Ionizable lipids and nanoparticles including same

Abstract

In some embodiments, one or more ionizable lipids and lipid nanoparticles are provided, the lipid nanoparticles including the ionizable lipids and further encapsulating a polynucleic acid. Also provided are pharmaceutical compositions comprising a therapeutically effective amount of the lipid nanoparticles encapsulating a therapeutically active polynucleic acid. Also provided are pharmaceutical compositions for delivering the polynucleic acids to lung tissue of a subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 437,810, filed on January 9, 2023, entitled “IONIZABLE LIPIDS AND NANOPARTICLES COMPRISING SAME,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] The present invention relates to ionizable lipids and lipid nanoparticles comprising the same and their use in pharmaceutical compositions. Background Art

[0003] New delivery methods for therapeutic and diagnostic compounds are constantly being developed. While lipid-based nanoparticles are a well-known delivery method, these agents are also undergoing continuous improvement. Among other concerns, the ability of therapeutic carriers to effectively load active agents and subsequently deliver them to the target site is crucial for reducing dosing and improving therapeutic efficiency.

[0004] Although various ionizable lipids are known to be capable of encapsulating hydrophilic agents (such as DNA and / or RNA), there is a continuing need for new and superior lipid nanoparticles (LNPs). In particular, there is a great need to develop new and superior LNPs that can enhance drug delivery to specific locations in the body. In particular, there is an unmet need for lipid nanoparticles characterized by enhanced lung specificity and suitable for the administration of drugs specific to the lung. Summary of the Invention

[0005] The present invention provides novel compounds suitable for use as ionizable lipids. In addition, nanoparticles comprising the same are provided. Compositions comprising the nanoparticles are also provided for delivering an active agent to a subject, such as for treating or preventing a disease or disorder in the subject.

[0006] According to one aspect, the lipid nanoparticle comprises a compound, a salt of the compound, or both, wherein the compound is represented by the following Formula 1: , wherein: each X independently represents -O-, -S-, CH2, or X is absent; each Z independently represents -OH or -SH; each A independently represents O or S; each n is independently between 0 and 5, and at least one n is not 0; each R is independently H, or comprises an optionally substituted C5-C30 alkyl; wherein the lipid nanoparticle further comprises an active agent, a helper lipid, a structural lipid, and a modifying lipid, wherein: the helper lipid is a cationic lipid; the proportion of the compound relative to the total lipid content of the lipid nanoparticle is between 10 mol% and 50 mol%; and the proportion of the structural lipid relative to the total lipid content of the lipid nanoparticle is between 5 mol% and 50 mol%; and wherein the lipid nanoparticle is characterized by an average zeta potential in the range of between -5 mV and +40 mV at a pH of 6 to 8.

[0007] In one embodiment, the molar ratio of the helper lipid to the modifying lipid is between 1:0.2 and 1:0.01.

[0008] In one embodiment, the molar ratio of the compound to the helper lipid is between 0.2:1 and 5:1.

[0009] In one embodiment, the molar ratio of the structural lipid to the modified lipid is between 1:0.01 and 1:0.2.

[0010] In one embodiment, the active agent comprises a polynucleic acid; and the ratio between N:P within the LNP is between 1 and 120.

[0011] In one embodiment, the weight ratio between the total amount of (i) the compound, the helper lipid, the structural lipid and the modified lipid and (ii) the polynucleic acid in the lipid nanoparticle is between 0.001:1 and 10:1.

[0012] In one embodiment, the lipid nanoparticles have a size distribution between 50 nm and 500 nm; and wherein the cationic lipid is selected from DOTAP, DDAB, 18:1 EPC (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) and 18:0 EPC (1,2-distearoyl-sn-glycero-3-ethylphosphocholine), including any salts and any combinations thereof.

[0013] In one embodiment, the lipid nanoparticles are characterized by any of the following: an average zeta potential between 0 mV and +5 mV at a pH of 6 to 8; an N:P ratio between 3 and 9; the structural lipid is cholesterol; the modifying lipid is a PEG-lipid; the proportion of the helper lipid relative to the total lipid content of the lipid nanoparticle is between 35 mol% and 45 mol%; and the proportion of the PEG-lipid relative to the total lipid content of the lipid nanoparticle is between 0.1 mol% and 3 mol%.

[0014] In one embodiment, the lipid nanoparticle is characterized by a pKa between 5 and 9; and wherein the compound comprises any one of MB-212 and MB 222: .

[0015] In one embodiment, upon administration to a subject, the LNP is characterized by at least 10-fold higher expression in the lung compared to any of the liver, spleen, and kidney of the subject.

[0016] In one embodiment, the ratio between N:P is about 5; wherein the cationic lipid is DOTAP; and wherein the ratio of the compound relative to the total lipid content of the lipid nanoparticle is between about 15 mol% and about 25 mol%.

[0017] In another aspect, a pharmaceutical composition is provided, comprising a plurality of lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0018] In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the active agent.

[0019] In another aspect, a method for delivering an active agent to lung tissue of a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention, thereby delivering the active agent to the lung tissue.

[0020] In one embodiment, the therapeutically effective amount is between 0.01 mg / kg / day and 5 mg / kg / day.

[0021] The method of the present invention, wherein said administration is via intravenous, intratracheal, intranasal administration or via inhalation.

[0022] According to another aspect, a lipid nanoparticle is provided, comprising a compound, a salt of the compound, or both, wherein the compound is represented by the following Formula I: , wherein: each L is independently R1, or ; Each L1 is independently R1, 、 ;or , represents a single, triple, or double bond; Z independently represents -OH or -SH; A independently represents O or S; Each k is independently between 0 and 10; As long as valence permits, each Y is independently absent or comprises CH2, CHR'2, NR'2, NH, O, S, -CONH-, -CONR'-, -C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-, or phosphate; Each T independently represents an optionally substituted C5-C30 alkyl group or an optionally substituted C5-C30 alkenyl group; each R' is independently H or includes optionally substituted C1-C10 alkyl, C1-C10 alkyl-aryl, C1-C10 alkyl-cycloalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; Each X independently represents a heteroatom, CH2, optionally substituted C1-C10 alkyl, or X is absent; Each of n and p is independently between 0 and 5, and at least one of n is not 0; m is between 1 and 3; Each R is independently H, or includes optionally substituted C5-C30 alkyl; Each R1 is an optionally substituted C1-C24 alkyl group, and at least one L or L1 is or includes ; The lipid nanoparticles further comprise an active agent, an auxiliary lipid, a structural lipid, and a modified lipid: wherein the ratio of the compound relative to the total lipid content of the lipid nanoparticles is between 15 mol% and 55 mol%; wherein the ratio of the structural lipid relative to the total lipid content of the lipid nanoparticles is between 20 mol% and 60 mol%.

[0023] In some embodiments, the molar ratio of the helper lipid to the modifying lipid ranges between 1:0.2 and 1:0.01.

[0024] In some embodiments, the molar ratio of the compound to the helper lipid ranges between 0.5:1 and 5:1.

[0025] In some embodiments, the molar ratio of the structural lipid to the modified lipid ranges between 1:0.01 and 1:0.2.

[0026] In some embodiments, the active agent comprises a polynucleic acid.

[0027] In some embodiments, the weight ratio between the total amount of (i) the compound, the helper lipid, the structural lipid, and the modified lipid and (ii) the polynucleic acid in the lipid nanoparticle is between 0.001:1 and 10:1.

[0028] In some embodiments, the size distribution of the lipid nanoparticles ranges between 50 nm and 500 nm.

[0029] In some embodiments, the lipid nanoparticles are characterized by a zeta potential ranging between -5 mV and 40 mV at a pH of 6 to 8.

[0030] In some embodiments, the lipid nanoparticles are characterized by a pKa between 5 and 9.

[0031] In some embodiments, the ratio of N:P within the lipid nanoparticles ranges between 3 and 20.

[0032] In some embodiments, the ratio between N:P is about 12.

[0033] In another aspect, a pharmaceutical composition is provided, comprising a plurality of lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0034] In some embodiments, the pharmaceutical composition includes a therapeutically effective amount of the active agent.

[0035] In some embodiments, the pharmaceutical composition is used to deliver the active agent to lung tissue.

[0036] In some embodiments, the pharmaceutical composition is used to treat a lung disease or lung disorder.

[0037] In another aspect, a method for delivering an active agent to lung tissue of a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention, thereby delivering the active agent to the lung tissue.

[0038] In some embodiments, the therapeutically effective amount is between 0.01 mg / kg / day and 1 mg / kg / day. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Some embodiments of the present invention are described herein by way of example only, with reference to the accompanying drawings. With specific reference now to the accompanying drawings in detail, it is emphasized that these details are shown by way of example only and are intended to be illustrative of various embodiments of the present invention. In this regard, the description taken in conjunction with the accompanying drawings will make apparent to those skilled in the art how to practice various embodiments of the present invention.

[0040] Figure 1 FIG2 is a bar graph showing in vivo expression analysis of three LNP compositions, namely FMB-1143, FMB-748 and FMB-745. The bar graph shows the fluorescence signal intensity in lung tissue compared with heart tissue, spleen tissue, kidney tissue and liver tissue. DETAILED DESCRIPTION

[0041] The present invention is based, at least in part, on the surprising discovery that specific lipid nanoparticles (LNPs) of the present invention exhibit lung-specific activity in vivo. Thus, the LNPs of the present invention can be used to specifically deliver active agents to the lung tissue of a subject in need thereof.

[0042] The compounds disclosed herein were discovered using computational screening methods. Numerous LNP compositions were generated computer-generated using a machine learning algorithm and ranked according to their predicted activity. After several cycles of computer optimization, a library containing several LNP compositions was obtained. RNA was encapsulated into the LNP compositions and then tested for RNA activity in vivo. The disclosed LNP compositions were selected based on the results obtained from in vivo experiments, as exemplified below.

[0043] lipid nanoparticles In one aspect of the present invention, lipid nanoparticles (LNPs) are provided, comprising (i) a compound, including any salt thereof; (ii) a helper lipid, (iii) a structural lipid, and (iv) a modified lipid; wherein the molar ratio of the compound relative to the total lipid content of the composition is between about 15 mol% and 55 mol%, or between about 10 mol% and 55 mol%, and wherein the compound is represented by the following Formula I: , wherein: each L is independently R1, or ; Each L1 is independently R1, 、 ;or , represents a single bond, a triple bond or a double bond; Z independently represents -OH or -SH; A independently represents O or S; each k is independently between 0 and 10; as long as valence permits, each Y independently does not exist or includes CH2, CHR'2, NR'2, NH, O, S, -CONH-, -CONR'-, C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N- or a phosphate group; each T independently represents an optionally substituted C5-C30 alkyl group or an optionally substituted substituted C5-C30 alkenyl; each R' is independently H or includes optionally substituted C1-C10 alkyl, C1-C10 alkyl-aryl, C1-C10 alkyl-cycloalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; each X independently represents a heteroatom, CH2, optionally substituted C1-C10 alkyl, or X is absent; each n and p are independently between 0 and 5, and at least one n is not 0; m is between 1 and 3; each R is independently H, or includes optionally substituted C1-C30 alkyl, or C5-C30 alkyl; each R1 is optionally substituted C1-C24 alkyl, and at least one L or L1 is or includes .

[0044] In some embodiments, a composition comprising a plurality of LNPs is provided, wherein each LNP comprises (i) a compound, including any salt thereof; (ii) a helper lipid, (iii) a structural lipid, and (iv) a modifying lipid; wherein the molar ratio of the compound relative to the total lipid content of the composition is between about 105 mol % and 55 mol %, and wherein the compound is represented by the following Formula I: , wherein: each L is independently R1, or; ; Each L1 is independently R1, 、 ;or , represents a single bond, a triple bond or a double bond; Z independently represents -OH or -SH; A independently represents O or S; each k is independently between 0 and 10; as long as valence permits, each Y independently does not exist or includes CH2, CHR'2, NR'2, NH, O, S, -CONH-, -CONR'-, C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N- or a phosphate group; each T independently represents an optionally substituted C5-C30 alkyl group or an optionally substituted substituted C5-C30 alkenyl; each R' is independently H or includes optionally substituted C1-C10 alkyl, C1-C10 alkyl-aryl, C1-C10 alkyl-cycloalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; each X independently represents a heteroatom, CH2, optionally substituted C1-C10 alkyl, or X is absent; each n and p are independently between 0 and 5, and at least one n is not 0; m is between 1 and 3; each R is independently H, or includes optionally substituted C5-C30 alkyl, or C1-C30 alkyl; each R1 is optionally substituted C1-C24 alkyl, and at least one L or L1 is or includes .

[0045] In some embodiments, the composition of the present invention is an LNP composition (e.g., a solid composition comprising dried LNPs, or a liquid dispersion comprising LNPs dispersed or suspended in a solvent, such as an aqueous solvent), comprising (i) a compound, including any salt thereof; (ii) a helper lipid, (iii) a structural lipid, and (iv) a modified lipid; wherein the molar ratio of the compound relative to the total lipid content of the composition is between about 15 mol% and 55 mol%, or between about 10 mol% and 55 mol%, and wherein the compound is represented by the following Formula I: , wherein: each L is independently R1, or ; Each L1 is independently R1, 、 ;or , represents a single bond, a triple bond or a double bond; Z independently represents -OH or -SH; A independently represents O or S; each k is independently between 0 and 10; as long as valence permits, each Y independently does not exist or includes CH2, CHR'2, NR'2, NH, O, S, -CONH-, -CONR'-, C(=NH)NR'-, -C(=S)NR'-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N- or a phosphate group; each T independently represents an optionally substituted C5-C30 alkyl group or an optionally substituted C5-C30 alkenyl; each R' is independently H or includes optionally substituted C1-C10 alkyl, C1-C10 alkyl-aryl, C1-C10 alkyl-cycloalkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; each X independently represents a heteroatom, CH2, optionally substituted C1-C10 alkyl, or X is absent; each n and p is independently between 0 and 5, and at least one n is not 0; m is between 1 and 3; each R is independently H, or includes optionally substituted C5-C30 alkyl or C1-C30 alkyl; and wherein each R1 is optionally substituted C1-C24 alkyl, and at least one L or L1 is or includes .

[0046] In some embodiments, the compound is represented by the following Formula II: , where each of L and L1 is wherein X, Z, A, and R are as described above, and wherein each n is independently between 0 and 5, and at least one n is not 0. In some embodiments, the compound is represented by Formula II, wherein each X is selected from -O-, -S-, and CH2; and wherein at least two R represent optionally substituted C1-C30 alkyl.

[0047] In some embodiments, the compound is represented by Formula II or by Formula I, wherein the total number of C atoms in the R group is between 5 and 50, between 10 and 50, between 20 and 40, between 20 and 35, between 10 and 35, including any ranges therebetween.

[0048] In some embodiments, the compound is represented by Formula II or by Formula I, wherein at least one R represents an optionally substituted alkyl group of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, 5 to 10 carbon atoms in length, including any range therebetween.

[0049] In some embodiments, the compound is represented by Formula II or by Formula I, wherein at least two R represent an optionally substituted alkyl group of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, 5 to 10 carbon atoms in length, including any range therebetween.

[0050] In some embodiments, the compound is represented by Formula II or by Formula I, wherein each R represents an optionally substituted alkyl group of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, 5 to 10 carbon atoms in length, including any range therebetween.

[0051] In some embodiments, the compound is represented by the following Formula 1: , wherein Z, A, R, and n are as described above; wherein each X independently represents -O-, -S-, methylene, or X is absent; wherein at least one n is not 0. In some embodiments, the compound is represented by Formula 1, wherein at least one X is absent, and wherein at least one R is not H. In some embodiments, the compound is represented by Formula 1, wherein at least two Rs are not H.

[0052] In some embodiments, the compound is represented by Formula 1, wherein the total number of C atoms in the R group is between 10 and 60, between 5 and 50, between 5 and 60, between 510 and 40, between 20 and 40, between 20 and 35, between 10 and 35, including any range therebetween. In some embodiments, the compound is represented by Formula 1, wherein at least one, or at least two R groups represent an optionally substituted alkyl group of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, 5 to 10 carbon atoms in length, including any range therebetween.

[0053] In some embodiments, the compound is represented by Formula 1, wherein each A is -O- and each Z is -OH; and wherein each R is an optionally substituted alkyl group of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, 5 to 10 carbon atoms in length, including any range therebetween.

[0054] In some embodiments, the compound is represented by the following Formula 2: , wherein X is as described above, and each n is between 1 and 5, wherein m is between 0 and 3, and each R is an optionally substituted alkyl group of 1 to 30, 5 to 30, 5 to 20, 1 to 20, 1 to 10, or 5 to 10 carbon atoms in length, including any range therebetween. In some embodiments, the compound is represented by Formula 2, wherein each R is an optionally substituted C5-C10 alkyl group. In some embodiments, the compound includes either or both of MB-212 and MB 222: .

[0055] In some embodiments, each LNP comprises (i) a compound represented by Formula 1 or Formula 2, including any salt thereof; (ii) a helper lipid, (iii) a structural lipid, and (iv) a modifying lipid; wherein the helper lipid is a cationic lipid; wherein the structural lipid is a sterol; wherein the modifying lipid is a PEG lipid; and wherein the plurality of LNPs are characterized by a positive or neutral average zeta potential; and wherein each LNP encapsulates an active agent, the active agent comprising a polynucleic acid.

[0056] In some embodiments, the molar concentration of one or more compounds of the invention within the LNP is between 10 mol% and 55 mol%, between 10 mol% and 25 mol%, between 10 mol% and 20 mol%, between 20 mol% and 25 mol%, between 15 mol% and 30 mol%, between 20 mol% and 35 mol%, between 30 mol% and 40 mol%, between 30 mol% and 35 mol%, between 20 mol% and 55 mol%, including any range therebetween. As used herein, the term "concentration" or "molar concentration" refers to the molar ratio relative to the total lipid content of the nanoparticle. It will be understood by those skilled in the art that the molar ratios of the essential components (i.e., the compounds of the invention, helper lipids, structural lipids, and modified lipids) within the LNP and within the compositions of the invention are the same. Thus, for example, the molar concentrations and molar ratios disclosed herein with respect to the LNP also encompass the corresponding molar concentrations and molar ratios within the compositions of the invention, and vice versa.

[0057] In some embodiments, the total lipid content refers to the combined content of the compound of the invention and structural lipids, modifying lipids, and helper lipids.

[0058] In some embodiments, the molar ratio of the structural lipid relative to the total lipid content of the composition is between 5 mol% and 50 mol%, including any range or value therebetween. In some embodiments, the molar ratio of the compound (represented by any of Formulas 1-2) relative to the total lipid content of the lipid nanoparticle is between 10 mol% and 55 mol%.

[0059] In some embodiments, the molar ratio of the compound of the present invention relative to the total lipid content of the composition is between 10 mol% and 55 mol%, between 15 mol% and 25 mol%, between 15 mol% and 20 mol%, between 20 mol% and 25 mol%, between 15 mol% and 30 mol%, between 20 mol% and 35 mol%, between 30 mol% and 40 mol%, between 30 mol% and 35 mol%, between 20 mol% and 55 mol%, between 20 mol% and 55 mol%, including any range therebetween. In some embodiments, the molar ratio of the compound of the present invention relative to the total lipid content of the composition is between about 20 mol% and about 40 mol%.

[0060] In some embodiments, the molar concentration of the helper lipid relative to the total lipid content of the composition is between 5 mol% and about 60 mol%, between 5 mol% and 10 mol%, between 5 mol% and 15 mol%, between 10 mol% and 40 mol%, between 10 mol% and 30 mol%, between 30 mol% and 40 mol%, between 40 mol% and 45 mol%, between 30 mol% and 55 mol%, between 30 mol% and 60 mol%, including any range therebetween.

[0061] In some embodiments, the molar concentration of the helper lipid within the LNP is between about 35 mol% and about 45 mol%, between about 38 mol% and about 45 mol%, between about 38 mol% and about 42 mol%, or about 40 mol%, including any range therebetween.

[0062] In some embodiments, the molar concentration of the structured lipid relative to the total lipid content of the composition is between 5 mol% and 50 mol%, between 15 mol% and 60 mol%, between 15 mol% and 50 mol%, between 20 mol% and 25 mol%, between 20 mol% and 30 mol%, between 20 mol% and 40 mol%, between 25 mol% and 45 mol%, between 35 mol% and 40 mol%, between 35 mol% and 45 mol%, between 35 mol% and 50 mol%, between 40 mol% and 50 mol%, between 45 mol% and 50 mol%, between 35 mol% and 50 mol%, including any range or value therebetween.

[0063] In some embodiments, the molar concentration of structural lipids within the LNP is between 20 mol% and 40 mol%, between 20 mol% and 23 mol%, between 20 mol% and 25 mol%, between 35 mol% and 40 mol%, between 37 mol% and 40 mol%, between 25 mol% and 35 mol%, including any range therebetween.

[0064] In some embodiments, the molar concentration of the modified lipid (e.g., PEG-lipid) within the LNP is between 0.5 mol% and 10 mol%, between 0.1 mol% and 10 mol%, between 0.1 mol% and 0.5 mol%, between 0.5 mol% and 1 mol%, between 1 mol% and 5 mol%, between 0.5 mol% and 2 mol%, between 5 mol% and 10 mol%, between 5 mol% and 7 mol%, between 7 mol% and 10 mol%, including any range therebetween.

[0065] In some embodiments, the LNP or composition comprises a molar concentration of: (i) between about 20 mol% and about 40 mol% of a structural lipid, and (ii) between about 1.5 mol% and about 2.5 mol% of a modifying lipid, and (iii) between about 35 mol% and about 40 mol% of a helper lipid, and (iv) between about 20 mol% and 35 mol% of a compound of the invention.

[0066] In some embodiments, the weight ratio between the compound within the LNP / composition and the polynucleic acid is between 0.001:1 and 10:1, between 0.001:1 and 0.1:1, between 0.1:1 and 1:1, between 1:1 and 10:1, including any range therebetween.

[0067] In some embodiments, the N:P ratio within the LNP or within the composition of the invention ranges from 1 to 20, from 3 to 6, from 3 to 9, from 6 to 8, from 6 to 8, from 8 to 10, from 10 to 12, from 6 to 12, from 6 to 13, from 10 to 20, from 8 to 20, from 8 to 15, and from 4 to 5, including any ranges therebetween. The term "N:P ratio" refers to the ratio between N atoms of the compound of the invention and P atoms of the polynucleotide within the lipid nanoparticle or within the composition of the invention.

[0068] In some embodiments, the N:P ratio is about 5, or is selected from 3, 4, 5, and 6, or is between 3 and 9, including any value or range therebetween.

[0069] In some embodiments, LNP is mainly composed of the compound of the present invention, helper lipids, structural lipids and modified lipids. In some embodiments, LNP further encapsulates an active agent. In some embodiments, LNP includes a shell and an aqueous core, and the aqueous core includes an active agent. In some embodiments, the shell of LNP includes a compound of the present invention, helper lipids, structural lipids and modified lipids. In some embodiments, the active agent is incorporated into the shell, incorporated into the aqueous core or incorporated into the interface between the shell and the core. As used herein, the term "shell" refers to the outside of the particle, and its composition is different from the core.

[0070] In some embodiments, the LNP consists essentially of a compound of Formula I, a cationic helper lipid, a sterol, and a PEG-lipid; wherein the LNP further encapsulates an active agent; the molar ratio of the compound relative to the total lipid content of the composition is between about 15 mol% and 40 mol%; the molar ratio of the cationic helper lipid relative to the total lipid content of the composition is between about 30 mol% and 50 mol%, and wherein the LNP within the composition is characterized by an average zeta potential in the range of between -5 mV and +40 mV when measured at a pH of 6 to 8 (e.g., about 7).

[0071] In some embodiments, the combined molar concentration of the helper lipid and the compound of the invention within the LNP is between about 40 mol% and about 75 mol%, or between about 60 mol% and about 75 mol%; wherein the ratio between N:P is between 3 and 9; wherein the average particle size of the LNPs within the composition is between about 50 nm and 300 nm; and wherein the LNPs within the composition are characterized by an average zeta potential in the range of between -5 mV and +40 mV, or between 0 mV and +10 mV, when measured at a pH of 6 to 8 (e.g., about 7).

[0072] In some embodiments, the compound of the invention is a compound of Formula 1 or Formula 2; the combined molar concentration of the helper lipid and the compound of the invention within the LNP is between about 40 mol% and about 75 mol% or between about 60 mol% and about 75 mol%; the ratio of the helper lipid relative to the total lipid content of the lipid nanoparticle is between 35 mol% and 45 mol%; the ratio of the PEG-lipid relative to the total lipid content of the lipid nanoparticle is between 1 mol% and 5 mol%; wherein the N:P ratio is between 8 and 12; wherein the average particle size of the LNPs within the composition is between about 50 nm and 300 nm; and wherein the LNPs within the composition are characterized by an average zeta potential in the range of between -1 mV and +5 mV when measured at a pH of 6 to 8 (e.g., about 7).

[0073] In some embodiments, the compound of the invention is a compound of Formula 1 or Formula 2; the combined molar concentration of the helper lipid and the compound of the invention within the LNP is between about 40 mol% and about 75 mol%; the ratio of the helper lipid relative to the total lipid content of the lipid nanoparticle is between 35 mol% and 45 mol%; the ratio of the PEG-lipid relative to the total lipid content of the lipid nanoparticle is between 1 mol% and 5 mol%; wherein the ratio between N:P is about 12; wherein the average particle size of the LNPs in the composition is between about 50 nm and 300 nm; and wherein the LNPs in the composition are characterized by an average zeta potential in the range of between 0 mV and +5 mV when measured at a pH of 6 to 8 (e.g., about 7).

[0074] In some embodiments, the compound of the invention is a compound of Formula 1 or Formula 2; wherein the helper lipid is DOTAP; the combined molar concentration of the helper lipid and the compound of the invention within the LNP is between about 40 mol% and about 75 mol%; the ratio of the helper lipid relative to the total lipid content of the lipid nanoparticle is between 30 mol% and 50 mol%; the ratio of the compound relative to the total lipid content of the lipid nanoparticle is between about 10 mol% and about 30 mol%; the ratio of PEG-lipid relative to the total lipid content of the lipid nanoparticle is between 0.1 mol% and 5 mol%; wherein the ratio between N:P is between about 4 and about 5; wherein the average particle size of the LNPs within the composition is between about 50 nm and 300 nm; and wherein the LNPs within the composition are characterized by an average zeta potential in the range of between 0 mV and +5 mV when measured at a pH between 6 and 8 (e.g., about 7).

[0075] In some embodiments, under suitable conditions, at least one compound of the present invention, active agent, helper lipid, structural lipid and modified lipid spontaneously undergo self-assembly in aqueous solution to form LNP. In some embodiments, lipid nanoparticles are formulated to deliver one or more agents to one or more target cells.

[0076] In some embodiments, LNP of the present invention has spherical geometry or shape.In some embodiments, LNP has expansion or deflated shape.In some embodiments, multiple core-shell particles lack any characteristic geometry or shape.In some embodiments, LNP has spherical shape, quasi-spherical shape, quasi-elliptical shape, deflated shape, concave shape, irregular shape or its any combination.

[0077] In some embodiments, the multiple core-shell particles in the composition of the present invention are substantially spherical, wherein substantially as described herein. In some embodiments, the multiple core-shell particles are substantially elliptical, wherein substantially as described herein. It will be appreciated by those skilled in the art that the exact shape of each of the multiple core-shell particles can be different because of the particle. In addition, the exact shape of the LNP can be drawn from any of the geometric forms listed above, so the shape of the particle is not fully suitable for specific geometric form. It will be appreciated by those skilled in the art that the exact shape of the LNP can have considerable deviation (such as at least 5%, at least 10%, at least 20% deviation) from specific geometric shape (e.g., sphere or ellipse).

[0078] In some embodiments, the proportion of the compound relative to the total lipid content of the LNP of the present invention is between 10% and 55%, between 15% and 25%, between 25% and 30%, between 25% and 35%, between 30% and 35%, between 35% and 45%, and between 45% and 55%, including any ranges therebetween.

[0079] In some embodiments, the ratio between the structural lipids relative to the total lipid content of the LNP of the present invention is between 5% and 50%, between 20% and 30%, between 25% and 30%, between 25% and 35%, between 30% and 35%, between 35% and 45%, between 45% and 50%, between 35% and 50%, including any range therebetween.

[0080] In some embodiments, the molar ratio of the helper lipid to the modifying lipid is between 1:0.2 and 1:0.01, between 1:0.15 and 1:0.01, between 1:0.1 and 1:0.01, between 1:0.05 and 1:0.01, including any range therebetween.

[0081] In some embodiments, the molar ratio of the compound to the helper lipid ranges from 1:0.5 to 1:5, from 1:0.5 to 4:1, from 1:0.5 to 3:1, from 1:0.5 to 2:1, from 1:0.5 to 1:1, from 1:0.1 to 5:1, from 1:0.25 to 5:1, from 1:0.5 to 2:1, from 1:1 to 1:2, from 1:1 to 1:5, including any ranges therebetween.

[0082] In some embodiments, the molar ratio of the structural lipid to the modifying lipid ranges from 200:1 to 2:1, from 100:1 to 5:1, from 100:1 to 10:1, from 100:1 to 30:1, from 100:1 to 50:1, from 100:1 to 70:1, from 100:1 to 90:1, from 100:1 to 100:3, from 100:1 to 20:1, including any ranges therebetween.

[0083] Lipid nanoparticle compositions In some embodiments, the compounds of the present invention are represented by Formula I as described above. In some embodiments, the compounds of the present invention are ionizable lipids. In some embodiments, the terms "compound," "compound of the present invention," and "ionizable lipid" are used interchangeably herein. In some embodiments, heteroatoms include O, N, NH, NR1, or S. In some embodiments, each X is independently O, or absent.

[0084] In some embodiments, one of R and R1 each independently represents a linear or branched alkyl group.

[0085] In some embodiments, L is , wherein R is as described herein. In some embodiments, each R represents the same or different alkyl groups.

[0086] In some embodiments, L is , wherein R is as described herein. In some embodiments, each R represents the same or different alkyl groups.

[0087] In some embodiments, L is , wherein R is as described herein. In some embodiments, each R represents the same or different alkyl groups.

[0088] In some embodiments, L is , wherein R is as described herein. In some embodiments, each R represents the same or different alkyl groups.

[0089] As used herein, the term "(C3-C 10 )cycloalkyl" refers to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or C10 ring. In some embodiments, (C3-C 10 ) ring includes optionally substituted cyclopropane, cyclobutene, cyclopentane, cyclohexane or cycloheptane.

[0090] As used herein, the term "C3-C 10"Heterocyclyl" refers to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or C10 heterocyclic aromatic and / or aliphatic ring, or an unsaturated ring.

[0091] As used herein, the term "alkyl" describes aliphatic hydrocarbons including straight and branched chain groups. In some embodiments, the alkyl group has 1 to 10 carbon atoms, 1 to 30 carbon atoms, or 5 to 30 carbon atoms. Whenever a numerical range is stated herein (e.g., "5-30"), it means that the group (in this case, the alkyl group) may contain 5 carbon atoms, 6 carbon atoms, 10 carbon atoms, 5 to 20 carbon atoms, 5 to 25 carbon atoms, 5 to 30 carbon atoms, 10 to 20 carbon atoms, 10 to 25 carbon atoms, 10 to 30 carbon atoms, including any range between, up to, and including 30 carbon atoms. As defined herein, an alkyl group may be substituted or unsubstituted.

[0092] As used herein, the term "alkyl" also includes saturated or unsaturated hydrocarbons, and thus the term also includes alkenyl and alkynyl. In some embodiments, the alkyl group is a C1-C10 alkyl group.

[0093] As used herein, the term "C1-C10 alkyl" (including any compounds related to C1-C10 alkyl) refers to any linear or branched alkyl chain comprising 1 to 6, 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10 carbon atoms, including any range therebetween. In some embodiments, the C1-C10 alkyl group includes any of the following: methyl, ethyl, propyl, butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, and tert-butyl, or any combination thereof. In some embodiments, the C1-C10 alkyl group as described herein further includes an unsaturated bond, wherein the unsaturated bond is located at position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the C1-C10 alkyl group.

[0094] The term "alkenyl" describes an unsaturated alkyl group as defined herein having from 2 to 30 carbon atoms and at least one carbon-carbon double bond. As noted above, an alkenyl group may be unsubstituted or substituted with one or more substituents.

[0095] As defined herein, the term "alkynyl" is an unsaturated alkyl group having from 2 to 30 carbon atoms and at least one carbon-carbon triple bond. As described above, an alkynyl group may be substituted or unsubstituted with one or more substituents.

[0096] In some embodiments, the ionizable lipid is capable of undergoing ionization (protonation or positive ionization) in a solution at a pH below the pKa value of the ionizable lipid. In some embodiments, the ionizable lipid is capable of undergoing protonation in a solution at a pH below the pKa value of the ionizable lipid. In some embodiments, at least 50 mol% of the ionizable moieties in a composition of the invention at a pH below the pKa value of the ionizable lipid are positively charged (or protonated).

[0097] In some embodiments, the MW of the compounds of the invention is between 100 Da and 2000 Da, between 100 Da and 300 Da, between 100 Da and 500 Da, between 100 Da and 800 Da, between 300 Da and 500 Da, between 100 Da and 1,000 Da, between 500 Da and 800 Da, between 500 Da and 1,000 Da, between 800 Da and 1,200 Da, between 1000 Da and 2,000 Da, including any ranges therebetween.

[0098] In some embodiments, the compounds of the present invention comprise substantially a single enantiomer of any one of the compounds described herein, wherein substantially is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 95%, at least 97%, at least 98%, at least 99%, or any value therebetween.

[0099] In some embodiments, the compounds of the present invention also include any structurally similar functional derivatives of the compounds disclosed herein, wherein structural similarity is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% structural similarity, including any ranges therebetween.

[0100] In some embodiments, the term "structural similarity" refers to fingerprint similarity between two molecules. The term "fingerprint similarity" is well known to those skilled in the art. In some embodiments, fingerprint similarity is calculated based on circular fingerprints, substructure keys-based fingerprints, and / or topological or path-based fingerprints.

[0101] Exemplary circular fingerprints include, but are not limited to, Molprint 2D, ECFP (or Morgan fingerprint), FCFP, etc. In some embodiments, the term "structural similarity" as used herein is calculated by Morgan fingerprint.

[0102] In some embodiments, the functional derivative is an ionizable lipid having a pKa value between 6.2 and 6.8 and capable of undergoing self-assembly in water to stably bind and / or encapsulate a polynucleic acid. In some embodiments, the functional derivative is further configured to internalize the polynucleic acid into a cell (e.g., by forming lipid nanoparticles as described herein). Cellular internalization can be determined as described below.

[0103] In some embodiments, the compound is MB-212 or any salt thereof, as described below.

[0104] In some embodiments, the compound is MB-222 or any salt thereof, as described below.

[0105] In some embodiments, the active agent is a small molecule and / or a biomolecule, such as a polypeptide, a polynucleotide, etc. In some embodiments, the active agent is selected from a therapeutic agent, a prophylactic agent, and a diagnostic agent, including any combination thereof. In some embodiments, the one or more active agents are selected from a protein, a peptide, a nucleic acid, a small molecule, and an antibody.

[0106] In some embodiments, the active agent is a lung therapeutic agent. In some embodiments, the active agent is an anticancer drug. In some embodiments, the active agent is an immunotherapy. In some embodiments, the active agent is an anti-infective agent. In some embodiments, the active agent is an anti-inflammatory agent.

[0107] In some embodiments, the active agent is a polynucleic acid.

[0108] In some embodiments, the terms "polynucleic acid" and "polynucleotide" are used interchangeably herein. In some embodiments, a polynucleotide comprises 60 to 15,000 nucleobases, 15,000 to 10,000 nucleobases, 10,000 to 4,700 nucleobases, 200 to 5,000 nucleobases, 300 to 5,000 nucleobases, 400 to 5,000 nucleobases, 400 to 2,500 nucleobases, 200 to 3,000 nucleobases, 400 to 2,000 nucleobases, 400 to 1,000 nucleobases, including any range therebetween.

[0109] In some embodiments, the polynucleotide comprises at least 10 nucleobases, at least 250 nucleobases, at least 300 nucleobases, at least 350 nucleobases, at least 400 nucleobases, at least 450 nucleobases, at least 475 nucleobases, or at least 500 nucleobases. Each possibility represents a separate embodiment of the invention.

[0110] In some embodiments, the polynucleotide comprises at most 500 nucleobases, at most 750 nucleobases, at most 1,000 nucleobases, at most 1,250 nucleobases, at most 1,750 nucleobases, at most 2,500 nucleobases, at most 3,000 nucleobases, at most 4000 nucleobases, or at most 5000 nucleobases. Each possibility represents a separate embodiment of the invention.

[0111] In some embodiments, polynucleotides include multiple polynucleotide types. In some embodiments, LNPs include multiple polynucleotide types. In some embodiments, compositions include multiple nanoparticle types, and each type of LNP includes specific polynucleotides.

[0112] In some embodiments, a particular polynucleotide comprises a plurality of polynucleotide molecules carrying the same or identical nucleic acid sequence. In some embodiments, a particular polynucleotide comprises a plurality of polynucleotide molecules carrying substantially the same nucleic acid sequence.

[0113] As used herein, the term "plurality" includes any integer equal to or greater than 2. In some embodiments, a plurality includes at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[0114] As used herein, the term "polynucleotide type" refers to a plurality of polynucleotides, each of which comprises a nucleic acid sequence that differs from any other polynucleotide in the plurality of polynucleotides by at least 1 nucleobase, at least 1 nucleobase, at least 1 nucleobase, at least 1 nucleobase, at least 1 nucleobase, or at least 10 nucleobases (or any value or range therebetween). Each possibility represents a separate embodiment of the present invention.

[0115] In some embodiments, the polynucleotide comprises RNA, DNA, a synthetic analog of RNA, a synthetic analog of DNA, a DNA / RNA hybrid, or any combination thereof. In some embodiments, the LNP of the present invention comprises a polynucleotide selected from the group consisting of RNA, DNA, a synthetic analog of RNA, a synthetic analog of DNA, a DNA / RNA hybrid, or any combination thereof.

[0116] In some embodiments, the polynucleotide comprises or consists of RNA. The polynucleotide comprises or consists of messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide. The basic components of an mRNA molecule generally include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly(A) tail. The polynucleotide can function as mRNA but can differ from wild-type mRNA in its functional and / or structural design features, which are used to overcome existing problems with effective polypeptide expression using nucleic acid-based therapeutics.

[0117] As provided herein, mRNA comprises at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one polypeptide of interest. In some embodiments, the RNA polynucleotides of the mRNA encode 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 polypeptides. In some embodiments, the RNA polynucleotides of the mRNA encode at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 polypeptides. In some embodiments, the RNA polynucleotide of the mRNA encodes at least 100 or at least 200 polypeptides.

[0118] In some embodiments, the nucleic acid is a therapeutic polynucleotide. As used herein, the term "therapeutic polynucleotide" refers to a polynucleotide encoding a therapeutic protein. Therapeutic proteins mediate a variety of effects in host cells or subjects to treat diseases or improve the signs and symptoms of diseases. For example, therapeutic proteins can replace defective or abnormal proteins, enhance the function of endogenous proteins, provide new functions to cells (e.g., inhibit or activate endogenous cellular activities), or serve as a delivery agent for another therapeutic compound (e.g., antibody-drug conjugates). Therapeutic mRNA can be used to treat the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, and autoimmune diseases.

[0119] In some embodiments, the therapeutic polynucleotide is an mRNA. In some embodiments, the mRNA is used to treat lung disease. In some embodiments, the mRNA encodes a therapeutic protein to treat lung disease. In some embodiments, the mRNA has a sequence complementary to a mutant gene associated with a lung disease.

[0120] Therefore, the polynucleotides of the present invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine. For example, mRNA of the structures described herein can be administered to a subject, wherein the polynucleotides are translated in vivo to produce therapeutic peptides.

[0121] In some embodiments, the polynucleotide comprises an inhibitory nucleic acid.In some embodiments, the polynucleotide comprises an antisense oligonucleotide.

[0122] As used herein, "antisense oligonucleotide" refers to a nucleic acid sequence that is opposite and complementary to a DNA or RNA sequence.

[0123] As referred to herein, a "reverse and complementary nucleic acid sequence" is a nucleic acid sequence that is capable of hybridizing to another nucleic acid sequence consisting of complementary nucleotide bases. "Hybridization" refers to the formation of a double-stranded molecule between complementary nucleotide bases under appropriately stringent conditions (e.g., adenine (A) forms base pairs with thymine (T) (or uracil (U) in the case of RNA), and guanine (G) forms base pairs with cytosine (C)) (see, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507). For the purposes of the present methods, the inhibitory nucleic acid need not be complementary to the entire sequence, as long as it is sufficient to provide specific inhibition; for example, in some embodiments, the sequence is 100% complementary to at least 2-7 or 2-8 nucleotides (nt), e.g., 2-7 or 20 nt, at the 5' end of the microRNA itself (e.g., the "seed sequence").

[0124] In some embodiments, the inhibitory nucleic acid has one or more chemical modifications to the backbone or side chains. In some embodiments, the inhibitory nucleic acid has at least one locked nucleotide, and / or has a phosphorothioate backbone.

[0125] Non-limiting examples of inhibitory nucleic acids useful in accordance with the invention disclosed herein include, but are not limited to, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds (e.g., siRNA compounds), modified base / locked nucleic acids (LNA), antagomirs, peptide nucleic acids (PNA), ribozymes (catalytic RNA molecules capable of cleaving other specific sequences of RNA molecules), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of a target nucleic acid and modulate its function. In some embodiments, the inhibitory nucleic acid comprises antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified bonds, interfering RNA (RNAi), short interfering RNA (siRNA); microinterfering RNA (miRNA); small sequential RNA (strRNA); or short hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNA (saRNA), or a combination thereof.

[0126] In some embodiments, the inhibitory nucleic acid is an RNA interference molecule (RNAi). In some embodiments, the RNAi is or comprises double-stranded RNA (dsRNA).

[0127] As used herein, "interfering RNA" refers to any double-stranded or single-stranded RNA sequence that is capable of inhibiting or downregulating gene expression, either directly or indirectly (i.e., upon transformation), by mediating RNA interference. Interfering RNAs include, but are not limited to, small interfering RNAs ("siRNAs") and small hairpin RNAs ("shRNAs"). "RNA interference" refers to the selective degradation of sequence-compatible messenger RNA transcripts.

[0128] In some embodiments, the polynucleotide is chemically modified. In some embodiments, the chemical modification is a modification of the backbone of the polynucleotide. In some embodiments, the chemical modification is a modification of the sugar of the polynucleotide. In some embodiments, the chemical modification is a modification of the core base of the polynucleotide. In some embodiments, the chemical modification improves the stability of the polynucleotide in cells. In some embodiments, the chemical modification improves the stability of the polynucleotide in vivo. In some embodiments, the chemical modification improves the stability of the polynucleotide in the open air, in the field, on surfaces exposed to air, etc. In some embodiments, the chemical modification improves the ability of the polynucleotide to trigger silencing of a target gene or sequence, including but not limited to RNA molecules derived from pathogens as described herein or RNA derived from plant cells. In some embodiments, the chemical modification is selected from the group consisting of: a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a phosphorothioate-deoxyribose backbone, a 2'-O-methylphosphorothioate backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethylphosphorothioate backbone, a constrained ethyl backbone, an alternating locked nucleic acid backbone, a phosphorothioate backbone, an N3'-P5' aminophosphorothioate, a 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid, a cyclohexene nucleic acid backbone, a tricyclic DNA (tcDNA) nucleic acid backbone, a ligand-conjugated antisense, and any combination thereof.

[0129] In some embodiments, the structured lipid is or includes a sterol. In some embodiments, the term "structured lipid" includes non-liposome-forming lipids as described herein. In some embodiments, the term "non-liposome-forming lipid" is understood to refer to lipids that do not spontaneously form vesicles when introduced into an aqueous medium. There are various types of lipids that do not spontaneously form vesicles but are used or can be incorporated into vesicles. In some embodiments, the non-liposome-forming lipid is or includes a sterol.

[0130] In some embodiments, the structured lipids include any one of the following: avenasterol, betulin, brassicasterol, calcipotriol, campesterol, campestanol, cholesterol, cholesterol hemisuccinate, cholesterol sulfate, sulfate), carrot sterol, DC-cholesterol, dehydroergosterol, DMAPC-Chol, DMHAPC-Chol, ergosterol, fuccasterol, HAPC-Chol, lupeol, MHAPC-Chol, OH-C-Chol, OH-Chol, oleanolic acid, stigmasterol, stigmasterol, ursolic acid, hydrophobic vitamins (e.g., vitamin D2, vitamin D3, vitamin E, etc.), β-sitosterol, β-sitosterol-acetate, β-sitosterol-arginine, β-sitosterol-cysteine, β-sitosterol-glycine, β-sitosterol-histidine, β-sitosterol-serine or steroids, including any salts or any combinations thereof.

[0131] In some embodiments, the structured lipid is cholesterol.

[0132] In some embodiments, the helper lipid is or includes a phospholipid. In some embodiments, the helper lipid is or includes a liposome-forming lipid. As used herein, the term "liposome-forming lipid" includes lipids (e.g., phospholipids) that have a transition temperature (T m ) or higher, after being dispersed or dissolved in an aqueous solution, they undergo self-assembly to form stable vesicles (e.g., lipid nanoparticles). As used herein, the term Tm refers to the temperature at which a lipid undergoes a phase transition from a solid (ordered phase, also known as a gel phase) to a fluid (disordered phase, also known as a fluid crystalline phase). Tm also refers to the temperature (or temperature range) at which the maximum change in heat capacity occurs during a phase transition.

[0133] In some embodiments, the phospholipids include a single phospholipid species or a plurality of chemically distinct phospholipids.

[0134] In some embodiments, the liposome-forming lipids are phospholipids having one or two C12-C24 hydrocarbon tails (generally, acyl, alkyl, or alkenyl chains) and having varying degrees of unsaturation, ranging from fully saturated to fully hydrogenated, partially hydrogenated, or non-hydrogenated lipids (the level of saturation can affect the rigidity of the liposomes formed therefrom (generally, liposomes formed from lipids having saturated chains are more rigid than liposomes formed from lipids of the same chain length having unsaturated chains (specifically, having cis double bonds)). In some embodiments, at least one of the liposome-forming lipids is a phospholipid having one or two C12 to C20, C16 to C20, or C16 to C18 hydrocarbon tails, including any values ​​and ranges therebetween. In some embodiments, the liposome-forming lipids are fully saturated, linear, or branched.

[0135] Furthermore, phospholipids can be of natural origin (eg, naturally occurring phospholipids), semisynthetic, or fully synthetic lipids, and be neutrally charged (eg, zwitterionic), negatively charged, or positively charged.

[0136] Non-limiting examples of neutral phospholipids include, but are not limited to, diacylphosphatidylcholines, dialkylphosphatidylcholines, sphingomyelins, and diacylphosphatidylethanolamines. Phosphatidylcholines (PC), including those obtained from eggs, soybeans, or other plant sources, or partially or fully synthesized, or with varying lipid chain lengths and degrees of unsaturation, are suitable for use in the compositions of the present invention. Synthetic, semisynthetic, and natural product phosphatidylcholines, including but not limited to POPC, DOPC, DMPC, distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (soy PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC), and dipalmitoylphosphatidylcholine (DPPC), are suitable phosphatidylcholines for use in preparing liposomes. Charged phospholipids may include phosphatidylglycerol, cardiolipin, or headgroup modified lipids such as N-succinyl-phosphatidylethanolamine, N-glutaryl-phosphatidylethanolamine, and PEG-derivatized phosphatidylethanolamine.

[0137] In some embodiments, the helper lipid is a cationic lipid. In some embodiments, the helper lipid does not contain a non-cationic lipid.

[0138] In some embodiments, the cationic lipid comprises at least one permanent positive charge (i.e., at least one non-ionizable cationic moiety). In some embodiments, the cationic lipid is a trialkylammonium-based lipid. In some embodiments, the cationic lipid comprises 1 to 5, or 1 to 3, positive charges.

[0139] Non-limiting examples of cationic lipids include, but are not limited to, 5-carboxyspermylglycinedioctadecylamide or "DOGS," N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA," 2,3-dioleyloxy-N-[2-(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanetrifluoroammonium acetate or "DOSPA," 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP," and 1,2-dioleoyl-3-trimethylammonium-propane or "DOTAP." Cationic lipids contemplated also include 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinolen ... "DLenDMA", N,N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-stearoyl-N,N-dimethylammonium bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane 1-(cis,cis-9',1-2'-octadecadienoxy)propane) or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane or "DLincarbDAP", 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or "DLin-K-DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-K-XTC2-DMA" and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethylamine (DLin-KC2-DMA).

[0140] In some embodiments, the helper lipid is or includes a non-cationic lipid. As used herein, the term "non-cationic lipid" refers to any neutral or zwitterionic lipid. Non-cationic lipids include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOP-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE) or a mixture thereof.

[0141] In some embodiments, the helper lipid is a cationic lipid. In some embodiments, the cationic lipid is or includes any one of DOTAP, DDAB, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 18:1 EPC (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine), and 18:0 EPC (1,2-distearoyl-sn-glycero-3-ethylphosphocholine), or any combination thereof.

[0142] In some embodiments, the modified lipid is a PEG-modified lipid. In some embodiments, the modified lipid comprises a single PEG moiety covalently bound to the head group of the lipid. In some embodiments, the modified lipid comprises multiple PEG moieties covalently bound to the head group of the lipid. In some embodiments, the PEG moiety comprises an alkylated PEG, such as methoxypoly(ethylene glycol) (mPEG). The molecular weight of the head group of the PEG moiety can be from about 750 Da to about 10,000 Da, sometimes from about 750 Da to about 6,000 Da, and generally from about 1,000 Da to 5,000 Da, or about 2000 Da, including any range therebetween.

[0143] In some embodiments, the modified lipid is or includes DMG-PEG2000.

[0144] Lipid nanoparticle properties In some embodiments, the pKa value of the LNP of the present invention is between 5 and 9, including any range therebetween. In some embodiments, the pKa value of the LNP is between 5 and 8, between 5 and 6, between 6 and 8, between 6 and 7, between 7 and 9, including any range therebetween.

[0145] In some embodiments, the LNPs of the present invention have a pKa value between 6 and 7, between 6 and 6.2, between 6.2 and 6.4, between 6.4 and 6.6, between 6.6 and 6.8, and between 6.8 and 7, including any ranges therebetween.

[0146] In some embodiments, the LNP is characterized by an average particle size of less than 500 nm to facilitate its entry into cells through the extracellular matrix. In one embodiment, the carrier is characterized by an average particle size of less than 300 nm in diameter to facilitate its entry into cells through the extracellular matrix.

[0147] In some embodiments, the LNPs are characterized by an average particle size of less than 500 nm, less than 400 nm, less than 300 nm, less than 350 nm, less than 200 nm, less than 100 nm, less than 50 nm, including any range therebetween.

[0148] In some embodiments, the LNPs are characterized by an average particle size between 50 and 500 nm, between 50 and 250 nm, between 50 and 400 nm, between 100 and 300 nm, between 200 and 400 nm, between 250 and 350 nm, including any range therebetween.

[0149] In some embodiments, the LNP is characterized by a positive zeta potential when measured at a pH of about 7 (e.g., between about 6.5 and 7.5). In some embodiments, the LNP is characterized by a zeta potential in the range of between -5 mV and +40 mV, including any ranges therein. In some embodiments, the LNP is characterized by a zeta potential in the range of between -3 mV and +20 mV, between -3 mV and +10 mV, between -2 mV and +10 mV, between -1 mV and +10 mV, between 0 mV and +20 mV, between -0 mV and +10 mV, between -0 mV and +5 mV, including any ranges therein, when measured at physiological pH (e.g., a pH between 6.5 and 7.5, including any ranges therein).

[0150] In some embodiments, the LNP is stable for a period of time ranging from 1 day to 1 year, or longer, including any range therebetween. In some embodiments, the term "stable" refers to the physical and chemical stability of the dry nanoparticles under appropriate storage conditions (e.g., substantially no phase separation, aggregation, disintegration, and / or substantially maintaining the initial loading of the active agent). In some embodiments, the term "stable" refers to the physical and chemical stability (e.g., dispersion stability) of the nanoparticles in aqueous solution or within the compositions of the present invention.

[0151] The present inventors have surprisingly discovered that, upon administration to a subject (i.e., an animal subject), the LNP compositions of the present invention are characterized by a different activity profile in vivo compared to similar LNP compositions that lack structural lipids and / or helper lipids, or that have different ratios between the LNP components. As shown in the Examples section, the LNPs of the present invention are characterized by enhanced specificity and, therefore, enhanced activity in lung tissue compared to other organs of a subject (e.g., kidney, liver, heart, etc.).

[0152] In some embodiments, the LNPs of the present invention are characterized by enhanced specificity for the lung (e.g., lung tissue, lung cells) of a subject. In some embodiments, the LNPs of the present invention are characterized by enhanced specificity for the lung compared to a similar LNP comprising a commercial ionizable lipid (DliN-MC3-DMA). As used herein, the term "enhanced specificity" encompasses the property of an LNP to undergo specific expression in the lung of a subject compared to other organs (e.g., kidney, liver, heart, etc.). In some embodiments, enhanced specificity includes at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, or at least 1000-fold greater expression of an mRNA in the lung compared to other organs (e.g., kidney, spleen, liver, or heart). In some embodiments, upon administration to a subject, the LNPs of the invention are characterized by at least 10-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 1000-fold, or between 10-fold and 500-fold, between 10-fold and 200-fold, including any ranges therebetween, higher expression in the lung compared to any of the liver, spleen, and kidney of the subject.

[0153] For example, the specificity of the LNPs can be determined by performing tissue expression analysis in vivo (e.g., by using LNPs encapsulating signaling probes or using appropriate imaging techniques such as luminescence).

[0154] Pharmaceutical composition On the other hand, the LNPs of the present invention can be used as therapeutic or prophylactic agents. In some embodiments, the LNPs are in a composition. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a plurality of LNPs and a pharmaceutically acceptable carrier, wherein the LNPs are the same or different. In some embodiments, "different LNPs" refer to lipid nanoparticles containing different active agents. In some embodiments, the pharmaceutically acceptable carrier of the active agent is an LNP of the present invention.

[0155] In some embodiments, a pharmaceutically acceptable carrier is also referred to as an excipient or adjuvant. As used herein, the terms "carrier," "excipient," or "adjuvant" refer to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid, semi-solid, liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous medium, such as saline. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol solutions, and phosphate buffered saline, as well as other nontoxic, compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can serve as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffered solution, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible materials used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier can be used to prepare the compositions contemplated herein.In this regard, suitable pharmaceutically acceptable carriers, excipients, and diluents are well known to those skilled in the art, such as those described in The Merck Index, 13th Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, 10th Edition (2004); and "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management (the entire contents of which are hereby incorporated by reference in their entirety). Examples of pharmaceutically acceptable excipients, carriers, and diluents for use in the present compositions include distilled water, physiological saline, Hartmann's solution, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive ingredients, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21. stEd., LippincottWilliams & Wilkins, Philadelphia, Pa., (2005) (each of which is incorporated herein by reference in its entirety). The compositions described herein may also be contained in artificially created structures such as liposomes, ISCOMS, slow-release particles, and other vehicles that increase the half-life of the peptide or polypeptide in serum. The liposomes used with the peptides described herein are formed by standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid is generally determined by factors such as liposome size and blood stability. For example, Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, summarizes various methods that can be used to prepare liposomes, and also see U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0156] In some embodiments, the LNPs of the present invention, comprising a compound of the present invention, a structural lipid, a helper lipid, and a modified lipid, and in relative molar ratios to the compound of the present invention as described above in each embodiment, are based on the active agent to be delivered and its target. In some embodiments, the relative molar ratios to the compound of the present invention as described above in each embodiment enable specific targeting of the LNP to the lung.

[0157] Carriers may comprise in total from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0158] In some embodiments, the pharmaceutical composition comprising the LNPs of the present invention further comprises an effective amount of an active agent as described herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an active agent as described herein. In some embodiments, the therapeutically effective amount is sufficient to reduce at least one symptom, or to substantially lessen the severity and / or inhibit the progression of the aforementioned disease, disorder, or condition.

[0159] In some embodiments, the pharmaceutical composition is used to treat or prevent a lung disease. In some embodiments, the lung disease is selected from lung cancer, ARDS, pulmonary fibrosis, viral infection, bacterial infection, COPD, asthma, bronchiectasis, bronchiolitis, bronchitis, cystic fibrosis, emphysema, mesothelioma, pleural effusion, pleurisy, pneumonia, pneumothorax, RSV, SARS, SARS-CoV-2, silicosis, tuberculosis, pertussis, and influenza. Lung cancer is well known in the art and includes, for example, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma, Pancoast tumor, and carcinoid tumor. The term "effective amount" or "therapeutically effective amount" refers to an amount effective at the dosage and time period required to achieve the desired therapeutic outcome. It will be apparent to one of ordinary skill in the art that the therapeutically effective amount of a molecule according to the present invention will depend, among other things, on the administration schedule, the unit dose of the administered molecule, whether the molecule is administered in combination with other therapeutic agents, the patient's immune status and health status, the therapeutic activity of the administered molecule, and the discretion of the treating physician. In some embodiments, the therapeutically effective amount of an active agent administered daily to a subject is reduced compared to IV administration of a pharmaceutical composition comprising the same active agent not encapsulated in an LNP of the present invention. In some embodiments, the therapeutically effective amount of an active agent administered daily to a subject is reduced compared to a similar pharmaceutical composition comprising the same active agent encapsulated in a commercial LNP (e.g., DliN-MC3-DMA). In some embodiments, the reduced therapeutically effective amount is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, including any range therebetween.

[0160] In some embodiments, the LNPs of the present invention are composed of pharmaceutically acceptable ingredients (such as phospholipids, lipids and sterols) or pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical composition includes the LNPs of the present invention, pharmaceutically acceptable salts thereof, or both.

[0161] In some embodiments, the pharmaceutical composition material is characterized as pharmaceutical grade. In some embodiments, pharmaceutical grade is characterized by a chemical purity of at least 90%, at least 95%, at least 99%, including any range therebetween.

[0162] The term "pharmaceutically acceptable" may mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0163] In some embodiments, the pharmaceutical compositions of the present invention are used as a medicament. In some embodiments, the pharmaceutical compositions are formulated for administration to the lungs, lung tissue, or lung cells. In some embodiments, the pharmaceutical compositions are formulated for systemic administration. In some embodiments, the pharmaceutical compositions are formulated for intravenous (IV) administration. In some embodiments, the pharmaceutically acceptable carrier comprises a buffer.

[0164] In some embodiments, administration to the lungs (eg, tissue, cells) is by IV administration. In some embodiments, administration to the lungs (eg, tissue, cells) is systemic.

[0165] As used herein, the terms "administering," "administration," and similar terms refer to any method of delivering a composition comprising an active agent to a subject in a manner that provides a therapeutic effect in reasonable medical practice. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration may include parenteral, intravenous, subcutaneous, oral, intramuscular, intrathecal, inhalation, intracerebroventricular, intravitreal, transdermal, or intraperitoneal administration. In some embodiments, the pharmaceutical composition is used in a method of treatment. In some embodiments, the method of treatment is a method of treatment. In some embodiments, the pharmaceutical composition is used to treat a medical condition. In some embodiments, the medical condition is a condition, disease, or disorder. In some embodiments, the pharmaceutical composition is used in a method of diagnosis.

[0166] In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the method comprises administering a pharmaceutical composition of the present invention to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is in need of a pharmaceutical composition. In some embodiments, the pharmaceutical composition is used to treat or prevent a disease or condition in humans and other mammals. In some embodiments, the subject is in need of treatment. In some embodiments, the subject is a volunteer for a diagnostic method. In some embodiments, the subject is in need of a diagnosis.

[0167] The active therapeutic agents of the present invention include lipid nanoparticles, or proteins translated from polynucleotides contained within lipid nanoparticles.

[0168] In some embodiments, the mammal is a laboratory animal. Examples of laboratory animals include, but are not limited to, mice, rats, rabbits, hamsters, dogs, pigs, and monkeys. In some embodiments, the mammal is a mouse or a rat.

[0169] In some embodiments, the pharmaceutical composition of the present invention is in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, gel, cream, ointment, foam, paste, sustained-release formulation, etc. In some embodiments, the pharmaceutical composition of the present invention can be formulated into a suppository together with traditional binders and carriers such as triglycerides, microcrystalline cellulose, tragacanth gum or gelatin. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0170] How to use According to another aspect, a method of delivering an active agent to the lung, lung tissue, or lung cells of a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present invention described above, thereby delivering the active agent to the lung. In some embodiments, the pharmaceutical composition is used to deliver the active agent to the lung (e.g., lung tissue, lung cells) of a subject.

[0171] In some embodiments, the pharmaceutical composition is used to treat a lung disease, lung disorder, or lung condition.In some embodiments, the pharmaceutical composition includes a therapeutically effective amount of an active agent.

[0172] In some embodiments, the pharmaceutical composition is a lung-targeted composition. In some embodiments, the pharmaceutical composition is used to treat a lung disease. In some embodiments, the lung disease comprises lung inflammation. Examples of lung diseases include, but are not limited to, lung cancer, pulmonary fibrosis, viral infections, bacterial infections, COPD, asthma, bronchiectasis, bronchiolitis, bronchitis, cystic fibrosis, emphysema, mesothelioma, pleural effusion, pleurisy, pneumonia, pneumothorax, RSV, SARS, SARS-CoV-2, silicosis, tuberculosis, whooping cough, and influenza. Lung cancer is well known in the art and includes, for example, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma, Pancoast tumor, and carcinoid tumor.

[0173] In some embodiments, the method comprises administering an effective amount of a pharmaceutical composition. In some embodiments, the effective amount is the human equivalent of a rat dose of 0.01 mg / kg body weight / day to 5 mg / kg body weight / day. In some embodiments, the human equivalent of a rat dose depends on the route of administration. In some embodiments, the effective amount is the human equivalent of a rat dose of 0.01 mg / kg body weight to 5 mg / kg body weight, 0.1 mg / kg body weight to 5 mg / kg body weight, 0.1 mg / kg body weight to 2 mg / kg body weight, 0.01 mg / kg body weight to 2 mg / kg body weight, 0.01 mg / kg body weight to 3 mg / kg body weight, 0.01 mg / kg body weight to 1 mg / kg body weight, 0.5 mg / kg body weight to 5 mg / kg body weight, including any values ​​therebetween.

[0174] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0175] definition As used herein, the term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or parts, but only if the additional ingredients, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0176] As used herein, the term "substantially" means at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or between 60% and 99.9%, between 70% and 80%, between 70% and 90%, between 80% and 90%, between 90% and 95%, between 95% and 99.9%, including any range or value therebetween.

[0177] As used herein, the term "substituent" includes hydrogen, halogen, -NO2, -CN, -OH, oxo, imino, -CONH2, -CONR'2, -CNNR'2, -CSNR'2, -CONH-OH, -CONH-NH2, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR', ​​-NC(=S)OR', -NC(=S)NR', ​​-SO2R', -SOR', -SR', -SO2OR', -SON(R')2, -NHNR'2, -NNR', C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR'2, -NH( C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(C1-C6 alkyl), hydroxy(C1-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), C1-C6 alkyl-NR'2, C1-C6 alkyl-SR', -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -CO2H, -CO2R', -OCOR, -OCOR', -OC(=O)OR', -OC(=O)NR', ​​-OC(=S)OR', -OC(=S)NR', ​​or a combination thereof; wherein each R' independently represents H, or is selected from optionally substituted C1-C 10 Alkyl, optionally substituted C3-C 10 Cycloalkyl, optionally substituted C3-C 10heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, hydroxy, amino, -NH2, -NR'2‌-NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(C1-C6 alkyl), hydroxy(C1-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(C1-C6 alkoxy), C1-C6 alkyl-NR'2, C1-C6 alkyl-SR', or a combination thereof.

[0178] As used herein, the term "alkyl" describes aliphatic hydrocarbons including straight and branched chain groups. As used herein, the term "alkyl" also includes saturated or unsaturated hydrocarbons, and thus this term also includes alkenyl and alkynyl groups.

[0179] The term "alkenyl" describes an unsaturated alkyl group as defined herein having at least two carbon atoms and at least one carbon-carbon double bond. As noted above, an alkenyl group may be unsubstituted or substituted with one or more substituents.

[0180] As defined herein, the term "alkynyl" is an unsaturated alkyl group having at least two carbon atoms and at least one carbon-carbon triple bond. As described above, an alkynyl group may be substituted or unsubstituted with one or more substituents.

[0181] The term "cycloalkyl" describes an all-carbon monocyclic or fused ring (i.e., rings that share a pair of adjacent carbon atoms) group in which one or more of the rings does not have a completely conjugated pi electron system. As indicated herein, a cycloalkyl group may be substituted or unsubstituted.

[0182] The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (ie, rings that share adjacent pairs of carbon atoms) group having a completely conjugated pi-electron system. As indicated herein, an aryl group can be substituted or unsubstituted.

[0183] The term "alkoxy" describes O-alkyl and -O-cycloalkyl groups as defined herein. The term "aryloxy" describes -O-aryl groups as defined herein.

[0184] As used herein, each of the alkyl, cycloalkyl, and aryl groups in the general formula may be substituted with one or more substituents, whereby each substituent may independently be, for example, a halide, an alkyl, an alkoxy, a cycloalkyl, a nitro, an amino, a hydroxyl, a thiol, a thioalkoxy, a carboxyl, an amide, an aryl, and an aryloxy group, depending on the substituted group and its position in the molecule. Other substituents are also contemplated.

[0185] The terms "halide," "halogen," or "halo / halo" describe fluorine, chlorine, bromine, or iodine. The term "haloalkyl" describes an alkyl group, as defined herein, further substituted with one or more halides. The term "haloalkoxy" describes an alkoxy group, as defined herein, further substituted with one or more halides. The term "hydroxyl" or "hydroxy" describes an -OH group. The term "mercapto" or "thiol" describes an -SH group. The term "thioalkoxy" describes an -S-alkyl and -S-cycloalkyl group, as defined herein. The term "thioaryloxy" describes an -S-aryl and -S-heteroaryl group, as defined herein. The term "amino" describes an -NR'R" group, or a salt thereof, wherein R' and R" are as described herein.

[0186] The term "heterocyclyl" describes a monocyclic or fused ring group having one or more atoms such as nitrogen, oxygen, and sulfur in the ring(s). These rings may also have one or more double bonds. However, these rings do not have a completely conjugated π electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino, and the like.

[0187] The term "carboxy" describes a -C(O)OR' group, or a carboxylate salt thereof, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon), or heterocyclyl (bonded through a ring carbon), or "carboxylate," as defined herein.

[0188] The term "carbonyl" describes a -C(O)R' group, wherein R' is as defined above. The above term also includes its thio derivatives (thiocarboxy and thiocarbonyl).

[0189] The term "thiocarbonyl" describes a -C(S)R' group, where R' is as defined above. A "thiocarboxy" group describes a -C(S)OR' group, where R' is as defined herein. A "sulfinyl" group describes a -S(O)R' group, where R' is as defined herein. A "sulfonyl" or "sulfonic acid" group describes a -S(O)2R' group, where R' is as defined herein.

[0190] A "carbamoyl" or "carbamate" group describes a -OC(O)NR'R" group, where R' is as defined herein and R" is as defined for R'. A "nitro" group refers to a -NO2 group. As used herein, the term "amide" includes C-amides and N-amides. The term "C-amide" describes a -C(O)NR'R" terminal group or a -C(O)NR'- linking group, as defined above for these phrases, where R' and R" are as defined herein. The term "N-amide" describes a -NR"C(O)R' terminal group or a -NR'C(O)- linking group, as defined above for these phrases, where R' and R" are as defined herein.

[0191] "Cyano" or "nitrile" refers to a -CN group. The term "azo" or "diazo" describes an -N=NR' terminal group or an -N=N- linking group, as defined above for these phrases, where R' is as defined above. The term "guanidine" describes an -R'NC(N)NR"R"' terminal group or an -R'NC(N)NR"- linking group, as defined above for these phrases, where R', R"', and R"' are as defined herein. As used herein, the term "azide" refers to an -N3 group. The term "sulfonamide" refers to an -S(O)2NR'R"' group, where R' and R"' are as defined herein.

[0192] The term "phosphono" or "phosphonate" describes a -OP(O)-(OR')2 group, where R' is as defined above. The term "phosphinyl" describes a -PR'R" group, where R' and R" are as defined above. The term "alkylaryl" describes an alkyl group, as defined herein, substituted with an aryl group, as described herein. An exemplary alkylaryl group is benzyl.

[0193] The term "heteroaryl" describes a monocyclic or fused ring (i.e., a ring that shares a pair of adjacent atoms) group having one or more atoms such as, for example, nitrogen, oxygen, and sulfur in the ring(s), and further having a completely conjugated pi electron system. As used herein, the term "heteroaryl" refers to an aromatic ring in which at least one of the atoms forming the aromatic ring is a heteroatom. Heteroaryl rings can be formed by three, four, five, six, seven, eight, nine, and more than nine atoms. Heteroaryl groups may be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C 2-1 ... 3-8 Heterocyclic groups, and their substituted and benzo-fused and pyrido-fused derivatives - for example, linked through one of the carbon atoms forming the ring. In certain embodiments, the heteroaryl group is selected from Azolyl, iso Azolyl, oxadiazolyl, thiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinazolinyl or quinoxalinyl.

[0194] In some embodiments, heteroaryl is selected from pyrrolyl, furanyl (furyl), phenylthio (thienyl), imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3- Azolyl ( oxazolyl), 1,2- Azolyl (iso oxazolyl), oxadiazolyl, 1,3-thiazolyl (thiazolyl), 1,2-thiazolyl (isothiazolyl), tetrazolyl, pyridyl (pyridyl) pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, indazolyl, indolyl, benzothiophenyl, benzofuranyl, benzothiazolyl, benzimidazolyl, benzodioxolyl, acridinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, thienophenylthio, 1,8-naphthyridinyl, other naphthyridinyl, pteridinyl, or phenothiazinyl. When a heteroaryl group includes more than one ring, each additional ring is saturated (perhydro) or partially unsaturated (e.g., dihydro or tetrahydro) or maximally unsaturated (non-aromatic). Thus, the term heteroaryl includes bicyclic radicals in which both rings are aromatic and bicyclic radicals in which only one ring is aromatic. Examples of such heteroaryl groups include 3H-indolyl, 2(1H)-quinolyl, 4-oxo-1,4-dihydroquinolyl, 2H-1-oxoisoquinolyl, 1,2-dihydroquinolyl, (2H)quinolyl N-oxide, 3,4-dihydroquinolyl, 1,2-dihydroisoquinolyl, 3,4-dihydro-isoquinolyl, chromonyl, 3,4-dihydroiso-quinoxalinyl, 4-(3H)quinazolinyl, 4H-chromenyl, 4-chromanon ... omanonyl), oxindolyl, 1,2,3,4-tetrahydroisoquinolyl, 1,2,3,4-tetrahydro-quinolyl, 1H-2,3-dihydroisoindolyl, 2,3-dihydrobenzo[f]isoindolyl, 1,2,3,4-tetrahydrobenzo-[g]isoquinolyl, 1,2,3,4-tetrahydro-benzo[g]isoquinolyl, chromanyl, isochromanonyl, 2,3-dihydrochromononyl, 1,4-benzo-dihydro Alkyl, 1,2,3,3,4-tetrahydro-quinoxalinyl, 5,6-dihydro-quinolinyl, 5,6-dihydroiso-quinolinyl, 5,6-dihydroquinoxalinyl, 5,6-dihydroquinazolinyl, 4,5-dihydro-1H-benzimidazolyl, 4,5-dihydro-benzo oxazolyl, 1,4-naphthoquinolinyl, 5,6,7,8-tetrahydro-quinolinyl, 5,6,7,8-tetrahydro-isoquinolinyl, 5,6,7,8-tetrahydroquinoxalinyl, 5,6,7,8-tetrahydroquinazolinyl, 4,5,6,7-tetrahydro-1H-benzimidazolyl, 4,5,6,7-tetrahydro-benzo oxazolyl, 1H-4-oxa-1,5-diaza-naphthalen-2-onyl, 1,3-dihydroimidizolo-[4,5]-pyridin-2-one, 2,3-dihydro-1,4-dinaphthoquinone, 2,3-dihydro-1H-pyrrolo[3,4-b]quinolinyl, 1,2,3,4-tetrahydrobenzo[b]-[1,7]naphthopyridinyl, 1,2,3,4-tetrahydrobenzo[b][1,6]naphthopyridinyl, 1,2,3,4-tetrahydro-9H-pyrido[3 ,4-b]indolyl, 1,2,3,4-tetrahydro-9H-pyrido[4,3-b]indolyl, 2,3-dihydro-1H-pyrrolo[3,4-b]indolyl, 1H-2,3,4,5-tetrahydro-azepino[3,4-b]indolyl, 1H-2,3,4,5-tetrahydro-azepino[4,3-b]indolyl, 1H-2,3,4,5-tetrahydro-azepino[4,5-b]indolyl, 5,6,7,8-tetrahydro[1,7]naphthyridinyl, 1,2,3,4-tetrahydro-[2,7]-naphthyridinyl, 2,3-dihydro[1,4]di 2,3-dihydro[1,4]dioxino[2,3-b]pyridyl, 2,3-dihydro[1,4]-dioxino[2,3-b]pyridyl , 1,2,3,4-tetrahydro[1,5]-naphthyridinyl, 1,2,3,4-tetrahydro[1,6]naphthyridinyl, 1,2,3,4-tetrahydro[1,7]naphthyridinyl, 1,2,3,4-tetrahydro-[1,8]naphthyridinyl or 1,2,3,4-tetrahydro[2,6]naphthyridinyl. In some embodiments, heteroaryl is optionally substituted. In one embodiment, one or more substituents are each independently selected from halo, hydroxy, amino, cyano, nitro, alkylamido, acyl, C 1-6 -alkyl, C 1-6 -haloalkyl, C 1-6 -Hydroxyalkyl, C 1-6 -aminoalkyl, C 1-6 -alkylamino, alkylsulfinyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl or trifluoromethyl.

[0195] Examples of heteroaryl groups include, but are not limited to, furan, benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole, Azoles, benzophenones Azoles, isocyanates azole, benzyl isocyanate azole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3- oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, triazole, benzotriazole, pteridine, benzene Phenoxazole, Non-substituted and mono- or di-substituted derivatives of oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline, and quinoxaline. In some embodiments, the substituent is a halo, hydroxyl, cyano, O—C 1-6 -alkyl, C 1-6 -alkyl, hydroxy-C 1-6 -alkyl and amino-C 1-6 -alkyl.

[0196] As used herein, the terms "halo" and "halide" are used interchangeably herein to describe a halogen atom, ie, fluorine, chlorine, bromine, or iodine, also referred to herein as fluoride, chloride, bromide, and iodide.

[0197] As used herein, the term "substituted" or the term "substituent" refers to one or more (eg, 2, 3, 4, 5, or 6) substituents, wherein the substituent(s) are as described herein.

[0198] General considerations As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm +- 100 nm.

[0199] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polynucleotide" includes a plurality of such polynucleotides, and reference to a "polypeptide" includes one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely" and "only" in connection with a limitation of claim elements, or for the use of a "negative" limitation.

[0200] In those instances where a convention similar to "at least one of A, B, and C, etc." is used, such construction is generally intended to be in a sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One skilled in the art would further understand that virtually any disjunctive conjunction and / or phrase presenting two or more alternative terms, whether in the specification or in the claims, should be understood to contemplate the possibility of including one, either, or both of the terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0201] It should be understood that certain features of the present invention that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present invention that are described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination. All combinations of embodiments related to the present invention are specifically encompassed by the present invention and disclosed herein, just as each combination is individually and explicitly disclosed. In addition, all subcombinations of various embodiments and elements thereof are also specifically encompassed by the present invention and disclosed herein, just as each such subcombination is individually and explicitly disclosed herein.

[0202] Other objects, advantages and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, as described above and as claimed in the claims section below, each of the various embodiments and aspects of the present invention is experimentally supported in the following examples.

[0203] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0204] Example In general, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are explained in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); Methodologies described in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Vols. I-III, Cellis, JE, ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), 3rd ed.; "Current Protocols in Immunology", Vols. I-III, Coligan JE, ed. (1994); Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

[0205] Example 1 Exemplary compounds of the present invention (MB-212 and MB-222, described below) were synthesized according to the synthetic schemes presented below. The present inventors successfully formed lipid nanoparticles of the present invention using MB-212 or MB-222 and tested their ability to deliver active agents to the lungs in vivo. Exemplary compounds of the present invention (ionizable lipids) are presented below. Here is a general synthesis scheme for some of the exemplary compounds of the present invention. Other possible synthesis strategies are well known to those skilled in the art. Some of the exemplary compositions of the present invention are characterized by enhanced specificity for lung cells, as determined in in vivo studies in mice. For example, LNPs of the present invention comprising MB-212 or MB-222 as the ionizable lipid exhibited enhanced specificity for lung tissue compared to similar compositions comprising DliN-MC3-DMA as the ionizable lipid ( Figure 1 Surprisingly, the inventors discovered that exemplary LNPs comprising about 30% to about 50% helper lipid (e.g., DOTAP), about 20% to about 50% structural lipid (cholesterol), and about 0.1% to about 5% modifying lipid (e.g., PEG-lipid), and about 15% to about 50% ionizable lipid (e.g., MB-212 or MB-222) resulted in enhanced LNP lung specificity, as assessed by in vivo mRNA expression analysis. Exemplary LNP compositions of the present invention exhibited high lung specificity, resulting in significantly enhanced expression of the encapsulated polynucleic acid (mRNA F-LUC) in the lung compared to commercial controls ( Figure 1Furthermore, a particular LNP composition of the invention (FMB-1143) comprising about 15% to about 30% ionizable lipids exhibits excellent lung specificity (see Figure 1 ).

[0206] Preparation of FMB-745 40% DOTAP, 22.5% cholesterol, 2.5% DMG-PEG2000, and 35% commercial ionizable lipid (DliN-MC3-DMA) were dissolved in ethanol (EtOH) at 55°C-60°C. mRNA and F-LUC were added to a citrate buffer at a pH of 5.0 (range 4.5-5.5). Lipid mixing was performed using microfluidic mixing or by injecting the lipid-EtOH into the citrate buffer containing mRNA and firefly luciferase (F-LUC) under constant mixing conditions. The mixture was then diluted with PBS to raise its pH, and residual EtOH was removed by dialysis before injection. The FMB-745 prepared by the present inventors is characterized by an average particle size ranging from approximately 60 nm to approximately 130 nm.

[0207] Preparation of FMB-748 40% DOTAP, 22.5% cholesterol, 2.5% DMG PEG2000, and 35% ionizable MB-222 were dissolved in ethanol (EtOH) at 55°C-60°C. mRNA and F-LUC were added to a citrate buffer at a pH of 5.0 (range 4.5-5.5). Lipid mixing was performed under microfluidic mixing or by injecting the lipid-EtOH into the citrate buffer containing mRNA and F-LUC under constant mixing conditions. The pH of the mixture was then raised by dilution with PBS, and residual EtOH was removed by dialysis before injection. The FMB-748 prepared by the present inventors is characterized by an average particle size ranging from approximately 60 nm to approximately 130 nm, an N:P ratio of 8-9, and an average Z of approximately +4 mV.

[0208] Preparation of FMB-1143 40% DOTAP, 38.5% cholesterol, 1.5% DMG-PEG2000, and 20% ionizable MB-212 were dissolved in ethanol (EtOH) at 55°C-60°C. mRNA and F-LUC were added to a citrate buffer at a pH of 5.0 (range 4.5-5.5). Lipid mixing was performed under microfluidic mixing or by injecting the lipid-EtOH into the citrate buffer containing mRNA and F-LUC under constant mixing conditions. The pH of the mixture was then raised by dilution with PBS, and residual EtOH was removed by dialysis before injection. The FMB-1143 prepared by the present inventors is characterized by an average particle size ranging from approximately 60 nm to approximately 130 nm, an N:P ratio of 4-5, and an average Z value of approximately +3 mV.

[0209] The present inventors determined the in vivo expression profile of an exemplary LNP by assessing the expression of encapsulated mRNA (mRNA F-LUC). LNPs of the present invention encapsulating mRNA F-LUC were intravenously injected into BALB / c mice at a dose of 13 μg / mouse (0.52-0.365 mg / kg). In vivo imaging was performed using IVIS imaging. Ex vivo tissue analysis of the lungs, heart, spleen, kidneys, and liver was performed using IVIS. Histological assessments were confirmed by H&E staining and a pathologist's toxicity report. The histological assessments concluded that the morphology was normal, with no treatment-related pathological changes.

[0210] Figure 1 The expression profiles presented in show that the luciferase signal in the lung is more than 100-fold greater than in the heart, liver, spleen, and kidney, indicating that the exemplary LNPs of the present invention have excellent lung specificity.

[0211] Furthermore, a similar formulation (FMB-393) that includes a zwitterionic helper lipid (DOPE) and is characterized by a negative zeta potential (approximately -22 mV) exhibits predominantly hepatic expression / accumulation (unlike the positive zeta potential of cationic helper lipids and exemplary formulations of the present invention).

[0212] FMB-393 was prepared as described above. The chemical composition of FMB-393 is disclosed in Table 1.

[0213] Table 1: Ionizable lipids [mol%] Helper lipids [mol%] Structural lipids [mol%] PEG lipid [mol%] N:P MB-212

[50] DOPE

[10] Cholesterol [38.5] DMG-PEG 2000 [1.5] 8 FMB-393 was injected intravenously into BALB / c mice and the in vivo expression profile was assessed as disclosed above.

[0214] The expression profile of FMB-393 is shown in Table 2. organ Fluorescence intensity (from luciferase expression) heart 0.00E+00 lung 4.04E+06 spleen 4.57E+07 kidney 4.78E+06 liver 6.41E+08 As is apparent from Table 2, FMB-393 showed predominant liver accumulation, with the luciferase signal in the liver being over 100-fold greater compared to the lung.

[0215] The present inventors determined the in vivo expression profile of an exemplary LNP by assessing the expression of encapsulated mRNA (mRNA mCherry). mRNA mCherry-encapsulated LNPs of the present invention were administered intravenously to cynomolgus monkeys at a dose of 0.6 mg / kg. Tissue samples were collected 24 hours after administration from the following tissues: lung, brain, liver, heart, spleen, kidney, testis, and mesenteric lymph node. The samples were analyzed for mCherry signal using immunohistochemistry and compared to the same samples collected from control animals injected with PBS / sucrose. Tissue analysis revealed moderate to strong positive mCherry staining in lung tissue samples.

[0216] Although the present invention has been described in conjunction with specific embodiments of the present invention, it is apparent that various alternatives, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A lipid nanoparticle comprising a compound, a salt of the compound, or both, wherein the compound is represented by the following formula 1: ,in: Each X independently represents -O-, -S-, CH2, or X is absent; Each Z independently represents -OH or -SH; Each A independently represents O or S; Each n is independently between 0 and 5, and at least one n is not 0; Each R is independently H, or includes optionally substituted C5-C30 alkyl; The lipid nanoparticles further comprise an active agent, a helper lipid, a structural lipid, and a modified lipid, wherein: The helper lipid is a cationic lipid; The ratio of the compound relative to the total lipid content of the lipid nanoparticle is between 10 mol% and 50 mol%; and The proportion of the structural lipids relative to the total lipid content of the lipid nanoparticle is between 5 mol% and 50 mol%; and wherein the lipid nanoparticle is characterized by an average zeta potential in the range of between -5 mV and +40 mV at a pH of 6 to 8.

2. The lipid nanoparticle of claim 1, wherein the molar ratio of the helper lipid to the modifying lipid is between 1:0.2 and 1:0.

01.

3. The lipid nanoparticle of claim 1 or 2, wherein the molar ratio of the compound to the helper lipid is between 0.2:1 and 5:

1.

4. The lipid nanoparticle according to any one of claims 1 to 3, wherein the molar ratio of the structural lipid to the modifying lipid is between 1:0.01 and 1:0.

2.

5. The lipid nanoparticle of any one of claims 1 to 4, wherein the active agent comprises a polynucleic acid; and the ratio between N:P within the LNP is between 1 and 20.

6. The lipid nanoparticle according to any one of claims 1 to 5, wherein the weight ratio between the total amount of (i) the compound, the helper lipid, the structural lipid and the modified lipid and (ii) the polynucleic acid in the lipid nanoparticle is between 0.001:1 and 10:

1.

7. The lipid nanoparticle according to any one of claims 1 to 6, wherein the size distribution of the lipid nanoparticles is within a range between 50 nm and 500 nm; and wherein the cationic lipid is selected from DOTAP, DDAB, 18:1 EPC (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine) and 18:0 EPC (1,2-distearoyl-sn-glycero-3-ethylphosphocholine), including any salts and any combination thereof.

8. The lipid nanoparticle according to any one of claims 5 to 7, wherein: The lipid nanoparticles are characterized by an average zeta potential between 0 mV and +5 mV at a pH of 6 to 8; The N:P ratio is between 3 and 9; The structural lipid is cholesterol; The modified lipid is a PEG-lipid; The ratio of the helper lipid to the total lipid content of the lipid nanoparticle is between 35 mol% and 45 mol%; and The ratio of the PEG-lipid relative to the total lipid content of the lipid nanoparticles is between 0.1 mol% and 3 mol%.

9. The lipid nanoparticle of any one of claims 1 to 8, wherein the lipid nanoparticle is characterized by a pKa between 5 and 9; and wherein the compound comprises any one of MB-212 and MB 222: 。 10. The lipid nanoparticle of any one of claims 1 to 9, wherein upon administration to a subject, the LNP is characterized by at least 10-fold higher expression in the lung compared to any one of the liver, spleen, and kidney of the subject.

11. The lipid nanoparticle of claim 10, wherein the ratio between N:P is about 5; wherein the cationic lipid is DOTAP; and wherein the proportion of the compound relative to the total lipid content of the lipid nanoparticle is between about 15 mol % and about 25 mol %.

12. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12, comprising a therapeutically effective amount of the active agent.

14. The pharmaceutical composition according to claim 12 or 13, for use in delivering the active agent to lung tissue.

15. The pharmaceutical composition according to claim 12 or 13, which is used for treating lung diseases or lung disorders.

16. A method for delivering an active agent to lung tissue of a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 12 to 13, thereby delivering the active agent to the lung tissue.

17. The method of claim 16, wherein the therapeutically effective amount is between 0.01 mg / kg / day and 5 mg / kg / day.

18. The method of claim 16 or 17, wherein the administration is via intravenous, intratracheal, intranasal administration or via inhalation.

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