Ionizable lipids useful in nucleic acid delivery

Abstract

The present invention provides lipids and lipid nanoparticle formulations comprising these lipids, alone or in combination with other lipids These lipid nanoparticles may be formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications. The present invention also provides methods of chemical synthesis of these lipids, lipid nanoparticle preparation and formulation with nucleic acids.

Description

[0001] IONIZABLE LIPIDS USEFUL IN NUCLEIC ACID DELIVERY

[0002] FIELD OF THE INVENTION

[0003] The present invention provides lipids and lipid nanoparticle formulations comprising these lipids, alone or in combination with other lipids. These lipid nanoparticles may be formulated with nucleic acids to facilitate their intracellular delivery both in vitro and for in vivo therapeutic applications. The present invention also provides methods of chemical synthesis of these lipids, lipid nanoparticle preparation and formulation with nucleic acids.

[0004] BACKGROUND OF THE INVENTION

[0005] Therapeutic nucleic acids including small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, messenger RNA (mRNA), ribozymes, pDNA and immune stimulating nucleic acids act via a variety of mechanisms. Specific proteins can be downregulated by siRNA or miRNA through RNA interference (RNAi). Hematopoietic cells, such as leukocytes in general, and primary T lymphocytes and B-cells in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs). Modulating immune cell function, such as T cells and B cells, by downregulating specific genes using RNA interference (RNAi) holds tremendous potential in advancing targeted therapies in many immune-related disorders including cancer, inflammation, autoimmunity and viral infections. The therapeutic applications of RNAi are extremely broad, since siRNA and miRNA constructs can be synthesized with any nucleotide sequence directed against a target protein. To date, siRNA constructs have shown the ability to specifically silence target proteins in both in vitro and in vivo models. These are currently being evaluated in clinical studies.

[0006] Messenger RNA (mRNA) is the family of large RNA molecules which convey the genetic information from DNA to the ribosome. Some nucleic acids, such as mRNA or plasmids, can be used to effect expression of specific cellular products. Such nucleic acids would be useful in the treatment of diseases related deficiency of a protein or enzyme. However, there are many problems associated with nucleic acids in therapeutic contexts. One of the major problems with therapeutic nucleic acids is the stability of the phosphodiester inter nucleotide link and its susceptibility to nucleases. Apart from that these nucleic acids have limited ability to cross the cell membrane. Various lipids, e.g., cationic lipids, have proved to be excellent carriers of nucleic acids to treat different diseases in gene therapy applications. Lipid nanoparticles formed from cationic lipids and other co-lipids such as cholesterol, DSPC and PEGylated lipids encapsulated oligonucleotides which protect them from degradation and facilitate the cellular uptake.

[0007] WO 2018 / 087753 discloses a cationic lipid comprising a functional group represented by the structure: -W-(T=0)m-X-(CH2)z-Y, wherein X and Y are each independently O, N or NH, wherein X and Y cannot both be O; W is a bond, O, NH or S; T is C or S; m is 0 or 1; and z is 0 or 2, wherein said functional group is linked to at least one saturated or unsaturated fatty acid residue. WO 2022 / 168085 discloses a cationic lipid comprising a functional group represented by the structure:

[0008]

[0009] (II) wherein each one of R1and R13is independently selected from the group consisting of: OH, C1-3 alkyl-OH, C4-14 alkyl and C4-14 alkenyl; R12is selected from the group consisting of: C1-13 alkyl; C2-15 alkenyl, C1-6 alkyl-CO2-C0-3 alkylene– –N(C1-8 alkyl)2 and C1-6 alkyl-CO2- C0-3 alkylene-NH-C1-8 alkyl; R14is selected from the group consisting of: C1-13 alkyl; C2-15 alkenyl and

[0010]

[0011] wherein each L is an alkylene ester linker represented by: La-Xa-Lb; Xais selected from the group consisting of: -O2C-, -CO2-C2-4 alkylene-O2C-, O2C-C2-4 alkylene-O2C-, -CO2-C2-4 alkylene-CO2-, and O2C-C2-4 alkylene-CO2-; Lais selected from the group consisting of: C1-3 alkylene, C4-12 alkylene, C2-10 alkenylene and absent; and Lbis selected from the group consisting of: C1-3 alkylene, C2-10 alkenylene and C4-12 alkylene. Nevertheless, there remains a need in the art for suitable and efficient delivery platforms for delivery of oligonucleotides.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention relates to novel lipids which can be used in lipid nanoparticle preparation. These lipid nanoparticles protect nucleic acids from degradation, clearance from circulation and intracellular release. In addition, the nucleic acid encapsulated lipid nanoparticles advantageously are well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and / or risk to the patient. The present invention also provides the methods of chemical synthesis of these lipids, lipid nanoparticle preparation and formulations with nucleic acids.

[0014] In some embodiments, the present invention relates to novel lipids, and formulations of such lipids with siRNA and pDNA. These lipid nanoparticles (LNPs) were further characterized by DLS and assessed for their in vitro activity in various cancer cell lines.

[0015] According to some embodiments, there is provided a lipid represented by the structure of Formula (II), Formula (III), or salts thereof, wherein the structures of Formula (II) and Formula (III) are represented below:

[0016]

[0017] Formula (II)

[0018] wherein

[0019] Y is H or -xc-Zc-Lc;

[0020] each one of xa, Xb, and xcis independently selected from the group consisting of: Co-12 alkylene, C2-12 alkenylene, C2-12 alkynylene, and (CH2CH2O)n3;

[0021] each one of Za, Zb, and Zcis independently selected from the group consisting of: absent,

[0022]

[0023] each one of La, Lb, and Lcis independently selected from the group consisting of: C1-20 alkyl, C2- 20 alkenyl, C2-20 alkynyl, (CH2CH2O)n3R3, Co-12 alkylene-Ze-R3, Co-12 alkylene-Ze-NR'R2, Co-12 alkylene-N(Co-i2 alkylene-Ze-R3)2;

[0024] Zeis independently in each instance selected from the group consisting of: absent,

[0025]

[0026] each one of R1and R2is independently selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl, or wherein R1and R2, together with the nitrogen to which they are bound, form a heterocycle;

[0027] R3is selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl, C1-12 alkynyl, C0-12alkylene-N(C1-12alkyl)2and P(O)(OH)2;

[0028] each one of nl and n2 and m is 1, 2, 3, 4 or 5;

[0029] and n3 is an integer in the range of 1 to 45;

[0030] La–Za

[0031]

[0032] Formula (III)

[0033] wherein Y is H or -xc-Zc-Lc;

[0034] each one of xa, Xb, and xcis independently selected from the group consisting of: Co-12 alkylene, C2-12 alkenylene, C2-12 alkynylene and (CH2CH2O)n3;

[0035] each one of Za, Zb, and Zcis independently selected from the group consisting of: absent,

[0036]

[0037] each one of La, Lb, and Lcis independently selected from the group consisting of: C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl, (CH2CH2O)n3R3, Co-12 alkylene-Ze-R3, Co-12 alkylene-Ze-NR'R2, Co-12 alkylene-N(Co-i2 alkylene-Ze-R3)2;

[0038] Zeis independently in each instance selected from the group consisting of: absent,

[0039]

[0040] each one of R1and R2is independently selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl, or wherein R1and R2, together with the nitrogen to which they are bound, form a heterocycle;

[0041] R3selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl, C1-12 alkynyl, Co-12 alkylene-N(Ci-i2alkyl)2and P(O)(OH)2;

[0042] each one of nl and n2 and m is, independently, 1, 2, 3, 4 or 5; and

[0043] n3 is an integer in the range of 1 to 45. Each possibility represents a separate embodiment of the invention.

[0044] According to some embodiments, there is provided a lipid represented by the structure of Formula (Ila), wherein Y is H:

[0045]

[0046] Formula (Ila).

[0047] According to some embodiments, there is provided a lipid represented by the structure of Formula (lib), wherein Y is -xc-Zc-Lc:

[0048]

[0049] Formula (lib).

[0050] According to some embodiments, there is provided a lipid represented by the structure of Formula (lie):

[0051]

[0052] Formula (lie)

[0053] wherein each one of na and nb is each independently an integer in the range of 0-20, including each value within the specified range.

[0054] According to some embodiments, there is provided a lipid represented by the structure of Formula (lid):

[0055]

[0056] Formula (lid). According to some embodiments, there is provided a lipid represented by the structure of Formula (lie): La-N(¥)-(CH2)ni-(OCH2CH2)m-(CH2)n2-NH-Lb.

[0057] According to some embodiments, xais the same as Xb. According to some embodiments, xais the same as xc. According to some embodiments, xa, xb, and xcare the same.

[0058] According to some embodiments, Zais the same as Zb. According to some embodiments, Zais the same as Zc.

[0059] According to some embodiments, Lais the same as Lb. According to some embodiments, Lais the same as Lc.

[0060] According to some embodiments, xa-Za-Lais the same as Xb-Zb-Lb. According to some embodiments, xa-Za-Lais the same as xc-Zc-Lc.

[0061] According to some embodiments, xais a Co-12 linear alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xais an unsubstituted Co-12 alkylene. According to some embodiments, xais absent, -CH2(CH2)8CH2- or -CH2(CH2)7CH2-. According to some embodiments, xais absent or -CH2(CH2)7CH2-.

[0062] According to some embodiments, Xb is a Co-12 linear alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xb is an unsubstituted Co-12 alkylene. According to some embodiments, xb is absent, -CH2(CH2)8CH2- or -CH2(CH2)7CH2-. According to some embodiments, xcis absent. According to some embodiments, Xb is absent or -CH2(CH2)7CH2-.

[0063] According to some embodiments, xcis a Co-12 linear alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xcis an unsubstituted Co-12 alkylene. According to some embodiments, xcis absent. According to some embodiments, xcis absent or -CH2(CH2)8CH2-. According to some embodiments, xcis absent. According to some embodiments, xcis absent or -CH2(CH2)7CH2-.

[0064] According to some embodiments, Zais selected from the group consisting of: absent, |-C(O)O-, and -C(O)-. Each possibility represents a separate embodiment.

[0065] According to some embodiments, Zb is selected from the group consisting of: absent, -C(O)O-, and -C(O)-. Each possibility represents a separate embodiment. According to some embodiments, Zcis absent. According to some embodiments, Zcis absent or -C(O)O-. Each possibility represents a separate embodiment.

[0066] According to some embodiments, Zeis absent or -C(O)O-. Each possibility represents a separate embodiment.

[0067] According to some embodiments, Lais C4-18alkyl or C0-6alkylene-N(C1-12alkyl)2. Each possibility represents a separate embodiment. According to some embodiments, Lais C8-14alkyl or C2-4alkylene-N(CH2(CH2)10CH3)2. According to some embodiments, Lais selected from the group consisting of: C4-18 alkyl, C4-18 alkenyl, (CH2CH2O)-C0-12alkylene-N(C1-12alkyl)2, and Co-12 alkylene-Ze-N(Ci-i2 alkyl)2. According to some embodiments, Lais selected from the group consisting of: -(CH2)nCH3, -CH2CH2-N(CH2(CH2)IOCH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -(CH2)3-N(CH2(CH2)10CH3)2, and -(CH2)4-N(CH2(CH2)10CH3)2.

[0068] According to some embodiments, Lais selected from the group consisting of: -(CH2)11CH3, - CH2CH2-N(CH2(CH2)1OCH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -(CH2)3-N(CH2(CH2)10CH3)2, -CH2(CH2)7CH2C(O)O-N((CH2)5CH3)2, -(CH2)4-N(CH2(CH2)10CH3)2, -CH2CH2-N(CH2(CH2)5CH3)2, -CH2CH2OCH2CH2N((CH2)5CH3)2, -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3, and -N((CH2)5CH3)2.

[0069] According to some embodiments, Lbis C4-18alkyl or C0-6alkylene-N(C1-12alkyl)2. Each possibility represents a separate embodiment. According to some embodiments, Lb is C4-18 alkyl, (CH2CH2O)-Co-i2-alkylene-N(Ci-i2alkyl)2, or Co-6 alkylene-N(Ci-i2 alkyl)2. According to some embodiments, Lbis C8-14alkyl or C0-4alkylene-N(C6-12alkyl)2. According to some embodiments, Lb is (CH2CH2O)-C0-6-alkylene-N(C1-9alkyl)2, C8-14alkyl, or C0-4alkylene-N(C4-12alkyl)2. According to some embodiments, Lb is selected from the group consisting of: -CH2CH2-N(CH2(CH2)IOCH3)2, -CH[(CH2)5CH3](CH2)4CH3, -N((CH2)5CH3)2, -(CH2)3-N((CH2)11CH3)2, and -(CH2)4-N((CH2)11CH3)2. According to some embodiments, Lb is selected from the group consisting of: -CH2CH2-N(CH2(CH2)10CH3)2, -CH[(CH2)5CH3](CH2)4CH3, -N((CH2)5CH3)2, -(CH2)3-N((CH2)11CH3)2, -(CH2)4-N((CH2)HCH3)2, -CH2CH2-N((CH2)5CH3)2, and -CH2CH2OCH2CH2N((CH2)5CH3)2. According to some embodiments, Lcis C4-18 alkyl. According to some embodiments, Lcis C4-18 alkyl, C4-18 alkenyl, or Co-6 alkylene-N(Ci-i2 alkyl)2. Each possibility represents a separate embodiment. According to some embodiments, Lcis -(CH2)nCH3. According to some embodiments, Lcis selected from the group consisting of: -(CH2)nCH3, CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3, and -N((CH2)5CH3)2.

[0070] According to some embodiments, xb is absent; Zb is -C(O)O-; Lb is Co-12 alkyl ene-Zc-NR'R2; nl and n2 are each independently 1; and m is 1. According to some embodiments, Lb is C1-4 alkylene-NR'R2. According to some embodiments, Lb is C2 alkylene-N(C12H25)2.

[0071] According to some embodiments, Y is -xc-Zc-Lc; each one of xaand xcis absent; each one of Zaand Zcis independently absent; and each one of Laand Lcis independently C1-20 alkyl; or wherein Y is H; xais absent; Zais -C(O)O-; Lais Co-12 alkylene-Ze-NR'R2; wherein Zeis -C(O)O-; and each one of R1and R2is independently C1-12 alkyl. Each possibility represents a separate embodiment. According to some embodiments, xais absent; Zais absent or -C(O)O-; Lais selected from the group consisting of: Ce-18 alkyl, C6-20 alkenyl, and Co-12 alkylene-Ze-NR'R2; nl and n2 are each independently 1; and m is 1. Each possibility represents a separate embodiment of the invention.

[0072] According to some embodiments, Y is H; or Y is -xc-Zc-Lcand wherein xcis absent; Zcis absent or -C(O)O-; and Lcis selected from the group consisting of: Ce-18 alkyl, C6-20 alkenyl, and Co-12 alkylene-Ze-NR1R2. Each possibility represents a separate embodiment of the invention.

[0073] According to some embodiments, xb is Co-12 alkylene; Zb is -C(O)O-; and Lb is selected from the group consisting of: Co-12 alkylene-Ze-NR1R2and (CEhCEhO^R3, wherein n3 is 1-3 and R3is Co-9 alkylene-N(Ci-i2alkyl)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, each one of R1and R2is independently C1-12 alkyl and wherein Zeis absent or -C(O)O. Each possibility represents a separate embodiment of the invention.

[0074] According to some embodiments, the lipid is selected from the group consisting of: Lipid 101, Lipid 102, Lipid 103, Lipid 104, Lipid 105, Lipid 106, and Lipid 107, Lipid 108, Lipid 109, Lipid 110, Lipid 111, Lipid 112, Lipid 113, Lipid 114, Lipid 115, Lipid 116, Lipid 117, Lipid 118, Lipid 119, Lipid 120, Lipid 121, Lipid 122, Lipid 123, Lipid 124, Lipid 125, Lipid 126, Lipid 127, Lipid 128, Lipid 129, Lipid 130, Lipid 131, Lipid 132, Lipid 133, Lipid 134, Lipid 135, Lipid 136, Lipid 137, Lipid 138, Lipid 139, Lipid 140, Lipid 141, Lipid 142, Lipid 143, Lipid 144, Lipid 145, Lipid 146, Lipid 147, Lipid 147, Lipid 149, Lipid 150, Lipid 151, Lipid 152, Lipid 153, Lipid 154, Lipid 155, Lipid 156, Lipid 157, Lipid 158, Lipid 159, Lipid 160, Lipid 161, Lipid 162, Lipid 163, Lipid 164, Lipid 165, Lipid 166, Lipid 167, Lipid 168, Lipid 169, Lipid 170, Lipid 171, Lipid 172, and Lipid 173, and salts thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid is selected from the group consisting of: Lipid 101, Lipid 102, Lipid 103, Lipid 104, Lipid 105, Lipid 106, and Lipid 107. Each possibility represents a separate embodiment of the invention. The structures of the above lipids are shown in the Exemplary Lipids section.

[0075] According to some embodiments, the lipid is selected from the group consisting of: Lipid 101, Lipid 102, and Lipid 103. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid is selected from the group consisting of: Lipid 101, and Lipid 102. Each possibility represents a separate embodiment of the invention.

[0076] According to some embodiments, the lipid is selected from the group consisting of: Lipid 103, Lipid 104, Lipid 105, Lipid 106, and Lipid 107. Each possibility represents a separate embodiment of the invention.

[0077] According to some embodiments, the lipid is selected from the group consisting of: Lipid 102, Lipid 110, Lipid 101, Lipid 105, Lipid 111, and Lipid 115. Each possibility represents a separate embodiment of the invention.

[0078] According to some embodiments, the lipid is selected from the group consisting of: Lipid 102 and Lipid 110. Each possibility represents a separate embodiment of the invention.

[0079] According to some embodiments, the present invention provides a particle comprising the lipid according to the present invention and a membrane stabilizing lipid. According to some embodiments, the particle comprises the membrane stabilizing lipid and a lipid membrane comprising the lipid.

[0080] According to some embodiments, the membrane stabilizing lipid is selected from the group consisting of cholesterol, phospholipids, cephalins, sphingolipids and glycoglycerolipids. Each possibility represents a separate embodiment of the invention. According to some embodiments, the membrane stabilizing lipid comprises cholesterol. According to some embodiments, the particle further comprises one or more additional components selected from the group consisting of: a PEG-lipid conjugate, a neutral lipid and a charged lipid. According to some embodiments, the additional component comprises l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Each possibility represents a separate embodiment of the invention. According to some embodiments, the additional component comprises 1,2-Di stearoyl -sn-glycero-3 -phosphocholine (DSPC). According to some embodiments, the additional component comprises l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). According to some embodiments, the additional component comprises 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG). According to some embodiments, the particle comprises the lipid, cholesterol, l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG). According to some embodiments, the lipid comprises cholesterol, 1,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) and 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG). According to some embodiments, the particle is conjugated to a targeting moiety. According to some embodiments, the particle further comprises a nucleic acid. According to some embodiments, the nucleic acid is encapsulated within a particle comprising the lipid. According to some embodiments, the nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), an antisense oligo nucleotide, a messenger RNA (mRNA), a ribozyme, a pDNA, a CRISPR mRNA, a gRNA, a circular RNA and an immune-stimulating nucleic acid. Each possibility represents a separate embodiment of the invention.

[0081] According to some embodiments, the particle further comprises a therapeutic agent. According to some embodiments, the therapeutic agent is encapsulated within a particle comprising the lipid. According to some embodiments, the therapeutic agent is an RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein or an immunogenic fragment or variant thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the therapeutic agent is an RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein, an immunogenic fragment of SARS-CoV-2 or a SARS-CoV-2 variant. According to some embodiments, the therapeutic agent is an RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein or an immunogenic fragment or variant thereof.

[0082] According to some embodiments, there is provided a pharmaceutical composition comprising a plurality of particles as disclosed herein and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the pharmaceutical composition is a liposomal composition. According to some embodiments, the pharmaceutical composition is for use in treating a leukocyte associated condition.

[0083] According to some embodiments, there is provided a method of gene silencing, comprising contacting a cell with the pharmaceutical composition according to the present invention.

[0084] According to some embodiments, there is provided a method of gene silencing, comprising the step of contacting a cell with the pharmaceutical composition according to the present invention. According to some embodiments, the cell is a cancer cell.

[0085] According to some embodiments, the compositions of the present invention may be used as a delivery system to administer a therapeutic agent to a target location in the body. According to some embodiments, there is provided method for administering a therapeutic agent, the method comprising administering the pharmaceutical composition according to the present invention to a subject in need thereof.

[0086] The lipids of the present invention can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including, for example, sterols) and / or their analogs, and / or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents. In some instances, the lipid nanoparticles are used to deliver nucleic acids for the treatment of various diseases or conditions, in particular leukocyte associated conditions such as inflammation and / or lack of sufficient protein.

[0087] Thus, according to some embodiments, the present invention relates to a method of treating a leukocyte-associated condition, the method comprising the step of administering to a subject in need thereof the pharmaceutical composition according to the present invention. According to some embodiments, the leukocyte associated condition may be selected from the group consisting of cancer, infection, autoimmune diseases, neurodegenerative diseases and inflammation. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0088] BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1A is a 'H NMR spectrum of Lipid 108 (NV5-001).

[0090] Figure IB is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 108 (NV5-001).

[0091] Figure 2A is a 'H NMR spectrum of Lipid 109 (NV5-002).

[0092] Figure 2B is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 109 (NV5-002).

[0093] Figure 3A is a 'H NMR spectrum of Lipid 105 (NV5-003).

[0094] Figure 3B is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 105 (NV5-003).

[0095] Figure 4A is a 'H NMR spectrum of Lipid 101 (NV5-006).

[0096] Figure 4B is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 101 (NV5-006).

[0097] Figure 5A is a 'H NMR spectrum of Lipid 102 (NV5-007).

[0098] Figure 5B is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 102 (NV5-007).

[0099] Figure 6A is a 'H NMR spectrum of Lipid 110 (NV5-035).

[0100] Figure 6B is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 110 (NV5-035).

[0101] Figure 7A is a 'H NMR spectrum of Lipid 111 (NV5-039). Figure 7B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 111 (NV5-039).

[0102] Figure 8A is aJH NMR spectrum of Lipid 112 (NV5-040).

[0103] Figure 8B is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 112 (NV5-040).

[0104] Figure 9A is aJH NMR spectrum of Lipid 113 (NV5-041).

[0105] Figure 9B is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 113 (NV5-041).

[0106] Figure 10A is aJH NMR spectrum of Lipid 114 (NV5-042).

[0107] Figure 10B is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 114 (NV5-042).

[0108] Figure 11A is aJH NMR spectrum of Lipid 115 (NV5-043).

[0109] Figure 11B is an Electrospray Ionization Mass Spectrometry (ESLMS) spectrum of Lipid 115 (NV5-043).

[0110] Figure 12A is a bar chart that represents the transfection efficiencies of luciferase mRNA-encapsulated LNPs, formulated with Lipid 102 (NV5-007), Formulation Fl (left group), Lipid 102 (NV5-007), Formulation F2 (middle group) and Lipid 110 (NV5-035) Formulation F2 (right group) in HepG2 cells, at concentration of 25ng / ml (horizontal stripes), 50ng / ml (squares), lOOng / ml (diagonal stripes) and 200ng / ml (solid fill).

[0111] Figure 12B is a bar chart that represents the transfection efficiencies of luciferase mRNA-encapsulated LNPs, formulated with Lipid 102 (NV5-007), Formulation Fl (left group), Lipid 102 (NV5-007), Formulation F2 (middle group) and Lipid 110 (NV5-035) Formulation F2 (right group) in Mutu DC 1340 mouse dendritic cells (DC) cells, at concentration of 25ng / ml (horizontal stripes), 50ng / ml (squares), lOOng / ml (diagonal stripes) and 200ng / ml (solid fill).

[0112] Figure 12C is a bar chart that represents the transfection efficiencies of luciferase mRNA-encapsulated LNPs, formulated with Lipid 102 (NV5-007), Formulation Fl (left group), Lipid 102 (NV5-007), Formulation F2 (middle group) and Lipid 110 (NV5-035) Formulation F2 (right group) in HTB11 human neuroblastoma cells (HTB11), at concentration of 25ng / ml (horizontal stripes), 50ng / ml (squares), lOOng / ml (diagonal stripes) and 200ng / ml (solid fill).

[0113] Figure 13 is a bar graph that represents the viability of HepG2 (left group), DC (right group) and HTB11 (middle group) cells treated with LNPs formulated with Lipid 102 (NV5-007) Formulation Fl (squares), Lipid 102 (NV5-007) Formulation F2 (horizontal lines) and Lipid 110 (NV5-035) Formulation F2 (vertical lines) versus control (solid fill).

[0114] Figures 14A-J are bar graphs representing Luciferase expression detected in BALB / c mice liver, spleen, lungs, heart and kidneys by IVIS imaging following systemic injection of LNPs formulated with F2 Formulation of Lipid 102 (NV5-007), Lipid 110 (NV5-035), Lipid 101 (NV5-006), Lipid 105 (NV5-003), Lipid 111 (NV5-039), and Lipid 115 (NV5-039) and loaded with luciferase mRNA. Figure 14A: Luciferase expression in liver following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 102 and Lipid 110; Figure 14B: Luciferase expression in liver following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 101, Lipid 105, Lipid 111, and Lipid 115; Figure 14C: Luciferase expression in spleen following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 102 and Lipid 110; Figure 14D: Luciferase expression in spleen following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 101, Lipid 105, Lipid 111, and Lipid 115; Figure 14E: Luciferase expression in lungs following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 102 and Lipid 110; Figure 14F:

[0115] Luciferase expression in lungs following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 101, Lipid 105, Lipid 111, and Lipid 115; Figure 14G: Luciferase expression in heart following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 102 and Lipid 110; Figure 14H: Luciferase expression in heart following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 101, Lipid 105, Lipid 111, and Lipid 115; Figure 141: Luciferase expression in kidneys following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 102 and Lipid 110;

[0116] Figure 14J: Luciferase expression in kidneys following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 101, Lipid 105, Lipid 111, and Lipid 115. Naive mice in all experiments represent untreated mice that served as controls. DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0117] The present invention based on the discovery of lipids useful in preparing lipid nanoparticles to deliver active agents in vitro and in vivo. The lipids of the present invention are useful in delivery of nucleic acids such as siRNA, miRNA and mRNA etc.

[0118] Lipids

[0119] As contemplated herein, the present invention relates to a lipid represented by the structure of Formula (II):

[0120]

[0121] Formula (II)

[0122] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention.

[0123] According to some embodiments, Y is H or xc-Zc-Lc.

[0124] According to some embodiments, the lipid of Formula (II), wherein Y is H, is represented by the structure of Formula (Ila):

[0125]

[0126] Formula (Ila)

[0127] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the lipid of Formula (II), wherein Y is -xc-Zc-Lc, is represented by the structure of Formula (lib):

[0128]

[0129] Formula (lib)

[0130] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention.

[0131] According to some embodiments, the lipid of Formula (II) is represented by the structure of Formula (lie):

[0132]

[0133] Formula (lie)

[0134] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y is H or (CH2)nc-Zc-Lc. According to some embodiments, Y is H. According to some embodiments, Y is (CH2)nc-Zc-Lc.

[0135] According to some embodiments, na is an integer in the range of 0-20. According to some embodiments, na is an integer in the range of 1-20. According to some embodiments, na is an integer in the range of 1-12. According to some embodiments, nb is an integer in the range of 0-20. According to some embodiments, nb is an integer in the range of 1-20. According to some embodiments, nb is an integer in the range of 1-12. According to some embodiments, na is 0. According to some embodiments, na is 9. According to some embodiments, na is 10. According to some embodiments, nb is 0. According to some embodiments, nb is 9. According to some embodiments, nb is 10. According to some embodiments, nc is an integer in the range of 1-20. According to some embodiments, nc is an integer in the range of 1-12. According to some embodiments, nc is an integer in the range of 0-20.

[0136] According to some embodiments, the lipid of Formula (II), is represented by the structure of Formula (lid):

[0137]

[0138] Formula (lid)

[0139] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y is H or -Zc-Lc.

[0140] According to some embodiments, the lipid of Formula (II), is represented by the structure of Formula (lie):

[0141] La-N(Y)-(CH2)ni-(OCH2CH2)m-(CH2)n2-NH-Lb (Formula (lie))

[0142] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y is H or -Lc.

[0143] As contemplated herein, the present invention relates to a lipid represented by the structure of Formula (III):

[0144]

[0145] Formula (III)

[0146] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y is H or xc-Zc-Lc.

[0147] According to some embodiments, the lipid of Formula (III) is represented by the structure of Formula (Illa):

[0148]

[0149] Formula (Illa)

[0150] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, Y is H or (CH2)nc-Zc-Lc. According to some embodiments, Y is H. According to some embodiments, Y is (CH2)nc-Zc-Lc.

[0151] According to some embodiments, the lipid of Formula (III) is represented by the structure of Formula (Illb):

[0152]

[0153] Formula (Illa)

[0154] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the invention.

[0155] According to some embodiments, xa(e.g., in Formulae (II), (Ila), (lib) and (III)) is selected from an alkylene, an alkenylene, and an alkynylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xais selected from absent, an alkylene, an alkenylene, an alkynylene, and (CH2CH2O)n3, wherein n3 is a positive integer or zero. Each possibility represents a separate embodiment of the invention. According to some embodiments, xais selected from an alkylene, an alkenylene, an alkynylene, and (CH2CH2O)n3, wherein n3 is a positive integer or zero. Each possibility represents a separate embodiment of the invention. According to some embodiments, xais an alkylene. According to some embodiments, xais a linear alkylene or a branched alkylene. According to some embodiments, xais a linear alkylene. According to some embodiments, xais a branched alkylene. According to some embodiments, xais an unsubstituted alkylene. According to some embodiments, xais Co-12 alkylene. According to some embodiments, xais C0-9 alkylene. It is to be understood that Co connotes absent. According to some embodiments, xais selected from the group consisting of: absent, -CH2(CH2)7CH2-, and -CH2(CH2)8CH2-. According to some embodiments, xais selected from the group consisting of: absent and -CH2(CH2)7CH2-. According to some embodiments, xais absent. According to some embodiments, xais -CH2(CH2)7CH2-. According to some embodiments, xais -CH2(CH2)8CH2-. According to some embodiments, xais an alkenylene. According to some embodiments, xais a linear alkenylene or a branched alkenylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xais a linear alkenylene. According to some embodiments, xais a branched alkenylene. According to some embodiments, xais an unsubstituted alkenylene. According to some embodiments, xais C2-12 alkenylene. According to some embodiments, xais C2-9 alkenylene. According to some embodiments, xais C2-6 alkenylene.

[0156] According to some embodiments, xais an alkynylene. According to some embodiments, xais a linear alkynylene or a branched alkynylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xais a linear alkynylene. According to some embodiments, xais a branched alkynylene. According to some embodiments, xais an unsubstituted alkynylene. According to some embodiments, xais C2-12 alkynylene. According to some embodiments, xais C2-9 alkynylene. According to some embodiments, xais C2-6 alkynylene.

[0157] According to some embodiments, xaincludes a monoethylene glycol or polyethylene glycol (PEG), e.g., (CH2CH2O)n3, wherein n3 is a positive integer. According to some embodiments, xais (CH2CH2O)n3, wherein n3 is an integer in the range of 0 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 45, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 30, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 15, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 10, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 5, including each integer value within the specified range. According to some embodiments, n3 is 1. According to some embodiments, n3 is 2. According to some embodiments, xais -CH2CH2O-. According to some embodiments, xais -CH2CH2OCH2CH2O-.

[0158] According to some embodiments, xb (e.g., in Formulae (II), (Ila), (lib) and (III)) is selected from an alkylene, an alkenylene, and an alkynylene. Each possibility represents a separate embodiment. According to some embodiments, Xb is selected from absent, an alkylene, an alkenylene, an alkynylene, and (CH2CH2O)n3, wherein n3 is a positive integer or zero. Each possibility represents a separate embodiment of the invention. According to some embodiments, xb is selected from an alkylene, an alkenylene, an alkynylene, and (CH2CH2O)n3, wherein n3 is a positive integer or zero. Each possibility represents a separate embodiment. According to some embodiments, xb is an alkylene. According to some embodiments, Xb is a linear alkylene or a branched alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, Xb is a linear alkylene. According to some embodiments, Xb is a branched alkylene. According to some embodiments, Xb is an unsubstituted alkylene. According to some embodiments, Xb is Co-12 alkylene. According to some embodiments, Xb is C0-9 alkylene. According to some embodiments, xb is selected from the group consisting of: absent, -CH2(CH2)8CH2-, and -CH2(CH2)8CH2-. According to some embodiments, xb is selected from the group consisting of: absent and -CH2(CH2)7CH2-. According to some embodiments, xb is absent. According to some embodiments, xbis -CH2(CH2)7CH2-. According to some embodiments, xb is -CH2(CH2)8CH2-.

[0159] According to some embodiments, Xb is an alkenylene. According to some embodiments, Xb is a linear alkenylene or a branched alkenylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, Xb is a linear alkenylene. According to some embodiments, Xb is a branched alkenylene. According to some embodiments, Xb is an unsubstituted alkenylene. According to some embodiments, Xb is C2-12 alkenylene. According to some embodiments, Xb is C2-9 alkenylene. According to some embodiments, Xb is C2-6 alkenylene. According to some embodiments, Xb is an alkynylene. According to some embodiments, Xb is a linear alkynylene or a branched alkynylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, Xb is a linear alkynylene. According to some embodiments, Xb is a branched alkynylene. According to some embodiments, xais an unsubstituted alkynylene. According to some embodiments, Xb is C2-12 alkynylene. According to some embodiments, Xb is C2-9 alkynylene. According to some embodiments, Xb is C2-6 alkynylene.

[0160] According to some embodiments, Xb includes a monoethylene glycol or polyethylene glycol (PEG) e.g., (CH2CH2O)n3, wherein n3 is a positive integer. According to some embodiments, Xb is (CH2CH2O)n3, wherein n3 is an integer in the range of 0 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 45, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 30, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 15, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 10, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 5, including each integer value within the specified range. According to some embodiments, n3 is 1. According to some embodiments, n3 is 2. According to some embodiments, Xb is -CH2CH2O-. According to some embodiments, Xb is -CH2CH2OCH2CH2O-.

[0161] According to some embodiments, xc(e.g., in Formulae (II), (lib) and (III)) is selected from an alkylene, an alkenylene, and an alkynylene. Each possibility represents a separate embodiment. According to some embodiments, xcis selected from absent, an alkylene, an alkenylene, an alkynylene, and (CH2CH2O)n3, wherein n3 is a positive integer or zero. Each possibility represents a separate embodiment of the invention. According to some embodiments, xcis selected from an alkylene, an alkenylene, an alkynylene, and (CH2CH2O)n3, wherein n3 is a positive integer or zero. Each possibility represents a separate embodiment. According to some embodiments, xcis an alkylene. According to some embodiments, xcis a linear alkylene or a branched alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xcis a linear alkylene. According to some embodiments, xcis a branched alkylene. According to some embodiments, xcis an unsubstituted alkylene. According to some embodiments, xcis Co-12 alkylene. According to some embodiments, xcis C0-9 alkylene. According to some embodiments, xcis Co-6 alkylene. According to some embodiments, xcis Co-4 alkylene. According to some embodiments, xcis absent. According to some embodiments, xcis -CH2(CH2)8CH2-.

[0162] According to some embodiments, xcis an alkenylene. According to some embodiments, xcis a linear alkenylene or a branched alkenylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xcis a linear alkenylene. According to some embodiments, xcis a branched alkenylene. According to some embodiments, xcis an unsubstituted alkenylene. According to some embodiments, xcis C2-12 alkenylene. According to some embodiments, xcis C2-9 alkenylene. According to some embodiments, xcis C2-6 alkenylene. According to some embodiments, xcis an alkynylene. According to some embodiments, xcis a linear alkynylene or a branched alkenylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xcis a linear alkynylene. According to some embodiments, xcis a branched alkynylene. According to some embodiments, xcis an unsubstituted alkynylene. According to some embodiments, xcis C2-12 alkynylene. According to some embodiments, xcis C2-9 alkynylene. According to some embodiments, xcis C2-6 alkynylene. According to some embodiments, xcincludes a monoethylene glycol or polyethylene glycol (PEG) e.g., (CH2CH2O)n3, wherein n3 is a positive integer. According to some embodiments, xcis (CH2CH2O)n3, wherein n3 is an integer in the range of 0 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 45, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 30, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 15, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 10, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 5, including each integer value within the specified range. According to some embodiments, n3 is 1. According to some embodiments, n3 is 2. According to some embodiments, xcis -CH2CH2O-. According to some embodiments, xcis -CH2CH2OCH2CH2O-. According to some embodiments, each one of xa, Xb, and xcis independently alkylene. It is to be understood that embodiments which refer to a number of substituents and define each of the substituents in one sentence, do not necessarily require that the substituents are the same. For example, the phrase “each one of xa, Xb, and xcis independently Co-12 alkylene” requires that the aforementioned substituents are each Co-12 alkylene, however, it does not require that xa, xb, and xcare the same alkylene, e.g., xaand xb may be alkylenes of the same or different lengths or branching / linear pattern. For example, in Lipid 102, shown herein, each one of xa, Xb, and xcis independently Co-12 alkylene, wherein xa, Xb, and xcare the same, i.e., Co alkylene. In contrast, for example, in Lipid 101, shown herein, each one of xa, Xb, and xcis independently Co-12 alkylene, wherein xaand xb are different, i.e., xaand xcare absent and xb is -CFh CFh^CFh-.

[0163] According to some embodiments, each one of xa, Xb, and xcis independently Co-12 alkylene. According to some embodiments, each one of xa, Xb, and xcis independently unsubstituted Co- 12 alkylene. According to some embodiments, each one of xa, xb, and xcis independently C0-9 alkylene. According to some embodiments, the alkylene group of each one of xa, Xb, and xcis independently linear. According to some embodiments, the alkylene group of each one of xa, Xb, and xcis independently branched. According to some embodiments, each one of xa, xb, and xcis independently absent.

[0164] According to some embodiments, each one of xa, Xb, and xcis independently alkenylene. According to some embodiments, each one of xa, Xb, and xcis independently C2-12 alkenylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, each one of xa, Xb, and xcis independently C2-9 alkenylene. According to some embodiments, each one of xa, Xb, and xcis independently C2-6 alkenylene. According to some embodiments, the alkenylene in each one of xa, xb, and xcis independently unsubstituted. According to some embodiments, the alkenylene in each one of xa, Xb, and xcis independently linear. According to some embodiments, the alkenylene in each one of xa, Xb, and xcis independently branched.

[0165] According to some embodiments, each one of xa, Xb, and xcis independently alkynylene. According to some embodiments, each one of xa, Xb, and xcis independently C2-12 alkynylene. According to some embodiments, the alkynylene in each one of xa, Xb, and xcis independently unsubstituted. According to some embodiments, the alkynylene in each one of xa, Xb, and xcis independently linear. According to some embodiments, the alkynylene in each one of xa, Xb, and xcis independently branched.

[0166] According to some embodiments, each one of xa, xb, and xcis independently (CH2CH2O)n3, wherein n3 is a positive integer. According to some embodiments, each one of xa, Xb, and xcis independently (CH2CH2O)n3, wherein n3 is an integer in the range of 0 to 45, including each integer value within the specified range. According to some embodiments, each one of xa, xb, and xcis independently (CH2CH2O)n3, wherein n3 is an integer in the range of 1 to 45, including each integer value within the specified range. According to some embodiments, each one of xa, xb, and xcis independently (CH2CH2O)n3, wherein n3 is an integer in the range of 1 to 20, including each integer value within the specified range. According to some embodiments, each one of xa, xb, and xcis independently -CH2CH2O-. According to some embodiments, each one of xa, xb, and xcis independently -CH2CH2OCH2CH2O-.

[0167] According to some embodiments, xais the same as Xb. According to some embodiments, xais the same as xc. According to some embodiments, xaand xcare the same and are different from xb. According to some embodiments, xais different from Xb. According to some embodiments, Xb is different from xc. It is to be understood that the term “the same” means that the specified x substituents have the same chemical structure whereas the term “different” refers to chemical structures that are not the same (e.g., -CH2CH2CH2CH2- and -CH(CH3)CH2CH2-). For example, Lipid 107, shown herein, has the same xaand Xb, each of which is absent. According to some embodiments, each one of xaand Xb is independently absent. In another example, Lipid 101, shown herein, has the same xaand xc, each of which is absent. Also in Lipid 101, xb is -CH2(CH2)7CH2-, which is different from xaand xc. According to some embodiments, each one of xaand xcis independently absent. According to some embodiments, each one of xaand xcis independently -CH2(CH2)7CH2-. According to some embodiments, each one of xaand xb is independently absent. According to some embodiments, each one of xaand Xb is independently -CH2(CH2)7CH2-. According to some embodiments, xais -CH2(CH2)7CH2- and xbis -CH2(CH2)8CH2-. According to some embodiments, xais -CH2(CH2)7CH2- and xb is absent. According to some embodiments, xais -CH2(CH2)8CH2- and xbis -CH2(CH2)7CH2-. According to some embodiments, each one of xaand xcis independently absent and xbis -CH2(CH2)7CH2-. According to some embodiments, each one of xaand xcis independently -CH2(CH2)7CH2- and Xb is absent. According to some embodiments, xa, Xb, and xcare the same. It is to be understood that the term “the same” means that the three specified x substituents have the same chemical structure. For example, Lipid 102, shown herein, has the same xa, Xb, and xc, each of which is absent. According to some embodiments, each one of xa, xb, and xcis independently absent.

[0168] According to some embodiments, Zais selected from the group consisting of absent, -C(O)O-, -C(O)NH-, -OC(O)O-, -OC(O)NH-, -OC(O)-, -NHC(O)-, -NHC(O)O-, -O-P(O)(OH)O-, -O-, -S-, -S-S-, and -C(O)-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Zais selected from the group consisting of absent, -C(O)O-, -OC(O)-, and -C(O)- According to some embodiments, Zais selected from the group consisting of absent, -C(O)O-, and -C(O)-. According to some embodiments, Zais absent. According to some embodiments, Zais -C(O)O-. According to some embodiments, Zais -C(O)-.

[0169] According to some embodiments, Zb is selected from the group consisting of absent, -C(O)O-, -C(O)NH-, -OC(O)O-, -OC(O)NH-, -OC(O)-, -NHC(O)-, -NHC(O)O-, -O-P(O)(OH)O-, -O-, -S-, -S-S-, and -C(O)-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Zb is selected from the group consisting of absent, -C(O)O-, -OC(O)-, and -C(O)- According to some embodiments, Zb is selected from the group consisting of absent, -C(O)O-, and -C(O)-. According to some embodiments, Zb is absent. According to some embodiments, Zb is -C(O)O-. According to some embodiments, Zb is -C(O)-.

[0170] According to some embodiments, Zcis selected from the group consisting of absent, -C(O)O-, -C(O)NH-, -OC(O)O-, -OC(O)NH-, -OC(O)-, -NHC(O)-, -NHC(O)O-, -O-P(O)(OH)O-, -O-, -S-, -S-S-, and -C(O)-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Zcis absent. According to some embodiments, Zcis -C(O)O-.

[0171] According to some embodiments, each one of Za, Zb, and Zcis independently selected from the group consisting of absent, -C(O)O-, -C(O)NH-, -OC(O)O-, -OC(O)NH-, -OC(O)-, -NHC(O)-, -NHC(O)O-, -O-P(O)(OH)O-, -O-, -S-, -S-S-, and -C(O)-. Each possibility represents a separate embodiment of the invention. According to some embodiments, each one of Za, Zb, and Zcis independently selected from the group consisting of absent, -C(O)O-, and -C(O)-. According to some embodiments, each one of Za, Zb, and Zcis independently selected from the group consisting of absent, and -C(O)O-. According to some embodiments, each one of Za, Zb, and Zcis independently absent. According to some embodiments, each one of Za, Zb, and Zcis independently -C(O)O-.

[0172] According to some embodiments, Zais the same as Zb. According to some embodiments, Zais the same as Zc. According to some embodiments, Zaand Zcare the same and are different from Zb. It is to be understood that the term “the same” means that the specified Z substituents have the same chemical structure whereas the term “different” refers to chemical structures that are not the same (e.g., -C(O)O- and -OC(O)-). For example, Lipid 104, shown herein, has the same Zaand Zb, each of which is -C(O)O-. According to some embodiments, each one of Zaand Zb is independently selected from the group consisting of: absent, -C(O)O-, and -C(O)-. Each possibility represents a separate embodiment of the invention. According to some embodiments, each one of Zaand Zb is independently absent. According to some embodiments, each one of Zaand Zb is independently -C(O)O-. According to some embodiments, each one of Zaand Zb is independently -C(O)-. In another example, Lipid 101, shown herein, has the same Zaand Zc, each of which is absent. Also in Lipid 101, Zb is -C(O)O-, which is different from Zaand Zc. According to some embodiments, each one of Zaand Zcis independently absent. According to some embodiments, each one of Zaand Zcis independently absent and Zb is -C(O)O-.

[0173] According to some embodiments, Lais selected from the group consisting of: alkyl, alkenyl, alkynyl, alkylene-NRlaR2a, alkenylene-NR1aR2a, alkynylene-NRlaR2a, alkylene-OR3a, alkenylene-OR3a, and alkynylene-OR3a. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lais selected from the group consisting of: alkyl, alkenyl, alkynyl, (CH2CH2O)n3R3a, alkylene-Zea-R3a, alkylene-Zea-NRlaR2a, and alkylene-N(alkylene-Zea-R3a)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lais selected from the group consisting of: C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl, (CH2CH2O)n3R3a, Co-12 alkyl ene-Zea-R3a, Co-12 alkylene-Zea-NRlaR2a, and Co-12 alkylene-N(Co-i2 alkylene-Zea-R3a)2. According to some embodiments, Lais selected from the group consisting of: C1-20 alkyl, C2-20 alkenyl, Co-6 alkylene-NRlaR2a, (CH2CH2O)n3R3a, Co-12 alkylene-Zea-R3a, Co-12 alkylene-Zea-NRlaR2a, and Co-12 alkylene-N(Co-i2 alkylene-Zea-R3a)2.

[0174] According to some embodiments, Lais an alkyl. According to some embodiments, Lais a linear alkyl or a branched alkyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lais a linear alkyl. According to some embodiments, Lais an unsubstituted alkyl. According to some embodiments, Lais C1-20 alkyl. According to some embodiments, Lais C4-18 alkyl. According to some embodiments, Lais C8-14alkyl. According to some embodiments, Lais C9-12 alkyl. According to some embodiments, Lais -(CH2)11CH3or -CH[(CH2)5CH3](CH2)4CH3. According to some embodiments, Lais -(CH2)11CH3. According to some embodiments, Lais -CH[(CH2)5CH3](CH2)4CH3.

[0175] According to some embodiments, Lais an alkenyl. According to some embodiments, Lais a linear alkenyl or a branched alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lais a linear alkenyl. According to some embodiments, Lais an unsubstituted alkenyl. According to some embodiments, Lais an monoenyl. According to some embodiments, Lais C2-20monoenyl. According to some embodiments, Lais C4-18 monoenyl. According to some embodiments, Lais Cio-18 monoenyl. According to some embodiments, Lais C15-18 monoenyl. According to some embodiments, Lais a dienyl. According to some embodiments, Lais C2-20dienyl. According to some embodiments, Lais C4-18 dienyl. According to some embodiments, Lais Cio-18 dienyl. According to some embodiments, Lais C15-18 dienyl. According to some embodiments, Lais -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3.

[0176] According to some embodiments, Lais alkylene-Zea-NRlaR2a. According to some embodiments, Zeais absent. According to some embodiments, Lais an alkylene-NRlaR2a. According to some embodiments, Lais a linear or branched alkylene-Zea-NRlaR2a. According to some embodiments, Lais a linear or branched alkylene-NRlaR2a. It is to be understood that linear or branched alkylene-NRlaR2aor alkylene-Zea-NRlaR2ameans a linear or branched alkylene, which is bonded to Zea-NRlaR2aor an NRlaR2agroup. According to some embodiments, Lais a linear alkylene-Zea-NRlaR2a. According to some embodiments, Lais a linear alkylene-NRlaR2a. According to some embodiments, Lais Co-12 alkylene-Zea-NRlaR2a. According to some embodiments, Lais Co-6 alkylene-NRlaR2a. It is to be understood that Co alkylene-Zea-NRlaR2arefers to -Zea-NRlaR2aand C0alkylene-NR1aR2arefers to -NR1aR2a. According to some embodiments, Lais selected from the group consisting of:, -CH2CH2-NRlaR2a, -(CH2)3-NRlaR2a, -(CH2)4-NRlaR2a. Each possibility represents a separate embodiment. According to some embodiments, Lais selected from the group consisting of:, -CH2(CH2)7CH2-Zea-NRlaR2a, -CH2CH2-Zea-NRlaR2a, -(CH2)3-Zea-NRlaR2a, -(CH2)4-Zea-NRlaR2a, -CH2CH2-NRlaR2a, -(CH2)3-NRlaR2a, and -(CH2)4-NRlaR2a. Each possibility represents a separate embodiment. According to some embodiments, Lais CH2(CH2)7CH2-Zea-NRlaR2a. According to some embodiments, Lais -CH2CH2-Zea-NRlaR2a. According to some embodiments, Lais -(CH2)3-Zea-NRlaR2a. According to some embodiments, Lais -(CH2)4-Zea-NRlaR2a. According to some embodiments, Lais -CH2CH2-NRlaR2a. According to some embodiments, Lais -(CH2)3-NRlaR2a. According to some embodiments, Lais -(CH2)4-NRlaR2a. According to some embodiments, Laincludes a monoethylene glycol or polyethylene glycol (PEG). According to some embodiments, Lais (CH2CH2O)n3R3a, wherein n3 is an integer. According to some embodiments, Lais (CH2CH2O)n3R3a, wherein n3 is an integer in the range of 0 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 45, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 30, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 15, including each integer value within the specified range. n3 is an integer in the range of 1 to 10, including each integer value within the specified range. n3 is an integer in the range of 1 to 5, including each integer value within the specified range. According to some embodiments, n3 is 1. According to some embodiments, n3 is 2. According to some embodiments, Lais -CH2CH2OR3a. According to some embodiments, Lais -CH2CH2OCH2CH2OR3a. According to some embodiments, Lais -CH2CH2OCH2CH2OCH2CH2OR3a

[0177] According to some embodiments, Lais alkylene-Zea-R3a. According to some embodiments, Lais Co-12 alkylene-Zea-R3a. According to some embodiments, Lais C0-9 alkylene-Zea-R3a. According to some embodiments, Lais Co-6 alkylene-Zea-R3a.

[0178] According to some embodiments, Lais alkylene-N(alkylene-Zea-R3a)2. According to some embodiments, Lais Co-12 alkylene-N(Co-i2-alkylene-Zea-R3a)2. According to some embodiments, Lais C0-9 alkylene-N(Co-i2 alkylene-Zea-R3a)2. According to some embodiments, Lais Co-6 alkylene-N(Co-i2 alkyl ene-Zea-R3a)2. According to some embodiments, Lais Co-12 alkylene-N(Co-9 alkylene-Zea-R3a)2. According to some embodiments, Lais Co-12 alkylene-N(Co-6 alkylene-Zea-R3a)2. According to some embodiments, Lais C0-9 alkylene-N(Co-9 alkylene-Zea-R3a)2. According to some embodiments, Lais Co-6 alkylene-N(Co-6 alkylene-Zea-R3a)2.

[0179] According to some embodiments, each one of Rlaand R2ais independently selected from the group consisting of: H, alkyl, alkenyl and alkynyl. According to some embodiments, each one of Rlaand R2ais independently C1-12 alkyl. According to some embodiments, each one of Rlaand R2ais independently C4-12 alkyl. According to some embodiments, each one of R1aand R2ais independently -(CH2)11CH3. According to some embodiments, each one of Rlaand R2ais independently -(CH2)5CH3.

[0180] According to some embodiments, Rlais selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Rlais selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl. According to some embodiments, Rlais a linear alkyl or a branched alkyl. According to some embodiments, Rlais a linear alkyl. According to some embodiments, Rlais an unsubstituted alkyl. According to some embodiments, Rlais C1-12 alkyl. According to some embodiments, Rlais C4-12 alkyl. According to some embodiments, R1ais -(CH2)11CH3. According to some embodiments, Rlais (CH2)5CH3.

[0181] According to some embodiments, R2ais selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2ais selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl. According to some embodiments, R2ais a linear alkyl or a branched alkyl. According to some embodiments, R2ais a linear alkyl. According to some embodiments, R2ais an unsubstituted alkyl. According to some embodiments, R2ais C1-12 alkyl. According to some embodiments, R2ais C4-12 alkyl. According to some embodiments, R2ais -(CH2)11CH3. According to some embodiments, R2ais (CH2)5CH3. According to some embodiments, Rlaand R2a, together with the nitrogen to which they are bound, form a heterocycle.

[0182] According to some embodiments, R3ais selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment. According to some embodiments, R3ais selected from the group consisting of: H, alkyl, alkenyl alkynyl, alkylene-N(alkyl)2 and P(0)(0H)2. Each possibility represents a separate embodiment. According to some embodiments, R3ais selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl, C1-12 alkynyl, Co-12 alkylene-N(Ci-i2alkyl)2 and P(0)(0H)2. According to some embodiments, R3ais -CH2CH2-N((CH2)5CH3)2.

[0183] According to some embodiments, each one of Rlband R2bis independently selected from the group consisting of: H, alkyl, alkenyl and alkynyl. According to some embodiments, each one of Rlband R2bis independently C1-12 alkyl. According to some embodiments, each one of Rlband R2bis independently C4-12 alkyl. According to some embodiments, each one of Rlband R2bis independently -(CH2)nCH3. According to some embodiments, each one of Rlband R2bis independently -(CH2)5CH3.

[0184] According to some embodiments, Rlbis selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Rlbis selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl. According to some embodiments, Rlbis a linear alkyl or a branched alkyl. According to some embodiments, Rlbis a linear alkyl. According to some embodiments, Rlbis an unsubstituted alkyl. According to some embodiments, Rlbis C1-12 alkyl. According to some embodiments, Rlbis C4-12 alkyl. According to some embodiments, Rlbis -(CH2)nCH3 or -(CH2)5CH3. According to some embodiments, Rlbis -(CE^iiCFE. According to some embodiments, Rlbis -(CH2)5CH3.

[0185] According to some embodiments, R2bis selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2bis selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl. According to some embodiments, R2bis a linear alkyl or a branched alkyl. According to some embodiments, R2bis a linear alkyl. According to some embodiments, R2bis an unsubstituted alkyl. According to some embodiments, R2bis C1-12 alkyl. According to some embodiments, R2bis C4-12 alkyl. According to some embodiments, R2bis -(CH2)11CH3or -(CH2)5CH3. According to some embodiments, R1bis -(CH2)11CH3. According to some embodiments, R1bis -(CH2)5CH3. According to some embodiments, Rlband R2b, together with the nitrogen to which they are bound, form a heterocycle.

[0186] According to some embodiments, R3bis selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment. According to some embodiments, R3bis selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl. According to some embodiments, R3bis selected from the group consisting of: H, alkyl, alkenyl alkynyl, alkylene-N(alkyl)2 and P(0)(0H)2. Each possibility represents a separate embodiment. According to some embodiments, R3bis selected from the group consisting of: H, C1-12 alkyl, Ci-12 alkenyl, C1-12 alkynyl, Co-12 alkylene-N(Ci-i2alkyl)2 and P(0)(0H)2. According to some embodiments, R3bis -CH2CH2-N((CH2)5CH3)2. According to some embodiments, R3bis -CH2CH2-N(CH3)2. For example, in Lipid 171, R3bis -CH2CH2-N(CH3)2.

[0187] According to some embodiments, each one of Rlcand R2cis independently selected from the group consisting of: H, alkyl, alkenyl and alkynyl. According to some embodiments, each one of Rlcand R2Cis independently C1-12 alkyl. According to some embodiments, each one of Rlcand R2cis independently C4-12 alkyl. According to some embodiments, each one of R1cand R2cis independently -(CH2)5CH3.

[0188] According to some embodiments, Rlcis selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Rlcis selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl. According to some embodiments, Rlcis a linear alkyl or a branched alkyl. According to some embodiments, Rlcis a linear alkyl. According to some embodiments, Rlcis an unsubstituted alkyl. According to some embodiments, Rlcis C1-12 alkyl. According to some embodiments, R1cis -(CH2)5CH3.

[0189] According to some embodiments, R2cis selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2cis selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl. According to some embodiments, R2cis a linear alkyl or a branched alkyl. According to some embodiments, R2cis a linear alkyl. According to some embodiments, R2cis an unsubstituted alkyl. According to some embodiments, R2cis C1-12 alkyl. According to some embodiments, R2cis -(CH2)5CH3. According to some embodiments, Rlcand R2c, together with the nitrogen to which they are bound, form a heterocycle.

[0190] According to some embodiments, R3cis selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment. According to some embodiments, R3Cis selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl. According to some embodiments, R3bis selected from the group consisting of: H, alkyl, alkenyl alkynyl, alkylene-N(alkyl)2 and P(0)(0H)2. Each possibility represents a separate embodiment. According to some embodiments, R3cis selected from the group consisting of: H, C1-12 alkyl, Ci-12 alkenyl, C1-12 alkynyl, Co-12 alkylene-N(Ci-i2alkyl)2 and P(0)(0H)2. According to some embodiments, Rlaand R2aare the same. For example, in Lipid 101, shown herein, each one of Rlaand R2ais -(CH2)11CH3.

[0191] According to some embodiments, Rlband R2bare the same. For example, in Lipid 101, shown herein, each one of Rlband R2bis -(CH2)5CH3.

[0192] According to some embodiments, Rlcand R2care the same. For example, in Lipid 115, shown herein, each one of Rlband R2bis -(CH2)5CH3.

[0193] According to some embodiments, Rlaand R2ais the same as Rlband R2b. For example, in Lipid 105, shown herein, each one of Rlaand R2ais -(CH2)11CH3and each one of Rlband R2bis - (CH2)IICH3. According to some embodiments, Rlaand R2ais the same as Rlcand R2c. For example, in Lipid 113, shown herein, each one of Rlaand R2ais -(CH2)5CH3and each one of Rlband R2bis -(CH2)5CH3.

[0194] According to some embodiments, Zeais selected from the group consisting of: absent,

[0195] o0O O O O

[0196] 0 o

[0197] / II \11O

[0198]

[0199] °7, OH ’ W \ ■ \andA < Each possibility represents a separate embodiment. According to some embodiments, Zeais selected from the group consisting of: absent and -C(O)O-. According to some embodiments, Zeais absent. According to some embodiments, Zeais -C(O)O-.

[0200] According to some embodiments, Zebis selected from the group consisting of: absent,

[0201] o0 02 o O

[0202] ° o / H \ " °

[0203] -dfos

[0204]

[0205] represents a separate embodiment. According to some embodiments, Zebis selected from the group consisting of: absent, -C(O)O-, and -O-. According to some embodiments, Zebis absent. According to some embodiments, Zebis -C(O)O-.

[0206] According to some embodiments, Zecis selected from the group consisting of: absent,

[0207] ° O

[0208] Ha-dAEach possiblllty

[0209]

[0210] represents a separate embodiment. According to some embodiments, Zecis selected from the group consisting of: absent, and -C(O)O-. According to some embodiments, Zecis absent. According to some embodiments, Zecis -C(O)O-.

[0211] According to some embodiments, Lais selected from the group consisting of: -(CH2)nCH3, -CH2CH2-N(CH2(CH2)10CH3)2, -CH[(CH2)5CH3](CH2)4CH3, -(CH2)4-N((CH2)IICH3)2, -(CH2)3-N((CH2)HCH3)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lais selected from the group consisting of: -N((CH2)5CH3)2, -(CH2)11CH3, -CH2CH2-N(CH2(CH2)10CH3)2, -CH[(CH2)5CH3](CH2)4CH3, -(CH2)4-N((CH2)11CH3)2, -(CH2)3-N((CH2)11CH3)2, -CH2CH2-N(CH2(CH2)5CH3)2, CH2CH2OCH2CH2N((CH2)5CH3)2, -CH2(CH2)7CH2C(O)O-N((CH2)5CH3)2,

[0212] and -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3. According to some embodiments, Lais selected from the group consisting of: -N((CH2)5CH3)2, -(CH2)11CH3, and -CH2CH2-N(CH2(CH2)10CH3)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lais -N((CH2)5CH3)2. According to some embodiments, Lais -(CH2)nCH3. According to some embodiments, Lais -CH2CH2-N(CH2(CH2)10CH3)2. According to some embodiments, Lais -CH[(CH2)5CH3](CH2)4CH3. According to some embodiments, Lais -(CH2)4-N((CH2)11CH3)2. According to some embodiments, Lais -(CH2)3-N((CH2)11CH3)2. According to some embodiments, Lais -CH2CH2-N(CH2(CH2)5CH3)2. According to some embodiments, Lais -CH2CH2OCH2CH2N((CH2)5CH3)2. According to some embodiments, Lais -CH2(CH2)7CH2C(O)O-N((CH2)5CH3)2. According to some embodiments, Lais -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3.

[0213] According to some embodiments, Lb is selected from the group consisting of: alkyl, alkenyl, alkynyl, alkylene-NR1R2, alkenylene-NR1bR2b, alkynylene-NR1bR2b, alkylene-OR3, alkenylene-OR3b, and alkynylene-OR3b. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lb is selected from the group consisting of: alkyl, alkenyl, alkynyl, (CH2CH2O)n3R3b, alkylene-Zeb-R3b, alkylene-Zeb-NR1bR2b, and alkylene-N(alkylene-Zeb-R3b)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lb is selected from the group consisting of: C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl, (CH2CH2O)n3R3b, C0-12alkylene-Zeb-R3b, C0-12alkylene-Zeb-NR1bR2b, and C0-12alkylene-N(C0-12alkylene-Zeb-R3b)2. According to some embodiments, Lb is selected from the group consisting of: C1-20alkyl, C2-20alkenyl, C0-6alkylene-NR1bR2b, (CH2CH2O)n3R3b, C0-12alkylene-Zeb-R3b, C0-12alkylene-Zeb-NR1bR2b, and C0-12alkylene-N(C0-12alkylene-Zeb-R3b)2.

[0214] According to some embodiments, Lb is an alkyl. According to some embodiments, Lb is a linear alkyl or a branched alkyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lb is a linear alkyl. According to some embodiments, Lb is an unsubstituted alkyl. According to some embodiments, Lb is C1-20 alkyl. According to some embodiments, Lb is C4-18 alkyl. According to some embodiments, Lb is C8-14alkyl. According to some embodiments, Lb is C9-12 alkyl. According to some embodiments, Lb is -CH[(CH2)5CH3](CH2)4CH3.

[0215] According to some embodiments, Lb is an alkenyl. According to some embodiments, Lb is a linear alkenyl or a branched alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lb is a linear alkenyl. According to some embodiments, Lb is an unsubstituted alkenyl. According to some embodiments, Lb is an monoenyl. According to some embodiments, Lais C2-20monoenyl. According to some embodiments, Lb is C4-18 monoenyl. According to some embodiments, Lb is Cio-18 monoenyl. According to some embodiments, Lb is C15-18 monoenyl. According to some embodiments, Lb is a dienyl. According to some embodiments, Lais C2-20dienyl. According to some embodiments, Lb is C4-18 dienyl. According to some embodiments, Lb is Cio-18 dienyl. According to some embodiments, Lb is C15-18 dienyl. According to some embodiments, Lb is alkylene-Zeb-NRlbR2b. According to some embodiments, Zebis absent. According to some embodiments, Lb is an alkylene-NRlbR2b. According to some embodiments, Lb is a linear or branched alkylene-Zeb-NRlbR2b. According to some embodiments, Lb is a linear or branched alkylene-NR1R2. Each possibility represents a separate embodiment of the invention. It is to be understood that linear or branched alkylene-NR1R2or alkylene-Zeb-NR1bR2bmeans a linear or branched alkylene, which is bonded to an NR1R2group. According to some embodiments, Lb is a linear alkylene-Zeb-NRlbR2b. According to some embodiments, Lb is a linear alkylene-NRlbR2b. According to some embodiments, Lais C0-12alkylene-Zeb-NR1bR2b. According to some embodiments, Lb is Co-6 alkylene-NRlbR2b. It is to be understood that Co alkylene-Zeb-NRlbR2brefers to -Zeb-NRlbR2band that Co alkylene-NRlbR2brefers to -NRlbR2b. According to some embodiments, Lb is selected from the group consisting of: -CH2CH2-NRlbR2b, -NRlbR2b, -(CH2)4-NRlbR2band -(CH2)3-NRlbR2b. Each possibility represents a separate embodiment. According to some embodiments, Lb is selected from the group consisting of: -Zeb-NRlbR2b, -CH2CH2-Zeb-NRlbR2b, -(CH2)3-Zeb-NRlbR2b, and -(CH2)4-Zeb-NRlbR2b. Each possibility represents a separate embodiment. According to some embodiments, Lb is -Zeb-NRlbR2b. According to some embodiments, Lb is -CH2CH2-Zeb-NRlbR2b. According to some embodiments, Lb is -(CH2)3-Zeb-NR1bR2b. According to some embodiments, Lb is -(CH2)4-Zeb-NRlbR2b. According to some embodiments, Lb is -CH2CH2-NRlbR2b. According to some embodiments, Lb is. According to some embodiments, Lb is -NRlbR2b. According to some embodiments, Lb is -(CH2)4-NRlbR2b. According to some embodiments, Lb is -(CH2)3-NRlbR2b. According to some embodiments, Lb includes a monoethylene glycol or polyethylene glycol (PEG). According to some embodiments, Lb is (CH2CH2O)n3R3b, wherein n3 is an integer. According to some embodiments, Lb is (CH2CH2O)n3R3b, wherein n3 is an integer in the range of 0 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 45, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 30, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 15, including each integer value within the specified range. n3 is an integer in the range of 1 to 10, including each integer value within the specified range. n3 is an integer in the range of 1 to 5, including each integer value within the specified range. According to some embodiments, n3 is 1. According to some embodiments, n3 is 2. According to some embodiments, Lb is -CH2CH2OR3b. According to some embodiments, Lb is -CH2CH2OCH2CH2OR3b. According to some embodiments, Lb is -CH2CH2OCH2CH2OCH2CH2OR3b. According to some embodiments, Lb is -(CH2CH2O)sR3bwherein R3bis CH2CH2N(CH3)2, as shown, for example, in Lipid 172. According to some embodiments, Lb is alkylene-Zeb-R3b. According to some embodiments, Lb is Co-12 alkylene-Zeb-R3b. According to some embodiments, Lb is C0-9 alkylene-Zeb-R3b. According to some embodiments, Lais C0-6alkylene-Zeb-R3b.

[0216] According to some embodiments, Lb is alkylene-N(alkylene-Zeb-R3b)2. According to some embodiments, Lb is Co-12 alkylene-N(Co-i2 alkylene-Zeb-R3b)2. According to some embodiments, Lb is Co-12 alkylene-N(Co-i2 alkylene-Zeb-R3b)2. According to some embodiments, Lb is C0-9 alkylene-N(Co-i2 alkylene-Zeb-R3b)2. According to some embodiments, Lb is Co-6 alkylene-N(Co-i2 alkylene-Zeb-R3b)2. According to some embodiments, Lb is Co-12 alkylene-N(Co-9 alkylene-Zeb-R3b)2. According to some embodiments, Lb is Co-12 alkylene-N(Co-6 alkylene-Zeb-R3b)2. According to some embodiments, Lb is C0-9 alkylene-N(Co-9 alkylene-Zeb-R3b)2. According to some embodiments, Lb is C0-6alkylene-N(C0-6alkylene-Zeb-R3b)2.

[0217] According to some embodiments, Lb is selected from the group consisting of: -CH2CH2-N(CH2(CH2)10CH3)2, -N((CH2)5CH3)2, -(CH2)4-N((CH2)IICH3)2, -(CH2)3-N((CH2)I ICH3)2, and -CH[(CH2)SCH3](CH2)4CH3. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lb is selected from the group consisting of: -CH2CH2-N(CH2(CH2)10CH3)2, -CH2CH2OCH2CH2N((CH2)5CH3)2, -N((CH2)5CH3)2, -(CH2)4-N((CH2)11CH3)2, -(CH2)3-N((CH2)11CH3)2, -CH[(CH2)5CH3](CH2)4CH3, and -CH2CH2-N((CH2)5CH3)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lb is selected from the group consisting of: -CH2CH2-N(CH2(CH2)10CH3)2and -CH2CH2OCH2CH2N((CH2)5CH3)2. According to some embodiments, Lb is -CH2CH2-N(CH2(CH2)10CH3)2. According to some embodiments, Lb is -N((CH2)5CH3)2. According to some embodiments, Lb is -(CH2)4-N((CH2)11CH3)2. According to some embodiments, Lb is -(CH2)3-N((CH2)11CH3)2. According to some embodiments, Lb is -CH[(CH2)5CH3](CH2)4CH3. According to some embodiments, Lb is -CH2CH2-N((CH2)5CH3)2. According to some embodiments, Lb is -CH2CH2OCH2CH2N((CH2)5CH3)2.

[0218] According to some embodiments, Lcis selected from the group consisting of: alkyl, alkenyl, alkynyl, alkylene-NR1cR2c, alkenylene-NR1cR2c, alkynylene-NR1cR2c, alkylene-OR3c, alkenylene-OR3c, and alkynylene-OR3c. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lcis selected from the group consisting of: alkyl, alkenyl, alkynyl, (CH2CH2O)n3R3c, alkylene-Zec-R3c, alkylene-Zec-NR1aR2a, and alkylene-N(alkylene-Zec-R3c)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lcis selected from the group consisting of: Ci-20 alkyl, C2-20 alkenyl, C0-6alkylene-NR1R2, C1-3alkylene-OR3, (CH2CH2O)n3R3c, C0-12alkylene-Zec-R3c, C0-12alkylene-Zec-NR1cR2c, and C0-12alkylene-N(C0-12alkylene-Zec-R3c)2. According to some embodiments, Lcis an alkyl. According to some embodiments, Lcis a linear alkyl or a branched alkyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lcis a linear alkyl. According to some embodiments, Lcis an unsubstituted alkyl. According to some embodiments, Lcis C1-20 alkyl. Lcis C4-18 alkyl. According to some embodiments, Lcis C8-14alkyl. According to some embodiments, Lcis C9-12 alkyl. According to some embodiments, Lcis -(CH2)11CH3.

[0219] According to some embodiments, Lcis an alkenyl. According to some embodiments, Lcis a linear alkenyl or a branched alkenyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lcis a linear alkenyl. According to some embodiments, Lcis an unsubstituted alkenyl. According to some embodiments, Lcis an monoenyl. According to some embodiments, Lcis C2-20 monoenyl. According to some embodiments, Lcis C4-18 monoenyl. According to some embodiments, Lcis C10-18monoenyl. According to some embodiments, Lcis C15-18monoenyl. According to some embodiments, Lcis a dienyl. According to some embodiments, Lcis C2-20dienyl. According to some embodiments, Lcis C4-18dienyl. According to some embodiments, Lcis Cio-18 dienyl. According to some embodiments, Lcis C15-18 dienyl. According to some embodiments, Lcis -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3.

[0220] According to some embodiments, Lcis alkylene-Zec-NRlcR2c. According to some embodiments, Zecis absent. According to some embodiments, Lcis an alkylene-NRlcR2c. According to some embodiments, Lais a linear or branched alkylene-Zec-NR1cR2c. According to some embodiments, Lcis a linear or branched alkylene-NRlcR2c. It is to be understood that linear or branched alkylene-NR1cR2cor alkylene-Zec-NR1cR2cmeans a linear or branched alkylene, which is bonded to Zec-NR1cR2cor an NR1cR2cgroup. According to some embodiments, Lcis a linear alkylene-Zec-NR1cR2c. According to some embodiments, Lcis a linear alkylene-NRlcR2c. According to some embodiments, Lcis Co-12 alkylene-Zec-NRlcR2c. According to some embodiments, Lcis Co-6 alkylene-NRlcR2c. It is to be understood that Co alkylene-Zec-NRlcR2crefers to -Zec-NRlcR2cand Co alkylene-NRlcR2crefers to -NRlcR2c. According to some embodiments, Lcis -Zec-NRlcR2c. According to some embodiments, Lcincludes a monoethylene glycol or polyethylene glycol (PEG). According to some embodiments, Lcis (CH2CH2O)n3R3c, wherein n3 is an integer. According to some embodiments, Lcis (CH2CH2O)n3R3c, wherein n3 is an integer in the range of 0 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 50, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 45, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 30, including each integer value within the specified range. According to some embodiments, n3 is an integer in the range of 1 to 15, including each integer value within the specified range. n3 is an integer in the range of 1 to 10, including each integer value within the specified range. n3 is an integer in the range of 1 to 5, including each integer value within the specified range. According to some embodiments, n3 is 1. According to some embodiments, n3 is 2. According to some embodiments, Lcis -CH2CH2OR3c. According to some embodiments, Lcis -CH2CH2OCH2CH2OR3c. According to some embodiments, Lcis -CH2CH2OCH2CH2OCH2CH2OR3c. According to some embodiments, Lcis alkylene-Zec-R3c. According to some embodiments, Lcis Co-12 alkylene-Zec-R3c. According to some embodiments, Lcis C0-9 alkyl ene-Zec-R3c. According to some embodiments, Lcis Co-6 alkylene-Zec-R3c.

[0221] According to some embodiments, Lcis alkylene-N(alkylene-Zec-R3c)2. According to some embodiments, Lcis Co-12 alkyl ene-N(Co- 12 alkylene-Zec-R3c)2. According to some embodiments, Lcis C0-9 alkyl ene-N(Co- 12 alkylene-Zec-R3c)2. According to some embodiments, Lcis Co-6 alkylene-N(Co-i2 alkylene-Zec-R3c)2. According to some embodiments, Lcis Co-12 alkylene-N(Co-9 alkylene-Zec-R3c)2. According to some embodiments, Lcis Co-12 alkylene-N(Co-6 alkylene-Zec-R3c)2. According to some embodiments, Lcis C0-9 alkylene-N(Co-9 alkylene-Zec-R3c)2. According to some embodiments, Lcis C0-6alkylene-N(C0-6alkylene-Zec-R3c)2. According to some embodiments, Lcis an alkylene-OH. According to some embodiments, Lcis a linear or branched alkylene-OH. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lcis a linear alkylene-OH. According to some embodiments, Lcis an unsubstituted alkylene-OH. According to some embodiments, Lcis Co-6 alkylene-OH. It is to be understood that Co alkylene-OH refers to hydroxyl group.

[0222] According to some embodiments, Lcis selected from the group consisting of: -N((CH2)5CH3)2, -(CH2)11CH3, and -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3. Each possibility represents a separate embodiment. According to some embodiments, Lcis -(CH2)11CH3. According to some embodiments, Lcis -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3. According to some embodiments, Lcis -N((CH2)5CH3)2.

[0223] According to some embodiments, Lais the same as Lb. According to some embodiments, Lais is different from Lb. According to some embodiments, Lais the same as Lc. According to some embodiments, Laand Lcare different from Lb. It is to be understood that the term “the same” means that the specified L substituents have the same chemical structure whereas the term “different” refers to chemical structures that are not the same (e.g., -CH2CH2CH2CH2-and -CH(CH3)CH2CH2-). For example, Lipid 105, shown herein, has the same Laand Lb, each of which is CH2CH2-N(CH2(CH2)10CH3)2. In another example, Lipid 101, shown herein, has the same Laand Lc, each of which is -(CH2)nCH3. Also in Lipid 101, Lb is different from Laand Lc. In yet another example, in Lipid 112, shown herein, Lais different from Lb. According to some embodiments, each one of Laand Lb is independently selected from the group consisting of: -CH2CH2-N(CH2(CH2)10CH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -(CH2)3-N(CH2(CH2)10CH3)2, and -(CH2)4-N(CH2(CH2)10CH3)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, each one of Laand Lb is independently selected from the group consisting of: -CH2CH2N(CH2(CH2)10CH3)2, -CH2CH2OCH2CH2N((CH2)5CH3)2, -CH2CH2N((CH2)5CH3)2-CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -(CH2)3-N(CH2(CH2)10CH3)2, and -(CH2)4-N(CH2(CH2)10CH3)2. According to some embodiments, each one of Laand Lcis independently selected from the group consisting of: -N((CH2)5CH3)2, -(CH2)11CH3, and -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3. According to some embodiments, each one of Laand Lcis independently -N((CH2)5CH3)2. According to some embodiments, each one of Laand Lc is independently -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3. According to some embodiments, each one of Laand Lcis independently -(CH2)nCH3.

[0224] According to some embodiments, m is 1, 2, 3, 4 or 5. Each possibility represents a separate embodiment of the invention. For example, in Lipid 101, shown herein, m is 1. For example, in Lipid 117, shown herein, m is 2. For example, in Lipid 118, shown herein, m is 4. According to some embodiments, m is 1, 2 or 3. According to some embodiments, m is 1 or 2. According to some embodiments, m is 1. According to some embodiments, m is 2. According to some embodiments, m is 3. According to some embodiments, m is 4. According to some embodiments, m is 5.

[0225] According to some embodiments, each one of nl and n2 is independently 1, 2, 3, 4 or 5. Each possibility represents a separate embodiment. According to some embodiments, nl is 1, 2, 3, 4 or 5. According to some embodiments, n2 is 1, 2, 3, 4 or 5. According to some embodiments, nl is 1. According to some embodiments, n2 is 1. According to some embodiments, nl is the same as n2. According to some embodiments, nl and n2 are each independently 1. According to some embodiments, each one of nl and n2 is independently 1. According to some embodiments, each one of nl and n2 is independently 2. For example, in Lipid 170, shown herein, nl and n2 is each independently 2.

[0226] According to some embodiments, xa-Za-Lais the same as Xb-Zb-Lb. According to some embodiments, xa-Za-Lais the same as xc-Zc-Lc. According to some embodiments, xa-Za-Lais different from Xb-Zb-Lb. According to some embodiments, xa-Za-Laand xc-Zc-Lcare different from Xb-Zb-Lb. It is to be understood that the term “the same” means that the specified x-Z-L substituents have the same chemical structure whereas the term “different” refers to chemical structures that are not the same (e.g., -CH2CH2CH2CH2- and -CH(CH3)CH2CH2-). For example, Lipid 105, shown herein, has the same xa-Za-Laand Xb-Zb-Lb, each of which is -C(0)0-CH2CH2-N(CH2(CH2)IOCH3)2. In another example, Lipid 101, shown herein, has the same xa-Za-Laand xc-Zc-Lc, each of which is -(CH2)nCH3. Also in Lipid 101, xa-Za-Laand xc-Zc-Lcare different from Xb-Zb-Lb. In yet another example, as shown in Lipid 112, xa-Za-Lais different from Xb-Zb-Lb. According to some embodiments, xa-Za-Lais selected from the group consisting of: -(CH2)11CH3, -CH2CH2-N(CH2(CH2)10CH3)2, -(CH2)4-N(CH2(CH2)10CH3)2, -CH2(CH2)8CH2-C(O)O-CH[CH2(CH2)4CH3]CH2(CH2)3CH3,

[0227] -CH2(CH2)7CH2-C(O)O-CH[CH2(CH2)4CH3]CH2(CH2)3CH3,

[0228] -C(O)O-CH2CH2-N(CH2(CH2)10CH3)2,

[0229] and C(O)-(CH2)3-N(CH2(CH2)10CH3)2. Each possibility represents a separate embodiment. According to some embodiments, xa-Za-Lais selected from the group consisting of: -(CH2)nCH3, -CH2CH2-N(CH2(CH2)10CH3)2, -CH2(CH2)7CH2-C(O)O-CH[CH2(CH2)4CH3]CH2(CH2)3CH3,

[0230] -C(O)O-CH2CH2-N(CH2(CH2)10CH3)2, -(CH2)4-N(CH2(CH2)IOCH3)2, C(0)-(CH2)3-N(CH2(CH2)IOCH3)2, -C(O)O-CH2CH2-N((CH2)5CH3)2, -C(O)O-CH2CH2OCH2CH2-N((CH2)5CH3)2,

[0231] -C(O)O-CH2(CH2)7CH2-C(O)O-N((CH2)5CH3)2,

[0232] and -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3. Each possibility represents a separate embodiment.

[0233] According to some embodiments, Xb-Zb-Lb is selected from the group consisting of: -CH2(CH2)7CH2-C(O)O-N((CH2)5CH3)2, -C(O)O-CH2CH2-N(CH2(CH2)10CH3)2, -CH2CH2-N(CH2(CH2)10CH3)2,

[0234] -CH2(CH2)8CH2-C(O)O-CH[CH2(CH2)4CH3]CH2(CH2)3CH3,

[0235] -CH2(CH2)7CH2-C(O)O-CH[CH2(CH2)4CH3]CH2(CH2)3CH3,

[0236] -(CH2)4-N(CH2(CH2)IOCH3)2, and C(O)-(CH2)3-N(CH2(CH2)10CH3)2. Each possibility represents a separate embodiment. According to some embodiments, Xb-Zb-Lb is selected from the group consisting of: -CH2(CH2)7CH2-C(O)O-N((CH2)5CH3)2, -C(O)O-CH2CH2-N(CH2(CH2)10CH3)2, -CH2CH2-N(CH2(CH2)10CH3)2,

[0237] -CH2(CH2)7CH2-C(O)O-CH[CH2(CH2)4CH3]CH2(CH2)3CH3,

[0238] -(CH2)4-N(CH2(CH2)IOCH3)2, C(0)-(CH2)3-N(CH2(CH2)IOCH3)2, -C(O)O-CH2CH2-N((CH2)5CH3)2, and -C(O)O-CH2CH2O-CH2CH2-N((CH2)5CH3)2. Each possibility represents a separate embodiment.

[0239] According to some embodiments, xc-Zc-Lcis selected from the group consisting of: -CH2(CH2)7CH2-C(O)O-N((CH2)5CH3)2, -(CH2)11CH3, and -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3. Each possibility represents a separate embodiment. According to some embodiments, xc-Zc-Lcis -CH2(CH2)7CH2-C(O)O-N((CH2)5CH3)2. According to some embodiments, xc-Zc-Lcis -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3. According to some embodiments, xc-Zc-Lcis -(CH2)11CH3.

[0240] According to some embodiments, each one of xa-Za-Laand xb-Zb-Lb is independently selected from the group consisting of: -CH2CH2-N(CH2(CH2)IOCH3)2, -CH2(CH2)8CH2-C(O)O-CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -C(0)0-CH2CH2-N(CH2(CH2)IOCH3)2, -CH2(CH2)7CH2-C(O)O-CH[CH2(CH2)4CH3]CH2(CH2)3CH3,

[0241] -(CH2)4-N(CH2(CH2)IOCH3)2, and C(0)-(CH2)3-N(CH2(CH2)IOCH3)2. Each possibility represents a separate embodiment. According to some embodiments, each one of xa-Za-Laand xb-Zb-Lb is independently selected from the group consisting of: -CH2CH2-N(CH2(CH2)ioCH3)2, -C(O)O-CH2CH2-N(CH2(CH2)10CH3)2, -CH2(CH2)7CH2-C(O)O-CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -CH2(CH2)7CH2-C(O)O-N((CH2)5CH3)2, -(CH2)4-N(CH2(CH2)IOCH3)2, and C(O)-(CH2)3- N(CH2(CH2)IOCH3)2. Each possibility represents a separate embodiment.

[0242] According to some embodiments, each one of xa-Za-Laand xc-Zc-Lcis independently selected from the group consisting of: -(CH2)nCH3, -CH2(CH2)7CH2-C(O)O-N((CH2)5CH3)2, and CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3. Each possibility represents a separate embodiment. According to some embodiments, each one of xa-Za-Laand xc-Zc-Lcis independently -(CH2)nCH3. According to some embodiments, each one of xa-Za-Laand xc-Zc-Lcis independently -CH2(CH2)7CH2-C(O)O-N((CH2)5CH3)2. According to some embodiments, each one of xa-Za-Laand xc-Zc-Lcis independently CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3.

[0243] The chemical structures of each of the specific lipids are detailed below in the “Exemplary Lipids” Section and in the claims.

[0244] Exemplary Lipids

[0245] Exemplary lipids according to Formula (II), specifically according to Formula (Ila), Formula (lib), Formula (lie), Formula (lid), Formula (lie), as well as exemplary lipids according to Formula (III), specifically according to Formula (Illa) and Formula (Illb) of the present invention are shown below. It is to be understood that, according to some embodiments, the invention is not limited to any one or more of the following exemplary lipids. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: Lipid 101, Lipid 102, Lipid 103, Lipid 104, Lipid 105, Lipid 106, and Lipid 107, Lipid 108, Lipid 109, Lipid 110, Lipid 111, Lipid 112, Lipid 113, Lipid 114, Lipid 115, Lipid 116, Lipid 119, Lipid 122, Lipid 123, Lipid 124, Lipid 125, Lipid 126, Lipid 127, Lipid 128, Lipid 129, Lipid 130, Lipid 131, Lipid 132, Lipid 133, Lipid 134, Lipid 135, Lipid 136, Lipid 137, Lipid 138, Lipid 139, Lipid 140, Lipid 141, Lipid 142, Lipid 143, Lipid 144, Lipid 145, Lipid 146, Lipid 147, Lipid 147, Lipid 149, Lipid 150, Lipid 151, Lipid 152, Lipid 153, Lipid 154, Lipid 155, Lipid 156, Lipid 157, Lipid 158, Lipid 159, Lipid 160, Lipid 161, Lipid 162, Lipid 163, Lipid 164, Lipid 165, Lipid 166, Lipid 167, Lipid 168, Lipid 169, Lipid 170, Lipid 171, Lipid 172, and Lipid 173, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: Lipid 101, Lipid 102, Lipid 103, Lipid 104, Lipid 105, Lipid 106, Lipid 107, Lipid 108, Lipid 109, Lipid 110, Lipid 111, Lipid 112, Lipid 113, Lipid 114, and Lipid 115, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid is Lipid 101. According to some embodiments, the lipid is Lipid 102. According to some embodiments, the lipid is Lipid 103. According to some embodiments, the lipid is Lipid 104. According to some embodiments, the lipid is Lipid 105. According to some embodiments, the lipid is Lipid 106. According to some embodiments, the lipid is Lipid 107. According to some embodiments, the lipid is Lipid 108. According to some embodiments, the lipid is Lipid 109. According to some embodiments, the lipid is Lipid 110. According to some embodiments, the lipid is Lipid 111. According to some embodiments, the lipid is Lipid 112. According to some embodiments, the lipid is Lipid 113. According to some embodiments, the lipid is Lipid 114. According to some embodiments, the lipid is Lipid 115. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: Lipid 101, Lipid 102, Lipid 103, Lipid 104, Lipid 105, Lipid 106, and Lipid 107, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: Lipid 101, Lipid 102, Lipid 103, Lipid 105, Lipid 108, Lipid 108, Lipid 109, Lipid 110, Lipid 111, Lipid 112, Lipid 113, Lipid 114, and Lipid 115, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: Lipid 101, Lipid 102, and Lipid 103, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: Lipid 101, Lipid 102, Lipid 103, Lipid 105, Lipid 108, Lipid 109, Lipid 110, Lipid 111, Lipid 112, Lipid 113, Lipid 114, and Lipid 115, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: Lipid 102, Lipid 110, Lipid 101, Lipid 105, Lipid 111, and Lipid 115. According to some embodiments, the lipid of Formula (II) is selected from the group consisting of: Lipid 102 and Lipid 110.

[0246] According to some embodiments, the lipid of Formula (Ila) is selected from the group consisting of: Lipid 103, Lipid 104, Lipid 105, Lipid 106, and Lipid 107 including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid of Formula (Ila) is selected from the group consisting of: Lipid 103, Lipid 104, Lipid 105, Lipid 106, Lipid 107, Lipid 108, Lipid 109, Lipid 110, and Lipid 112, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0247] According to some embodiments, the lipid of Formula (lib) is Lipid 101 or Lipid 102, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid of Formula (lib) is Lipid 101, Lipid 102, Lipid 113, Lipid 114, and Lipid 115, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0248] According to some embodiments, the lipid of Formula (lie) is selected from the group consisting of: Lipid 101, Lipid 102, Lipid 103, Lipid 105, Lipid 108, Lipid 108, Lipid 109, Lipid 110, Lipid 111, Lipid 112, Lipid 113, Lipid 114, and Lipid 115, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0249] According to some embodiments, the lipid of Formula (lid) is selected from the group consisting of: Lipid 102, Lipid 103, Lipid 105, Lipid 107, Lipid 108, Lipid 109, and Lipid 111, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0250] According to some embodiments, the lipid of Formula (lie) is selected from the group consisting of: Lipid 103 and Lipid 106. Each possibility represents a separate embodiment of the present invention.

[0251] According to some embodiments, the lipid of Formula (III) is selected from the group consisting of: Lipid 117, Lipid 118, Lipid 120, and Lipid 121, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the lipid is Lipid 117. According to some embodiments, the lipid is Lipid 118. According to some embodiments, the lipid is Lipid 120. According to some embodiments, the lipid is Lipid 121.

[0252] According to some embodiments, the lipid of Formula (Illa) is selected from the group consisting of: Lipid 117, Lipid 118, Lipid 120, and Lipid 121, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0253] According to some embodiments, the lipid of Formula (Illb) is selected from the group consisting of: Lipid 117, Lipid 118, Lipid 120, and Lipid 121, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0254] The following exemplary lipids are portrayed as non-limiting examples of the lipids of the present invention. The substituents of each lipid are identified below, sometimes within curly brackets, { }, for identification of most specific embodiments.

[0255] It is to be understood that any lipid or lipid component described herein may potentially fall within the scope of a generic formula (e.g., Formula (II), (Ila), (lib), (lie), (lid), (lie), (III), (Illa), (Illb) etc.) of the present invention in multiple ways, depending on the assignment of various substituents (e.g., xa, Zb, Lc) or structural variations permitted by the formulae. Furthermore, any single lipid or lipid component may satisfy the structural requirements of more than one of the generic formulae or specific structures disclosed herein.

[0256] All such lipids and all possible interpretations and representations of such structures that satisfy the claimed or described generic formulae, whether described specifically or by example, are intended to be encompassed within the scope of the present specification and claims. The following presentation of specific lipids, examples, or structural assignments under a particular formula is merely for illustration and should not be construed as limiting the scope of the present invention to that specific assignment, formula, or illustrative embodiment.

[0257] Specifically, Lipid 101, also known as NV5-006, is shown below,

[0258]

[0259] It is represented by Formula (II), wherein Y is -xc-Zc-Lc; wherein each one of xaand xcis independently Co-12 alkylene {Co alkylene, i.e., absent} and wherein Xb is Co-12 alkylene {C9 alkylene}; wherein each one of Zaand Zcis independently absent and wherein Zb is -C(O)O-; wherein each one of Laand Lcis independently C1-20 alkyl {C12 alkyl} and wherein Lbis C0-12alkylene-Ze-NR1R2{C0alkylene-Ze-NR1R2, i.e., -Ze-NR1R2} wherein Zeis absent and wherein each one of R1and R2is independently C1-12alkyl {C6alkyl}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0260] Specifically, Lipid 102, also known as NV5-007, is shown below,

[0261]

[0262] It is represented by Formula (II), wherein Y is -xc-Zc-Lc; wherein each one of xa, Xb, and xcis independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Zaand Zcis independently absent and wherein Zb is -C(O)O-; wherein each one of Laand Lcis independently C1-20 alkyl {C12 alkyl} and wherein Lbis C0-12alkylene-Ze-NR1R2{C2alkylene-Ze-NR1R2} wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0263] Specifically, Lipid 103, also known as NV5-008, is shown below,

[0264]

[0265] It is represented by Formula (II), wherein Y is H; wherein each one of xaand Xb is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Zaand Zb is independently absent; wherein each one of Laand Lb is independently Co-12 alkylene-Ze-NR1R2{C2 alkylene-Ze-NR1R2} wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0266] Specifically, Lipid 104 is shown below,

[0267]

[0268] It is represented by Formula (II), wherein Y is H; wherein each one of xaand Xb is independently Co- 12 alkylene {C9 alkylene}; wherein each one of Zaand Zb is independently -C(O)O-; wherein each one of Laand Lb is independently C1-20 alkyl {C12 alkyl}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0269] Specifically, Lipid 105, also known as NV5-003, is shown below,

[0270]

[0271] It is represented by Formula (II), wherein Y is H; wherein each one of xaand Xb is independently Co-12 alkylene {Co, absent}; wherein each one of Zaand Zb is independently -C(O)O-, wherein each one of Laand Lb is independently Co-12 alkylene-Ze-NR'R2{C2 alkylene- ZC-NR'R2} wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0272] Specifically, Lipid 106 is shown below,

[0273]

[0274] It is represented by Formula (II), wherein Y is H; wherein each one of xaand Xb is independently Co- 12 alkylene {Co, absent}; wherein each one of Zaand Zb is independently absent; wherein each one of Laand Lb is Co-12 alkylene-Ze-NR'R2{C4 alkylene-Zc-NR'R2} wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0275] Specifically, Lipid 107 is shown below,

[0276]

[0277] It is represented by Formula (II), wherein Y is H; wherein each one of xaand Xb is independently Co-12 alkylene {Co, absent}; wherein each one of Zaand Zb is independently -C(O)-; wherein each one of Laand Lb is independently Co-12 alkylene-Ze-NR^2{C3 alkylene-Ze- NR1R2} wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0278] Specifically, Lipid 108, also known as NV5-001, is shown below,

[0279]

[0280] It is represented by Formula (II), wherein each one of xaand Xb is independently Co-12 alkylene {Co, absent}; wherein each one of Zaand Zb is independently -C(O)O-; wherein each one of Laand Lb is independently Co-12 alkylene-Ze-NR1R2{C2 alkylene-Ze-NR1R2} wherein Zeis absent and wherein each one of R1and R2is independently C1-12alkyl {C6alkyl}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0281] Specifically, Lipid 109, also known as NV5-002, is shown below,

[0282]

[0283] It is represented by Formula (II), wherein Y is H; wherein each one of xaand Xb is independently Co-12 alkylene {Co, absent}; wherein each one of Zaand Zb is independently -C(O)O-; wherein each one of Laand Lbis independently (CH2CH2O)n3R3, wherein n3 is 1 {CH2CH2OR3} and wherein R3is Co-12 alkylene-N(Ci-i2alkyl)2 {C2 alkylene-N(Ce alkyl)2}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0284] Specifically, Lipid 110, also known as NV5-035, is shown below,

[0285]

[0286] 4

[0287] It is represented by Formula (II), wherein Y is H;

[0288] wherein xais Co-12 alkylene {Co, absent}; wherein Zais -C(O)O-; and wherein Lais Co-12 alkylene-Ze- NR'R2{C9 alkyl ene-Zc-NR'R2}, wherein Zeis -C(O)O- and wherein each one of R1and R2is independently C1-12alkyl {C6alkyl};

[0289] wherein Xb is Co-12 alkylene {Co, absent}; wherein Zb is -C(O)O-; and wherein Lb is Co-12 alkylene-Ze- NR'R2{C2 alkyl ene-Zc-NR'R2}, wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl};

[0290] wherein nl and n2 are each independently 1; and wherein m is 1.

[0291] Specifically, Lipid 111, also known as NV5-039, is shown below,

[0292]

[0293] NVILOJS

[0294] It is represented by Formula (II), wherein Y is -xc-Zc-Lc; wherein each one of xa, Xb and xcis independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Zaand Zcis independently absent and wherein Zb is -C(O)O-; wherein each one of Laand Lcis independently C2-20 alkenyl {Cis dienyl} and wherein Lb is Co-12 alkylene-Ze-NR'R2{C2 alkylene-Zc-NR'R2}, wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl}; wherein nl and n2 are each independently 1; and wherein m is 1.

[0295] Specifically, Lipid 112, also known as NV5-040, is shown below,

[0296]

[0297] NVU;J U4:j It is represented by Formula (II), wherein Y is H;

[0298] wherein xais Co-12 alkylene {C9 alkylene}; wherein Zais -C(O)O-; and wherein Lais Co-12 alkylene-Ze-NR1R2{Co alkylene-Ze-NR'R2, i.e., ZC-NR'R2} wherein Zeis absent and wherein each one of R1and R2is independently C1-12alkyl {C6alkyl};

[0299] wherein Xb is Co-12 alkylene {Co alkylene, i.e., absent}; wherein Zb is -C(O)O-; wherein Lb is Co-12 alkylene-Ze-NR1R2{C2 alkylene-Ze-NR1R2}, wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl};

[0300] wherein nl and n2 are each independently 1; and wherein m is 1.

[0301] Specifically, Lipid 113, also known as NV5-041, is shown below,

[0302]

[0303] NVUt: J41 It is represented by Formula (II), wherein Y is -xc-Zc-Lc;

[0304] wherein xais Co-12 alkylene {C9 alkylene}; wherein Zais -C(O)O-; and wherein Lais Co-12 alkylene-Ze-NR1R2{Co alkylene-Ze-NR'R2, i.e., ZC-NR'R2}, wherein Zeis absent and wherein R1and R2are each independently C1-12 alkyl {Ce alkyl};

[0305] wherein Xb is Co-12 alkylene {Co alkylene, i.e., absent}; wherein Zb is -C(O)O-; and wherein Lb is Co-12 alkylene-Ze-NR'R2{C2 alkylene-Ze-NR'R2}, wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl};

[0306] wherein xcis Co-12 alkylene {C9 alkylene}; wherein Zcis -C(O)O-; and wherein Lcis Co-12 alkylene-Ze-NR1R2{Co alkylene-Ze-NR'R2, i.e., ZC-NR'R2}, wherein Zeis absent and wherein R1and R2are each independently C1-12 alkyl {Ce alkyl};

[0307] wherein nl and n2 are each independently 1; and wherein m is 1.

[0308] Specifically, Lipid 114, also known as NV5-042, is shown below,

[0309]

[0310] It is represented by Formula (II), wherein Y is -xc-Zc-Lc;

[0311] wherein xais Co-12 alkylene {C9 alkylene}; wherein Zais -C(O)O-; and wherein Lais Co-12 alkylene-Ze-NR'R2{Co alkyl ene-Zc-NR'R2, i.e., ZC-NR'R2}, wherein Zeis absent and wherein R1and R2are each independently C1-12 alkyl {Ce alkyl};

[0312] wherein Xb is Co-12 alkylene {Co alkylene, i.e., absent}; wherein Zb is -C(O)O-; and wherein Lb is Co-12 alkyl ene-Ze-NR'R2{C2 alkylene-Zc-NR'R2}, wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C12 alkyl};

[0313] wherein xcis Co-12 alkylene {C9 alkylene}; wherein Zcis -C(O)O-; and wherein Lcis Co-12 alkylene-ZC-NR'R2{CO alkyl ene-Ze-NR'R2, i.e., ZC-NR'R2}, wherein Zeis absent and wherein R1and R2are each independently C1-12 alkyl {Ce alkyl};

[0314] wherein nl and n2 are each independently 1; and wherein m is 2.

[0315] Specifically, Lipid 115, also known as NV5-043, is shown below,

[0316]

[0317] It is represented by Formula (II), wherein Y is -xc-Zc-Lc;

[0318] wherein xais Co-12 alkylene {C9 alkylene}; wherein Zais -C(O)O-; and wherein Lais Co-12 alkylene-ZC-NR'R2{CO alkyl ene-Ze-NR'R2, i.e., ZC-NR'R2}, wherein Zeis absent and wherein R1and R2are each independently C1-12 alkyl {Ce alkyl};

[0319] wherein Xb is Co-12 alkylene {Co alkylene, i.e., absent}; wherein Zb is -C(O)O-; and wherein Lb is (CH2CH2O)n3R3, wherein n3 is ^CFLCFLOR3} and wherein R3is Co-12 alkylene-N(Ci-i2alkyl)2 {C2 alkylene-N(Ce alkyl )2 }; wherein xcis Co-12 alkylene {C9 alkylene}; wherein Zcis -C(0)0-; and wherein Lcis Co-12 alkylene-Ze-NR'R2{Co alkyl ene-Zc-NR'R2, i.e., ZC-NR'R2J, wherein Zeis absent and wherein R1and R2are each independently C1-12 alkyl {Ce alkyl};

[0320] wherein nl and n2 are each independently 1; and wherein m is 1.

[0321] According to some embodiments, the lipid is selected from the group selected from: Lipid 101, Lipid 102, Lipid 103, Lipid 104, Lipid 105, Lipid 106, and Lipid 107, Lipid 108, Lipid 109, Lipid 110, Lipid 111, Lipid 112, Lipid 113, Lipid 114, and Lipid 115,

[0322] Lipid 116

[0323] Lipid 117

[0324]

[0325] Lipid 118 .0 N„

[0326] Lipid 119

[0327] Lipid 120

[0328] Lipid 121

[0329] Lipid 122

[0330]

[0331] Lipid 123 Lipid 124

[0332] Lipid 125

[0333] Lipid 126

[0334] Lipid 127

[0335] Lipid 128

[0336]

[0337] Lipid 129 Lipid 130

[0338] Lipid 130 Lipid 131

[0339] Lipid 132

[0340]

[0341] Lipid 133 Lipid 134

[0342] Lipid 135 Lipid 136

[0343]

[0344] Lipid 138 Lipid 139

[0345] Lipid 140

[0346] Lipid 141

[0347]

[0348] Lipid 142 Lipid 144

[0349]

[0350] Lipid 146 Lipid 147

[0351]

[0352] Lipid 150 Lipid 152

[0353] Lipid 153

[0354]

[0355] Lipid 154 Lipid 155

[0356] Lipid 156

[0357]

[0358] Lipid 158

[0359]

[0360] Lipid 161 Lipid 162

[0361] Lipid 163

[0362] Lipid 164

[0363]

[0364] Lipid 165 Lipid 166

[0365] Lipid 167

[0366]

[0367] Lipid 168 Lipid 170

[0368]

[0369] Lipid 172

[0370]

[0371] Lipid 173

[0372] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0373] Chemical Definitions

[0374] The term “cationic lipid”, as used herein refers to lipid species that carries a net positive charge at a selected pH. Selected pH values include, but not limited to physiological pH, pH=7 and the like. It is to be understood by the person having ordinary skill in the art that the lipids of the present invention may be considered as cationic lipids, since they bear 2 or more nitrogen atoms, where these atoms are typically basic and protonizable at the selected pH, so that the lipid may carry a net positive charge. According to some embodiments, the lipid of the present invention is a cationic lipid.

[0375] According to some embodiments, “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glyccro-3- phosphocholine (DPPC), l,2-Dimyristoyl-sn-glyccro-3-phosphocholine (DMPC), l-Palmitoyl-2-olcoyl-sn-glyccro-3 -phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phophatidyl ethanolamines such as 1,2-Diolcoyl-sn-glyccro-3 -phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived. The term “ionizable lipid”, as used herein refers to lipid species that carries a charge at a selected pH. Selected pH values include, but not limited to physiological pH, pH=7 and the like. It is to be understood by the person having ordinary skill in the art that the lipids of Formulae (II), and (III) may be considered as ionizable lipids. An “alkyl” group refers to any saturated aliphatic hydrocarbon, including straight-chain and branched-chain alkyl groups. The term “linear alkyl” refers to a straight chain of methylene groups terminated by CH3, which is substituted in any one of its carbon atoms. Linear alkyls include, but are not limited to –(CH2)7CH3, –(CH2)8CH3, -(CH2)10CH3,

[0376] -(CH2)11CH3and -CH[CH2(CH2)4CH3]CH2(CH2)3CH3. The alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl. The term “Co” connotes absent, i.e., no alkyl chain present. The term " Cn-m alkyl", refers to an alkyl group having n to m carbon atoms. An alkyl group formally corresponds to an alkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologs such as 2- methyl- 1 -butyl, 3 -pentyl, hexyl, 1,2,2-trimethylpropyl and the like. The term "alkylene," employed alone or in combination with other terms, refers to a divalent alkyl linking group. An alkylene group formally corresponds to an alkane with two C-H bonds replaced by points of attachment of the alkylene group to the remainder of the compound. The term " Cn-m alkylene" refers to an alkylene group having n to m carbon atoms. Examples of alkylene groups include, but are not limited to, ethan-1, 2-diyl, ethan-1, 1-diyl, propan-1, 3-diyl, propan-1, 2-diyl, propan-1, 1-diyl, butan-1, 4-diyl, butan-1, 3-diyl, butan-1, 2-diyl, 2-methyl-propan-l, 3-diyl and the like. It is to be understood that Co-alkylene means that the specified substituent is absent. For example, when referring to the substituent C0-4 alkylene-aryl-Co-4 alkylene, if both numerals are 0, the substituent is divalent aryl (e.g., phenylene, C6H4). In various section of the present application ranges of alkyl chains are presented, e.g., C0-4 alkyl, C4-20 alkyl, C4-14 alkyl etc. It is to be understood that such ranges include any sub range thereof, for example, C4-14 alkyl may include and / or be directed to: C4-8 alkyl, C8-14alkyl, Ce-12 alkyl, C9 alkyl etc.

[0377] An "alkenyl" group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond including straight-chain, branched-chain and cyclic alkenyl groups. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, i-butenyl, 3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, cyclohexyl-butenyl and decenyl. The alkenyl group can be unsubstituted or substituted through available carbon atoms with one or more groups defined hereinabove for alkyl. Alkenyls according to the present invention may include more than one carbon-carbon double bond. Thus, dienes and trienes are within the definition of alkenyl. According to some embodiments, the alkenyl is a monoenyl, i.e., an alkenyl, which includes a single carbon-carbon double bond. According to some embodiments, the alkenyl is a dienyl. The term " Cn-m alkenyl", refers to an alkyl group having n to m carbon atoms. An alkenyl group formally corresponds to an alkene with one C-H bond replaced by the point of attachment of the alkenyl group to the remainder of the lipid. Examples of alkenyl moieties include, but are not limited to, chemical groups such as ethenyl, propenyl, isopropenyl, n- butenyl, sec-butenyl the like. The term "alkenylene," employed alone or in combination with other terms, refers to a divalent alkenyl linking group. An alkenylene group formally corresponds to an alkane with two C-H bonds replaced by points of attachment of the alkenylene group to the remainder of the lipid. The term " Cn-m alkenylene" refers to an alkenylene group having n to m carbon atoms. In various section of the present application ranges of alkenyl chains are presented, e.g., C2-8 alkenyl, C4-20 alkenyl etc. It is to be understood that such ranges include any sub range thereof, for example, C4-14 alkenyl may include and / or be directed to: C4-8 alkenyl, C8-14alkenyl, C6-12 alkenyl, C9 alkenyl etc.

[0378] One or more of the lipids of the invention, may be present as a salt. The term "salt" encompasses both basic and acid addition salts, including but not limited to, carboxylate salts, phosphate salts, or salts with amine nitrogen atoms, and include salts formed with the organic and inorganic anions and cations discussed below. Furthermore, the term includes salts that form by standard acid-base reactions with basic groups (such as amino groups) and organic or inorganic acids. Such acids include hydrochloric, hydrofluoric, trifluoroacetic, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids. Each possibility represents a separate embodiment of the invention.

[0379] The term "organic or inorganic cation" refers to counter-ions for the anion of a salt. The counterions include, but are not limited to, alkali and alkaline earth metals (such as lithium, sodium, potassium, barium, aluminum and calcium); ammonium and mono-, di- and tri-alkyl amines such as trimethylamine, cyclohexylamine; and the organic cations, such as dibenzylammonium, benzylammonium, 2 -hydroxy ethylammonium, bis(2-hydroxyethyl)ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations. See, for example, Berge et al., J. Pharm. Sci. (1977), 66:1-19, which is incorporated herein by reference.

[0380] The term “zeta potential” (Q refers to a physical measurement of a colloidal system by electrophoresis. It gives the value of the potential (in mV) of a colloid in a suspension at the boundary between the Stem layer and the diffuse layer. In other words, the zeta potential in a colloidal system is the difference in potential between the immovable layer attached to the surface of the dispersed phase and the dispersion medium. The zeta potential is related to stability of suspensions of particles. Zeta potential may be adjusted, in part, for example, by adjusting the concentration of an electrolyte in the buffer system.

[0381] Particles, Compositions and Uses

[0382] According to some embodiments, the present invention provides a particle comprising the lipid according to the present invention and a membrane stabilizing lipid. Thus, in some aspects, the present invention provides a composition comprising a lipid according to any Formula (II), e.g., any one of Lipid 101 to Lipid 107, and a pharmaceutically acceptable excipient. Each possibility represents a separate embodiment. In other aspects, the present invention provides a composition comprising a lipid according to any Formula (II), e.g., any one of Lipid 101 to Lipid 115, or a lipid according to any Formula (III) and a pharmaceutically acceptable excipient. Each possibility represents a separate embodiment. In other aspects, the present invention provides a composition comprising a lipid according to any Formula (II) or any Formula (III), e.g., any one of Lipid 101 to Lipid 173, and a pharmaceutically acceptable excipient. Each possibility represents a separate embodiment.

[0383] According to some embodiments, there is provided a composition comprising a plurality of particles as discloses herein and a pharmaceutically acceptable carrier, diluent or excipient. According to some embodiments, the composition is a liposomal composition. According to some embodiments, the particles of the present invention are in the form of liposomes. In other embodiments, the composition further comprises one or more components selected from the group consisting of a neutral lipid, a charged lipid, a steroid, and a polymer-conjugated lipid. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the composition is a pharmaceutical composition.

[0384] According to some embodiments, the particle comprises the membrane stabilizing lipid and a lipid membrane comprising the lipid. According to some embodiments, the membrane stabilizing lipid is selected from the group consisting of cholesterol, phospholipids, cephalins, sphingolipids and glycoglycerolipids. According to some embodiments, the membrane stabilizing lipid comprises cholesterol.

[0385] In some embodiments, the membrane stabilizing lipids may be selected from, but not limited to: cholesterol, phospholipids (such as, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerols), cephalins, sphingolipids (sphingomyelins and glycosphingolipids), glycoglycerolipids, and combinations thereof. Each possibility represents a separate embodiment of the present invention. In some embodiments, the phosphatidylethanolamines may be selected from, but not limited to: 1.2-dilauroyl-L-phosphatidyl-ethanolamine (DLPE), 1,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-Diphytanoyl-sn-glycero-3 -phosphoethanolamine (DPhPE) 1.3-Dipalmitoyl-sn-glycero-2-phosphoethanolamine (1,3-DPPE), l-Palmitoyl-3-oleoyl-sn-glycero-2-phosphoethanolamine (1,3-POPE), Biotin-Phosphatidylethanolamine, 1,2-Dimyristoyl-sn-glycero-3 -phosphoethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) or combinations thereof. According to some embodiments, the phosphatidylethanolamine is selected from l,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and l,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). In some embodiments, the Phosphatidylethanolamines may be conjugated to a PEG-Amine derivative. Each possibility represents a separate embodiment of the present invention.

[0386] According to some embodiments, “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-sn-glycero-3 -phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glyccro-3- phosphocholine (DPPC), l,2-Dimyristoyl-sn-glyccro-3-phosphocholine (DMPC), l-Palmitoyl-2-olcoyl-sn-glyccro-3 -phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), phophatidyl ethanolamines such as 1,2-Di oleoyl - sn-glyccro-3 -phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0387] According to some embodiments, the particle further comprising one or more additional components selected from the group consisting of a PEG-lipid conjugate, a neutral lipid and a charged lipid. According to some embodiments, the additional component comprises 1,2-Distearoyl-sn-glycero-3 -phosphocholine (DSPC). According to some embodiments, the additional component comprises 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG). According to some embodiments, the particles (lipid phase thereof), may further include one or more PEG derivatives. In some embodiments, the PEG derivatives may be conjugated to one or more additional molecules, such as, a lipid. In some embodiments, the PEG derivative is selected from, but not limited to: PEG-DMG 3 -7V-(-m ethoxy poly(ethylene glycol)2000)carbamoyl-l,2-dimyrisyl glycerol, PEG-cDMA 3-A-(-methoxy poly(ethylene glycol)2000)carbamoyl-l,2-dimyristyloxy-propylamine; PEG-cDSA, 3 -7V-(-m ethoxy polyethylene glycol)2000)carbamoyl-1,2-distearyloxy-propylamine, DSPE-PEG, PEG-maleimide, DSPE-PEG-maleimide, or combinations thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the particle comprises the lipid, cholesterol, 1,2-Distearoyl-sn-glycero-3 -phosphocholine (DSPC) and 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG). According to some embodiments, the particle comprises the lipid, cholesterol, 1,2-Diolcoyl-sn-glyccro-3-phosphoethanolamine (DOPE) and 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG). According to some embodiments, particle comprises the lipid according to the present invention, a membrane stabilizing lipid, an additional phospholipid and PEG-lipid conjugate.

[0388] According to some embodiments, the ratio between the various lipids in the particle may vary. In some embodiments, the ratio is a molar ratio. In some embodiments, the ratio is a weight ratio. In some embodiments, each of the lipid groups may be at molar ratio / a weight ratio of about 1%-99%. According to some embodiments, particle comprises 10-70% mol% of the lipid according to the present invention, 20-80% mol% of the membrane stabilizing lipid, 5-50% of the additional phospholipid and 0.5-10% of the PEG-lipid conjugate. According to some embodiments, the molar percentage of the lipid is at least 10 mol% of the particle. According to some embodiments, the molar percentage of the lipid is at least 15 mol% of the particle. According to some embodiments, the molar percentage of the lipid is at least 20 mol% of the particle. According to some embodiments, the molar percentage of the lipid is at least 25 mol% of the particle. According to some embodiments, the molar percentage of the lipid is no more than 50 mol% of the particle. According to some embodiments, the molar percentage of the lipid is no more than 45 mol% of the particle. According to some embodiments, the molar percentage of the lipid is no more than 40 mol% of the particle. According to some embodiments, the molar percentage of the lipid is no more than 35 mol% of the particle.

[0389] It is to be understood that by the phrase “the molar percentage of the lipid is at least x mol% of the particle” it is meant that at least x% of the particle molecules are of the lipid. The same terminology is reflected with other components of the present particle. Similarly, the phrase “the molar percentage of the lipid is no more than x mol% of the particle” it is meant that no more than x% of the particle molecules are of the lipid. The unit “mol%” is also sometimes referred as “mol:mol” or “% mol:mol”.

[0390] According to some embodiments, the molar percentage of the additional phospholipid is at least 5 mol% of the particle. According to some embodiments, the molar percentage of the additional phospholipid is at least 10 mol% of the particle. According to some embodiments, the molar percentage of the additional phospholipid is no more than 35 mol% of the particle. According to some embodiments, the molar percentage of the additional phospholipid is no more than 25 mol% of the particle. According to some embodiments, the molar percentage of the membrane stabilizing lipid is at least 30 mol% of the particle. According to some embodiments, the molar percentage of the membrane stabilizing lipid is at least 40 mol% of the particle. According to some embodiments, the molar percentage of the membrane stabilizing lipid is no more than 70 mol% of the particle. According to some embodiments, the molar percentage of the membrane stabilizing lipid is no more than 60 mol% of the particle. According to some embodiments, the molar percentage of the PEG-lipid conjugate is at least 1 mol% of the particle. According to some embodiments, the molar percentage of the PEG-lipid conjugate is at least 1.5 mol% of the particle. According to some embodiments, the molar percentage of the PEG-lipid conjugate is no more than 5 mol% of the particle. According to some embodiments, the molar percentage of the PEG-lipid conjugate is no more than 3.5 mol% of the particle. According to some embodiments, the particles of the present intention are nanoparticles. According to some embodiments, the lipidic particles of the present intention are lipid nanoparticles.

[0391] In some embodiments, the particles (including any nucleic acid, therapeutic agent and the like encapsulated within and any targeting moiety conjugated thereto) have a particle size (diameter) in the range of about 10 to about 500 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 10 to about 350 nm. In some embodiments, the particles have a particle size (diameter) in the range of about 40 to about 270 nm. In some embodiments, the particles have a particle size (diameter) in the range of over about 10 nm. In some embodiments, the particles have a particle size (diameter) of over about 20 nm. In some embodiments, the particles have a particle size (diameter) of over about 30 nm. In some embodiments, the particles have a particle size (diameter) of over about 40 nm. In some embodiments, the particles have a particle size (diameter) of over about 45 nm. In some embodiments, the particles have a particle size (diameter) of over about 50 nm. In some embodiments, the particles have a particle size (diameter) of over about 60 nm. In some embodiments, the particles have a particle size (diameter) of over about 70 nm. In some embodiments, the particles have a particle size (diameter) of over about 80 nm. In some embodiments, the particles have a particle size (diameter) of over about 90 nm. In some embodiments, the particles have a particle size (diameter) of over about 100 nm. In some embodiments, the particles have a particle size (diameter) of over about 150 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 500 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 400 nm. In some embodiments, the particles have a particle size (diameter) of not more than about 300 nm. In some embodiments, the size is a hydrodynamic diameter.

[0392] According to some embodiments, the nanoparticle has a positive zeta potential, the term “zeta potential” having been defined hereinabove. According to some embodiments, the nanoparticle has a zeta potential of about ImV to about 20m V, including each value within the specified range. According to some embodiments, the nanoparticle has a zeta potential of about 5 mV to about 15mV. According to some embodiments, the nanoparticle has a zeta potential of about 7.5mV to about 12.5mV. According to some embodiments, the nanoparticle has a zeta potential of at least 5mV. According to some embodiments, the nanoparticle has a zeta potential of not more than 20mV. According to some embodiments, the nanoparticle has a negative zeta potential. According to some embodiments, the nanoparticle has a zeta potential of about -ImV to about -20m V, including each value within the specified range. According to some embodiments, the nanoparticle has a zeta potential of about -1 mV to about -lOmV. According to some embodiments, the nanoparticle has a zeta potential of about -1 mV to about -5 mV. According to some embodiments, the nanoparticle has a zeta potential of no more than -5mV. According to some embodiments, the nanoparticle has a zeta potential of at least -20mV.

[0393] According to some embodiments, the lipid nanoparticle composition has poly dispersity index (PDI) of no more than 0.75. According to some embodiments, the lipid nanoparticle composition has PDI of no more than 0.5. According to some embodiments, the lipid nanoparticle composition has PDI of no more than 0.25. According to some embodiments, the lipid nanoparticle composition has PDI of no more than 0.2. According to some embodiments, the lipid composition has PDI of no more than 0.15.

[0394] According to some embodiments, the particle further comprises a nucleic acid. According to some embodiments, the nucleic acid is encapsulated within a particle comprising the lipid. According to some embodiments, the nucleic acid is selected from the group consisting of small interfering RNA (siRNA), microRNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. In some embodiments, the composition may further comprise a nucleic acid. Examples of nucleic acids include small interfering RNA (siRNA), microRNA (miRNA), antisense oligo nucleotides, messenger RNA (mRNA), ribozymes, pDNA, CRISPR mRNA, gRNA, circular RNA and immune stimulating nucleic acids. Each possibility represents a separate embodiment of the present invention.

[0395] According to some embodiments, the weight ratio between the nucleic acid and the lipid mixture may be adjusted so as to achieve maximal biological effect by the nucleic acid on the target site. In some embodiments, the ratio between the nucleic acid and the lipid phase may be 1:1. For example, the weight ratio between the nucleic acid and the lipid phase may be 1:2. For example, the weight ratio between the nucleic acid and the lipid phase may be 1:5. For example, the weight ratio between the nucleic acid and the lipid phase may be 1:10. For example, the weight ratio between the nucleic acid and the lipids phase may be 1:16. For example, the weight ratio between the nucleic acid and the lipid phase may be 1:20. In some embodiments, the weight ratio between the nucleic acid and the lipid phase is about 1:1 to 1:20 (w:w).

[0396] According to some embodiments, the particle further comprises a therapeutic agent. According to some embodiments, the therapeutic agent is encapsulated within a particle comprising the lipid. According to some embodiments, the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein or an immunogenic fragment or variant thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein, an immunogenic fragment of SARS-CoV-2 or a SARS-CoV-2 variant. Each possibility represents a separate embodiment of the invention. According to some embodiments, the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein.

[0397] According to some embodiments, there is provided a method of gene silencing, comprising contacting a cell with a plurality of particles as disclosed herein. According to some embodiments, there is provided a method of gene silencing, comprising the step of contacting a cell with a composition comprising a plurality of particles according to the present invention and a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the present invention provides a method of gene silencing, comprising the step of contacting a cell with a composition comprising a lipid of the present invention. In some embodiments, the cell is a cancer cell.

[0398] In other embodiments, the pharmaceutical compositions of the present invention may be used as a delivery system to administer a therapeutic agent to its target location in the body. Thus, in some embodiments, the present invention relates to a method for administering a therapeutic agent, by preparing a pharmaceutical composition comprising a lipid as described herein and a therapeutic agent, and administering the composition to a subject in need thereof. According to some embodiments, the present invention relates to a method for administering a therapeutic agent, by preparing a particle as described herein comprising a therapeutic agent, and administering the pharmaceutical composition to a subject in need thereof. According to some embodiments, the method further comprises encapsulating the therapeutic agent within a particle comprising the lipid. According to some embodiments, the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein or an immunogenic fragment or variant thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein or an immunogenic fragment or variant thereof. Each possibility represents a separate embodiment of the invention. According to some embodiments, the therapeutic agent is RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein, an immunogenic fragment of SARS-CoV-2 or a SARS-CoV-2 variant. Each possibility represents a separate embodiment of the invention.

[0399] In particular embodiments, the present invention provides novel lipids that enable the formulation of improved compositions for the in vitro and in vivo delivery of IVT- mRNA and / or other oligonucleotides.

[0400] In some embodiments, these lipid nanoparticle compositions are useful for expression of protein encoded by mRNA.

[0401] In other embodiments, these improved lipid nanoparticles compositions are useful for upregulation of endogenous protein expression by delivering miRNA inhibitors targeting one specific miRNA or a group of miRNA regulating one target mRNA or several mRNA.

[0402] In other embodiments, these improved lipid nanoparticle compositions are useful for downregulating (e.g., silencing) the protein levels and / or mRNA levels of target genes.

[0403] In some other embodiments, the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes.

[0404] In yet other embodiments, the lipid nanoparticle compositions are useful for inducing a pharmacological effect resulting from expression of a protein, e.g., increased production of red blood cells through the delivery of a suitable erythropoietin mRNA, or protection against infection through delivery of mRNA encoding for a suitable antibody.

[0405] According to some embodiments, the lipid may be in the form of nanoparticles and administered as is. In some embodiments, the nanoparticles may be administered in a solution. In some embodiments, the nanoparticles may be formulated to a suitable pharmaceutical composition to be administered by any desired route of administration. Exemplary routes of administration include such routes as, but not limited to: topical, oral or parenteral. Depending on the intended mode of administration, the pharmaceutical compositions used may be in the form of solid, semi-solid or liquid dosage forms, such, as for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, or the like, preferably in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical compositions may include the particles, a pharmaceutical acceptable excipient, and, optionally, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, and the like. It is preferred that the pharmaceutically acceptable carrier be one which is inert to the nucleic acid encapsulated within the particles and which has no detrimental side effects or toxicity under the conditions of use. In some embodiments, the administration is localized. In some embodiments, the administration is systemic.

[0406] In some embodiments, injectable formulations for parenteral administration can be prepared as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and the like. Aqueous injection suspensions may also contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. Optionally, the suspension may also contain stabilizers. The parenteral formulations can be present in unit dose or multiple dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, such as, for example, water, for injections immediately prior to use. In some embodiments, parenteral administration includes intravenous administration.

[0407] In other embodiments, for oral administration, a pharmaceutically acceptable, non-toxic composition may be formed by the incorporation of any of the normally employed excipients, such as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate, and the like. Such compositions include solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations and the like. Formulations suitable for oral administration can consist of liquid solutions such as effective amounts of the compound(s) dissolved in diluents such as water, saline, or orange juice; sachets, lozenges, and troches, each containing a predetermined amount of the active ingredient as solids or granules; powders, suspensions in an appropriate liquid; and suitable emulsions. Liquid formulations may include diluents such as water and alcohols, (such as, for example ethanol, benzyl alcohol, and the polyethylene alcohols), either with or without the addition of a pharmaceutically acceptable surfactant, suspending agents, or emulsifying agents.

[0408] In determining the dosages of the particles to be administered, the dosage and frequency of administration may be selected in relation to the pharmacological properties of the specific nucleic acids encapsulated within the particles.

[0409] The lipids of the present invention can be used alone or in combination with other lipid components such as neutral lipids, charged lipids, steroids (including, for example, sterols) and / or their analogs, and / or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents. In some instances, the lipid nanoparticles are used to deliver nucleic acids for the treatment of various diseases or conditions, in particular leukocyte associated conditions such as inflammation and / or lack of sufficient protein.

[0410] Thus, in some embodiments, the present invention relates to a method of treating a leukocyte associated condition, the method comprising the step of administering to a subject in need thereof a pharmaceutical composition according to the present invention. The leukocyte associated condition may be selected from the group consisting of cancer, infection, autoimmune diseases, neurodegenerative diseases and inflammation.

[0411] In some representative embodiments, the particle comprises a nucleic acid, such as, for example, siRNA, miRNA, shRNA, anti-sense RNA, and the like, may be used in the treatment of various leukocyte-associated conditions, depending on the identity of the nucleic acid, the specific target leukocyte, and the like. In some embodiments, the nucleic acid encapsulated within the particles may be a nucleic acid capable of inducing silencing of a target gene. In some embodiments, the target gene may be any gene, the expression of which is related to the condition to be treated. In some embodiments, the target gene may be a gene selected from, but not limited to: growth factors (such as EGFR, PDGFR), genes related to angiogenesis pathways (such as VEGF, Integrins), genes involved in intracellular signaling pathways and cell cycle regulation (such as PI3K / AKT / mT0R, Ras / Raf / MAPK, PDK1, CHK1, PLK1, Cyclins). In some embodiments, a combination of nucleic acids, each having one or more targets may be encapsulated within the particles.

[0412] According to some embodiments, exemplary leukocyte-associated conditions that may be treated by the targeted particles may be selected from, but not limited to: various types of cancer, various infections (such as, for example, viral infection, bacterial infection, fungal infection, and the like), autoimmune diseases, neurodegenerative diseases, inflammations, and the like.

[0413] In some representative embodiments, the targeted particles comprising a nucleic acid (such as, siRNA or miRNA, shRNA, anti-sense RNA, or the like), may be used for the treatment of cancer. In some embodiments, cancer is a disorder in which a population of cells has become, in varying degrees, unresponsive to the control mechanisms that normally govern proliferation and differentiation. In some embodiments, the cancer is a blood cancer. Non-limiting examples of blood cancers are lymphoma, leukemia and myeloma. Lymphomas may be divided into two categories: Hodgkin lymphoma and non-Hodgkin lymphoma. Most non-Hodgkin lymphomas are B-cell lymphomas, that grow quickly (high-grade) or slowly (low-grade). There are 14 types of B-cell non-Hodgkin lymphomas. The others are T-cell lymphomas.

[0414] In some representative embodiments, the nucleic acid that may be used for the treatment of cancer is directed against a target gene, which is involved in the regulation of cell cycle. In some representative embodiments, the target gene may be Polo-like Kinase 1 (PLK), Cyclin DI, CHK1, Notch pathway genes.

[0415] According to some exemplary embodiments, the plurality of lipids of the lipid particles may be of natural or synthetic source and may be selected from, but not limited to: cationic lipids, phosphatidylethanolamines, ionized lipids, membrane stabilizing lipids, phospholipids, and the like, or combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0416] According to some embodiments, the particle further comprises a targeting moiety connected to a component of the composition. According to some embodiments, the particle is conjugated to a targeting moiety. According to some embodiments, the targeting moiety may by conjugated to any one of the lipids included in the present particle. According to some embodiments, the targeting moiety is selected from the group consisting of: liver targeting moiety, spleen targeting moiety, lung targeting moiety, heart targeting moiety and kidney targeting moiety. Each possibility represents a separate embodiment of the invention. According to some embodiments, the particles may be comprised of any one or more of the lipids of the present invention, a phospholipid (e.g. DSPC), a membrane stabilizing lipid (e.g. cholesterol), a PEG-lipid conjugate (e.g. DMG-PEG); at various mol mol ratios, and further conjugated to a targeting moiety, wherein the targeting moiety is conjugated, linked or attached to any one of the particle’s components.

[0417] According to some embodiments, the LNP is devoid of targeting moieties.

[0418] Advantageously, it was found that the present LNPs are highly effective in targeting specific organs, even in the absence of organ-specific targeting moieties. Accordingly, the incorporation of such moieties may be avoided. This enables a simplified formulation process at significantly reduced costs.

[0419] According to some embodiments, the LNP selectively targets an organ selected from the group consisting of: liver, spleen, lungs, heart, and kidneys. Each possibility represents a separate embodiment of the invention. According to some embodiments, the LNP selectively targets an organ selected from the group consisting of: liver, spleen, and lungs. Each possibility represents a separate embodiment of the invention. According to some embodiments, the LNP selectively targets the liver. According to some embodiments, the LNP selectively targets the spleen. According to some embodiments, the LNP selectively targets the lungs. According to some embodiments, the LNP selectively targets the heart. According to some embodiments, the LNP selectively targets the kidneys.

[0420] Definitions

[0421] To facilitate an understanding of the present invention, a number of terms and phrases are defined below. It is to be understood that these terms and phrases are for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art. As used herein the term “pharmaceutical composition” refers to a mixture containing a therapeutic compound to be administered to a mammal, e.g., a human, in order to prevent, treat or control a particular disease or condition affecting the mammal.

[0422] As referred to herein, the terms "nucleic acid", "nucleic acid molecules" “oligonucleotide”, "polynucleotide", and "nucleotide" may interchangeably be used herein. The terms are directed to polymers of deoxy ribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof in the form of a separate fragment or as a component of a larger construct, linear or branched, single stranded, double stranded, triple stranded, or hybrids thereof. The term also encompasses RNA / DNA hybrids. The polynucleotides may include sense and antisense oligonucleotide or polynucleotide sequences of DNA or RNA. The DNA or RNA molecules may be, for example, but not limited to: complementary DNA (cDNA), genomic DNA, synthesized DNA, recombinant DNA, or a hybrid thereof or an RNA molecule such as, for example, mRNA, shRNA, siRNA, miRNA, Antisense RNA, and the like. Each possibility represents a separate embodiment of the present invention. The terms further include oligonucleotides composed of naturally occurring bases, sugars, and covalent inter nucleoside linkages, as well as oligonucleotides having non-naturally occurring portions, which function similarly to respective naturally occurring portions. The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

[0423] The term "construct", as used herein, refers to an artificially assembled or isolated nucleic acid molecule which may include one or more nucleic acid sequences, wherein the nucleic acid sequences may include coding sequences (that is, sequence which encodes an end product), regulatory sequences, non-coding sequences, or any combination thereof. The term construct includes, for example, vector but should not be seen as being limited thereto.

[0424] " Expression vector" refers to constructs that have the ability to incorporate and express heterologous nucleic acid fragments (such as, for example, DNA), in a foreign cell. In other words, an expression vector comprises nucleic acid sequences / fragments (such as DNA, mRNA, tRNA, rRNA), capable of being transcribed. Many prokaryotic and eukaryotic expression vectors are known and / or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art. In some representative embodiments, the expression vector may encode for a double stranded RNA molecule in the target site.

[0425] The term "expression", as used herein, refers to the production of a desired end-product molecule in a target cell. The end-product molecule may include, for example an RNA molecule; a peptide or a protein; and the like; or combinations thereof.

[0426] As used herein, the terms "introducing" and "transfection" may interchangeably be used and refer to the transfer of molecules, such as, for example, nucleic acids, polynucleotide molecules, vectors, and the like into a target cell(s), and more specifically into the interior of a membrane-enclosed space of a target cell(s). The molecules can be "introduced" into the target cell(s) by any means known to those of skill in the art, for example as taught by Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001), the contents of which are incorporated by reference herein. Means of "introducing" molecules into a cell include, for example, but are not limited to: heat shock, calcium phosphate transfection, PEI transfection, electroporation, lipofection, transfection reagent(s), viral-mediated transfer, and the like, or combinations thereof. The transfection of the cell may be performed on any type of cell, of any origin, such as, for example, human cells, animal cells, plant cells, virus cell, and the like. The cells may be selected from isolated cells, tissue cultured cells, cell lines, cells present within an organism body, and the like.

[0427] The term “treating” and "treatment" as used herein refers to abrogating, inhibiting, slowing or reversing the progression of a disease or condition, ameliorating clinical symptoms of a disease or condition or preventing the appearance of clinical symptoms of a disease or condition. The term “preventing” is defined herein as barring a subject from acquiring a disorder or disease or condition.

[0428] The term "treatment of cancer" is directed to include one or more of the following: a decrease in the rate of growth of the cancer (i.e. the cancer still grows but at a slower rate); cessation of growth of the cancerous growth, i.e., stasis of the tumor growth, and, the tumor diminishes or is reduced in size. The term also includes reduction in the number of metastases, reduction in the number of new metastases formed, slowing of the progression of cancer from one stage to the other and a decrease in the angiogenesis induced by the cancer. In most preferred cases, the tumor is totally eliminated. Additionally included in this term is lengthening of the survival period of the subject undergoing treatment, lengthening the time of diseases progression, tumor regression, and the like. In some embodiments, the cancer is a blood cancer.

[0429] The term “leukocytes” is directed to white blood cells (WBCs), produced and derived from a multipotent, hematopoietic stem cell in the bone marrow. The white blood cells have nuclei, and types of white blood cells can be classified into five main types, including, neutrophils, eosinophils, basophils, lymphocytes, and monocytes, based on functional or physical characteristics. The main types may be classified into subtypes. For example, lymphocytes include B cells, T cells, and NK cells. B-cells, for example, release antibodies and assist activation of T cells. T cells, for example, can be classified to several subtypes, including: T-helper cells (CD4+ Th) which activate and regulate T and B cells; cytotoxic T cells (CD8+) that can target and kill virus-infected cells and tumor cells; Gamma-delta T cells (y5 T cells) which can bridge between innate and adaptive immune responses and be involved in phagocytosis; and Regulatory (suppressor) T cells which modulate the immune system, maintain tolerance to self-antigens, and abrogate autoimmune conditions.

[0430] EXAMPLES EXAMPLE 1: Synthesis of ionizable lipids

[0431] Abbreviations: STAB: Sodium triacetoxyborohydride; DCM: Dichloromethane; TLC: thin-layer chromatography; SM: starting material; THF: tetrahydrofuran; CDI: 1,1' -Carbonyldiimidazole; DMAP: 4-Dimethylaminopyridine; MeCN: acetonitrile; TBDPS-C1: tert- Butyl(chloro)diphenylsilane; TBAF: tetra-n-butylammonium fluoride; EtOAc: ethyl acetate; rt: room temperature; Synthesis of Lipid 108 (also referred to as NV5-001): Synthetic scheme for Lipid 108 (NV5-001):

[0432]

[0433] Experimental procedure for Lipid 108 (NV5-001):

[0434] 5 Synthesis of 2-(dihexylamino)ethan-l-ol (2)

[0435]

[0436] HO ' ■ '

[0437] 2

[0438] Chemical Formula: C14H31NO

[0439] fv'olm-tjOr '.‘Oiqht -1*

[0440] To a stirred solution of hexanal (6.0 g, 60.21 mmol, 1.0 equiv.) in dry DCM (100 mL) was added ethanolamine (1.9 mL, 31.49 mmol, 0.5 equiv.) at room temperature and then stirred for 2h. Then STAB (25.41 g, 119.86 mmol, 2.0 equiv.) was charged at room temperature and left to stir overnight. The progress of the reaction was monitored by TLC (10% MeOH in CHCh). After completion of SM, the reaction was quenched with sat. NaHCCh solution and extracted into DCM (2x120 mL). The combined organic layers washed with brine solution, dried over anhydrous Na2SO4 and the solvent evaporated. The crude was purified by Buchi flash using 0-3% MeOH in CHCI3 to give 1.50 g of 2- (dihexylamino)ethan-l-ol (2) as a pale-yellow liquid.

[0441] ’H NMR (400 MHz, CDCI3) 83.54 (t, J = 5.2 Hz, 2H), 2.60 (t, J = 5.4 Hz, 2H), 2.47 (dt, J= 13.2, 7.6 Hz, 4H), 1.48 - 1.38 (m, 4H), 1.30 - 1.20 (m, 12H), 0.85 (t, J= 6.4 Hz, 6H).

[0442] ESI-MS: m / z 230.6 [M+H]+.

[0443] Synthesis of bis(2-(dihexylamino)ethyl) ((ethane-l,2-diylbis(oxy))bis(ethane-2, l-diyl))dicarbamate (Lipid 108, NV5-001)

[0444] 1 1li ' '

[0445] . ■. ’ \.0„N■, %..lJ•. >, ■ 0 • ' ■ ■ ’ -

[0446] t::?, • >! <1-111'10 ‘ ihh.nio ’? (iivb-iv> bu fll',110: 2 1 toyl tt Gifhrn.il?

[0447]

[0448] WA’ r.w 11

[0449] To a stirred solution of 2-(dihexylamino)ethan-l-ol (2) (1.00 g, 4.35 mmol, 1.00 equiv.) in THF (10 mL) was added CDI (0.72 g, 4.44 mmol, 1.02 equiv.) at room temperature, stirred for 2h. The progress of the reaction was monitored by TLC (10% MeOH in CHCI3). After completion of SM, DMAP (0.2458 g, 2.01 mmol, 0.4 equiv.) and 2,2'-(ethane-l,2-diylbis(oxy))bis(ethan-l-amine) (4) (0.25 mL, 1.71 mmol, 0.39 equiv.) was charged, and reaction left to stir overnight. The reaction was monitored by TLC (10% MeOH in CHCI3) and, after completion, it was quenched with sat. NaHCOs and extracted with ethyl acetate (2x100 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and the solvent evaporated. The obtained crude was then purified by Buchi flash using 0-5% MeOH in CHCI3 to give 0.1523 g of bis(2-(dihexylamino)ethyl) ((ethane-l,2-diylbis(oxy))bis(ethane-2,l-diyl))dicarbamate (NV5-001) as a clear oil.

[0450] ’H NMR (400 MHz, CDCI3): d 4.11 (t, J= 6.1 Hz Hz, 4H), 3.60 (s, 4H), 3.55 (t, J= 5.1 Hz, 4H), 3.40 – 3.36 (q, J = 5.2 Hz, 4H), 2.67 (t, J= 6.5 Hz, 4H), 2.45 (t, J= 7.4 Hz, 8H), 1.46 - 1.37 (m, 8H), 1.34 - 1.24 (m, 24H), 0.88 (t, J= 6.5 Hz 12H) (Figure 1A).

[0451] ESI-MS: m / z 659.9 [M-H]+(Figure IB).

[0452] HPLC-CAD purity: 89.5%

[0453] Synthesis of Lipid 109 (also referred to as NV5-002):

[0454] Synthetic scheme for Lipid 109 (NV5-002): ,■,. ■. •.. I,

[0455] 1

[0456] f

[0457] :i i > > iJ. 2fcPP f

[0458] MV= n?: E 2-: ■ T ■.:< r ": -E: II: ■: 2-: dbh:: E rd'iane-l. '-c ”::aio? ' ~E

[0459] J L> I - ■ II ll.-l

[0460]

[0461] :.. ■ ■:

[0462] Experimental procedure for Lipid 109 (NV5-002):

[0463] Synthesis of 2-(2-(dihexylamino)ethoxy)ethan-l-ol (2)

[0464]

[0465] Cheti’ idi I cnnula G-; H:.5KIV

[0466] Molecular Weight: 273.46

[0467] To a stirred solution of 1 -bromohexane 3 (7.40 g, 44.88 mmol, 2.73 equiv.) in MeCN: THF (1:1, 50 mL) was added 2-)2-aminoethoxy)ethan-l-ol (1.65 g, 16.45 mmol, 1.00 equiv.) at room temperature, stirred for Ih. Then reaction mixture was heated to 60°C and left to stir overnight. The progress of the reaction was monitored by TLC (10% MeOH in CHCI3). After completion of SM, the solvent was evaporated off and the mixture dissolved in ethyl acetate (100 mL) and washed with water (2x100 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and the solvent evaporated. The obtained crude was then purified by Buchi flash using 0-5% MeOH in CHCI3 to give 1.37 g of 2-(2- (dihexylamino)ethoxy)ethan-l-ol as an orange oil.

[0468] ’H NMR (400 MHz, CDCh): 33.74 (t, J= 4.7 Hz, 2H), 3.70 (t, J= 4.7 Hz, 2H ), 3.61 - 3.58 (m, 2H), 2.96 - 2.90 (m, 2H), 2.76 (dt, J= 7.8 Hz, 4H), 1.62 - 1.56 (m, 4H), 1.35 - 1.21 (m, 12H), 0.86 (m, 6H).

[0469] ESI-MS: m / z 274.6 [M-H]+.

[0470] Synthesis of bis(2-(2-(dihexylamino)ethoxy)ethyl) ((ethane-1, 2-diylbis(oxy))bis(ethane-2,l- diyl))dicarbamate (Lipid 109, NV5-002).

[0471]

[0472] bis(2-(2-(dihexylamino)ethoxy)ethyl) ((ethane-1,2- diylbis(oxy))bis(ethane-2,1-diyl))dicarbamate

[0473] Molecular Weight: 747.12

[0474] To a stirred solution of 2-(2-(dihexylamino)ethoxy)ethan-l-ol (2) (1.0 g, 3.66 mmol, 1.00 equiv.) in THF (20 mL) was added CDI (0.73 g, 4.50 mmol, 1.23 equiv.) at room temperature, and stirred for 2h. The progress of the reaction was monitored by TLC (10% MeOH in CHCI3). After completion of SM, 2,2'-(ethane-l,2-diylbis(oxy))bis(ethan-l-amine) (4) (0.30 mL, 2.05 mmol, 0.56 equiv.) was charged, and reaction left to stir at 50°C overnight. The reaction was monitored by TLC (10% MeOH in CHCI3) and, after completion, it was quenched with sat. NaHCOs and extracted with ethyl acetate (2x50 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and the solvent evaporated. The obtained crude was then purified by Buchi flash using 0-5% MeOH in CHCI3 to give 0.1592 g of 2 bis(2-(2- (dihexylamino)ethoxy)ethyl) ((ethane- 1,2-diylbis(oxy))bis(ethane-2, 1 -diyl))dicarbamate (Lipid 109, NV5-002) as a yellow oil. ’H NMR (400 MHz, CDCh): <5 4.21 (t, J= 4.5 Hz, 4H), 3.64 (t, J= 4.5 Hz, 4H), 3.60 (s, 4H), 3.56 - 3.52 (m, 8H), 3.38 (q, J = 5.6 Hz, 4H), 2.64 (t, J= 6.2 Hz, 4H), 2.44 (t, J = 7.3 Hz, 8H), 1.45 - 1.37 (m, 8H), 1.34 - 1.25 (m, 26H), 0.88 (t, J= 6.7 Hz 12H) (Figure 2A).

[0475] ESI-MS: m / z 747.6 [M-H]+(Figure 2B).

[0476] HPLC-CAD purity: 92.6%

[0477] Synthesis of Lipid 105 (also referred to as NV5-003):

[0478] Synthetic scheme for Lipid 105 (NV5-003):

[0479]

[0480] Experimental procedure for Lipid 105 (NV5-003)

[0481] Synthesis of 2-(didodecylamino)ethan-l-ol:

[0482]

[0483] To a clean flask, 1-Dodecanal (18.0g, 0.0982 mmol, 1.5 eq.) and 2-aminoethanol-l-ol (4 mL, 0.065 mmol, 1.0 equiv.) were charged, dissolved in 500mL DCM and stirred at room temperature for 2h-4h. The progress of the reaction was monitored by TLC (10% MeOH in CHCI3) and LCMS. Once the formation of intermediate was seen, 3 eq. STAB (20.81g, 0.0983 mmol), alongside another 1.5eq 1-Dodecanal were added and stirred at room temperature for 6-12h. After completion of SM, the reaction mixture was quenched with 350mL NaHCOs. The phases were separated, and aqueous layer was washed with DCM 3 x 300mL. DCM layers were combined and washed with 500mL brine solution. The DCM layer was dried with Na2SO4, filtered over cotton plug and the solvent evaporated. The crude was purified by Buchi Flash system using 0-10% MeOH in CHCI3 to give the 3.1 g of 2-(dodecylamino) ethan-l-ol as a white solid. Product eluted at 2% MeOH.

[0484] ’H NMR (400 MHz, CDCh): 53.65 (t, J = 5.0, 3.3 Hz, 2H), 2.75 (t, 2H), 2.6 (t, 2H), 2.25 (s, 2H), 1.47 (q, J = 7.0 Hz, 2H), 1.31-1.25 (m, 18H), 0.88 (t, J = 6.8 Hz, 3H).

[0485] ESI-MS analysis: m / z 398.73 [M+H],

[0486] Synthesis of bis(2-(didodecylamino)ethyl) ((ethane-l,2-diylbis(oxy))bis(ethane-2,l-diyl))dicarbamate (Lipid 105, NV5-003):

[0487]

[0488] To a stirred solution of 2-(didodecylamino)ethan-l-ol (3.2 g, 32.8 mmmol, 2.2 equiv.) in 1: 1 ACN: THF (60 mL) was added CDI (1.5 g, 9.26 mmol, 2.5 equiv.) at room temperature, stirred for 2h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh). After intermediate formation, 0.122g DMAP (1.03 mmol, 0.2 equiv.) and 0.5g 2, 2' -(ethane- 1,2- diylbis(oxy))bis(ethan-l -amine) (3.4mmol, 1.0 eq.) was charged, and reaction left to stir at room temperature overnight. The reaction was checked by TLC (10% MeOH in CHCh) and mass of product confirmed by mass spectrometry. After 12h, the reaction mixture was quenched with sat. NaHCCh and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with brine solution and dried over anhydrous Na2SO4 and the solvent evaporated, to obtain 3.9g yellow crude. The obtained crude was then purified by Buchi flash using 0-2% MeOH in CHCI3 to give 0.652g (18% yield, 0.6mmol) of bis(2-(didodecylamino)ethyl) ((ethane-1,2-diylbis(oxy))bis(ethane-2,l-diyl))dicarbamate (Lipid 105, NV5-003) as a pale yellow oil.

[0489] ’H NMR (400 MHz, CDCI3): 52.25 (s, 2H), 4.15 (4H, t), 3.65(s, 4H), 3.55 (t, 4H), 2.7 (dt, 4H), 2.5(t, 4H), 1.4-1.5 (m, 8H), 1.31-1.25 (m, 74H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 3A).

[0490] ESI-MS: m / z: 997.5 [M + H]+, 498.8 [M-2]+, 1017 [M+Na]+(Figure 3B).

[0491] HPLC-CAD purity: 96%

[0492] Synthesis of Lipid 101 (also referred to as NV5-006):

[0493] Synthetic scheme for Lipid 101 (NV5-006): lrr.l:c:-T DCM

[0494] EEXXHO

[0495] _...

[0496] i v.i

[0497]

[0498] NV05-CC6 Experimental procedure for Lipid 101 (NV5-006)

[0499] Synthesis of TBDPS protection of 10-hydroxy decanoic acid:

[0500] TBDPSO^ / ^^^

[0501]

[0502] I0-((tart..butylcliphenylsilyl)oxy)cl®cano« acid

[0503] Molecular Weight: 426.66 To a clean vessel were charged 10-hydroxydecanoic acid (1 wt, 1.0 equiv) and imidazole (0.82 wt, 2.25 equiv). The solids were dissolved in anhydrous DMF (2.5 vol) and TBDPS-C1 (1.8 vol, 1.3) was charged to the resulting solution in one portion. The reaction mixture was stirred at 18 to 25 °C for at least 12 h. On reaction completion, the reaction mixture was diluted with hexane (15 vol) and cooled to 0 to 5 °C. Purified water (15 vol) was charged to the mixture maintaining 0 to 5 °C and stirred at this temperature for at least 5 min. The aqueous layer was back-extracted with 20% (v / v) ethyl acetate in hexane (4x 15 vol). The organic layers were combined and washed with brine (15 vol). The organic layer was dried further with sodium sulfate (1 wt) before concentration to a residue under reduced pressure at <45 °C to obtain a clear oil. The crude compound was purified by column chromatography with silica gel (60-120 mesh) and a gradient of 0-5% (v / v) ethyl acetate in hexane to obtain pure 10-((tert-butyldiphenylsilyl)oxy)decanoic acid.

[0504] ¹H NMR (400 MHz, CDCl3): δ 7.69 (4 H, m), 7.40 (6 H, m), 3.65 (2 H, t), 2.34 (2 H, t), 1.58 (6 H, m), 1.30 (8 H, m), 1.04 (9 H, s).

[0505] Synthesis of N, N-dihexylhydroxylamine:

[0506]

[0507] I K:

[0508] • dihnx lhy roxylaminp

[0509] Molecular Weight 201.35

[0510] To a suspension of hydroxylamine hydrochloride (1 wt, 1.0 equiv) in anhydrous DCM (15 vol) was charged triethylamine (2 vol, 1.0 equiv). The reaction mixture was stirred under a nitrogen atmosphere at 18 to 25 °C for 15 min. Hexanal (4.3 wt, 3.0 equiv) was charged to the reaction vessel and a line rinse of anhydrous DCM (3 vol) was applied. The reaction mixture was stirred for at least 60 min at 18 to 25 °C. Sodium triacetoxyborohydride (9.2 wt, 3.0 equiv) was charged to the reaction mixture portion-wise maintaining <25 °C. The reaction mixture was stirred at 18 to 25 °C for at least 10 hours or until completion. On reaction completion, a saturated solution of sodium hydrogen carbonate (15 vol) was charged to the reaction vessel over at least 10 min to quench residual borohydride. DCM (13 vol) was charged, and the resulting biphasic mixture was stirred for at least 10 min at 18 to 25 °C before extracting the organic layer. The aqueous layer was back-extracted with DCM (2x 10 vol). The recovered organic layers were combined and washed with brine solution (15 vol). The organic layer was separated and dried with sodium sulfate (1 wt). The dried organic layer was concentrated to a residue under reduced pressure at <45 °C to obtain a clear to white semi-solid. The crude residue was purified by column chromatography with silica gel (60-120 mesh) and a gradient of 0-10% (v / v) ethyl acetate in hexane to obtain pure / f-dihexylhydroxylamine.

[0511] ¹H NMR (400 MHz, CDCl3): 2.64 (4 H, t), 1.58 (4 H, m), 1.40-1.18 (12 H, m), 0.87 (6 H, t).

[0512] Synthesis of O-(10-((tert-butyldiphenylsilyl)oxy)decanoyl)-N, N-dihexylhydroxylamine:

[0513] O IBDPSO ■- -■ -■ J- N

[0514]

[0515] O HO {(for f biitvMiphenylhilyncixy.anoylj M

[0516]

[0517] W dihoxylhyci

[0518] Molecular Weight: 610.00

[0519] To a clean vessel was charged 10-(( / c77-butyldiphenylsilyl)oxy)decanoic acid (2.46 wt, 1.2 equiv) and N, N-dihexylhydroxylamine (1.0 wt, 1.0 equiv). The reagents were dissolved in anhydrous DCM (5 vol) followed by charging of EDC. HC1 (2.29 wt, 2.0 equiv) and DMAP (0.12 wt, 0.2 equiv) to the reaction vessel. The reaction mixture was stirred at 18 to 25 °C for at least 3 h. On reaction completion, the reaction mixture was quenched with saturated sodium hydrogen carbonate solution (10 vol). The layers were separated and the aqueous layer back-extracted with DCM (2 x 10 vol). The combined organic layers were washed with brine solution (27 vol). The organic layer was dried with sodium sulfate (1.0 wt) and inorganics filtered off through filter paper. The organic layers were concentrated to a residue under reduced pressure at <45 °C to obtain a pale-yellow oil.

[0520] 1H NMR (400 MHz, CDCl3): δ 3.63 (2 H, t), 2.80 (4 H, t), 2.28 (2 H, t), 1.71-1.44 (8 H, m), 1.30 (22 H, s), 0.86 (6 H, t).

[0521] Synthesis of 10-((dihexylamino)oxy)-10-oxodecan-l-ol:

[0522] O

[0523]

[0524] zX / x

[0525] To a clean vessel were charged -(IO-(( / c77-butyldiphenylsilyl)oxy)decanoyl)-W, W- dihexylhydroxylamine (1 wt, 1.0 equiv) and THF (3.6 vol). The mixture was stirred at 18 to 25 °C until full dissolution was observed. TBAF, 1 M in THF (3 vol, 2.0 equiv) was charged in a single portion to the vessel. The reaction mixture was stirred at 18 to 25 °C for at least 2 h. On reaction completion, the reaction mixture was diluted with ethyl acetate (15 vol) and quenched with saturated ammonium chloride solution (15 vol). The reaction quench was stirred at 18 to 25 °C for at least 10 min. The layers were separated and the aqueous back-extracted with ethyl acetate (3 x 15 vol). The organic layers were combined and concentrated to a residue under reduced pressure at <45 °C to obtain 10-((dihexylamino)oxy)- 10-oxodecan- 1 -ol.

[0526] Synthesis of mono-substituted 10-((dihexylamino)oxy)-10-oxodecyl (2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate:

[0527]

[0528] To a clean flask 0.28g 10-((dihexylamino)oxy)-l 0-oxodecan- l-ol (0.8mmol, 1.0 eq.) and 0.18g CDI (l.lmmol, 1.5 eq.) were dissolved in a 1:1 mixture of acetonitrile and THF. The reaction was stirred at room temperature for 3h until formation of the intermediate was observed by mass spectrometry. ImL of 2-(2-(2-(12-azaneyl)ethoxy)ethoxy)ethan-l -amine was charged and stirred at room temperature for a further 2h. The solvent was evaporated and redissolved in ethyl acetate. The reaction crude was quenched with water and phases were separated. The aqueous layer was washed with ethyl acetate 3 x 30mL. Organics were combined and washed with 50mL brine, dried with Na2SO4and filter over a cotton plug and solvent reduced under pressure, affording 0.4g yellow oil as crude.

[0529] Synthesis of 10-((dihexylamino)oxy)-10-oxodecyl (2-(2-(2- (didodecylamino)ethoxy)ethoxy)ethyl)carbamate (Lipid 101, NV5-006)

[0530] 1 i

[0531] fl o c.

[0532] o ri o • ■ *i

[0533] H A I

[0534]

[0535] To a clean flask, 0.38g 10-((dihexylamino)oxy)-10-oxodecyl (2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate (0.7mmol, 1.0 eq.) and 0.32g dodecanal (1.7mmol, 2.5 eq.) were dissolved in 15ml anh. DCM and stirred at rt for l-2h. After intermediate formation, 0.36g STAB (1.7mmol, 2.5 eq.) was charged and stirred at rt for 2h. The reaction was quenched with 30mL NaHCCh and stirred for 5min to degas. The phases were separated and aqueous was washed with 3 x 50 mL DCM. DCM layers were then washed with brine, dried with Na2SO4, filtered over a cotton plug and reduced under pressure giving 0.72g yellow crude. This was then purified using Flash Pure Systems with a 12g Silica Select cartridge, Chloroform / Methanol as mobile phase. The crude was dry loaded with DCM. Product eluted at 1% Methanol. 214mg (35% yield) of product was isolated as a single spot on the TLC.

[0536] ’H NMR (400 MHz, CDCh): 53.63 (4 H, s), 3.5 (4H, t), 3.33 (2H, q), 2.85 (4 H, t), 2.75 (2 H, t), 2.4 (4H, t), 2.3 (2H,t), 1.75 (s, 2H), 1.65 (4H,q), 1.5 (4H, m) 1.3 (4H, m), 1.30 (66 H, s), 0.86 (6 H, t) (Figure 4A).

[0537] ESLMS: 882.5 (M+H)+, 442.5 (M’2)+, 905.3 (M+Na)+(Figure 4B).

[0538] HPLC-CAD purity: 98%

[0539] Synthesis of Lipid 102 (also referred to as NV5-007):

[0540] Synthetic scheme for Lipid 102 (NV5-007):

[0541] r-r

[0542]

[0543] Experimental procedure for Lipid 102 (NV5-007) Synthesis of 2-(didodecylamino)ethyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate:

[0544] H

[0545] (7 -Ni.

[0546] v--Ox

[0547] NHj

[0548]

[0549] 2-(didodecylamino)ethan-l-ol was synthesized according to the protocol provided in the above section. To a clean flask 2.0g of 2-(didodecylamino)ethan-l-ol (5.0 mmol, 1.0 eq.) and 1.04g CDI (6.4 mmol, 1.5 eq.) were dissolved in a 1:1 mixture of acetonitrile and THF. The reaction was stirred at room temperature for 3h until formation of the intermediate was observed by mass spectrometry. l.lmL of 2-(2-(2-(12-azaneyl)ethoxy)ethoxy)ethan-l -amine (7.5 mmol, 1.5 eq.) was charged and stirred at room temperature for further 2h. The solvent was evaporated and redissolved in DCM. The reaction crude was quenched with water and phases were separated. The aqueous layer was washed with DCM 3 x 30mL. Organics were combined and washed with 50mL brine, dried with Na2SO4and filter over a cotton plug and solvent reduced under pressure affording 2.3g yellow oil as crude.

[0550] Synthesis of 2-(didodecylamino)ethyl (2-(2-(2-(didodecylamino)ethoxy)ethoxy)ethyl)carbamate (Lipid 102, NV5-007):

[0551]

[0552] NV05-007’

[0553] To a clean flask, 0.19g 2-(didodecylamino)ethyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (0.3 mmol, 1.0 eq.) and 0.1g dodecanal (1.7mmol, 0.5 eq.) were dissolved in 15ml anh. DCM and stirred at rt for l-2h. After intermediate formation, 0.13g STAB (0.6 mmol, 2.5 eq.) was charged and stirred at rt for 2h. The reaction was quenched with 30mL NaHCCh and stirred for 5min to degas. The phases were separated and aqueous was washed with 3 x 50 mL DCM. DCM layers were then washed with brine, dried with Na2SO4, filtered over a cotton plug and reduced under pressure, giving 0.33g yellow crude. This was then purified using Flash Pure Systems with a 12g Silica Select cartridge, Chloroform / Methanol as mobile phase. The crude was dry loaded with DCM. Product eluted at 1% Methanol. 186mg (68% yield) of product was isolated as a pale yellow oil and single spot on the TLC. ’H NMR (400 MHz, CDCh): 54.1 (2H, t), 3.63 (4 H, s), 3.5 (4H, t), 3.33 (2H, q), 2.85 (4 H, t), 2.4 (8H, t), 1.35 (8H, s), 1.30 (74 H, s), 0.86 (12 H, t) (Figure 5A).

[0554] ESI-MS: 909.3 (M+H)+, 455.4 (M’2)+(Figure 5B).

[0555] HPLC-CAD purity: 95%

[0556] Synthesis of Lipid 110 (also referred to as NV5-035):

[0557] Synthetic scheme for Lipid 110 (NV5-035):

[0558] 1 COI

[0559] jw««m

[0560]

[0561] Experimental procedure for Lipid 110 (NV5-035)

[0562] Synthesis of 10-((dihexylamino)oxy)-10-oxodecyl IH-imidazole-l-carboxylate:

[0563] -.. ij....

[0564] ■ \ ■ ■ - 4 ■

[0565]

[0566] To a clean flask, 0.28g of 10-((dihexylamino)oxy)-10-oxodecan-l-ol (0.8mmol, 1.0 eq.; synthesized according to the above-mentioned protocol) and 0.18g CDI (l.lmmol, 1.5 eq.) were dissolved in a 1:1 mixture of acetonitrile and THF. The reaction was stirred at room temperature for 3h until formation of the desired material was observed by mass spectrometry. The reaction crude was quenched with water and phases were separated. The aqueous layer was washed with ethyl acetate 3 x 30mL. Organics were combined and washed with 50mL brine, dried with Na2SO4, filtered over a cotton plug and solvent reduced under pressure, affording 0.4g yellow oil as crude. Synthesis of 2-(didodecylamino)ethyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate:

[0567] 1

[0568]

[0569] To a clean flask, 2.0g 2-(didodecylamino)ethan-l-ol (5.0 mmol, 1.0 eq.; synthesized according to the above-mentioned protocol) and 1.04g CDI (6.4 mmol, 1.5 eq.) were dissolved in a 1:1 mixture of acetonitrile and THF. The reaction was stirred at room temperature for 3h until formation of the intermediate was observed by mass spectrometry. l.lmL of 2-(2-(2-(12- azaneyl)ethoxy)ethoxy)ethan-l -amine (7.5 mmol, 1.5 eq.) was charged and stirred at room temperature for a further 2h. The solvent was evaporated and redissolved in DCM. The reaction crude was quenched with water and phases were separated. The aqueous layer was washed with DCM 3 x 30mL. Organics were combined and washed with 50mL brine, dried with Na2SO4, filtered over a cotton plug and solvent reduced under pressure, affording 2.3g yellow oil as crude. Synthesis of 10-((dihexylamino)oxy)-10-oxodecyl (14-dodecyl-10-oxo-3,6,l l-trioxa-9,14- diazahexacosyl)carbamate (Lipid 110, NV5-035):

[0570] 10-i uil’e- v airirc so yi-10-o □dec.111 j-c ■ o-'4-m leiyl-36 U-trcx-9 '4-lazjl’e ■ acosd zaio.i^iate

[0571]

[0572] Vuiuii'ldr V / s-y Id I j'j

[0573] To a clean flask, 0.3g 10-((dihexylamino)oxy)-10-oxodecyl IH-imidazole-l-carboxylate (0.6 mmol, 1.0 eq.), 25mg DMAP (0.2 eq., 1.6mmol) and 0.45g 2-(didodecylamino)ethyl (2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate (8.0 mmol, 1.0 eq.) were dissolved in 30mL anh. DCM stirred at rt for l-2h. No progress was seen in 2h, hence the reaction was heated gently at 45°C overnight. The full conversion of the SM was confirmed by mass spectrometry. The reaction was quenched with 30mL NaHCCf and stirred at room temperature for 5 minutes. The phases were separated and aqueous was washed with 3x 50 mL DCM. DCM layers were then washed with brine, dried with Na2SO4, filtered over a cotton plug and reduced under pressure, giving 0.93g yellow crude. This was then purified using Flash Pure Systems with a 25g Silica Select cartridge, Chloroform / Methanol as mobile phase. The crude was dry loaded with DCM. Product eluted at 0- 1% Methanol. 346mg (47% yield) of product was isolated as a sticky / dense translucent oil and single spot on the TLC.

[0574] ’H NMR (400 MHz, CDCk): 54.15 (2H, t), 4.10 (2H, t), 3.63 (4 H, s), 3.5 (4H, t), 3.33 (4H, q), 2.85 (4 H, t), 2.4 (91H, s),1.8(s,4H), 1.7(m,4H), 1.5 (t,4H), 1.35 (4H, s), 1.30 (62 H, s), 0.86 (12 H, t) (Figure 6A).

[0575] ESI-MS: 970.4 (M+H)+, 485.6 (M’2)+(Figure 6B).

[0576] HPLC-CAD purity: 96.7%

[0577] Synthesis of Lipid 111 (also referred to as NV5-039):

[0578] Synthetic scheme for Lipid 111 (NV5-039)

[0579] H

[0580] r’J■ '■ ’M

[0581] 2- •: sr- C- 1- -.c > le: -.1

[0582] A ■_ I -

[0583] Ur-E- X - 1 DCM

[0584] H

[0585] < N. •. ■ _ _ ■

[0586] ’I 'j

[0587] NV*t> UJS

[0588] •I if|. \ i ' ' •':1 '." I','.. i-i-'-y, lv-t1< " -,.'l

[0589]

[0590] Experimental procedure for Lipid 111 (NV5-039)

[0591] Synthesis of 2-(didodecylamino)ethyl (2-(2-(2-(di((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethoxy)ethoxy)ethyl)carbamate (Lipid 111, NV5-039):

[0592]

[0593] MV05-033 To a clean flask, 0.3g 2-(didodecylamino)ethyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate(0.5 mmol, 1.0 eq.; synthesized according to the protocol mentioned above) and 0.34g (9Z,12Z)- octadeca-9,12-dienal (1.3mmol, 2.5 eq.) were dissolved in 15ml anh. DCM and stirred at rt for 1-2h. After intermediate formation, 0.27g STAB (1.3 mmol, 2.5 eq.) was charged and stirred at rt for 2-3h. The reaction was quenched with 30mL NaHCCh and stirred for 5 min to degas. The phases were separated and aqueous was washed with 3 x 50 mL DCM. DCM layers were then washed with brine, dried with Na2SO4, filtered over a cotton plug and reduced under pressure, giving 0.73g yellow crude. This was then purified using Flash Pure Systems with a 25g Silica Select cartridge, Chloroform / Methanol as mobile phase. The crude was dry loaded with DCM. Product eluted at 1-2% Methanol. 390mg (74% yield) of product was isolated as a pale-yellow oil and single spot on the TLC.

[0594] ’H NMR (400 MHz, CDCk): 5 5.35(8H, m), 4.1 (2H, t), 3.63 (4 H, s), 3.5 (4H, t), 3.33 (2H, s), 2.85 (42H, t), 2.65(s, 4H), 2.4 (8H, t),2.01(t,8H), 1.35 (10H, s), 1.30 (75 H, s), 0.86 (12 H, t) (Figure 7A).

[0595] ESI-MS: 1069.9 (M+H)+, 535.5 (M’2)+(Figure 7B).

[0596] HPLC-CAD purity: 95.8%

[0597] Synthesis of Lipid 112 (also referred to as NV5-040):

[0598] Synthetic scheme for Lipid 112 (NV5-040)

[0599] ME- j J r ’ ■ / • • ’ -. 4 '>; i. f

[0600] NV5 04tJ.. C ' > ’. M, J.. >• f y •

[0601]

[0602] Synthesis of 2-(didodecylamino)ethyl (21 -hexyl- 19-oxo-3,6,20-trioxa-9,21-diazaheptacosyl)carbamate (Lipid 112, NV5-040):

[0603] X ■■ X X -‘*'v "j «

[0604] I iii - j Ji

[0605] n.tn

[0606] ......,....... • ■. -,.....

[0607]

[0608] ■ ■: r:..:■ j.«

[0609] To a clean flask, 0.33g of 2-(didodecylamino)ethyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate(0.6 mmol, 1.0 eq.; Synthesized according to the above-mentioned protocol.) and 0.11g of 10-((dihexylamino)oxy)-10-oxodecanal (0.3mmol, 0.5 eq.; synthesized according to the above-mentioned protocol.) were dissolved in 15ml anh. DCM and stirred at rt for l-2h. After intermediate formation, 0.21g STAB (0.6 mmol, 2.0 eq.) was charged and stirred at rt for 2-3h. The reaction was quenched with 30mL NaHCCh and stirred for 5min to degas. The phases were separated and aqueous was washed with 3 x 50 mL DCM. DCM layers were then washed with brine, dried with Na2SO4, filtered over a cotton plug and reduced under pressure giving 0.43g yellow crude. A mix of mono and disubstituted was identified by MS. This was then purified using Flash Pure Systems with a 25g Silica Select cartridge, Chloroform / Methanol as mobile phase. The crude was dry loaded with DCM. Product eluted at 1% Methanol. 90mg (16% yield) of product was isolated as a white semisolid and single spot on the TLC.

[0610] ’H NMR (400 MHz, CDCh): 55.45(1H, NH, br), 4.1 (2H, t), 3.63 (4 H, s), 3.5 (2H, t), 3.33 (2H, d), 2.85 (2H, t), 2.65 (s, 4H), 2.4 (4H, t), 2.25 (t,2H), 2.0-1.8 (br,5H), 1.6 (2H,t), 1.4(5H, t), 1.35 (2H, s), 1.30 (58H, s), 0.86 (12 H, t) (Figure 8A).

[0611] ESI-MS: 926.4 (M+H)+, 463.8 (M’2)+(Figure 8B).

[0612] HPLC-CAD purity: 87.1%

[0613] Synthesis of Lipid 113 (also referred to as NV5-041):

[0614] Synthetic scheme for Lipid 113 (NV5-041) H

[0615] o 'I

[0616] 2 eq L.-PI '

[0617] ' _. ” 'vt • ’

[0618] i -- x / XZ\Z - -. |. '.

[0619] H NV05 041. ‘S jf. / < n. 's ' I.‘>11 -i, 'I i ’ -'I' -. 1 t'.. •

[0620] -J’ u..e.

[0621]

[0622] V: ’2~e ' '

[0623] To a clean flask, 0.33g 2-(didodecylamino)ethyl (2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate(0.6 mmol, 1.0 eq.) and 0.22g 10-((dihexylamino)oxy)-10- oxodecanal (0.6mmol, 1.2 eq,) were dissolved in 15ml anh. DCM and stirred at rt for l-2h. After intermediate formation, 0.21g STAB (0.6 mmol, 2.0 eq.) was charged and stirred at rt for 2-3h. The reaction was quenched with 30mL NaHCCh and stirred for 5min to degas. The phases were separated and aqueous was washed with 3 x 50 mL DCM. DCM layers were then washed with brine, dried with Na2SO4, filtered over a cotton plug and reduced under pressure giving 0.73g yellow crude. A mix of mono and disubstituted was identified by MS. This was then purified using Flash Pure Systems with a 25g Silica Select cartridge, Chloroform / Methanol as mobile phase. The crude was dry loaded with DCM. Product eluted at 1-2% Methanol. 50mg (8% yield) of product was isolated as a yellow oil and single spot on the TLC.

[0624] ’H NMR (400 MHz, CDCh): 5 4.1 (2H, t), 3.63 (2H, s), 3.5 (2H, t), 3.33 (2H, d), 2.85 (5H, t), 2.65 (s, 2H), 2.45 (5H, t), 2.25 (t,3H), 1.6 (2H,t), 1.4 (5H, t), 1.35 (6H, s), 1.30 (56H, s), 0.86 (12 H, t) (Figure 9A).

[0625] ESI-MS: 1279.8 (M+H)+, 640.5 (M’2)+(Figure 9B).

[0626] HPLC-CAD purity: 80.8%

[0627] Synthesis of Lipid 114 (also referred to as NV5-042): Nvns ni?

[0628]

[0629] Synthetic scheme for Lipid 114 (NV5-042)

[0630] Experimental procedure for Lipid 114 (NV5-042):

[0631] Synthesis of 2-(didodecylamino)ethyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate:

[0632] zXz°

[0633] 2.:iiriod<\"vlriniir.n;pthvl:2 i? <>’ anun«pth<:xy;fMhnxvh?lh;rfyjnthvl:c< Hh.irv<iln

[0634]

[0635] Molecular Weight 6’6.99

[0636] To a clean flask 2.0g 2-(didodecylamino)ethan-l-ol (5.0 mmol, 1.0 eq.; synthesized according to the protocol mentioned above) and 1.11g CDI (7.5 mmol, 1.5 eq.) were dissolved in a 1: 1 mixture of acetonitrile and THF. The reaction was stirred at rt for 3h until formation of the intermediate was observed by MS. 1.93mL of 2,2'-((oxybis(ethane-2,l-diyl))bis(oxy))bis(ethan-l -amine) (7.5 mmol, 1.5 eq.) was charged and stirred at rt for further 2h. The solvent was evaporated and redissolved in DCM. The reaction crude was quenched with water and phases were separated. The aqueous layer was washed with DCM 3 x 30mL. Organics were combined and washed with 50mL brine, dried with Na2SO4and filter over a cotton plug and solvent reduced under pressure affording 2.8g yellow oil as crude. The crude was dry loaded with DCM and purified using a 40g column and Chloroform / Methanol as a mobile phase. 1.5g of product was isolated. (50%yield)

[0637] ’H NMR(400 MHz, CDCh): 55.5 (s, 1H), 10 (t,2H), 3.65 (s,8H), 3.55 (t,4H), 3.45 (s, 2H),2.85 (t,2H), 2.75 (t,2H), 2.42 (d, 2H), 1.63 - 1.59 (br., 1H), 1.35 - 1.32 (s, 5H), 1.2 (s,38), 0.88 (t, 6H).

[0638] ESI-MS: m / z 309.1 [M-2]+, 616.9 [M+H]+.

[0639] Synthesis of 2-(didodecylamino)ethyl (12-(10-((dihexylamino)oxy)-10-oxodecyl)-24-hexyl-22- oxo-3,6,9,23-tetraoxa-12,24-diazatriacontyl)carbamate (Lipid 114, NV5-042):

[0640] NVU'j U47

[0641] . • ■. ■, i i " ■’ s ‘ i -!>, ' <..;

[0642] -■ n,;. i i wu v.-

[0643]

[0644] To a clean flask, 0.25g 2-(didodecylamino)ethyl (2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (0.4 mmol, 1.0 eq.) and 0.32g 10- ((dihexylamino)oxy)-10-oxodecanal (0.9mmol, 2.0 eq.) were dissolved in 15ml anh. DCM and stirred at rt for l-2h. After intermediate formation, 0.17g STAB (0.8 mmol, 2.0 eq.) was charged and stirred at rt for 2-3h. The reaction was quenched with 30mL NaHCCh and stirred for 5 min to degas. The phases were separated and aqueous was washed with 3 x 20 mL DCM. DCM layers were then washed with brine, dried with Na2SO4, filtered over a cotton plug and reduced under pressure giving 0.45g yellow crude. A mix of mono and disubstituted was identified by MS. This was then purified using Flash Pure Systems with a 12g Silica Select cartridge, DCM / Methanol as mobile phase. The crude was dry loaded with DCM. Product eluted at 1-2% Methanol. 71 mg (15% yield) of product was isolated as a yellow oil and single spot on the TLC.

[0645] ’H NMR (400 MHz, CDCh): 5 4.1 (1H, t), 3.63 (5H, s), 3.5 (3H, t), 3.33 (1H, d), 2.85 (5H, t), 2.65 (m, 2H), 2.45 (5H, q), 2.25 (t,3H), 1.6 (2H, t), 1.5 (s, 2H), 1.4 (5H, t), 1.35 (5H, s), 1.30 (54H, s), 0.86 (12 H, t) (Figure 10A).

[0646] ESI-MS: 1320.9(M+H)+, 662.6 (M’2)+, 442.0 (M’3)+(Figure 10B). HPLC-CAD purity: 88.5%

[0647] Synthesis of Lipid 115 (also referred to as NV5-043):

[0648] Synthetic scheme for Lipid 115 (NV5-043)

[0649] □0.j HCM wi - ' ~ o • V

[0650] -,..f)t\. ■, I C h:"Jon'' 0 N, - ■ U. K,

[0651] NV05-043? •? dihf--yM’,nnnt!‘thn.y:t.th;:. Tin-y Tnn^ n>yt If n>nt’cr-, J:

[0652] 21-'w«yl-19-a- 62‘C’tr:-a-9 Z'-darahepiacosyi catbamate

[0653]

[0654] Mue-.ird’ *1’1311: ' 15484

[0655] To a clean flask, 0.20g 2-(2-(dihexylamino)ethoxy)ethyl (2-(2-(2- aminoethoxy)ethoxy)ethyl)carbamate (0.4 mmol, 1.0 eq.; Synthesized according to the above- mentioned protocol) and 0.29g 10-((dihexylamino)oxy)-10-oxodecanal (0.8mmol, 2.0 eq.; Synthesized according to the above-mentioned protocol) were dissolved in 15ml anh. DCM and stirred at rt for l-2h. After intermediate formation, 0.17g STAB (0.8 mmol, 2.0 eq.) was charged and stirred at rt for 2-3h. The reaction was quenched with 30mL NaHCCh and stirred for 5 min to degas. The phases were separated and aqueous was washed with 3 x 20 mL DCM. DCM layers were then washed with brine, dried with Na2SO4, filtered over a cotton plug and reduced under pressure giving 0.48g yellow crude. A mix of mono and disubstituted was identified by MS. This was then purified using Flash Pure Systems with a 12g Silica Select cartridge, CHCh / Methanol as mobile phase. The crude was dry loaded with DCM. Product eluted at 1-2% Methanol. 88mg (19% yield) of product was isolated as a yellow oil and single spot on the TLC. ’H NMR (400 MHz, CDCh): 5 4.1 (1H, t), 3.63 (2H, s), 3.5 (3H, t), 3.33 (2H, t), 2.85 (5H, t), 2.65 (m, 2H), 2.45 (5H, q), 2.25 (t,3H), 1.6 (2H, t), 1.5 (s, 2H), 1.4 (5H, t), 1.35 (5H, s), 1.30 (39H, s), 0.86 (12 H, t) (Figure 11A).

[0656] ESI-MS:1154.8 (M+H)+, 578.4 (M’2)+(Figure 11B).

[0657] HPLC-CAD purity: 89.3%

[0658] EXAMPLE 2: Lipid nanoparticle (LNP) formulation and physico-chemical properties Various formulations of LNPs using Lipid 102 (NV5-007) and Lipid 110 (NV5-035) were prepared and their physico-chemical properties assessed.

[0659] The LNPs were formulated using the microfluidic mixing device (NanoAssemblr Ignite, Cytiva). To prepare the LNPs organic phase, lipids were mixed in the following molar ratios: 40% ionizable lipid, 49.5% Cholesterol, 1.5% PEG-DMG, 9% DSPC or DOPE. One volume of lipid mix in ethanol solution and three volumes of firefly Luciferase mRNA (total lipid to mRNA ratio at 40: 1 w / w) in citrate buffer (pH 4.5) were mixed using a microfluidic mixing device at a total flow rate of 12 ml / min. LNPs were dialyzed against lOmM Tris pH 7.4 buffer.

[0660] As shown in Table 1 below, all the mRNA-LNP formulations were uniformly distributed with a 100 d.nm size with more than 90% mRNA encapsulation. Interestingly the zeta potential of 5-007 LNPs were positive.

[0661] The hydrodynamic diameter and zeta potential of the LNPs were measured by dynamic light scattering using disposable cuvettes in the Malvern Zetasizer (Malvern Instruments). Encapsulation was quantified by Quant-it™ RiboGreen RNA Assay Kit (Thermo fisher) according to the manufacturer protocol.

[0662] Table 1: Lipid Formula Molar ratio Composition Size PolyZeta EE*( tion (d.nm) dispersion Potentia %)

[0663] index (PDI) l (mV)

[0664] Lipid Fl 40:9:49.5:1 Lipid 102: 95.3 0.07 9.15 90

[0665] 102.5 DSPC:

[0666] (NV5- Cholesterol:

[0667] 007) DMG-PEG

[0668] F2 40:9:49.5:1 Lipid 102: 110.6 0.15 9.08 90

[0669] .5 DOPE:

[0670] Cholesterol:

[0671] DMG-PEG

[0672] Lipid F2 40:9:49.5:1 Lipid 110: 100 0.08 -2.8 93

[0673] 110.5 DOPE:

[0674] (NV5- Cholesterol:

[0675] 035) DMG-PEG

[0676]

[0677] * EE=Encapsulation efficiency

[0678] EXAMPLE 3: LNPs in vitro testing

[0679] The transfection efficiency of the formulations was tested in a human hepatocarcinoma cell line (HepG2), a mouse dendritic cell line (Mutu DC 1340), and a human neuroblastoma cell line (HTB11) by encapsulating firefly Luciferase mRNA (mLuc) as a reporter gene.

[0680] Cells were cultured according to producer’s instructions. HepG2 (14000cells / well) and HTB11 (16000 cells / well) cells were treated with LNPs, formulated as shown in Table 1, at an mLuc mRNA concentration of 25ng / ml, 50ng / ml, lOOng / ml, and 200ng / ml. Mutu DC1340 (DC) cells were treated with LNPs at an mLuc mRNA concentration of 62.5ng / ml, 125ng / ml, 250ng / ml, and 500ng / ml. After 24hrs, cells were lysed and analysed for luciferase expression using a Promega plate reader.

[0681] Results are shown in Figures 12A-C. As shown in Figure 12A-C, respectively, the transfection efficiency of both Lipid 102 and Lipid 110 were efficient to deliver the mRNA into HepG2 cells, into DC cells, and into HTB 11 cells. In all cells, the transfection efficiency of Lipid 102 was higher compared to Lipid 110. Unexpectedly, LNPs formulated with Lipid 102 and DOPE, demonstrated improved transfection efficiency in HTB11 cells over LNPs formulated with Lipid 102 and DSPC.

[0682] EXAMPLE 4: LNPs in vitro toxicity testing

[0683] Luciferase mRNA encapsulated LNPs were transfected in HepG2, HTB11 and mouse DCs cell lines at 1mg / mL mRNA concentration. The LNPs used were formulated with Lipid 102 and Lipid 110 as described in Table 1. As a control, the commercial benchmark ionizable lipid SM-102 was used and formulated with the following molar ratios 40:9:49.5:1.5 of SM-102: DSPC: Cholesterol: DMG-PEG.

[0684] After 24 hrs, cells were analyzed for viability using Alamar Blue (Invitrogen, cat# A50100). As shown in Figure 13, none of the lipid formulations affected cell viability in any of the tested cell lines, demonstrating their overall safety.

[0685] EXAMPLE 5: In vivo biodistribution

[0686] Healthy BALB / c mice were intravenously (i.v) injected with LNPs encapsulated Luciferase mRNA at a dose of 5 pg per mouse. After 6 hours, animals were sacrificed; organs (heart, lung, spleen, liver, kidney) were excised and ex vivo embedded in D-luciferin solution for 5 minutes. Luciferase expression was measured as total flux of bioluminescent light (photons / minute) by using the IVIS imaging system.

[0687] The experiment was performed with LNPs formulated with Lipid 102 and Lipid 110. The same experiment was performed independently with LNPs formulated with Lipid 101, Lipid 105, Lipid 111, and Lipid 115. All LNPs were formulated according to Formulation F2 as described in Table 1. In both sets of these experiments, untreated mice served as the control (naive).

[0688] Luciferase expression in the liver, spleen, lungs, heart, and kidneys with LNPs formulated with Lipid 102 and Lipid 110 are shown in Figures 14A, C, E, G, and I, respectively. Luciferase expression in the liver, spleen, lungs, heart, and kidneys with LNPs formulated with Lipid 101, Lipid 105, Lipid 111, and Lipid 115 are shown in Figures 14B, D, F, H, and J, respectively. The results indicate that the tested lipids facilitated efficient in vivo delivery of Luciferase mRNA to the liver, spleen and lungs.

[0689] Ill

Claims

CLAIMSWhat is claimed is:

1. A lipid represented by the structure of Formula (II), Formula (III), or salts thereof, wherein the structures of Formula (II) and Formula (III) are represented below:Formula (II)whereinY is H or -xc-Zc-Lc;each one of xa, Xb, and xcis independently selected from the group consisting of: Co-12 alkylene, C2-12 alkenylene, C2-12 alkynylene and (CH2CH2O)n3;each one of Za, Zb, and Zcis independently selected from the group consisting of: absent,each one of La, Lb, and Lcis independently selected from the group consisting of: C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl, (CH2CH2O)n3R3, Co-12 alkylene-Ze-R3, C0-12alkylene-Ze-NR1R2, C0-12alkylene-N(C0-12alkylene-Ze-R3)2;Zeis independently in each instance selected from the group consisting of: absent,each one of R1and R2is independently selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl, or wherein R1and R2, together with the nitrogen to which they are bound, form a heterocycle;R3is selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl, C1-12 alkynyl, Co-12 alkylene-N(Ci-i2alkyl)2 and P(0)(0H)2;each one of nl and n2 and m is, independently, 1, 2, 3, 4 or 5; andn3 is an integer in the range of 1 to 45;Formula (III)whereinY is H or -xc-Zc-Lc;each one of xa, Xb, and xcis independently selected from the group consisting of: Co-12 alkylene, C2-12 alkenylene, C2-12 alkynylene and (CFkCFfcO)^;each one of Za, Zb, and Zcis independently selected from the group consisting of: absent,each one of La, Lb, and Lcis independently selected from the group consisting of: Ci-20 alkyl, C2-20 alkenyl, C2-20 alkynyl, (CH2CH2O)n3R3, Co-12 alkylene-Ze-R3, C0-12alkylene-Ze-NR1R2, C0-12alkylene-N(C0-12alkylene-Ze-R3)2;Zeis independently in each instance selected from the group consisting of: absent,each one of R1and R2is independently selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl, or wherein R1and R2, together with the nitrogen to which they are bound, form a heterocycle;R3selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl, C1-12 alkynyl, Co-12 alkylene-N(Ci-i2alkyl)2 and P(0)(0H)2;each one of nl and n2 and m is, independently, 1, 2, 3, 4 or 5; andn3 is an integer in the range of 1 to 45.

2. The lipid of claim 1, wherein Y is H, and the lipid is represented by Formula (Ila):Formula (Ila).

3. The lipid of claim 1, wherein Y is -xc-Zc-Lc, and the lipid is represented by Formula (lib):Formula (lib).

4. The lipid of claim 1, wherein the lipid is represented by Formula (lie):Z|..jFormula (lie)wherein each one of na and nb is each independently an integer in the range of 0-20.

5. The lipid of claim 1, wherein the lipid is represented by Formula (lid):Formula (lid).

6. The lipid of claim 1, wherein the lipid is represented by Formula (lie):La-N(Y)-(CH2)n1-(OCH2CH2)m-(CH2)n2-NH-Lb.

7. The lipid of any one of claims 1 to 4, wherein xais an unsubstituted Co-12 alkylene.

8. The lipid of claim 7, wherein xais absent, -CH2(CH2)8CH2- or -CH2(CH2)7CH2-.

9. The lipid of any one of claims 1 to 4, wherein Xb is an unsubstituted Co-12 alkylene.

10. The lipid of claim 9, wherein Xb is absent, -CH2(CH2)8CH2- or -CH2(CH2)7CH2-.

11. The lipid of any one of claims 1 to 4, wherein xcis an unsubstituted Co-12 alkylene.

12. The lipid of claim 11, wherein xcis absent or -CH2(CH2)7CH2-.

13. The lipid of any one of claims 1 to 4, wherein Zais selected from the group consisting of: absent, - C(O)O-, and -C(O)-.

14. The lipid of any one of claims 1 to 4, wherein Zb is selected from the group consisting of: absent, - C(O)O-, and -C(O)-.

15. The lipid of any one of claims 1 to 4, wherein Zcis absent or -C(O)O-.

16. The lipid of any one of claims 1 to 15, wherein Zeis absent or -C(O)O-.

17. The lipid of any one of claims 1 to 16, wherein Lais selected from the group consisting of: C4-18 alkyl, C4-18 alkenyl, (CH2CH2O)-C0-12alkylene-N(C1-12alkyl)2, and Co-12 alkylene-Ze-N(Ci-i2 alkyl)2.

18. The lipid of claim 17, wherein Lais selected from the group consisting of: -(CH2)11CH3, -CH2CH2-N(CH2(CH2)I0CH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -(CH2)3-N(CH2(CH2)IOCH3)2, -CH2(CH2)7CH2C(O)O-N((CH2)5CH3)2, -(CH2)4-N(CH2(CH2)1OCH3)2, -CH2CH2-N(CH2(CH2)5CH3)2, CH2CH2OCH2CH2N((CH2)5CH3)2, - CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3, and -N((CH2)5CH3)2.

19. The lipid of any one of claims 1 to 18, wherein Lb is C4-18 alkyl, (CH2CH20)-Co-i2-alkylene-N(Ci-i2alkyl)2, or Co-6 alkylene-N(Ci-i2 alkyl)2.

20. The lipid of claim 19, wherein Lb is (CH2CH20)-Co-6-alkylene-N(Ci-9alkyl)2, C8-14alkyl, or Co-4 alkylene-N(C4-i2 alkyl)2.

21. The lipid of claim 20, wherein Lb is selected from the group consisting of:-CH2CH2-N(CH2(CH2)10CH3)2, -CH[(CH2)5CH3](CH2)4CH3, -N((CH2)5CH3)2, -(CH2)3-N((CH2)IICH3)2, and -(CH2)4-N((CH2)nCH3)2, -CH2CH2-N((CH2)5CH3)2, and -CH2CH2OCH2CH2N((CH2)5CH3)2.

22. The lipid of any one of claims 1 to 21, wherein Lcis C4-18 alkyl, C4-18 alkenyl, or Co-6 alkylene-N(Ci- 12 alkyl )2.

23. The lipid of claim 22, wherein Lcis selected from the group consisting of:-(CH2)11CH3, -CH2(CH2)7(CH=CH)CH2(CH=CH)(CH2)4CH3, and -N((CH2)5CH3)2.

24. The lipid of claim 1, wherein xais absent; Zais absent or -C(O)O-;Lais selected from the group consisting of: Ce-18 alkyl, C6-20 alkenyl, and Co-12 alkylene-Zc-NR'R2; nl and n2 are each independently 1; and m is 1.

25. The lipid of claim 24, wherein Y is H; orY is -xc-Zc-Lcand wherein xcis absent; Zcis absent or -C(O)O-; and Lcis selected from the group consisting of: Ce-18 alkyl, C6-20 alkenyl, and Co-12 alkylene-Ze- NR'R2.

26. The lipid of claim 24 or 25, wherein Xb is Co-12 alkylene; Zb is -C(O)O-; and Lb is selected from the group consisting of: Co-12 alkylene-Zc-NR'R2and (CH2CH2O)n3R3, wherein n3 is 1-3 and R3is C0-9 alkylene-N(Ci-i2alkyl)2.

27. The lipid of any one of claims 24 to 26, wherein each one of R1and R2is independently C1-12 alkyl and wherein Zeis absent or -C(O)O-.

28. The lipid of claim 1, which is selected from the group consisting of:Lipid 101Lipid 102NV5-007Lipid 103Lipid 104NV5-003Lipid 105Lipid 106Lipid 107Lipid 108Lipid 109Lipid 110NV05-039Lipid 111NV05-040Lipid 112NV05-041Lipid 113Lipid 114Lipid 115Lipid 116Lipid 117Lipid 118Lipid 119Lipid 120Lipid 121Lipid 122Lipid 123Lipid 124Lipid 125 Lipid 126 Lipid 127 Lipid 128Lipid 129Lipid 130 Lipid 131Lipid 132Lipid 133Lipid 134Lipid 135 Lipid 136 Lipid 137Lipid 138Lipid 139Lipid 140Lipid 141Lipid 142Lipid 143 Lipid 144Lipid 145Lipid 146Lipid 147Lipid 150Lipid 151Lipid 152 Lipid 153Lipid 154Lipid 155Lipid 156 Lipid 157Lipid 158Lipid 159 Lipid 160Lipid 161Lipid 162Lipid 163Lipid 164Lipid 165Lipid 166Lipid 167Lipid 168Lipid 169Lipid 172Lipid 173and salts thereof.

29. The lipid according to claim 28, which is selected from the group consisting of: Lipid 102, Lipid 110, Lipid 101, Lipid 105, Lipid 111, and Lipid 115.

30. A particle comprising the lipid according to any one of claims 1 to 29 and a membrane stabilizing lipid.

31. The particle according to claim 30 comprising the membrane stabilizing lipid and a lipid membrane comprising the lipid.

32. The particle according to any one of claims 30 to 31, wherein the membrane stabilizing lipid is selected from the group consisting of cholesterol, phospholipids, cephalins, sphingolipids and glycoglycerolipids.

33. The particle according to any one of claims 30 to 32, wherein the membrane stabilizing lipid comprises cholesterol.

34. The particle according to any one of claims 30 to 33, further comprising one or more additional components selected from the group consisting of: a PEG-lipid conjugate, a neutral lipid and a charged lipid.

35. The particle according to claim 34, wherein the additional component comprises 1,2-Distearoyl-sn- glycero-3 -phosphocholine (DSPC) or 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

36. The particle according to claim 34 or 35, wherein the additional component comprises 1,2- Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG).

37. The particle according to any one of claims 30 to 36, comprising the lipid, cholesterol, 1,2-Distearoyl- sn-glycero-3 -phosphocholine (DSPC) and 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG).

38. The particle according to any one of claims 30 to 36, comprising the lipid, cholesterol, 1,2-Dioleoyl- sn-glycero-3 -phosphoethanolamine (DOPE) and 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG).

39. The particle according to any one of claims 30 to 38, which is conjugated to a targeting moiety.

40. The particle according to any one of claims 30 to 39, further comprising a nucleic acid encapsulated within a particle comprising the lipid.

41. The particle according to claim 40, wherein the nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), an antisense oligo nucleotide, a messenger RNA (mRNA), a ribozyme, a pDNA, a CRISPR mRNA, a gRNA, a circular RNA and an immune- stimulating nucleic acid.

42. The particle according to any one of claims 30 to 41, further comprising a therapeutic agent, wherein the therapeutic agent is encapsulated within a particle comprising the lipid.

43. The particle according to claim 42, wherein the therapeutic agent is an RNA comprising an open reading frame encoding a polypeptide that comprises a SARS-CoV-2 spike protein or an immunogenic fragment or variant thereof.

44. A pharmaceutical composition comprising a plurality of particles according to any one of claims 30 to 43 and a pharmaceutically acceptable carrier, diluent or excipient.

45. The pharmaceutical composition according to claim 44, being a liposomal composition.

46. A method of gene silencing, comprising contacting a cell with the pharmaceutical composition according claim 44 or 45.

47. The method according to claim 46, wherein the cell is a cancer cell.

48. A method for administering a therapeutic agent, the method comprising administering the pharmaceutical composition according to claim 44 or 45 to a subject in need thereof.

49. A method of treating a leukocyte associated condition, the method comprising the step of administering to a subject in need thereof the pharmaceutical composition according to claim 44 or 45.

50. The method according to claim 49, wherein the leukocyte associated condition is selected from the group consisting of cancer, infection, autoimmune diseases, neurodegenerative diseases and inflammation.

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