositions of the present invention can be formulated with one or more carriers, stabilizing agents or other excipients.These compositions are pharmaceutical compositions, and therefore they contain one or more pharma-ceutically acceptable excipients.The one or more pharma-ceutically acceptable excipients can be selected from buffers, sugars, salts, surfactants or combinations thereof.

[0281] In certain embodiments, the compositions of the present invention include a buffer. In certain embodiments, the compositions of the present invention include a salt, such as sodium chloride. In some embodiments, the compositions of the present invention include a sugar, such as a disaccharide, such as sucrose or trehalose, at a suitable concentration, such as about 4% w / v, about 6% w / v, about 8% w / v, or about 10% w / v.

[0282] In some embodiments, the compositions of the present invention are stable at room temperature (e.g., at least 12 hours or 24 hours) or at -20°C (e.g., at least 6 months or 1 year). In a particular embodiment, the compositions of the present invention are provided in lyophilized form and are reconstituted in an aqueous solution (e.g., water for injection) before inhalation administration. Such compositions typically include a lyoprotectant. A suitable lyoprotectant for use with the lipid nanoparticles of the present invention is a disaccharide (e.g., sucrose or trehalose).

[0283] Pharmaceutically acceptable excipients In some embodiments, the pharmaceutical composition is formulated with a diluent. In some embodiments, the diluent is selected from the group consisting of ethylene glycol, glycerol, propylene glycol, sucrose, trehalose, or combinations thereof. In some embodiments, the formulation comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% diluent.

[0284] In some embodiments, the LNPs are suspended in an aqueous solution containing a disaccharide. Suitable disaccharides for use with the present invention include trehalose and sucrose. For example, in some embodiments, the LNPs are suspended in an aqueous solution containing trehalose, for example, 10% (w / v) trehalose in water. In other embodiments, the LNPs are suspended in an aqueous solution containing sucrose, for example, 10% (w / v) sucrose in water.

[0285] In some embodiments, the aqueous solution further comprises a buffer, a salt, a surfactant, or a combination thereof.

[0286] In some embodiments, the salt is selected from the group consisting of NaCl, KCl, and CaCl2. Thus, in some embodiments, the salt is NaCl. In some embodiments, the salt is KCl. In some embodiments, the salt is CaCl2.

[0287] In some embodiments, the buffer is selected from the group consisting of phosphate buffer, citrate buffer, imidazole buffer, histidine buffer and Good's buffer. Thus, in some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer is a citrate buffer. In some embodiments, the buffer is an imidazole buffer. In some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is Good's buffer. In some embodiments, Good's buffer is a Tris buffer or a HEPES buffer.

[0288] In particular embodiments, the buffer is a phosphate buffer (eg, a citrate-phosphate buffer), a Tris buffer, or an imidazole buffer.

[0289] In particular embodiments, compositions according to the invention include a buffer and a salt (typically in addition to a suitable diluent, such as a disaccharide or optionally propylene glycol), for example to improve the stability of the composition during storage. In some embodiments, the total concentration of the buffer and the salt is selected from about 40 mM Tris buffer and about 75-125 mM NaCl, about 50 mM Tris buffer and about 50 mM-100 mM NaCl, about 100 mM Tris buffer and about 100 mM-200 mM NaCl, about 40 mM imidazole and about 100 mM-125 mM NaCl, and about 50 mM imidazole and 75 mM-100 mM NaCl.

[0290] disaccharide Disaccharides, such as trehalose and sucrose, are excipients that can maintain the stability of the lipid nanoparticles of the present invention during inhalation administration and, in some embodiments, during lyophilization.

[0291] A sugar:lipid ratio of about 7 to about 9 is typically sufficient to maintain the stability of lipid nanoparticles.In some embodiments, even lower ratios may be acceptable.For example, as can be seen from the examples, a disaccharide concentration of less than 10%, for example about 4% to about 8%, is effective in maintaining the size and encapsulation efficiency of lipid nanoparticles of the present invention after inhalation administration.Such lipid nanoparticle formulations can also be lyophilized without significant increase in size or decrease in encapsulation efficiency after lyophilization and reconstitution.

[0292] Sucrose has been found to be a particularly effective excipient.In some embodiments, sucrose can be used as the sole excipient, for example, at a concentration of less than 10%, for example, between about 4% and about 8%, for example, about 8%.In other embodiments, sucrose can be combined with a buffer (for example, phosphate buffer) and a salt (for example, NaCl).

[0293] Illustrative Compositions In some embodiments, the compositions comprising lipid nanoparticles of the present invention comprise mRNA encapsulated in lipid nanoparticles (typically at a concentration of 0.4-0.8 mg / ml), a disaccharide, such as trehalose or sucrose, at a concentration of about 3-10% (w / v), and optionally TPGS at a concentration of about 0.1-1% (w / v). In a particular embodiment, the compositions of the present invention comprise mRNA encapsulated in lipid nanoparticles at a concentration of about 0.6 mg / ml, trehalose at a concentration of about 8% (w / v), and TPGS at a concentration of about 0.5% (w / v). In another particular embodiment, the compositions of the present invention comprise mRNA encapsulated in lipid nanoparticles at a concentration of about 0.6 mg / ml, and sucrose at a concentration of about 8% (w / v).

[0294] In some embodiments, the composition comprising the lipid nanoparticles of the present invention comprises an mRNA encapsulated in the lipid nanoparticles, a disaccharide, such as trehalose or sucrose, at a concentration of about 3-8% (w / v), a buffer (e.g., a phosphate buffer), and a salt (e.g., sodium chloride). In some embodiments, the composition comprises an mRNA encapsulated in the lipid nanoparticles at a concentration of 0.4-0.8 mg / ml, trehalose or sucrose at a concentration of about 4%-6% (w / v), a phosphate buffer at 1 mM-10 mM (pH 5-5.5), and a sodium chloride at a concentration of at least 75 mM (e.g., about 75 mM to 200 mM). In a particular embodiment, the composition comprises an mRNA encapsulated in the lipid nanoparticles at a concentration of about 0.4 mg / ml, sucrose at a concentration of about 4% (w / v), a phosphate buffer at about 2.5 mM (pH 5.5), and a sodium chloride at a concentration of about 150 mM. In another specific embodiment, the composition comprises mRNA encapsulated in lipid nanoparticles at a concentration of about 0.4 mg / ml, trehalose at a concentration of about 4% (w / v), phosphate buffer at about 10 mM (pH 5), and sodium chloride at a concentration of about 150 mM.

[0295] In certain embodiments, a composition comprising the mRNA-encapsulated lipid nanoparticles of the present invention comprises mRNA at a concentration of about 0.6 mg / mL, sucrose at a concentration of about 8% (w / v), and the molar ratios of the lipid components of the lipid nanoparticles are: a. about 50% SY-3-E14-DMAPr; b. about 15% DOPE; c. about 5% DMG-PEG2K; and d. about 30% cholesterol.

[0296] In a particular embodiment, a composition comprising the mRNA-encapsulated lipid nanoparticles of the present invention comprises mRNA at a concentration of about 0.4 mg / ml, a disaccharide, sucrose, at a concentration of about 4% (w / v), phosphate buffer at a concentration of about 2.5 mM (pH 5.5), sodium chloride at a concentration of about 150 mM, and the molar ratios of the lipid components of the lipid nanoparticles are: a. about 47% TL1-01D-DMA; b. about 22.5% DOPE; c. about 3% DMG-PEG2K; and d. about 27.5% cholesterol.

[0297] Dry Powder Formulation The present invention also provides a dry powder formulation comprising a plurality of spray-dried particles comprising the lipid nanoparticles of the present invention. Typically, the dry powder formulation suitable for use in the present invention comprises one or more polymers. In addition, it comprises one or more other excipients, such as one or more sugars or sugar alcohols, and / or one or more surfactants. In a typical embodiment of the present invention, the dry powder formulation comprises the lipid nanoparticles of the present invention, one or more polymers (e.g., polymethacrylate-based polymers, such as Eudragit EPO), one or more sugars or sugar alcohols, or combinations thereof (e.g., mannitol, or mannitol and lactose, or mannitol and trehalose), and optionally one or more surfactants (e.g., poloxamer, e.g., poloxamer 407). Providing the lipid nanoparticles of the present invention as a dry powder formulation is advantageous because such formulations are stable. In a particular embodiment, the spray-dried particles are administered via inhalation administration.

[0298] Sugar or sugar alcohol Illustrative sugars or sugar alcohols suitable for use in dry powder formulations according to the invention are mono-, di- and polysaccharides selected from the group consisting of glucose, fructose, galactose, mannose, sorbose, lactose, sucrose, cellobiose, trehalose, raffinose, starch, dextran, maltodextrin, cyclodextrin, inulin, xylitol, sorbitol, lactitol, and mannitol.

[0299] In some embodiments, the suitable sugar or sugar alcohol is lactose and / or mannitol. In some embodiments, the suitable sugar is mannitol. In some embodiments, mannitol is added at a concentration of about 1-10%. In some embodiments, mannitol is added at a concentration of about 2-10%. In some embodiments, mannitol is added at a concentration of about 3-10%. In some embodiments, mannitol is added at a concentration of about 4-10%. In some embodiments, mannitol is added at a concentration of about 5-10%.

[0300] In some embodiments, the suitable sugar is trehalose, hi some embodiments, both mannitol and trehalose are added.

[0301] Surfactants In some embodiments, the dry formulation according to the present invention further comprises one or more surfactants. The surfactant increases the surface tension of the composition. In some embodiments, the surfactant used in spray drying the mRNA-lipid composition is selected from the group consisting of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), phospholipid, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, Triton X-100, cocamide monoethanolamine, cocamide diethanolamine, glycerol monostearate, glycerol monolaurate, sorbitan monolaurate, sorbitan monostearate, Tween 20, Tween 40, Tween 60, Tween 80, alkyl polyglucoside, and poloxamer.

[0302] In particular embodiments, the surfactant is a poloxamer (eg, poloxamer 407).

[0303] Inhalation The composition comprising lipid nanoparticles of the present invention is typically administered by pulmonary delivery, particularly by inhalation administration.Inhalation administration results in an aerosolized composition that can be inhaled.Upon inhalation, lipid nanoparticles are distributed throughout the nose, airways and lungs, and are taken up by the epithelial cells of these tissues.As a result, the mRNA encapsulated in lipid nanoparticles is delivered into cells and expressed, for example, in the cells or tissues associated with the nasal cavity, trachea, bronchi, bronchioles and / or other pulmonary system.

[0304] Additional teachings of pulmonary delivery and inhalation administration are described, for example, in WO2018089790A1 and WO2018213476A1, each of which is incorporated by reference in its entirety.

[0305] Inhaled aerosol droplets with a particle size of less than 8 μm (e.g., 1-5 μm) can penetrate into the narrow branches of the lower respiratory tract. Aerosol droplets with a larger diameter are typically absorbed by epithelial cells lining the oral cavity and upper respiratory tract and are unlikely to reach the epithelium and deep alveolar tissue of the lower respiratory tract. Thus, a method comprising administering mRNA encapsulated in lipid nanoparticles of the present invention as an aerosol, particularly in the context of pulmonary delivery to the pulmonary epithelium, typically comprises the step of generating droplets with a particle size of less than 8 μm (e.g., 1-5 μm) by inhalation administration of the composition of the present invention, for example, by using a nebulizer suitable for use with the composition of the present invention.

[0306] In certain embodiments, the compositions of the present invention are nebulized to generate nebulized particles for inhalation by a subject. In particular embodiments, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of greater than about 12 ml / h. In particular embodiments, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of greater than about 15 ml / h. In other particular embodiments, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of greater than about 30 ml / h. In some embodiments, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of 12-50 ml / h. In some embodiments, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of 12-40 ml / h. In some embodiments, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of 15-50 ml / h. In some embodiments, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of 15-40 ml / h. In typical embodiments, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of between 12 ml / h and about 30 ml / h, for example, between 15 ml / h and about 30 ml / h. For example, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of about 12 ml / h or about 15 ml / h. In a specific embodiment, the lipid nanoparticles of the present invention can be nebulized at an inhalation throughput of about 30 ml / h.

[0307] Typically, in the context of the present invention, lipid nanoparticles that can be nebulized at higher inhalation dosage throughputs, such that the majority of lipid nanoparticles in the composition of the present invention (e.g., at least 80%, e.g., at least 85%, particularly at least 90%) encapsulate mRNA after being nebulized, retain the ability to effectively encapsulate mRNA after inhalation administration. Thus, in some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention changes less than about 20% upon inhalation administration. In particular embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention changes less than about 15% upon inhalation administration. In certain embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention changes less than about 10% upon inhalation administration. For example, in some embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 20% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In particular embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 15% or less, which is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration. In certain embodiments, the encapsulation efficiency of the lipid nanoparticles of the present invention after inhalation administration is about 10% or less, lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration.

[0308] The spray particles for inhalation by a subject typically have an average size of less than 8 μm. In some embodiments, the spray particles for inhalation by a subject have an average size of about 1-8 μm. In a particular embodiment, the spray particles for inhalation by a subject have an average size of about 1-5 μm. In a particular embodiment, the average particle size of the spray composition of the present invention is between 4 μm and 6 μm, for example, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm or about 6 μm.

[0309] Particle size in aerosols is commonly described in references to mass median aerodynamic diameter (MMAD). MMAD, together with geometric standard deviation (GSD), describes the particle size distribution of any aerosol based statistically on the weight and size of the particles. Means of calculating the MMAD of an aerosol are well known in the art. For example, the MMAD extrusion of a nebulizer using the composition of the present invention can be determined using a next-generation impactor. Another parameter describing particle size in an aerosol is the volume median diameter (VMD). VMD also describes the particle size distribution of an aerosol based on the volume of the particles. Means of calculating the VMD of an aerosol are well known in the art. The specific method used to determine VMD is laser diffraction, which is used herein to measure the VMD of the composition of the present invention (see, for example, Clark, 1995, Int J Pharm. 115:69-78).

[0310] Thus, in some embodiments, inhalation administration according to the present invention is carried out to generate fine particle fraction (FPF), which is defined as the proportion of particles in the aerosol that have MMAD or VMD less than a certain value.In a particular embodiment, the FPF of the nebulized composition of the present invention with particle size <5 μm is at least about 30%, more typically at least about 40%, for example at least about 50%, more typically at least about 60%.In another particular embodiment, inhalation administration is carried out in such a manner that the average respirable emitted dose (i.e., the percentage of FPF with particle size <5 μm; for example, determined by a next-generation impactor at 15 L / min extraction) is at least about 30% of the emitted dose, for example at least about 31%, at least about 32%, at least about 33%, at least about 34% or at least about 35% of the emitted dose. In yet another specific embodiment, inhalation administration is performed in a manner such that the mean respirable delivered dose (i.e., the percentage of FPFs with a particle size <5 μm; e.g., as determined by a next generation impactor at 15 L / min extraction) is at least about 15% of the emitted dose, e.g., at least 16% or 16.5% of the emitted dose.

[0311] In some embodiments, inhalation administration is performed using a nebulizer. One type of nebulizer is a jet nebulizer that includes a tube connected to a compressor that causes a high-velocity flow of compressed air or oxygen across the drug solution, turning it into an aerosol, which is then inhaled by the subject. Another type of nebulizer is an ultrasonic nebulizer that includes a high-frequency generator that generates high-frequency ultrasonic waves in contact with a liquid reservoir, which causes mechanical vibration of a piezoelectric element. The high-frequency vibration of the liquid is sufficient to generate a vapor mist. Illustrative ultrasonic nebulizers are the Omron NE-U17 and the Beurer Nebulizer IH30. A third type of nebulizer includes a vibrating mesh technology (VMT). VMT nebulizers typically include a mesh / membrane with 1000-7000 holes that vibrates on top of a liquid reservoir, thereby pushing a mist of extremely fine aerosol droplets through the holes in the mesh / membrane. Exemplary VMT nebulizers include eFlow (PARI Medical Ltd.), i-Neb (Respironics Respiratory Drug Delivery Ltd), Nebulizer IH50 (Beurer Ltd.), AeroNeb Go (Aerogen Ltd.), InnoSpire Go (Respironics Respiratory Drug Delivery Ltd), Mesh Nebulizer (Shenzhen Homed Medical Device Co, Ltd), Portable Nebulizer (Microbase Technology Corporation), and Airworks (Convexity Scientific LLC). In some embodiments, the mesh or membrane of the VMT nebulizer is made to vibrate with a piezoelectric element. In some embodiments, the mesh or membrane of the VMT nebulizer is made to vibrate with ultrasound.

[0312] VMT nebulizers have been found to be particularly suitable for the practice of the present invention because they do not affect the mRNA integrity of the encapsulated mRNA within the LNPs of the present invention present in the composition. Typically, at least about 60%, e.g., at least about 65% or at least about 70% of the mRNA in the compositions of the present invention maintains its integrity following inhalation administration.

[0313] In some embodiments, the inhalation administration is continuous during inhalation and exhalation. More typically, the inhalation administration is breath actuated. Suitable nebulizers for use with the present invention have an inhalation dosage volume of greater than 0.2 mL / min. In some embodiments, the inhalation dosage volume is greater than 0.25 mL / min. In other embodiments, the inhalation dosage volume is greater than 0.3 mL / min. In certain embodiments, the inhalation dosage volume is greater than 0.45 mL / min. In typical embodiments, the inhalation dosage volume ranges between 0.2 mL / min and 0.5 mL / min.

[0314] Therapeutic Uses of the Composition The present invention provides a method for in vivo delivery of lipid nanoparticles of the present invention, comprising administering the composition of the present invention to a subject via pulmonary delivery.In some embodiments, the subject is a human.In certain embodiments, pulmonary delivery can be by intranasal administration or inhalation.In certain embodiments, the composition is nebulized before inhalation.

[0315] The mRNA encapsulated in the lipid nanoparticles of the present invention encodes a protein. In some embodiments, the mRNA is delivered to the lung. In particular embodiments, the protein encoded by the mRNA is expressed in the lung. The protein may be, for example, a secreted protein, such as an antibody. In some embodiments, the protein may be a membrane protein, such as a viral surface antigen, a cell surface receptor, or a membrane channel (e.g., the cystic fibrosis transmembrane conductance regulator (CFTR)). The protein expressed by the mRNA typically has a therapeutic activity. For example, the expressed protein can be used to treat or prevent a disease or disorder. Thus, the lipid nanoparticles and compositions of the present invention are for use in the treatment or prevention of a disease or disorder. Typically, such use includes pulmonary administration of the lipid nanoparticles or compositions, for example, via inhalation administration. In some embodiments, the lipid nanoparticles and compositions are for use in the manufacture of a medicament for treating or preventing a disease or disorder. In typical embodiments, such manufacture includes the formulation of lipid nanoparticles in a composition suitable for pulmonary administration, for example, via inhalation administration. The present invention also provides a method of treating or preventing a disease or disorder in a subject, comprising administering a composition of the present invention to the subject via pulmonary delivery. In some embodiments, pulmonary delivery is via inhalation administration.

[0316] The method of the present invention can be used to treat various diseases and disorders in subjects, such as lung diseases, e.g., chronic respiratory diseases; protein deficiencies, e.g., protein deficiencies affecting the lungs; neoplastic diseases, e.g., tumors; and infectious diseases. In a particular embodiment, the disease or disorder is a protein deficiency. In some embodiments, the subject is healthy when the treatment is for the prevention of the disease or disorder (e.g., by immunization with the antigen encoded by the mRNA to prevent infectious disease). In other embodiments, the subject is suffering from a disease or disorder when the treatment is aimed at reducing or relieving one or more symptoms of the disease or disorder and / or addressing the root cause of the disease, e.g., by providing the defective protein via delivery of the mRNA encoding the protein, or by providing an agent that targets diseased tissue, e.g., an antibody that interferes with tumor growth.

[0317] In certain embodiments, the mRNA encodes a protein that is deficient in the subject. Examples of protein deficiencies that can be treated are cystic fibrosis, primary ciliary dyskinesia or surfactant deficiency. Expression of the mRNA in the lung can partially or completely restore the level of the protein in the subject. For example, the method of the present invention allows the subject to have a protein level equivalent to that of a healthy subject. In certain embodiments, the method of the present invention results in 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in the production of protein.

[0318] In some embodiments, the present invention provides a method for treating cystic fibrosis in a subject, comprising administering a composition of the present invention to the subject via pulmonary delivery. In some embodiments, the mRNA encodes CFTR. In some embodiments, the present invention provides a method for treating primary ciliary dyskinesia in a subject, comprising administering a composition of the present invention to the subject via pulmonary delivery. In some embodiments, the mRNA encodes DNAI1. In some embodiments, the present invention provides a method for treating surfactant deficiency in a subject, comprising administering a composition of the present invention to the subject via pulmonary delivery. In some embodiments, the mRNA encodes a surfactant protein.

[0319] In some embodiments, the present invention provides a method for treating chronic respiratory disease in a subject, comprising administering the composition of the present invention to the subject via pulmonary delivery.Examples of chronic respiratory disease that can be treated by the method of the present invention include chronic obstructive pulmonary disease (COPD), asthma, pulmonary arterial hypertension or idiopathic pulmonary fibrosis.In some embodiments, the mRNA encodes a protein for treating the symptoms of pulmonary disease or disorder.In certain embodiments, the mRNA encodes an antibody against proinflammatory cytokines.

[0320] In some embodiments, the present invention provides a method for treating or preventing neoplastic disease, such as tumor, in a subject, comprising administering the composition of the present invention to the subject via pulmonary delivery.In some embodiments, the tumor is a lung tumor or lung cancer, such as non-small cell lung cancer or small cell lung cancer.In certain embodiments, the mRNA encodes an antibody that targets a protein expressed on the surface of cells that constitute the tumor.In other embodiments, the mRNA encodes an antigen derived from the tumor, such as a tumor neoantigen.

[0321] In some embodiments, the present invention provides a method of treating or preventing an infectious disease in a subject, comprising administering a composition of the present invention to a subject via pulmonary delivery. In some embodiments, the infectious disease is caused by a virus. In some embodiments, the infectious disease is a pulmonary infectious disease or disorder. In some embodiments, the mRNA encodes a soluble decoy receptor that binds to a surface protein of a virus. In some embodiments, the mRNA encodes an antibody that is directed against a surface protein of a virus. In some embodiments, the infectious disease is caused by a bacterium. In a particular embodiment, the mRNA encodes an antibody that is directed against a surface protein of a bacterium. In some embodiments, the mRNA encodes an antigen derived from a causative agent of an infectious disease (e.g., a surface protein derived from a virus or bacterium that causes an infectious disease). For example, the lipid nanoparticles of the present invention encapsulating an mRNA that encodes an antigen, or a composition comprising the lipid nanoparticles, can be used to immunize a subject to prevent an infectious disease in the subject.

[0322] Working Example While certain lipid nanoparticles, compositions and methods of the present invention have been specifically described according to certain embodiments, the following examples serve only to illustrate the invention and are not intended to limit it. EXAMPLES

[0323] Production of lipid nanoparticles The lipid nanoparticles in Tables 1A to 1F were prepared to investigate the effect of decreasing the molar ratio of non-cationic lipid on the inhalation delivery properties of lipid nanoparticles. As a control, lipid nanoparticles having a molar ratio of non-cationic lipid of 30% were also prepared.

[0324] Cationic lipids (SY-3-E14-DMAPr), non-cationic lipids (DOPE or DPPC), cholesterol or cholesterol analogs (β-sitosterol or stigmastanol) and PEG-modified lipids (DMG-PEG2K) were dissolved in ethanol and mixed with citrate buffer using a pump system. Instantaneous mixing of the two streams led to the formation of empty 4-component lipid nanoparticles through a self-assembly process. The formulation was then subjected to a TFF purification process to remove the citrate buffer and alcohol, which was exchanged for a storage buffer (10% trehalose). The resulting suspension of preformed empty lipid nanoparticles was then mixed with firefly luciferase (FFL) mRNA to encapsulate the FFL mRNA according to methods known in the art. The N / P ratio was about 4.

[0325] The amount of lipid in the table indicates the total amount of lipid per mg of mRNA.

[0326] [Table 9]

[0327] [Table 10]

[0328] [Table 11]

[0329] [Table 12]

[0330] [Table 13]

[0331] [Table 14] EXAMPLES

[0332] Improving the extrusion rate of lipid nanoparticles administered by inhalation This example demonstrates that reducing the molar ratio of non-cationic lipid in mRNA-encapsulated lipid nanoparticles, relative to other lipids in the lipid component, improves inhalation dose output when the lipid nanoparticles are nebulized.

[0333] The effect of the molar ratio of non-cationic lipids in the test lipid nanoparticles produced in Example 1 on the inhalation dose output (ml / h) was investigated. Inhalation doses were performed using a nebulizer containing vibrating mesh technology (VMT). The specific nebulizer model used in this experiment was the Aerogen Solo. 1-6 ml of each test formulation containing 0.4-0.8 mg / ml mRNA in 4%-10% disaccharide was nebulized. Mice served as test animals in the in vivo expression experiments. The results of the experiments are shown in Figures 1, 4 and 7A.

[0334] As can be seen from FIG. 1, decreasing the molar ratio of the non-cationic lipid DOPE in the test lipid nanoparticles from 30% (LNP12) to 15% (LNP3) improved the inhalation dose output from about 10 ml / h to about 15 ml / h.

[0335] Further improvement in inhaled output was achieved by a still further reduction in the molar ratio of non-cationic lipid from 15% (LNP3) to 10% (LNP9), which effectively doubled the inhaled output from about 15 ml / h to about 30 ml / h.

[0336] The effect of decreasing the molar ratio of non-cationic lipid on inhaled extrusion was also investigated using lipid nanoparticles containing different cholesterol analogues (β-sitosterol and stigmastanol) instead of cholesterol. As can be seen in Figure 4, a similar improved inhaled extrusion was observed with lipid nanoparticles containing a lower molar ratio of non-cationic lipid. By decreasing the molar ratio of non-cationic lipid from 30% (LNP13 and LNP15) to 15% (LNP14 and LNP16), the inhaled extrusion was improved. This observation was made for both cholesterol analogues β-sitosterol and stigmastanol.

[0337] The effect of the molar ratio of non-cationic lipids on the inhalation extrusion rate was further investigated using DPPC as an alternative non-cationic lipid in the lipid nanoparticles. As can be seen in Figure 7A, the inhalation extrusion rate was also improved by decreasing the molar ratio of the non-cationic lipid DPPC from 30% to 15%. This improvement was observed with cholesterol, as well as the cholesterol analogues β-sitosterol and stigmastanol. This indicates that the findings in this example are broadly applicable to lipid nanoparticles with lipid components consisting of cationic lipids, non-cationic lipids, PEG-modified lipids, and cholesterol or cholesterol analogues.

[0338] Therefore, this embodiment shows that lipid nanoparticles with low molar ratio of non-cationic lipid to other lipids in lipid composition are particularly suitable for delivering mRNA to a subject via inhalation administration, and thus lipid nanoparticles can be nebulized with improved inhalation administration output.These improved lipid nanoparticles are therefore more effective in delivering mRNA to the lungs in therapeutic settings.The shortened inhalation administration time provides patients with significant benefits of treatment with mRNA therapy, which is delivered via pulmonary administration. EXAMPLES

[0339] Maintained encapsulation efficiency following inhalation administration This example demonstrates that reducing the molar ratio of non-cationic lipids in lipid nanoparticles encapsulating mRNA relative to other lipids in the lipid component maintains or improves encapsulation efficiency following inhalation administration of the lipid nanoparticles.

[0340] The effect of the molar ratio of non-cationic lipid in the lipid nanoparticles prepared in Example 1 on the encapsulation efficiency of the lipid nanoparticles after inhalation administration was investigated.

[0341] The encapsulation efficiency of lipid nanoparticles before inhalation administration was determined according to methods known in the art. Briefly, mRNA encapsulation was evaluated by performing Ribogreen assay (Invitrogen) in the presence or absence of 0.1% Triton-X 100, which disturbs lipid nanoparticles and releases their contents, to show the percentage of mRNA encapsulation in lipid nanoparticles relative to the total mRNA in the sample. This was performed for each lipid nanoparticle composition before and after inhalation administration to evaluate the loss in encapsulation efficiency due to inhalation administration. The loss in encapsulation efficiency after inhalation administration was calculated as a percentage. The results of the experiment are shown in Figures 2, 5 and 7B.

[0342] As can be seen from Figure 2, decreasing the molar ratio of non-cationic lipid from 30% (LNP12) to 15% (LNP3) reduced the change in lipid nanoparticle encapsulation efficiency after inhalation administration from a loss of more than 30% to a loss of less than 20%. Further reduction in the molar ratio of non-cationic lipid from 15% (LNP3) to 10% (LNP9) further reduced the loss in lipid nanoparticle encapsulation efficiency after inhalation administration.

[0343] The effect of the molar ratio of non-cationic lipid on the encapsulation efficiency after inhalation administration was also investigated by using two different cholesterol analogues (β-sitosterol and stigmastanol) instead of cholesterol. As can be seen in Figure 5, a similar reduced loss in encapsulation efficiency after inhalation administration was observed for lipid nanoparticles with a lower molar ratio of non-cationic lipid. By decreasing the molar ratio of non-cationic lipid from 30% (LNP13 and LNP15) to 15% (LNP14 and LNP16), the loss in encapsulation efficiency of lipid nanoparticles after inhalation administration was suppressed. This observation was made for both cholesterol analogues β-sitosterol and stigmastanol.

[0344] The effect of the molar ratio of non-cationic lipid on the encapsulation efficiency after inhalation administration was further investigated using DPPC as the non-cationic lipid. As can be seen from FIG. 7B, the loss in the encapsulation efficiency of lipid nanoparticles after inhalation administration was suppressed by decreasing the molar ratio of non-cationic lipid DPPC from 30% to 15%. This improvement was observed with cholesterol and cholesterol analogues β-sitosterol and stigmastanol. This indicates that the findings in this example are broadly applicable to lipid nanoparticles with lipid components consisting of cationic lipids, non-cationic lipids, PEG-modified lipids and cholesterol or cholesterol analogues.

[0345] Therefore, this embodiment shows that lipid nanoparticles with a low molar ratio of non-cationic lipids are particularly suitable for delivering mRNA to a subject via inhalation administration, as they preserve more mRNA after inhalation administration, compared to other lipids in lipid composition.This is significant because the reduced encapsulation efficiency after inhalation administration means that less mRNA is encapsulated by lipid nanoparticles, and therefore less mRNA is delivered intact to the lung to induce the expression of the protein encoded by the mRNA.Improved lipid nanoparticles are therefore expected to be more effective in delivering intact mRNA to the lung, where it can be expressed. EXAMPLES

[0346] Expression of firefly luciferase (FFL) mRNA in the lung This example demonstrates that reducing the molar ratio of non-cationic lipids in lipid nanoparticles encapsulating mRNA can result in improved in vivo expression of the protein encoded by the mRNA relative to other lipids in the lipid component when the nebulized lipid nanoparticle composition is delivered to the lungs of a test animal.

[0347] The effect of the molar ratio of non-cationic lipids in the lipid nanoparticles prepared in Example 1 on protein expression in the lung was investigated.

[0348] Lipid nanoparticles containing FFL mRNA were administered to mice via inhalation administration. Approximately 5 hours after administration, animals were administered luciferin via intraperitoneal injection, and all animals were imaged using an IVIS imaging system to measure luciferase production in the lungs. The results of the experiment are shown in Figures 3, 6, and 7C.

[0349] As can be seen from Figure 3, decreasing the molar ratio of non-cationic lipid from 30% (LNP12) to 15% (LNP3) and 10% (LNP9) improved the amount of mRNA delivered and therefore expressed in the lung.

[0350] The effect of lowering the molar ratio of non-cationic lipids on protein expression in the lungs was also investigated using two different cholesterol analogues (β-sitosterol and stigmastanol) instead of cholesterol. As can be seen in Figure 6, lowering the molar ratio of non-cationic lipids from 30% (LNP12, LNP13 and LNP15) to 15% (LNP3, LNP14 and LNP16) improved the amount of mRNA delivered and therefore expressed in the lungs. This observation was observed with both cholesterol analogues β-sitosterol and stigmastanol.

[0351] The effect of reducing the molar ratio of non-cationic lipids on protein expression in the lung was further investigated using DPPC as the non-cationic lipid. As can be seen from Figure 7C, reducing the molar ratio of the non-cationic lipid DPPC from 30% to 15% improves the amount of mRNA delivered and thus expressed in the lung. This improvement was observed with cholesterol, as well as the cholesterol analogues β-sitosterol and stigmastanol. This indicates that the findings in this example are broadly applicable to lipid nanoparticles with lipid components consisting of cationic lipids, non-cationic lipids, PEG-modified lipids and cholesterol or cholesterol analogues.

[0352] Thus, this example demonstrates that lipid nanoparticles having a low molar ratio of non-cationic lipids relative to other lipids in the lipid component are particularly suitable for delivering mRNA to a subject via inhalation administration, as they achieve elevated levels of expression of the encoded mRNA in the lung, as would be expected in light of the findings made in Examples 2 and 3. EXAMPLES

[0353] Optimizing total lipid content for inhaled administration This example demonstrates that, for encapsulated mRNA, reducing the overall lipid content of lipid nanoparticles can result in improved expression of the protein encoded by the mRNA after pulmonary delivery in vivo, while also improving inhaled dose output and encapsulation efficiency after inhaled administration.

[0354] The experiments described in Examples 2-4 were repeated with the lipid nanoparticle formulations shown in Table 2A. In addition to various molar ratios of non-cationic lipids, different molar ratios of cationic lipids were also tested. Cationic lipids are important for effective encapsulation of mRNA in lipid nanoparticles, as well as for endosomal release of mRNA after lipid nanoparticles are taken up by target cells. Both encapsulation efficiency and endosomal release affect the efficacy of lipid nanoparticles, i.e., their ability to effectively deliver intact mRNA and induce the expression of mRNA-encoded proteins in vivo.

[0355] Lipid nanoparticle formulations were prepared as described in Example 1. The resulting lipid nanoparticles were nebulized with a vibrating mesh nebulizer as described in Example 2. The inhalation dose output was measured and the results are shown in Figure 8A. As can be seen from the first three bars in this figure, decreasing the molar concentration of non-cationic lipid from 30% to 25% to 15%, while keeping the molar concentration of cationic lipid constant at 40%, resulted in an increase in the inhalation dose output. However, no improvement in encapsulation efficiency was observed after inhalation dose (see the first three bars in Figure 8B).

[0356] [Table 15]

[0357] Decreasing the non-cationic lipid content to 18% or less (molar ratio) to reach a total lipid:mRNA ratio (mg:mg) of 19:1 or less, while increasing the cationic lipid content to greater than 40% (molar ratio), maintained the improvement in inhaled dose output but led to a dramatic increase in encapsulation efficiency after inhaled administration (FIG. 8B; see bars 4-10). The improvement in maintaining effective encapsulation of mRNA after inhaled administration was at least partially reflected by the improved in vivo efficacy of the lipid nanoparticles, as shown in FIG. 8C (see especially bars 4-6).

[0358] Surprisingly, this result was achieved by reducing the total lipid content of the lipid nanoparticles per gram of mRNA delivered. Surprisingly, none of the lipid nanoparticles with reduced total lipid content were inferior in in vivo efficacy, despite maintaining their superior encapsulation efficiency relative to particles with higher lipid content, and despite being more effectively nebulized. Indeed, as can be seen from the last column in Table 2A, lipid nanoparticle formulations with total lipid:mRNA ratios (mg:mg) of 19:1 or less and cationic lipid molar ratios greater than 40% were superior in all three parameters (inhaled dose output, encapsulation efficiency after inhaled dose, and in vivo efficacy). Lipid nanoparticles with low lipid content routinely achieved inhaled dose output of 12 ml / h or more, while the change in encapsulation efficiency before and after inhaled dose was typically about 10% or less.

[0359] To better understand whether the findings with the lipid nanoparticle formulations in Table 2A, which used DOPE as the non-cationic lipid component, were broadly applicable, a second series of lipid nanoparticle formulations containing different non-cationic lipids were tested. The formulations tested in this second series are shown in Table 2B.

[0360] [Table 16]

[0361] The lipid nanoparticle formulations in Table 2B were prepared as described in Example 1. Instead of DOPE as the non-cationic lipid component, either DLPC (12:0PC), DMPC (14:0PC) or DOPC (18:1PC) was used. In each of the lipid formulations tested, the total lipid:mRNA ratio (mg:mg) was 19:1 or less, and the molar ratio of cationic lipid was greater than 40%. Furthermore, in this particular experiment, the non-cationic lipid was less than 18% (molar ratio) of the lipid component.

[0362] The resulting lipid nanoparticles were nebulized with a vibrating mesh nebulizer as described above to measure the inhalation throughput and encapsulation efficiency after inhalation administration of each test formulation. As can be seen from Figure 9A, the inhalation throughput was significantly improved for each of the lipid formulations tested. Indeed, all of the test formulations exceeded 12 ml / h, and many even exceeded 20 ml / h. Similarly, the change in encapsulation efficiency was typically about 10% or less (see Figure 9B). Although an increase in size was observed, all lipid nanoparticles tested maintained a size of less than 150 nm. Typically, the size before and after inhalation administration ranged from about 50 to 125 nm (see Figure 9C).

[0363] Although the PC lipids tested generally had reduced in vivo potency, the mRNA expression achieved following pulmonary delivery of the lipid nanoparticle formulations to test animals was in many cases comparable to that achieved with the original DOPE test formulation (see FIG. 10). Indeed, the use of DMPC in place of DOPE resulted in significantly improved mRNA expression levels relative to the DOPE control in formulations containing 50% cationic lipid and 10% DMPC (molar ratio).

[0364] A more significant increase in efficacy was observed when DLPE(12:0PE) was used in place of DOPE. The lipid nanoparticle formulations shown in Table 2C were administered to the lungs of test animals via inhalation administration using a vibrating mesh nebulizer.

[0365] [Table 17]

[0366] The results of this experiment are summarized in Figure 11. The original DOPE-containing lipid nanoparticle formulation was included as a positive control. Lipid nanoparticle formulations with DLPE as the non-cationic lipid generally performed better than the DOPE-containing control. The optimally performing formulations had a cationic lipid content of greater than 50% (molar ratio), a non-cationic lipid content of less than 15% (molar ratio), and a total lipid:mRNA ratio (mg:mg) of less than 19:1.

[0367] This example demonstrates that the inhalation administration characteristics and in vivo efficacy of mRNA-encapsulated lipid nanoparticles can be improved by adjusting the total lipid:mRNA ratio (mg:mg) to 19:1 or less. This is achieved by increasing the molar ratio of cationic lipid to more than 40% (molar ratio) and decreasing the molar ratio of non-cationic lipid content. Lipid nanoparticles containing cationic lipid at a molar ratio of 18% or less perform particularly well, and in some examples, further improvement is achieved by decreasing the non-cationic lipid content to less than 15% (molar ratio). The resulting formulations have excellent inhalation administration output and encapsulation efficiency after inhalation administration, which is found to result in improved in vivo efficacy as measured by the expression level of the protein encoded by the mRNA in the lungs of test animals. EXAMPLES

[0368] Optimizing the cationic lipid:non-cationic lipid ratio This example confirms that lipid nanoparticles with reduced total lipid content and cationic lipid molar ratios greater than 40% show significantly higher expression of mRNA-encoded proteins in vivo.

[0369] The mRNA encoding the mCherry protein was encapsulated into lipid nanoparticles having the composition shown in Table 3A.

[0370] [Table 18]

[0371] Test formulations were administered to CD-1 mice by inhalation. The various treatment groups are shown in Table 3B. Test animals were sacrificed either immediately after treatment (baseline) or 24 hours after exposure. Mice treated with saline served as controls. Lungs were isolated and homogenized, and mCherry expression levels were determined by ELISA.

[0372] [Table 19]

[0373] The results of this experiment are summarized in Figure 12. mCherry expression levels in the lungs were highest when a 24 mg dose of mRNA was administered by inhalation over a 4 hour period. Mice treated with lipid nanoparticle formulations containing 50% or 60% (molar ratio) cationic lipid had significantly higher expression levels than mice treated with lipid nanoparticles containing 40% (molar ratio) cationic lipid. Mice in groups 3, 4, 6 and 7 stably expressed more than 250 ng mCherry protein / mg total lung protein.

[0374] Expression levels were higher in the group receiving 24 mg over a 4-hour period compared to the group receiving 6 mg over a 1-hour period. Surprisingly, mCherry expression in mice treated with a 24 mg dose of lipid nanoparticle formulation containing 50% (molar ratio) cationic lipid (group 3) was about 25-fold higher than in mice treated with the same dose of lipid nanoparticle formulation containing 40% (molar ratio) cationic lipid (group 2). Mice in group 3 averaged about 1.15 ng mCherry protein / mg total protein in lungs. mCherry expression was undetectable at selected 24-hour time points in the group receiving a 6 mg dose of lipid nanoparticle formulation containing 40% (molar ratio) cationic lipid (group 5). As can be seen from the comparison of groups 3 and 6, there was a clear dose-dependence, as mice in group 3 had about 3-fold more mCherry protein in the lungs than mice in group 6. The difference in expression levels was smaller between the groups of mice that received lipid nanoparticle formulations containing 60% (molar ratio) cationic lipid (groups 4 and 7).

[0375] This example confirms that increasing the molar ratio of cationic lipid to more than 40% (e.g., 50% or 60%) while lowering the lipid:mRNA ratio to 19:1 or less through the reduction of non-cationic lipid content results in lipid nanoparticle formulations with improved in vivo efficacy.The data also suggest that the optimal cationic lipid molar ratio in lipid nanoparticles with a total lipid:mRNA ratio (mg:mg) of 19:1 or less is around 50%. EXAMPLES

[0376] Optimized lipid formulation with improved inhalation delivery properties This example demonstrates that by adjusting cationic lipid content while reducing non-cationic lipid content, the inhalation administration of lipid nanoparticles can be improved regardless of the cationic lipid contained in the formulation.These optimized lipid nanoparticle formulations with improved inhalation administration properties are better at maintaining mRNA encapsulation after inhalation administration and have greater in vivo efficacy.

[0377] To assess whether the findings with SY-3-E14-DMAPr are broadly applicable, experiments corresponding to those described in Examples 1-6 were repeated with lipid nanoparticle formulations containing the structurally different cationic lipid TL1-01D-DMA. The composition of the lipid nanoparticles tested is shown in Table 4A.

[0378] [Table 20]

[0379] As can be seen from Figures 13A and 13B, adjusting the molar ratio of both cationic and non-cationic lipids results in improved inhalation extrusion of the resulting lipid nanoparticles while maintaining mRNA encapsulation. Increasing the amount of TL1-01D-DMA to 47% (molar ratio) and decreasing the DOPE content to about 22.5% (molar ratio) results in lipid nanoparticles with a total lipid to mRNA ratio (mg:mg) of 17.1 that can nebulize more effectively at extrusion rates above 15 ml / h while retaining less than 10% change in encapsulation efficiency after inhalation administration. This optimized lipid nanoparticle formulation also exhibited improved in vivo efficacy over other cationic lipid formulations previously identified as highly effective for pulmonary delivery via inhalation administration (see Figure 13C).

[0380] This example confirms that increasing the molar ratio of cationic lipid to more than 40% while decreasing the total lipid:mRNA ratio to 19:1 or less through the reduction of non-cationic lipid content leads to lipid nanoparticle formulations with improved in vivo efficacy.It also confirms that the optimal molar ratio of cationic lipid in lipid nanoparticles with a total lipid:mRNA ratio (mg:mg) of 19:1 or less is around 50%. EXAMPLES

[0381] Lipid nanoparticles using PE lipids outperform those of PC lipids This example demonstrates that lipid nanoparticles having PE lipid as non-cationic lipid component generally have improved inhalation administration characteristics and in vivo efficacy compared to lipid nanoparticles having PC lipid as non-cationic lipid component.This example also confirms that lipid nanoparticles with reduced non-cationic lipid content, and therefore generally lower total lipid content, perform better during inhalation administration and result in improved mRNA expression in vivo.

[0382] To evaluate the relative performance of PC and PE lipids, the experiments described in Examples 2-4 were repeated with lipid nanoparticles having DPPC (PE lipid) as the cationic lipid component. Standard lipid nanoparticles having 30% DOPE as the non-cationic lipid component were included as a comparator. Lipid nanoparticles were prepared as described in Example 1. Test formulations are listed in Table 5A.

[0383] [Table 21]

[0384] Inhalation administration was performed as described in Example 2, and inhalation extrusion was measured. As expected, a decrease in the amount of DPPC from 30% to 15% (molar ratio) resulted in an increase in inhalation extrusion (see FIG. 14A). A decrease in DPPC from 30% to 15% (molar ratio) was accompanied by an increase in SY-3-E14-DMAPR percentage from 40% to 50%. FIG. 14B also showed that inhalation extrusion was slightly improved for lipid nanoparticles with 15% DPPC compared to those with 30% DOPE.

[0385] The change in encapsulation efficiency was then determined for each of the compositions. The relative change in encapsulation efficiency after inhalation administration was calculated as described in Example 3. As can be seen from Figure 14B, reducing the level of DPPC from 30% to 15% improved the encapsulation efficiency after inhalation administration. Lipid nanoparticles containing DPPC as a non-cationic lipid improved the encapsulation efficiency after inhalation administration compared to lipid nanoparticles containing standard DOPE.

[0386] To evaluate the in vivo efficacy of lipid nanoparticles, the expression of luciferase mRNA in the lungs of mice was measured as described in Example 4. In this experiment, lipid nanoparticles containing ML2 as a cationic lipid component were included as an additional comparator. The results are shown in Figure 14C. Decreasing the DPPC percentage from 30% to 15% or 10% (molar ratio), while increasing the relative cationic lipid molar ratio from 40% to 50% or 60%, respectively, resulted in an increase in the average radiance detected in the lungs of test animals. This finding was consistent with the observations made in the previous examples, which demonstrated that the improvement of the inhalation administration characteristics associated with the reduction of the total lipid content of mRNA-encapsulated lipid nanoparticles typically results in higher mRNA expression in vivo.

[0387] Surprisingly, the data in Figure 15 also show that standard lipid nanoparticles containing 30% DOPE as the non-cationic lipid result in significantly higher mRNA expression than optimized lipid nanoparticles containing 15% or 10% DPPC. To assess whether this finding can be extended to other PC lipids, and to determine whether PE lipids are generally more potent at inducing mRNA expression in the lungs of test animals, the in vivo potency of various PE and PC lipids was compared. Specifically, the PC lipids DPPC, DSPC, and DOPC, and the PE lipids DLPE, DMPE, DLoPE, and DOPE were evaluated.

[0388] Lipid nanoparticles were produced according to the method described in Example 1. The composition of the lipid nanoparticle formulations tested is shown in Table 5B. A non-optimized lipid nanoparticle formulation was used in this experiment. The results of the experiment are summarized in Figure 15.

[0389] [Table 22]

[0390] As can be seen from FIG. 15, administration of lipid nanoparticles having PE lipid as the non-cationic lipid, except for DOPC, by inhalation resulted in higher mRNA expression levels in mouse lungs than lipid nanoparticles having PC lipid as the non-cationic lipid.

[0391] Consistent with the observations made in the previous examples, this example confirms that lipid nanoparticles with reduced non-cationic lipid content and therefore generally lower total lipid:mRNA ratio (19:1 or less) perform better during inhalation administration and result in improved mRNA expression in vivo.In addition, this example demonstrates that lipid nanoparticles that contain PE lipid as a non-cationic lipid component generally have greater in vivo efficacy compared to lipid nanoparticles that contain PC lipid as a non-cationic lipid component. EXAMPLES

[0392] Synthesis of lipids for use in pulmonary delivery The synthesis of cationic lipids for use in the lipid nanoparticles of the present invention is described in this Example.

[0393] 1.GL-TES-SA-DME-E18-2 [ka]

[0394] Synthesis scheme [ka]

[0395] Synthesis Protocol Synthesis of (9Z,12Z)-Octadeca-9,12-dienoyl chloride (2) [ka] To a solution of linolenic acid (1.0 g, 3.6 mmol) in 10 mL of dichloromethane was added N,N-dimethylformamide (0.1 mL) and oxalyl chloride (1.2 mL, 14.3 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 3 h. The solvent was removed under reduced pressure and the crude was used in the next step without further purification.

[0396] Synthesis of 2-((1,3-bis(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propan-2-yl)amino)ethane-1-sulfonic acid (3) [ka] To a solution of (9Z,12Z)-octadeca-9,12-dienoyl chloride 2 (1.1 g, 3.6 mmol) in anhydrous N,N-dimethylacetamide (5.0 mL) and N-methylmorpholine (3.0 mL) was added 2-((1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl)amino)ethane-1-sulfonic acid (1, TES) (200 mg, 0.87 mmol). The reaction mixture was heated to 55° C. for 3 h. MS analysis indicated the formation of the desired product. The reaction mixture was cooled to room temperature, diluted with water (100 mL) and extracted with dichloromethane (2×100 mL). The combined organic layers were washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. The solvent was removed in vacuo and the residue was purified by column chromatography (40 g SiO: 0% to 10% methanol in dichloromethane gradient) to give 2-((1,3-bis(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propan-2-yl)amino)ethane-1-sulfonic acid as a colorless solid (562 mg, 47% yield).

[0397] Synthesis of 2-((2-(chlorosulfonyl)ethyl)amino)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate)(3-Cl) [ka] To a solution of 2-((1,3-bis(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propan-2-yl)amino)ethane-1-sulfonic acid 3 (210 mg, 0.82 mmol) in anhydrous dichloromethane (5.0 mL) was added N,N-dimethylformamide (0.05 mL) and oxalyl chloride (0.08 mL, 2.1 mmol) at 0° C. The reaction mixture was allowed to warm to room temperature and stirred for 3 h. The solvent was removed under reduced pressure to dryness to give 2-((2-(chlorosulfonyl)ethyl)amino)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propane-1,3-diyl (9Z,9′Z,12Z,12′Z)-bis(octadeca-9,12-dienoate), which was used in the next step without further purification.

[0398] Synthesis of 2-((2-(N-(2-(dimethylamino)ethyl)sulfamoyl)ethyl)amino)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate) (Compound I) [ka] To a solution of 2-((2-(chlorosulfonyl)ethyl)amino)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate)3-Cl (210 mg, 0.82 mmol) in anhydrous dichloromethane (5.0 mL) at 0 °C was added N 1 ,N 1-Dimethylethane-1,2-diamine (182 mg, 2.1 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction was quenched by the addition of water and the mixture was extracted with dichloromethane (2×100 mL). The combined organic layers were washed with saturated brine (100 mL) and dried over anhydrous sodium sulfate. The solvent was removed and the crude material was purified by column chromatography (40 g SiO: 0% to 15% methanol in dichloromethane gradient) to give 2-((2-(N-(2-(dimethylamino)ethyl)sulfamoyl)ethyl)amino)-2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propane-1,3-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate) as a yellow oil (139 mg, 62% yield). 1H NMR (300 MHz, Chloroform-d) δ 5.26-5.44 (m, 12H), 4.09 (s, 6H), 3.06-3.18 (m, 6H), 2.75 (t, 6H), 2.47 (t, 2H), 2.32 (t, 6H), 2.24 (s, 6H), 2.00-2.10 (m, 12H), 1.52-1.65 (m, 4H), 1.20-1.40 (m, 44H), 0.88 (t, 9H). APCI-MS analysis: Calculated C64H115N3O8S, [M+H] = 1186.7, observed = 1186.8.

[0399] 2.GL-TES-SA-DMP-E18-2 [ka]

[0400] Synthetic Route [ka]

[0401] Synthesis Protocol Compound II was prepared following the representative procedure above in yields similar to those obtained for Compound I.

[0402] Linoleic acid is treated with a chlorinating reagent, such as oxalyl chloride, to provide the acyl chloride compound 2. Reaction of compound 2 with a nucleophilic compound, such as buffer compound 1, gives compound 3. Compound 3 is treated with a chlorinating agent, such as oxalyl chloride, to provide the electrophilic compound 3-Cl. Reaction of 3-Cl with a nucleophilic reagent, such as compound 4b, then gives compound II.

[0403] The reaction conditions used were as follows:

[0404] [Table 23] 1H NMR (300 MHz, Chloroform-d) δ 5.24-5.42 (m, 12H), 4.08 (s, 6H), 3.17 (t, 2H), 3.06 (bs, 4H), 2.75 (t, 6H), 2.43 (t, 2H), 2.31 (t, 6H), 2.23 (s, 6H), 1.98-2.08 (m, 12H), 1.70 (quint, 2H), 1.52-1.63 (m, 4H), 1.17-1.45 (m, 44H), 0.87 (t, 9H). APCI-MS analysis: Calculated C65H117N3O8S, [M+H] = 1100.7, observed = 1100.8.

[0405] 3.TL1-01D-DMA [ka]

[0406] Synthesis scheme [ka]

[0407] Synthesis Protocol Synthesis of (Trioctyl 2-hydroxypropane-1,2,3-tricarboxylate) [ka] To a solution of citric acid A1 (2.1 g, 11.0 mmol) and 1-octanol A2-1 (9.4 g, 72.6 mmol) in dichloromethane (40 mL), DMAP (1.34 g, 11.0 mmol) and EDCI (14.3 g, 72.6 mmol) were added, and the resulting mixture was stirred at room temperature for 24 h. The reaction mixture was evaporated under vacuum. The residue was dissolved in dichloromethane (200 mL) and washed with brine (100 mL x 3). After drying over anhydrous Na2SO4, the solvent was evaporated and the crude was purified by column chromatography (220 g SiO2: 0 to 20% ethyl acetate in hexane gradient) to give (trioctyl 2-hydroxypropane-1,2,3-tricarboxylate) as a colorless oil (5.2 g, 90%).

[0408] Synthesis of (trioctyl 2-((3-(dimethylamino)propanoyl)oxy)propane-1,2,3-tricarboxylate) To a solution of trioctyl 2-hydroxypropane-1,2,3-tricarboxylate A3-1 (0.528 g, 1.0 mmol), DMAP (122 mg, 1.0 mmol) and pyridine (316 mg, 4.0 mmol) in 10 mL of dichloromethane, 3-(dimethylamino)propanoyl chloride A4-1 (271 mg, 2.0 mmol) was added at 0° C., and then the resulting mixture was stirred at room temperature for 24 h. The reaction mixture was evaporated under vacuum. The residue was dissolved in dichloromethane (100 mL) and washed with brine (80 mL×3). After drying over anhydrous Na2SO4, the solvent was evaporated and the crude was purified by column chromatography (80 g SiO2: 0 to 10% methanol in dichloromethane gradient) to give trioctyl 2-((3-(dimethylamino)propanoyl)oxy)propane-1,2,3-tricarboxylate as a colorless oil (210 mg, 33%).

[0409] Alternatively, to a suspension of 3-(dimethylamino)propanoic acid (8.02 g, 68.5 mmol) in 150 mL of dichloromethane, EDCI (13.1 g, 68.5 mmol) and DMAP (2.09 g, 17.1 mmol) were added at 0° C., and the resulting mixture was stirred at this temperature for 5 min. A solution of trioctyl 2-hydroxypropane-1,2,3-tricarboxylate A3-1 (9.05 g, 17.1 mmol) in 10 mL of dichloromethane was added, and then the resulting mixture was stirred at room temperature for 48 h. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate and brine. After drying over sodium sulfate, the organic layer was evaporated under vacuum. The residue was purified by column chromatography (220 g SiO2: 0 to 10% methanol in dichloromethane gradient) to give trioctyl 2-((3-(dimethylamino)propanoyl)oxy)propane-1,2,3-tricarboxylate as a colorless oil (4.2 g, 38%). 1 H NMR (300 MHz, CDCl3) δ 4.56 (s, br., 6H), 4.24 (t, 2H), 4.12 (s, 2H), 2.55 (t, 2H), 2.28-2.17 (m, 14H), 1.63-1.48 (m, 8H), 1.25 (s, br., 32H), 0.86 (t, 12H). APCI-MS analysis: Calculated C35H65NO8, [M+H] = 627.9, Observed = 628.5.

[0410] 4.TL1-04D-DMA [ka] TD1-04D-DMA can be made in a similar manner as TD-01D-DMA described above.

[0411] 5.SY-3-E14-DMAPr [ka]

[0412] Synthesis scheme [ka]

[0413] Synthesis of 3-(dimethylamino)propyl 4-hydroxy-3,5-dimethoxybenzoate (6) [ka] To a suspension of syringic acid 5 (7.5 g, 0.04 mol) in 100 mL of dichloromethane at 0 °C, oxalyl chloride (12.8 mL, 0.15 mol) was added followed by dimethylformamide (5 drops) and the resulting mixture was stirred at this temperature for 2 h. The reaction mixture was evaporated to dryness and the residue was dissolved in 100 mL of dichloromethane. After cooling to 0 °C, 3-(dimethylamino)propan-1-ol 2 (4.5 mL, 40 mmol) was added slowly and the reaction mixture was stirred at room temperature overnight. The precipitate was filtered to give 3-(dimethylamino)propyl 4-hydroxy-3,5-dimethoxybenzoate 6 as a white solid (6.2 g, 58%).

[0414] 6.TL1-10D-DMA [ka]

[0415] TD1-04D-DMA can be made in a similar manner as TD-01D-DMA described above.

[0416] 7.HEP-E3-E10 [ka]

[0417] Synthesis scheme [ka]

[0418] Synthesis Protocol Synthesis of [3] As presented in Scheme 1: To a solution containing HEP[1] (0.100 g, 0.494 mmol, 1.0 equiv), E3-E10[2] (0.668 g, 1.038 mmol, 2.1 equiv), 1 ml of dimethylformamide, 3 ml of dichloroethane, diisopropylethylamine (0.344 μL, 1.98 mmol, 4.0 equiv), and N,N-dimethylaminopyridine (0.024 g, 0.198 mmol, 0.4 equiv) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.285 g, 1.48 mmol, 3.0 equiv) and allowed to react at room temperature overnight (18 h). The reaction mixture was then concentrated using a rotary evaporator and purified using a Buchi Combi-flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (70% yield).

[0419] Synthesis of HEP-E3-E10[4] As presented in Scheme 1: To a 20 ml polypropylene scintillation vial equipped with a PTFE stir bar, [3] (0.500 g, 0.344 mmol, 1.0 equiv) was added along with 4 ml of dry tetrahydrofuran. The vial was cooled to 0-5 °C in an ice bath and HF / pyridine (1.76 ml, 67.86 mmol, 197.3 equiv) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi-flash system on a 12 g, 40 μm size silica gel column using dichloromethane / methanol (3% methanol) as the mobile phase to give a colorless oil (60% yield). 1H NMR (400 MHz, CDCl3) 4.16 (m, 4H), 3.60 (m, 4H), 2.97 (m, 3H), 2.78 (d, 3H), 2.58 (m, 9H), 2.37 (m, 12H), 2.15 (m, 2H), 1.78 (m, 4H), 1.44 (m, 7H), 1.36 (m, 9H), 1.26 (br, 45H), 1.05 (d, 6H), 0.87 (t, 12H). Expected M / Z = 998.59, Observed = 998.0.

[0420] 8.HEP-E4-E10 [ka]

[0421] Synthesis scheme [ka]

[0422] Synthesis Protocol Synthesis of

[12] As presented in Scheme 2: To a solution of HEP[1] (0.100 g, 0.494 mmol, 1.0 equiv), E4-E10

[11] (0.683 g, 1.038 mmol, 2.1 equiv), 1 ml of dimethylformamide, 3 ml of dichloroethane, diisopropylethylamine (0.344 μL, 1.98 mmol, 4.0 equiv), and N,N-dimethylaminopyridine (0.024 g, 0.198 mmol, 0.4 equiv) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.285 g, 1.48 mmol, 3.0 equiv) and allowed to react at room temperature overnight (18 h). The reaction mixture was then concentrated using a rotary evaporator and purified using a Buchi Combi-flash system on a 12 g, 40 μm size silica gel column using hexane / ethyl acetate as the mobile phase to give a colorless oil (63.3% yield).

[0423] Synthesis of HEP-E4-E10

[13] As presented in Scheme 2: To a 20 ml polypropylene scintillation vial equipped with a PTFE stir bar,

[12] (0.450 g, 0.303 mmol, 1.0 equiv) was added along with 4 ml of dry tetrahydrofuran. The vial was cooled in an ice bath to 0-5 °C and HF / pyridine (1.55 ml, 59.920 mmol, 197.3 equiv) was added dropwise. After addition, the reaction vial was allowed to warm to room temperature and stirred overnight (18 h). The reaction mixture was then neutralized with saturated sodium bicarbonate at 0 °C. Ethyl acetate was used for extraction (3x). The organic layers were combined, washed with saturated sodium chloride (4x), dried over sodium sulfate, filtered, and rotary evaporated to give an off-yellow oil. This oil was further purified using a Buchi Combi-flash system on a 12 g, 40 μm size silica gel column using dichloromethane / methanol (3%) as the mobile phase to give a colorless oil (48.4% yield). 1H NMR (400 MHz, CDCl3) 4.16 (t, 4H), 3.62 (br, 4H), 2.96 (q, 3H), 2.76 (d, 4H), 2.56 (m, 8H), 2.40 (m, 4H), 2.32 (t, 4H), 2.13 (t, 2H), 1.61 (m, 4H), 1.46 (m, 8H), 1.37 (m, 8H), 1.28 (br, 44H), 1.03 (d, 6H), 0.87 (t, 12H), 13C NMR (400 MHz, CDCl3) 173.65 (2C), 69.65 (2C), 68.04 (2C), 62.84 (2C), 61.82 (2C), 61.44 (2C), 60.89 (2C), 55.57 (4C), 51.55 (2C), 35.35 (4C), 34.20 (2C), 32.09 (7C), 30.00 (5C), 29.77 (6C), 29.47 (6C), 26.93 (2C), 25.84 (5C), 22.84 (9C), 17.77 (2C), 14.30 (7C). Expected M / Z = 1025.64, Observed = 1025.8. EXAMPLES

[0424] Evaluation of cationic lipids for pulmonary delivery This example demonstrates that a structurally diverse group of cationic lipids are effective in inducing expression of proteins encoded by mRNA encapsulated in lipid nanoparticles produced with these cationic lipids.

[0425] In this example, various cationic lipids were tested for in vivo efficacy when mRNA encapsulated in lipid nanoparticles (mRNA-LNP) was administered to mice by pulmonary delivery. The cationic lipids were tested for both efficacy, as determined by the level of protein production, and tolerability, as determined by side effects related to clearance and metabolism.

[0426] Approximately 150 cationic lipids were tested (FIG. 16). Each cationic lipid was used to prepare lipid nanoparticles encapsulating mRNA encoding firefly luciferase protein (FFL mRNA) according to methods known in the art. For example, suitable methods for mRNA encapsulation include those described in International Publication Nos. WO2016 / 004318 and WO2018 / 089801, the entire contents of which are incorporated herein by reference. The lipid nanoparticles tested contained lipid components consisting of cationic lipids, non-cationic lipids (DOPE), PEG-modified lipids (DMG-PEG2K), and optionally cholesterol.

[0427] Lipid nanoparticle formulations containing FFL mRNA were administered to mice.Approximately 5 hours after administration, animals were administered luciferin by intraperitoneal injection, and all animals were imaged using IVIS imaging system to measure luciferase production in lungs.Figure 16 shows that each cationic lipid has different efficacy of in vivo protein expression in lungs.Some cationic lipids notably have more than 50-fold increase in lung protein expression compared to other cationic lipids.

[0428] Based on their performance in this in vivo screen, eight cationic lipids were selected for further investigation in lipid nanoparticles using lipid components consisting of cationic lipids, non-cationic lipids, PEG-modified lipids, and cholesterol or cholesterol analogs: GL-TES-SA-DME-E18-2, TL1-01D-DMA, SY-3-E14-DMAPr, TL1-10D-DMA, GL-TES-SA-DMP-E18-2, HEP-E4-E10, HEP-E3-E10, and TL1-04D-DMA. Of these, HEP-E4-E10, HEP-E3-E10, GL-TES-SA-DME-E18-2, GL-TES-SA-DMP-E18-2, TL1-01D-DMA, and TL1-04D-DMA exhibited particularly high potency as determined by the mean radiance detected in mouse lungs.

[0429] This example shows that various cationic lipids can be usefully used to prepare lipid nanoparticles with lipid components consisting of cationic lipids, non-cationic lipids, PEG-modified lipids, and cholesterol or cholesterol analogs.These lipid nanoparticles can be effectively used to encapsulate and deliver mRNA to the lungs of a subject to induce the expression of the protein encoded by the mRNA. EXAMPLES

[0430] Excipient Optimization This example demonstrates that optimization of lipid nanoparticle formulations can reduce the amount of additional excipients required to improve inhalation administration characteristics or to maintain lipid nanoparticle size and encapsulation efficiency during lyophilization.

[0431] The preceding experiments demonstrate that the inhalation administration characteristics and in vivo efficacy of mRNA-encapsulated lipid nanoparticles can be improved by adjusting the total lipid:mRNA ratio (mg:mg) to 19:1 or less. This is achieved by increasing the molar ratio of cationic lipid to more than 40% (molar ratio) and decreasing the molar ratio of non-cationic lipid. To determine how changes in total lipid content and associated changes in sugar-to-lipid ratio affect the size of lipid nanoparticles before and after lyophilization, standard and optimized lipid nanoparticles were suspended in aqueous solutions containing either trehalose or sucrose at either 8% or 10% (w / v). The composition of the lipid nanoparticles is shown in Table 6A. The sugar-to-lipid ratio is shown in Table 6B.

[0432] [Table 24]

[0433] [Table 25]

[0434] The size and encapsulation efficiency of lipid nanoparticle formulations were determined before and after lyophilization. The size and encapsulation efficiency after lyophilization were determined by reconstituting the lyophilized composition in water. As can be seen in Figure 17A, a standard lipid formulation containing 40% cationic lipid and 30% cationic lipid (molar ratio) dramatically changed the size after lyophilization when suspended in either 10% or 8% trehalose. Moreover, as can be seen in Figure 17B, the change in size was accompanied by a loss of more than 50% in encapsulation efficiency. In contrast, no such change in size was observed when sucrose was used.

[0435] Surprisingly, the two optimized lipid nanoparticle formulations were able to maintain their size and encapsulation efficiency after lyophilization, regardless of whether trehalose or sucrose was used as an excipient. Reconstituted lipid nanoparticles with total lipid:mRNA ratios of 19:1 or less maintained a size of less than 150 nm after lyophilization (see Figures 17C and 17E). Moreover, the encapsulation efficiency remained above 90% (see Figures 17D and 17F).

[0436] In the lipid nanoparticle formulations tested, sucrose was more effective than trehalose in maintaining the size of lipid nanoparticles during lyophilization (see Figures 17A, 17C and 17E). This was true even when the sugar-to-lipid ratio was decreased by lowering the disaccharide concentration from 10% to 8% (see Table 6B). Therefore, a concentration of 8% (w / v) sucrose was selected for the optimized lipid nanoparticle formulation.

[0437] Next, the inhalation delivery properties of the optimized lipid nanoparticle formulation were compared with previous lipid nanoparticle formulations. Previous lipid nanoparticle formulations optimized for inhalation delivery contained trehalose as an excipient. Furthermore, the inclusion of surfactant D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) in these trehalose-based formulations showed improved inhalation delivery output. These optimization steps were described in U.S. Provisional Patent Application No. 63 / 217,633, filed July 1, 2021, and incorporated herein by reference, and used a standard lipid formulation containing DMG-PEG2k:cationic lipid:Chol:DOPE in a molar ratio of 5:40:25:30.

[0438] A standard lipid nanoparticle formulation containing SY-3-E14-DMAPr as the cationic lipid component and DOPE as the non-cationic lipid component, encapsulating two different mRNAs, was compared to an optimized lipid nanoparticle formulation as specified in Example 5. The lipid nanoparticles for the standard formulation contained 0.5% TPGS in addition to sucrose to improve inhalation dose output. The optimized lipid nanoparticle formulation contained sucrose as the only excipient.

[0439] As can be seen in Table 6C below, the optimized lipid nanoparticle formulation maintained an encapsulation efficiency after inhalation administration of nearly 90%, but the overall mRNA:lipid ratio was reduced. Furthermore, the inhalation output of the optimized formulation was comparable to that of the standard formulation, despite the absence of TPGS.

[0440] [Table 26]

[0441] Changes in salt and buffer concentration can also affect how effective lipid nanoparticle formulations are nebulized.For example, optimization of salt and buffer concentration can reduce the amount of trehalose required to maintain favorable inhalation administration properties (see US Provisional Patent Application No. 63 / 217,633).Standard lipid nanoparticle formulations that contain TL1-01D-DMA as cationic lipid component and DOPE as non-cationic lipid component and encapsulate two different mRNAs were compared with the optimized lipid nanoparticle formulations specified in Example 7.These standard lipid nanoparticles were formulated with 4% trehalose, 10 mM phosphate buffer (pH 5) and 150 mM NaCl to improve inhalation administration output.

[0442] As can be seen in Table 6D below, the optimized lipid nanoparticle formulation containing sucrose instead of trehalose achieved an inhalation dose extrusion rate equivalent to that achieved with the standard formulation at a reduced buffer concentration of 75%, while maintaining an encapsulation efficiency after inhalation dose of approximately 90%.

[0443] [Table 27]

[0444] This example demonstrates that optimization of lipid composition can improve the inhalation administration properties of the formulation or reduce the need for excipients in lipid nanoparticle formulations that are otherwise required to maintain the size and encapsulation efficiency of the lipid nanoparticles during lyophilization.

[0445] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above description, but is set forth in the following claims.

Claims

1. (i) mRNA encapsulated within lipid nanoparticles, and (ii) the following components: a. a cationic lipid component; b. a non-cationic lipid component; c. a PEG-modified lipid component, and d. Cholesterol components Lipid components consisting of 1. A lipid nanoparticle comprising: (1) the cationic lipid component is greater than 40% (molar ratio); (2) the non-cationic lipid component is less than 25% (by molar ratio); (3) A lipid nanoparticle having a total lipid:mRNA ratio (mg:mg) of 19:1 or less.

2. The lipid nanoparticle of claim 1, wherein the total lipid:mRNA ratio (mg:mg) is between 11:1 and 19:1, and optionally the total lipid:mRNA ratio (mg:mg) is about 18:1 or less, about 17:1 or less, about 15:1 or less.

3. The lipid nanoparticle of claim 1, wherein the cationic lipid component is between 45% and 60% (molar ratio), optionally between 45% and 55% (molar ratio), about 50% (molar ratio), about 22.5% (molar ratio), less than 18% (molar ratio), about 15% (molar ratio), or less than 13% (molar ratio).

4. The molar ratio of the lipid components is: a. about 47% to 60% cationic lipid; b. about 10% to 22.5% non-cationic lipid; c. about 3% to 5% PEGylated lipid; d. the remainder is cholesterol or a cholesterol analog; or The molar ratio of the lipid components is: a. about 50% to 55% cationic lipid; b. about 10-15% non-cationic lipid; c. about 3-5% PEGylated lipid; d. the remainder is cholesterol or a cholesterol analog; or The molar ratios of the lipid components are: a. about 55% cationic lipid; b. about 10% non-cationic lipid; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues - 30% or The molar ratio of the lipid components is: a. about 50% cationic lipid; b. about 12.5% ​​non-cationic lipids; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues - 32.5% is; The molar ratio of the lipid components is: a. about 50% cationic lipid; b. about 12.5% ​​non-cationic lipids; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues - 32.5% or The molar ratio of the lipid components is: a. about 47% cationic lipid; b. about 22.5% non-cationic lipids; c. about 3% PEG-modified lipid, and d. Cholesterol or cholesterol analogues -27.5% The lipid nanoparticle of claim 1 ,

5. The lipid nanoparticle of claim 1, wherein the cationic lipid is SY-3-E14-DMAPr.

6. The lipid nanoparticle of claim 1, wherein (i) the lipid nanoparticles are capable of being nebulized at an inhalation throughput of greater than about 12 ml / h, greater than about 15 ml / h, or greater than about 20 ml / h, and / or (ii) the encapsulation efficiency of the lipid nanoparticles after inhalation administration is less than or equal to about 10% and is lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration, and / or (iii) the lipid nanoparticles have an encapsulation efficiency before and after inhalation administration of at least about 90%.

7. (i) mRNA encapsulated within lipid nanoparticles, and (ii) lipids having the following molar ratio: a. 41% to 70% cationic lipid; b. 9% to 18% non-cationic lipid; c. 2% to 6% PEG-modified lipid, and d. Cholesterol or cholesterol analogues 9% to 48% Lipid components consisting of A lipid nanoparticle comprising:

8. (i) the lipid nanoparticles are capable of being nebulized; or The lipid nanoparticle of claim 7, wherein (ii) the lipid nanoparticle is nebulizable with an inhalation throughput of greater than about 12 ml / h, in particular with an inhalation throughput of greater than about 15 ml / h, and / or (iii) the lipid nanoparticle is for pulmonary delivery by inhalation.

9. 8. The lipid nanoparticle of claim 7, wherein the molar ratio of cationic lipids is 45% to 70%, or 45% to 65%, or 50% to 70%, or 50% to 65%, or 50% to 60%, or about 50%, or about 55%, or about 60%, and / or (ii) the molar ratio of non-cationic lipids is 9% to 15%, or 10% to 15%, or about 15%, or about 12.5%, or about 10%, and / or (iii) the molar ratio of PEG-modified lipids is 3% to 6%, or 4% to 6%, or about 5%, or about 3%; and / or (iv) the molar ratio of cholesterol or cholesterol analogues is 10% to 45%, or 10% to 30%, or 25% to 30%, or about 25%, or about 30%.

10. The lipid / lipid component molar ratio is: a. 50% to 60% cationic lipid; b. 9% to 18% non-cationic lipid; c. 4% to 6% PEG-modified lipid, and d. Cholesterol or cholesterol analogues 20-35% is; or The lipid / lipid component molar ratio is: a. 50% to 60% cationic lipid; b. 9% to 15% non-cationic lipid; c. 4% to 6% PEG-modified lipid, and d. Cholesterol or cholesterol analogues 25-30% is; or The lipid / lipid component molar ratio is: a. about 50% cationic lipid; b. about 15% non-cationic lipid; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues - 30% is; or The lipid / lipid component molar ratio is: a. about 60% cationic lipid; b. about 10% non-cationic lipid; c. about 5% PEG-modified lipid, and d. about 25% cholesterol or cholesterol analogue is; or The lipid / lipid component molar ratio is: a. about 50% cationic lipid; b. about 10% non-cationic lipid; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues, about 35% is; or The lipid / lipid component molar ratio is: a. about 50% cationic lipid; b. about 12.5% ​​non-cationic lipids; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues - 32.5% is; or The lipid / lipid component molar ratio is: a. about 50% cationic lipid; b. about 17.5% non-cationic lipids; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues -27.5% is; or The lipid / lipid component molar ratio is: a. about 55% cationic lipid; b. about 10% non-cationic lipid; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues - 30% is; or The lipid / lipid component molar ratio is: a. about 55% cationic lipid; b. about 12.5% ​​non-cationic lipids; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues -27.5% is; or The lipid / lipid component molar ratio is: a. about 55% cationic lipid; b. about 15% non-cationic lipid; c. about 5% PEG-modified lipid, and d. about 25% cholesterol or cholesterol analogue is; or The lipid / lipid component molar ratio is: a. about 55% cationic lipid; b. about 17.5% non-cationic lipids; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues -22.5% is; or The lipid / lipid component molar ratio is: a. about 60% cationic lipid; b. about 12.5% ​​non-cationic lipids; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues -22.5% is; or The lipid / lipid component molar ratio is: a. about 60% cationic lipid; b. about 15% non-cationic lipid; c. about 5% PEG-modified lipid, and d. Cholesterol or cholesterol analogues, about 20% The lipid nanoparticle of claim 1 ,

11. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle has an encapsulation efficiency before and after inhalation administration of at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

12. The lipid nanoparticle of claim 1, wherein the encapsulation efficiency of the lipid nanoparticles after inhalation administration is about 20% or less, about 15% or less, about 10% or less, about 5% or less, or about 3% or less, lower than the encapsulation efficiency of the lipid nanoparticles before inhalation administration; and / or the encapsulation efficiency of the lipid nanoparticles after inhalation administration is approximately the same as the encapsulation efficiency of the lipid nanoparticles before inhalation administration.

13. The cationic lipid has the formula (IIA): 【Chemistry 1】 wherein X is O or S; Here, R′ is 【Chemistry 2】 and Here, R ６ teeth, 【Chemistry 3】 and wherein m and p are each independently 0, 1, 2, 3, 4, or 5; Here, R ７ is H, optionally substituted (C １ ~C ６ ) alkyl, optionally substituted (C ２ ~C ６ ) alkenyl, optionally substituted (C ２ ~C ６ ) alkynyl, optionally substituted (C １ ~C ６ ) acyl, -(CH ２ ) ｋ R Ａ Or - (CH ２ ) ｋ CH (OR １１ ) R Ａ Selected from: Here, R ８ is H, optionally substituted (C １ ~C ６ ) alkyl, optionally substituted (C ２ ~C ６ ) alkenyl, optionally substituted (C ２ ~C ６ ) alkynyl, optionally substituted (C １ ~C ６ ) acyl, -(CH ２ ) ｎ R Ｂ Or - (CH ２ ) ｎ CH (OR １２ ) R Ｂ Selected from: Here, R ９ is H, optionally substituted (C １ ~C ６ ) alkyl, optionally substituted (C ２ ~C ６ ) alkenyl, optionally substituted (C ２ ~C ６ ) alkynyl, optionally substituted (C １ ~C ６ ) acyl, -(CH ２ ) ｑ R Ｃ Or - (CH ２ ) ｑ CH (OR １３ ) R Ｃ Selected from: Here, R １０ is H, optionally substituted (C １ ~C ６ ) alkyl, optionally substituted (C ２ ~C ６ ) alkenyl, optionally substituted (C ２ ~C ６ ) alkynyl, optionally substituted (C １ ~C ６ ) acyl, -(CH ２ ) ｒ R Ｄ Or - (CH ２ ) ｒ CH (OR １４ ) R Ｄ Selected from: wherein k, n, q and r are each independently 1, 2, 3, 4 or 5; Or, (i) R ７ and R ８ or (ii) R ９ and R １０ together form an optionally substituted 5- or 6-membered heterocycloalkyl or heteroaryl, the heterocycloalkyl or heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; Here, R １１ , R １２ , R １３ and R １４ are each independently selected from H, methyl, ethyl, or propyl; Here, R Ａ , R Ｂ , R Ｃ and R Ｄ is optionally substituted (C ６ ~C ２０ ) alkyl, optionally substituted (C ６ ~C ２０ ) alkenyl, optionally substituted (C ６ ~C ２０ ) alkynyl, optionally substituted (C ６ ~C ２０ ) acyl, optionally substituted —OC(O) alkyl, optionally substituted —OC(O) alkenyl, optionally substituted (C １ ~C ６ ) monoalkylamino, optionally substituted (C １ ~C ６ )dialkylamino, optionally substituted (C １ ~C ６ ) Alkoxy, -OH, -NH ２ are each independently selected from Here, R ７ , R ８ , R ９ , R １０ At least one of Ａ , R Ｂ , R Ｃ Or R Ｄ Each of these R Ａ , R Ｂ , R Ｃ Or R Ｄ is optionally substituted (C ６ ~C ２０ ) alkyl, optionally substituted (C ６ ~C ２０ ) alkenyl, optionally substituted (C ６ ~C ２０ ) alkynyl, optionally substituted (C ６ ~C ２０ ) acyl, optionally substituted —OC(O)(C ６ ~C ２０ ) alkyl or optionally substituted —OC(O)(C ６ ~C ２０ ) alkenyl) 2. The lipid nanoparticle of claim 1, having a structure according to:

14. The cationic lipid has the formula (IIID): 【Chemistry 4】 wherein X is O or S; Here, R′ is 【Chemistry 5】 and Here, R ６ teeth, 【Chemistry 6】 and wherein m and p are each independently 0, 1, 2, 3, 4, or 5; Here, R ７ is H, optionally substituted (C １ ~C ６ ) alkyl, optionally substituted (C ２ ~C ６ ) alkenyl, optionally substituted (C ２ ~C ６ ) alkynyl, optionally substituted (C １ ~C ６ ) acyl, -(CH ２ ) ｋ R Ａ Or - (CH ２ ) ｋ CH (OR １１ ) R Ａ Selected from: Here, R ８ is H, optionally substituted (C １ ~C ６ ) alkyl, optionally substituted (C ２ ~C ６ ) alkenyl, optionally substituted (C ２ ~C ６ ) alkynyl, optionally substituted (C １ ~C ６ ) acyl, -(CH ２ ) ｎ R Ｂ Or - (CH ２ ) ｎ CH (OR １２ ) R Ｂ Selected from: Here, R ９ is H, optionally substituted (C １ ~C ６ ) alkyl, optionally substituted (C ２ ~C ６ ) alkenyl, optionally substituted (C ２ ~C ６ ) alkynyl, optionally substituted (C １ ~C ６ ) acyl, -(CH ２ ) ｑ R Ｃ Or - (CH ２ ) ｑ CH (OR １３ ) R Ｃ Selected from: Here, R １０ is H, optionally substituted (C １ ~C ６ ) alkyl, optionally substituted (C ２ ~C ６ ) alkenyl, optionally substituted (C ２ ~C ６ ) alkynyl, optionally substituted (C １ ~C ６ ) acyl, -(CH ２ ) ｒ R Ｄ Or - (CH ２ ) ｒ CH (OR １４ ) R Ｄ Selected from: wherein k, n, q and r are each independently 1, 2, 3, 4 or 5; Or, (i) R ７ and R ８ or (ii) R ９ and R １０ together form an optionally substituted 5- or 6-membered heterocycloalkyl or heteroaryl, the heterocycloalkyl or heteroaryl containing 1 to 3 heteroatoms selected from N, O and S; Here, R １１ , R １２ , R １３ and R １４ are each independently selected from H, methyl, ethyl, or propyl; Here, R Ａ , R Ｂ , R Ｃ and R Ｄ is optionally substituted (C ６ ~C ２０ ) alkyl, optionally substituted (C ６ ~C ２０ ) alkenyl, optionally substituted (C ６ ~C ２０ ) alkynyl, optionally substituted (C ６ ~C ２０ ) acyl, optionally substituted —OC(O) alkyl, optionally substituted —OC(O) alkenyl, optionally substituted (C １ ~C ６ ) monoalkylamino, optionally substituted (C １ ~C ６ )dialkylamino, optionally substituted (C １ ~C ６ ) Alkoxy, -OH, -NH ２ are each independently selected from Here, R ７ , R ８ , R ９ , R １０ At least one of Ａ , R Ｂ , R Ｃ Or R Ｄ Each of these R Ａ , R Ｂ , R Ｃ Or R Ｄ is optionally substituted (C ６ ~C ２０ ) alkyl, optionally substituted (C ６ ~C ２０ ) alkenyl, optionally substituted (C ６ ~C ２０ ) alkynyl, optionally substituted (C ６ ~C ２０ ) acyl, optionally substituted —OC(O)(C ６ ~C ２０ ) alkyl or optionally substituted —OC(O)(C ６ ~C ２０ ) alkenyl) 2. The lipid nanoparticle of claim 1, having a structure according to:

15. The cationic lipid has the formula (IIIE): 【Chemistry 7】 (In the formula, Each n is independently 0 or 1; X １Ａ are independently O or NR １Ａ and R １Ａ is H or C １ ~C ６ is alkyl; X １Ｂ is a covalent bond, C(O), CH ２ CO ２ or CH ２ C(O); X ２Ａ and X ２Ｂ one of is O and the other is a covalent bond; X ３Ａ and X ３Ｂ one of is O and the other is a covalent bond; X ４Ａ and X ４Ｂ one of is O and the other is a covalent bond; R １ is independently L １ -B １ , C ６ ~C ３０ Alkyl, C ６ ~C ３０ Alkenyl or C ６ ~C ３０ alkynyl; R ２ is independently L ２ -B ２ , C ６ ~C ３０ Alkyl, C ６ ~C ３０ Alkenyl, C ６ ~C ３０ alkynyl; R ３ is independently L ３ -B ３ , C ６ ~C ３０ Alkyl, C ６ ~C ３０ Alkenyl, C ６ ~C ３０ alkynyl; R ４ is independently L ４ -B ４ , C ６ ~C ３０ Alkyl, C ６ ~C ３０ Alkenyl, C ６ ~C ３０ alkynyl; L １ , L ２ , L ３ , and L ４ are each independently １ ~C ３０ Alkylene; C ２ ~C ３０ alkenylene; or C ２ ~C ３０ alkynylene; B １ , B ２ , B ３ and B ４ each is independently an ionizable nitrogen-containing group; Cationic lipids contain at least one ionizable nitrogen-containing group.

2. The lipid nanoparticle of claim 1, having a structure according to:

16. The cationic lipid has the formula (IIIF): 【Chemistry 8】 (In the formula, B １ is an ionizable nitrogen-containing group; L １ is C １ ~C １０ is alkylene; R ２ , R ３ and R ４ Each of the groups independently represents C ６ ~C ３０ Alkyl, C ６ ~C ３０ Alkenyl, C ６ ~C ３０ alkynyl) 2. The lipid nanoparticle of claim 1, having a structure according to:

17. The cationic lipid has the formula (IIIG): 【Chemistry 9】 (In the formula, B １ is an ionizable nitrogen-containing group; R ２ , R ３ and R ４ Each of the groups independently represents C ６ ~C ３０ Alkyl, C ６ ~C ３０ Alkenyl, C ６ ~C ３０ alkynyl) 2. The lipid nanoparticle of claim 1, having a structure according to:

18. The lipid nanoparticle of claim 1, wherein the non-cationic lipid is a PE lipid or a PC lipid, and / or the non-cationic lipid is selected from DOPE, DLoPE, DMPE, DLPE, DOPC, DEPE, DSPC, DPPC, DMPC, DOPC, 16:1PC, and 14:1PC, and / or the cholesterol or cholesterol analog is cholesterol, and / or the cholesterol analog is selected from β-sitosterol, stigmastanol, campesterol, fucosterol, stigmasterol, and dexamethasone, and / or the PEG-modified lipid is selected from DMG-PEG2K, 2[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, and DSPE-PEG2K-COOH.

19. (i) b. the non-cationic lipid is DOPE; c. The PEG-modified lipid is DMG-PEG2K; d. the cholesterol or cholesterol analog is cholesterol; or b. the non-cationic lipid is DSPC; c. The PEG-modified lipid is DMG-PEG2K; d. the cholesterol or cholesterol analog is cholesterol; or a. the cationic lipid is SY-3-E14-DMAPr; b. the non-cationic lipid is DOPE; c. The PEG-modified lipid is DMG-PEG2K; d. the cholesterol or cholesterol analog is cholesterol; or a. the cationic lipid is SY-3-E14-DMAPr; b. the non-cationic lipid is DSPC; c. The PEG-modified lipid is DMG-PEG2K; d. the cholesterol or cholesterol analog is cholesterol; and / or (ii) The lipid nanoparticle of claim 1, wherein the lipid nanoparticle has a total lipid:mRNA ratio of less than 19:1 (mg:mg), about 19:1 (mg:mg), about less than 17:1 (mg:mg), or the lipid nanoparticle is produced using a total lipid:mRNA ratio of 11:1 to 19:1 (mg:mg).

20. 2. The composition of claim 1, comprising the lipid nanoparticles of claim 1, wherein the composition is formulated for pulmonary delivery by inhalation, optionally wherein the inhalation is performed using a nebulizer comprising vibrating mesh technology (VMT), and / or wherein the composition further comprises one or more excipients, wherein the one or more excipients are selected from a buffer, a salt, a sugar, or a combination thereof.

21. a. mRNA encapsulated in lipid nanoparticles; b. a disaccharide such as trehalose or sucrose, at a concentration of about 3-10% w / v; c. a buffer, optionally a phosphate buffer, and d. salt, optionally including sodium chloride; Where: a. the mRNA is at a concentration of 0.4 to 0.8 mg / ml; b. the trehalose or sucrose is at a concentration of about 4% to 6% w / v; c. the buffer is a phosphate buffer at a concentration of 1 mM to 10 mM (pH 5-5.5); d. the salt is sodium chloride at a concentration of at least 75 mM; or a. The mRNA is at a concentration of about 0.4 mg / ml; b. the disaccharide is sucrose at a concentration of about 4% w / v; c. the buffer is a phosphate buffer at a concentration of about 2.5 mM (pH 5.5); d. The salt is sodium chloride at a concentration of about 150 mM; or a. The mRNA is at a concentration of about 0.4 mg / ml; b. the disaccharide is trehalose at a concentration of about 4% w / v; c. the buffer is a phosphate buffer at a concentration of about 10 mM (pH 5); d. The composition of claim 20, wherein the salt is sodium chloride at a concentration of about 150 mM.

22. 1. A pharmaceutical composition comprising mRNA for use in a method of delivering mRNA encoding a therapeutic protein in vivo, comprising administering the mRNA to a subject via pulmonary delivery, wherein the mRNA is encapsulated within a lipid nanoparticle, wherein the lipid nanoparticle comprises a cationic lipid, a non-cationic lipid, a PEG-modified lipid, and cholesterol, wherein the cationic lipid is greater than 40% (molar ratio) and the non-cationic lipid is less than 25% (molar ratio); the total lipid:mRNA ratio (mg:mg) is 19:1 or less, and wherein the pulmonary delivery is via inhalation, and the composition is nebulized prior to inhalation.

23. 11. A pharmaceutical composition comprising the lipid nanoparticles of claim 1 for use in a method of treating or preventing a disease or disorder in a subject, comprising administering the lipid nanoparticles of claim 1 via inhalation administration, wherein the mRNA encodes a therapeutic protein, and optionally the mRNA encodes cystic fibrosis transmembrane conductance regulator, ATP-binding cassette subfamily A member 3 protein, axonemal dynein intermediate chain 1 (DNAI1) protein, axonemal dynein heavy chain 5 (DNAH5) protein, alpha-1-antitrypsin protein, forkhead box P3 (FOXP3) protein, or one or more surfactant proteins.

24. The disease or disorder is: (i) a pulmonary disease or disorder, e.g., a chronic respiratory disease; (ii) a protein deficiency, e.g. a protein deficiency affecting the lung; (iii) neoplastic diseases, e.g., tumors, and (iv) Infectious diseases 24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is selected from:

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