Polynucleotide compositions, related formulations and methods of use thereof

Abstract

Disclosed are compositions of polynucleotide(s), pharmaceutical compositions thereof, and methods of use thereof. The polynucleotide may be or encode a synthetic transfer ribonucleic acid (tRNA). The polynucleotide may be assembled with a lipid composition for delivery to a cell or organ, such as a lung cell or lung, of a subject. Methods are provided for enhancing the expression or activity of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein in a cell. Methods are also provided for treating a subject having or suspected of having a CFTR-associated condition.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 208,957, filed June 9, 2021, which is incorporated by reference herein in its entirety. [Background technology]

[0002] Nucleic acids such as transfer RNA (tRNA) can be used by cells to express proteins and polypeptides. Some cells may be deficient in certain proteins or nucleic acids, resulting in disease states. Cells can also take up and use exogenous tRNAs that can be used in protein synthesis reactions, but many factors affect the efficient uptake and translation of tRNAs. For example, the immune system recognizes many exogenous RNAs as foreign RNA and triggers a response aimed at inactivating the RNA. Summary of the Invention

[0003] Compositions and methods for delivering nucleic acids are provided herein. Nucleic acids may be used as therapeutic agents. In particular, tRNAs may be delivered to cells of a subject. Once a nucleic acid is delivered to a cell, it can be used to synthesize a polypeptide. In the case of a cell or subject with a disease or disorder, the nucleic acid may be effective to act as a therapeutic agent by increasing expression of the polypeptide. When a disorder or disease is caused or correlated with abnormal expression or activity of a polypeptide, increasing expression of the polypeptide may be beneficial. However, cells may have limited uptake of exogenous nucleic acids, and delivery of nucleic acids may benefit from compositions that allow for increased uptake of the nucleic acid.

[0004] Furthermore, treatments can benefit from organ-specific delivery. Many different types of compounds, such as chemotherapeutic agents, exhibit significant cytotoxicity. If these compounds can be better directed for delivery to the desired organ, off-target effects are less likely to be seen.

[0005] In one aspect, the disclosure provides a composition comprising a synthetic transfer ribonucleic acid (tRNA) assembled with a lipid composition, wherein the lipid composition comprises a zwitterionic lipid, and the composition is an aerosol composition.

[0006] In another aspect, the disclosure provides a composition comprising a synthetic transfer ribonucleic acid (tRNA) assembled with a lipid composition, wherein the lipid composition comprises a zwitterionic lipid, and wherein the composition is formulated for aerosol administration. In some embodiments, the composition has a droplet size of 0.5 micrometers (μm) to 10 μm. In some embodiments, the composition has a median droplet size of 0.5 μm to 10 μm. In some embodiments, the composition has an average droplet size of 0.5 μm to 10 μm. In some embodiments, the synthetic tRNA is a folded tRNA. In some embodiments, the folded tRNA comprises a T arm, a D arm, an anticodon arm, a variable loop, an acceptor stem, or a combination thereof. In some embodiments, the synthetic tRNA comprises an anticodon arm configured to recognize a premature stop codon. In some embodiments, the synthetic tRNA comprises an acceptor stem configured to be operably linked to an arginine. In some embodiments, the tRNA comprises a polynucleotide sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NOs: 1-20. The composition of any one of claims 1-10, wherein the ratio (e.g., by weight or by mass) of zwitterionic lipid to synthetic tRNA is about 50:1, 40:1, 30:1, or 20:1 or less. In some embodiments, the lipid composition comprises a molar percentage of zwitterionic lipid of about 1% to about 60%. In some embodiments, the lipid composition further comprises a steroid or steroid derivative. In some embodiments, the composition comprises a molar percentage of a steroid or steroid derivative of about 20% to about 60%. In some embodiments, the lipid composition further comprises a polymer-conjugated lipid. In some embodiments, the lipid composition comprises a polymer-conjugated lipid at a molar percentage of about 0.5% to about 12%. In some embodiments, the molar ratio of nitrogen in the lipid composition to phosphate in the synthetic tRNA (N / P ratio) is about 50:1, 40:1, 30:1, 20:1, or 10:1 or less. In some embodiments, the zwitterionic lipid comprises a sulfonate anion.In some embodiments, the zwitterionic lipid further comprises a quaternary ammonium cation. In some embodiments, the zwitterionic lipid comprises an alkylated or alkenylated phosphate anion. In some embodiments, the alkylated or alkenylated phosphate anion has the structural formula: [ka] where R is alkyl or alkenyl, n is 1, 2, 3, 4, 5, or 6, and * indicates the point of attachment of the alkylated or alkenyl phosphate anion. In some embodiments, * indicates the point of attachment of the alkylated or alkenyl phosphate anion to the quaternary ammonium cation.

[0007] In another aspect, the disclosure provides a method of enhancing expression or activity of a cystic fibrosis transmembrane conductance regulator (CFTR) protein in a cell, comprising contacting the cell with a composition comprising a lipid composition and an assembled transfer ribonucleic acid (tRNA) to introduce an amino acid into a growing peptide chain of a CFTR protein in the cell, thereby producing a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell at least 48 hours after the contacting, wherein the therapeutically effective activity of the functional variant of the CFTR protein may be determined by measuring a change in transepithelial ion transport characteristic of a plurality of cells comprising the cell compared to that of a reference plurality of cells in the absence of the contacting.

[0008] In another aspect, the disclosure provides a method of enhancing expression or activity of a cystic fibrosis transmembrane conductance regulator (CFTR) protein in a cell of a subject exhibiting or suspected of exhibiting a mutation in the CFTR gene, comprising contacting the cell with a composition comprising a lipid composition and an assembled transfer ribonucleic acid (tRNA) to introduce an amino acid into a growing peptide chain of a CFTR protein in the cell at a position corresponding to the mutation in the CFTR gene of the subject, thereby resulting in a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell, the therapeutically effective activity of the functional variant of a CFTR protein being optionally determined by measuring a change in a transepithelial ion transport property of a plurality of cells comprising the cell compared to that of a reference plurality of cells in the absence of the contact. In some embodiments, the method results in a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell at least 72 hours after the contact. In some embodiments, the contact is repeated. In some embodiments, the contact is at least once a week. In some embodiments, the contact is at least twice a week. In some embodiments, the method provides a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell at least 24 hours after each contact. In some embodiments, the contact is a first contact and the method includes a second contact that may occur at least about 1, 2, or 3 days after the first contact. In some embodiments, the method further includes a third contact that may occur at least about 1, 2, or 3 days after the second contact. In some embodiments, the method provides a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell at least 24 hours after the second contact. In some embodiments, the method provides a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell at least 24 hours after the third contact. In some embodiments, the composition at each contact is the same. In some embodiments, the cell is a lung airway cell. In some embodiments, the cell is a lung secretory cell. In some embodiments, the cell is a bronchial epithelial cell. In some embodiments, the cell is undifferentiated.In some embodiments, the cells are differentiated. In some embodiments, the cells are derived from a subject. In some embodiments, the contacting is in vivo. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is ex vivo. In some embodiments, the functional variant of the CFTR protein is a wild-type CFTR protein. In some embodiments, the functional variant of the CFTR protein is a full-length CFTR protein. In some embodiments, the therapeutically effective activity of the functional variant of the CFTR protein corresponds to a transepithelial current of at least about 2 microamps (μA), e.g., as determined in an in vitro assay. In some embodiments, the therapeutically effective activity of the functional variant of the CFTR protein corresponds to a transepithelial current of about 2 microamps (μA) to about 30 μA, e.g., as determined in an in vitro assay. In some embodiments, the therapeutically effective activity of the functional variant of the CFTR protein corresponds to a transepithelial current of at least about 2 microamps (μA) per square centimeter per minute (μA·cm), e.g., as determined in an in vitro assay. -2 · minutes -1 ) corresponding to a transepithelial current of about 2 microamps (μA) per square centimeter per minute (μA cm ) in some embodiments. In some embodiments, the therapeutically effective activity of the functional variant of the CFTR protein corresponds to a transepithelial current of about 2 microamps (μA) per square centimeter per minute (μA cm ), as determined, for example, in an in vitro assay. -2 ·minute -1 ) ~ approx. 30 μA cm -2 ·minute -1In some embodiments, the method increases the amount or activity of a functional variant of a CFTR protein in the cell (e.g., at least about 1.1-fold) compared to a matched control (e.g., a cell of a non-contacted matched cell). In some embodiments, the method enhances ion transport (e.g., chloride) in the cell (e.g., at least about 1.1-fold) compared to a matched control (e.g., a cell of a non-contacted matched cell). In some embodiments, the mutation is a loss-of-function mutation. In some embodiments, the mutation is a nonsense mutation or a frameshift mutation. In some embodiments, the mutation is in one or more of exons 11-27 of the CFTR gene. In some embodiments, the mutation is R553X. In some embodiments, the tRNA is a suppressor tRNA. In some embodiments, the tRNA comprises a polynucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NOs: 1-20. In some embodiments, the composition comprising the tRNA assembled with the lipid composition is an aerosol. In some embodiments, the composition is formulated for apical delivery. In some embodiments, the composition is formulated for nebulization.

[0009] In another aspect, the disclosure provides a method of treating a subject having or suspected of having a cystic fibrosis transmembrane conductance regulator (CFTR)-associated condition, comprising administering to the subject a composition described elsewhere herein. In some embodiments, the CFTR-associated condition is cystic fibrosis, hereditary emphysema, or chronic obstructive pulmonary disease (COPD). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, administration comprises inhalation by nebulization.

[0010] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. Incorporation by Reference

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material.

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "FIG" and "FIG"). [Brief description of the drawings]

[0014] [Figure 1A] FIG. 1 shows rescue of CFTR function using a tRNA-LNP composition of the present disclosure with a single administration. [Figure 1B] FIG. 1 shows rescue of CFTR function using a tRNA-LNP composition of the present disclosure with a single administration.

[0015] [Figure 2A] FIG. 1 shows rescue of CFTR function using a tRNA-LNP composition of the present disclosure after repeated administration. [Figure 2B] FIG. 1 shows rescue of CFTR function using a tRNA-LNP composition of the present disclosure after repeated administration.

[0016] [Figure 3A] FIG. 1 shows rescue of CFTR function using tRNA compositions of the present disclosure over a time course study in G542X / F508del human bronchial epithelial cells (hBE). [Figure 3B] FIG. 1 shows rescue of CFTR function using tRNA compositions of the present disclosure over a time course study in G542X / F508del human bronchial epithelial cells (hBE). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may be made by those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be used.

[0018] The term "disease" as used herein generally refers to an abnormal physiological condition that affects some or all of a subject, e.g., a disease (e.g., primary ciliary dyskinesia) or another abnormality that causes defects in the action of cilia, for example, in the lining of the airways (upper and lower respiratory tract, sinuses, Eustachian tube, middle ear), in various lung cells, in the fallopian tubes, or in the flagella of sperm cells.

[0019] The term "polynucleotide" or "nucleic acid" as used herein generally refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, containing purine and pyrimidine bases, purine and pyrimidine analogs, chemically or biochemically modified, natural or non-natural, or derivatized nucleotide bases. Polynucleotides include sequences of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or DNA copies of ribonucleic acid (cDNA), all of which can be recombinantly produced, artificially synthesized, or isolated and purified from natural sources. Polynucleotides and nucleic acids can exist as single-stranded or double-stranded. The backbone of a polynucleotide can include sugar and phosphate groups, as typically found in RNA or DNA, or analogs or substituted sugar or phosphate groups. Polynucleotides can include naturally occurring or non-naturally occurring nucleotides, such as methylated nucleotides and nucleotide analogs (or analogs).

[0020] The term "polyribonucleotide" as used herein generally refers to a polynucleotide polymer that contains ribonucleic acid. This term also refers to a polynucleotide polymer that contains chemically modified ribonucleotides. Polyribonucleotides can be formed from d-ribose sugars that can be found in nature.

[0021] The term "polypeptide" as used herein generally refers to a polymeric chain composed of amino acid residue monomers linked together through amide bonds (peptide bonds). A polypeptide can be a chain of at least three amino acids, a protein, a recombinant protein, an antigen, an epitope, an enzyme, a receptor, or a structural analog or combination thereof. As used herein, the abbreviations for the l-enantiomeric amino acids forming the polypeptides are as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine ​​(C, Cys); glutamic acid (E, Glu); glutamine (Q, Gln); glycine (G, Gly); histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). X or Xaa can represent any amino acid.

[0022] The term "engineered" as used herein generally refers to polynucleotides, vectors, and nucleic acid constructs that have been genetically designed and engineered to provide a polynucleotide into a cell. Engineered polynucleotides can be partially or fully synthesized in vitro. Engineered polynucleotides can also be cloned. Engineered polyribonucleotides can contain one or more base or sugar analogs, such as ribonucleotides not naturally found in messenger RNA. Engineered polyribonucleotides can include nucleotide analogs present in transfer RNA (tRNA), ribosomal RNA (rRNA), guide RNA (gRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, spliced ​​leader RNA (SL RNA), CRISPR RNA, long non-coding RNA (lncRNA), microRNA (miRNA), or another suitable RNA.

[0023] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate (e.g., a non-human primate). In certain embodiments, the patient or subject is a human. Non-limiting examples of human subjects are adults, juveniles, infants, and fetuses.

[0024] The terms "assemble" or "assembled," as used herein, in the context of delivery of a payload to a target cell(s), generally refer to a covalent or non-covalent interaction(s) or association(s), such that, for example, a therapeutic or prophylactic agent is complexed to or encapsulated in a lipid composition.

[0025] As used herein, the term "lipid composition" generally refers to a composition comprising lipid compound(s), including, but not limited to, lipoplexes, liposomes, lipid particles. Examples of lipid compositions include suspensions, emulsions, and vesicle compositions.

[0026] As used herein, the term "detectable" refers to the occurrence or change of a signal that is directly or indirectly detectable, either by observation or measurement. Typically, the detectable response is the occurrence of a signal where the fluorophore is intrinsically fluorescent and does not result in a change in signal when bound to a metal ion or biological compound. Alternatively, the detectable response is an optical response that results in a change in the wavelength distribution pattern or the intensity of absorbance or fluorescence, or in light scattering, fluorescence lifetime, fluorescence polarization, or a combination of the above parameters. Other detectable responses include, for example, chemiluminescence, phosphorescence, radiation from radioisotopes, magnetic attraction, and electron density.

[0027] Unless otherwise indicated, all numbers expressing quantities, ranges, conditions, and the like used in the specification and claims are to be understood in all instances as being modified by the term "about". Thus, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present application. In general, the term "about" as used herein when referring to measurable values ​​such as amounts of weight, time, dose, and the like, is meant to encompass variations of ±20% or ±10% in one example, ±5% in another example, ±1% in another example, and ±0.1% from the specified amount, such variations being appropriate for carrying out the disclosed methods.

[0028] As used herein, the term "ratio" generally refers to the relative amount of one or more molecules to another molecule(s). Non-limiting examples of ratio(s) include molar ratio(s), weight ratio(s), or mass ratio(s).

[0029] When used in the context of a chemical group: "hydrogen" means -H, "hydroxy" means -OH, "oxo" means =O, "carbonyl" means -C(=O)-, "carboxy" means -C(=O)OH (also written as -COOH or -COH), "halo" means, independently, -F, -Cl, -Br or -I, and "amino" means -NH 2 "hydroxyamino" means -NHOH, and "nitro" means -NO 2 "imino" means =NH, "cyano" means -CN, "isocyanate" means -N=C=O, and "azido" means -N 3 In the monovalent context, "phosphate" means -OP(O)(OH) 2 or its deprotonated form, and in the divalent context, "phosphate" means -OP(O)(OH)O- or its deprotonated form, "mercapto" means -SH, "thio" means =S, and "sulfonyl" means -S(O) 2 - and "hydroxysulfonyl" is -S(O)2 OH, and "sulfonamide" is -S(O) 2 NH 2 "Sulfinyl" means -S(O)-.

[0030] In the context of chemical formulas, the symbol "-" denotes a single bond, "=" denotes a double bond, and "≡" denotes a triple bond. [ka] " represents any bond, if present, which is either a single bond or a double bond. [ka] " represents a single or double bond. Thus, for example, the formula [ka] teeth, [ka] , [ka] , [ka] , [ka] and [ka] and it is understood that no such ring atom forms part of more than one double bond. It should further be noted that the covalent bond symbol "-" does not indicate any preferred stereochemistry when linking one or two stereogenic atoms. Instead, it encompasses all stereoisomers as well as mixtures thereof. The symbol " [ka] " is a bond (e.g., for methyl [ka] When drawn vertically across a group, it indicates the point of attachment of the group. Note that points of attachment are typically only identified in this manner relative to a larger group to help the reader clearly identify the point of attachment. [ka] " means a single bond where the group attached to the thick end of the wedge is "off the page". [ka] " means a single bond where the group attached to the thick end of the wedge is "into the page." [ka] " means a single bond with no defined geometry around the double bond (e.g., either E or Z). Thus, both options and combinations thereof are contemplated. Any undefined valence on an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom. A bold dot on a carbon atom indicates that the hydrogen bonded to that carbon is oriented out of the plane of the paper.

[0031] When the group "R" is depicted as a "floating group" on a ring system, for example, the formula: [ka] , R may replace any hydrogen atom attached to any of the ring atoms, including hydrogens shown, implied, or explicitly defined, so long as a stable structure is formed. When the group "R" is shown as a "floating group" on a fused ring system, for example, in the formula: [ka] , R may replace any hydrogen bonded to any ring atom of any of the fused rings, unless otherwise specified. Substitutable hydrogens include depicted hydrogens (e.g., hydrogens bonded to nitrogens in the formula above), implied hydrogens (e.g., hydrogens in the formula above that are not depicted but are understood to be present), explicitly defined hydrogens, and any hydrogens whose presence depends on the identity of the ring atom (e.g., hydrogens bonded to group X when X is equal to -CH-), so long as a stable structure is formed. In the illustrated example, R may be present in either the 5-membered or 6-membered ring of the fused ring system. In the formula above, the subscript "y" immediately following the bracketed group "R" represents a numerical variable. Unless otherwise specified, this variable may be 0, 1, 2, or any integer greater than 2, limited only by the maximum number of substitutable hydrogen atoms in the ring or ring system.

[0032] For chemical groups and compound classes, the number of carbon atoms in the group or class is indicated as follows: "Cn" defines the exact number (n) of carbon atoms in the group / class. "C≦n" defines the maximum number (n) of carbon atoms that may be in the group / class, being the smallest possible minimum number for the group / class in question, e.g., the group "alkenyl (C≦8) " or class "Alkenes (C≦8) It is understood that the minimum number of carbon atoms in is 2. "Alkoxy" refers to an alkoxy group having 1 to 10 carbon atoms. (C≦10) "Cn-n'" defines both the minimum (n) and maximum (n') number of carbon atoms in the group. Thus, "alkyl (C2-10) " denotes an alkyl group having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical group or class they modify, and may or may not be enclosed in parentheses without indicating a change in meaning. Thus, "C5 olefin," "C5-olefin," "olefin (C5) " and "Olefin C5 " are all synonymous.

[0033] The term "saturated", when used to modify a compound or chemical group, means that the compound or chemical group has no carbon-carbon double bonds and carbon-carbon triple bonds, except as described below. When the term is used to modify an atom, it means that the atom is not part of a double or triple bond. In the case of substituted forms of saturated groups, one or more carbon-oxygen or carbon-nitrogen double bonds may be present. And, when such bonds are present, carbon-carbon double bonds that may occur as part of keto-enol or imine / enamine tautomerism are not excluded. When the term "saturated" is used to modify a solution of a substance, it means that the substance is no longer soluble in the solution.

[0034] The term "aliphatic", when used without the "substituted" modifier, means that the compound or chemical group so modified is an acyclic or cyclic, but non-aromatic, hydrocarbon compound or group. In an aliphatic compound / group, the carbon atoms can be bonded together in a straight chain, branched chain, or non-aromatic ring (alicyclic). An aliphatic compound / group can be saturated, bonded by a single carbon-carbon bond (alkane / alkyl), or unsaturated, with one or more carbon-carbon double bonds (alkene / alkenyl) or one or more carbon-carbon triple bonds (alkyne / alkynyl).

[0035] The term "aromatic," when used to modify an atom of a compound or chemical moiety, means that the compound or chemical moiety contains a planar unsaturated ring of atoms stabilized by the interaction of the bonds that form the ring.

[0036] The term "alkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group that contains a carbon atom as the point of attachment, has a straight or branched non-cyclic structure, and contains no atoms other than carbon and hydrogen. 3 (Me), -CH 2 CH 3 (Et), -CH 2 CH 2 CH3 (n-Pr or propyl), -CH(CH 3 ) 2 (i-Pr 、i Pr or isopropyl), -CH 2 CH 2 CH 2 CH 3 (n-Bu), -CH(CH 3 )CH 2 CH 3 (sec-Butyl), -CH 2 CH(CH 3 ) 2 (isobutyl), -C(CH 3 ) 3 (tert-Butyl, t-Butyl, t-Bu or t Bu) and -CH 2 C(CH 3 ) 3 The group (neopentyl) is a non-limiting example of an alkyl group. The term "alkanediyl", when used without the "substituted" modifier, refers to a divalent saturated aliphatic group having one or two saturated carbon atoms as the point(s) of attachment, a straight or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The group -CH 2 -(methylene), -CH 2 CH 2 -, -CH 2 C(CH 3 ) 2 CH 2 - and -CH 2 CH 2 CH 2 - is a non-limiting example of an alkanediyl group. "Alkane" refers to the class of compounds having the formula HR, where R is alkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2 The following groups are non-limiting examples of substituted alkyl groups: -CH 2 OH, -CH 2 Cl, -CF 3 , -CH 2 CN, -CH 2 C(O)OH, -CH 2 C(O)OCH 3 , -CH 2 C(O)NH 2 , -CH 2 C(O)CH 3 , -CH 2 OCH 3 , -CH 2 OC(O)CH 3 , -CH 2 NH 2 , -CH 2 N(CH 3 ) 2 , and -CH 2 CH 2 The term "haloalkyl" is a subset of substituted alkyl, where the replacement of hydrogen atoms is limited to halo (i.e., -F, -Cl, -Br, or -I) such that no other atoms other than carbon, hydrogen, and halogen are present. -CH 2 The Cl group is a non-limiting example of a haloalkyl. The term "fluoroalkyl" is a subset of substituted alkyl, in which the replacement of hydrogen atoms is limited to fluoro, such that no other atoms other than carbon, hydrogen, and fluorine are present. The group -CH 2 F, -CF 3 and -CH 2 CF3 are non-limiting examples of fluoroalkyl groups.

[0037] The term "cycloalkyl," when used without the "substituted" modifier, refers to a monovalent saturated aliphatic group that contains a carbon atom as the point of attachment, that forms part of one or more non-aromatic ring structures, that does not contain carbon-carbon double or triple bonds, and that does not contain any atoms other than carbon and hydrogen. Non-limiting examples include -CH(CH 2 ) 2 (cyclopropyl), cyclobutyl, cyclopentyl or cyclohexyl (Cy). The term "cycloalkanediyl" when used without the "substituted" modifier refers to a divalent saturated aliphatic group having two carbon atoms as points of attachment, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. [ka] is a non-limiting example of a cycloalkanediyl group. "Cycloalkane" refers to the class of compounds having the formula HR, where R is cycloalkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O)2 OH or -S(O) 2 NH 2 has been replaced by

[0038] The term "alkenyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group that contains a carbon atom as the point of attachment, a straight or branched acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. Non-limiting examples include -CH=CH 2 (vinyl), -CH=CHCH 3 , -CH=CHCH 2 CH 3 , -CH 2 CH=CH 2 (allyl), -CH 2 CH=CHCH 3 and -CH=CHCH=CH 2 The term "alkenediyl," when used without the "substituted" modifier, refers to a divalent unsaturated aliphatic group with two carbon atoms as points of attachment, a straight or branched chain, a straight or branched chain acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bonds, and no atoms other than carbon and hydrogen. The groups -CH=CH-, -CH=C(CH 3 )CH 2 -, -CH=CHCH 2 -, and-CH 2 CH=CHCH 2- is a non-limiting example of an alkenediyl group. It should be noted that although alkenediyl groups are aliphatic, once attached at both ends, this does not exclude the group from forming part of an aromatic structure. The terms "alkene" and "olefin" are synonymous and refer to a class of compounds having the formula HR, where R is alkenyl as this term is defined above. Similarly, the terms "terminal alkene" and "α-olefin" are synonymous and refer to an alkene with only one carbon-carbon double bond, which is part of a vinyl group at the end of the molecule. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH, or-S(O) 2 NH 2 The groups -CH=CHF, -CH=CHCl, and -CH=CHBr are non-limiting examples of substituted alkenyl groups.

[0039] The term "alkynyl," when used without the "substituted" modifier, refers to a monovalent unsaturated aliphatic group that contains a carbon atom as the point of attachment, a straight or branched non-cyclic structure, contains at least one carbon-carbon triple bond, and contains no atoms other than carbon and hydrogen. As used herein, the term alkynyl does not preclude the presence of one or more non-aromatic carbon-carbon double bonds. The groups -C≡CH, -C≡CCH 3 , and -CH 2 C≡CCH 3 is a non-limiting example of an alkynyl group. "Alkyne" refers to the class of compounds having the formula HR where R is alkynyl. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH, or-S(O) 2 NH 2 has been replaced by

[0040] The term "aryl", when used without the "substituted" modifier, refers to a monovalent unsaturated aromatic group having an aromatic carbon atom as the point of attachment, the carbon atom forming part of one or more six-membered aromatic ring structures, the ring atoms being all carbon, and the group consisting of no atoms other than carbon and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl or aralkyl groups (possibly limited in number of carbon atoms) attached to the first aromatic ring or to any additional aromatic rings present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl, (dimethyl)phenyl, -C 6 H 4 CH 2 CH 3 (ethylphenyl), naphthyl, and biphenyl. The term "arenediyl", when used without the "substituted" modifier, refers to a divalent aromatic group having two aromatic carbon atoms as attachment points, the carbon atoms forming part of one or more six-membered aromatic ring structures, the ring atoms being all carbon, and the monovalent group consisting of no atoms other than carbon and hydrogen. As used herein, the term does not preclude the presence of one or more alkyl, aryl, or aralkyl groups (carbon number limitations possible) attached to the first aromatic ring or to any additional aromatic rings present. When two or more rings are present, the rings may be fused or unfused. Non-fused rings may be linked via one or more of a covalent bond, an alkanediyl, or an alkenediyl group (carbon number limitations possible). Non-limiting examples of arenediyl groups include: [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , and [ka] . "Arene" refers to the class of compounds having the formula HR, where R is aryl, as that term is defined above. Benzene and toluene are non-limiting examples of arenes. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH, or-S(O) 2 NH 2 has been replaced by

[0041] The term "aralkyl" when used without the "substituted" modifier refers to the monovalent group -alkanediyl-aryl, where the terms alkanediyl and aryl are each used in a manner consistent with the definitions provided above. Non-limiting examples are phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term aralkyl is used with the "substituted" modifier, one or more hydrogen atoms from the alkanediyl and / or aryl groups are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2 Non-limiting examples of substituted aralkyls are (3-chlorophenyl)-methyl and 2-chloro-2-phenyl-ethyl-1-yl.

[0042] The term "heteroaryl", when used without the "substituted" modifier, refers to a monovalent aromatic group having an aromatic carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the heteroaryl group consisting of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. The heteroaryl ring may contain 1, 2, 3 or 4 ring atoms selected from nitrogen, oxygen and sulfur. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not exclude the presence of one or more alkyl, aryl and / or aralkyl groups (which may be limited in number of carbon atoms) attached to the aromatic ring or aromatic ring system. Non-limiting examples of heteroaryl groups include furanyl, imidazolyl, indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl, phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers to a heteroaryl group having a nitrogen atom as the point of attachment. The term "heteroarenediyl," when used without the "substituted" modifier, refers to a divalent aromatic group having two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon atom and one aromatic nitrogen atom as the points of attachment, the atoms forming part of one or more aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen, or sulfur, and the divalent group consisting of no atoms other than carbon, hydrogen, aromatic nitrogen, aromatic oxygen, and aromatic sulfur. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be linked via one or more covalent bonds, alkanediyl, or alkenediyl groups (carbon number limit possible). As used herein, the term does not preclude the presence of one or more alkyl, aryl, and / or aralkyl groups (carbon number limit possible) attached to an aromatic ring or aromatic ring system. Non-limiting examples of heteroarenediyl groups include: [ka] , [ka] and [ka] . "Heteroarene" refers to the class of compounds having the formula HR, where R is heteroaryl. Pyridine and quinoline are non-limiting examples of heteroarenes. When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2 has been replaced by

[0043] The term "heterocycloalkyl", when used without the "substituted" modifier, refers to a monovalent non-aromatic group having a carbon or nitrogen atom as the point of attachment, the carbon or nitrogen atom forming part of one or more non-aromatic ring structures, at least one of the ring atoms being nitrogen, oxygen or sulfur, and the heterocycloalkyl group not consisting of atoms other than carbon, hydrogen, nitrogen, oxygen and sulfur. Heterocycloalkyl rings may contain 1, 2, 3 or 4 ring atoms selected from nitrogen, oxygen or sulfur. When more than one ring is present, the rings may be fused or unfused. As used herein, the term does not preclude the presence of one or more alkyl groups (which may limit the number of carbon atoms) attached to a ring or ring system. Also, the term does not preclude the presence of one or more double bonds in the ring or ring system, provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl, oxiranyl, and oxetanyl. The term "N-heterocycloalkyl" refers to a heterocycloalkyl group having a nitrogen atom as the point of attachment. N-pyrrolidinyl is an example of such a group. The term "heterocycloalkanediyl", when used without the "substituted" modifier, refers to a divalent cyclic group having two carbon atoms, two nitrogen atoms, or one carbon atom and one nitrogen atom as the point of attachment, which atoms form part of one or more ring structures, at least one of the ring atoms is nitrogen, oxygen, or sulfur, and the divalent group is composed of no atoms other than carbon, hydrogen, nitrogen, oxygen, and sulfur. When more than one ring is present, the rings may be fused or unfused. Non-fused rings may be linked via one or more covalent bonds, alkanediyl, or alkenediyl groups (which may be limited in number of carbon atoms). As used herein, the term does not preclude the presence of one or more alkyl groups (which may be limited in number of carbon atoms) attached to the ring or ring system.Nor does the term preclude the presence of one or more double bonds in the ring or ring system provided that the resulting group remains non-aromatic. Non-limiting examples of heterocycloalkanediyl groups include: [ka] , [ka] and [ka] . When these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2 has been replaced by

[0044] The term "acyl," when used without the "substituted" modifier, refers to the group -C(O)R, where R is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aralkyl, or heteroaryl, as these terms are defined above. -CHO, -C(O)CH 3(Acetyl, Ac), -C(O)CH 2 CH 3 , -C(O)CH 2 CH 2 CH 3 , -C(O)CH(CH 3 ) 2 , -C(O)CH(CH 2 ) 2 , -C(O)C 6 H 5 , -C(O)C 6 H 4 CH 3 , -C(O)CH 2 C 6 H 5 , -C(O) (imidazolyl) groups are non-limiting examples of acyl groups. "Thioacyl" is similarly defined except that the oxygen atom of the group -C(O)R is replaced with a sulfur atom, -C(S)R. The term "aldehyde" corresponds to an alkane, as defined above, where at least one of the hydrogen atoms has been replaced with a -CHO group. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms (including the hydrogen atom directly bonded to the carbon atom of the carbonyl or thiocarbonyl group, if present) are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2The group -C(O)CH 2 CF 3 , -CO 2 H(carboxyl), -CO 2 CH 3 (Methylcarboxyl), CO 2 CH 2 CH 3 , -C(O)NH 2 (carbamoyl) and -CON(CH 3 ) 2 is a non-limiting example of a substituted acyl group.

[0045] The term "alkoxy," when used without the "substituted" modifier, refers to the group --OR, where R is alkyl, as that term is defined above. Non-limiting examples include, --OCH 3 (Methoxy), -OCH 2 CH 3 (ethoxy), -OCH 2 CH 2 CH 3 , -OCH(CH 3 ) 2 (isopropoxy), -OC(CH 3 ) 3 (tert-butoxy), -OCH(CH 2 ) 2, -O-cyclopentyl, and -O-cyclohexyl. The terms "cycloalkoxy", "alkenyloxy", "alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy", "heterocycloalkoxy", and "acyloxy", when used without the "substituted" modifier, refer to the group defined as -OR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl, respectively. The term "alkoxydiyl" refers to the divalent groups -O-alkanediyl-, -O-alkanediyl-O-, or -alkanediyl-O-alkanediyl-. The terms "alkylthio" and "acylthio", when used without the "substituted" modifier, refer to the group -SR, where R is alkyl and acyl, respectively. The term "alcohol" corresponds to an alkane, as defined above, where at least one of the hydrogen atoms has been replaced with a hydroxy group. The term "ether" corresponds to an alkane, as defined above, where at least one of the hydrogen atoms has been replaced with an alkoxy group. When any of these terms are used with the "substituted" modifier, one or more hydrogen atoms are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH or -S(O) 2 NH 2has been replaced by

[0046] The term "alkylamino," when used without the "substituted" modifier, refers to the group --NHR, where R is alkyl, as that term is defined above. Non-limiting examples include --NHCH 3 and -NHCH 2 CH 3 The term "dialkylamino," when used without the "substituted" modifier, refers to the group --NRR', where R and R' can be the same or different alkyl groups, or R and R' together can represent an alkanediyl. Non-limiting examples of dialkylamino groups include --N(CH 3 ) 2 and -N(CH 3 )(CH 2 CH 3 The terms "cycloalkylamino", "alkenylamino", "alkynylamino", "arylamino", "aralkylamino", "heteroarylamino", "heterocycloalkylamino", "alkoxyamino" and "alkylsulfonylamino", when used without the "substituted" modifier, refer to the group defined as -NHR, where R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. A non-limiting example of an arylamino group is -NHC 6 H 5 The term "alkylaminodiyl" refers to the divalent group -NH-alkanediyl-, -NH-alkanediyl-NH-, or -alkanediyl-NH-alkanediyl-. The term "amido" (acylamino), when used without the "substituted" modifier, refers to the group -NHR, where R is acyl, as that term is defined above. A non-limiting example of an amido group is -NHC(O)CH 3The term "alkylimino," when used without the "substituted" modifier, refers to the divalent group =NR, where R is alkyl, as that term is defined above. When any of these terms are used with the "substituted" modifier, one or more of the hydrogen atoms bonded to the carbon atom are independently replaced with -OH, -F, -Cl, -Br, -I, -NH 2 , -NO 2 , -CO 2 H, -CO 2 CH 3 , -CN, -SH, -OCH 3 , -OCH 2 CH 3 , -C(O)CH 3 , -NHCH 3 , -NHCH 2 CH 3 , -N(CH 3 ) 2 , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)N(CH 3 ) 2 , -OC(O)CH 3 , -NHC(O)CH 3 , -S(O) 2 OH, or-S(O) 2 NH 2 The group -NHC(O)OCH 3 and -NHC(O)NHCH 3 is a non-limiting example of a substituted amide group.

[0047] The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0048] As used in this application, the term "average molecular weight" refers to the relationship between the number of moles of each polymer species and the molar mass of that species. In particular, each polymer molecule may have a different level of polymerization and therefore a different molar mass. Average molecular weight can be used to describe the molecular weight of multiple polymer molecules. Average molecular weight is typically synonymous with average molar mass. In particular, there are three main types of average molecular weight: number average molar mass, weight (mass) average molar mass, and Z-average molar mass. In the context of this application, unless otherwise specified, average molecular weight refers to either the number average molar mass or the weight average molar mass of the formula. In some embodiments, the average molecular weight is the number average molar mass. In some embodiments, average molecular weight can be used to describe the PEG components present in the lipid.

[0049] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but also encompasses other unlisted steps.

[0050] The term "effective," as that term is used in the specification and / or claims, means sufficient to achieve a desired, expected, or intended result. An "effective amount," "therapeutically effective amount," or "pharmacologically effective amount," when used in the context of treating a patient or subject with a compound, means the amount of the compound that, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease.

[0051] As used herein, "IC 50The term "inhibitory dose" refers to an inhibitory dose that is 50% of the maximum response obtained. This quantitative measure indicates the amount of a particular drug or other substance (inhibitor) required to inhibit half a given biological, biochemical, or chemical process (or a component of the process, i.e., an enzyme, cell, cell receptor, or microorganism).

[0052] An "isomer" of a first compound is a distinct chemical compound in which each molecule contains the same constituent atoms as the first compound, but the atoms are arranged in a different spatial configuration.

[0053] As used herein, the term "patient" or "subject" refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human subjects are adults, juveniles, infants, and fetuses.

[0054] As generally used herein, "pharmacologically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues, organs, and / or body fluids of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0055] "Pharmaceutically acceptable salt" refers to a salt of a compound of the present disclosure that is pharma- ceutically acceptable as defined above and has the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4'-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acid, aromatic sulfuric acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethylenediaminetetraacetic ... Pharmaceutically acceptable salts include acid addition salts formed with organic acids such as ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, lauryl sulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiary butyl acetic acid, trimethyl acetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts that may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide, and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. It should be recognized that the particular anion or cation forming part of any salt of the present disclosure is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Further examples of pharmaceutically acceptable salts and their methods of preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C.G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).

[0056] "Prevention" or "preventing" includes (1) inhibiting the onset of a disease in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the pathology or symptomology of the disease, and / or (2) delaying the onset of disease symptoms or symptomology in a subject or patient who may be at risk and / or predisposed to the disease, but who has not yet experienced or displayed any or all of the pathology or symptomology of the disease.

[0057] A "repeat unit" is the simplest structural entity of a particular material, e.g., a framework and / or polymer, whether organic, inorganic, or metal-organic. In the case of a polymer chain, the repeat units are linked together consecutively along the chain, like beads on a necklace. For example, in polyethylene, -[-CH 2 CH 2 -] n -The repeating unit is -CH 2 CH 2 -. The subscript "n" indicates the degree of polymerization, i.e., the number of repeat units linked together. If the value of "n" is left undefined or is absent, it simply indicates the repetition of the formula within the brackets and the polymeric nature of the material. The concept of repeat units applies equally when the connectivity between repeat units extends in three dimensions, such as in metal organic frameworks, modified polymers, thermosetting polymers, etc. Within the context of dendrimers, the repeat units may be described as branch units, internal layers, or generations. Similarly, the terminal groups may be described as surface groups.

[0058] "Stereoisomers" or "optical isomers" are isomers of a given compound that have the same atoms bonded to the same other atoms but differ in the arrangement of those atoms in three dimensions. "Enantiomers" are stereoisomers of a given compound that are mirror images of each other, like left and right hands. "Diastereomers" are stereoisomers of a given compound that are not enantiomers. Chiral molecules contain a chiral center, also called a stereogenic center or stereogenic center, which is any point in a molecule that has groups such that the exchange of any two groups results in a stereoisomer, but not necessarily an atom. In organic compounds, chiral centers are typically carbon, phosphorus, or sulfur atoms, although other atoms can be stereogenic centers in organic and inorganic compounds. Molecules can have multiple stereocenters, giving many stereoisomers. In compounds where stereoisomerism is due to tetrahedral stereocenters (e.g., tetrahedral carbon), the total number of hypothetical possible stereoisomers is 2 n n is the number of tetrahedral stereocenters. Molecules with symmetry often have fewer than the maximum number of possible stereoisomers. A 50:50 mixture of enantiomers is called a racemic mixture. Alternatively, a mixture of enantiomers can be enantiomerically enriched such that one enantiomer is present in an amount greater than 50%. Typically, enantiomers and / or diastereomers can be resolved or separated using techniques known in the art. For any stereocenter or chirality axis with undefined stereochemistry, it is contemplated that the stereocenter or chirality axis may be present in its R form, S form, or as a mixture of R and S forms, including racemic and non-racemic mixtures. As used herein, the phrase "substantially free of other stereoisomers" means that the composition contains 15% or less, more preferably 10% or less, even more preferably 5% or less, or most preferably 1% or less of another stereoisomer(s).

[0059] "Treatment" or "treating" includes (1) inhibiting a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease (e.g., preventing further development of the pathology and / or symptomology), (2) ameliorating a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease (e.g., reversing the pathology and / or symptoms), and / or (3) causing any measurable decrease in a disease in a subject or patient experiencing or exhibiting the pathology or symptomology of the disease.

[0060] The term "mole percent" or "mole %" as used herein in reference to lipid composition(s) generally refers to the molar ratio of that component lipid compared to all lipids formulated or present in the lipid composition.

[0061] The above definitions supersede any conflicting definitions in any references incorporated herein by reference.However, the fact that a particular term is defined should not be taken to indicate that the undefined term is unclear.Rather, all terms used are considered to describe the present disclosure in terms that allow a person skilled in the art to understand the scope and implementation of the present disclosure. Cystic fibrosis transmembrane conductance regulator (CFTR)

[0062] Cystic fibrosis transmembrane conductance regulator (CFTR) is a vertebrate membrane protein and chloride channel encoded by the CFTR gene. The CFTR gene is located on the long arm of chromosome 7 at position q31.2. Mutations in the CFTR gene that affect chloride ion channel function result in dysregulation of epithelial fluid transport in the lungs, pancreas, and other organs, resulting in cystic fibrosis (CF).

[0063] Cystic fibrosis (CF) affects approximately 1 in every 2,500 infants in the United States. Within the general U.S. population, up to 10 million people carry a single copy of the defective gene with no apparent ill effects. In contrast, individuals with two copies of the CF-associated gene suffer the debilitating and fatal effects of CF, including chronic lung disease. Complications of cystic fibrosis include thickening of mucus in the lungs with frequent respiratory infections, and pancreatic insufficiency leading to malnutrition and diabetes. These conditions result in chronic disability and shortened life expectancy. In male patients, progressive obstruction and destruction of the developing vas deferens (spermatic cord) and epididymis, likely due to abnormal intraluminal secretions, causes congenital absence of the vas deferens and male infertility.

[0064] Nearly 1000 cystic fibrosis-causing mutations have been described so far. Most mutations are rare. The distribution and frequency of mutations vary between different populations. Mutations consist of substitutions, duplications, deletions or truncations in the CFTR gene. This can result in a dysfunctional protein that is less active, degraded more rapidly, or present in insufficient numbers. The most common mutation, DeltaF508 (ΔF508), is due to a deletion (Δ) of three nucleotides that results in the loss of the 508th amino acid phenylalanine (F) of the protein. As a result, the protein does not fold normally and is degraded more rapidly. composition

[0065] In one aspect, the present disclosure provides a composition comprising a synthetic transfer ribonucleic acid (tRNA) as described herein in combination with a lipid composition as described herein. The lipid composition may comprise a zwitterionic lipid. The composition may be an aerosol composition. The composition may be formulated for aerosol administration. Transfer RNA (tRNA)

[0066] As used herein, the term transfer RNA or tRNA refers to both conventional tRNA molecules and tRNA molecules with one or more modifications, unless otherwise specified. Transfer RNA is an RNA polymer about 70-100 nucleotides long. During protein synthesis, tRNA delivers amino acids to the ribosome for addition to the growing peptide chain. Active tRNA has a 3' CCA tail that can be transcribed into the tRNA during its synthesis or added later during post-transcriptional processing. An amino acid is covalently attached to the 2' or 3' hydroxyl group of the 3'-terminal ribose to form an aminoacyl-tRNA (aa-tRNA), which is incorporated into the growing protein chain at the ribosome from the 3'-OH position, although the amino acid can spontaneously migrate from the 2'-OH to the 3'-OH and vice versa. The loop at the other end of the folded aa-tRNA molecule contains a three-base sequence known as the anticodon. When this anticodon sequence base pairs with a three-base codon sequence in the ribosome-bound messenger RNA (mRNA), the aa-tRNA binds to the ribosome and incorporates that amino acid into the nascent protein chain. Translation of the genetic code is carried out by tRNAs, as every tRNA that base pairs with a particular codon is aminoacylated with a single specific amino acid: each of the 61 non-terminating codons in the mRNA directs the binding of its cognate aa-tRNA and the addition of a single specific amino acid to the growing protein polymer. In some embodiments, the tRNA may contain a sequence in the anticodon region of the tRNA such that the aa-tRNA base pairs with a different codon on the mRNA. In certain embodiments, the mutant tRNA introduces an amino acid into the growing protein chain that is different from the amino acid encoded by the mRNA. In other embodiments, the mutant tRNA base pairs with a stop codon and introduces an amino acid instead of terminating protein synthesis, thereby allowing the nascent peptide to continue growing. In some embodiments, a wild-type or mutant tRNA can read a stop codon and introduce an amino acid instead of terminating protein synthesis, hi some embodiments, a tRNA can include a full-length tRNA that includes a 3' terminal CCA nucleotide.In another embodiment, a tRNA lacking a 3'-terminal -A, -CA or CCA is made full length in vivo by a CCA-adding enzyme.

[0067] In other embodiments, the composition may further comprise one or more modified tRNA molecules including: acylated tRNA; alkylated tRNA; tRNA containing one or more bases other than adenine, cytosine, guanine, or uracil; tRNA covalently modified by attachment of a specific ligand or an antigenic, fluorescent, affinity, reactive, spectral or other probing moiety; tRNA containing one or more ribose moieties that are methylated or otherwise modified; tRNA aminoacylated with an amino acid other than the 20 naturally occurring amino acids, including unnatural amino acids that function as reagents, carriers for specific ligands, or as antigenic, fluorescent, reactive, affinity, spectral or other probes; or any combination of these compositions. Some examples of modified tRNA molecules are taught by Soll, et al., 1995; El Yacoubi, et al., 2012; Grosjean and Benne, et al., 1998; Hendrickson, et al., 2004; Ibba and Soll, 2000; Johnson, et al., 1995; Johnson, et al., 1982; Crowley, et al., 1994; Beier and Grimm, 2001; Tones, et al., 2014; and Bjork, et al., 1987, all of which are incorporated by reference herein.

[0068] In some embodiments, the synthetic tRNA is a folded tRNA. The folded tRNA may be folded so that the tRNA can perform a function. For example, the folded tRNA may include a folded shape that allows recognition of a codon or allows loading of an amino acid. The folded tRNA may perform a function that an unfolded tRNA cannot perform. The folded tRNA may include a T arm, a D arm, an anticodon arm, a variable loop, an acceptor stem, or a combination thereof. The folded tRNA may include a motif, structure, or sequence that can perform a specific function. The synthetic tRNA may include an anticodon arm that is configured to recognize a premature stop codon. The synthetic tRNA may include an anticodon arm that can recognize a codon that would normally code for an amino acid or a stop codon, and the acceptor stem may be configured to be operably linked to a non-corresponding amino acid or a stop codon. For example, a synthetic tRNA can include an anticodon arm capable of recognizing a stop codon, and the acceptor stem can be configured to be operably linked to an amino acid. This can allow the tRNA to recognize a premature stop codon and add an amino acid to a polypeptide chain instead of causing or allowing translation to terminate. This can allow the tRNA to prevent premature termination of polypeptide translation.

[0069] In some embodiments, the synthetic tRNA comprises an acceptor stem configured to be operably linked to an amino acid. The synthetic tRNA may comprise an acceptor stem configured to be operably linked to an arginine. The synthetic tRNA may comprise an acceptor stem configured to be operably linked to alanine. The synthetic tRNA may comprise an acceptor stem configured to be operably linked to cysteine. The synthetic tRNA may comprise an acceptor stem configured to be operably linked to aspartic acid. The synthetic tRNA may comprise an acceptor stem configured to be operably linked to glutamic acid. The synthetic tRNA may comprise an acceptor stem configured to be operably linked to phenylalanine. The synthetic tRNA may comprise an acceptor stem configured to be operably linked to histidine. The synthetic tRNA may comprise an acceptor stem configured to be operably linked to isoleucine. The synthetic tRNA may include an acceptor stem configured to be operably linked to a lysine. The synthetic tRNA may include an acceptor stem configured to be operably linked to a leucine. The synthetic tRNA may include an acceptor stem configured to be operably linked to a methionine. The synthetic tRNA may include an acceptor stem configured to be operably linked to an asparagine. The synthetic tRNA may include an acceptor stem configured to be operably linked to a proline. The synthetic tRNA may include an acceptor stem configured to be operably linked to a glutamine. The synthetic tRNA may include an acceptor stem configured to be operably linked to a serine. The synthetic tRNA may include an acceptor stem configured to be operably linked to a threonine. The synthetic tRNA may include an acceptor stem configured to be operably linked to a valine. The synthetic tRNA may include an acceptor stem configured to be operably linked to a tryptophan. The synthetic tRNA can include an acceptor stem configured to be operably linked to a tyrosine.

[0070] In some embodiments, the tRNA is a tRNA amber suppressor. In other embodiments, the tRNA is a tRNA opal suppressor. In other embodiments, the tRNA is a tRNA ochre suppressor. In some embodiments, the tRNA is a tRNA frameshift suppressor.

[0071] In some embodiments, the synthetic tRNA comprises a nucleic acid sequence selected from SEQ ID NOs: 1-20. In some embodiments, the synthetic tRNA comprises a nucleic acid sequence having at least about 80% identity to a sequence selected from SEQ ID NOs: 1-20. In some embodiments, the synthetic tRNA comprises a nucleic acid sequence having at least about 85% identity to a sequence selected from SEQ ID NOs: 1-20. In some embodiments, the synthetic tRNA comprises a nucleic acid sequence having at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to a sequence selected from SEQ ID NOs: 1-20. In some embodiments, the synthetic tRNA comprises a nucleic acid sequence identical to a sequence selected from SEQ ID NOs: 1-20. [Table 1-1] [Table 1-2] lipid composition Zwitterionic lipids

[0072] In some embodiments, the lipid compositions of the present application comprise a molar percentage of zwitterionic lipids of about 1% to about 60%. In some embodiments, the lipid compositions comprise a molar percentage of zwitterionic lipids of at least (about) 1%, at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, at least (about) 55%, or at least (about) 60%. In some embodiments, the lipid composition comprises a molar percentage of zwitterionic lipids of up to (about) 60%, up to (about) 55%, up to (about) 50%, up to (about) 45%, up to (about) 40%, up to (about) 35%, up to (about) 30%, up to (about) 25%, up to (about) 20%, at least (about) 15%, up to (about) 10%, or up to (about) 5%. In some embodiments, the lipid composition comprises a molar percentage of zwitterionic lipids of (about) 1%, (about) 2%, (about) 5%, (about) 10%, (about) 15%, (about) 20%, (about) 25%, (about) 30%, (about) 35%, (about) 40%, (about) 45%, (about) 50%, (about) 55%, or (about) 60%, or a range between any two of the foregoing values.

[0073] In some embodiments of the lipid composition, the zwitterionic lipid is a zwitterionic phospholipid.

[0074] In some embodiments of the lipid composition, the zwitterionic lipid comprises a sulfonate anion. The zwitterionic lipid may further comprise a quaternary ammonium cation.

[0075] In some embodiments, the zwitterionic lipid has structural formula (I): [ka] (I) or a pharma- ceutically acceptable salt thereof; X 1 -S(O) 2 O - OR e O- where: R e is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) and Y 1 is an alkanediyl (C≦12) , Alkene Diyl (C≦12) or a substitution thereof, A is -NR a -, -S-, or -O-; R a , R 3 and R 4 are each independently hydrogen, alkyl, (C≦6) or substituted alkyl (C≦6) or Or, R a is R 3 or R 4 Together with Alkandiil (C≦8) or substituted alkanediyl (C≦8) Forming R 2 is hydrogen, alkyl (C≦8) ,-Alkanediyl (C≦6) -NH 2 ,-Alkanediyl (C≦6) -Alkylamino (C≦8) ,-Alkanediyl (C≦6) -Dialkylamino (C≦12) ,-Alkanediyl (C≦6) -NR'R", substituted embodiments of any of these groups, and -Z 3 A”R 8 is selected from the group consisting of R 5 is hydrogen, alkyl (C≦8) ,-Alkanediyl (C≦6) -NH 2 ,-Alkanediyl (C≦6) -Alkylamino (C≦8) ,-Alkanediyl (C≦6) -Dialkylamino (C≦12) ,-Alkanediyl (C≦6) -NR'R", substituted embodiments of any of these groups, and -Z 3 A”R 8 is selected from the group consisting of R 6 is hydrogen, alkyl (C≦8) ,-Alkanediyl (C≦6) -NH 2 ,-Alkanediyl (C≦6) -Alkylamino (C≦8) ,-Alkanediyl (C≦6) -Dialkylamino (C≦12) ,-Alkanediyl (C≦6) -NR'R", substituted embodiments of any of these groups, and -Z 3 A”R 8 is selected from the group consisting of During the ceremony, R' and R" are each independently hydrogen, alkyl, or (C≦8) , substituted alkyl (C≦8) or Z 2 A'R 7 and During the ceremony, Z 2 is an alkanediyl (C≦4) or substituted alkanediyl (C≦4) and A' is -CHR j -, -C(O)O-, or -C(O)NR b - in which R b is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) and R j is hydrogen, halo, hydroxy, acyloxy (C≦24) or substituted acyloxy (C≦24) and R 7 is an alkyl (C6~24) , substituted alkyl (C6~24) , alkenyl (C6~24) or substituted alkenyl (C6~24) and Z 3 is an alkanediyl (C≦4) or substituted alkanediyl (C≦4) and A” is -CHR k -, -C(O)O-, or -C(O)NR l - and Rl is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) and R k is hydrogen, halo, hydroxy, acyloxy (C≦24) or substituted acyloxy (C≦24) and R 8 is an alkyl (C6~24) , substituted alkyl (C6~24) , alkenyl (C6~24) or substituted alkenyl (C6~24) and q is 1 or 2; r is 1, 2, or 3; and m and p are each independently 0, 1, 2 or 3.

[0076] In some embodiments of the zwitterionic lipid of formula (I), X 1 -S(O) 2 O - It is.

[0077] In some embodiments of the zwitterionic lipid of formula (I), Y 1 Alkanediyl (C≦12) or alkene diyl (C≦12) In some embodiments, A is -NR a In some embodiments, R a is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) It is.

[0078] In some embodiments of the zwitterionic lipid of formula (I), R 3 is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) It is.

[0079] In some embodiments of the zwitterionic lipid of formula (I), R 4 is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) It is.

[0080] In some embodiments of the zwitterionic lipid of formula (I), R 2 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or -Z 3 A”R 8 R 2 In some embodiments, Z 3 Alkanediyl (C≦4) and A" is -CHR k -, -C(O)O- or -C(O)NR l -, wherein R l is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) and R k is hydrogen, halo, hydroxy, acyloxy (C≦24) or substituted acyloxy (C≦24) and R 8 is alkyl (C6~24) or alkenyl (C6~24) It is.

[0081] In some embodiments of the zwitterionic lipid of formula (I), R 5 is hydrogen, alkyl (C≦8) , substituted alkyl (C≦8) or -Z 3 A”R 8 R 5 In some embodiments, Z 3 Alkanediyl (C≦4) and A" is -CHR k -, -C(O)O- or -C(O)NR l -, wherein R l is hydrogen, alkyl (C≦6) or substituted alkyl (C≦6) and R k is hydrogen, halo, hydroxy, acyloxy (C≦24) or substituted acyloxy (C≦24) and R 8 is alkyl (C6~24) or alkenyl (C6~24) It is.

[0082] In some embodiments of the zwitterionic lipid of formula (I), R 6 is an alkyl(C≦8 ), -Alkanediyl (C≦6) -NH 2 ,-Alkanediyl (C≦6 )-Alkylamino (C≦8) ,-Alkanediyl (C≦6) -Dialkylamino (C≦12) ,-Alkanediyl (C≦6) -NR'R", or substituted embodiments of any of these groups. In some embodiments, R 6 is an alkyl (C≦8) or substituted alkyl (C≦8) In some embodiments, R 6 -alkanediyl (C≦6) -NR'R". 6 In some embodiments, R' is -Z 2 A'R 7 In some embodiments, Z 2 is an alkanediyl (C≦4) and A' is -CHR j -, -C(O)O-, or -C(O)NR b - where R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and R j is hydrogen, halo, hydroxy, acyloxy (C≦24) or substituted acyloxy (C≦24) and R 7 is an alkyl (C6~24) or alkenyl (C6~24) In some embodiments, R″ is -Z 2 A'R 7 In some embodiments of R″, Z 2 is an alkanediyl (C≦4) and A' is -CHR j -, -C(O)O-, or -C(O)NR b - where R b is hydrogen, alkyl (C≦6) , or substituted alkyl (C≦6) and R j is hydrogen, halo, hydroxy, acyloxy (C≦24) or substituted acyloxy (C≦24) and R7 is an alkyl (C6~24) or alkenyl (C6~24) It is.

[0083] In some embodiments of the zwitterionic lipid of formula (I), q is 2.

[0084] In some embodiments of the zwitterionic lipid of formula (I), r is 2 or 3.

[0085] In some embodiments, the zwitterionic lipid is [ka] , [ka] , [ka] , [ka] , [ka] , [ka] and pharma- ceutically acceptable salts thereof, wherein R is H, -CH 2 CH(OH)R 8 , -CH 2 CH 2 C(O)OR 8 , and -CH 2 CH 2 C(O)NHR 8 wherein R 8 is selected from the group consisting of octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl.

[0086] In some embodiments, the zwitterionic lipid has the structural formula: [ka] or a pharma- ceutically acceptable salt thereof.

[0087] In some embodiments, the zwitterionic lipid is [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , and pharma- ceutically acceptable salts thereof.

[0088] In some embodiments of the lipid composition, the zwitterionic lipid comprises an alkylated or alkenylated phosphate anion. The alkylated or alkenylated phosphate anion has the structural formula: [ka] where R can be alkyl or alkenyl, n is 1, 2, 3, 4, 5, or 6, and the * may indicate the point of attachment of the alkylated or alkenyl phosphate anion to the quaternary ammonium cation.

[0089] In some embodiments, the lipid composition comprises two or more zwitterionic lipids, including a zwitterionic lipid and a second zwitterionic lipid that is separate from the (first) zwitterionic lipid. The second zwitterionic lipid can be a phospholipid.

[0090] In some embodiments, the (e.g., first or second) zwitterionic lipid or phospholipid has one or two long chains (e.g., C 6 ~C 24 ) alkyl or alkenyl groups, glycerol or sphingosine, one or two phosphate groups, and a small organic molecule. The small organic molecule can be an amino acid, a sugar, or an amino-substituted alkoxy group, such as choline or ethanolamine. In some embodiments, the (e.g., first or second) zwitterionic lipid or phospholipid is a phosphatidylcholine. In some embodiments, the (e.g., first or second) zwitterionic lipid or phospholipid is a distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipids define lipids and lipid-like molecules that have both positive and negative charges.

[0091] In some embodiments, the two or more zwitterionic lipids are present in the lipid composition at a molar percentage of about 1% to about 60%. In some embodiments, the two or more zwitterionic lipids are present in the lipid composition at a molar percentage of at least (about) 5%, at least (about) 10%, at least (about) 15%, at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, at least (about) 50%, at least (about) 55%, or at least (about) 60%. In some embodiments, two or more zwitterionic lipids are present in the lipid composition at a molar percentage of at most (about) 60%, at most (about) 55%, at most (about) 50%, at most (about) 45%, at most (about) 40%, at most (about) 35%, at most (about) 30%, at most (about) 25%, at most (about) 20%, at least (about) 15%, at most (about) 10%, or at most (about) 5%. In some embodiments, two or more zwitterionic lipids are present in the lipid composition at a molar percentage of at most (about) 5%, at most (about) 10%, at most (about) 15%, at most (about) 20%, at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at most (about) 45%, at most (about) 50%, at most (about) 55%, or at most (about) 60%, or a range between any two of the foregoing values.

[0092] In some embodiments, the ratio (e.g., weight or mass) of zwitterionic lipid to synthetic tRNA is about 50:1, 40:1, 30:1, 20:1, 10:1, 7.5:1, or 5:1 or less. In some embodiments, the ratio (e.g., weight or mass) of zwitterionic lipid to synthetic tRNA is at least about 1:1 or 2:1. In some embodiments, the ratio (e.g., weight or mass) of zwitterionic lipid to synthetic tRNA is about 1:1 to about 50:1 or about 2:1 to about 50:1. In some embodiments, the ratio (e.g., weight or mass) of zwitterionic lipid to synthetic tRNA is about 1:1 to about 40:1 or about 2:1 to about 40:1. In some embodiments, the ratio (e.g., weight or mass) of zwitterionic lipid to synthetic tRNA is about 1:1 to about 30:1 or about 2:1 to about 30:1. In some embodiments, the ratio (eg, by weight or mass) of zwitterionic lipid to synthetic tRNA is from about 1:1 to about 20:1, or from about 2:1 to about 20:1. Additional lipids

[0093] In some embodiments of the lipid compositions of the present application, the lipid composition further comprises an additional lipid, including, but not limited to, a steroid or steroid derivative, a polymer-conjugated lipid (e.g., a polyethylene glycol (PEG)-conjugated lipid), or a combination thereof.

[0094] In some embodiments, the molar ratio of nitrogen in the lipid composition to phosphate in the synthetic polynucleotide (N / P ratio) is about 50:1 or less, about 40:1 or less, about 30:1 or less, or about 20:1 or less. In some embodiments, the molar ratio of nitrogen in the lipid composition to phosphate in the synthetic polynucleotide (N / P ratio) is at least about 1:1, at least about 2:1, at least about 3:1, at least about 4:1, or at least about 5:1. In some embodiments, the molar ratio of nitrogen in the lipid composition to phosphate in the synthetic polynucleotide (N / P ratio) is about 1:1 to about 50:1, at least about 2:1 to about 50:1, at least about 3:1 to about 50:1, at least about 4:1 to about 50:1, or at least about 5:1 to about 50:1. In some embodiments, the molar ratio of nitrogen in the lipid composition to phosphate in the synthetic polynucleotide (N / P ratio) is about 1:1 to about 40:1, at least about 2:1 to about 40:1, at least about 3:1 to about 40:1, at least about 4:1 to about 40:1, or at least about 5:1 to about 40:1. In some embodiments, the molar ratio of nitrogen in the lipid composition to phosphate in the synthetic polynucleotide (N / P ratio) is about 1:1 to about 30:1, at least about 2:1 to about 30:1, at least about 3:1 to about 30:1, at least about 4:1 to about 30:1, or at least about 5:1 to about 30:1. Steroids or steroid derivatives

[0095] In some embodiments of the lipid composition of the present application, the lipid composition further comprises a steroid or a steroid derivative. In some embodiments, the steroid or steroid derivative comprises any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" refers to a class of compounds having a tetracyclic 17-carbon ring structure that may further include one or more substitutions including an alkyl group, an alkoxy group, a hydroxy group, an oxo group, an acyl group, or a double bond between two or more carbon atoms. In one aspect, the ring structure of the steroid has the formula: [ka] As shown in Figure 1, the steroid derivative comprises three fused cyclohexyl rings and a fused cyclopentyl ring. In some embodiments, the steroid derivative comprises the above ring structure with one or more non-alkyl substitutions. In some embodiments, the steroid or steroid derivative is a sterol, and the formula is further defined as: [ka] In some embodiments of the present application, the steroid or steroid derivative is cholestane or a cholestane derivative. In cholestane, the ring structure has the formula: [ka] As described above, cholestane derivatives include one or more non-alkyl substitutions of the ring system described above. In some embodiments, the cholestane or cholestane derivative is cholestane or a cholestane derivative or a sterol or a sterol derivative. In other embodiments, the cholestane or cholestane derivative is both cholestare and a sterol or a derivative thereof.

[0096] In some embodiments of the lipid composition, the composition may further comprise a molar percentage of steroid relative to the total lipid composition of about 40 to about 46. In some embodiments, the molar percentage is about 40, 41, 42, 43, 44, 45 to about 46, or any range derivable therein. In other embodiments, the molar percentage of steroid relative to the total lipid composition is about 15 to about 40. In some embodiments, the molar percentage is 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40, or any range derivable therein.

[0097] In some embodiments, the lipid composition comprises a molar percentage of a steroid or steroid derivative of about 1% to about 60%, about 5% to about 60%, about 10% to about 60%, or about 20% to about 60%.

[0098] In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 15% to about 46%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 20% to about 40%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 25% to about 35%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 30% to about 40%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of about 20% to about 30%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative at a molar percentage of at least (about) 15%, at least (about) 20%, at least (about) 25%, at least (about) 30%, at least (about) 35%, at least (about) 40%, at least (about) 45%, or at least (about) 46%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a steroid or steroid derivative in a molar percentage of at most (about) 15%, at most (about) 20%, at most (about) 25%, at most (about) 30%, at most (about) 35%, at most (about) 40%, at most (about) 45%, or at most (about) 46%. Polymer-conjugated lipids

[0099] In some embodiments of the lipid composition of the present application, the lipid composition further comprises a polymer-conjugated lipid. In some embodiments, the polymer-conjugated lipid is a PEG lipid. In some embodiments, the PEG lipid is a diglyceride that also comprises a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a diglyceride that also comprises one or more C linked to a linker group having a PEG chain. 6 ~C 24 Long chain alkyl or alkenyl group or C 6 ~C 24It is a compound containing a fatty acid group. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates, PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified diastearoylphosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, PEG-modified phosphatidylethanolamine (PE). In some embodiments, the PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight is about 200 to about 500, about 400 to about 5,000, about 500 to about 3,000, or about 1,200 to about 3,000. The molecular weight of the PEG modification is about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present application are taught by U.S. Patent No. 5,820,873, WO 2010 / 141069, or U.S. Patent No. 8,450,298, which are incorporated herein by reference.

[0100] In some embodiments of the lipid compositions of the present application, the PEG lipid has the structural formula: [ka] wherein R 12 and R 13 are each independently an alkyl (C≦24) , alkenyl (C≦24) or substituted embodiments of any of these groups, R e is hydrogen, alkyl (C≦8) or substituted alkyl (C≦8)and x is 1 to 250. In some embodiments, R e is an alkyl group such as methyl (C≦8) R 12 and R 13 are each independently an alkyl (C≦4~20) In some embodiments, x is 5 to 250. In one embodiment, x is 5 to 125, or x is 100 to 250. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol.

[0101] In some embodiments of the lipid compositions of the present application, the PEG lipid has the structural formula: [ka] wherein n 1 is an integer from 1 to 100, and n 2 and n 3 are each independently selected from an integer of 1 to 29. In some embodiments, n 1 is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, or any range derivable therein. 1 is about 30 to about 50. In some embodiments, n 2 In some embodiments, n is 5 to 23. 2 is from 11 to about 17. In some embodiments, n 3 In some embodiments, n is 5 to 23. 3 is between 11 and about 17.

[0102] In some embodiments of the lipid composition of the present application, the composition may further comprise a molar percentage of PEG lipid of about 4.0 to about 4.6 relative to the total lipid composition. In some embodiments, the molar percentage is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 to about 4.6, or any range derivable therein. In other embodiments, the molar percentage is about 1.5 to about 4.0. In some embodiments, the molar percentage is about 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75 to about 4.0, or any range derivable therein.

[0103] In some embodiments, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 0.5% to about 12%. In some embodiments, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 1% to about 12%. In some embodiments, the lipid composition comprises a polymer-complex lipid at a molar percentage of about 1.5% to about 12%.

[0104] In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-conjugated lipid at a molar percentage of about 0.5% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-conjugated lipid at a molar percentage of about 1% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-conjugated lipid at a molar percentage of about 2% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-conjugated lipid at a molar percentage of about 3% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-conjugated lipid at a molar percentage of about 4% to about 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-conjugated lipid in a molar percentage of at least (about) 0.5%, at least (about) 1%, at least (about) 1.5%, at least (about) 2%, at least (about) 2.5%, at least (about) 3%, at least (about) 3.5%, at least (about) 4%, at least (about) 4.5%, at least (about) 5%, at least (about) 5.5%, at least (about) 6%, at least (about) 6.5%, at least (about) 7%, at least (about) 7.5%, at least (about) 8%, at least (about) 8.5%, at least (about) 9%, at least (about) 9.5%, or at least (about) 10%. In some embodiments of the lipid composition of the present application, the lipid composition comprises a polymer-conjugated lipid in a molar percentage of at most (about) 0.5%, at most (about) 1%, at most (about) 1.5%, at most (about) 2%, at most (about) 2.5%, at most (about) 3%, at most (about) 3.5%, at most (about) 4%, at most (about) 4.5%, at most (about) 5%, at most (about) 5.5%, at most (about) 6%, at most (about) 6.5%, at most (about) 7%, at most (about) 7.5%, at most (about) 8%, at most (about) 8.5%, at most (about) 9%, at most (about) 9.5%, or at most (about) 10%. formulation

[0105] In some embodiments, when the composition is an aerosol composition or is formulated for aerosol administration, the composition has a droplet size of 0.5 micrometers (μm) to 10 μm. In some embodiments, the composition has a median droplet size of 0.5 μm to 10 μm. In some embodiments, the composition has a mean droplet size of 0.5 μm to 10 μm. The droplet size may be determined by cascade impactor analysis or laser diffraction, or other suitable technique for measuring aerosol droplets. The aerosol administration may be delivered to the respiratory epithelium.

[0106] In some embodiments of the composition, the composition can be formulated as any suitable dosage known in the art. In some embodiments, the composition is formulated into nanoparticles or nanocapsules. In some embodiments, the composition is formulated for administration by any suitable route known in the art, including, for example, oral, rectal, vaginal, transmucosal, intratracheal or pulmonary, including inhalation, or intestinal administration; intramuscular, subcutaneous, intramedullary injection, and parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0107] In some embodiments of the method, the compositions of the present application are formulated for administration in a local rather than systemic manner, for example, by injecting the pharmaceutical composition directly into the target tissue, preferably in a sustained release formulation. Local delivery can be affected in a variety of ways depending on the tissue being targeted.

[0108] In some embodiments of the method, an aerosol containing the composition of the present application can be inhaled (for nasal, tracheal, or bronchial delivery). In some embodiments, the composition of the present application can be injected, for example, at the site of injury, disease manifestation, or pain. In some embodiments, the composition of the present application can be provided in a lozenge for oral, tracheal, or esophageal application. In some embodiments, the composition of the present application can be provided in liquid, tablet, or capsule form for administration to the stomach or intestines. In some embodiments, the composition of the present application can be provided in suppository form for rectal or vaginal application. In some embodiments, the composition of the present application can even be delivered to the eye by use of creams, drops, or injections. kit

[0109] In one aspect, provided herein is a kit comprising a composition described herein. In some embodiments, the kit further comprises a container and a label or package insert on or associated with the container. method Methods for enhancing CFTR expression or activity in a cell(s)

[0110] In one aspect, provided herein is a method of enhancing expression or activity of a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein in a cell. The method can include contacting a cell with a composition as described herein, e.g., a composition comprising a transfer ribonucleic acid (tRNA) as described herein assembled with a lipid composition as described herein, to introduce an amino acid into a growing peptide chain of a CFTR protein in the cell, thereby resulting in a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell at least 24 hours, 48 ​​hours, or 72 hours after contacting.

[0111] In one aspect, provided herein is a method of enhancing expression or activity of a Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein in a cell of a subject exhibiting or suspected of exhibiting a mutation in the CFTR gene. The method can include contacting the cell with a composition comprising a lipid composition and an assembled transfer ribonucleic acid (tRNA) to introduce an amino acid into a growing peptide chain of a CFTR protein in the cell at a position corresponding to the mutation in the subject's CFTR gene, thereby providing a therapeutically effective amount or activity of a functional variant of the CFTR protein in the cell.

[0112] The therapeutically effective activity of a functional variant of a CFTR protein can be determined by measuring a change in a transepithelial ion transport property of a plurality of cells comprising the cells, e.g., in the absence of contact, compared to a transepithelial ion transport property (e.g., transepithelial current or transepithelial voltage) of a reference plurality of cells.

[0113] In some embodiments of the methods described herein, the contacting is repeated. The contacting may be repeated once, twice, three times, or more. In some embodiments, the contacting is at least once a week. In some embodiments, the contacting is at least twice a week. In some embodiments, the method provides a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell at least 24 hours after each contacting. In some embodiments, the second contacting may be performed at least about 1, 2, or 3 days after the first contacting. In some embodiments, the method further includes a third contacting, which may be performed at least about 1, 2, or 3 days after the second contacting. In some embodiments, the method provides a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell at least 24 hours after the second contacting. In some embodiments, the method provides a therapeutically effective amount or activity of a functional variant of a CFTR protein in the cell at least 24 hours after the third contacting. The composition in each contacting may be the same or identical. The therapeutically effective amount or activity of the functional variant of the CFTR protein can be increased following repeated contact.

[0114] The contacting(s) may be performed in vivo. The contacting(s) may be performed in vitro. The contacting(s) may be performed ex vivo.

[0115] In some embodiments, the method achieves a therapeutically effective activity of a functional variant of a CFTR protein. In some embodiments, the therapeutically effective activity may be measured by a transepithelial assay. A transepithelial assay may measure a voltage or current that may correspond to a function of a functional protein. In some embodiments, the therapeutically effective activity of a functional variant of a CFTR protein corresponds to a transepithelial current of about 2 microamps (μA) to about 30 μA. In some embodiments, the therapeutically effective activity of a functional variant of a CFTR protein corresponds to a transepithelial current of at least about 2 microamps (μA). In some embodiments, the therapeutically effective activity of a functional variant of a CFTR protein corresponds to a transepithelial current of at least about 2 microamps (μA) per square centimeter per minute (μA·cm -2 ·minute -1 ) corresponding to a transepithelial current of about 2 microamperes (μA) per square centimeter per minute (μA cm -2 ·minute -1 ) ~ approx. 30 μA cm -2 ·minute -1 Transepithelial currents can be determined via equivalent transepithelial current assays using the TECC24 system, such as those described elsewhere herein.

[0116] In some embodiments of the methods described herein, the method increases the amount of a functional variant of a CFTR protein in a cell compared to a matched control. The functional variant can be a wild-type CFTR protein. The functional variant can be a full-length CFTR protein. In some embodiments, the method increases the amount of a functional variant CFTR protein in a cell compared to a matched control. In some embodiments, the control includes a matched cell in the absence of one or more steps of contacting(s). In some embodiments, the method increases the amount of a functional variant of a CFTR protein in a cell relative to a matched control by at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2.0 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, at least about 3.0 fold, at least about 3.1 fold, at least about 3.2 fold, at least about 3.3 fold, at least about 3.4 fold, at least about 3.5 fold, at least about 3.6 fold, at least about 3.7 fold, at least about 3.8 fold, at least about 3.9 fold, at least about 4.0 fold, at least about 4.1 fold, at least about 4.2 fold, at least about 4.3 fold, at least about 4.4 fold, at least about 4.5 fold, at least about 4.6 fold, at least about 4.7 fold, at least about 4.8 fold, at least about 4.9 fold, at least about 4.1 fold, at least about 4.1 fold, at least about 4.2 fold, at least about 4.3 fold, at least about 4.4 fold, at least about 4.5 fold, at least about 4.6 fold, at least about 4.7 fold, at least about 4.8 fold, at least 8-fold, at least about 2.9-fold, at least about 3.0-fold, at least about 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at least about 3.5-fold, at least about 3.6-fold, at least about 3.7-fold, at least about 3.8-fold, at least about 3.9-fold, at least about 4.0-fold, at least about 4.1-fold, at least about 4.2-fold, at least about 4.3-fold, at least about 4.4-fold, at least about 4.5-fold, at least about 4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at least about 4.9-fold, or at least about 5.0-fold increase.

[0117] In some embodiments, the methods result in a therapeutically effective amount of a functional variant of a CFTR protein in a cell. In some embodiments, the methods result in a therapeutically effective amount of a wild-type (WT) protein or a full-length CFTR protein in a cell.

[0118] In some embodiments, the method enhances ion transport in the cell compared to a matched control. In some embodiments, the method enhances chloride transport in the cell compared to a matched control. In some embodiments, the control comprises a non-contacted matched cell. In some embodiments, the method enhances ion transport in the cell compared to a matched control by at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2.0 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, at least about 3.0 fold, at least about 3.1 fold, at least about 3.2 fold, at least about 3.3 fold, at least about 3.4 fold, at least about 3.5 fold, at least about 3.6 fold, at least about 3.7 fold, at least about 3.8 fold, at least about 3.9 ... or at least about 2.9-fold, at least about 3.0-fold, at least about 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at least about 3.5-fold, at least about 3.6-fold, at least about 3.7-fold, at least about 3.8-fold, at least about 3.9-fold, at least about 4.0-fold, at least about 4.1-fold, at least about 4.2-fold, at least about 4.3-fold, at least about 4.4-fold, at least about 4.5-fold, at least about 4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at least about 4.9-fold, or at least about 5.0-fold. Methods for Treating Cystic Fibrosis

[0119] In one aspect, provided herein is a method of treating a subject having or suspected of having a cystic fibrosis transmembrane conductance regulator (CFTR)-associated condition. The method may include administering a composition described herein to the subject. In some embodiments, the CFTR-associated condition is cystic fibrosis, hereditary emphysema, or chronic obstructive pulmonary disease (COPD), or a combination thereof. The subject may be a mammal. The subject may be a human. In some embodiments, administering includes pulmonary administration. In some embodiments, administering includes inhalation by nebulization. In some embodiments, administering includes apical administration.

[0120] The methods of the present disclosure may be capable of treating a subject with cystic fibrosis based on the properties of the formulation or composition. In particular, the compositions described elsewhere herein may be able to penetrate the mucus associated with cystic fibrosis, thereby delivering polynucleotides to cells. Cell(s)

[0121] In some embodiments of the methods described herein, the cell is a lung cell. In some embodiments, the lung cell is a lung airway cell. Exemplary lung airway cells that may be targeted by delivery of the present application include, but are not limited to, basal cells, secretory cells, such as goblet cells and club cells, ciliated cells, ion cells, and any combination thereof. In some embodiments of the methods, the cell is an airway epithelial cell. In some embodiments, the cell is a bronchial epithelial cell. In some embodiments, the cell is an airway epithelial cell. In some embodiments, the cell is a basal cell characterized by expression of the p63 marker. In some embodiments, the cell is an ion cell characterized by expression of the FOXI1 marker. In some embodiments, the cell is undifferentiated. In some embodiments, the cell is differentiated. In some embodiments, the cell(s) are derived from a subject. The subject may be a mammal. The subject may be a human. Mutation(s)

[0122] In some embodiments, the cell or subject exhibits a mutation in the CFTR gene or transcript. In some embodiments, the cell or subject exhibits a mutation in one or more of exons 11-27 of the CFTR gene. The cell or subject exhibits a nonsense or frameshift mutation in one or more of exons 11-27 of the CFTR gene. In some embodiments, the mutation is located at a position in the CFTR gene where an alteration can result in a mutant protein having a mutation at F508, e.g., F508del. In some embodiments, the mutation is located at a position in the CFTR gene where an alteration can result in a mutant protein having a mutation at R553 in the CFTR protein, e.g., R553X, which corresponds to c.1657C>T in the CFTR gene. In some embodiments, the cell or subject can have multiple mutations. In some embodiments, the mutation is associated with cystic fibrosis, hereditary emphysema, or chronic obstructive pulmonary disease (COPD). [Example] [Example 1] Production of tRNA

[0123] To generate tRNA, DNA fragments encoding specific tRNA sequences behind a T7 RNA polymerase promoter sequence were chemically synthesized. The T7 promoter-tRNA DNA sequences were amplified by PCR and then transcribed in vitro using T7 RNA polymerase using standard techniques such as those described in Green and Sambrook, 2012; Rio et al., 2011; Flanagan et al., 2003; and Janiak et al., 1992. The resulting tRNA transcripts were extracted with phenol, precipitated in high salt and ethanol, and purified by HPLC using a MonoQ ion exchange column. The purified tRNA was precipitated, resuspended, and dialyzed against water. Example 2 Compensation of the R553X / F508del CFTR mutation with the suppressor tRNA LNP formulation of the present application in differentiated primary hBE cells from R553X / F508del subjects.

[0124] Suppressor tRNA encoding arginine encapsulated by LNPs showed significant rescue of CFTR in R553X / F508del CFTR hBE models. Briefly, suppressor tRNA was encapsulated in LNP compositions and delivered to R553X / F508del CFTR hBE cells as an apical liquid bolus or as apical exposure of ALI hBEs to nebulized LNP aerosols. hBE cells isolated from cystic fibrosis patients with the R553X / F508del CFTR genotype at passage 3 were seeded in 24-well Transwell® plates and airlifted after 96 hours. Cells were grown following a 3 day / week feeding routine with Vertex ALI medium. After 5 weeks, hBE cell cultures were considered fully differentiated, polarized, and ready for TECC24 functional assays. Four days prior to treatment, the apical side of the hBE cultures were washed with 3 mM DTT in PBS to remove mucus. 24 hours prior to treatment, cells were washed with PBS and on the day of treatment, treated apically with liquid bolus or VitroCell nebulized formulations and tested after 24 or 24+n24 hours of CO2 incubation as scheduled. Specifically, the hBE assay sequence included a background current / resistance recording interval (~25 min), a baseline Cl- current recording interval after inhibition of Na+ conductance with 6 μM benzamyl (~15 min), a 10 μM forskolin + 1 μM VX-770-induced CFTR activation interval (~25 min), and a 20 μM bumetanide-induced Cl- current inhibition interval (~25 min). hBE transepithelial equivalent current traces [Ieq=Vt / (Rt-50), μA / cm] versus time were reconstructed. Cl- current induced by forskolin / VX-770 was measured using a 10 μM CFTR activation interval (~25 min). - Current responses were calculated as the area under the Ieq curve (IeqAUC) for the time points between forskolin / VX770 and INH-172 addition. Ieq AUC / min values ​​were statistically validated and compared across experimental samples. As shown in Figure 1A, apical bolus treatment with LNPs containing suppressor tRNA suppressed forskolin-dependent Cl -Currents were collected and demonstrate rescue of CFTR function in R553X / F508del hBE. Assays were performed at three time points (24 hours post-dose, 48 hours post-dose, and 72 hours post-dose), all of which demonstrated rescue of CFTR function. Figures 1A and 1B show functional CFTR compared to cells treated with DMSO, demonstrating that the increase in CFTR function was significant compared to the negative control.

[0125] In a similar assay, the suppressor tRNA LNP formulation was repeatedly administered on a twice-weekly dosing schedule. Using a similar protocol to determine CFTR function, repeated dosing showed improved CFTR function after each dose. Figures 2A and 2B show that the first and second doses can improve CFTR function over the negative control, and the third dose further increases CFTR function.

[0126] In the time course assay, the tRNA formulations (as apical bolus formulations and as CFTR modulators) were added to the cell culture medium. After 24 hours, the cell culture medium was replaced. Figure 3A and Figure 3B show improved CFTR function at the 72 hour time point.

[0127] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in carrying out the present invention. It is therefore contemplated that the present invention encompasses any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. A composition comprising a synthetic transfer ribonucleic acid (tRNA) assembled with a lipid composition, wherein the lipid composition comprises an amphiphilic lipid, and the composition is an aerosol composition.

2. The composition according to claim 1, having a droplet size of 0.5 micrometers (μm) to 10 μm, having a central droplet size of 0.5 μm to 10 μm, having an average droplet size of 0.5 μm to 10 μm, or having any combination thereof.

3. The composition according to claim 1, wherein the synthetic tRNA is folded tRNA.

4. The composition according to claim 3, wherein the synthetic tRNA comprises an acceptor stem configured to be operably linked to arginine.

5. The composition according to claim 1, wherein the tRNA comprises a polynucleotide sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequence selected from SEQ ID NOs: 1-20.

6. The composition according to claim 1, wherein the mass ratio or weight ratio of the amphiphilic lipid to the synthetic tRNA is about 50:1, 40:1, 30:1, or 20:1 or less.

7. The composition according to claim 1, wherein the lipid composition comprises the amphiphilic lipid in a molar percentage of about 1% to about 60%, and the molar percentage is determined based on the total lipids present in the lipid composition.

8. The composition according to claim 1, wherein the lipid composition further comprises a steroid or a steroid derivative.

9. The composition according to claim 8, wherein the lipid composition contains the steroid or steroid derivative in a molar percentage of about 20% to about 60%, and the molar percentage is determined based on the total lipids present in the lipid composition.

10. The composition according to claim 1, wherein the lipid composition further comprises a polymer complex lipid.

11. The composition according to claim 10, wherein the lipid composition contains the polymer complex lipid in a molar percentage of about 0.5% to about 12%, and the molar percentage is determined based on the total lipids present in the lipid composition.

12. The composition according to claim 1, wherein the molar ratio (N / P ratio) of nitrogen in the lipid composition to phosphate in the synthetic tRNA is about 50:1, 40:1, 30:1, 20:1, or 10:1 or less.

13. The zwitterionic lipid has the structural formula (I) (I) [wherein, X1 is -S(O)2O- or -OP(O)OR e O-, wherein, R e is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6), Y1 is alkanediyl (C≤12), alkenediyl (C≤12) or a substituted form thereof, A is -NR a -, -S-, or -O-, R a, R3 and R4 are each independently hydrogen, alkyl (C≤6) or substituted alkyl (C≤6), or alternatively, R a together with R3 or R4 forms alkanediyl (C≤8) or substituted alkanediyl (C≤8), R2 is selected from the group consisting of hydrogen, alkyl (C≤8), -alkanediyl (C≤6)-NH2, -alkanediyl (C≤6)-alkylamino (C≤8), -alkanediyl (C≤6)-dialkylamino (C≤12), -alkanediyl (C≤6)-NR'R", a substituted form of any of these groups, and -Z3A"R8, R5 is selected from the group consisting of hydrogen, alkyl (C≤8), -alkanediyl (C≤6)-NH2, -alkanediyl (C≤6)-alkylamino (C≤8), -alkanediyl (C≤6)-dialkylamino (C≤12), -alkanediyl (C≤6)-NR'R", a substituted form of any of these groups, and -Z3A"R8, R6 is selected from the group consisting of hydrogen, alkyl (C≤8), -alkanediyl (C≤6)-NH2, -alkanediyl (C≤6)-alkylamino (C≤8), -alkanediyl (C≤6)-dialkylamino (C≤12), -alkanediyl (C≤6)-NR'R", a substituted form of any of these groups, and -Z3A"R8, Here, R' and R" are each independently hydrogen, alkyl (C≤8), substituted alkyl (C≤8) or -Z2A'R7, In the formula, Z2 is alkanediyl (C≤4) or substituted alkanediyl (C≤4), A' is -CHRj-, -C(O)O-, or -C(O)NRb-, Here, Rb is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6), and Rj is hydrogen, halo, hydroxy, acyloxy (C≤24) or substituted acyloxy (C≤24), R7 is alkyl (C6-24), substituted alkyl (C6-24), alkenyl (C6-24) or substituted alkenyl (C6-24), Z3 is alkanediyl (C≤4) or substituted alkanediyl (C≤4), A” is -CHRk-, -C(O)O-, or -C(O)NRl-, Rl is hydrogen, alkyl (C≤6) or substituted alkyl (C≤6), and Rk is hydrogen, halo, hydroxy, acyloxy (C≤24) or substituted acyloxy (C≤24), and R8 is alkyl (C6-24), substituted alkyl (C6-24), alkenyl (C6-24) or substituted alkenyl (C6-24), q is 1 or 2, r is 1, 2, or 3, and m and p are each independently 0, 1, 2 or 3], or a pharmaceutically acceptable salt thereof, the composition according to claim 1.

14. The zwitterionic lipid is , , , , , and has a structural formula selected from the group consisting of these pharmaceutically acceptable salts: [Wherein, R is selected from the group consisting of H, -CH2CH(OH)R8, -CH2CH2C(O)OR8, and -CH2CH2C(O)NHR8, Here, R8 is selected from the group consisting of octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl], the composition according to claim 13.

15. The zwitterionic lipid has the structural formula: or a pharmaceutically acceptable salt thereof, the composition according to claim 14.

16. The zwitterionic lipid is , , , , , , The composition according to claim 15, which is a compound selected from the group consisting of and pharmaceutically acceptable salts thereof.

17. The composition according to claim 1, formulated as an aerosol.

18. The composition according to claim 1, formulated for apical delivery.

19. The composition according to claim 1, formulated for nebulization.

20. The composition according to any one of claims 1 to 19 for use as a medicament for treating cystic fibrosis transmembrane conductance regulator (CFTR)-related symptoms in a subject.

21. The composition according to claim 20, wherein the CFTR-related condition is cystic fibrosis, hereditary emphysema, or chronic obstructive pulmonary disease (COPD).

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