Novel lipids for delivery of nucleic acid therapeutics

Abstract

The present disclosure relates generally to novel lipids and lipid nanoparticles or their compositions for the delivery of nucleic acid therapeutics. Further, the present disclosure relates generally to methods of treating or preventing a disease comprising administering to a subject in need thereof the novel lipids and lipid nanoparticle compositions described herein.

Description

[0001] PVX-PAT-2402-WO

[0002] NOVEL LIPIDS FOR DELIVERY OF NUCLEIC ACID THERAPEUTICS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 575,934, filed April 8, 2024, which is incorporated herein by reference in its entirety.

[0004] Provided are lipids and lipid nanoparticle (LNP) composition or formulation for nucleic acid therapeutics.

[0005] Small molecules and protein based drugs or therapeutics have been used since several decades. Nucleic acid based therapies are emerging as a promising class of drugs in the recent times. These drugs comprise a segment of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Delivering nucleic acid to the target cells has been a challenge due to lack of suitable nucleic acid delivery vectors. Viral vectors have been used for this purpose, but immune responses to these vectors and their transgene product presents substantial hurdles to their wider use.

[0006] In recent years lipid nanoparticles (LNPs) have received a lot of attention as delivery vehicle for nucleic acid therapeutics. Patisiran is the first FDA approved short interfering RNA (siRNA) therapeutic encapsulated in a lipid nanoparticle. Lipid nanoparticles typically comprise of four different types of lipids viz., cationic lipid / ionizable lipid, phospholipid, sterol and PEG-lipid. Cationic or ionizable lipid play a crucial role in the delivery of nucleic acid therapeutics to the cells. Efforts are being made to develop novel cationic lipids or improve the existing cationic lipids for the delivery of nucleic acid therapeutics that offers protection to nucleic acid against degradation, reduced toxicity, improved encapsulation efficiency, efficient endosomal escape, and biodegradability. Design of efficient cationic lipids for delivery of nucleic acid therapeutics remains a challenging area.

[0007] Therefore, there is a need for novel cationic lipids and lipid nanoparticle composition for the delivery of nucleic acid based therapeutics.

[0008] Accordingly, provided are novel cationic lipids for the delivery of nucleic acid therapeutics, including, isomers, and salts thereof. They can be used either alone or in combination with other lipid and polymeric components for the delivery of nucleic acid therapeutics.

[0009] In one embodiment, the disclosure relates to a cationic lipid represented by formula (I)PVX-PAT-2402-WO

[0010] R1 — L5

[0011] AI L4

[0012] R2L6A L3Q 1—j

[0013] O - P = O

[0014] O - L2

[0015]

[0016] X2— R4

[0017] formula (I)

[0018] or isomer, or salt thereof, wherein:

[0019] Ri and R2 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?, or

[0020] Ri and R2 may combine together to form a saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;

[0021] R3 and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26;

[0022] Rs, Re, and R7 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms;

[0023] each of L1, L2, L3, L4, L5, L6, and L7 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene;

[0024] Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-;

[0025] A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;PVX-PAT-2402-WO

[0026] — HC

[0027] A1is \, -CH-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, S, or N provided that when A1is -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or S, one of R1or R2is absent; and

[0028] wherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

[0029] In some embodiments, the disclosure relates to a cationic lipid represented by formula

[0030]

[0031] X2R4

[0032] formula (II)

[0033] or isomer, or salt thereof, wherein:

[0034] = represents either a single bond or a double bond;

[0035] Ri and R2are independently chosen from H, -OH, Ci-io alkyl, C2-io alkenyl, C2-io alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?, or

[0036] Ri and R2may combine together to form a saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;

[0037] R3 and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26alkenyl, C2-26alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26;

[0038] Rs, Re, and R7 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4

[0039] heteroatoms;PVX-PAT-2402-WO

[0040] each of L1, L2, L3, L4, L5, L6, and L7 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene;

[0041] Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-;

[0042] A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;

[0043] —HC\

[0044] Ai is \, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, S, or N provided that when Ai is -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or S, one of Ri or R2 is absent; and

[0045] wherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

[0046] In some embodiments, the disclosure relates to a lipid nanoparticle composition comprising any of the cationic lipids described herein, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid.

[0047] In some embodiments, the disclosure relates to a lipid nanoparticle composition comprising any of the cationic lipids described herein, an ionizable polymer, a phospholipid, a sterol, a PEG-lipid and a nucleic acid.

[0048] In some embodiments, the nucleic acid is a DNA, an RNA or a combination thereof. The RNA may be a messenger RNA (mRNA), a non-coding RNA (ncRNA) or a combination thereof. The non-coding RNA (ncRNA) may be long non-coding RNA (IncRNA), micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and PIWI-interacting RNA (piRNA), transfer RNA (tRNA) or ribosomal RNA (rRNA) or a combination thereof.

[0049] In some embodiments, the nucleic acid encodes an antigen. The antigen may be derived from an infectious agent, such as a virus or a bacterium.

[0050] In some embodiments, the nucleic acid regulates or modulates cellular functions. In some embodiments, the nucleic acid comprises at least one chemical modification. In some aspects, provided herein is a nucleic acid vaccine or therapeutic comprising a lipid nanoparticle composition described herein.PVX-PAT-2402-WO

[0051] In some aspects, provided herein is a method of treating or preventing a disease, comprising administering to a subject in need thereof the lipid nanoparticle composition comprising a cationic lipid described herein, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid. In some aspects, the disclosure relates to use of a lipid nanoparticle composition comprising a cationic lipid described herein, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid in the manufacture of a medicament for the treatment or prevention of a disease in a subject. In some embodiments, the disease is cancer, an infectious disease or a disease and / or disorder ameliorated by humoral and / or cellular immune response.

[0052] BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 - depicts expression of luciferase mRNA by different lipid nanoparticle compositions or formulations

[0054] DESCRIPTION

[0055] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of person skilled in the art. Some of the terms are defined briefly here below; the definitions should not be construed in a limiting sense.

[0056] The singular forms “a”, “an”, and “the” as used in the specification also include plural aspects unless the context dictates otherwise. Similarly, any singular term used in the specification also mean plural or vice versa unless the context dictates otherwise. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.

[0057] It must be noted that the words “comprising” or any of its form such as “comprise” or “comprises”, “having” or any of its forms such as “have” or “has”, “including” or any of its forms such as “include” or “includes”, or “containing” or any of its forms such as “contain” or “contains” are open-ended and do not exclude additional unrecited elements or method steps.

[0058] Wherever any quantity or range is stated one skilled in the art will recognize that quantity or range within 10 or 20 percent (%) of the stated values can also be expected to be appropriate and reasonable and included within the scope of the invention.

[0059] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by thosePVX-PAT-2402-WO

[0060] skilled in the art. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, protein, adjuvant, pharmaceutical biotechnology, and biopharmaceutical manufacturing described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The cationic lipids of the present disclosure can be synthesized using the methods described herein, as well as alternative synthetic methods well known to those skilled in the art. The starting materials can be procured from various suppliers or produced using conventional methods known in the art. Various organic synthesis methods are disclosed in standard textbooks, for example, Smith, Michael B.

[0061] Compendium of Organic Synthetic Methods, Vol 13, 2014, John Wiley & Sons, Inc. During the synthesis, it may be necessary and / or desirable to protect sensitive or reactive groups on any molecule of interest. This can be accomplished by using standard techniques which are known to one skilled in the art, for example, Wuts, Peter G. M. Greene’s Protective Groups in Organic Synthesis, 5thedition, 2014, John Wiley & Sons, Inc. The exemplary cationic lipids described herein can be prepared according to the reaction schemes described herein. These methods can be adjusted or modified in a manner known to one skilled in the art.

[0062] The term “isomer” as used herein means another compound that has the same molecular formula but differs in the arrangement or configuration of atoms, and includes isomers, stereoisomers, diastereomers, or enantiomers.

[0063] The term “salts” as used herein means salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids. In case the compounds of the present invention contain one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically utilizable salts.

[0064] The “= ’’symbol used herein represents either a single bond or a double bond.

[0065] The term “alkyl” as used herein means, a straight (unbranched) or branched, substituted or unsubstituted, hydrocarbon chain containing saturated carbon chain.

[0066] The term “alkenyl” as used herein means, a straight (unbranched) or branched, substituted or unsubstituted, hydrocarbon chain containing at least one carbon-carbon double bond.PVX-PAT-2402-WO

[0067] The term “alkynyl” as used herein means, a straight (unbranched) or branched, substituted or unsubstituted, hydrocarbon chain containing at least one carbon-carbon triple bond.

[0068] The term “alkylene” as used herein means, a bivalent straight (unbranched) or branched saturated, substituted or unsubstituted, hydrocarbon chain.

[0069] The term “alkenylene” as used herein means, a bivalent straight (unbranched) or branched, substituted or unsubstituted, unsaturated hydrocarbon chain containing at least one carbon-carbon double bond.

[0070] The term “alkynylene” as used herein means, a bivalent straight (unbranched) or branched, substituted or unsubstituted hydrocarbon chain containing at least one carboncarbon triple bond.

[0071] The term “hydrocarbon chain” as used herein means, simplest chain made up of only carbon and hydrogen atoms which may be straight (unbranched) or branched, substituted or unsubstituted, saturated or unsaturated.

[0072] The term “cyclic”, “cyclo”, or “cycle” has been used interchangeably, whether as prefix, infix, or suffix, herein to mean a structure characterized by one or more rings. In some embodiments, two or more rings may be joined to form bicyclic, tricyclic, tetracyclic, pentacyclic, or polycyclic structures. These cyclic structures may assume different configurations, for example, fused, bridged, spiro, trans etc.

[0073] The term “cycloalkyl” as used herein means, saturated or unsaturated, optionally substituted, non-aromatic carbon-based cyclic compound composed of at least three carbon atoms.

[0074] The term “heterocycloalkyl” as used herein means, a cycloalkyl where at least one of the carbon atoms of the cycloalkyl is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. In some embodiments, the heterocycloalkyl may be substituted with one or more substituents. In some embodiments, the substitution may be on one or more carbon atoms of the heterocycloalkyl or on one or more heteroatoms of the heterocycloalkyl.

[0075] The term “aryl” as used herein means, univalent organic radical derived from an aromatic hydrocarbon by removing a hydrogen atom.

[0076] The term “heteroaryl” as used herein means, a group or part of a group that contains an aromatic carbon based cyclic moiety of 5 to 10 ring atoms in which one or more, in some embodiments, one, two, or three of the ring atom(s) is(are) selected from nitrogen, oxygen,PVX-PAT-2402-WO

[0077] phosphorus, or sulfur, the remaining ring atoms being carbon. In some embodiments, the heteroaryl may be substituted with one or more substituents. In some embodiments, the substitution may be on one or more carbon atoms of the heteroaryl or on one or more heteroatoms of the heteroaryl.

[0078] The term “bivalent” as used herein means, a chemical moiety having two unoccupied valences to form bonds with another chemical moiety.

[0079] The term “monovalent’ ’ as used herein means, a chemical moiety having a single unoccupied valency to form bond with another chemical moiety.

[0080] The term “substituent”, “substitution”, or “substituted” has been used interchangeably to mean all permissible substitution of organic compounds. These modifications involve either an atom or group replacing another atom or group, or an atom or group carrying a substitution on it. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom or group and the substituent. In some embodiments, the substituent may include further substitution. In some embodiments, the substituent may be selected from, but not limited to, H, -OH, Cl, Br, I, O, S, N, P, Ci-6 alkoxy, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms, which may be further substituted.

[0081] The term “composition”, “formulation”, “lipid nanoparticle composition”, “LNP composition”, “LNP formulation”, “LNP”, or “lipid nanoparticle” has been used interchangeably to mean a nanoparticle, nanostructure, vesicle, liposome, composition or formulation comprising one or more lipid components (for example a cationic lipid, a phospholipid, a sterol, and a PEG-lipid), and / or ionizable polymer component. In some embodiments, the lipid nanoparticle comprises a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and nucleic acid. In some embodiments, the lipid nanoparticle comprises one or more lipid components, an ionizable polymer component, and a nucleic acid. In some embodiments, the nucleic acid associated with the lipid nanoparticle is a DNA, an mRNA, a micro RNA, a small interfering RNA, a small nucleolar RNA, a small nuclear RNA, a long non-coding RNA or a combination thereof. In some embodiments, the lipid nanoparticle composition may additionally contain pharmaceutical carriers or excipients, such as but not limited to, buffering agents, stabilizers, tonicity modifiers, surfactants, chelating agents, salts, anti-oxidants, diluents, and / or preservatives or combinations thereof. The term lipidPVX-PAT-2402-WO

[0082] nanoparticle also denotes lipid nanoparticles that are devoid of any encapsulated nucleic acid (empty lipid nanoparticles or ghost lipid nanoparticles). In some embodiments, the lipid nanoparticle composition comprises lipid nanoparticles with encapsulated nucleic acid as well as empty lipid nanoparticles.

[0083] The term “therapeutic”, “therapeutic agent”, “prophylactic”, “prophylactic agent”, or “drug” has been used interchangeably to mean a compound or composition (such as a lipid nanoparticle composition described herein) having a biological effect or a combination of biological effects that prevents, inhibits, eliminates or prevents the progression of a disease or other aberrant biological processes in a subject, for example, an animal, or human.

[0084] The term “preventing” is art-recognized, and when used in relation to a condition, such as an infection is well understood in the art, and includes administration of a composition, which reduces the frequency or severity, or delays the onset, of one or more symptoms of the medical condition in a subject relative to a subject who does not receive the composition. Thus, the prevention of a condition, such as an infection, includes, for example, the reduction of the frequency or severity of one or more symptoms of the medical condition in a population of patients receiving a therapy relative to a control population that did not receive the therapy, e.g., by a statistically and / or clinically significant amount. Similarly, the prevention of an infection includes reducing the likelihood that a patient receiving a therapy will develop the infection or related symptoms, relative to a patient who does not receive the therapy.

[0085] The term “stable” as used herein means a composition which retains an acceptable degree of physical stability, chemical stability and / or biological activity upon storage for a specified period of time at a given temperature. Stability of the therapeutic may be measured by techniques known to the person skilled in the art, for example, by SDS PAGE, dynamic light scattering, or immunogenicity assays. A composition may be stable even though the nucleic acid contained therein does not maintain 100% of its structure and / or function and / or biological activity after storage for a defined amount of time. Under certain circumstances, maintenance of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of nucleic acid’s structure and / or function and / or biological activity after storage for a defined amount of time may be regarded as “stable”. In some embodiments, maintenance of about 30% to about 40%, about 40% to about 50%, about 50% to about 60%,PVX-PAT-2402-WO

[0086] about 60% to about 70%, or about 70% to about 80% of nucleic acid’s structure and / or function and / or biological activity after storage for a defined amount of time may be regarded as “stable”.

[0087] The “specified period of time” as used herein means at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6, weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 16 weeks, at least about 18 weeks, at least about 20 weeks, at least about 22 weeks, at least about 24 weeks, at least about 28 weeks, at least about 32 weeks, at least about 36 weeks, at least about 40 weeks, at least about 44 weeks, at least about 48 weeks, at least about 52 weeks, or more. In some embodiments, specified period of time also means at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, at least about 14 months, at least about 16 months, at least about 18 months, at least about 20 months, at least about 22 months, at least about 24 months or more. In some embodiments, specified period of time also means at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days or more.

[0088] The term “molar percent”, “mol percent”, “molar %”, or “mol %” have been used interchangeably to mean number of moles of a component expressed as percentage relative to total moles of all lipid components present in the lipid nanoparticle compositions described herein. For example, 50 mol % cationic lipid means, 50 mol % of cationic lipid is present in the lipid nanoparticle composition and other lipid components together constitute remaining 50 mol % such that the total amount of all the lipid components constitute 100 mol %. In some embodiments, mol % also denotes to mean number of moles of a component expressed as percentage relative to total moles of all lipid components (such as cationic lipid, phospholipid, sterol and PEG-lipid) and ionizable polymer component(s) present in the lipid nanoparticle compositions described herein. For example, 50 mol % of cationic lipid means, 50 mol % of cationic lipid is present in the lipid nanoparticle composition and other lipids components and ionizable polymer components together constitute the remaining 50 mol %PVX-PAT-2402-WO

[0089] such that the total amount of all the lipid components and ionizable polymer components constitute 100 mol %.

[0090] The term “N / P ratio”, “N: P ratio”, “N / P”, “N: P”, “lipid to nucleic acid ratio”, or “cationic lipid to nucleic acid ratio” have been used interchangeably herein and means the ratio (molar ratio) of the positive charges in the cationic lipid relative to the negative charges in the nucleic acid in a lipid nanoparticle. In some embodiments, the N / P ratio refers to the ratio of protonable nitrogen present in the cationic lipid relative to the phosphate present in the nucleic acid in a lipid nanoparticle. In some embodiments, the N / P ratio is between 1 to 18 (i.e., 1:1 to 18:1). For example, a N / P ratio of 18 refers to the presence of 18 protonable nitrogen of the cationic lipid relative to 1 phosphate of the nucleic acid in a lipid nanoparticle.

[0091] The terms “protein”, “peptide” and “polypeptide” have been used interchangeably herein and mean a polymer of amino acids linked through peptide bonds, but does not imply any specific length. The term also includes fusion proteins, muteins, analogs or modified forms.

[0092] The terms “antibody” and “antibodies” have been used interchangeably herein and means any antibody or antibody fragment (whether produced naturally or recombinantly) which retains antigen binding activity. This includes a monoclonal or polyclonal antibody, a single chain antibody, a Fab fragment of a monoclonal or polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, a multispecific antibody, or a nanobody.

[0093] The term “buffer” as used herein means those agents that maintains the pH of a solution in a desired range.

[0094] The term “cell” as used herein means a single cell or a population of cells or plurality of cells.

[0095] The term “biologically effective amount” or “therapeutically effective amount” as used herein means an amount of an agent, for example, a therapeutic, drug, therapeutic agent, prophylactic agent, diagnostic agent, composition, etc., that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, prevent, diagnose, improve symptoms of, and / or delay the onset of the infection, disease, disorder, and / or condition. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient.

[0096] As used herein, the term “treating” or “treatment” includes reducing, arresting, or reversing the symptoms, clinical signs, or underlying pathology of a condition to stabilize orPVX-PAT-2402-WO

[0097] improve a subject’s condition or to reduce the likelihood that the subject’s condition will worsen as much as if the subject did not receive the treatment. Treatment may be administered to a subject who does not exhibit signs of a disease and / or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0098] The term “subject” as used herein refers to a living mammal and may be interchangeably used with the term “patient”. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. The term does not denote a particular age or gender.

[0099] As used herein, an individual “at risk” of developing a particular disease, disorder, or condition may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein. “At risk” denotes that an individual has one or more risk factors, which are measurable parameters that correlate with development of a particular disease, disorder, or condition, as known in the art. An individual having one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition than an individual without one or more of these risk factors.

[0100] The term “disease” as used herein, means an interruption, cessation, or disorder of body function, system, or organ. Non limiting examples of disease include malignant diseases, autoimmune diseases, inherited diseases, metabolic disorders, rare diseases, cardiovascular diseases, neurological diseases, gene or protein regulatory disorders, or infectious diseases.

[0101] As used herein, administration “conjointly” with another compound or composition includes simultaneous administration and / or administration at different times. Conjoint administration also encompasses administration as a co-formulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.

[0102] Cationic LipidsPVX-PAT-2402-WO

[0103] Cationic lipid refers to a lipid that has a net positive charge at a selected pH. Cationic lipids generally consist of a hydrophilic head group that carries the charge and a hydrophobic tail.

[0104] In some embodiments, the cationic lipid is a cationic lipid with an amine head group. The amine head group can be primary, secondary, or tertiary. The cationic lipid may comprise one (monoamine) or more (polyamine) such amine groups.

[0105] In some embodiments, the cationic lipids are positively charged at pH below the pKa of the cationic lipid. In certain embodiments, the cationic lipids are neutral i.e., when pH is same or above the pKa of the cationic lipid. In some embodiment the cationic lipids are positively charged at acidic pH i.e., pH 1.0 to pH 6.9. In certain embodiments, the cationic lipids are neutral at certain pH i.e., around physiological pH (pH 7.0 to pH 7.5). A cationic lipid that can exist in a positively charged or neutral form depending on the pH is commonly referred to as ionizable lipid. In some embodiments, the cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on the pH.

[0106] In one embodiment, the disclosure relates to a cationic lipid represented by formula (I)

[0107] Ri — L5

[0108] A-| L4X1R3

[0109] R2Lg A L3Q L-|

[0110] O - P = O

[0111] O - L2

[0112]

[0113] \2— R4

[0114] formula (I)

[0115] or isomer, or salt thereof, wherein:

[0116] Ri and R2 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?, or

[0117] Ri and R2 may combine together to form a saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;PVX-PAT-2402-WO

[0118] Rs and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26;

[0119] Rs, Re, and R7 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms;

[0120] each of Li, L2, L3, L4, L5 Le, and L7 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene;

[0121] Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-;

[0122] A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatom, or C5-10 heteroaryl containing 1-4 heteroatom;

[0123] —HC\

[0124] Ai is \, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, S, or N provided that when Ai is -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or S, one of Ri or R2 is absent; and

[0125] wherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

[0126] In some embodiments of the cationic lipid represented by formula (I), R3 and R4 are independently chosen from, optionally substituted, branched or unbranched, C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26.

[0127] In some embodiments of the cationic lipid represented by formula (I), R3 and R4 are independently chosen from, optionally substituted, branched or unbranched, C2-26, C2-22, and C2-20 alkenyl.

[0128] In some embodiments of the cationic lipid represented by formula (I), R3 and R4 are independently chosen from, optionally substituted, branched or unbranched, C2-26, C2-22, and C2-20 alkynyl.PVX-PAT-2402-WO

[0129] In some embodiments of the cationic lipid represented by formula (I), R3 and R4 are independently chosen from, optionally substituted, branched or unbranched, Ci-26, Ci-22, and C1-20 alkyl.

[0130] In some embodiments of the cationic lipid represented by formula (I), R3 and R4 are each independently, optionally substituted, branched or unbranched, C1-17 alkyl.

[0131] In some embodiments of the cationic lipid represented by formula (I), R3 and R4 are independently chosen from -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26, 0 to 22, 0 to 20, 0 to 18, 0 to 16, 0 to 14, or 0 to 12.

[0132] In some embodiments of the cationic lipid represented by formula (I), R3 and R4 are independently -CH((CH2)m-A-(CH2)n-(CH3)y)2, and each of m, n, and y is independently an integer ranging from 0 to 26, 0 to 22, 0 to 20, 0 to 18, 0 to 16, 0 to 14, or 0 to 12.

[0133] In some embodiments of the cationic lipid represented by formula (I), R3 and R4 are each independently -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 8, or 0 to 10.

[0134] In some embodiments of the cationic lipid represented by formula (I), R3 and R4 are each independently -(CH2)m-A-(CH2)n-(CH3)y, wherein each of m, n, and y is independently an integer ranging from 0 to 26, 0 to 22, 0 to 20, 0 to 18, 0 to 16, 0 to 14, or 0 to 12.

[0135] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-.

[0136] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-.

[0137] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are each independently -C(O)O-, or -OC(O)-.

[0138] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are both -C(O)O-.

[0139] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are both -OC(O)-.

[0140] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are each independently -C(O)NH-, or -NHC(O)-.PVX-PAT-2402-WO

[0141] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are both -C(O)NH-.

[0142] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are each independently -C(S)NH-, or -NHC(S)-.

[0143] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are both -C(S)NH-.

[0144] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are each independently -C(S)O-, or -OC(S)-.

[0145] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are both -C(S)O-.

[0146] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are each independently -OC(O)NH-, or -NHC(O)O-.

[0147] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are both -OC(O)NH-.

[0148] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are each independently -OP(O)(OH)O-, or -OP(O)(O-)O-.

[0149] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are both -OP(O)(O-)O-.

[0150] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are -C(O)O- or -OC(O)- and -OP(O)(OH)O- or -OP(O)(O-)O- respectively.

[0151] In some embodiments of the cationic lipid represented by formula (I), Xi and X2 are -OP(O)(OH)O- or -OP(O)(O-)O- and -C(O)O- or -OC(O)- respectively.

[0152] In some embodiments of the cationic lipid represented by formula (I), each of L1, L2, L3, L4, L5, L6, and L7 is either absent or independently chosen from, optionally substituted, C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene.

[0153] In some embodiments of the cationic lipid represented by formula (I), each of L1 and L2 is independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-3 alkylene.

[0154] In some embodiments of the cationic lipid represented by formula (I), each of Li and L2 is independently, optionally substituted, C1-8 alkylene.

[0155] In some embodiments of the cationic lipid represented by formula (I), each of L3 and L4 is independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-2 alkylene.PVX-PAT-2402-WO

[0156] In some embodiments of the cationic lipid represented by formula (I), each of L3 and L4 is independently chosen from, optionally substituted, C1-5 and C1-3 alkylene.

[0157] In some embodiments of the cationic lipid represented by formula (I), each of L5 and L / > is independently absent.

[0158] In some embodiments of the cationic lipid represented by formula (I), each of L5 and L / > is independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-2 alkylene.

[0159] In some embodiments of the cationic lipid represented by formula (I), each of L5 and L / > is independently chosen from, optionally substituted, C1-5 and C1-3 alkylene.

[0160] In some embodiments of the cationic lipid represented by formula (I), L7 is absent. In some embodiments of the cationic lipid represented by formula (I), L7 is, optionally substituted, C1-10, C1-8, C1-6, C1-4, or C1-2 alkylene.

[0161] In some embodiments of the cationic lipid represented by formula (I), L7 is, optionally substituted, C1-5, or C1-3 alkylene.

[0162] In some embodiments of the cationic lipid represented by formula (I), A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms.

[0163] In some embodiments of the cationic lipid represented by formula (I), A is H.

[0164] In some embodiments of the cationic lipid represented by formula (I), A is a bond. In some embodiments of the cationic lipid represented by formula (I), A is, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 cycloalkyl.

[0165] In some embodiments of the cationic lipid represented by formula (I), A is, optionally substituted, saturated or unsaturated, C3-6 cycloalkyl.

[0166] In some embodiments of the cationic lipid represented by formula (I), A is, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0167] In some embodiments of the cationic lipid represented by formula (I), A is, optionally substituted, saturated or unsaturated, C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0168] In some embodiments of the cationic lipid represented by formula (I), A is, optionally substituted, C5-10, C5-8, or C5-7 heteroaryl containing 1-4 heteroatoms.

[0169] In some embodiments of the cationic lipid represented by formula (I), A is, optionally substituted, C5-6 heteroaryl containing 1-4 heteroatoms.PVX-PAT-2402-WO

[0170] In some embodiments of the cationic lipid represented by formula (I), Ai is

[0171]

[0172] -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, S, or N.

[0173] — HC^ In some embodiments of the cationic lipid represented by formula (I), Ai is

[0174]

[0175] \. In some embodiments of the cationic lipid represented by formula (I), Ai is -CH2-. In some embodiments of the cationic lipid represented by formula (I), Ai is -C(O)O-. In some embodiments of the cationic lipid represented by formula (I), Ai is -C(O)NH. In some embodiments of the cationic lipid represented by formula (I), Ai is S.

[0176] In some embodiments of the cationic lipid represented by formula (I), Ai is N.

[0177] In some embodiments of the cationic lipid represented by formula (I), Ri and R2are independently chosen from H, -OH, optionally substituted C1-10 alkyl, C2-io alkenyl, C2-io alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?.

[0178] In some embodiments of the cationic lipid represented by formula (I), Ri and R2may combine together to form an optionally substituted, saturated or unsaturated, C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms.

[0179] In some embodiments of the cationic lipid represented by formula (I), Ri and R2are independently chosen from H, and -OH.

[0180] In some embodiments of the cationic lipid represented by formula (I), Ri and R2are both H.

[0181] In some embodiments of the cationic lipid represented by formula (I), Ri and R2are both -OH.

[0182] In some embodiments of the cationic lipid represented by formula (I), Ri and R2are independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-2 alkyl.

[0183] In some embodiments of the cationic lipid represented by formula (I), Ri and R2are independently chosen from, optionally substituted, C1-4, C1-3 and C1-2 alkyl.

[0184] In some embodiments of the cationic lipid represented by formula (I), Ri and R2are independently chosen from, optionally substituted, saturated or unsaturated, C3-10, C3-8, and C3-6 cycloalkyl.

[0185] In some embodiments of the cationic lipid represented by formula (I), Ri and R2are independently chosen from, optionally substituted, Ce-io, Ce-8, and Ce-7 aryl.PVX-PAT-2402-WO

[0186] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 are independently chosen from, optionally substituted, saturated or unsaturated, C3-10, C3-8, and C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0187] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 are independently chosen from, optionally substituted, C5-10, and C5-8 heteroaryl containing 1-4 heteroatoms.

[0188] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 are independently chosen from H, and -O-L7-R5.

[0189] In some embodiments of the cationic lipid represented by formula (I), Ri is H and R2 is -O-L7-R5.

[0190] In some embodiments of the cationic lipid represented by formula (I), Ri is -O-L7-R5 and R2 is H.

[0191] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 are independently chosen from H, and -NReR?.

[0192] In some embodiments of the cationic lipid represented by formula (I), Ri is H and R2 is -NReR?.

[0193] In some embodiments of the cationic lipid represented by formula (I), Ri is -NReR? and R2 is H.

[0194] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 combine together to form, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 cycloalkyl.

[0195] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 combine together to form, optionally substituted, saturated or unsaturated, C3-6 cycloalkyl.

[0196] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 combine together to form, optionally substituted, Ce-io, Ce-8, or Ce-7 aryl.

[0197] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 combine together to form, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0198] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 combine together to form, optionally substituted, saturated or unsaturated, C3-6 heterocycloalkyl containing 1-4 heteroatoms.PVX-PAT-2402-WO

[0199] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 combine together to form, optionally substituted, C5-10, C6-8, or C6-7 heteroaryl containing 1-4 heteroatoms.

[0200] In some embodiments of the cationic lipid represented by formula (I), Ri and R2 combine together to form, optionally substituted, C5-6 heteroaryl containing 1-4 heteroatoms.

[0201] In some embodiments of the cationic lipid represented by formula (I), R5, Re, and R7 are independently chosen from H, -OH, optionally substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms.

[0202] In some embodiments of the cationic lipid represented by formula (I), R5 is H In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, C1-10, C1-8, C1-6, C1-4, or C1-2 alkyl.

[0203] In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, C1-5, or C1-3 alkyl.

[0204] In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, C2-10, C2-8, C2-6, C2-4, or C2 alkenyl.

[0205] In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, C2-5, or C2-3 alkenyl.

[0206] In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, C2-10, C2-8, C2-6, C2-4, or C2 alkynyl.

[0207] In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, C2-5, or C2-3 alkynyl.

[0208] In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 cycloalkyl.

[0209] In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, saturated or unsaturated, C3-6 cycloalkyl.

[0210] In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, Ce-io, Ce-8, or Ce-7 aryl.

[0211] In some embodiments of the cationic lipid represented by formula (I), R5 is, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 heterocycloalkyl containing 1-4 heteroatoms.PVX-PAT-2402-WO

[0212] In some embodiments of the cationic lipid represented by formula (I), Rs is, optionally substituted, saturated or unsaturated, C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0213] In some embodiments of the cationic lipid represented by formula (I), Rs is, optionally substituted, C5-10, C5-8, or C5-7 heteroaryl containing 1-4 heteroatoms.

[0214] In some embodiments of the cationic lipid represented by formula (I), Rs is, optionally substituted, C5-6 heteroaryl containing 1-4 heteroatoms.

[0215] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are independently chosen from H, and -OH.

[0216] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-2 alkyl.

[0217] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are independently chosen from, optionally substituted, C1-5, and C1-3 alkyl.

[0218] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are independently chosen from, optionally substituted, C2-10, C2-8, C2-6, C2-4, and C2 alkenyl.

[0219] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are independently chosen from, optionally substituted, C2-5, and C2-3 alkenyl.

[0220] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are independently chosen from, optionally substituted, C2-10, C2-8, C2-6, C2-4, and C2 alkynyl.

[0221] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are independently chosen from, optionally substituted, C2-5, and C2-3 alkynyl.

[0222] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are independently chosen from, optionally substituted, saturated or unsaturated, C3-10, C3-8, and C3-6 cycloalkyl.

[0223] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are each independently, optionally substituted, saturated or unsaturated, C3-6 cycloalkyl.

[0224] In some embodiments of the cationic lipid represented by formula (I), R6 and R7 are independently chosen from, optionally substituted, C6-10, C6-8, and C6-7 aryl.

[0225] In some embodiments of the cationic lipid represented by formula (I), Re and R7 are independently chosen from, optionally substituted, saturated or unsaturated, C3-10, C3-8, and C3-6 heterocycloalkyl containing 1-4 heteroatoms.PVX-PAT-2402-WO

[0226] In some embodiments of the cationic lipid represented by formula (I), Re and R7 are each independently, optionally substituted, saturated or unsaturated, C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0227] In some embodiments of the cationic lipid represented by formula (I), Re and R7 are independently chosen from, optionally substituted, C5-10, C5-8, C5-7 and C5-6 heteroaryl containing 1-4 heteroatoms.

[0228] In some embodiments, the disclosure relates to a cationic lipid represented by formula

[0229]

[0230] X2R4

[0231] formula (II)

[0232] or isomer, or salt thereof, wherein:

[0233] = represents either a single bond or a double bond;

[0234] Ri and R2 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?, or

[0235] Ri and R2 may combine together to form a saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;

[0236] R3 and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26;

[0237] Rs, Re, and R7 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms;

[0238] each of L1, L2, L3, L4, L5, L6, and L7 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene;PVX-PAT-2402-WO

[0239] Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-;

[0240] A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatom, or C5-10 heteroaryl containing 1-4 heteroatom;

[0241] — HC^ A

[0242]

[0243] i is \, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, S, or N provided that when Ai is -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or S, one of Ri or R2 is absent; and

[0244] wherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

[0245] In some embodiments of the cationic lipid represented by formula (II), R3 and R4 are independently chosen from, optionally substituted, branched or unbranched, C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26.

[0246] In some embodiments of the cationic lipid represented by formula (II), R3 and R4 are independently chosen from, optionally substituted, branched or unbranched, C2-26, C2-22, and C2-20 alkenyl.

[0247] In some embodiments of the cationic lipid represented by formula (II), R3 and R4 are independently chosen from, optionally substituted, branched or unbranched, C2-26, C2-22, and C2-20 alkynyl.

[0248] In some embodiments of the cationic lipid represented by formula (II), R3 and R4 are independently chosen from, optionally substituted, branched or unbranched, Ci-26, Ci-22, and C1-20 alkyl.

[0249] In some embodiments of the cationic lipid represented by formula (II), R3 and R4 are each independently, optionally substituted, branched or unbranched, C1-17 alkyl.

[0250] In some embodiments of the cationic lipid represented by formula (II), R3 and R4 are independently chosen from -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26, 0 to 22, 0 to 20, 0 to 18, 0 to 16, 0 to 14, or 0 to 12.PVX-PAT-2402-WO

[0251] In some embodiments of the cationic lipid represented by formula (II), R3 and R4 are each independently -(CH2)m-A-(CH2)n-(CH3)y, wherein each of m, n, and y is independently an integer ranging from 0 to 26, 0 to 22, 0 to 20, 0 to 18, 0 to 16, 0 to 14, or 0 to 12.

[0252] In some embodiments of the cationic lipid represented by formula (II), R3 and R4 are each independently -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26, 0 to 22, 0 to 20, 0 to 18, 0 to 16, 0 to 14, or 0 to 12.

[0253] In some embodiments of the cationic lipid represented by formula (II), R3 and R4 are each independently -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 8, or 0 to 10.

[0254] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-.

[0255] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-.

[0256] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are each independently -C(O)O-, or -OC(O)-.

[0257] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are both -C(O)O-.

[0258] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are both -OC(O)-.

[0259] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are each independently -C(O)NH-, or -NHC(O)-.

[0260] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are both -C(O)NH-.

[0261] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are each independently -C(S)NH-, or -NHC(S)-.

[0262] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are both -C(S)NH-.

[0263] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are each independently -C(S)O-, or -OC(S)-.PVX-PAT-2402-WO

[0264] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are both -C(S)O-.

[0265] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are each independently -OC(O)NH-, or -NHC(O)O-.

[0266] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are both -OC(O)NH-.

[0267] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are each independently -OP(O)(OH)O-, or -OP(O)(O-)O-.

[0268] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are both -OP(O)(O-)O-.

[0269] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are -C(O)O- or -OC(O)- and -OP(O)(OH)O- or -OP(O)(O-)O- respectively.

[0270] In some embodiments of the cationic lipid represented by formula (II), Xi and X2 are -OP(O)(OH)O- or -OP(O)(O-)O- and -OC(O)- or -C(O)O- respectively.

[0271] In some embodiments of the cationic lipid represented by formula (II), each of L1, L2, L3, L4, L5, L6, and L7 is either absent or independently chosen from, optionally substituted, C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene.

[0272] In some embodiments of the cationic lipid represented by formula (II), each of L1 and L2 is independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-3 alkylene.

[0273] In some embodiments of the cationic lipid represented by formula (II), each of Li and L2 is independently, optionally substituted, C1-8 alkylene.

[0274] In some embodiments of the cationic lipid represented by formula (II), each of L3 and L4 is independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-2 alkylene.

[0275] In some embodiments of the cationic lipid represented by formula (II), each of L3 and L4 is independently chosen from, optionally substituted, C1-5 and C1-3 alkylene.

[0276] In some embodiments of the cationic lipid represented by formula (II), each of L5 and Le is independently absent.

[0277] In some embodiments of the cationic lipid represented by formula (II), each of L5 and Le is independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-2 alkylene.PVX-PAT-2402-WO

[0278] In some embodiments of the cationic lipid represented by formula (II), each of Ls and L / > is independently chosen from, optionally substituted, C1-5 and C1-3 alkylene.

[0279] In some embodiments of the cationic lipid represented by formula (II), L7 is absent. In some embodiments of the cationic lipid represented by formula (II), L7 is, optionally substituted, C1-10, C1-8, C1-6, C1-4, or C1-2 alkylene.

[0280] In some embodiments of the cationic lipid represented by formula (II), L7 is, optionally substituted, C1-5, or C1-3 alkylene.

[0281] In some embodiments of the cationic lipid represented by formula (II), A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms.

[0282] In some embodiments of the cationic lipid represented by formula (II), A is H.

[0283] In some embodiments of the cationic lipid represented by formula (II), A is a bond. In some embodiments of the cationic lipid represented by formula (II), A is, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 cycloalkyl.

[0284] In some embodiments of the cationic lipid represented by formula (II), A is, optionally substituted, saturated or unsaturated, C3-6 cycloalkyl.

[0285] In some embodiments of the cationic lipid represented by formula (II), A is, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0286] In some embodiments of the cationic lipid represented by formula (II), A is, optionally substituted, saturated or unsaturated, C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0287] In some embodiments of the cationic lipid represented by formula (II), A is, optionally substituted, C5-10, C5-8, or C5-7 heteroaryl containing 1-4 heteroatoms.

[0288] In some embodiments of the cationic lipid represented by formula (II), A is, optionally substituted, C5-6 heteroaryl containing 1-4 heteroatoms.

[0289] — HC^ In some embodiments of the cationic lipid represented by formula (II), Ai is \, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, S, or N.

[0290] —HC\ In some embodiments of the cationic lipid represented by formula (II), Ai is \. In some embodiments of the cationic lipid represented by formula (II), Ai is -CH2-. In some embodiments of the cationic lipid represented by formula (II), Ai is -C(O)O-.PVX-PAT-2402-WO

[0291] In some embodiments of the cationic lipid represented by formula (II), Ai is -C(O)NH-.

[0292] In some embodiments of the cationic lipid represented by formula (II), Ai is S.

[0293] In some embodiments of the cationic lipid represented by formula (II), Ai is N.

[0294] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are independently chosen from H, -OH, optionally substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?.

[0295] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 may combine together to form a saturated or unsaturated, optionally substituted, C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms.

[0296] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are independently chosen from H, and -OH.

[0297] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are both H.

[0298] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are both -OH.

[0299] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-2 alkyl.

[0300] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are independently chosen from, optionally substituted, C1-4, C1-3, and C1-2 alkyl.

[0301] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are independently chosen from, optionally substituted, saturated or unsaturated, C3-10, C3-8, and C3-6 cycloalkyl.

[0302] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are independently chosen from, optionally substituted, C6-10, C6-8, and C6-7 aryl.In some embodiments of the cationic lipid represented by formula (II), R1 and R2 are independently chosen from, optionally substituted, saturated or unsaturated, C3-10, C3-8, and C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0303] 1PVX-PAT-2402-WO

[0304] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are independently chosen from, optionally substituted, C5-10 and C5-8 heteroaryl containing 1-4 heteroatoms.

[0305] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are independently chosen from H, and -O-L7-R5.

[0306] In some embodiments of the cationic lipid represented by formula (II), Ri is H and R2 is -O-L7-R5.

[0307] In some embodiments of the cationic lipid represented by formula (II), Ri is -O-L7-R5 and R2 is H.

[0308] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 are independently chosen from H, and -NReR?.

[0309] In some embodiments of the cationic lipid represented by formula (II), Ri is H and R2 is -NReR?.

[0310] In some embodiments of the cationic lipid represented by formula (II), Ri is -NReR? and R2 is H.

[0311] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 combine together to form, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 cycloalkyl.

[0312] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 combine together to form, optionally substituted, saturated or unsaturated, C3-6 cycloalkyl.

[0313] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 combine together to form, optionally substituted C6-10, C6-8, or C6-7 aryl.In some embodiments of the cationic lipid represented by formula (II), R1 and R2 combine together to form, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0314] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 combine together to form, optionally substituted, saturated or unsaturated, C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0315] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 combine together to form, optionally substituted, C5-10, C6-8, or C6-7 heteroaryl containing 1-4 heteroatoms.

[0316] In some embodiments of the cationic lipid represented by formula (II), Ri and R2 combine together to form, optionally substituted, C5-6 heteroaryl containing 1-4 heteroatoms.PVX-PAT-2402-WO

[0317] In some embodiments of the cationic lipid represented by formula (II), R5, R6, and R7 are independently chosen from H, -OH, optionally substituted C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms.

[0318] In some embodiments of the cationic lipid represented by formula (II), Rs is H. In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, C1-10, C1-8, C1-6, C1-4, or C1-2 alkyl.

[0319] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, C1-5, or C1-3 alkyl.

[0320] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, C2-10, C2-8, C2-6, C2-4, or C2 alkenyl.

[0321] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, C2-5, or C2-3 alkenyl.

[0322] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, C2-10, C2-8, C2-6, C2-4, or C2 alkynyl.

[0323] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, C2-5, or C2-3 alkynyl.

[0324] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 cycloalkyl.

[0325] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, saturated or unsaturated, C3-6 cycloalkyl.

[0326] In some embodiments of the cationic lipid represented by formula (II), Rs is, optionally substituted, C6-10, C6-8, or C6-7 aryl.In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, saturated or unsaturated, C3-10, C3-8, or C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0327] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, saturated or unsaturated, C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0328] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, C5-10, C5-8, or C5-7 heteroaryl containing 1-4 heteroatoms.PVX-PAT-2402-WO

[0329] In some embodiments of the cationic lipid represented by formula (II), R5 is, optionally substituted, C5-6 heteroaryl containing 1-4 heteroatoms.

[0330] In some embodiments of the cationic lipid represented by formula (II), R6, and R7 are independently chosen from H, and -OH.

[0331] In some embodiments of the cationic lipid represented by formula (II), R6 and R7 are independently chosen from, optionally substituted, C1-10, C1-8, C1-6, C1-4, and C1-2 alkyl.

[0332] In some embodiments of the cationic lipid represented by formula (II), R6 and R7 are independently chosen from, optionally substituted, C1-5, and C1-3 alkyl.

[0333] In some embodiments of the cationic lipid represented by formula (II), R6 and R7 are independently chosen from, optionally substituted, C2-10, C2-8, C2-6, C2-4, and C2 alkenyl.

[0334] In some embodiments of the cationic lipid represented by formula (II), R6 and R7 are independently chosen from, optionally substituted, C2-5, and C2-3 alkenyl.

[0335] In some embodiments of the cationic lipid represented by formula (II), R6 and R7 are independently chosen from, optionally substituted, C2-10, C2-8, C2-6, C2-4, and C2 alkynyl.

[0336] In some embodiments of the cationic lipid represented by formula (II), R6 and R7 are independently chosen from, optionally substituted, C2-5, and C2-3 alkynyl.

[0337] In some embodiments of the cationic lipid represented by formula (II), R6 and R7 are independently chosen from, optionally substituted, saturated or unsaturated, C3-10, C3-8, and C3-6 cycloalkyl.

[0338] In some embodiments of the cationic lipid represented by formula (II), R6 and R7 are each independently, optionally substituted, saturated or unsaturated, C3-6 cycloalkyl.

[0339] In some embodiments of the cationic lipid represented by formula (II), R6 and R7 are independently chosen from, optionally substituted, C6-10, C6-8, and C6-7 aryl.

[0340] In some embodiments of the cationic lipid represented by formula (II), Re and R7 are independently chosen from, optionally substituted, saturated or unsaturated, C3-10, C3-8, and C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0341] In some embodiments of the cationic lipid represented by formula (II), Re and R7 are each independently, optionally substituted, saturated or unsaturated, C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0342] In some embodiments of the cationic lipid represented by formula (II), Re and R7 are independently chosen from, optionally substituted, C5-10, C5-8, C5-7, and C5-6 heteroaryl containing 1-4 heteroatoms.PVX-PAT-2402-WO

[0343] In some embodiments, cationic lipid represented by formula (I) or formula (II) are substituted with substituents independently chosen from H, -OH, Cl, Br, I, O, S, N, P, optionally substituted Ci-6 alkoxy, optionally substituted Ci-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted saturated or unsaturated C3-10 cycloalkyl, optionally substituted Ce-io aryl, optionally substituted saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatom, optionally substituted C5-10 heteroaryl containing 1-4 heteroatom or combinations thereof.

[0344] In some embodiments, N:P ratio or cationic lipid to nucleic acid ratio in LNP formulation is between 1 to 18, between 1 to 17, between 1 to 16, between 1 to 15, between 1 to 14, between 1 to 13, between 1 to 12, between 1 to 11, between 1 to 10, between 1 to 9, between 1 to 8, between 1 to 7, between 1 to 6, between 1 to 5, between 1 to 4, between 1 to 3, between 1 to 2, or any range therein.

[0345] In some embodiments, N:P ratio or cationic lipid to nucleic acid ratio in LNP formulation is about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, or any portion or fraction thereof.

[0346] The proportion of cationic lipid present in the lipid nanoparticle compositions is from about 10 mol % to about 70 mol %, from about 10 mol % to about 65 mol %, from about 10 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, from about 10 mol % to about 50 mol %, or any range therein. In some embodiments, the cationic lipid present in the lipid nanoparticle composition is about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about 14 mol %, about 15 mol %, about 16 mol %, about 17 mol %, about 18 mol %, about 19 mol %, about 20 mol %, about 21 mol %, about 22 mol %, about 23 mol %, about 24 mol %, about 25 mol %, about 26 mol %, about 27 mol %, about 28 mol %, about 29 mol %, about 30 mol %, about 31 mol %, about 32 mol %, about 33 mol %, about 34 mol %, about 35 mol %, about 36 mol %, about 37 mol %, about 38 mol %, about 39 mol %, about 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %, about 45 mol %, about 46 mol %, about 47 mol %, about 48 mol %, about 49 mol %, about 50 mol %, about 51 mol %, about 52 mol %, about 53 mol %, about 54 mol %, about 55 mol %, about 56 mol %, about 57 mol %, about 58 mol %, about 59 mol %, about 60 mol %, about 61 mol %, about 62 mol %, about 63 mol %, about 64 mol %, about 65 mol %, about 66 mol %, about 67 mol %, about 68 mol %, about 69 mol %, about 70 mol %, or any portion or fraction thereof.PVX-PAT-2402-WO

[0347] In some embodiments, the cationic lipid of the present disclosure comprises one of the structures set forth in Table 1, including isomers and salt thereof.

[0348] Table 1: Cationic Lipids

[0349] Cationic Lipid Structure

[0350] HO.

[0351] y y

[0352] PVXL-76

[0353] 1 U 0 1

[0354] 6

[0355] \ I

[0356] 1 °> \

[0357] A j

[0358] PVXL-77

[0359] 1 U ^0 U 1

[0360] 6

[0361] PVXL-78 \u /

[0362] 1 U ^0 U 1

[0363] II

[0364] 0

[0365] HO.

[0366] S J PVXL-79

[0367] 1 U ^0 U 1

[0368] 6

[0369] \ 1 / y k— °x y

[0370] PVXL-80

[0371] 1 U ^0? f

[0372] "■ — " X^ — XAA^ / X^A^°\^ / X / XAOA^ —

[0373] II

[0374] 0

[0375] \ 0 /

[0376] 1 N I PVXL-81

[0377] 1 U ^0 U 1

[0378] 6

[0379]

[0380] PVX-PAT-2402-WO

[0381]

[0382] PVX-PAT-2402-WO

[0383]

[0384] PVX-PAT-2402-WO

[0385] HN"^|

[0386] \ PNH / PVXL-101

[0387] 1 U fl f

[0388] II o

[0389] 0 ° / = HN''"X|

[0390] PVXL-102

[0391] o O>

[0392] 1 fl p oo \-=- fl I / \ ° — tz0

[0393] ^N''"X|

[0394] / c

[0395] PVXL-103

[0396] \ °

[0397] o

[0398] ^ ^ 1 flA ^ ^ ^0^ ^ ^ A fl I ^ ^

[0399] 0

[0400] \ <5\ / PVXL-104

[0401] 1 fl ^0 fl 1

[0402] 6

[0403] ^1 CzN. _ S' s ^N^NO y PVXL-105

[0404] '''''~Z-"''''-'-Z--'" Xz---'\zzA 10A fls^^^ ^o fl 1

[0405] 0

[0406] PVXL-106

[0407] , N=N

[0408] > Vk N < S 1 L ^N z'^x ' N. N "> NJ r PVXL-107

[0409] 1 fl fl 1

[0410] II

[0411] 0

[0412]

[0413] PVX-PAT-2402-WO

[0414] JL i JL i i °> i i PVXL-161 Y J

[0415] 1 U ^0 U 1

[0416] 6

[0417] 1 i i £

[0418] I I °> I I PVXL-162 L J

[0419] o

[0420] \°=

[0421] 1 u \ u I / \Yz 0

[0422] 1

[0423] °>

[0424] - / !

[0425] OO-0=- PVXL-163 b

[0426] 1 U ^0 U \

[0427] 6

[0428] ^ °=

[0429] o

[0430] PVXL-170

[0431] | N^N r < X > 1 N / ^NH ] PVXL-176 1 1

[0432] 1 U >>? [

[0433] II

[0434] 0

[0435] OH

[0436] \ 0 / PVXL-251

[0437] 1 0 L 0 <

[0438] 'px - - - - ^0 - - - - 0

[0439]

[0440] PVX-PAT-2402-WO

[0441]

[0442] Other Lipid Components

[0443] Other lipid components of the lipid nanoparticle compositions may include one or more lipids, such as a phospholipid, a sterol and a PEG-lipid.

[0444] Phospholipids

[0445] Phospholipid includes a lipid containing a hydrophilic head with a phosphate group and a hydrophobic tail composed of fatty acid chains attached to a glycerol or sphingosine backbone.

[0446] Exemplary phospholipids for use in the lipid nanoparticle compositions include, but are not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-PVX-PAT-2402-WO

[0447] phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-Phosphocholine (SMPC), 1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC), sphingomyelin, or a combination thereof.

[0448] The proportion of phospholipid present in the lipid nanoparticle compositions is from about 2 mol % to about 65 mol %, from about 5 mol % to about 65 mol %, from about 10 mol % to about 65 mol %, from about 10 mol % to about 50 mol %, or any range therein.

[0449] In some embodiments, the proportion of phospholipid present in the lipid nanoparticle composition is about 65 mol %, about 60 mol %, about 55 mol %, about 50 mol %, about 45 mol %, about 44 mol %, about 43 mol %, about 42 mol %, about 41 mol %, about 40 mol %, about 39 mol %, about 38 mol %, about 37 mol %, about 36 mol %, about 35 mol %, about 34 mol %, about 33 mol %, about 32 mol %, about 31 mol %, about 30 mol %, about 29 mol %, about 28 mol %, about 27 mol %, about 26 mol %, about 25 mol %, about 24 mol %, about 23 mol %, about 22 mol %, about 21 mol %, about 20 mol %, about 19 mol %, about 18 mol %, about 17 mol %, about 16 mol %, about 15 mol %, about 14 mol %, about 13 mol %, about 12 mol %, about 11 mol %, about 10 mol %, about 9 mol %, about 8 mol %, about 7 mol %, about 6 mol %, about 5 mol %, about 4 mol %, about 3 mol %, about 2 mol % or any portion or fraction thereof.

[0450] Sterol

[0451] Lipid nanoparticle composition disclosed herein may include sterol and / or sterol derivatives. The term “sterol” as used herein include, but not limited to, cholesterol, sitosterol, fecosterol, ergosterol, campesterol, stigmasterol or their derivatives. In some embodiments, lipid nanoparticle composition comprises cholesterol and / or cholesterol derivatives. Non-limiting examples of cholesterol and cholesterol derivatives include 5 a-cholestanol, 5P-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5a-cholestane, cholestenone, 5a-cholestanone, 5P-PVX-PAT-2402-WO

[0452] cholestanone, cholesteryl decanoate, or mixtures thereof. Methods of making cholesterol and cholesterol derivatives are well known in the art.

[0453] The proportion of sterol present in the lipid nanoparticle compositions is from about 20 mol % to about 65 mol %, from about 20 mol % to about 60 mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 50 mol %, from about 20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, or any range therein.

[0454] In some embodiments, the proportion of sterol present in the lipid nanoparticle composition is about 65 mol %, about 60 mol %, about 55 mol %, about 50 mol %, about 45 mol %, about 44 mol %, about 43 mol %, about 42 mol %, about 41 mol %, about 40 mol %, about 39 mol %, about 38 mol %, about 37 mol %, about 36 mol %, about 35 mol %, about 34 mol %, about 33 mol %, about 32 mol %, about 31 mol %, about 30 mol %, about 29 mol %, about 28 mol %, about 27 mol %, about 26 mol %, about 25 mol %, about 24 mol %, about 23 mol %, about 22 mol %, about 21 mol %, about 20 mol %, or any portion or fraction thereof.

[0455] PEG-lipid

[0456] The terms “PEG-lipid”, “pegylated lipid”, “PEG linked lipid”, “PEG conjugated lipid”, “PEG-lipid conjugate”, or “PEG modified lipid” have been used interchangeably to mean polyethylene glycol linked to a lipid moiety. The lipid moiety may be linked directly to the PEG molecule or through a linker. In some embodiments, a PEG-lipid comprises a PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and / or PEG-modified cholesterol, and / or mixtures thereof. The methods of making PEG-lipid are well known to persons skilled in the art.

[0457] In some embodiments, PEG-lipid is selected from, but not limited to, mPEG-Dimyristoyl glycerol (mPEG-DMG), mPEG-N, N-Ditetradecylacetamide (mPEG-DTA or ALC0159), mPEG-Cholesterol (mPEG-CLS), mPEG-DSPE, mPEG-DMPE, mPEG-DPPE, mPEG-DLPE, mPEG-DOPE, mPEG-DPPC, mPEG-DSPC, l,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine with conjugated methoxyl poly(ethylene glycol) (mPEG-DSPE),, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (mPEG2000-DMG), a-(3’-{[l,2-di(myristyloxy)propanoxy]carbonylamino}propyl)-co-methoxy, polyoxyethylene (mPEG2000C-DMG) or mixtures thereof.

[0458] The PEG moiety of the PEG-lipid may comprise an average molecular weight ranging from about 0.5 kDa to 10 kDa. In some embodiments, the PEG-lipid has an averagePVX-PAT-2402-WO

[0459] molecular weight of about 0.5 kDa to 5 kDa, about 0.5 kDa to 4 kDa, about 0.5 kDa to 3 kDa, about 0.5 kDa to 2 kDa, or any range therein. In some embodiments, the PEG-lipid has an average molecular weight of about 0.5 kDa to about 2 kDa.

[0460] The proportion of PEG-lipid present in the lipid nanoparticle compositions may be from about 0.2 mol % to about 2.0 mol %, from about 0.2 mol % to about 1.9 mol %, from about 0.2 mol % to about 1.8 mol %, from about 0.2 mol % to about 1.7 mol %, from about 0.2 mol % to about 1.6 mol %, from about 0.2 mol % to about 1.5 mol %, or any range therein.

[0461] In some embodiments, the proportion of PEG-lipid present in the lipid nanoparticle composition is about 2.0 mol %, about 1.9 mol %, about 1.8 mol %, about 1.7 mol %, about 1.6 mol %, about 1.5 mol %, about 1.4 mol %, about 1.3 mol %, about 1.2 mol %, about 1.1 mol %, about 1.0 mol %, about 0.9 mol %, about 0.8 mol %, about 0.7 mol %, about 0.6 mol %, about 0.5 mol %, about 0.4 mol %, about 0.3 mol %, about 0.2 mol % or any portion or fraction thereof.

[0462] Lipid nanoparticle composition comprising the cationic lipid represented by formula (I) or formula (II) may optionally comprise an ionizable polymer.

[0463] Ionizable polymer

[0464] As used herein the term “polymer” means a compound formed from a plurality of repeating units called monomers. Polymers are produced through a process called polymerization wherein two or more monomers are linked through chemical bonds to form the polymer. In some embodiments, the polymer is branched or unbranched. In some embodiments, the polymer may be homopolymer, i.e., consisting of same type of repeat units or monomers, or heteropolymer, i.e., consisting of more than one type of repeat units or monomers. The terms heteropolymer and copolymer have been used interchangeably herein.

[0465] The term “ionizable polymer” as used herein means, a polymer that can exist in a positively charged or neutral form depending on the pH of the solution or environment, for example, ionizable polymer will be cationic (positively charged) when pH of the solution is below the pKa of the ionizable polymer and neutral (no charge) when pH of the solution is same or above the pKa of the ionizable polymer. In some embodiments, ionizable polymer is positively charge in acidic pH i.e., pH 1.0 to pH 6.9. In some embodiments, ionizable polymer is neutral (no charge) around physiological pH (pH 7.0 to pH 7.5).PVX-PAT-2402-WO

[0466] In some embodiments, the ionizable polymer is a biocompatible polymer or biodegradable polymer. The term “biocompatible polymer” and “biodegradable polymer” have been used interchangeably to mean a polymer that is substantially free from any deleterious effects when introduced into a living or biological system. Such polymers are capable of undergoing degradation when introduced into the living or biological systems and are not expected to produce significant toxicity or immunological response.

[0467] In some embodiments, the lipid nanoparticle compositions comprise an ionizable polymer. The ionizable polymer may be selected from chitosan, chitosan derivatives, cellulose derivatives, poly-L-lysine (PLL), protamine, polyethyleneimine, their derivatives, or combinations thereof.

[0468] In some embodiments, the ionizable polymer is positively charged at acidic pH i.e., pH 1.0 to 6.9 and is neutral around physiological pH (pH 7.0 to 7.5).

[0469] The proportion of ionizable polymer present in the lipid nanoparticle compositions may be from about 1 mol % to about 25 mol %.

[0470] In some embodiments, the preferred ionizable polymer comprises chitosan, chitosan derivatives, cellulose derivatives, or combinations thereof.

[0471] Lipid Nanoparticle (LNP) or Lipid Nanoparticle (LNP) composition or formulation Lipid nanoparticle or lipid nanoparticle composition described herein typically comprises a cationic lipid represented by formula (I) or formula (II), a phospholipid, a sterol, a PEG-lipid, and a nucleic acid.

[0472] In some aspects, a lipid nanoparticle composition comprises a cationic lipid represented by formula (I) or formula (II), an ionizable polymer, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid.

[0473] In some aspects, a lipid nanoparticle composition comprising a cationic lipid represented by (I) or formula (II), a nucleic acid, a biocompatible polymer, a cationic lipid, a phospholipid, a sterol and a PEG-lipid is disclosed herein.

[0474] In some aspects, a lipid nanoparticle or lipid nanoparticle composition described herein comprises a cationic lipid represented by formula (I) or formula (II), a phospholipid, a sterol, and a PEG-lipid.

[0475] In some aspects, a lipid nanoparticle composition comprising a cationic lipid represented by formula (I) or formula (II) in an amount from 10 mol % to 70 mol %, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid is disclosed herein.PVX-PAT-2402-WO

[0476] In some aspects, a lipid nanoparticle composition comprising a cationic lipid represented by formula (I) or formula (II) in an amount from 10 mol % to 70 mol %, a phospholipid from 2 mol % to 65 mol %, a sterol, a PEG-lipid, and a nucleic acid is disclosed herein.

[0477] In some aspects, a lipid nanoparticle composition comprising a cationic lipid represented by formula (I) or formula (II) in an amount from 10 mol % to 70 mol %, a phospholipid from 2 mol % to 65 mol %, a sterol from 20 mol % to 65 mol %, a PEG-lipid, and a nucleic acid is disclosed herein.

[0478] In some aspects, a lipid nanoparticle composition comprising a cationic lipid represented by formula (I) or formula (II) in an amount from 10 mol % to 70 mol %, a phospholipid from 2 mol % to 65 mol %, a sterol from 20 mol % to 65 mol %, a PEG-lipid from 0.2 mol % to 2.0 mol %, and a nucleic acid is disclosed herein.

[0479] In some aspects, a lipid nanoparticle composition comprising a cationic lipid represented by formula (I) to formula (II) in an amount from 10 mol % to 70 mol %, a phospholipid from 2 mol % to 65 mol %, a sterol from 20 mol % to 65 mol %, a PEG-lipid from 0.2 mol % to 2.0 mol %, ionizable polymer from 1 mol % to 25 mol %, and a nucleic acid is disclosed herein.

[0480] In some embodiments, the disclosure relates to a composition comprising lipid nanoparticle described herein and pharmaceutically acceptable carrier or excipient, such as but not limited to, buffering agents, stabilizers, tonicity modifiers, surfactants, chelating agents, salts, anti-oxidants, diluents, and / or preservatives or a combination thereof.

[0481] In some embodiments, lipid to nucleic acid ratio or N: P ratio in lipid nanoparticle composition or formulation is between 1 to 18, between 1 to 17, between 1 to 16, between 1 to 15, between 1 to 14, between 1 to 13, between 1 to 12, between 1 to 11, between 1 to 10, between 1 to 9, between 1 to 8, between 1 to 7, between 1 to 6, between 1 to 5, between 1 to 4, between 1 to 3, between 1 to 2, or any range therein.

[0482] In some embodiments, lipid to nucleic acid ratio or N: P ratio in lipid nanoparticle composition or formulation is about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, or any portion or fraction thereof.

[0483] In some embodiments, the disclosure relates to a method of delivering a nucleic acid comprising administering the lipid nanoparticle composition comprising a nucleic acid, aPVX-PAT-2402-WO

[0484] cationic lipid represented by formula (I) or formula (II), a phospholipid, a sterol, and a PEG-lipid of the present disclosure.

[0485] In some embodiments, the disclosure relates to a method of delivering a nucleic acid comprising administering the lipid nanoparticle composition comprising a nucleic acid, a cationic lipid represented by formula (I) or formula (II), a phospholipid, a sterol, a PEG-lipid, and an ionizable polymer of the present disclosure.

[0486] In some embodiments, the disclosure relates to a method of treating or preventing a disease, comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a cationic lipid represented by formula (I) or formula (II), a phospholipid, a sterol, a PEG-lipid, and a nucleic acid.

[0487] In some embodiments, the disclosure relates to a method of treating or preventing a disease, comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a cationic lipid represented by formula (I) or formula (II), a phospholipid, a sterol, a PEG-lipid, an ionizable polymer, and a nucleic acid.

[0488] In some embodiments, the disclosure relates to use of a lipid nanoparticle composition comprising a cationic lipid represented by formula (I) or formula (II), a phospholipid, a sterol, a PEG-lipid, and a nucleic acid in the manufacture of a medicament for the treatment or prevention of a disease in a subject.

[0489] In some embodiments, the disclosure relates to use of a lipid nanoparticle composition comprising a cationic lipid represented by formula (I) or formula (II), a phospholipid, a sterol, a PEG-lipid, an ionizable polymer and a nucleic acid in the manufacture of a medicament for the treatment or prevention of a disease in a subject.

[0490] The lipid nanoparticle composition described herein can be used as a platform for therapeutic or prophylactic delivery of nucleic acids. The nucleic acids may encode one or more antigens, one or more proteins, one or more antibodies or combinations thereof. The nucleic acids may regulate or modulate cellular functions. The nucleic acid may belong to any organism such as a prokaryote or a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a mycoplasma, a protozoan, an animal, or a human.

[0491] In some embodiments, the lipid nanoparticle compositions may be used to treat or prevent diseases, such as but not limited to:

[0492] diseases caused by viruses belonging to families, for example, Picomaviride, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Arteriviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae,PVX-PAT-2402-WO

[0493] Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Pepadnaviridae, Adenoviridae, Parvoviridae, Hepeviridae, Circoviridae or a combination thereof.

[0494] • diseases caused by bacteria belonging to genera, for example, Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia or a combination thereof.

[0495] • cancers, for example, bladder cancer, breast cancer, colon and rectal cancer, endometrial cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, non-hodgkin lymphoma, pancreatic cancer, prostate cancer, or thyroid cancer.

[0496] Nucleic acid

[0497] The term “nucleic acid” as used herein means a polymer comprising two or more nucleotides for example, deoxyribonucleotides or ribonucleotides, either in an unmodified or modified form. The nucleic acid may be either single stranded or double stranded, linear or circular.

[0498] The term "nucleotide” as used herein means a ribonucleotide or a deoxyribonucleotide. If the term nucleotide is used in the context of RNA, it refers to ribonucleotide, and if it is used in the context of DNA, it refers to deoxyribonucleotide. The term “ribonucleic acid” or “RNA” has been used interchangeably herein and means a polymer of ribonucleotides. The RNA may be either single stranded or double stranded, linear or circular. The term RNA also includes messenger RNA (mRNA), and noncoding RNA (ncRNA).

[0499] The term “deoxyribonucleic acid” or “DNA” has been used interchangeably herein and means a polymer of deoxyribonucleotides. The DNA may be either single stranded or double stranded, linear or circular.

[0500] In some embodiments, the nucleic acid encodes an antigen such as, but not limited to: those derived from Cholera toxoid, tetanus toxoid, diphtheria toxoid, hepatitis B surface antigen, hemagglutinin, neuraminidase, influenza M protein, PfHRP2, pLDH, aldolase, MSP1, MSP2, AMA1, Der-p- 1, Der-f-1, Adipophilin, AFP, AIM-2, ART-4, BAGE, alphafetoprotein, BCL-2, Bcr-Abl, BING-4, CEA, CPSF, CT, cyclin DIEp-CAM, EphA2, EphA3,PVX-PAT-2402-WO

[0501] ELF-2, FGF-5, G250, Gonadotropin Releasing Hormone, HER-2, intestinal carboxyl esterase (iCE), IL13Ralpha2, MAGE-1, MAGE-2, MAGE-3, MART-1, MART-2, M-CSF, MDM-2, MMP-2, MUC-1, NY- EOS-1, MUM-1, MUM-2, MUM-3, p53, PBF, PRAME, PSA, PSMA, RAGE-1, RNF43, RU1, RU2AS, SART-1, SART-2, SART-3, SAGE-1, SCRN 1, SOX2, SOXIO, STEAP1, survivin (BIRC5), Telomerase, TGFbetaRl 1, TRAG-3, TRP-1, TRP-2, TERT, or WT1; those derived from a virus, such as Cowpoxvirus, Vaccinia virus, Pseudocowpox virus, Human herpesvirus 1, Human herpesvirus 2, Cytomegalovirus, Human adenovirus A-F, Polyomavirus, Human papillomavirus, Parvovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus, Orthoreo virus, Rotavirus, Ebolavirus, parainfluenza virus, influenza virus (e.g., influenza A virus, influenza B virus, influenza C virus), Measles virus, Mumps virus, Rubella virus, Pneumovirus, Human respiratory syncytial virus, Rabies virus, California encephalitis virus, Japanese encephalitis virus, Hantaan virus, Lymphocytic choriomeningitis virus, Coronavirus (e.g., MERS-CoV, SARS-CoV-1, SARS-CoV-2, OC43, HKU1, bat coronavirus, other betacoronavirus), Enterovirus, Rhino virus, Poliovirus, Norovirus, Flavivirus, Dengue virus, West Nile virus, Yellow fever virus and varicella; those derived from a bacterium, such as Anthrax (Bacillus anthracis), Brucella, Bordetella pertussis, Candida, Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus, Diphtheria, Escherichia coli 0151: H7, Enterohemorrhagic Escherichia coli, Enterotoxigenic Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Legionella, Leptospira, Listeria, Meningococcus, Mycoplasma pneumoniae, Mycobacterium, Pertussis, Pneumonia, Salmonella, Shigella, Staphylococcus, Streptococcus pneumoniae and Yersinia enterocolitica, or those derived from a protozoa, e.g., of the genus Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale or Plasmodium knowlesi).

[0502] The antigen may be an allergen derived from, without limitation, cells, cell extracts, proteins, polypeptides, peptides, peptide mimics of polysaccharides and other molecules, such as small molecules, lipids, glycolipids, carbohydrates of plants, animals, fungi, insects, food, drugs, dust, and mites. Allergens include but are not limited to environmental aeroallergens; plant pollens (e.g., ragweed / hayfever); weed pollen allergens; grass pollen allergens; Johnson grass; tree pollen allergens; ryegrass; arachnid allergens (e.g., house dust mite allergens); storage mite allergens; Japanese cedar pollen / hay fever; mold / fungal spore allergens; animal allergens (e.g., dog, guinea pig, hamster, gerbil, rat, mouse, etc., allergens); food allergens (e.g., crustaceans; nuts; citrus fruits; flour; coffee); insect allergens (e.g., fleas,PVX-PAT-2402-WO

[0503] cockroach); venoms (Hymenoptera, yellow jacket, honey bee, wasp, hornet, fire ant); bacterial allergens (e.g., streptococcal antigens; parasite allergens such as Ascaris antigen); viral antigens; drug allergens; hormones (e.g., insulin); enzymes (e.g., streptokinase); and drugs or chemicals capable of acting as incomplete antigens or haptens (e.g., the acid anhydrides and the isocyanates). Where a hapten is used in a composition of the disclosure, it may be attached to a carrier to form a hapten-carrier adduct. The hapten-carrier adduct is capable of initiating a humoral immune response, whereas the hapten itself would not elicit antibody production. Non-limiting examples of haptens are aniline, urushiol (a toxin in poison ivy), hydralazine, fluorescein, biotin, digoxigenin and dinitrophenol.

[0504] In some embodiments, the antigen is derived, without limitation, from viruses belonging to the families, for example, Picomaviride, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Arteriviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Pepadnaviridae, Adenoviridae, Parvoviridae, Hepeviridae, Circoviridae, or a combination thereof.

[0505] In some embodiments, the antigen is derived, without limitation, from bacteria belonging to genera, for example, Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof.

[0506] In other embodiments, the antigen is an antigen associated with a disease where it is desirable to sequester the antigen in circulation, such as for example an amyloid protein (e.g., Alzheimer's disease).

[0507] In some embodiments, the nucleic acid regulates or modulates cellular functions. The term “cellular functions” as used herein means various cellular or biological processes such as, but not limited to, biosynthesis, cell division, cell cycle regulation, cellular metabolism, ion transport, absorption, secretion, homeostasis, replication, transcription, translation, cell signalling, endocytosis, exocytosis, phagocytosis, apoptosis, DNA replication, DNA repair, protein synthesis, gene regulation, cell repair, cell growth, cell differentiation, cellular trafficking, cell proliferation, metabolic pathways etc.PVX-PAT-2402-WO

[0508] The terms “regulate” or “modulate” or “regulation” or “modulation” have been used interchangeably herein and means an act of controlling a cellular or biological process or to exert a modifying or controlling influence on cellular or biological process.

[0509] Messenger RNA (mRNA)

[0510] Messenger RNA (mRNA) is a polymer of ribonucleotides that encodes at least a protein or polypeptide or peptide. Typically, an mRNA includes at least a coding region, a 5’ UTR, a 3’ UTR, a 5’ cap and a poly(A) tail. UTR (untranslated regions) flanks the coding region or open reading frame (ORF). The 5’ UTR and the 3’ UTR are sections of the mRNA before the start codon and after the stop codon respectively. The 5’ UTR has a cap (5’ cap) consisting of altered nucleotides. mRNA also contains a poly adenylated region at its 3’ end having adenine nucleotides called poly(A) tail.

[0511] In some embodiments, the mRNA may be unmodified or modified or a combination of both. The modification may be in the nucleobase of the nucleotide, or sugar moiety of the nucleotide, or the phosphate of the nucleotide. In some embodiments, unmodified mRNA may comprise naturally occurring nucleosides, for example, adenosine, guanosine, cytidine, and uridine. mRNA may comprise one or more modified nucleosides, for example, adenosine analog, guanosine analog, cytidine analog, uridine analog, or a combination thereof.

[0512] In some embodiments, the one or more modified nucleosides is a nucleoside analog selected from 2-aminoadenosine, 3-methyl adenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, or a combination thereof.

[0513] In some embodiments, the one or more modified nucleosides is a uridine analog selected from propynyl-uridine, pseudouridine, C5 -bromouridine, C5-fluoro uridine, C5-iodouridine, C5-propynyl-uridine, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 3-methyl-uridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 2-thio-2’-O-methyl-uridine, 5-methoxycarbonylmethyl-2’-O-methyl-uridine, 5-carboxymethylaminomethyl-2’ -O-methyl-uridine, 3,2’ -O-dimethyl-uridine, 5 -propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurino-4-thio-pseudouridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-Ideaza-pseudouridine, 2-thio-l -methyl- 1-deaza-pseudouridine, dihydro-uridine, dihydropseudouridine, 2-thio-dihydro-uridine, 2-thio-dihydro-pseudouridine, 2-methoxy-uridine, 2-PVX-PAT-2402-WO

[0514] methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, or a combination thereof.

[0515] In some embodiments, the one or more modified nucleosides is a cytidine analog selected from 5-methylcytidine, C5-propynyl-cytidine, C5-methylcytidine, pseudoisocytidine, 1-methyl-pseudoisocytidine, pyrrolo-pseudoisocytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1-deaza-pseudoisocytidine, 1-methyl-l-ldeaza-pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine, or a combination thereof.

[0516] Methods for making modified nucleosides are well known in the art.

[0517] In some embodiments, the modified nucleoside is pseudouridine, for example, 1-methyl-pseudouridine, 1-propynyl-pseudouridine, 1 -carboxymethyl-pseudo uridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 4-methoxy -pseudouridine, or 4-methoxy-2-thio-pseudouridine 4-thio-pseudouridine, 2-thio-pseudouridine, 4-thio- 1-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, dihydro-pseudouridine, or a combination thereof.

[0518] In some embodiments, mRNA is obtained from natural sources (i.e., isolated from the cells), produced using recombinant expression system, or chemically synthesized.

[0519] mRNAs according to the present disclosure may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions may vary according to the specific application. Methods of making mRNA through IVT reaction is well known in the art (see for example, Beckert, Bertrand and Masquida, Benoit Methods in Molecular Biology (2011) 703, 29-41; Brunelle, Julie L. and Green Rachel Methods in Enzymology (2013) 530, 101-114; Kamakaka, Rohinton T. and Kraus W. Lee Current Protocols in Cell Biology (1999) 11.6.1-11.6.17; Kanwal, Fariha et al. Cellular Physiology and Biochemistry (2018) 48:1915-1927; WO2018157153; W02020185811;

[0520] W02022082001).

[0521] In some embodiments, the in vitro transcription occurs in a single batch. In some embodiments, IVT reaction includes capping and tailing reactions either co-transcriptionally or separately. A cap analog is added to the in vitro transcription reaction and will be incorporated at the 5’ end of the mRNA during the reaction. Alternative method of capping involves adding the cap post-transcriptionally through an enzymatic reaction. The poly (A)PVX-PAT-2402-WO

[0522] tail can be incorporated into the DNA template sequence, and thus the poly (A) tail will be incorporated into the mRNA by T7 RNA polymerase during the in vitro transcription.

[0523] Alternative method of tailing involves adding the poly (A) tail post-transcriptionally through an enzymatic reaction. In some embodiments, capping and tailing reactions are performed co-transcriptionally i.e., during the IVT reaction. In some embodiments, capping and tailing reactions are performed separately from IVT reaction.

[0524] mRNA produced as a result of IVT reaction may be purified using techniques well known in the art, such as, centrifugation, filtration and / or chromatographic techniques. The purification of mRNA may be accomplished before capping and tailing steps are performed or after capping and tailing. The synthesized mRNA may be purified by ethanol precipitation or filtration or chromatography methods. In some embodiments, tangential flow filtration is used to purify mRNA. In some embodiments, mRNA is purified by chromatographic step. In other embodiments, mRNA is purified by a combination of filtration and chromatography steps.

[0525] In some embodiments, a suitable mRNA sequence is an mRNA sequence encoding a protein, peptide, polypeptide or an antibody. In some embodiments, a suitable mRNA sequence is codon optimized for efficient expression in a host cell or organism. Codon optimization typically includes modifying a naturally-occurring or wild-type nucleic acid sequence encoding a peptide, polypeptide or protein to achieve the highest possible expression of peptide, polypeptide, protein or an antibody without altering the amino acid sequence.

[0526] Any length of mRNA can be encapsulated or formulated in the lipid nanoparticles of the present disclosure. The length of the mRNA used in the lipid nanoparticle of the present disclosure depends on the gene product or protein or protein fragment to be incorporated in the lipid nanoparticle. Thus, mRNA can be very short extending to about a few hundred nucleotides in length or very long extending to about several thousand nucleotides in length. In some embodiments, mRNA is about 0.5 kb, about 1 kb, about 1.5 kb, about 2 kb, about 2.5 kb, about 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, about 6 kb, about 6.5 kb, about 7 kb, about 7.5 kb, about 8 kb, about 8.5 kb, about 9 kb, about 9.5 kb, about 10 kb, about 11 kb, about 12 kb, about 13 kb, about 14 kb, about 15 kb, about 16 kb, about 17 kb, about 18 kb, about 19 kb, about 20 kb, about 22 kb, about 24 kb, about 26 kb, about 28 kb, or about 30 kb in length. In other embodiments, mRNA is about 0.5 to 30 kb, about 0.5 to 25 kb, about 0.5 to 20 kb in length. In still other embodiments, mRNA is about 1PVX-PAT-2402-WO

[0527] to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 1 to 9 kb, about 1 to 8 kb, about 1 to 7 kb, about 1 to 6 kb, about 1 to 5 kb, about 1 to 4 kb, about 1 to 3 kb, or about 1 to 2 kb in length.

[0528] In some embodiments, the mRNA is circular. In other embodiments, the mRNA is linear.

[0529] In some embodiments, the mRNA is self-amplifying or self-replicating. Selfamplifying or self-replicating mRNA as used herein means an mRNA that self-replicate upon delivery into the cells. Such mRNAs typically contain a replicase, usually derived from an alphavirus, which enables amplification of the original strand of mRNA encoding the protein of interest upon delivery into the cells.

[0530] mRNA present in the lipid nanoparticle composition may be present in a biologically effective amount or therapeutically effective amount. In some embodiments, the biologically effective amount of mRNA present in the lipid nanoparticle composition is between 0.1 pg to 1000 pg, 0.1 pg to 950 pg, 0.1 pg to 900 pg, 0.1 pg to 850 pg, 0.1 pg to 800 pg, 0.1 pg to 750 pg, 0.1 pg to 700 pg, 0.1 pg to 650 pg, 0.1 pg to 600 pg, 0.1 pg to 550 pg, 0.1 pg to 500 pg, 0.1 pg to 450 pg, 0.1 pg to 400 pg, 0.1 pg to 350 pg, 0.1 pg to 300 pg, 0.1 pg to 250 pg, 0.1 pg to 200 pg, 0.1 pg to 150 pg, 0.1 pg to 100 pg, 0.1 pg to 75 pg, 0.1 pg to 50 pg, 0.1 pg to 25 pg, 0.1 pg to 20 pg, 0.1 pg to 15 pg, 0.1 pg to 10 pg, 0.1 pg to 5 pg, or any range therein. In some embodiments, the biologically effective amount of mRNA present in the lipid nanoparticle composition is from about 0.1 pg to 1000 pg, 0.1 pg to 950 pg, 0.1 pg to 900 pg, 0.1 pg to 850 pg, 0.1 pg to 800 pg, 0.1 pg to 750 pg, 0.1 pg to 700 pg, 0.1 pg to 650 pg, 0.1 pg to 600 pg, 0.1 pg to 550 pg, 0.1 pg to 500 pg or any range therein.

[0531] In some embodiments, the biologically effective amount of mRNA present in the lipid nanoparticle composition is 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7, pg, 0.8 pg, 0.9 pg, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 15 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 55 pg, 60 pg, 65 pg, 70 pg, 75 pg, 80 pg, 85 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg, 200 pg, 220 pg, 240 pg, 260 pg, 280 pg, 300 pg, 350 pg, 400 pg, 450 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1000 pg or any portion or fraction thereof.

[0532] In some embodiments, the mRNA encodes one or more proteins, one or more antibodies, or combinations thereof. The mRNA encoding one or more proteins, or one or more antibodies may belong to any organism such as a prokaryote or a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a mycoplasma, a protozoan, an animal, or a human.PVX-PAT-2402-WO

[0533] Non-coding RNA (ncRNA)

[0534] The term “non-coding RNA” or “ncRNA” has been used interchangeably to mean any RNA molecule that is not generally translated, but sometimes can, into a polypeptide or protein and includes, long non-coding RNA (IncRNA), micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and PIWI-interacting RNA (piRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). The term ncRNA also includes such RNAs that encode small peptides such as IncRNA.

[0535] In some embodiments, the nucleic acid component of the lipid nanoparticle compositions comprises ncRNA. The ncRNA may be naturally occurring or wild type. In other embodiments, the ncRNA may be synthetically produced.

[0536] In some embodiments, the ncRNA is single stranded or double stranded.

[0537] In some embodiments, the ncRNA is a few nucleotides long to several thousand nucleotides long.

[0538] In some embodiments, the ncRNA comprises long non-coding RNA (IncRNA), micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and PIWI-interacting RNA (piRNA), transfer RNA (tRNA), ribosomal RNA (rRNA) or a combination thereof.

[0539] In some embodiments, the long non-coding RNA (IncRNA) is more than 200 nucleotide long.

[0540] Deoxyribonucleic Acid (DNA)

[0541] Any DNA molecule capable of transferring a gene into a cell, for example to express a transcript, can be incorporated into the lipid nanoparticle compositions described herein.

[0542] The term “DNA sequence” or “DNA segment” or "gene" has been used interchangeably and mean a segment or sequence of DNA capable of being used to produce a transcript which may either be a messenger RNA (mRNA) or non-coding RNAs (ncRNAs) such as long non-coding RNA (IncRNA), micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and PIWI-interacting RNA (piRNA), transfer RNA (tRNA) or ribosomal RNA (rRNA).

[0543] In some embodiments, the DNA molecule is obtained from natural sources. In other embodiments, the DNA molecule is recombinantly or synthetically produced.

[0544] In some embodiments, the DNA molecule is modified or unmodified, linear or circular.PVX-PAT-2402-WO

[0545] In some embodiments, the DNA molecule is double stranded or single stranded. In some embodiments, the DNA molecule includes a coding sequence or a noncoding sequence.

[0546] In some embodiments, the DNA molecule is a few nucleotides long to several thousand nucleotides long.

[0547] Methods of Treatment

[0548] Disclosed herein are methods of treating or preventing a disease. The method may comprise administering to a subject in need thereof the lipid nanoparticle composition comprising the cationic lipid represented by formula (I) or formula (II) disclosed herein. The disease may be cancer, an infectious disease or a disease and / or disorder ameliorated by humoral and / or cellular immune response.

[0549] The term “infectious disease” or “infectious diseases” have been used interchangeably and means the disorders caused by organisms such as a bacterium, a virus, a fungus, a protozoan, a worm, a mycoplasma, a parasite, or a combination thereof. Non-limiting examples of infectious diseases are those caused by a virus of the family Picornaviride, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Arteriviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Pepadnaviridae, Adenoviridae, Parvoviridae, Hepeviridae, Circoviridae; those caused by a bacterium of the genus Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, those caused by a protozoan of the genus Plasmodium, Trypanosoma, Leishmania, Toxoplasma, Entamoeba, Trichomonas or a combination thereof.

[0550] As used herein, the terms “cancer”, “cancer cells”, “tumor” and “tumor cells”, (used interchangeably) refer to cells that exhibit abnormal growth, characterized by a significant loss of control of cell proliferation or cells that have been immortalized. The term “cancer” or “tumor” includes metastatic as well as non-metastatic cancer or tumors. A cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.PVX-PAT-2402-WO

[0551] “Humoral immune response” as referred to herein relates to antibody production and the accessory processes that accompany it, such as for example T-helper 2 (Th2) cell activation and cytokine production, isotype switching, affinity maturation and memory cell activation. It also refers to the effector functions of an antibody, such as for example toxin neutralization, classical complement activation, and promotion of phagocytosis and pathogen elimination. The humoral immune response is aided by CD4+Th2 cells and therefore the activation or generation of this cell type is also indicative of a humoral immune response as referred to herein.

[0552] A “humoral immune response” as referred to herein may also encompass the generation and / or activation of T-helper cells. Helper T cells are a subset of helper effector T-lymphocytes characterized by the secretion of host defense cytokines such as interferon-y (IFN-y), tumor necrosis factor-a (TNF-a), IL-17, IL-17F, IL-21, and IL-22. Helper T cells have been postulated to facilitate the humoral immune response, such as for example, providing an important function in anti-microbial immunity and protecting against infections. Their production of IL-22 is thought to stimulate epithelial cells to produce antimicrobial proteins and production of IL- 17 may be involved in the recruitment, activation and migration of neutrophils to protect against host infection by various bacterial and fungal species.

[0553] In some embodiments, the antigen encoded by the nucleic acid in the composition of the disclosure may be a cancer or tumor-associated protein, such as for example, a membrane surface-bound cancer antigen which is capable of being recognized by an antibody.

[0554] Cancers that may be treated and / or prevented by the use or administration of a composition of the disclosure include, without limitation, carcinoma, adenocarcinoma, lymphoma, leukemia, sarcoma, blastoma, myeloma, and germ cell tumors. In one embodiment, the cancer may be caused by a pathogen, such as a virus. Viruses linked to the development of cancer are known to the skilled person and include, but are not limited to, Human Papillomaviruses (HPV), John Cunningham virus (JCV), Human herpes virus 8, Epstein Barr Virus (EBV), Merkel cell polyomavirus, Hepatitis C Virus and Human T cell leukemia virus- 1. A composition of the disclosure may be used for either the treatment or prophylaxis of cancer, for example, in the reduction of the severity of cancer or the prevention of cancer recurrences. Cancers that may benefit from the compositions of the disclosure include any malignant cell that expresses one or more tumor specific antigens.PVX-PAT-2402-WO

[0555] In some embodiments, the antigen may be a toxin or an allergen that is capable of being neutralized by an antibody.

[0556] In some embodiments, the antigen may be an antigen associated with a disease where it is desirable to sequester the antigen in circulation, such as for example an amyloid protein (e.g., Alzheimer's disease). Thus, a composition of the disclosure may be suitable for use in the treatment and / or prevention of a neurodegenerative disease in a subject in need thereof, wherein the neurodegenerative disease is associated with the expression of an antigen. The subject may have a neurodegenerative disease or may be at risk of developing a neurodegenerative disease. Neurodegenerative diseases that may be treated and / or prevented by the use or administration of a composition of the disclosure include, without limitation, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). For example, Alzheimer's disease is characterized by the association of B-amyloid plaques and / or tau proteins in the brains of patients with Alzheimer's disease (see, for example, Goedert and Spillantini, Science, 314: 777-781, 2006). Herpes simplex virus type 1 has also been proposed to play a causative role in people carrying the susceptible versions of the apoE gene (Itzhaki and Wozniak, J Alzheimers Dis 13: 393-405, 2008).

[0557] In some embodiments, the composition may comprise a mixture of B cell epitopes as antigens for inducing a humoral immune response. The B cell epitopes may be linked to form a single polypeptide.

[0558] In some embodiments, the antigen may be any peptide or polypeptide that is capable of inducing a specific humoral immune response to a specific conformation on targeted tumor cells.

[0559] In some embodiments, the compositions of the present disclosure may be used to induce humoral and / or cellular immune response in a subject. Accordingly, compositions as described herein may be useful for treating or preventing diseases and / or disorders ameliorated by humoral immune responses (e.g., involving B-cells and antibody production). The compositions may find application in any instance in which it is desired to administer an antigen to a subject to induce a humoral immune response or antibody production.

[0560] A humoral immune response, as opposed to cell-mediated immunity, is mediated by secreted antibodies which are produced in the cells of the B lymphocyte lineage (B cells). Such secreted antibodies bind to antigens, such as for example those on the surfaces of foreign substances and / or pathogens (e.g., viruses, bacteria, etc.) and flag them for destruction.PVX-PAT-2402-WO

[0561] Antibodies are the antigen- specific glycoprotein products of a subset of white blood cells called B lymphocytes (B cells). Engagement of antigen with antibody expressed on the surface of B cells can induce an antibody response comprising stimulation of B cells to become activated, to undergo mitosis and to terminally differentiate into plasma cells, which are specialized for synthesis and secretion of antigen- specific antibody.

[0562] B cells are the sole producers of antibodies during an immune response and are thus a key element to effective humoral immunity. In addition to producing large amounts of antibodies, B cells also act as antigen-presenting cells and can present antigen to T cells, such as T helper CD4 or cytotoxic CD8, thus propagating the immune response. B cells, as well as T cells, are part of the adaptive immune response which is essential for vaccine efficacy. During an active immune response, induced either by vaccination or natural infection, antigen- specific B cells are activated and clonally expand. During expansion, B cells evolve to have higher affinity for the epitope. Proliferation of B cells can be induced indirectly by activated T-helper cells, and also directly through stimulation of receptors, such as the tolllike receptors (TLRs).

[0563] Antigen presenting cells, such as dendritic cells, macrophages and B cells, are drawn to vaccination sites and can interact with antigens and adjuvants contained in the vaccine. The adjuvant stimulates the cells to become activated and the antigen provides the blueprint for the target. Different types of adjuvants provide different stimulation signals to cells. For example, Poly EC (a TLR3 agonist) can activate dendritic cells, but not B cells. Adjuvants such as Pam3Cys, Pam2Cys and FSL-1 are especially adept at activating and initiating proliferation of B cells, which is expected to facilitate the production of an antibody response (Moyle et al., Curr Med Chem, 2008; So., J Immunol, 2012, which are incorporated hereby by reference in their entireties).

[0564] The compositions of the present disclosure, by stimulating strong antibody responses, may be capable of protecting a subject from a disease, disorder or ailment associated with an antigen capable of inducing a humoral immune response.

[0565] Without limitation, this includes for example, infectious diseases, cancers involving a membrane surface-bound cancer antigen which is recognized by an antibody, diseases where it is desirable to sequester antigen in circulation, like amyloid protein (e.g., Alzheimer's disease); neutralizing toxins with an antibody; neutralizing viruses or bacteria with an antibody; or neutralizing allergens (e.g., pollen) for the treatment of allergies.PVX-PAT-2402-WO

[0566] In some embodiments, the composition may be administered via oral, nasal, rectal or parenteral administration. Parenteral administration includes intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, transepithelial, intrapulmonary, intrathecal, and topical modes of administration. In some embodiments, the composition is administered via intramuscular, subcutaneous or intradermal injection.

[0567] The amount of composition used in a single treatment may vary depending on factor such as the nature of negatively charged molecule to be delivered, the type of formulation, and the size of the subject. One skilled in the art will be able to determine, without undue experimentation, the effective amount of composition to use in a particular application.

[0568] The skilled artisan can determine suitable treatment regimes, routes of administration, dosages, etc., for any particular application in order to achieve the desired result. Factors that may be taken into account include, e.g., the nature of a polypeptide to be expressed; the disease state to be prevented or treated; the age, physical condition, body weight, sex and diet of the subject; and other clinical factors.

[0569] The subject to be treated may be any vertebrate, preferably a mammal, more preferably a human.

[0570] Embodiments

[0571] Some of the embodiments of the present disclosure are set out in the following numbered paragraphs.

[0572] 1. A cationic lipid represented by formula (I)

[0573] R1 — L5

[0574] AI L4

[0575] R2L6A L3Q 1—j

[0576] O - P = O

[0577] O - L2

[0578]

[0579] X2— R4

[0580] formula (I)

[0581] or isomer, or salt thereof, wherein:

[0582] Ri and R2 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, Ce-io aryl, saturated or unsaturatedPVX-PAT-2402-WO

[0583] C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?, or

[0584] Ri and R2 may combine together to form a saturated or unsaturated C3-10 cycloalkyl, Ci>-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;

[0585] R3 and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26;

[0586] Rs, Re, and R7 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms;

[0587] each of L1, L2, L3, L4, L5, L6, and L7 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene;

[0588] Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-;

[0589] A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;

[0590] _

[0591] Ai is \, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, S or N provided that when Ai is -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or S, one of Ri or R2is absent; and wherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

[0592] A cationic lipid represented by formula (II)PVX-PAT-2402-WO

[0593]

[0594] x2— R4formula (II)

[0595] or isomer, or salt thereof, wherein:

[0596] = represents either a single bond or a double bond;

[0597] Ri and R2 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?, or

[0598] Ri and R2 may combine together to form a saturated or unsaturated C3-10 cycloalkyl, Ci>-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;

[0599] R3 and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26;

[0600] Rs, Re, and R7 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms;

[0601] each of L1, L2, L3, L4, L5, L6, and L7 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene;

[0602] Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-;

[0603] A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;

[0604] _

[0605] Ai is \, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, S, or N provided thatPVX-PAT-2402-WO

[0606] when Ai is -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, - C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or S, one of Ri or R2is absent; and

[0607] wherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

[0608] 3. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2are each independently H or -OH.

[0609] 4. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2are each independently Ci-io alkyl.

[0610] 5. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2are each independently C2-10 alkenyl.

[0611] 6. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2are each independently C2-10 alkynyl.

[0612] 7. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2are each independently saturated or unsaturated C3-10 cycloalkyl.

[0613] 8. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2are each independently saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms.

[0614] 9. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2are each independently H or -O-L7-R5.

[0615] 10. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri is H and R2is -O-L7-R5.

[0616] 11. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri is -O-L7-R5 and R2is H.

[0617] 12. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2are each independently -O-L7-R5.

[0618] 13. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2are each independently H or -NReR?.

[0619] 14. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri is H and R2is -NR6R7.

[0620] 15. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri is -NReR? and R2is H.PVX-PAT-2402-WO

[0621] 16. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2 are each independently -NReR?.

[0622] 17. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2 combine together to form a saturated or unsaturated C3-10 cycloalkyl.

[0623] 18. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2 combine together to form Ce-io aryl.

[0624] 19. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2 combine together to form a saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 hetero atoms.

[0625] 20. The cationic lipid according to any one of the paragraphs 1-2, wherein Ri and R2 combine together to form C5-10 heteroaryl containing 1-4 heteroatoms.

[0626] 21. The cationic lipid according to any one of the paragraphs 9-12, wherein L7 is either absent or C1-10 alkylene, C2-10 alkenylene, or C2-10 alkynylene.

[0627] 22. The cationic lipid according to paragraph 21, wherein L7 is absent.

[0628] 23. The cationic lipid according to any one of the paragraphs 9-12, wherein R5 is H, -OH, Ci-10 alkyl, saturated or unsaturated C3-10 cycloalkyl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms.

[0629] 24. The cationic lipid according to paragraph 23, wherein R5 is C3-6 heterocycloalkyl containing 1-4 heteroatoms.

[0630] 25. The cationic lipid according to paragraph 23, wherein R5 is C5-9 heteroaryl containing 1- 4 heteroatoms.

[0631] 26. The cationic lipid according to any one of the paragraphs 13-16, wherein Re and R7 are independently chosen from H, -OH, C1-10 alkyl, saturated or unsaturated C3-10 cycloalkyl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms.

[0632] 27. The cationic lipid according to paragraph 26, wherein Re and R7 are independently chosen from H, -OH, C1-6 alkyl, and saturated or unsaturated C3-8 heterocycloalkyl containing 1-4 heteroatoms.

[0633] 28. The cationic lipid according to any one of the preceding paragraphs, wherein R3 and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26.PVX-PAT-2402-WO

[0634] 29. The cationic lipid according to paragraph 28, wherein R3 and R4 are independently chosen from C1-20 alkyl, C2-20 alkenyl, and C2-20 alkynyl.

[0635] 30. The cationic lipid according to paragraph 28, wherein R3 and R4 are independently chosen from -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26.

[0636] 31. The cationic lipid according to paragraph 28 or paragraph 30, wherein the m, n, and y are each independently an integer ranging from 0 to 20, 0 to 16, 0 to 12, 0 to 10, or 0 to 8.

[0637] 32. The cationic lipid according to any one of the preceding paragraphs, wherein L1 and L2 are independently chosen from C1-10, C1-8, C1-6, C1-4, C1-3, and C1-2 alkylene.

[0638] 33. The cationic lipid according to any one of the preceding paragraphs, wherein Li and L2 are independently chosen from C2-10, C2-8, C2-6, and C2-4 alkenylene.

[0639] 34. The cationic lipid according to any one of the preceding paragraphs, wherein L3 and L4 are independently chosen from C1-10, C1-8, C1-6, C, C1-3, and C1-2 alkylene.

[0640] 35. The cationic lipid according to any one of the preceding paragraphs, wherein L3 and L4 are independently chosen from C2-10, C2-8, C2-6, C2-4, C3, and C2 alkenylene.

[0641] 36. The cationic lipid according to any one of the preceding paragraphs, wherein L5 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene.

[0642] 37. The cationic lipid according to any one of the preceding paragraphs, wherein L6 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene.

[0643] 38. The cationic lipid according to any one of the paragraphs 36-37, wherein L5 and L6 are absent.

[0644] 39. The cationic lipid according to any one of the preceding paragraphs, wherein Xi is - C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, - OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or -OP(O)(O-)O-.

[0645] 40. The cationic lipid according to paragraph 39, wherein Xi is -C(O)O-, -OC(O)-, - C(O)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or -OP(O)(O-)O-.

[0646] 41. The cationic lipid according to any one of the preceding paragraphs, wherein X2 is - C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, - OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or -OP(O)(O-)O-.PVX-PAT-2402-WO

[0647] 42. The cationic lipid according to paragraph 41, wherein X2is -C(O)O-, -OC(O)-, - C(0)NH-, -NHC(O)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or -OP(O)(O-)O-. 43. The cationic lipid according to any one of the preceding paragraphs, wherein A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms.

[0648] 44. The cationic lipid according to paragraph 43, wherein A is saturated or unsaturated C3- 10 cycloalkyl, or saturated or unsaturated C3-10 heterocyclo alkyl containing 1-4 heteroatoms.

[0649] 45. The cationic lipid according to any one of the preceding paragraphs, wherein in A 1 is _

[0650]

[0651] \, -CH2-, orN.

[0652] 46. The cationic lipid according to any one of the preceding paragraphs, wherein the heteroatom is selected from the group consisting of O, N, S, or P.

[0653] 47. The cationic lipid according to paragraph 46, wherein the heteroatom is selected from the group consisting of O, N, or S.

[0654] 48. The cationic lipid according to any one of the preceding paragraphs, wherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

[0655] 49. The cationic lipid according to paragraph 48, wherein the substituent is chosen from H, -OH, Cl, Br, I, O, S, N, P, optionally substituted C1-6 alkoxy, optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted saturated or unsaturated C3-10 cycloalkyl, optionally substituted Ce-io aryl, optionally substituted saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, optionally substituted C5-10 heteroaryl containing 1-4 heteroatoms or combinations thereof.

[0656] 50. A lipid nanoparticle composition comprising the cationic lipid according to any one of the paragraphs 1-49 and optionally additionally comprising a phospholipid, a sterol, a PEG-lipid, or a nucleic acid.

[0657] 51. A lipid nanoparticle composition comprising the cationic lipid according to any one of the paragraphs 1-49, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid.PVX-PAT-2402-WO

[0658] 52. The lipid nanoparticle composition according to any one of the paragraphs 50-51, wherein the cationic lipid is present in an amount from 10 mol percent to 70 mol percent.

[0659] 53. The lipid nanoparticle composition according to any one of the paragraphs 50-51, wherein the phospholipid is present in an amount from 2 mol percent to 65 mol percent.

[0660] 54. The lipid nanoparticle composition according to any one of the paragraphs 50-51, wherein the sterol is present in an amount from 20 mol percent to 65 mol percent.

[0661] 55. The lipid nanoparticle composition according to any one of the paragraphs 50-51, wherein the PEG-lipid is present in an amount from 0.2 mol percent to 2.0 mol percent.

[0662] 56. The lipid nanoparticle composition according to any one of the paragraphs 50-55, wherein the cationic lipid is present in an amount from 10 mol percent to 70 mol percent, the phospholipid is present in an amount from 2 mol percent to 65 mol percent, the sterol is present in an amount from 20 mol percent to 65 mol percent, and the PEG- lipid is present in an amount from 0.2 mol percent to 2.0 mol percent.

[0663] 57. The lipid nanoparticle composition according to any one of the paragraphs 50-56, additionally comprising an ionizable polymer.

[0664] 58. The lipid nanoparticle composition according to paragraph 57, wherein the ionizable polymer is present in an amount from 1 mol percent to 25 mol percent.

[0665] 59. The lipid nanoparticle composition according to any one of the paragraphs 50-58, wherein the nucleic acid is a DNA, an RNA, or a combination thereof.

[0666] 60. The lipid nanoparticle composition according to paragraph 59, wherein the RNA is an mRNA, a long non-coding RNA (IncRNA), a micro RNA (miRNA), a small interfering RNA (siRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), a PIWI-interacting RNA (piRNA), or a combination thereof.

[0667] 61. The lipid nanoparticle composition according to paragraph 60, wherein the RNA is an mRNA.

[0668] 62. The lipid nanoparticle composition according to paragraph 61, wherein the mRNA encodes one or more protein, one or more antibodies, or combinations thereof.

[0669] 63. The lipid nanoparticle composition according to paragraph 61, wherein the mRNA encodes an antigen derived from a prokaryote or a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a mycoplasma, a protozoan, an animal, or a human.PVX-PAT-2402-WO

[0670] 64. A method of delivering a nucleic acid comprising administering the lipid nanoparticle composition comprising the cationic lipid according to any one of the paragraphs 1-49, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid.

[0671] 65. A method of treating or preventing a disease, comprising administering to a subject in need thereof a lipid nanoparticle composition comprising the cationic lipid according to any one of the paragraphs 1-49 and optionally additionally comprising a phospholipid, a sterol, a PEG-lipid, or a nucleic acid.

[0672] 66. A method of treating or preventing a disease, comprising administering to a subject in need thereof a lipid nanoparticle composition comprising the cationic lipid according to any one of the paragraphs 1-49, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid.

[0673] 67. The method according to any one of the paragraphs 65-66, wherein the cationic lipid is present in an amount from 10 mol percent to 70 mol percent.

[0674] 68. The method according to any one of the paragraphs 65-66, wherein the phospholipid is present in an amount from 2 mol percent to 65 mol percent.

[0675] 69. The method according to any one of the paragraphs 65-66, wherein the sterol is present in an amount from 20 mol percent to 65 mol percent.

[0676] 70. The method according to any one of the paragraphs 65-66, wherein the PEG-lipid is present in an amount from 0.2 mol percent to 2.0 mol percent.

[0677] 71. The method according to any one of the paragraphs 65-70, wherein the cationic lipid is present in an amount from 10 mol percent to 70 mol percent, the phospholipid is present in an amount from 2 mol percent to 65 mol percent, the sterol is present in an amount from 20 mol percent to 65 mol percent, and the PEG-lipid is present in an amount from 0.2 mol percent to 2.0 mol percent.

[0678] 72. The method according to any one of the paragraphs 65-71, additionally comprising an ionizable polymer.

[0679] 73. The method according to paragraph 72, wherein the ionizable polymer is present in an amount from 1 mol percent to 25 mol percent.

[0680] 74. The method according to paragraphs 65-73, wherein the nucleic acid is a DNA, an RNA, or a combination thereof.

[0681] 75. The method according to paragraph 74, wherein the RNA is an mRNA, a long noncoding RNA (IncRNA), a micro RNA (miRNA), a small interfering RNA (siRNA), aPVX-PAT-2402-WO

[0682] small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), a PIWI-interacting RNA (piRNA), or a combination thereof.

[0683] 76. The method according to paragraph 75, wherein the RNA is an mRNA.

[0684] 77. The method according to paragraph 76, wherein the mRNA encodes one or more protein, one or more antibodies, or combinations thereof.

[0685] 78. The method according to paragraph 76, wherein the mRNA encodes an antigen derived from a prokaryote or a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a mycoplasma, a protozoan, an animal, or a human.

[0686] 79. Use of a lipid nanoparticle composition according to any one of the paragraphs 50-63 in the manufacture of a medicament for the treatment or prevention of a disease in a subject.

[0687] The present disclosure is further exemplified by the following non limiting examples. It should be understood that the examples are provided to illustrate the disclosure. From the description and the exemplified embodiments and examples, one skilled in the art can make various modifications or adaptations to the disclosure. Such modifications or adaptations are deemed to be within the scope of the spirit of the disclosure.

[0688] EXAMPLES

[0689] The reactions described herein are generally performed under inert conditions, for example N2 atmosphere, except those reactions that involves moisture, water, or acid.

[0690] Example 1: Synthesis of heptadecan-9-yl 8-hydroxy octanoate (Intermediate 5)PVX-PAT-2402-WO

[0691] DHP(1.2 eqv) / TsOH(O.l) / DCM / / RT / 3 h (59%) Step-1 2 (1.1 eqv) 3 (1.0 eqv)

[0692] EDC. HC1(1.6 eqv) / DMAP(0.2) /

[0693] PPTS (0.2 eq) / DTPEA(2.5) / DCM / / RT / 16 h 10 min MeOH / RT-60 °C / 3 h (60%) (53%)

[0694] Step-3 Step-2

[0695]

[0696] 5 Step 1: Synthesis of 8-((tetrahydro-2H-pyran-2-yl)oxy)octanoic acid (2)

[0697] To a stirred solution of 8 -hydroxy octanoic acid (1) (5 g, 31.2 mmol) and 3,4-dihydro-2H-pyran (3.2 g, 37.5 mmol) in DCM (100 mL) was added p-toluenesulfonic acid (0.5 g, 3.1 mmol) at room temperature and the reaction mixture was stirred for 3 h. After 3 h, the reaction mixture was concentrated under reduced pressure to obtain crude. To the crude saturated NaHCOa solution (25 mL) was added and extracted with EtOAc (3 X 70 mL). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain crude (6.7g), which was purified by combiflash chromatography using 22% EtOAc in hexane as eluent to obtain 8-((tetrahydro-2H-pyran-2-yl)oxy)octanoic acid (2) (4.5 g, 59%) as colorless liquid.

[0698] Step 2: Synthesis of heptadecan-9-yl 8-((tetrahydro-2H-pyran-2-yl)oxy)octanoate (4) To a stirred solution of 8-((tetrahydro-2H-pyran-2-yl)oxy)octanoic acid (2) (4.19 g, 17.15 mmol) in DCM (20 mL) were added EDC (3.87 g, 24.95 mmol), DMAP (381 mg, 3.11 mmol) and DIPEA (6.8 mL, 38.99 mmol) at room temperature and the reaction mixture was stirred for 10 min. Then heptadecan-9-ol (3) (4 g, 15.6 mmol) was added dropwise to the reaction mixture at room temperature and stirred for 16 h. After 16 h, the reaction mixture was concentrated under reduced pressure, diluted with water, and extracted with DCM (2 X 50 mL). The combined organic layer was washed with water (50 mL), saturated NaHCOa solution (2 X 50 mL) and brine (50 mL). The combined organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain crude (6 g), which wasPVX-PAT-2402-WO

[0699] purified by combiflash chromatography using 3-4% EtOAc in heptane as eluent to obtain heptadecane-9-yl 8-((tetrahydro-2H-pyran-2-yl)oxy)octanoate (4) (4 g, 53%) as colorless liquid.

[0700] Step 3: Synthesis of heptadecane-9-yl 8-hydroxy octanoate (5)

[0701] To a stirred solution of heptadecane-9-yl 8-((tetrahydro-2H-pyran-2-yl)oxy)octanoate (4) (4 g, 8.29 mmol) in MeOH (100 mL) was added PPTS (416 mg, 1.66 mmol) at room temperature. The reaction mixture was stirred for 3 h at 60 °C. After 3 h, the reaction mixture was concentrated under reduced pressure to obtain crude. The crude was diluted with EtOAc (125 mL) and washed with water (3 X 100 mL). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain crude (3.6 g), which was purified by combiflash chromatography using 25% EtOAc in heptane as eluent to obtain heptadecan-9-yl 8-hydroxyoctanoate (5) (2 g, 60%) as color less liquid.

[0702] Example 2: Synthesis of heptadecan-9-yl 8-[({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy}[3-(piperidin-l-yl)propoxy]phosphoryl)oxy]-octanoate (PVXL-83)

[0703] Step 1: POCI3 (1 eqv) / Et3N (8 eqv) / Et2O / RT-60 °C / 3h Step 2: 6 (1 eqv) / 5 (2 eqv) RT-60 °C / 13h 6 (1.0 eqv)

[0704] (44%)

[0705]

[0706] To a stirred solution of heptadecan-9-yl 8-hydroxyoctanoate (5) (synthesized according to the process described in example 1) (278 mg, 0.7 mmol) in Et2O (2 mL) were added POCh (54 mg, 0.35 mmol) and EhN (0.4 mL, 2.8 mmol) at room temperature. The reaction mixture was stirred for 3 h at 60 °C. After 3 h, the reaction mixture was allowed to attain room temperature and 3-(piperidin-l-yl)propan-l-ol (6) (50 mg, 0.35 mmol) was added to the reaction mixture and the reaction mixture was stirred for 13 h at 60 °C. After 13 h,PVX-PAT-2402-WO

[0707] reaction mixture was diluted with EtOAc (50 mL) and washed with water (3 X 50 mL). The combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain crude, which was purified by combiflash chromatography using 3% MeOH in DCM as eluent to obtain Heptadecan-9-yl 8-[({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy}[3-(piperidin-l-yl)propoxy]phosphoryl)oxy]-octanoate (PVXL-83) (150 mg, 44%) as light-yellow gummy substance.

[0708] H NMR (401 MHz, MeOD) 50.89 (t, J= 6.7 Hz, 12H), 1.23 - 1.42 (m, 60H), 1.48 - 1.56 (m, 10H), 1.58 - 1.73 (m, 12H), 1.89 - 1.97 (m, 2H), 2.30 (t, 7= 7.3 Hz, 4H), 2.41 - 2.69 (m, 6H), 4.00 - 4.14 (m, 6H), 4.86 - 4.89 (m, 2H)].

[0709] Example 3: Synthesis of 1-octybionyl 8-[[8-(l-octylnonoxy)-8-oxo-octoxy]-[5-(l- piperidyI)pentoxy]phosphoryH oxyoctanoate (PVXL-170)

[0710] ACN / THF / 70 °C / 6 h (50%) 23 (5.0 eqv) Step-1

[0711] Step 1: POCI3 (0.5 eqv) / Et3N (2.5 eqv) / Et2O / RT-60 °C / 3 h Step 2: 24-11 (1.0 eqv) / Et3N (2.5 eqv) / Et2O / RT-60 °C / 16h (2%) Step-2

[0712]

[0713] Step 1: Synthesis of 5-(piperidin-l-yl)pentan-l-ol (24-11)

[0714] To a stirred solution of 5 -bromopentan- l-ol (24) (1 g, 5.986 mmol) in ACN: THF (1:1) (20 mL) was added piperidine (23) (2.54 g, 29.9 mmol) at room temperature. The reaction mixture was stirred for 6 h at 70 °C. After 6 h, the reaction mixture was diluted with water (8 mL) and extracted with EtOAc (2 X 10 mL). The combined organic layer was dried over Na2S0q and concentrated under reduced pressure to obtain crude, which was purified byPVX-PAT-2402-WO

[0715] combiflash chromatography using 10% MeOH in EtOAc as eluent to obtain 5-(piperidin-l-yl)pentan-l-ol (24-11) (500 mg, 50%) as colorless liquid.

[0716] Step 2: Synthesis of 1-octylnonyl 8-[[8-(l-octylnonoxy)-8-oxo-octoxy]-[5-(l-piperidyl)pentoxy]phosphoryl]oxyoctanoate (PVXL-170)

[0717] To a stirred solution of heptadecan-9-yl 8-hydroxyoctanoate (5) (synthesized according to the process described in example 1) (250 mg, 0.63 mmol) in Et2O (2 mL) were added POCh (48 mg, 0.31 mmol) and EhN (0.22 mL, 1.8 mmol) at room temperature. The reaction mixture was stirred for 3 h at 60 °C. After 3 h, the reaction mixture was allowed to attain room temperature and 5-(piperidin-l-yl)pentan-l-ol (24-11) (107 mg, 0.63 mmol) and EhN (0.22 mL, 1.8 mmol) were added to the reaction mixture and stirred for 16 h at 60 °C. After 16 h, the reaction mixture was concentrated under reduced pressure, diluted with DCM (20 mL), and washed with water (10 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude, which was purified by combiflash chromatography using 5% MeOH in DCM as eluent, and followed by prep HPLC to obtain 1-octylnonyl 8-[[8-(l-octylnonoxy)-8-oxo-octoxy]-[5-(l-piperidyl)pentoxy]phosphoryl]oxyoctanoate (PVXL-170) (10 mg, 2%) as light-yellow gummy substance.

[0718] H NMR (401 MHz, DMSO) 50.85 (t, J = 6.7 Hz, 12H), 1.14 - 1.27 (m, 56H), 1.43 - 1.48 (m, 8H), 1.52 - 1.57 (m, 10H), 1.72 - 1.77 (m, 14H), 2.19 - 2.29 (m, 10H), 3.89 - 3.94 (m, 6H), 4.76 - 4.80 (m, 2H)].

[0719] Example 4: Synthesis of heptadecan-9-yl 8-[({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy}[3-(4-hydroxypiperidin-l-yl)propoxy]phosphoryl)oxy]octanoate (PVXL-251)PVX-PAT-2402-WO

[0720] NaH (3 eqv) / DMF / 0°C / 15 min, TFA (5 eqv) / then 2 / RT / 16 h DCM / 0 “C-RT / 6 h (69%) (Crude) 2 (1 eqv) Step-2

[0721] Step-1

[0722] K2CO3(2.5 eqv) / Cs2CO3(0.5 eqv) / KI (0.2 eqv) / ACN: THF(1: I) / RT-80 °C / 16 h (58%, 2 steps) Step-3

[0723] Step 1: POC13(1 eqv) / 6 (2 eqv) / Et3N (6 eqv) / Et2O / RT-60 °C / 3 h Step 2: 5A (1.5 eqv) / RT-60 °C / 16 h (20%) Step-4

[0724]

[0725] Step 1: Synthesis of tert-butyl 4-(benzyloxy)piperidine-l-carboxylate (3)

[0726] To an ice cooled solution of tert-butyl 4-hydroxypiperidine- 1 -carboxylate (1) (3 g,14.91 mmol) in DMF (15 mL) was added NaH (60%, 1 g, 44.7 mmol) and the reactionPVX-PAT-2402-WO

[0727] mixture was stirred for 15 min under ice cold conditions. To this reaction mixture (bromomethyl)benzene (2) (2.5 g, 14.91 mmol) was added dropwise. The reaction mixture was allowed to attain room temperature and stirred for 16 h. After 16 h, the reaction mixture was quenched with ice cold water (50 mL) and extracted with diethyl ether (3 X 50 mL). The combined organic layer was washed with ice cold water followed by brine and dried over Na2SO4 and concentrated to obtain crude (3.9 g), which was purified by combiflash chromatography using 10% EtOAc in hexane to obtain tert-butyl 4-(benzyloxy)piperidine-l-carboxylate (3) (3 g, 69%) as colorless liquid.

[0728] Step 2: Synthesis of 4-(benzyloxy)piperidine trifluoroacetate salt (4)

[0729] To an ice cooled solution of tert-butyl 4-(benzyloxy)piperidine-l -carboxylate (3) (1 g, 3.432 mmol) in DCM (2.0 mL) was added TFA (1.3 mL, 17.159 mmol) under N2 atmosphere. The reaction mixture was allowed to attain room temperature and stirred for 6 h. After 6 h, the reaction mixture was concentrated under reduced pressure to obtain crude 4-(benzyloxy)piperidine trifluoroacetate salt (4) (1 g).

[0730] Step 3: Synthesis of 3-(4-(benzyloxy)piperidin-l-yl)propan-l-ol (5A)

[0731] To a stirred solution of 4-(benzyloxy)piperidine trifluoroacetate salt (4) (1 g, 3.48 mmol) and 3-bromopropan-l-ol (5) (484 mg, 3.48 mmol) in ACN: THF (1:1) (8.0 mL) were added K2CO3 (1.2 g, 8.7 mmol), CS2CO3 (570 mg, 1.74 mmol), and KI (116 mg, 0.7 mmol) at room temperature under N2 atmosphere. Then the reaction mixture was stirred for 16 h at 80 °C. After 16 h, the reaction mixture was diluted with water (8.0 mL) and extracted with EtOAc (2 X 20 mL). Then combined organic layer was dried over Na2SO4 and concentrated under reduced pressure to obtain crude (800 mg), which was purified by combiflash chromatography using 50% EtOAc in heptane as eluent to obtain 3-(4-(benzyloxy)piperidin-l-yl)propan-l-ol (5A) (500 mg; 58%, 2 steps) as colorless liquid.

[0732] Step 4: Synthesis of heptadecan-9-yl 8-[({3-[4-(benzyloxy)piperidin-l-yl]propoxy}({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy})phosphoryl)oxy]octanoate (7)

[0733] To a solution of heptadecan-9-yl 8-hydroxyoctanoate (5) (400 mg, 1.003 mmol) (synthesized according to the process described in example 1) in 3.0 mL of Et20 were added POCI3 (77 mg, 0.502 mmol) and TEA (0.4 mL, 3.01 mmol) at room temperature. The reaction mixture was stirred for 3 h at 60 °C. After 3 h, the reaction mixture was allowed toPVX-PAT-2402-WO

[0734] attain room temperature and 3-[4-(benzyloxy)piperidin-l-yl]propan-l-ol (5A) (188 mg, 0.75 mmol) was added to the reaction mixture. Then the reaction mixture was stirred for 16 h at 60 °C. After 16 h, the reaction mixture was concentrated under reduced pressure to obtain crude, the crude was diluted with DCM (20 mL) and washed with water (10 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude (350 mg), which was purified by combiflash chromatography using 5% MeOH in DCM as eluent to obtain heptadecan-9-yl 8-[({3-[4-(benzyloxy)piperidin-l-yl]propoxy}({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy}) phosphoryl)oxy]octanoate (7) (215 mg, 20%) as colorless liquid.

[0735] Step 5: Synthesis of heptadecan-9-yl 8-[({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy}[3-(4-hydroxypiperidin-l-yl)propoxy]phosphoryl)oxy]octanoate (PVXL-251)

[0736] A solution of heptadecan-9-yl 8-[({3-[4-(benzyloxy)piperidin-l-yl]propoxy}({[8-(heptadecan-9-yloxy)-8-oxooctyl] oxy})phosphoryl)oxy]octanoate (7) (200 mg, 0.18 mmol) in EtOH (4 mL) was degassed under N2 atmosphere for 10 min. Then Pd-C (10% wt, 50% moist, 63 mg) and 0.1 mL AcOH were added to the reaction mixture at room temperature and the reaction mixture was stirred for 16 h under H2 atmosphere. After 16 h, the reaction mixture was filtered and concentrated under reduced pressure to obtain crude (150 mg), which was purified by combiflash chromatography using 5% MeOH in DCM as eluent to obtain heptadecan-9-yl 8-[({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy}[3-(4-hydroxypiperidin-l-yl)propoxy] phosphoryl) oxy]octanoate (PVXL-251) (100 mg, 55%) as colorless liquid.

[0737] [¹H NMR (401 MHz, MeOD) δ 0.89 (t, J = 7.0 Hz, 12H), 1.19 - 1.44 (m, 58H), 1.48 - 1.74 (m, 20H), 1.81 - 1.95 (m, 4H), 2.12 - 2.24 (m, 2H), 2.30 (t, 7= 7.2 Hz, 4H), 2.47 (t, 7= 7.6 Hz, 2H), 2.74 - 2.94 (m, 2H), 3.52 - 3.71 (m, 1H), 3.98 - 4.15 (m, 6H), 4.86 - 4.97 (m, 2H).]

[0738] Example 5: Synthesis of heptadecan-9-yl 8-[({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy}(2-{l-[2-(4-hydroxypiperidin-l-yl)ethyl]-lH-l,2,3-triazol-4-yl}ethoxy)phosphoryl)oxy]octanoate (253A)PVX-PAT-2402-WO

[0739] K2CO3(1 5 eqv) / Cs2CO3(0 5 eqv) / RT / lh, then 3 (1 eqv) / KI (0 5 eqv) / ACN: THF(l:l) / 80 °C / 8 h (71%) 2 (1 0 eqv) 3 (1.0 eqv) Step-2

[0740] NaN3(1 5 eqv) / MsCl (1 5 eqv) /

[0741] KI (1 5 eqv) Et3N (2 5 eqv) /

[0742] DMSO / RT-60 °C / DCM / 0 “C-RT / 4 h 4 5 h (60%) (78%)

[0743] 7 (1.0 eqv) Step-3 Step-4

[0744]

[0745] Step 1: Synthesis of 4-((tert-butyldimethylsilyl)oxy)piperidine (2)

[0746] To an ice cooled solution of 4-hydroxypiperidine (1) (3.0 g, 29.7 mmol) in DCM (50 mL) were added pyridine (4.8 mL, 59.4 mmol) and tert-butyldimethylsilyl chloride (5.4 g, 35.6 mmol), and the reaction mixture was allowed to attain room temperature and stirred for 5 h. After 5 h, the reaction mixture was concentrated under reduced pressure to obtain crude, which was purified by combiflash chromatography using 2-5% MeOH in DCM as eluent toPVX-PAT-2402-WO

[0747] obtain 4-((tert-butyldimethylsilyl)oxy)piperidine (2) (3.5 g, 55%) as light-yellow solid substance.

[0748] Step 2: Synthesis of 2-(4-((tert-butyldimethylsilyl)oxy)piperidin-l-yl)ethan-l-ol (4)

[0749] To a stirred solution of 4-((tert-butyldimethylsilyl)oxy)piperidine (2) (1.8 g, 8.35 mmol) in THF: ACN (1:1) (30.0 mL) were added K2CO3 (1.73 g, 12.5 mmol), CS2CO3 (1.34 g, 4.2 mmol) at room temperature and the reaction mixture was stirred for 1 h. Then 2-bromoethan-l-ol (3) (1.04 g, 8.35 mmol), and KI (694 mg, 4.2 mmol) were added to the reaction mixture, and the reaction mixture was stirred for 8 h at 80 °C. After 8 h, the reaction mixture was quenched with ice cold water (20 mL) and stirred for 10 min, then the reaction mixture was extracted with EtOAc (3 X 50 mL). The combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude, which was purified by combiflash chromatography using 80% EtOAc in heptane as eluent to obtain 2-(4-((tert-butyldimethylsilyl)oxy)piperidin-l-yl)ethan-l-ol (4) (1.55 g, 71 %) as brown-gummy substance.

[0750] Step 3: Synthesis of 4-((tert-butyldimethylsilyl)oxy)-l-(2-chloroethyl)piperidine (5) To an ice cooled solution of 2-{4-[(tert-butyldimethylsilyl)oxy]piperidin-l-yl}ethan-l-ol (4) (1.55 g, 5.9 mmol) in DCM (15.0 mL) were sequentially added EhN (2.1 mL, 14.75 mmol), and MsCl (0.7 mL, 8.9 mmol), and the reaction mixture was allowed to attain room temperature and stirred for 4 h. After 4 h, reaction mixture was quenched with ice cold water (20 mL) and stirred for 10 min, then the reaction mixture was extracted with EtOAc (3 X 50 mL). The combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude (2.1 g), which was purified by combiflash chromatography using 10-20% EtOAc in heptane as eluent to obtain 4-((tert-butyldimethylsilyl)oxy)-l-(2-chloroethyl)piperidine (5) (1.0 g, 60%) as colorless liquid.

[0751] Step 4: Synthesis of l-(2-azidoethyl)-4-((tert-butyldimethylsilyl)oxy)piperidine (6)

[0752] To a stirred solution of 4-[(tert-butyldimethylsilyl)oxy]-l-(2-chloroethyl)piperidine (5) (1.0 g, 3.6 mmol) in DMSO (15.0 mL) were added NaN3 (351 mg, 5.4 mmol) and KI (896 mg, 5.4 mmol) at room temperature and stirred for 30 min. The reaction mixture was stirred for 4 h at 60 °C. After 4 h, reaction mixture was quenched with water (50 mL) and extracted with EtOAc (3 X 50 mL) and combined organic layer was washed with brine (3 X 40 mL),PVX-PAT-2402-WO

[0753] dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude (1.8 g), which was purified by combiflash chromatography using 15-20% EtOAc in heptane as eluent to obtain l-(2-azidoethyl)-4-((tert-butyldimethylsilyl)oxy)piperidine (6) (800 mg, 78%) as colorless liquid.

[0754] Step 5: Synthesis of 2-(l-(2-(4-((tert-butyldimethylsilyl)oxy)piperidin-l-yl)ethyl)-lH-l,2,3-triazol-4-yl)ethan-l-ol (8)

[0755] To a stirred suspension of but-3-yn-l-ol (7) (202 mg, 2.9 mmol), 1 -(2- azidoethyl) -4-[(tert-butyldimethylsilyl)oxy]piperidine (6) (820 mg, 2.9 mmol) in tert-butanol / water (1:1) (11.2 mL) were added sodium ascorbate (0.29 mL, 0.29 mmol, freshly prepared IM solution in water), and copper (II) sulfate pentahydrate (15 mg, 0.06 mmol) at room temperature, and the heterogeneous mixture was stirred vigorously for 3 h. After 3 h, the reaction mixture was quenched with water (20 mL) and stirred for 10 min at room temperature, then the reaction mixture was extracted with EtOAc (3 X 50 mL). The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude (1.4 g), which was purified by combiflash chromatography using 2-4% MeOH in DCM as eluent to obtain 2-(l-(2-(4-((Tert-butyldimethylsilyl)oxy)piperidin-l-yl)ethyl)-lH-l,2,3-triazol-4-yl)ethan-l-ol (8) (800 mg, 78%) as an off-white powdery substance.

[0756] Step 6: Synthesis of heptadecan-9-yl 8-[({2-[l-(2-{4-[(tert-butyldimethylsilyl)oxy]piperidin-l-yl}ethyl)-lH-l,2,3-triazol-4-yl]ethoxy} ({[8-(heptadecane-9-yloxy)-8-oxooctyl]oxy})phosphoryl)oxy]octanoate ) (14)

[0757] To a stirred solution of heptadecan-9-yl 8-hydroxyoctanoate (5) (synthesized according to the process described in example 1) (494 mg, 1.24 mmol) was added POCh (95 mg, 0.62 mmol) at room temperature and reaction mixture was stirred for 10 min. Then the reaction mixture was stirred for 3 h at 60 °C. After 3 h, the reaction mixture was allowed to attain room temperature and concentrated under rotavapor to remove HC1 and excess POCI3. Then DCM (5 mL) was added to the reaction mixture. Subsequently, DIPEA (0.54 mL, 3.1 mmol) was added followed by 2-[l-(2-{4-[(tert-butyldimethylsilyl)oxy]piperidin-l-yl}ethyl)-lH-l,2,3-triazol-4-yl]ethan-l-ol (8) (330 mg, 0.93 mmol) in DCM (1.0 mL) at room temperature, and the reaction mixture stirred for 6 h. After 6 h, the reaction mixture was cooled to 0 °C and quenched with water (20 mL) and extracted with EtOAc (2 X 100 mL) and the combined organic layer was washed with brine (20 mL), dried over Na2SO4, filtered,PVX-PAT-2402-WO

[0758] and concentrated under reduced pressure to obtain crude (910 mg), which was purified by combiflash chromatography using 20-30% EtOAc in heptane as eluent to obtain heptadecan-9-yl 8- [( { 2- [ 1 -(2- { 4- [(tert-butyldimethylsilyl)oxy ]piperidin- 1 -yl } ethyl)- 1 H- 1,2,3-triazol-4-yl]ethoxy] ({[8-(heptadecane-9-yloxy)-8-oxooctyl]oxy})phosphoryl)oxy]octanoate (14) (150 mg, 20%) as colorless liquid.

[0759] Step 7: Synthesis of heptadecan-9-yl 8-[({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy}(2-{l-[2- (4-hydroxypiperidin- 1 -yl)ethyl] - 1H- 1,2,3 -triazol-4-yl}ethoxy)phosphoryl)oxy]octanoate (253A)

[0760] To a stirred solution of heptadecan-9-yl 8-[({2-[l-(2-{4-[(tert-butyldimethylsilyl)oxy]piperidin-l-yl}ethyl)-lH-l,2,3-triazol-4-yl]ethoxy}({[8-(heptadecan-9-yloxy)-8-oxooctyl]oxy})phosphoryl)oxy]octanoate (14) (120 mg, 0.1 mmol) in MeOH (2 mL) was added NH4F (40 mg, 1.0 mmol) at room temperature. The reaction mixture was stirred for 14 h at 65 °C. After 14 h, the reaction mixture was quenched by adding water (20 mL) at 0 °C and stirred for 10 min. Then the reaction mixture was extracted with EtOAc (2 X 50 mL) and the combined organic layer was washed with brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude (150 g), which was purified by combiflash chromatography using 30% EtOAc in heptane as eluent to obtain heptadecan-9-yl 8-[({ [8-(heptadecan-9-yloxy)-8-oxooctyl]oxy } (2- { l-[2-(4-hydroxypiperidin-l-yl)ethyl]-lH-l,2,3-triazol-4-yl}ethoxy)phosphoryl)oxy]octanoate (253A) (70 mg, 64%) as colorless gummy substance.

[0761] [¹H NMR (400 MHz, MeOD) δ 7.87 (s, 1H), 4.91 - 4.85 (m, 2H), 4.50 (t, J= 6.6 Hz, 2H), 4.33 - 4.24 (m, 2H), 4.04 - 3.95 (m, 4H), 3.64 - 3.54 (m, 1H), 3.09 (t, J = 6.5 Hz, 2H), 2.82 (t, J= 7.1 Hz, 4H), 2.30 (t, J= 7.3 Hz, 4H), 2.26 - 2.19 (m, 2H), 1.87 - 1.79 (m, 2H), 1.68 -1.60 (m, 8H), 1.52 (d, J = 7.4 Hz, 10H), 1.45 - 1.20 (m, 60H), 0.89 (t, J= 6.6 Hz, 12H).]

[0762] Example 6: Preparation of lipid nanoparticle composition

[0763] Cationic lipids (PVXL-83, PVXL-170, PVXL-251, and PVXL-253A) were synthesized according to the process described in examples 2 to 5. Phospholipids [1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 2-dioleoyl-sn-glycero-3-phosphoethanlamine (DOPE)] used in the LNP compositions were either procured from BOC Sciences, TCI America, or Sinopeg. Cholesterol was procured from BOC Sciences or Merck.PVX-PAT-2402-WO

[0764] PEG-lipids [l,2-dimyristoyl-rac-glycero-3-methoxypolytheyleneglycol 2000 (mPEG2000-DMG) or a-(3’-{[l,2-di(myristyloxy)propanoxy]carbonylamino}propyl) -co-methoxy, polyoxyethylene (mPEG2000C-DMG)] were procured from Sinopeg or BOC Sciences respectively.

[0765] Luciferase mRNA was synthesized through conventional invitro transcription (IVT) technique well known to a person skilled in the art.

[0766] Different lipid nanoparticle compositions were prepared with the molar percentages of different lipids as mentioned in table 2.

[0767] Table 2: Lipid nanoparticle compositions

[0768] Cationic mRNA Molar Percentage Total F-ID N: P

[0769] Lipid (pg / ml) CL % PL% Chol% PEG-L % Mol % F-l PVXL-83 5 pg 30 30 38.5 1.5 6:1 100 F-2 PVXL-170 5 pg 50 10 38.5 1.5 6:1 100 F-3 PVXL-251 5 pg 30 30 38.5 1.5 6:1 100 F-4 PVXL-253A 5 pg 17.6 35.3 45.3 1.8 6:1 100

[0770]

[0771] F-ID: Formulation ID

[0772] CL: Cationic Lipid

[0773] PL: Phospholipid

[0774] Choi: Cholesterol

[0775] PEG-L: Polyethylene Glycol Lipid

[0776] N / P: Cationic Lipid to Nucleic Acid Ratio

[0777] Nucleic acid (luciferase mRNA) was dissolved in aqueous phase (25mM Sodium acetate buffer, pH 5.0) and lipid components (cationic lipid, phospholipid, cholesterol & PEG-lipid) were dissolved in organic phase (IPEC grade 99.5% (v / v) ethanol). The aqueous phase (continuous) and organic phase (dispersed) were mixed using a dual syringe pump (Microlab 600 Advanced Dual Syringe Diluter, Hamilton Company, catalogue no. ML625-DIL-BZ) in a microfluidic device (AXF Mini, Micropore Technologies). The total flow rate was maintained at 100 mL / min or 300 mL / min, and a flow rate ratio (FRR) of 3:1 for aqueous phase to organic phase respectively. The lipid nanoparticles were buffer exchanged with either phosphate buffered saline (PBS) or tris acetate sucrose, pH 7.5 by using Amicon®PVX-PAT-2402-WO

[0778] Ultra 15 (catalogue no. UFC910096) or TFF Cassetes 100 kDa (catalogue no. VF05H4, Sartorius), followed by sterile filtration using 0.22 micron PES filters (catalogue no. SF-13-100NO, HiMedia Laboratories).

[0779] Example 7: Characterization of LNP compositions

[0780] Particle Size, Zeta Potential & Polydispersity Index (PDI)

[0781] Particle size, zeta potential, and polydispersity index (PDI) of different lipid nanoparticle compositions were determined by diluting the compositions in nuclease free water using particle analyzer (LiteSizer 500, catalogue no. 155761, Anton Paar).

[0782] Encapsulation Efficiency

[0783] Encapsulation efficiency of lipid nanoparticle compositions was quantified by Quant iT RiboGreen RNA Assay Kit (catalogue no. R11490, ThermoFisher Scientific) by measuring the free mRNA and total mRNA. Briefly, the LNP compositions were diluted with IX TE buffer and transferred to the 96 well plate in duplicates. Another set of LNP compositions were diluted with IX TE buffer containing 1% triton to lyse the LNPs and added to the adjacent wells in duplicates on the same plate. The plate was incubated at 37 °C for 10 min. The RNA standard from the assay kit was serially diluted in IX TE buffer and IX TE buffer containing 1% triton and added to the same plate in duplicates. To estimate background from buffers, IX TE buffer and IX TE buffer containing 1% triton was added in duplicates to the same plate and measured as blank. Ribogreen dye was thawed at room temperature in the dark, diluted with IX TE buffer and added to all the wells. The plate was read using a microplate reader (Spark Multimode Microplate Reader from TECAN, catalogue no. 30086376) and the fluorescence was measured with an excitation wavelength of 485 nm and emission wavelength of 528 nm. Two standard curves were plotted against standards in IX TE buffer and IX TE buffer containing 1% triton and free and total mRNA was calculated using the respective standard curves subtracting blank, if any. Encapsulation efficiency was determined as:

[0784] T otal mRNA — free mRNA Encapsulation Efficiency (EE%) = - — ~RNA - % 100

[0785]

[0786] LNP composition characterization results are summarized in Table 3.PVX-PAT-2402-WO

[0787] Table 3: Characterization of LNP compositions

[0788] Cationic Molar % EE Particle F-ID N: P Zeta PDI

[0789] Lipid [CL: PL: Chol: PEG-L] % Size F-l PVXL-83 30:30:38.5:1.5 6:1 97.6 -2.58 0.09 108.93 F-2 PVXL-170 50:10:38.5:1.5 6:1 92.2 -11.76 0.05 98.02 F-3 PVXL-251 30:30:38.5:1.5 6:1 93.2 -2.81 0.05 132.34 F-4 PVXL-253A 17.6:35.3:45.3:1.8 6:1 83.7 -8.42 0.15 131.56

[0790]

[0791] F-ID: Formulation ID

[0792] CL: Cationic Lipid

[0793] PL: Phospholipid

[0794] Choi: Cholesterol

[0795] PEG-L: Polyethylene Glycol Lipid

[0796] N / P: Cationic Lipid to Nucleic Acid Ratio

[0797] PDI: Polydispersity Index

[0798] Example 8: Translational efficacy of lipid nanoparticle composition by luciferase assay The translational efficacy of lipid nanoparticle composition was estimated by expression of luciferase, by luciferase assay. HEK 293 T cells from ATCC (catalogue no. CRL -1573) were cultured in DMEM high glucose media (catalogue no. 11995-065, ThermoFisher Scientific) [supplemented with L-Glutamine, sodium pyruvate, 10% FBS (catalogue no. 10270-106, ThermoFisher Scientific) and 1% Pen-strep (catalogue no.

[0799] 15140122, ThermoFisher Scientific)] at 37 °C, 5% CO2 under humid conditions. The cells were seeded in a 24-well plate at a density of 0.5 x 105cells per well 24 hours prior to transfection. 250 ng of luciferase mRNA encapsulated lipid nanoparticle (luciferase LNP-mRNA) was added to each well. The plates were incubated at 37 °C with 5% CO2 for 24 hours. After incubation, media is removed and cells were lysed with 125 pl reporter lysis buffer (catalogue no. E4030, Luciferase Assay System with Reporter Lysis Buffer, Promega) and incubated on thermomixer C (catalogue no. EP5382000023, Eppendorf) at room temperature under agitation at 600 rpm and supernatant was collected. 20 pl of supernatant was added to a well of black flat bottom 96-well plate and 100 pl of luciferin substrate (catalogue no. E4030, Luciferase Assay System with Reporter Lysis Buffer, Promega) wasPVX-PAT-2402-WO

[0800] added to the wells. Immediately after adding the substrate, luminescence was measured using microplate reader (catalogue no. 30086376, Spark Multimode Microplate Reader, TECAN). Results are shown in Fig 1.

[0801] INCORPORATION BY REFERENCE

[0802] Each of the patents, published patent applications, or provisional application and nonpatent references cited herein are hereby incorporated by reference in their entirety.

[0803] EQUIVALENTS

[0804] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

PVX-PAT-2402-WOWhat is claimed:

1. A cationic lipid represented by formula (I)formula (I)or isomer, or salt thereof, wherein:Ri and R2 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?, orRi and R2 may combine together to form a saturated or unsaturated C3-10 cycloalkyl, Ce- 10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;R3 and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26;Rs, Re, and R7 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms;each of L1, L2, L3, L4, L5, L6, and L7 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene;Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, - C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-;PVX-PAT-2402-WOA is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;— HcAi is \, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, - C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, S, or N provided that when Ai is -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, - C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or S, one of Ri or R2is absent; andwherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

2. A cationic lipid represented by formula (II)X2R4formula (II)or isomer, or salt thereof, wherein:= represents either a single bond or a double bond;Ri and R2are independently chosen from H, -OH, C1-10 alkyl, C2-io alkenyl, C2-io alkynyl, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, C5-10 heteroaryl containing 1-4 heteroatoms, -O-L7-R5, and -NReR?, orRi and R2may combine together to form a saturated or unsaturated C3-10 cycloalkyl, Ce- 10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;R3 and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26alkenyl, C2-26alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26;Rs, Re, and R7 are independently chosen from H, -OH, C1-10 alkyl, C2-10 alkenyl, C2-10 alkynyl, saturated or unsaturated C3-10 cycloalkyl, Ce-io aryl, saturated or unsaturatedPVX-PAT-2402-WOC3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms;each of L1, L2, L3, L4, L5, L6, and L7 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene;Xi and X2 are independently chosen from -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, - C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, and -OP(O)(O-)O-;A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms;— HcAi is \, -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, - C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, S, or N provided that when Ai is -CH2-, -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, - C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or S, one of Ri or R2is absent; andwherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

3. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 are each independently H or -OH.

4. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 are each independently C1-10 alkyl.

5. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 are each independently C2-10 alkenyl.

6. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 are each independently C2-10 alkynyl.

7. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 are each independently saturated or unsaturated C3-10 cycloalkyl.

8. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 are each independently saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms.

9. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 are each independently H or -O-L7-R5.PVX-PAT-2402-WO10. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 are each independently H or -NReR?.

11. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 combine together to form a saturated or unsaturated C3-10 cycloalkyl.

12. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 combine together to form Ce-io aryl.

13. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 combine together to form a saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms.

14. The cationic lipid according to any one of the claims 1-2, wherein Ri and R2 combine together to form C5-10 heteroaryl containing 1-4 heteroatoms.

15. The cationic lipid according to claim 9, wherein L7 is either absent or C1-10 alkylene, C2- 10 alkenylene, or C2-10 alkynylene.

16. The cationic lipid according to claim 15, wherein L7 is absent.

17. The cationic lipid according to claim 9, wherein R5 is H, -OH, C1-10 alkyl, saturated or unsaturated C3-10 cycloalkyl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms.

18. The cationic lipid according to claim 17, wherein R5 is C3-6 heterocycloalkyl containing 1-4 hetero atoms.

19. The cationic lipid according to claim 17, wherein R5 is C5-9 heteroaryl containing 1-4 heteroatoms.

20. The cationic lipid according to claim 10, wherein Re and R7 are independently chosen from H, -OH, C1-10 alkyl, saturated or unsaturated C3-10 cycloalkyl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, and C5-10 heteroaryl containing 1-4 heteroatoms.

21. The cationic lipid according to claim 20, wherein Re and R7 are independently chosen from H, -OH, C1-6 alkyl, and saturated or unsaturated C3-8 heterocycloalkyl containing 1-4 hetero atoms.

22. The cationic lipid according to any one of the preceding claims, wherein R3 and R4 are independently chosen from branched or unbranched C1-26 alkyl, C2-26 alkenyl, C2-26 alkynyl, -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26.PVX-PAT-2402-WO23. The cationic lipid according to claim 22, wherein R3 and R4 are independently chosen from Ci -20 alkyl, C2-20 alkenyl, and C2-20 alkynyl.

24. The cationic lipid according to claim 22, wherein R3 and R4 are independently chosen from -(CH2)m-A-(CH2)n-(CH3)y, and -CH((CH2)m-A-(CH2)n-(CH3)y)2, wherein each of m, n, and y is independently an integer ranging from 0 to 26.

25. The cationic lipid according to claim 22 or claim 24, wherein the m, n, and y are each independently an integer ranging from 0 to 20, 0 to 16, 0 to 12, 0 to 10, or 0 to 8.

26. The cationic lipid according to any one of the preceding claims, wherein Li and L2 are independently chosen from C1-10, C1-8, C1-6, C1-4, C1-3, and C1-2 alkylene.

27. The cationic lipid according to any one of the preceding claims, wherein Li and L2 are independently chosen from C2-10, C2-8, C2-6, and C2-4 alkenylene.

28. The cationic lipid according to any one of the preceding claims, wherein L3 and L4 are independently chosen from C1-10, C1-8, C1-6, C1-4, C1-3, and C1-2 alkylene.

29. The cationic lipid according to any one of the preceding claims, wherein L3 and L4 are independently chosen from C2-10, C2-8, C2-6, C2-4, C3, and C2 alkenylene.

30. The cationic lipid according to any one of the preceding claims, wherein L5 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene.

31. The cationic lipid according to any one of the preceding claims, wherein L6 is either absent or independently chosen from C1-10 alkylene, C2-10 alkenylene, and C2-10 alkynylene.

32. The cationic lipid according to any one of the claims 30-31, wherein L5 and L6 are absent.

33. The cationic lipid according to any one of the preceding claims, wherein Xi is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or -OP(O)(O-)O-.

34. The cationic lipid according to claim 33, wherein Xi is -C(O)O-, -OC(O)-, -C(O)NH-, - NHC(O)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or -OP(O)(O-)O-.

35. The cationic lipid according to any one of the preceding claims, wherein X2 is -C(O)O-, -OC(O)-, -C(O)NH-, -NHC(O)-, -C(S)NH-, -NHC(S)-, -C(S)O-, -OC(S)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or -OP(O)(O-)O-.

36. The cationic lipid according to claim 35, wherein X2 is -C(O)O-, -OC(O)-, -C(O)NH-, - NHC(O)-, -OC(O)NH-, -NHC(O)O-, -OP(O)(OH)O-, or -OP(O)(O-)O-.PVX-PAT-2402-WO37. The cationic lipid according to any one of the preceding claims, wherein A is H, a bond, saturated or unsaturated C3-10 cycloalkyl, C6-10 aryl, saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, or C5-10 heteroaryl containing 1-4 heteroatoms.

38. The cationic lipid according to claim 37, wherein A is saturated or unsaturated C3-10 cycloalkyl, or saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms.— Hc 39. The cationic lipid according to any one of the preceding claims, wherein in Ai is X, -CH2-, or N.

40. The cationic lipid according to any one of the preceding claims, wherein the heteroatom is selected from the group consisting of O, N, S, or P.

41. The cationic lipid according to claim 40, wherein the heteroatom is selected from the group consisting of O, N, or S.

42. The cationic lipid according to any one of the preceding claims, wherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl is independently optionally substituted with one or more substituent.

43. The cationic lipid according to claim 42, wherein the substituent is chosen from H, - OH, Cl, Br, I, O, S, N, P, optionally substituted C1-6 alkoxy, optionally substituted C1-6 alkyl, optionally substituted C2-6 alkenyl, optionally substituted C2-6 alkynyl, optionally substituted saturated or unsaturated C3-10 cycloalkyl, optionally substituted Ce-io aryl, optionally substituted saturated or unsaturated C3-10 heterocycloalkyl containing 1-4 heteroatoms, optionally substituted C5-10 heteroaryl containing 1-4 heteroatoms or combinations thereof.

44. A lipid nanoparticle composition comprising the cationic lipid according to any one of the claims 1-43 and optionally additionally comprising a phospholipid, a sterol, a PEG- lipid, or a nucleic acid.

45. A lipid nanoparticle composition comprising the cationic lipid according to any one of the claims 1-43, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid.

46. The lipid nanoparticle composition according to any one of the claims 44-45, wherein the cationic lipid is present in an amount from 10 mol percent to 70 mol percent.

47. The lipid nanoparticle composition according to any one of the claims 44-45, wherein the phospholipid is present in an amount from 2 mol percent to 65 mol percent.PVX-PAT-2402-WO48. The lipid nanoparticle composition according to any one of the claims 44-45, wherein the sterol is present in an amount from 20 mol percent to 65 mol percent.

49. The lipid nanoparticle composition according to any one of the claims 44-45, wherein the PEG-lipid is present in an amount from 0.2 mol percent to 2.0 mol percent.

50. The lipid nanoparticle composition according to any one of the claims 44-49, wherein the cationic lipid is present in an amount from 10 mol percent to 70 mol percent, the phospholipid is present in an amount from 2 mol percent to 65 mol percent, the sterol is present in an amount from 20 mol percent to 65 mol percent, and the PEG-lipid is present in an amount from 0.2 mol percent to 2.0 mol percent.

51. The lipid nanoparticle composition according to any one of the claims 44-50, additionally comprising an ionizable polymer.

52. The lipid nanoparticle composition according to claim 51, wherein the ionizable polymer is present in an amount from 1 mol percent to 25 mol percent.

53. The lipid nanoparticle composition according to any one of the claims 44-52, wherein the nucleic acid is a DNA, an RNA, or a combination thereof.

54. The lipid nanoparticle composition according to claim 53, wherein the RNA is an mRNA, a long non-coding RNA (IncRNA), a micro RNA (miRNA), a small interfering RNA (siRNA), a small nucleolar RNA (snoRNA), a small nuclear RNA (snRNA), a PIWI-interacting RNA (piRNA), or a combination thereof.

55. The lipid nanoparticle composition according to claim 54, wherein the RNA is an mRNA.

56. The lipid nanoparticle composition according to claim 55, wherein the mRNA encodes one or more protein, one or more antibodies, or combinations thereof.

57. The lipid nanoparticle composition according to claim 55, wherein the mRNA encodes an antigen derived from a prokaryote or a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a mycoplasma, a protozoan, an animal or a human.

58. A method of treating or preventing a disease, comprising administrating to a subject in need thereof the lipid nanoparticle composition according to any one of the claims 44- 57.

59. Use of a lipid nanoparticle composition according to any one of the claims 44-57 in the manufacture of a medicament for the treatment or prevention of a disease in a subject.PVX-PAT-2402-WO60. A method of delivering a nucleic acid comprising administering a lipid nanoparticle composition comprising the cationic lipid according to any one of the claims 1-43, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid.

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