Ionizable lipids suitable for 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 SUITABLE FOR 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-C02-Co-3 alkylene- -N(CI-8 alkyl)2 and C1-6 alkyl-CO2- C0-3 alkylene-NH-Ci-s alkyl; R14is selected from the group consisting of: C1-13 alkyl; C2-15 alkenyl and

[0010] R1

[0011]

[0012] 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.

[0013] SUMMARY OF THE INVENTION

[0014] 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.

[0015] 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.

[0016] According to some embodiments, there is provided a lipid represented by the structure of Formula (I) or salt thereof:

[0017]

[0018] Formula (I)

[0019] wherein

[0020] each one of xa, Xb, xc, and xa is 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, Zc, and Za is independently selected from the group consisting of: absent,

[0022]

[0023]

[0024] each one of La, Lb, Lc, and La is independently selected from the group consisting of: C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl, (

[0025]

[0026] CEECEEC^nsR3, Co-12 alkylene-Ze-R3, Co-12 alkylene-Ze-NR1R2, Co-12 alkylene-N(Co-i2 alkylene-Ze-R3)2;

[0027] Zeis independently in each instance selected from the group consisting of: absent,

[0028]

[0029] 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;

[0030] 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; and

[0031] each one of nl and n2 and m is, independently, 1, 2, 3, 4 or 5;

[0032] and n3 is an integer in the range of 1 to 45. Each possibility represents a separate embodiment of the invention.

[0033] According to some embodiments, the lipid is represented by Formula (la):

[0034]

[0035] Formula (la) wherein each one of na, nb, nc, and nd is independently an integer in the range of 1 to 20, including each value within the specified range.

[0036] According to some embodiments, the lipid is represented by Formula (lb):

[0037]

[0038] Formula (lb).

[0039] According to some embodiments, xais the same as Xb, and xcis the same as xa. According to some embodiments, xa, Xb, xcand xa are the same.

[0040] According to some embodiments, Zais the same as Zb, and Zcis the same as Za. According to some embodiments, Za, Zb, Zcand Z are the same.

[0041] According to some embodiments, Lais the same as Lb, and Lcis the same as La. According to some embodiments, La, Lb, Lcand La are the same.

[0042] According to some embodiments, xa-Za-Lais the same as Xb-Zb-Lb, and xc-Zc-Lcis the same as xa-Za-La. According to some embodiments, xa-Za-La, Xb-Zb-Lb, xc-Zc-Lcand xa-Za-La are the same. According to some embodiments, xais a Co-12 linear alkylene. According to some embodiments, xais an unsubstituted Co-12 alkylene. According to some embodiments, xais an unsubstituted C0-9 alkylene. According to some embodiments, xais an unsubstituted C0-4 alkylene. According to some embodiments, xais selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, -CH2(CH2)4CH2- and -CH2(CH2)7CH2-. Each possibility represents a separate embodiment of the invention.

[0043] According to some embodiments, Xb is a Co-12 linear alkylene. According to some embodiments, Xb is an unsubstituted Co-12 alkylene. According to some embodiments, Xb is an unsubstituted C0-9 alkylene. According to some embodiments, Xb is an unsubstituted C0-4 alkylene. According to some embodiments, xb is selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, -CH2(CH2)4CH2- and -CH2(CH2)7CH2-. Each possibility represents a separate embodiment of the invention. According to some embodiments, xcis a Co-12 linear alkylene. According to some embodiments, xcis an unsubstituted Co-12 alkylene. According to some embodiments, xcis an unsubstituted Co-6 alkylene. According to some embodiments, xcis an unsubstituted Co-4 alkylene. According to some embodiments, xcis selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, and -CH2(CH2)4CH2-. Each possibility represents a separate embodiment of the invention. According to some embodiments, xcis -CH2CH2-.

[0044] According to some embodiments, xa is a Co-12 linear alkylene. According to some embodiments, xa is an unsubstituted Co-12 alkylene. According to some embodiments, xa is an unsubstituted Co-6 alkylene. According to some embodiments, xa is an unsubstituted Co-4 alkylene. According to some embodiments, xa is selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, and -CH2(CH2)4CH2-. Each possibility represents a separate embodiment of the invention. According to some embodiments, xa is -CH2CH2-.

[0045] According to some embodiments, Zais selected from the group consisting of: absent, -OC(O)-, -OC(O)NH-, -NHC(O)O-, and -C(O)O-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Zais -OC(O)-.

[0046] According to some embodiments, Zb is selected from the group consisting of: absent, -OC(O)-, -OC(O)NH-, -NHC(O)O-, and -C(O)O-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Zb is -OC(O)-.

[0047] According to some embodiments, Zcis selected from the group consisting of: absent, -O-, -OC(O)-, -OC(O)NH-, -NHC(O)O-, and -C(O)O-. Each possibility represents a separate embodiment. According to some embodiments, Zcis selected from absent, -O-and -OC(O)-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Zcis selected from -OC(O)- and -O-. Each possibility represents a separate embodiment of the invention.

[0048] According to some embodiments, Za is selected from the group consisting of: absent, -O-, -OC(O)-, -OC(O)NH-, -NHC(O)O-, and -C(O)O-. Each possibility represents a separate embodiment. According to some embodiments, Za is selected from -OC(O)- and -O-. Each possibility represents a separate embodiment of the invention. According to some embodiments, Zeis selected from the group consisting of: absent, -O-, and -C(O)O-. Each possibility represents a separate embodiment of the invention. Lais selected from the group consisting of: C4-18 alkyl, C4-18 alkenyl, and Co-6 alkylene-Ze-N(Ci-i2 alkyl)2. Each possibility represents a separate embodiment of the invention.

[0049] According to some embodiments, Lais selected from the group consisting of: C4-18 alkyl, C4-18 alkenyl, and Co-6 alkyl ene-N(C 1-12 alkyl)2. Each possibility represents a separate embodiment. According to some embodiments, Lais selected from the group consisting of: Cs-14 alkyl, C15-18 alkenyl and C0-4 alkyl ene-N(Ce- 12 alkyl)2. According to some embodiments, Lais selected from the group consisting of: -(CE^ioCEE, -(CH2)nCH3, -(CEh^CEE, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -CH2CH2-N((CH2)7CH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -N((CH2)7CH3)2, -(CH2)4-N((CH2)? CH3)2, and -(CH₂)₇-CH=CH-(CH₂)₇CH₃. Each possibility represents a separate embodiment of the invention.

[0050] According to some embodiments, Lb is selected from the group consisting of: C4-18 alkyl, C4-18 alkenyl, and Co-6 alkylene-Ze-N(Ci-i2 alkyl)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lb is selected from the group consisting of: C4-18 alkyl, C4-18 alkenyl, and Co-6 alkylene-N(Ci-i2 alkyl)2. Each possibility represents a separate embodiment. According to some embodiments, Lb is selected from the group consisting of: Cs-i4 alkyl, C15-18 alkenyl and C0-4 alkylene-N(Ce-i2 alkyl)2. According to some embodiments, Lb is selected from the group consisting of: -(CH2)IOCH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)IICH3, -(CH2)8CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -CH2CH2N((CH2)7CH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -N((CH2)7CH3)2, -(CH2)4-N((CH2)7CH3)2, and -(CH2)7-CH=CH-(CH2)? CH3. Each possibility represents a separate embodiment of the invention.

[0051] According to some embodiments, Lcis selected from the group consisting of: Co-6 alkylene-Ze-R3, C4-18 alkyl, C4-18 alkenyl, and Co-6 alkylene-Ze-N(Ci-i2 alkyl)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, Lcis selected from the group consisting of: Co-6 alkylene-OH, C4-18 alkyl, C4-18 alkenyl, and Co-6 alkylene-N(Ci-i2 alkyl)2. Each possibility represents a separate embodiment. According to some embodiments, Lcis selected from the group consisting of: Cs-14 alkyl, C15-18 alkenyl, C0-4 alkylene-N(Ce-i2 alkyl)2, and - CH2CH2OH. According to some embodiments, Lcis selected from the group consisting of: -CH2CH2-OH, -(CH2)4OH, -(CH2)3OH, -(CH2)IOCH3, -(CH2)7-CH=CH-CH2- CH=CH-(CH2)4CH3, -(CH2)HCH3, -(CH2)8CH3, -CH2CH2N((CH2)7CH3)2, H[CH2(CH2)4CH3]CH2(CH2)3CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3 and -(CH2)7-CH=CH-(CH2)7CH3. Each possibility represents a separate embodiment of the invention.

[0052] According to some embodiments, La is selected from the group consisting of: Co-6 alkylene-Ze- R3, C4-i8alkyl, C4-i8alkenyl, and Co-6 alkylene-Ze-N(Ci-i2alkyl)2. According to some embodiments, La is selected from the group consisting of: Co-6 alkylene-OH, C4-is alkyl, C4-18 alkenyl, and Co-6 alkyl ene-N(C 1-12 alkyl)2. Each possibility represents a separate embodiment. According to some embodiments, La is selected from the group consisting of: C8-i4alkyl, C15-18 alkenyl, Co-4alkylene-N(Ce-i2 alkyl)2, and -CH2CH2OH. According to some embodiments, La is selected from the group consisting of: - CH2CH2-OH, -(CH2)4OH, -(CH2)3OH, -(CH2)10CH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -CH2CH2N((CH2)7CH3)2, -(CH2)IICH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)7-CH=CH-(CH2)7CH3, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, and -(CEDsCEE. Each possibility represents a separate embodiment of the invention.

[0053] According to some embodiments, m is 1, 2, or 3 and each one of nl and n2 is independently 1 or 2.

[0054] According to some embodiments, xais absent; Zais absent; and Lais selected from the group consisting of: Ce-i8alkyl, Ce-18 alkynyl, and C2-4alkylene-Ze-R3, wherein Zeis selected from -O-, -OC(O)-, and -C(O)O- and R3is Ce-18 alkyl.

[0055] According to some embodiments, xb is absent; Zb is absent; and Lb is selected from the group consisting of: Ce-i8alkyl, Ce-18 alkynyl, and C2-4alkylene-Ze-R3, wherein Zeis selected from -O-, -OC(O)-, and -C(O)O- and R3is Ce-18 alkyl.

[0056] According to some embodiments, xcis absent; Zcis absent; and Lcis selected from the group consisting of: Ce-i8alkyl, C2-6 alkylene-Ze-R3, and (CELCEEO^R3, wherein n3 is 1-4, wherein Zeis selected from -O-, -OC(O)-, and -C(O)O-, and R3is H. According to some embodiments, xa is absent; Zais absent; and La is selected from the group consisting of: Ce-18 alkyl, C2-6 alkylene- Ze-R3, and (CH2CH2O)n3R3, wherein n3 is 1-4, wherein Zeis selected from -O-, -OC(O)-, and - C(O)O-, and R3is H.

[0057] According to some embodiments, the lipid is selected from the group consisting of: Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, and Lipid 20. Each possibility represents a separate embodiment of the invention.

[0058] According to some embodiments, the lipid is selected from the group consisting of: Lipid 3, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, and Lipid 17. Each possibility represents a separate embodiment of the invention.

[0059] According to some embodiments, the lipid is selected from the group consisting of: Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23, Lipid 24, Lipid 25, Lipid 26, Lipid 27, Lipid 28, Lipid 29, Lipid 30, Lipid 31, Lipid 32, Lipid 33, Lipid 34, Lipid 35, Lipid 36, Lipid 37, Lipid 38, Lipid 39, Lipid 40, Lipid 41, Lipid 42, Lipid 43, Lipid 44, Lipid 45, Lipid 46, Lipid 47, Lipid 48, Lipid 49, Lipid 50, Lipid 51, Lipid 52, Lipid 53, Lipid 54, Lipid 55, Lipid 56, Lipid 57, Lipid 58, and salts thereof. Each possibility represents a separate embodiment of the invention.

[0060] According to some embodiments, the lipid is selected from the group consisting of: Lipid 11, Lipid 20, Lipid 19, Lipid 13, Lipid 18, Lipid 21, Lipid 31, Lipid 32, Lipid 23, Lipid 24, Lipid 27, and salts thereof. Each possibility represents a separate embodiment of the invention.

[0061] 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.

[0062] 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-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE) or 1,2-Distearoyl-sn-glycero-3 -phosphocholine (DSPC). Each possibility represents a separate embodiment. According to some embodiments, the additional component comprises 1,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE). According to some embodiments, the additional component comprises l,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 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, l,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.

[0063] 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.

[0064] 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. 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.

[0065] 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.

[0066] 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.

[0067] According to some embodiments, the compositions of the present invention may be used as a delivery system to administer a therapeutic agent to its 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.

[0068] 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.

[0069] 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. The leukocyte associated condition may be selected from the group consisting of cancer, infection, autoimmune diseases, neurodegenerative diseases and inflammation.

[0070] 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.

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1A is a 'H NMR spectrum of Lipid 11 (NV5-009).

[0073] Figure 1B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 11 (NV5-009).

[0074] Figure 2A is a 'H NMR spectrum of Lipid 12 (NV5-010).

[0075] Figure 2B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 12 (NV5-010).

[0076] Figure 3A is a 'H NMR spectrum of Lipid 14 (NV5-011).

[0077] Figure 3B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 14 (NV5-011).

[0078] Figure 4A is a 'H NMR spectrum of Lipid 15 (NV5-012).

[0079] Figure 4B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 15 (NV5-012).

[0080] Figure 5A is a 'H NMR spectrum of Lipid 3 (NV5-013).

[0081] Figure 5B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 3 (NV5-013).

[0082] Figure 6A is a 'H NMR spectrum of Lipid 16 (NV5-014).

[0083] Figure 6B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 16 (NV5-014).

[0084] Figure 7A is a 'H NMR spectrum of Lipid 20 (NV5-015).

[0085] Figure 7B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 20 (NV5-015).

[0086] Figure 8A is a 'H NMR spectrum of Lipid 19 (NV5-016). Figure 8B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 19 (NV5-016).

[0087] Figure 9A is a1H NMR spectrum of Lipid 13 (NV5-017).

[0088] Figure 9B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 13 (NV5-017).

[0089] Figure 10A is a 'H NMR spectrum of Lipid 18 (NV5-018).

[0090] Figure 10B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 18 (NV5-018).

[0091] Figure 11A is a1H NMR spectrum of Lipid 23 (NV5-019).

[0092] Figure 11B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 23 (NV5-019).

[0093] Figure 12A is a1H NMR spectrum of Lipid 24 (NV5-020).

[0094] Figure 12B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 24 (NV5-020).

[0095] Figure 13A is a1H NMR spectrum of Lipid 27 (NV5-021).

[0096] Figure 13B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 27 (NV5-021).

[0097] Figure 14A is a1H NMR spectrum of Lipid 28 (NV5-022).

[0098] Figure 14B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 28 (NV5-022).

[0099] Figure 15A is a1H NMR spectrum of Lipid 21 (NV5-024).

[0100] Figure 15B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 21 (NV5-024).

[0101] Figure 16A is a1H NMR spectrum of Lipid 22 (NV5-025).

[0102] Figure 16B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 22 (NV5-025). Figure 17A is a1H NMR spectrum of Lipid 29 (NV5-026).

[0103] Figure 17B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 29 (NV5-026).

[0104] Figure 18A is a1H NMR spectrum of Lipid 30 (NV5-027).

[0105] Figure 18B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 30 (NV5-027).

[0106] Figure 19A is a1H NMR spectrum of Lipid 25 (NV5-028).

[0107] Figure 19B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 25 (NV5-028).

[0108] Figure 20A is a1H NMR spectrum of Lipid 31 (NV5-029).

[0109] Figure 20B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 31 (NV5-029).

[0110] Figure 21A is a1H NMR spectrum of Lipid 33 (NV5-034).

[0111] Figure 21B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 33 (NV5-034).

[0112] Figure 22A is a1H NMR spectrum of Lipid 34 (NV5-036).

[0113] Figure 22B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 34 (NV5-036).

[0114] Figure 23A is a1H NMR spectrum of Lipid 35 (NV5-037).

[0115] Figure 23B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 35 (NV5-037).

[0116] Figure 24A is a1H NMR spectrum of Lipid 36 (NV5-038).

[0117] Figure 24B is an Electrospray Ionization Mass Spectrometry (ESI-MS) spectrum of Lipid 36 (NV5-038).

[0118] Figure 25A is a bar chart that represents the transfection efficiencies of luciferase mRNA-encapsulated LNPs, formulated with (from left to right) Lipid 11 (NV5-009, Formulation F2.9), Lipid 20 (NV5-015, Formulation F2.3), Lipid 20 (NV5-015, Formulation F2.9), Lipid 19 (NV5- 016, Formulation F2.3), Lipid 19 (NV5-016, Formulation F2.9), Lipid 13 (NV5-017, Formulation F2.9), Lipid 23 (NV5-019, Formulation F2.9), Lipid 24 (NV5-020, Formulation F2.9), Lipid 27 (NV5-021, Formulation F2.9), Lipid 21 (NV5-024, Formulation F2.3), Lipid 21 (NV5-024, Formulation Fl.2), Lipid 32 (NV5-032, Formulation Fl.2), Lipid 32 (NV5-032, Formulation F2.3), Lipid 32 (NV5-032, Formulation F2.9) in HepG2 cells, at concentration of mRNA of 25ng / ml (horizontal stripes), 50ng / ml (solid grey fill), lOOng / ml (checkered fill) and 200ng / ml (dotted fill).

[0119] Figure 25B is a bar chart that represents the transfection efficiencies of luciferase mRNA-encapsulated LNPs, formulated with (from left to right) Lipid 11 (NV5-009, Formulation F2.9), Lipid 20 (NV5-015, Formulation F2.3), Lipid 20 (NV5-015, Formulation F2.9), Lipid 19 (NV5-016, Formulation F2.3), Lipid 19 (NV5-016, Formulation F2.9), Lipid 13 (NV5-017, Formulation F2.9), Lipid 21 (NV5-024, Formulation F2.3), Lipid 31 (NV5-029, Formulation Fl.2), Lipid 32 (NV5-032, Formulation Fl.2), Lipid 32 (NV5-032, Formulation F2.3), Lipid 32 (NV5-032, Formulation F2.9) in HTB11 cells, at mRNAs concentration of 25ng / ml (horizontal stripes), 50ng / ml (solid grey fill), lOOng / ml (checkered fill) and 200ng / ml (dotted fill).

[0120] Figure 26A is a bar chart that represents the viability of HepG2 cells treated with luciferase mRNA-encapsulated LNPs formulated with (from left to right) Lipid 11 (NV5-009, Formulation F2.9), Lipid 20 (NV5-015, Formulation F2.3), Lipid 20 (NV5-015, Formulation F2.9), Lipid 19 (NV5-016, Formulation F2.3), Lipid 19 (NV5-016, Formulation F2.9), Lipid 13 (NV5-017, Formulation F2.9), Lipid 23 (NV5-019, Formulation F2.9), Lipid 24 (NV5-020, Formulation F2.9), Lipid 27 (NV5-021, Formulation F2.9), Lipid 21 (NV5-024, Formulation F2.3), Lipid 21 (NV5-024, Formulation Fl.2), Lipid 32 (NV5-032, Formulation Fl.2), Lipid 32 (NV5-032, Formulation F2.3), Lipid 32 (NV5-032, Formulation F2.9) and 1 mg / mL mRNA.

[0121] Figure 26B is a bar chart that represents the viability of HTB11 cells treated with luciferase mRNA-encapsulated LNPs formulated with (from left to right) Lipid 11 (NV5-009, Formulation F2.9), Lipid 20 (NV5-015, Formulation F2.3), Lipid 20 (NV5-015, Formulation F2.9), Lipid 19 (NV5-016, Formulation F2.3), Lipid 19 (NV5-016, Formulation F2.9), Lipid 13 (NV5-017, Formulation F2.9), Lipid 18 (NV5-018, Formulation F2.3), Lipid 21 (NV5-024, Formulation F2.3), Lipid 31 (Formulation Fl.2), Lipid 32 (NV5-032, Formulation Fl.2), Lipid 32 (NV5-032, Formulation F2.3), Lipid 32 (NV5-032, Formulation F2.9) and 1 mg / mL mRNA. Figures 27A-J are bar graphs representing Luciferase expression detected in mice liver, spleen, lungs, heart and kidneys by IVIS imaging following systemic injection of LNPs formulated with F2.3 Formulation of Lipid 20 (NV5-015), Lipid 21 (NV5-024), Lipid 11 (NV5-009), and Lipid 32 (NV5-032) and loaded with luciferase mRNA to BALB / c mice. Figure 27A: Luciferase expression in liver following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 20; Figure 27B: Luciferase expression in liver following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 21, Lipid 11, and Lipid 32; Figure 27C: Luciferase expression in spleen following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 20; Figure 27D: Luciferase expression in spleen following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 21, Lipid 11, and Lipid 32; Figure 27E: Luciferase expression in lungs following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 20; Figure 27F: Luciferase expression in lungs following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 21, Lipid 11, and Lipid 32; Figure 27G: Luciferase expression in heart following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 20;

[0122] Figure 27H: Luciferase expression in heart following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 21, Lipid 11, and Lipid 32; Figure 271: Luciferase expression in kidneys following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 20; Figure 27 J: Luciferase expression in kidneys following systemic injection of luciferase mRNA-encapsulated LNPs formulated with Lipid 21, Lipid 11, and Lipid 32. Naive mice in all experiments represent untreated mice that served as controls.

[0123] DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0124] 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.

[0125] Lipids

[0126] As contemplated herein, the present invention relates to a lipid represented by the structure of Formula (I):

[0127]

[0128] Formula (I)

[0129] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.

[0130] According to some embodiments, the lipid of Formula (I) is represented by the structure of Formula (la):

[0131]

[0132] Formula (la)

[0133] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof.

[0134] 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, nc is an integer in the range of 0-20. 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, nd is an integer in the range of 0-20. According to some embodiments, nd is an integer in the range of 1-20. According to some embodiments, nd is an integer in the range of 1- 12. According to some embodiments, na is 0. According to some embodiments, na is 2. According to some embodiments, na is 4. According to some embodiments, na is 6. According to some embodiments, na is 9. According to some embodiments, nb is 0. According to some embodiments, nb is 2. According to some embodiments, nb is 4. According to some embodiments, nb is 6. According to some embodiments, nb is 9. According to some embodiments, nc is 0. According to some embodiments, nc is 2. According to some embodiments, nc is 4. According to some embodiments, nc is 6. According to some embodiments, nc is 9. According to some embodiments, nd is 0. According to some embodiments, nd is 2. According to some embodiments, nd is 4. According to some embodiments, nd is 6. According to some embodiments, nd is 9.

[0135] According to some embodiments, the lipid of Formula (I) is represented by the structure of Formula (lb):

[0136]

[0137] Formula (lb)

[0138] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. According to some embodiments, xa(e.g., in Formula (I)) is selected from an alkylene, an alkenylene, and an alkynylene. Each possibility represents a separate embodiment. 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. Each possibility represents a separate embodiment of the invention. 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 a substituted 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 Co-6 alkylene. According to some embodiments, xais Co-4 alkylene. According to some embodiments, xais Co. According to some embodiments, xais selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, -CH2(CH2)4CH2-, and -CH2(CH2)7CH2-. According to some embodiments, xais absent. According to some embodiments, xais -CH2CH2-. According to some embodiments, xais -CH2CH2CH2CH2-. According to some embodiments, xais -CH2(CH2)4CH2-. According to some embodiments, xais -CH2(CH2)7CH2-.

[0139] 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.

[0140] 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. 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-.

[0141] According to some embodiments, Xb (e.g., in Formula (I)) is selected from an alkylene, an alkenylene, and an alkynylene. Each possibility represents a separate embodiment. 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 of the invention. 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 a substituted 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 Co-6 alkylene. According to some embodiments, Xb is Co-4 alkylene. According to some embodiments, Xb is Co. According to some embodiments, xb is selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, -CH2(CH2)4CH2- and -CH2(CH2)7CH2-. According to some embodiments, xb is absent. According to some embodiments, Xb is -CH2CH2-. According to some embodiments, Xb is -CH2CH2CH2CH2-. According to some embodiments, Xb is -CH2(CH2)4CH2-. According to some embodiments, Xb is -CH2(CH2)7CH2-. 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 alkenylene. 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. 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-.

[0142] According to some embodiments, xc(e.g., in Formula (I)) is selected from an alkylene, an alkenylene, and an alkynylene. Each possibility represents a separate embodiment. 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 of the invention. 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 a substituted 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 Co. According to some embodiments, xcis selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, and -CH2(CH2)4CH2-. According to some embodiments, xcis absent. According to some embodiments, xcis -CH2CH2-. According to some embodiments, xcis -CH2CH2CH2CH2-. According to some embodiments, xcis -CH2(CH2)4CH2-.

[0143] 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.

[0144] According to some embodiments, xcis an alkynylene. According to some embodiments, xcis a linear alkynylene or a branched alkynylene. 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, xd(e.g., in Formula (I)) is selected from an alkylene, an alkenylene, and an alkynylene. Each possibility represents a separate embodiment. According to some embodiments, xdis 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, xdis an alkylene. According to some embodiments, xdis a linear alkylene or a branched alkylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xdis a linear alkylene. According to some embodiments, xdis a branched alkylene. According to some embodiments, xdis an unsubstituted alkylene. According to some embodiments, xdis a substituted alkylene. According to some embodiments, xdis C0-12 alkylene. According to some embodiments, xdis C0-9 alkylene. According to some embodiments, xdis C0-6 alkylene. According to some embodiments, xdis C0-4 alkylene. According to some embodiments, xdis C0. According to some embodiments, xdis selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, and -CH2(CH2)4CH2-. According to some embodiments, xdis absent. According to some embodiments, xdis -CH2CH2-. According to some embodiments, xdis -CH2CH2CH2CH2-. According to some embodiments, xdis -CH2(CH2)4CH2-.

[0145] According to some embodiments, xdis an alkenylene. According to some embodiments, xdis a linear alkenylene or a branched alkenylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xdis a linear alkenylene. According to some embodiments, xdis a branched alkenylene. According to some embodiments, xdis an unsubstituted alkenylene. According to some embodiments, xdis C2-12 alkenylene. According to some embodiments, xdis C2-9 alkenylene. According to some embodiments, xdis C2-6 alkenylene. According to some embodiments, xdis an alkynylene. According to some embodiments, xdis a linear alkynylene or a branched alkynylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, xdis a linear alkynylene. According to some embodiments, xdis a branched alkynylene. According to some embodiments, xdis an unsubstituted alkynylene. According to some embodiments, xdis C2-12 alkynylene. According to some embodiments, xdis C2-9 alkynylene. According to some embodiments, xdis C2-6 alkynylene. According to some embodiments, xdincludes a monoethylene glycol or polyethylene glycol (PEG) e.g., (CH2CH2O)n3, wherein n3 is a positive integer. According to some embodiments, xdis (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, xdis -CH2CH2O-. According to some embodiments, xdis -CH2CH2OCH2CH2O-.

[0146] According to some embodiments, each one of xa, Xb, xcand Xd is 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, xcand xd is independently Co-12 alkylene” requires that the aforementioned substituents are each Co-12 alkylene, however, it does not require that xa, xb, xcand xd are the same alkylene, e.g., xaand xd may be alkylenes of the same or different lengths or branching / linear pattern. For example, in Lipid 1, shown herein, each one of xa, Xb, xcand Xd is independently Co-12 alkylene, wherein xa, Xb, xcand Xd are the same, i.e., Co alkylene. In another example, Lipid 3, shown herein, each one of xa, Xb, xcand Xd is independently Co-12 alkylene, wherein xa, Xb, xcand Xd are the same, i.e., -CH2CH2-. In contrast, for example, Lipid 7, shown herein, each one of xa, Xb, xcand Xd is independently Co-12 alkylene, wherein xaand Xd are different, i.e., xais -CH2CH2CH2CH2- and xdis -CH2CH2-.

[0147] According to some embodiments, each one of xa, Xb, xcand Xd is independently Co-12 alkylene. According to some embodiments, each one of xa, Xb, xcand Xd is independently unsubstituted Co-12 alkylene. According to some embodiments, each one of xa, Xb, xcand Xd is independently C0-9 alkylene. According to some embodiments, each one of xa, Xb, xcand Xd is independently Co-6 alkylene. According to some embodiments, each one of xa, Xb, xcand Xd is independently C0-4 alkylene. According to some embodiments, each one of xa, Xb, xcand Xd is independently Co. According to some embodiments, the alkylene group of each one of xa, xb, xcand xdis independently linear. According to some embodiments, the alkylene group of each one of xa, xb, xcand xdis independently branched. According to some embodiments, each one of xa, xb, xcand xd is independently selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-and -CH2(CH2)4CH2-. According to some embodiments, each one of xa, xb, xcand xd is independently absent. According to some embodiments, each one of xa, xb, xcand xd is independently -CH2CH2-. According to some embodiments, each one of xa, xb, xcand xd is independently -CH2CH2CH2CH2-. According to some embodiments, each one of xa, xb, xcand xd is independently -CH2(CH2)4CH2-.

[0148] According to some embodiments, each one of xa, Xb, xcand Xd is independently alkenylene. According to some embodiments, each one of xa, Xb, xcand Xd is independently C2-12 alkenylene. Each possibility represents a separate embodiment of the invention. According to some embodiments, each one of xa, Xb, xcand Xd is independently C2-9 alkenylene. According to some embodiments, each one of xa, Xb, xcand Xd is independently C2-6 alkenylene. According to some embodiments, the alkenylene in each one of xa, Xb, xcand Xd is independently unsubstituted. According to some embodiments, the alkenylene in each one of xa, Xb, xcand Xd is independently linear. According to some embodiments, the alkenylene in each one of xa, Xb, xcand Xd is independently branched.

[0149] According to some embodiments, each one of xa, Xb, xcand Xd is independently alkynylene. According to some embodiments, each one of xa, Xb, xcand Xd is independently C2-12 alkynylene. According to some embodiments, the alkynylene in each one of xa, Xb, xcand Xd is independently unsubstituted. According to some embodiments, the alkynylene in each one of xa, Xb, xcand Xd is independently linear. According to some embodiments, the alkynylene in each one of xa, Xb, xcand Xd is independently branched.

[0150] According to some embodiments, each one of xa, xb, xc, and xdis 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 50, 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 50, 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, 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, each one of xa, xb, xc, and xdis independently -CH2CH2O-. According to some embodiments, each one of xa, xb, xc, and xdis independently -CH2CH2OCH2CH2O-.

[0151] According to some embodiments, xais the same as Xb. According to some embodiments, xcis the same as xd. According to some embodiments, xaand xbare different from xcand xd. It is to be understood that the term “the same” means that the two 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 7, shown herein, has the same xaand xb, each of which is -CH2CH2CH2CH2-, as well as the same xcand xd, each of which is -CH2CH2-. Also, in Lipid 7, xaand xbare different from xcand xd. According to some embodiments, xaand xbare each -CH2CH2CH2CH2- and xcand xdare each -CH2CH2-. According to some embodiments, xaand xbare each -CH2(CH2)7CH2- and xcand xdare each absent. According to some embodiments, xaand xbare each absent and xcand xdare each -CH2CH2-.

[0152] According to some embodiments, xa, xb, xcand xa are the same. It is to be understood that the term “the same” means that the four specified x substituents have the same chemical structure. For example, Lipid 2, shown herein, has the same xa, xb, xcand xd, each of which is -CH2CH2CH2CH2-

[0153] 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, -OC(O)-, -OC(O)NH-, -NHC(O)O-, -C(O)O-, and -O-. According to some embodiments, Zais selected from the group consisting of absent, -OC(O)-, -OC(O)NH-, -C(O)O-, and -O-. According to some embodiments, Zais absent. According to some embodiments, Zais -OC(O)-. According to some embodiments, Zais -OC(O)NH-. According to some embodiments, Zais -NHC(O)O-. According to some embodiments, Zais -C(O)O-. According to some embodiments, Zais -O-.

[0154] 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, -OC(O)-, -OC(O)NH-, -C(O)O-, and -O-. According to some embodiments, Zb is absent. According to some embodiments, Zb is -OC(O)-. According to some embodiments, Zb is -OC(O)NH-. According to some embodiments, Zb is -C(O)O-. According to some embodiments, Zb is -O-.

[0155] 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 selected from the group consisting of: absent, -OC(O)-, -OC(O)NH-, -NHC(O)O-, -C(O)O-, and -O-. According to some embodiments, Zcis selected from the group consisting of: absent, -OC(O)-, -OC(O)NH-, -C(O)O-, and -O-. According to some embodiments, Zcis absent. According to some embodiments, Zcis -OC(O)-. According to some embodiments, Zcis -C(O)O-. According to some embodiments, Zcis -O-. According to some embodiments, Zd 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, Zd is selected from the group consisting of: absent, -OC(O)-, -OC(O)NH-, -C(O)O-, and -O-. According to some embodiments, Zd is absent. According to some embodiments, Zd is -OC(O)-. According to some embodiments, Zd is -OC(O)NH-. According to some embodiments, Zdis -C(O)O-. According to some embodiments, Zdis -O-.

[0156] According to some embodiments, each one of Za, Zb, Zcand Zd is 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, Zcand Zd is independently selected from the group consisting of: absent, -OC(O)-, -OC(O)NH-, -C(O)O-, and -O-. According to some embodiments, each one of Za, Zb, Zcand Zdis independently absent. According to some embodiments, each one of Za, Zb, Zcand Zd is independently -OC(O)-. According to some embodiments, each one of Za, Zb, Zcand Zd is independently -OC(O)NH-. According to some embodiments, each one of Za, Zb, Zcand Zd is independently -C(O)O-. According to some embodiments, each one of Za, Zb, Zcand Zd is independently -O-.

[0157] According to some embodiments, Zais the same as Zb. According to some embodiments, Zcis the same as Zd. According to some embodiments, Zaand Zb are different from Zcand Zd. It is to be understood that the term “the same” means that the two 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 8, shown herein, has the same Zaand Zb, each of which is -C(O)O-, as well as the same Zcand Zd, each of which is -O-. Also in Lipid 8, Zaand Zd are different. According to some embodiments, Zaand Zb are both -C(O)O- and Zcand Zd are both -O-. According to some embodiments, Zaand Zb are both -C(O)O- and Zcand Zd are both absent. According to some embodiments, Zaand Zb are both absent and Zcand Zd are both -O-.

[0158] According to some embodiments, Za, Zb, Zcand Zd are the same. It is to be understood that the term “the same” means that each one of the four specified Z substituents have the same chemical structure. For example, Lipid 1, shown herein, has the same Za, Zb, Zcand Zd, each of which is absent.

[0159] According to some embodiments, Zeais selected from the group consisting of: absent,

[0160]

[0161] represents a separate embodiment. According to some embodiments, Zeais selected from the group consisting of: absent, -O-, -OC(O)-, and -C(O)O-. According to some embodiments, Zeais absent. According to some embodiments, Zeais -O-. According to some embodiments, Zeais -OC(O)-. According to some embodiments, Zeais -C(O)O-.

[0162] According to some embodiments, Zebis selected from the group consisting of: absent,

[0163] <3 O

[0164] , |J ■, Il 0

[0165] Iff "oj -H ^s's\a"

[0166]

[0167] d

[0168] H l:" ' ' '1 S? T‘. Each possibility represents a separate embodiment. According to some embodiments, Zebis selected from the group consisting of: absent, -O-, -OC(O)-, and -C(O)O-. According to some embodiments, Zebis absent. According to some embodiments, Zebis -O-. According to some embodiments, Zebis -OC(O)-. According to some embodiments, Zebis -C(O)O-.

[0169] According to some embodiments, Zecis selected from the group consisting of: absent,

[0170]

[0171] represents a separate embodiment. According to some embodiments, Zecis selected from the group consisting of: absent, -O-, -OC(O)-, and -C(O)O-. According to some embodiments, Zecis selected from absent and -O-. According to some embodiments, Zecis absent. According to some embodiments, Zecis -O-. According to some embodiments, Zecis -OC(O)-. According to some embodiments, Zecis -C(O)O-.

[0172] According to some embodiments, Zedis selected from the group consisting of: absent,

[0173]

[0174] c?0o

[0175] th' " O'). ted m |s V and

[0176]

[0177] H ' '• OH -1' < > ' 1 7 ' 1sI 1 /

[0178] ' '' Each possibility represents a separate embodiment. According to some embodiments, Zedis selected from the group consisting of: absent, -O-, -OC(O)-, and -C(O)O-. According to some embodiments, Zedis selected from absent and -O-. According to some embodiments, Zedis absent. According to some embodiments, Zedis -O-. According to some embodiments, Zedis -OC(O)-. According to some embodiments, Zedis -C(O)O-.

[0179] According to some embodiments, Lais selected from the group consisting of: alkyl, alkenyl, alkynyl, alkylene-NRlaR2a, alkenelyne-NRlaR2a, 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, 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. Each possibility represents a separate embodiment of the invention. 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 a substituted alkyl, as shown, for example, in Lipid 39 (a hydroxyalkyl: 2-hydroxydodecyl). According to some embodiments, Lais C1-20 alkyl. According to some embodiments, Lais C4-18 alkyl. According to some embodiments, Lais Cs-i4 alkyl. According to some embodiments, Lais C9-12 alkyl. According to some embodiments, Lais selected from the group consisting of: -(CH2)10CH3, -(CH2)11CH3, -(CH2)8CH3 and -CH[CH2(CH2)4CH3]CH2(CH2)3CH3. According to some embodiments, Lais -(CH2)10CH3. According to some embodiments, Lais -(CH2)11CH3. According to some embodiments, Lais -(CH2)8CH3. According to some embodiments, Lais -CH[CH2(CH2)4CH3]CH2(CH2)3CH3. 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 C2-20 monoenyl. 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 C2-20 dienyl. 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 selected from the group consisting of: -(CH2)?-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, and -(CH2)7-CH=CH-(CH2)7CH3. According to some embodiments, Lais -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, Lais -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, Lais -(CH₂)₇-CH=CH-(CH₂)₇CH₃.

[0180] 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-NRlaR2a. According to some embodiments, Lais a linear or branched alkylene-Zea-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 C0-9 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 Co alkylene-NRlaR2arefers to -NRlaR2a. According to some embodiments, Lais -CH2CH2-NRlaR2aor -CH₂CH₂CH₂CH₂-NR1aR2a. Each possibility represents a separate embodiment. According to some embodiments, Lais -CH2CH2-Zea-NRlaR2aor -CH2CH2CH2CH2-Zea-NRlaR2a. According to some embodiments, Lais -CH2CH2-NRlaR2a. According to some embodiments, Lais -CH₂CH₂CH₂CH₂-NR1aR2a. 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

[0181] According to some embodiments, Lais alkylene-Zea-R3a. According to some embodiments, Lais Co-12 alkylene-Zda-R3a. According to some embodiments, Lais C0-9 alkylene-Zea-R3a. According to some embodiments, Lais Co-6 alkylene-Zea-R3a. According to some embodiments, Lais Co-6 alkylene-OH.

[0182] 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 Co-12 alkylene-N(Co-i2-alkylene-Zea-R3a)2. According to some embodiments, Lais C0-9 alkylene-N(Co-i2 alkyl ene-Zea-R3a)2. According to some embodiments, Lais Co-6 alkylene-N(Co-i2 alkylene-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 alkyl ene-N(Co-6 alkylene-Zea-R3a)2.

[0183] 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, R3ais selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment of the invention. 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 Rlaand R2ais independently -CH₂(CH₂)₆CH₃. According to some embodiments, each one of R1aand R2ais independently -CH₂(CH₂)₅CH₃. According to some embodiments, each one of R1aand R2ais independently -CH₂(CH₂)₃CH₃.

[0184] 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 C4-10 alkyl. According to some embodiments, Rlais -CH₂(CH₂)₆CH₃. According to some embodiments, R1ais -CH₂(CH₂)₄CH₃.

[0185] 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 C4-10 alkyl. According to some embodiments, R2ais -CH₂(CH₂)₆CH₃. According to some embodiments, R2ais -CH₂(CH₂)₅CH₃. According to some embodiments, R2ais -CH₂(CH₂)₃CH₃.

[0186] According to some embodiments, Rlaand R2a, together with the nitrogen to which they are bound, form a heterocycle.

[0187] 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, Ci-12 alkyl, Ci-12 alkenyl, C1-12 alkynyl, Co-12 alkylene-N(Ci-i2alkyl)2 and P(0)(0H)2. According to some embodiments, R3ais H. According to some embodiments, R3ais P(0)(0H)2.

[0188] 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(CH₂)₆CH₃. According to some embodiments, each one of R1band R2bis independently -CH₂(CH₂)₅CH₃. According to some embodiments, each one of R1band R2bis independently -CH₂(CH₂)₃CH₃.

[0189] 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 C4-10 alkyl. According to some embodiments, Rlbis -CH₂(CH₂)₆CH₃. According to some embodiments, R1bis -CH₂(CH₂)₅CH₃. According to some embodiments, R1bis -CH₂(CH₂)₃CH₃.

[0190] 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 C4-10 alkyl. According to some embodiments, R2bis -CH₂(CH₂)₆CH₃. According to some embodiments, R2bis -CH₂(CH₂)₅CH₃. According to some embodiments, R2bis -CH₂(CH₂)₃CH₃.

[0191] According to some embodiments, Rlband R2b, together with the nitrogen to which they are bound, form a heterocycle. 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, 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, Ci-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 H. According to some embodiments, R3bis P(0)(0H)2.

[0192] 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 Rlcand R2cis independently -CH₂(CH₂)₆CH₃. According to some embodiments, each one of R1cand R2cis independently -CH₂(CH₂)₅CH₃. According to some embodiments, each one of R1cand R2cis independently -CH₂(CH₂)₃CH₃.

[0193] 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, Rlcis C4-10 alkyl. According to some embodiments, Rlcis -CH₂(CH₂)₆CH₃. According to some embodiments, R1cis -CH₂(CH₂)₅CH₃. According to some embodiments, R1cis -CH₂(CH₂)₃CH₃.

[0194] 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 C4-10 alkyl. According to some embodiments, R2cis - CH₂(CH₂)₆CH₃. According to some embodiments, R2cis -CH₂(CH₂)₅CH₃. According to some embodiments, R2cis -CH₂(CH₂)₃CH₃.

[0195] According to some embodiments, Rlcand R2c, together with the nitrogen to which they are bound, form a heterocycle.

[0196] 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, 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, C1-12 alkenyl, C1-12 alkynyl, Co-12 alkylene-N(Ci-i2alkyl)2 and P(0)(0H)2. According to some embodiments, R3cis H. According to some embodiments, R3cis P(0)(0H)2.

[0197] According to some embodiments, each one of Rldand R2dis independently selected from the group consisting of: H, alkyl, alkenyl and alkynyl. According to some embodiments, each one of Rldand R2dis independently C1-12 alkyl. According to some embodiments, each one of Rldand R2dis independently C4-12 alkyl. According to some embodiments, each one of Rldand R2dis independently -CH₂(CH₂)₆CH₃. According to some embodiments, each one of R1dand R2dis independently -CH₂(CH₂)₅CH₃. According to some embodiments, each one of R1dand R2dis independently -CH₂(CH₂)₃CH₃.

[0198] According to some embodiments, Rldis selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, Rldis selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C1-12 alkynyl. According to some embodiments, Rldis a linear alkyl or a branched alkyl. According to some embodiments, Rldis a linear alkyl. According to some embodiments, Rldis an unsubstituted alkyl. According to some embodiments, Rldis C1-12 alkyl. According to some embodiments, Rldis C4-10 alkyl. According to some embodiments, Rldis -CH₂(CH₂)₆CH₃. According to some embodiments, R1dis -CH₂(CH₂)₅CH₃. According to some embodiments, R1dis -CH₂(CH₂)₃CH₃.

[0199] According to some embodiments, R2dis selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment of the invention. According to some embodiments, R2dis selected from the group consisting of: H, C1-12 alkyl, C1-12 alkenyl and C 1-12 alkynyl. According to some embodiments, R2dis a linear alkyl or a branched alkyl. According to some embodiments, R2dis a linear alkyl. According to some embodiments, R2dis an unsubstituted alkyl. According to some embodiments, R2dis C1-12 alkyl. According to some embodiments, R2dis C4-10 alkyl. According to some embodiments, R2dis -CH₂(CH₂)₆CH₃. According to some embodiments, R2dis -CH₂(CH₂)₅CH₃. According to some embodiments, R2dis -CH₂(CH₂)₃CH₃.

[0200] According to some embodiments, Rldand R2d, together with the nitrogen to which they are bound, form a heterocycle.

[0201] According to some embodiments, R3dis selected from the group consisting of: H, alkyl, alkenyl and alkynyl. Each possibility represents a separate embodiment. According to some embodiments, R3dis 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, R3dis 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, R3dis H. According to some embodiments, R3dis P(0)(0H)2.

[0202] According to some embodiments, Rlaand R2aare the same. For example, in Lipid 8, shown herein, each one of R1aand R2ais -(CH₂)₇CH₃.

[0203] According to some embodiments, Rlband R2bare the same. For example, in Lipid 8, shown herein, each one of R1band R2bis -(CH₂)₇CH₃.

[0204] According to some embodiments, Rlcand R2care the same. For example, in Lipid 4, shown herein, each one of R1cand R2cis -(CH₂)₇CH₃.

[0205] According to some embodiments, Rldand R2dare the same. For example, in Lipid 4, shown herein, each one of R1dand R2dis -(CH₂)₇CH₃.

[0206] According to some embodiments, Rlaand R2ais the same as Rlband R2b. For example, in Lipid 4, shown herein, each one of Rlaand R2ais -(CEh^CEE and each one of R1band R2bis -(CH₂)₇CH₃. According to some embodiments, Rlcand R2cis the same as Rldand R2d. For example, in Lipid 4, shown herein, each one of R1cand R2cis -(CH₂)₇CH₃ and each one of R1dand R2dis -(CH₂)₇CH₃. According to some embodiments, Rlais the same as Rlb. According to some embodiments, Rlais the same as Rlc. According to some embodiments, Rlais the same as Rld. According to some embodiments, Rlbis the same as Rlc. According to some embodiments, Rlbis the same as Rld. According to some embodiments, Rlcis the same as Rld.

[0207] According to some embodiments, R2ais the same as R2b. According to some embodiments, R2ais the same as R2c. According to some embodiments, R2ais the same as R2d. According to some embodiments, R2bis the same as R2c. According to some embodiments, R2bis the same as R2d. According to some embodiments, R2cis the same as R2d.

[0208] According to some embodiments, R3ais the same as R3b. According to some embodiments, R3ais the same as R3c. According to some embodiments, R3ais the same as R3d. According to some embodiments, R3bis the same as R3c. According to some embodiments, R3bis the same as R3d. According to some embodiments, R3cis the same as R3d.

[0209] According to some embodiments, Rla, Rlb, Rlc, Rld, R2a, R2b, R2c, and R2dare the same. For example, in Lipid 4, shown herein, each one of Rla, Rlb, Rlc, Rld, R2a, R2b, R2c, and R2dis -(CH2)7CH3.

[0210] According to some embodiments, Lais selected from the group consisting of: -CH2CH2-N((CH2)7CH3)2, -N((CH2)7CH3)2, -(CH2)4-N((CH2)7CH3)2, and -CH[CH2(CH2)4CH3]CH2(CH2)3CH3. According to some embodiments, Lais -CH2CH2-N((CH2)7CH3)2. According to some embodiments, Lais -N((CH2)7CH3)2. According to some embodiments, Lais -(CH2)4-N((CH2)7CH3)2. According to some embodiments, Lais selected from the group consisting of: -(CH2)IOCH3, -(CH2)11CH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8CH3, -CH2CH2N((CH2)7CH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -N((CH2)7CH3)2, -(CH2)4-N((CH2)7CH3)2, and -(CH2)7-CH=CH-(CH2)7CH3. Each possibility represents a separate embodiment of the invention.

[0211] According to some embodiments, Lb is selected from the group consisting of: alkyl, alkenyl, alkynyl, alkylene-NR1R2, alkenylene-NRlbR2b, alkynylene-NRlbR2b, 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-NRlbR2b, 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, Co-6 alkylene-NR1bR2b, (CH₂CH₂O)n3R3b, Co-12 alkylene-Zeb-R3b, Co-12 alkylene-Zeb-NRlbR2b, and Co-12 alkylene-N(C0-12alkylene-Zeb-R3b)2. Each possibility represents a separate embodiment of the invention.

[0212] 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 a substituted alkyl. According to some embodiments, Lb is a substituted alkyl, as shown, for example, in Lipid 39 (a hydroxyalkyl: 2-hydroxy dodecyl). According to some embodiments, Lb is C1-20 alkyl. According to some embodiments, Lb is C4-18 alkyl. According to some embodiments, Lb is Cs-14 alkyl. According to some embodiments, Lb is C9-12 alkyl. According to some embodiments, Lb is selected from the group consisting of: -(CH₂)₁₀CH₃, -(CH₂)₁₁CH₃, -(CH₂)₈CH₃ and -CH[CH2(CH2)4CH3]CH2(CH2)3CH3. According to some embodiments, Lb is -(CH2)IOCH3. According to some embodiments, Lb is -(CEE^iCEE. According to some embodiments, Lb is -(CEh^CEE. According to some embodiments, Lb is -CH[CH2(CH2)4CH3]CH2(CH2)3CH3.

[0213] 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, Lb is C2-20 monoenyl. According to some embodiments, Lb is C4-18 monoenyl. According to some embodiments, Lb is C10-18 monoenyl. According to some embodiments, Lb is C15-18 monoenyl. According to some embodiments, Lb is a dienyl. According to some embodiments, Lb is C2-20 dienyl. 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 selected from the group consisting of: -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, and -(CH₂)₇-CH=CH-(CH₂)₇CH₃. According to some embodiments, Lb is -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, Lb is -(CH2)s- CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, Lb is -(CH₂)₇-CH=CH-(CH₂)₇CH₃.

[0214] 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. It is to be understood that linear or branched alkylene-NR1R2or alkyl ene-Zeb-NRlbR2bmeans a linear or branched alkylene, which is bonded to an NR1R2or Zeb-NRlbR2bgroup. According to some embodiments, Lb is a linear alkylene-NRlbR2b. According to some embodiments, Lb is Co-12 alkylene-Zeb-NRlbR2b. According to some embodiments, Lb is C0-9 alkylene-Zeb-NRlbR2b. According to some embodiments, Lb is Co-6 alkyl ene-NRlbR2b. It is to be understood that Co alkylene-Zeb-NRlbR2brefers to -Zeb-NRlbR2band Co alkylene-NRlbR2brefers to -NRlbR2b. According to some embodiments, Lb is -CH2CH2-NRlbR2bor -CH2CH2CH2CH2-NRlbR2b. Each possibility represents a separate embodiment. According to some embodiments, Lb is -CH2CH2-Zeb-NRlbR2bor -CH2CH2CH2CH2-Zeb-NRlbR2b. According to some embodiments, Lb is -CH2CH2-NRlbR2b. According to some embodiments, Lb is -CH2CH2CH2CH2-NRlbR2b.

[0215] 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.

[0216] According to some embodiments, Lb is alkylene-Zeb-R3b. According to some embodiments, Lb is C0-12alkylene-Zdb-R3b. According to some embodiments, Lb is C0-9 alkylene-Zeb-R3b. According to some embodiments, Lb is Co-6 alkylene-Zeb-R3b. According to some embodiments, Lb is Co-6 alkylene-OH.

[0217] According to some embodiments, Lb is alkylene-N(alkylene-Zeb-R3b)2. According to some embodiments, Lb is C0-12alkylene-N(C0-12-alkylene-Zeb-R3b)2. According to some embodiments, Lb is C0-12alkylene-N(C0-12-alkylene-Zeb-R3b)2. According to some embodiments, Lb is C0-9alkylene-N(C0-12alkylene-Zeb-R3b)2. According to some embodiments, Lb is C0-6alkylene-N(C0-12alkylene-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 Co-6 alkyl ene-N(Co-6 alkylene-Zeb-R3b)2.

[0218] According to some embodiments, Lb is selected from the group consisting of: -CH2CH2-N((CH2)7CH3)2, -N((CH2)7CH3)2, -(CH2)4-N((CH2)7CH3)2, and -CH[CH2(CH2)4CH3]CH2(CH2)3CH3. According to some embodiments, Lb is -CH2CH2-N((CH2)? CH3)2. According to some embodiments, Lb is -N((CH2)? CH3)2. According to some embodiments, Lb is -(CH2)4-N((CH2)? CH3)2. According to some embodiments, Lb is selected from the group consisting of: -(CH2)10CH3, -(CH2)11CH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -CH2CH2-N((CH2)7CH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -N((CH2)7CH3)2, -(CH2)4-N((CH2)7CH3)2, -(CH2)8CH3, and -(CH2)7-CH=CH-(CH2)7CH3. Each possibility represents a separate embodiment of the invention.

[0219] According to some embodiments, Lcis selected from the group consisting of: alkyl, alkenyl, alkynyl, alkylene-NRlcR2c, alkenylene-NRlcR2c, alkynylene-NRlcR2c, 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-NRlcR2c, 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: C1-20 alkyl, C2-20 alkenyl, C0-6alkylene-NR1cR2c, (CH2CH2O)n3R3c, Co-12 alkylene-Zec-R3c, Co-12 alkylene-Zec-NRlcR2c, and C0-12alkylene-N(C0-12alkylene-Zec-R3c)2. Each possibility represents a separate embodiment of the invention. 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 a substituted alkyl as shown, for example, in Lipid 39 (a hydroxyalkyl: 2-hydroxydodecyl). According to some embodiments, Lcis C2-20 alkyl. According to some embodiments, Lcis C4-18 alkyl. According to some embodiments, Lcis C8-14alkyl. According to some embodiments, Lcis C9-12 alkyl. According to some embodiments, Lcis selected from the group consisting of: -(CH2)ioCH3, -(CH₂)₁₁CH₃, -(CH₂)₈CH₃ and -CH[CH2(CH2)4CH3]CH2(CH2)3CH3. According to some embodiments, Lcis -(CH2)ioCH3. According to some embodiments, Lcis -(CEh^CEE. According to some embodiments, Lcis -CH[CH2(CH2)4CH3]CH2(CH2)3CH3.

[0220] 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 C10-18dienyl. According to some embodiments, Lcis C15-18dienyl. According to some embodiments, Lcis selected from the group consisting of: -(CH2)?-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, and -(CH2)7-CH=CH-(CH2)7CH3. According to some embodiments, Lcis -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, Lcis -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, Lcis -(CH₂)₇-CH=CH-(CH₂)₇CH₃.

[0221] 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, Lcis a linear or branched alkylene-Zec-NRlcR2c. According to some embodiments, Lcis a linear or branched alkylene-NR1cR2c. It is to be understood that linear or branched alkylene-NR1CR2Cor alkylene-Zec-NRlcR2cmeans a linear or branched alkylene, which is bonded to an NR1CR2Cor Zec-NRlcR2cgroup. According to some embodiments, Lcis a linear alkylene-NRlcR2c. According to some embodiments, Lcis Co-12 alkylene-Zec-NRlcR2c. According to some embodiments, Lcis C0-9 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 -(CH2)4-N((CH2)7CH3)2.

[0222] 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 -CH2CH2OCH2CH2OCH2CH2OR3cwherein R3cis H, as shown, for example, in Lipid 37. According to some embodiments, Lcis -CH2CH2OCH2CH2OCH2CH2OCH2CH2OR3cwherein R3cis H, as shown, for example, in Lipid 42. According to some embodiments, Lcis - CH2CH2OCH2CH2OCH2CH2OH. According to some embodiments, Lcis -CH2CH2OCH2CH2OCH2CH2OCH2CH2OH.

[0223] 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 alkylene-Zec-R3c. According to some embodiments, Lcis Co-6 alkylene-Zec-R3c. According to some embodiments, Lcis Co-6 alkylene-Zec-R3c, wherein Zecis -O-. According to some embodiments, Lcis Co-6 alkylene-Zec-R3c, wherein Zecis -O- and R3cis H. According to some embodiments, Lcis Co-6 alkylene-Zec-R3c, wherein Zecis -O- and R3cis P(0)(0H)2. For example, in Lipid 57, Lcis -CH2CH2OP(O)(OH)2. According to some embodiments, Lcis CH2CH2-Zec-R3c. According to some embodiments, Lcis CH2CH2CH2-Zec-R3c. According to some embodiments, Lcis CH2CH2CH2CH2-Zec-R3c. According to some embodiments, Lcis -CH2CH2OP(O)(OH)2. According to some embodiments, Lcis -CH2CH2OH. According to some embodiments, Lcis CH2CH2CH2OH. According to some embodiments, Lcis CH2CH2CH2CH2OH. 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.

[0224] According to some embodiments, Lcis selected from the group consisting of: -CH2CH2-OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, -(CH2)10CH3, -(CH2)11CH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -CH2CH2-N((CH2)7CH3)2, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -(CH2)8CH3, and -(CH₂)₇-CH=CH-(CH₂)₇CH₃. Each possibility represents a separate embodiment of the invention.

[0225] According to some embodiments, Ldis selected from the group consisting of: alkyl, alkenyl, alkynyl, alkylene-NR1dR2d, alkenylene-NR1dR2d, alkynylene-NR1dR2d, alkylene-OR3d, alkenylene-OR3d, and alkynylene-OR3d. Each possibility represents a separate embodiment of the invention. According to some embodiments, Ldis selected from the group consisting of: alkyl, alkenyl, alkynyl, (CH2CH2O)n3R3d, alkylene-Zed-R3d, alkylene-Zed-NR1dR2d, and alkylene-N(alkylene-Zed-R3d)2. Each possibility represents a separate embodiment of the invention. According to some embodiments, Ldis selected from the group consisting of: C1-20alkyl, C2-20alkenyl, C0-6alkylene-NR1dR2d, (CH2CH2O)n3R3d, C0-12alkylene-Zed-R3d, C0-12alkylene-Zed-NR1dR2d, and C0-12alkylene-N(C0-12alkylene-Zed-R3d)2. Each possibility represents a separate embodiment of the invention. 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, La is a substituted alkyl as shown, for example, in Lipid 39 (a hydroxyalkyl: 2-hydroxydodecyl). According to some embodiments, Lais C2-20 alkyl. According to some embodiments, Lais C4-18 alkyl. According to some embodiments, La is Cs-i4 alkyl. According to some embodiments, La is C9-12 alkyl. According to some embodiments, Lais selected from the group consisting of: -(CH₂)₁₀CH₃, -(CH2)nCH3, -(CEh^CEL and -CH[CH2(CH2)4CH3]CH2(CH2)3CH3. According to some embodiments, La is -(CH2)IOCH3. According to some embodiments, La is -(CH2)nCH3. According to some embodiments, La is -(CEh^CEL. According to some embodiments, La is -CH[CH2(CH2)4CH3]CH2(CH2)3CH3.

[0226] 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, La is C2-20 monoenyl. According to some embodiments, La is C4-18 monoenyl. According to some embodiments, Lais Cio-18 monoenyl. According to some embodiments, Lais C15-18 monoenyl. According to some embodiments, La is a dienyl. According to some embodiments, Lais C2-20 dienyl. According to some embodiments, Lais C4-18 dienyl. According to some embodiments, La is Cio-18 dienyl. According to some embodiments, La is C15-18 dienyl. According to some embodiments, Lais selected from the group consisting of: -(CH2)?-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, and -(CH2)7-CH=CH-(CH2)7CH3. According to some embodiments, La is -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, Lais -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, Lais -(CH₂)₇-CH=CH-(CH₂)₇CH₃.

[0227] According to some embodiments, Ldis alkylene-Zed-NR1dR2d. According to some embodiments, Zedis absent. According to some embodiments, Ldis an alkylene-NR1dR2d. According to some embodiments, Ldis a linear or branched alkylene-Zed-NR1dR2d. According to some embodiments, Ldis a linear or branched alkylene-NR1dR2d. According to some embodiments, Ldis a linear or branched alkylene-Zed-NR1dR2d. It is to be understood that linear or branched alkylene-NRldR2dor alkylene-Zed-NRldR2dmeans a linear or branched alkylene, which is bonded to an NRldR2dor Zed-NRldR2dgroup. According to some embodiments, La is a linear alkyl ene-NRldR2d. According to some embodiments, Lais Co-12 alkylene-Zed-NRldR2d. According to some embodiments, Lais C0-9 alkylene-Zed-NRldR2d. According to some embodiments, La is Co-6 alkylene-NRldR2d. It is to be understood that Co alkylene-Zec-NRlcR2crefers to -Zec-NRlcR2cand Co alkyl ene-NRldR2drefers to -NRldR2d. According to some embodiments, Lais -(CH2)4-N((CH2)7CH3)2.

[0228] According to some embodiments, Ldincludes a monoethylene glycol or polyethylene glycol (PEG). According to some embodiments, Ldis (CH2CH2O)n3R3d, wherein n3 is an integer. According to some embodiments, Ldis (CH2CH2O)n3R3d, 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, Ldis -CH2CH2OR3d. According to some embodiments, Ldis -CH2CH2OCH2CH2OR3d. According to some embodiments, Ldis -CH2CH2OCH2CH2OCH2CH2OR3d. According to some embodiments, Ldis -CH2CH2OCH2CH2OCH2CH2OR3dwherein R3dis H, as shown, for example, in Lipid 37. According to some embodiments, Ldis -CH2CH2OCH2CH2OCH2CH2OCH2CH2OR3dwherein R3dis H, as shown, for example, in Lipid 42. According to some embodiments, Ldis -CH2CH2OCH2CH2OCH2CH2OH. According to some embodiments, Ldis -CH2CH2OCH2CH2OCH2CH2OCH2CH2OH.

[0229] According to some embodiments, La is alkylene-Zed-R3d. According to some embodiments, La is Co-12 alkylene-Zed-R3d. According to some embodiments, La is C0-9 alkylene-Zed-R3d. According to some embodiments, La is Co-6 alkylene-Zed-R3d. According to some embodiments, La is Co-6 alkylene-Zed-R3d, wherein Zedis -O-. According to some embodiments, La is Co-6 alkylene-Zed-R3d, wherein Zedis -O- and R3dis H. According to some embodiments, La is Co-6 alkylene-Zed-R3d, wherein Zedis -O- and R3dis P(0)(0H)2. For example, in Lipid 57, La is -CH2CH2OP(O)(OH)2. According to some embodiments, La is CH2CH2-Zed-R3d. According to some embodiments, La is CH2CH2CH2-Zed-R3d. According to some embodiments, Lcis CH2CH2CH2CH2-Zed-R3d. According to some embodiments, La is -CH2CH2OP(O)(OH)2.

[0230] According to some embodiments, Ldis an alkylene-OH. According to some embodiments, Ldis a linear or branched alkylene-OH. Each possibility represents a separate embodiment of the invention. According to some embodiments, Ldis a linear alkylene-OH. According to some embodiments, Ldis an unsubstituted alkylene-OH. According to some embodiments, Ldis C0-6alkylene-OH. It is to be understood that C0alkylene-OH refers to hydroxyl group. According to some embodiments, Ldis -CH2CH2OH. According to some embodiments, Ldis CH2CH2CH2OH. According to some embodiments, Ldis CH2CH2CH2CH2OH.

[0231] According to some embodiments, Ldis selected from the group consisting of: -CH2CH2-OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, -(CH2)10CH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)11CH3, -(CH2)8CH3, -CH2CH2-N((CH2)7CH3)2, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, and -(CH2)7-CH=CH-(CH2)7CH3. Each possibility represents a separate embodiment of the invention.

[0232] According to some embodiments, Lais the same as Lb. According to some embodiments, Lcis the same as Ld. According to some embodiments, Laand Lbare different from Lcand Ld. It is to be understood that the term “the same” means that the two 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 7, shown herein, has the same Laand Lb, each of which is -CH2(CH2)9CH3, as well as the same Lcand La, each of which is -CH2CH2OH. Also, in Lipid 7, Laand Lb are different from Lcand La. According to some embodiments, Laand Lb are each -(CH₂)₁₀CH₃ and Lcand La are each -CH2CH2-N((CH2)7CH3)2. According to some embodiments, Laand Lb are each -N((CH2)? CH3)2 and Lcand La are each -CH2CH2OH. According to some embodiments, Laand Lb are each -(CH2)4-N((CH2)7CH3)2and Lcand La are each -CH2CH2OH. According to some embodiments, Laand Lb are each -(CH₂)₁₀CH₃ and Lcand La are each -CH2CH2OH. According to some embodiments, Laand Lbare each -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, and Lcand Ldare each -CH2CH2OH.

[0233] According to some embodiments, La, Lb, Lcand La are the same. It is to be understood that the term “the same” means that each one of the four specified L substituents have the same chemical structure. For example, Lipid 1, shown herein, has the same La, Lb, Lcand La, each of which is -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, each one of La, Lb, Lcand La is -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, each one of La, Lb, Lcand Lais -(CH₂)₁₀CH₃. According to some embodiments, each one of La, Lb, Lcand La is -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3. According to some embodiments, each one of La, Lb, Lcand La is -CFLCFLJSl^CFL^CHs^. According to some embodiments, each one of La, Lb, Lcand Lais -CH[CH2(CH2)4CH3]CH2(CH2)3CH3. According to some embodiments, each one of La, Lb, Lcand La is -(CEh^CEE. According to some embodiments, each one of La, Lb, Lcand La is -(CH2)7-CH=CH-(CH2)7CH3.

[0234] 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 1, shown herein, m is 1. For example, in Lipid 2, shown herein, m is 2. 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 1. According to some embodiments, m is 3. According to some embodiments, m is 4. According to some embodiments, m is 5.

[0235] According to some embodiments, n is 1, 2, 3, 4 or 5. Each possibility represents a separate embodiment of the invention. For example, in Lipid 1, shown herein, n is 1. For example, in Lipid 18, shown herein, n is 2. According to some embodiments, n is 1, 2 or 3. According to some embodiments, n is 1 or 2. According to some embodiments, n is 1. According to some embodiments, n is 2.

[0236] According to some embodiments, xa-Za-Lais the same as xb-Zb-Lb. According to some embodiments, xc-Zc-Lcis the same as xd-Zd-Ld. According to some embodiments, xa-Za-Laand xb-Zb-Lbare different from xc-Zc-Lcand xd-Zd-Ld. It is to be understood that the term “the same” means that the two 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 7, shown herein, has the same xa-Za-Laand Xb-Zb-Lb, each of which is -CH2CH2CH2CH2-OC(0)-(CH₂)₁₀CH₃, as well as the same xc-Zc-Lcand xd-Zd-Ld, each of which is -CH2CH2-O-CH2CH2OH. Also, in Lipid 7, xa-Za-Laand xb-Zb-Lbare different from xc-Zc-Lcand xd-Zd-Ld.

[0237] According to some embodiments, xa-Za-La, xb-Zb-Lb, xc-Zc-Lc, and xd-Zd-Ldare the same. It is to be understood that the term “the same” means that each one of the four specified x-Z-L substituents have the same chemical structure. For example, Lipid 1, shown herein, has the same xa-Za-La, xb-Zb-Lb, xc-Zc-Lc, and xd-Zd-Ld, each of which is CH2CH2-OC(O)-(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3.

[0238] The chemical structures of each of the specific lipid are detailed below in the “Exemplary Lipids” Section and in the claims.

[0239] Exemplary Lipids

[0240] Exemplary Lipids according to Formula (I), specifically according to Formula (Ia) or Formula (IIb) 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 is selected from the group consisting of: Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19 and Lipid 20, 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 selected from the group consisting of: Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23, Lipid 24, Lipid 25, Lipid 26, Lipid 27, Lipid 28, Lipid 29, Lipid 30, Lipid 31, Lipid 32, Lipid 33, Lipid 34, Lipid 35, and Lipid 36, 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 1. According to some embodiments, the lipid is Lipid 2. According to some embodiments, the lipid is Lipid 3. According to some embodiments, the lipid is Lipid 4. According to some embodiments, the lipid is Lipid 5. According to some embodiments, the lipid is Lipid 6. According to some embodiments, the lipid is Lipid 7. According to some embodiments, the lipid is Lipid 8. According to some embodiments, the lipid is Lipid 9. According to some embodiments, the lipid is Lipid 10. According to some embodiments, the lipid is Lipid 11. According to some embodiments, the lipid is Lipid 12. According to some embodiments, the lipid is Lipid 13. According to some embodiments, the lipid is Lipid 14. According to some embodiments, the lipid is Lipid 15. According to some embodiments, the lipid is Lipid 16. According to some embodiments, the lipid is Lipid 17. According to some embodiments, the lipid is Lipid 18. According to some embodiments, the lipid is Lipid 19. According to some embodiments, the lipid is Lipid 20. According to some embodiments, the lipid is Lipid 21. According to some embodiments, the lipid is Lipid 22. According to some embodiments, the lipid is Lipid 23. According to some embodiments, the lipid is Lipid 24. According to some embodiments, the lipid is Lipid 25. According to some embodiments, the lipid is Lipid 26. According to some embodiments, the lipid is Lipid 27. According to some embodiments, the lipid is Lipid 28. According to some embodiments, the lipid is Lipid 29. According to some embodiments, the lipid is Lipid 30. According to some embodiments, the lipid is Lipid 31. According to some embodiments, the lipid is Lipid 32. According to some embodiments, the lipid is Lipid 33. According to some embodiments, the lipid is Lipid 34. According to some embodiments, the lipid is Lipid 35. According to some embodiments, the lipid is Lipid 36.

[0241] According to some embodiments, the lipid is selected from the group consisting of: Lipid 11, Lipid 20, Lipid 19, Lipid 13, Lipid 18, Lipid 21, Lipid 31, Lipid 32, Lipid 23, Lipid 24, and Lipid 27, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0242] According to some embodiments, the lipid is selected from the group consisting of: Lipid 3, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, and Lipid 17, 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 selected from the group consisting of: Lipid 3, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23, Lipid 24, Lipid 25, Lipid 26, Lipid 27, Lipid 28, Lipid 29, Lipid 30, Lipid 31, Lipid 32, Lipid 33, Lipid 34, Lipid 35, and Lipid 36 including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0243] According to some embodiments, the lipid is according to Formula (la) and is selected from the group consisting of: Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23, Lipid 24, Lipid 25, Lipid 26, Lipid 27, Lipid 28, Lipid 29, Lipid 30, Lipid 31, Lipid 32, Lipid 33, Lipid 34, Lipid 35, and Lipid 36, 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 according to Formula (lb) and is selected from the group consisting of: Lipid 1, Lipid 10, Lipid 13, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23, Lipid 24, Lipid 25, Lipid 26, Lipid 27, Lipid 28, Lipid 29, Lipid 32, Lipid 33, Lipid 34, Lipid 35, and Lipid 36, including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0244] 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.

[0245] 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 (I), (la), (lb) 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.

[0246] 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.

[0247] Specifically, Lipid 1, also referred to as NV5-033, is shown below,

[0248]

[0249] NV5-033

[0250] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zcand Zd is independently absent; wherein each one of La, Lb, Lcand Ld is independently C2-20 alkenyl {C18dienyl}; wherein m is 1; and wherein nl and n2 are each independently 1.

[0251] Specifically, Lipid 2 is shown below,

[0252]

[0253] It is represented by Formula (I), wherein each one of xa, Xb, xcand Xd is independently Co-12 alkylene {C4 alkylene}; wherein each one of Za, Zb, Zcand Zd is independently -OC(O)-; wherein each one of La, Lb, Lcand Ld is independently C1-20 alkyl {C11alkyl}; wherein m is 3; and wherein nl and n2 are each independently 1.

[0254] Specifically, Lipid 3, also referred to as NV5-013, is shown below,

[0255]

[0256] NV05-013

[0257] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Za, Zb, Zcand Zais independently -OC(O)-; wherein each one of La, Lb, Lcand La is independently C2-20 alkenyl {C17 dienyl}; wherein m is 1; and wherein nl and n2 are each independently 1.

[0258] Specifically, Lipid 4 is shown below,

[0259]

[0260] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Za, Zb, Zcand Zais independently -OC(O)-; wherein each one of La, Lb, Lcand La is independently Co-12 alkylene-Ze-NR1R2{C2 alkylene-Zc-NR'R2}, wherein Zeis absent and each one of R1and R2is independently C1-12 alkyl {C8alkyl}; wherein m is 1; and wherein nl and n2 are each independently 1.

[0261] Specifically, Lipid 5 is shown below,

[0262]

[0263] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {C4 alkylene}; wherein each one of Za, Zb, Zcand Zais independently -OC(O)-; wherein each one of La, Lb, Lcand La is independently C1-20 alkyl {C12 alkyl}; wherein m is 1; and wherein nl and n2 are each independently 1.

[0264] Specifically, Lipid 6 is shown below,

[0265]

[0266] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {C4 alkylene}; wherein each one of Za, Zb, Zcand Za is independently -OC(O)NH-; wherein each one of La, Lb, Lcand La is independently C1-20 alkyl {C11alkyl}; wherein m is 3; and wherein nl and n2 are each independently 1.

[0267] Specifically, Lipid 7 is shown below,

[0268]

[0269] It is represented by Formula (I), wherein each one of xaand Xb is independently Co-12 alkylene {C4 alkylene}; wherein each one of Zaand Zb is independently -OC(O)-; wherein each one of Laand Lb, is independently C1-20 alkyl {Cn alkyl};

[0270] wherein each one of xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Zcand Za is independently -O-; wherein each one of Lcand La is independently Co-12 alkylene-Ze- R3{C2alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H;

[0271] wherein m is 1; and wherein nl and n2 are each independently 1.

[0272] Specifically, Lipid 8 is shown below,

[0273]

[0274] It is represented by Formula (I), wherein each one of xaand Xb is independently Co-12 alkylene {C4 alkylene}; wherein each one of Zaand Zb is independently -C(O)O-; wherein each one of Laand Lb, is independently Co-12 alkylene-Ze-NR1R2{Co alkylene-Ze-NR1R2, i.e., Ze-NR1R2}, wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C8alkyl}; wherein each one of xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Zcand Za is independently -O-; wherein each one of Lcand La is independently Co-12 alkylene-Ze- R3{C2alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H;

[0275] wherein m is 1; and wherein nl and n2 are each independently 1.

[0276] Specifically, Lipid 9 is shown below,

[0277]

[0278] It is represented by Formula (I), wherein each one of xaand Xb is independently Co-12 alkylene {C4 alkylene}; wherein each one of Zaand Zb is independently -C(O)O-; and wherein each one of Laand Lb is independently Co-12- Ze-alkylene-NR1R2{C4 alkylene-Zc-NR'R2}, wherein Zeis absent and wherein each one of R1and R2is independently C1-12 alkyl {C8alkyl};

[0279] wherein each one of xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Zcand Za is independently -O-; wherein each one of Lcand La is independently Co-12 alkylene-Ze-R3{C2alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H;

[0280] wherein m is 1; and wherein nl and n2 are each independently 1.

[0281] Specifically, Lipid 10 is shown below,

[0282]

[0283] It is represented by Formula (I), 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};

[0284] wherein each one of xcand xa is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Zcand Za is independently absent; wherein each one of Lcand La is independently Co- 12 alkyl ene-Ze-R3{C2 alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H;

[0285] wherein m is 1; and wherein nl and n2 are each independently 1.

[0286] Specifically, Lipid 11, also referred to as NV5-009, is shown below,

[0287]

[0288] NV05-009 It is represented by Formula (I), 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 C2-20 alkenyl {C18dienyl};

[0289] wherein each one of xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Zcand Za is independently -O-; and wherein each one of Lcand La is independently Co-12 alkylene- Ze-R3{C2 alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H;

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

[0291] Specifically, Lipid 12, also referred to as NV5-010, is shown below,

[0292]

[0293] NV05-010

[0294] It is represented by Formula (I), 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 C1-20 alkyl {C12 alkyl};

[0295] wherein each one of xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Zcand Za is independently -O-; wherein each one of Lcand La is independently Co-12 alkylene-Ze-R3{C2alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H; wherein m is 1; and wherein nl and n2 are each independently 1.

[0296] Specifically, Lipid 13, also referred to as NV5-017, is shown below,

[0297]

[0298] NV05-017

[0299] It is represented by Formula (I), wherein each one of xa, Xb, xc, and xa is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Zd is independently absent; wherein each one of Laand Lb is independently C1-20 alkyl {C12 alkyl} and wherein each one of Lcand Ld is independently Co-12 alkylene-Ze-R3{C2 alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H; wherein m is 1; and wherein nl and n2 are each independently 1.

[0300] Specifically, Lipid 14, also referred to as NV5-011, is shown below,

[0301]

[0302] It is represented by Formula (I), wherein each one of xa, Xb, xcand Xd is independently Co -12 alkylene {C2 alkylene}, wherein each one of Za, Zb, Zc and Zd is independently -OC(O)-; wherein each one of La, Lb, Lcand Ld is independently C1-20 alkyl {C11alkyl}; wherein m is 1; and wherein nl and n2 are each independently 1.

[0303] Specifically, Lipid 15, also referred to as NV5-012, is shown below, 0

[0304]

[0305] It is represented by Formula (I), wherein each one of xa, Xb, xcand Xd is independently Co-12 alkylene {C2 alkylene}; wherein each one of Za, Zb, Zcand Zd is independently -OC(O)-; wherein each one of La, Lb, Lcand Ld is independently C1-20 alkyl {C9 alkyl}; wherein m is 1; and wherein nl and n2 are each independently 1.

[0306] Specifically, Lipid 16, also referred to as NV5-014, is shown below,

[0307] o

[0308]

[0309] NV05-014

[0310] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Za, Zb, Zcand Zd is independently -OC(O)-; wherein each one of La, Lb, Lcand Ld is independently C2-20 alkenyl {C17 monoenyl}; wherein m is 1; and wherein nl and n2 are each independently 1.

[0311] Specifically, Lipid 17 is shown below,

[0312]

[0313] It is represented by Formula (I), wherein each one of xa, Xb, xcand Xd is independently Co-12 alkylene {C6alkylene}; wherein each one of Za, Zb, Zcand Zd is independently -OC(O)-; wherein each one of La, Lb, Lcand La is independently C1-20 alkyl {C9 alkyl}; wherein m is 1; and wherein nl and n2 are each independently 1.

[0314] Specifically, Lipid 18, also referred to as NV5-018, is shown below,

[0315] o

[0316]

[0317] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Za, Zb, Zcand Za is independently -OC(O)-; wherein each one of La, Lb, Lcand La is independently C1-20 alkyl {C11alkyl}; wherein m is 2; and wherein nl and n2 are each independently 2.

[0318] Specifically, Lipid 19, also referred to as NV5-016, is shown below,

[0319]

[0320] NV05-016

[0321] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {Co alkylene, absent}; wherein each one of Za, Zb, Zcand Za is independently absent; wherein each one of La, Lb, Lcand La is independently C1-20 alkyl {C12 alkyl}; wherein m is 2; and wherein nl and n2 are each independently 2.

[0322] Specifically, Lipid 20, also referred to as NV5-015, is shown below,

[0323]

[0324] NV05-015

[0325] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {Co alkylene, absent}; wherein each one of Za, Zb, Zcand Za is independently absent; wherein each one of La, Lb, Lcand La is independently C1-20 alkyl {C12 alkyl}; wherein m is 1; and wherein nl and n2 are each independently 1. Specifically, Lipid 21, also referred to as NV5-024, is shown below,

[0326]

[0327] NV05-024 It is represented by Formula (I), wherein each one of xa, Xb, xcand Xd is independently Co-12 alkylene {Co alkylene, absent}; wherein each one of Za, Zb, Zcand Zd is independently absent; wherein each one of La, Lb, Lcand Ld is independently C1-20 alkyl {C12 alkyl}; wherein m is 2; and wherein nl and n2 are each independently 1.

[0328] Specifically, Lipid 22, also referred to as NV5-025, is shown below,

[0329]

[0330] NV05-025

[0331] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {Co alkylene, absent}; wherein each one of Za, Zb, Zcand Zd is independently absent; wherein each one of La, Lb, Lcand Ld is independently C1-20 alkyl {C12 alkyl}; wherein m is 3; and wherein nl and n2 are each independently 1.

[0332] Specifically, Lipid 23, also referred to as NV5-019, is shown below,

[0333] . ■ -. t J 11

[0334]

[0335] NV05-019

[0336] It is represented by Formula (I), wherein each one of xa, Xb, xc, and Xd is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Zd is independently absent; wherein each one of Laand Lb is independently C1-20 alkyl {C12 alkyl} and wherein each one of Lcand La is independently Co-12 alkylene-Ze-R3{C4 alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H; wherein m is 1; and wherein nl and n2 are each independently 1.

[0337] Specifically, Lipid 24, also referred to as NV5-020, is shown below,

[0338]

[0339] NV05-020

[0340] It is represented by Formula (I), wherein each one of xa, Xb, xc, and xa is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Za is independently absent; wherein each one of Laand Lb is independently C1-20 alkyl {C12 alkyl} and wherein each one of Lcand La is independently Co-12 alkylene-Ze-R3{C3 alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H; wherein m is 1; and wherein nl and n2 are each independently 1.

[0341] Specifically, Lipid 25, also referred to as NV5-028, is shown below,

[0342]

[0343] NV5-028

[0344] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Za, Zb, Zcand Zais independently absent; wherein each one of La, Lb, Lcand La is independently C2-20 alkenyl {Cis dienyl}; wherein m is 2; and wherein nl and n2 are each independently 1.

[0345] Specifically, Lipid 26 is shown below,

[0346]

[0347] It is represented by Formula (I), wherein each one of xa, Xb, xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Za, Zb, Zcand Zais independently absent; wherein each one of La, Lb, Lcand La is independently C2-20 alkenyl {Cis dienyl}; wherein m is 3; and wherein nl and n2 are each independently 1.

[0348] Lipid 27, also referred to as NV5-021, is shown below,

[0349] OH OH

[0350] L..

[0351]

[0352] NV05-021

[0353] It is represented by Formula (I), wherein each one of xa, Xb, xc, and xa is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Za is independently absent; wherein each one of Laand Lb is independently C1-20 alkyl {C12 alkyl} and wherein each one of Lcand La is independently Co-12 alkylene-Ze-R3{C2 alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H; wherein m is 2; and wherein nl and n2 are each independently 1.

[0354] Specifically, Lipid 28, also referred to as NV5-022, is shown below,

[0355] an

[0356] J

[0357]

[0358] NV05-022

[0359] It is represented by Formula (I), wherein each one of xa, Xb, xc, and xa is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Za is independently absent; wherein each one of Laand Lb is independently C1-20 alkyl {C12 alkyl} and wherein each one of Lcand La is independently Co-12 alkylene-Ze-R3{C2 alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H; wherein m is 3; and wherein nl and n2 are each independently 1.

[0360] Specifically, Lipid 29, also referred to as NV5-026, is shown below,

[0361] r:> -

[0362]

[0363] NV05-026

[0364] It is represented by Formula (I), wherein each one of xa, Xb, xc, and xa is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Za is independently absent; wherein each one of Laand Lb is independently C1-20 alkyl {C12 alkyl} and wherein each one of Lcand La is independently Co-12 alkylene-Ze-R3{C2 alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H; wherein m is 4; and wherein nl and n2 are each independently 1.

[0365] Specifically, Lipid 30, also referred to as NV5-027, is shown below,

[0366] 1 J

[0367]

[0368] It is represented by Formula (I), 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 C2-20 alkenyl {Ci8 dienyl};

[0369] wherein each one of xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Zcand Zais independently -O-; wherein each one of Lcand La is independently Co-12 alkylene-Ze- R3{C2alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H; wherein m is 2; and wherein nl and n2 are each independently 1.

[0370] Specifically, Lipid 31, also referred to as NV5-029, is shown below,

[0371] J

[0372] f" r.-.i-s:;::.

[0373] .

[0374]

[0375] ■. r,.:

[0376] It is represented by Formula (I), 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 C2-20 alkenyl {Ci8 dienyl};

[0377] wherein each one of xcand xa is independently Co-12 alkylene {C2 alkylene}; wherein each one of Zcand Zais independently -O-; wherein each one of Lcand La is independently Co-12 alkylene-Ze- R3{C2alkylene-Ze-R3}, wherein Zeis -O- and wherein R3is H;

[0378] wherein m is 3; and wherein nl and n2 are each independently 1. Specifically, Lipid 32, also referred to as NV5-032, is shown below,

[0379]

[0380] It is represented by Formula (I), wherein each one of xa, Xb, xc, and Xd is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Zd is independently absent; wherein each one of Laand Lb is independently C2-20 alkenyl {Ci8 dienyl} and wherein each one of Lcand Ld is independently Co-12 alkylene-Ze-R3{C2 alkylene-Ze-R3}, wherein Zeis - O- and wherein R3is H; wherein m is 2; and wherein nl and n2 are each independently 1.

[0381] Specifically, Lipid 33, also referred to as NV5-034, is shown below,

[0382]

[0383] NV05-034

[0384] It is represented by Formula (I), wherein each one of xa, Xb, xc, and xa is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Zd is independently absent; wherein each one of Laand Lb is independently C2-20 alkenyl {Ci8 dienyl} and wherein each one of Lcand Ld is independently Co-12 alkylene-Ze-R3{C4 alkylene-Ze-R3}, wherein Zeis - O- and wherein R3is H; wherein m is 2; and wherein nl and n2 are each independently 1.

[0385] Specifically, Lipid 34, also referred to as NV5-036, is shown below,

[0386]

[0387] (j r - <--.r

[0388] It is represented by Formula (I), wherein each one of xa, Xb, xc, and Xd is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Zd is independently absent; wherein each one of Laand Lb is independently C2-20 alkenyl {Ci8 dienyl} and wherein each one of Lcand Ld is independently Co-12 alkylene-Ze-R3{C2 alkylene-Ze-R3}, wherein Zeis - O- and wherein R3is H; wherein m is 3; and wherein nl and n2 are each independently 1.

[0389] Specifically, Lipid 35, also referred to as NV5-037, is shown below,

[0390]

[0391] 'j.::

[0392] It is represented by Formula (I), wherein each one of xa, Xb, xc, and xa is independently Co-12 alkylene {Co alkylene, i.e., absent}; wherein each one of Za, Zb, Zc, and Zd is independently absent; wherein each one of Laand Lb is independently C2-20 alkenyl {Ci8 dienyl} and wherein each one of Lcand Ld is independently Co-12 alkylene-Ze-R3{C2 alkylene-Ze-R3}, wherein Zeis - O- and wherein R3is H; wherein m is 4; and wherein nl and n2 are each independently 1.

[0393] Specifically, Lipid 36, also referred to as NV5-038, is shown below,

[0394]

[0395] It is represented by Formula (I), wherein each one of xa, Xb, xc, and Xd is independently Co-12 alkylene {Co alkylene, i.e., absent}, wherein each one of Za, Zb, Zc, and Zd is independently absent; wherein each one of Laand Lb is independently C2-20 alkenyl {Ci8 dienyl} and wherein each one of Lcand Ld is independently Co-12 alkylene-Ze-R3{C3 alkylene-Ze-R3}, wherein Zeis - O- and wherein R3is H; wherein m is 2; and wherein nl and n2 are each independently 1. According to some embodiments, the lipid is selected from the group selected from: Lipid 1, Lipid 2, Lipid 3, Lipid 4, Lipid 5, Lipid 6, Lipid 7, Lipid 8, Lipid 9, Lipid 10, Lipid 11, Lipid 12, Lipid 13, Lipid 14, Lipid 15, Lipid 16, Lipid 17, Lipid 18, Lipid 19, Lipid 20, Lipid 21, Lipid 22, Lipid 23, Lipid 24, Lipid 25, Lipid 26, Lipid 27, Lipid 28, Lipid 29, Lipid 30, Lipid 31, Lipid 32, Lipid 33, Lipid 34, Lipid 35, Lipid 36,

[0396]

[0397] Lipid 37 Lipid 38

[0398] Lipid 39

[0399]

[0400] Lipid 40

[0401] 61 Lipid 41

[0402] Lipid 42

[0403] Lipid 43

[0404]

[0405] Lipid 44 Lipid 45

[0406] Lipid 46

[0407] Lipid 47

[0408]

[0409] Lipid 48 Lipid 49

[0410] Lipid 50

[0411]

[0412] Lipid 51 Lipid 53

[0413]

[0414] Lipid 54

[0415]

[0416] Lipid 57

[0417]

[0418] Lipid 58

[0419] including salts, hydrates, solvates, polymorphs, optical isomers, geometrical isomers, enantiomers, diastereomers, and mixtures thereof. Each possibility represents a separate embodiment of the present invention.

[0420] Chemical Definitions

[0421] 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 atom, 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.

[0422] 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 1,2-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.

[0423] 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 Formula (I) may be considered as ionizable lipids.

[0424] 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, -(CH2)11CH3and -CH[(CH2)4CH3](CH2)5CH3. 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 lipid. 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 lipid. 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-l,l-diyl, propan-1,3 -diyl, propan-1, 2-diyl, propan-1, 1-diyl, butan-l,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). Also, when referring to L is La-Xa-Lb; and each one of Laand Lbis Co alkylene, then L is Xa. 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, C6-12alkyl, C9 alkyl etc.

[0425] 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 (see e.g., Lipid 3) 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 carboncarbon double bond (see e.g., Lipid 16). 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.

[0426] 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.

[0427] 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.

[0428] 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.

[0429] Particles, Compositions and Uses

[0430] 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 (I), e.g., any one of Lipid 1 to Lipid 58, and a pharmaceutically acceptable excipient.

[0431] 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.

[0432] 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. 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). According to some embodiments, the phosphatidylethanolamine is l,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). According to some embodiments, the phosphatidylethanolamine is,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.

[0433] 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 1,2-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.

[0434] 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 particle comprises the lipid, cholesterol, l,2-Diolcoyl-sn-glyccro-3 -phosphoethanolamine (DOPE) and 1.2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG). According to some embodiments, the additional component comprises 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG).

[0435] 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 - / ' / -(-meth oxy 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, particle comprises the lipid according to the present invention, a membrane stabilizing lipid, an additional phospholipid and PEG-lipid conjugate.

[0436] 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.

[0437] 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”.

[0438] 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.

[0439] According to some embodiments, the particles of the present invention are nanoparticles. According to some embodiments, the lipidic particles of the present invention are lipid nanoparticles.

[0440] 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.

[0441] 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.

[0442] 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.

[0443] 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.

[0444] 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.

[0445] 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).

[0446] 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.

[0447] 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.

[0448] 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.

[0449] 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.

[0450] In some embodiments, these lipid nanoparticle compositions are useful for expression of protein encoded by mRNA.

[0451] 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.

[0452] 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.

[0453] In some other embodiments, the lipid nanoparticles are also useful for delivery of mRNA and plasmids for expression of transgenes.

[0454] 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.

[0455] 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.

[0456] 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.

[0457] 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 croscarmellose, 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.

[0458] 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.

[0459] 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.

[0460] 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.

[0461] 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.

[0462] 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.

[0463] 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.

[0464] 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.

[0465] 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.

[0466] 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.

[0467] 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.

[0468] According to some embodiments, the LNP is devoid of targeting moieties.

[0469] 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.

[0470] 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.

[0471] Definitions

[0472] 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.

[0473] 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.

[0474] 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.

[0475] 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.

[0476] " 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.

[0477] 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. 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.

[0478] 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.

[0479] 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.

[0480] 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.

[0481] Examples

[0482] EXAMPLE 1: Synthesis of ionizable lipids

[0483] Abbreviations: DCM: dichloromethane, TLC: thin layer chromatography; EtOAc: ethyl acetate; SM: starting material; DMF: dimethyl formamide; MS: mass spectrometry; LCMS: liquid chromatography -mass spectrometry; ACN: acetonitrile; EDC: l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; DMAP: 4-Dimethylaminopyridine; IPA: isopropyl alcohol;

[0484] Synthesis of Lipid 11 (also referred to as NV5-009):

[0485] Synthetic scheme: NV05 (Kill

[0486] d I " j;: 11 i I. r:: \l\ d: ’ I ’ y \ l.1Io

[0487] :i 3:= d -xic:: - dr? " "s; di?

[0488]

[0489] \l:i?:. idxi:| ■ I” o”JJd

[0490] Experimental procedure:

[0491] Synthesis of (6Z,9Z)-18-bromooctadeca-6,9-diene

[0492] ... _.."..... Br

[0493] :3Z? Z<- ' -i-l> -ij 'i see: a sec a-s ere

[0494]

[0495] ’.■Ifikvi. m rJ-- 3?f. "■ ■'

[0496] To a stirred solution of linoleyl alcohol (4.0g, 15.03 mmol, 1.0 equiv.) in DCM (100 mL) was added triphenylphosphine (4.72g, 18 mmol, 1.2 equiv.) and stirred for 10 min. Then, carbon tetrabromide (5.98g, 18.03 mmol, 1.2 equiv.) was added in one portion. The reaction mixture was then stirred at room temperature overnight. The progress of the reaction was monitored by TLC analysis (20% EtOAc in Hexane). After completion of the reaction, the solvent was removed and the product purified by column chromatography using 0-5% EtOAc in Hexane to afford 5.34g of (6Z,9Z)-18-bromooctadeca-6,9-diene (1) as a pale-brown liquid.

[0497] ’H NMR (400 MHz, CDCh): 3 5.44 - 5.28 (m, 4H), 3.41 (t, J = 6.9 Hz, 2H), 2.78 (t, J = 6.5 Hz, 2H), 2.05 (q, J = 6.9 Hz, 4H), 1.91 - 1.80 (m, 2H), 1.46 - 1.35 (m, 4H), 1.35 - 1.22 (m, 12H), 0.93 - 0.85 (m, 3H).

[0498] Synthesis of 2-(2-(((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)amino)ethoxy)ethan- 1 -ol

[0499] ■' ■ rv-«rlr-.^ ~ ■! -I ■ ir-.n - - -,n ■> - >1 ■ ■; -i ' n

[0500]

[0501] r \ r.- iL-.

[0502] To a stirred solution of (6Z,9Z)-18-bromooctadeca-6,9-diene 1 (5.31 g, 16.12 mmol, 1.0 equiv.) in ACN: THF (1:1, 30 mL) was added 2-(2-aminoethoxy)ethan-l-ol (13.4 mL, 224.0 mmol, 20.0 equiv.) at room temperature and then stirred for 24 h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh). After completion of SM, the solvent was removed and diluted with EtOAc (100 mL) and washed with water (2x100 mL). The organic layer was washed with brine solution, dried over anhydrous Na₂SO₄ and the product concentrated under reduced pressure. The crude was purified by Buchi Flash Pure System using 0-10% MeOH in CHCI3 to give the 3.95 g (75% yield) of 2-(2-(((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethoxy)ethan-l-ol as palebrown liquid.

[0503] ’H NMR (400 MHz, CDCh): 35.44 - 5.27 (m, 4H), 3.69 - 3.61 (m, 2H), 3.70(t,2H), 3.54(t,4H), 2.83 - 2.74 (m, 4H), 2.67 - 2.59 (m, 2H), 2.05 (q, J = 6.9 Hz, 4H), 1.49 (q, J = 7.2 Hz, 2H), 1.42 - 1.24 (m, 17H), 0.93 - 0.84 (m, 2H).

[0504] ESI-MS: m / z 354.3 [M+H]+.

[0505] Synthesis of 6, 15-di((9Z, 12Z)-octadeca-9, 12-dien- l-yl)-3, 9, 12, 18-tetraoxa-6, 15-diazaicosane- 1,20-diol (Lipid 11, NV5-009) NV05 009

[0506] ?. I r:.l. J d ' 7 2 S I Io lr?<

[0507] :i " ">;; d dit::: -dr? ' 70 dm

[0508]

[0509] \1:. i.lxi -Ae:ri". iC:'

[0510] To a clean flask, 2-(2-(((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethoxy)ethan-l-ol (0.7g, 2.0 mmol, 1 eq.) BR-PEG2-BR (0.2g, 0.5 mmol, 1.0 eq.) were charged and dissolved in lOmL DMF.

[0511] 0.31g KI (2.2mmol, 2.5 eq.) and 0.65g K2CO3 was added to the flask, and this was stirred at rt for 4h. After 12h days, full conversion of the SM was observed. The reaction mixture was then quenched with sodium metabisulphite, treated water and hexane, and the phases were separated. The aqueous phase was washed with 3 x 60 mL hexane. The organic layers were combined and washed with 50mL brine, dried with Na₂SO₄ and product concentrated under reduced pressure, giving the crude material as 0.8g yellow oil. This was subsequently purified using Flash Buchi Pure System 25g Silica Select column and Methanol / Chloroform as a mobile phase. The product eluted with 0-1% MeOH. After purification 187 mg (38% yield) of pure compound were isolated and confirmed by MS,JH NMR and TLC.

[0512] ’H NMR (400 MHz, CDCh) 85.4-5.3 (m, 8H), 3.7 (t, 4H), 3.6 (m,16H), 2.8 (t,12H) 2.55 (m,4H), 2.0 (q,8H), 1.5 (m, 4H), 1.40-1.20 (34m, H), 0.8 (t, 6H) (Figure 1A).

[0513] ESI-MS: m / z 821.83 [M+1]+;411.8[M’2+H]+’ 843.5[M+Na]+(Figure IB).

[0514] HPLC-CAD purity: 92.2%

[0515] Synthesis of Lipid 12 (also referred to as NV5-010):

[0516] Synthetic scheme: i - N i i i

[0517] z iduvei yliPr. I ID r,•;ethan * u’

[0518] KI I All

[0519] final

[0520]

[0521] NV05-010

[0522] i: Hi din:idi:-:: Yl J 'J 12 1S lelmtv. J ii ’ h di;j / au:: K uric? 1;l diol

[0523] W ilur WM]hl 6ii1 II -'

[0524] Experimental procedure:

[0525] Synthesis of 2-(2-(dodecylamine)ethoxy)ethan-l-ol:

[0526] : ■. ■■ i-: ■: ■; y I-:

[0527]

[0528] Me i-c.jyi' 'A'I- / <■ ■!::

[0529] To a clean flask, 1-Bromo dodecane (4.0g, 16.048mmol, leq.) and 2-(2-aminoethoxy)ethanol-l- ol (16 mL, 160.48 mmol, 10.0 equiv.) were charged and stirred at room temperature for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh) and LCMS. After completion of SM, the solvent was removed and the reaction mixture diluted with EtOAc (100 mL) and washed with water (2x100 mL). The organic layer was washed with brine solution, dried over anhydrous Na₂SO₄ and the solvent was evaporated. The crude product was purified by Buchi Flash system using 0-10% MeOH in CHCI3 to give the 3.1 g of 2-(2-(dodecylamino)ethoxy)ethan-l-ol as a white solid. Product eluted at 2% MeOH.

[0530] ¹H NMR (400 MHz, CDCl₃): δ 3.88 (t, J = 4.9 Hz, 2H), 3.80 - 3.73 (m, 2H), 3.65 (dd, J = 5.0, 3.3 Hz, 2H), 3.19 (t, J = 4.9 Hz, 2H), 3.05 - 2.96 (m, 2H), 1.95 - 1.83 (m, 2H), 1.31-1.25 (m, 18H), 0.88 (t, J = 6.8 Hz, 3H).

[0531] Synthesis of 6,15-didodecyl-3,9,12,18-tetraoxa-6,15-diazaicosane-l,20-diol (Lipid 12, NV5-010):

[0532] NV05-010

[0533] B 1!; didcdi:-:? / ’ 3 9 12 18 Intraoxa ii 1 h diazaicnsaru? 1 / I! di6

[0534]

[0535] MD ci.ii:ir •■'•'i' ■:;til L: I:

[0536] To a clean flask, Br PEG2 (0.25g, 0.9 mmol, 1 eq.), 2-(2-(dodecyl amino) ethoxy)ethan-l-ol (0.65g, 2.4mmol, 2.5 eq.), K2CO3 (0.62g, 3.6mmol, 5 eq.) and KI (0.38g, 2.3mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70-80°C for 12h. In the morning, TLC and MS performed on the reaction mixture showed full consumption of starting material. The reaction mixture was then quenched with H2O and diluted with ethyl acetate. The phases were separated and the aqueous layer was washed with ethyl acetate 3 x 50 mL. The organic layers were washed with 50mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 1.56g crude yellow oil (small traces of DMF present). The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 12g Silica Select Cartridge and CHCI3 and MeOH as a mobile phase. The compound eluted with 0-2% MeOH as 250mg (42% yield) yellow oil. ¹H NMR (400 MHz, CDCl₃): δ 3.65(t, 4H), 3.55 (t, 15H), 2.7 (dt, J = 4.9 Hz, 8H), 2.5(t, 4H), 1.4-1.5 (m, 4H), 1.31-1.25 (m, 37H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 2A).

[0537] ESI-MS: m / z 661.7 [M + H]⁺, 683.7 [M + Na]⁺, 331.4 [M⁻²]⁺ (Figure 2B).

[0538] HPLC-CAD purity: 97%

[0539] Synthesis of Lipid 14 (also referred to as NV5-011):

[0540] Synthetic scheme:

[0541]

[0542] Experimental procedure

[0543] Synthesis of 3,12-bis(2 -hydroxy ethyl)-6,9-dioxa-3,12-diazatetradecane- 1,14-diol:

[0544]

[0545] To a clean flask, 1g 2, 2’ (ethane- l,2-diybis(oxy)bix(ethan-l -amine) (6.75mmol, 1.0 eq.), 4.8mL 2-Bromoethanol (67.48mmol, 10.0 eq.), 5.6g KI (33.74mmol, 5.0 eq.), 5.58g K2CO3 (40.5mmol, 6.0 eq.) were charged. These were dissolved in lOmL ACN and lOmL THF. The reaction mixture was stirred at 40-50°C for 24h. The reaction progress was monitored using TLC and MS direct injection analysis. After 24h, the SM was fully consumed. The reaction mixture was filtered over a celite plug, and this was washed several times with ethyl acetate. The ethyl acetate filtrate solution was evaporated under reduced pressure, and the crude mixture was obtained as a yellow semisolid (m = 3.0 g). The crude mixture was purified using Buchi Flash Pure machine with an 12g Silica select column using 0-20% Methanol in Chloroform. The product eluted at 10% Methanol in Chloroform and 0.5g yellow gummy liquid was obtained (25% yield), and appeared as a single spot by TLC.

[0546] ¹H NMR (CDCl₃, 400 mHz): 3.71 (t, J = 4.9 Hz, 8H), 3.64 (s, 4H), 3.59 (q, J = 3.9 Hz, 4H), 2.66 (dt, J = 9.6, 4.8 Hz, 12H).

[0547] ESI-MS: m / z 325.7 [M + H]+; 347.7 [M + Na]

[0548] Synthesis of Lipid 14, NV5-011:

[0549] J r Nv:ib 1111

[0550]

[0551] o

[0552] To a clean flask, 0.15g tetra alcohol of the previous step (0.5 mmol, 1.0 eq.), 0.74g dodecanoic acid (3.7 mmol, 8.0 eq.), 1.32g EDC HC1 (6.9mmol, 15.0 eq.), and 56mg DMAP (0.5 mmol, 1.0 eq.) were dissolved lOmL anhydrous DCM and stirred at 30-40°C for 24h. The reaction mixture was monitored using MS analysis and the mass of the desired product was observed. The reaction mixture was then quenched with 20mL IM HC1 water and diluted with another 20mL DCM. Phases were separated and the aqueous layer was washed with 3 x 30mL DCM. DCM layers were combined and then washed with brine, dried using Na₂SO₄ and filtered over paper. The filtrate was evaporated under reduced pressure and 860mg of white solid was obtained. This was then purified using Buchi Flash Pure System using a 40g Silica Select column and ethyl acetate in petroleum ether as a mobile phase. The product eluted at 40% Ethyl acetate and 140mg (26% yield) was obtained.

[0553] ¹H NMR (400 MHz, CDCl₃): δ 4.20 (t, 8H), 3.6 (s, 4H), 3.5 (t, 4H), 2.29 (t, 8h), 1.79-1.67 (m, 16H), 1.06 (s, 70H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 3A).

[0554] ESI-MS: m / z 528 [M2‘], 1054.6 [M+H], 1075.9 [M+Na] (Figure 3B).

[0555] Synthesis of Lipid 15 (also referred to as NV5-012):

[0556] Synthetic scheme:

[0557] 3.12-D4a2-Hd c / ,.,einyl -69-aiJ'>:a-3 ’ 2-:l azateti a ilesare- ' '4-UiC-l

[0558] k’jk'UJ'.r ‘A'e|.y'i:.i?44?

[0559] Decanoic acid

[0560] EDC HCL DMAP O

[0561] L; CM

[0562] VlnniM'd; 94' -1 i Synthesis of Lipid 15 (NV5-012):

[0563]

[0564] WCKJ’I* 94* To a clean flask, 0.15g 3,12-bis(2-hydroxyethyl)-6,9-dioxa-3,12-diazatetradecane-l,14-diol from Stage 1(0.5 mmol, 1.0 eq.), 0.89g decanoic acid (3.7 mmol, 8.0 eq.), 1.32g EDC HC1 (6.9mmol, 15.0 eq.), and 56mgDMAP (0.51 mmol, 1.0 eq.) were dissolved lOmL anhydrous DCM and stirred at 30-40°C for 24h. The reaction mixture was monitored using MS analysis and the mass of the desired product was observed. The reaction mixture was then quenched with 20mL IM HC1 water and diluted with another 20mL DCM. Phases were separated and the aqueous layer was washed with 3 x 30mL DCM. DCM layers were then combined, washed with brine, dried Na₂SO₄ and filtered over paper. The filtrate was evaporated under reduced pressure and 1g of white solid was obtained. This was then purified using Buchi Flash Pure System using a 40g Silica Select column and ethyl acetate in petroleum ether as a mobile phase. The product eluted at 30-33% Ethyl acetate and 182mg (33% yield) was obtained.

[0565] ’H NMR (400 MHz, CDCh): 54.20 (t, 19H), 3.6 (s, 3H), 3.5 (t, 4H), 2.29 (t, 5h), 1.79-1.67 (m, 10H), 1.06 (s, 58H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 4A).

[0566] ESI-MS: m / z 472.2 [M2‘], 942.7 [M+H], 964.6 [M+Na] (Figure 4B).

[0567] Synthesis of Lipid 3 (also referred to as NV5-013):

[0568] Synthetic scheme:

[0569] |- ~ & 4 J-"'*-. O •’V OH kj3*1 " L< • 2- ■ i- ■:? >• 1-?.■

[0570]

[0571] MaleHJarWagftt: 1374.17 Synthesis of Lipid 3 (NV5-013):

[0572]

[0573] MaleHJarWagftt: 1374.17

[0574] To a clean flask, 0.25g 3,12-bis(2-hydroxyethyl)-6,9-dioxa-3,12-diazatetradecane-l,14-diol (0.7 mmol, 1.0 eq.), 1.29g linoleic acid (4.6 mmol, 8.0 eq.), 0.7g EDC HC1 (36.51mmol, 5.0 eq.), and 90mg DMAP (0.73 mmol, 1.0 eq.) were dissolved lOmL anhydrous DCM and stirred at 30-40°C for 24h. The reaction mixture was monitored using MS analysis and the mass of the desired product was observed. The reaction mixture was then quenched with 20mL IM HC1 and diluted with another 20mL DCM. Phases were separated and the aqueous layer was washed with 3 x 30mL DCM. DCM layers combined and were then washed with brine, dried with Na₂SO₄ and filtered over paper. The filtrate was evaporated under reduced pressure and 1.5g of cloudy oil was obtained. This was then purified using Buchi Flash Pure System using a 40g Silica Select column and ethyl acetate in petroleum ether as a mobile phase. The product eluted at 30-40% Ethyl acetate and 190mg (20% yield) was obtained as a clear pale-yellow oil.

[0575] ’H NMR (400 MHz, CDCk): 55.35(m, 14H), 4.20 (t, 8H), 3.6 (s, 3H), 3.5 (t, 4H), 2.29 (t, 8H), 2.10 (t,16H), 1.79-1.67 (m, 13H), 1.06 (s, 58H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 5A).

[0576] ESI-MS: m / z 688.1 [M2‘], 1374.6 [M+H], 1396.7 [M+Na] (Figure 5B).

[0577] Synthesis of Lipid 16 (also referred to as NV5-014):

[0578] Synthetic scheme: I: -

[0579] Synthesis of Lipid 16 (NV5-014):

[0580] a

[0581]

[0582] To a clean flask, 50mg 3,12-bis(2-hydroxyethyl)-6,9-dioxa-3,12-diazatetradecane-l,14-diol (0.15 mmol, 1.0 eq.), 338mg oleic acid (1.2 mmol, 8.0 eq.), 0.38g EDC HC1 (13.51mmol, 5.0 eq.), and 20mg DMAP (0.16 mmol, 1.0 eq.) were dissolved lOmL anhydrous DCM and stirred at 40°C for 3 days. The reaction mixture was monitored using MS analysis and the mass of the desired product was observed. The reaction mixture was then quenched with 20mL IM HC1 and diluted with another 20mL DCM. Phases were separated and the aqueous layer was washed with 3 x 30mL DCM. DCM layers were combined and then washed with brine, dried with Na₂SO₄ and filtered over paper. The filtrate was evaporated under pressure and 0.54g of cloudy white oil was obtained. This was then purified using Buchi Flash Pure System using a 40g Silica Select column and ethyl acetate in petroleum ether as a mobile phase. The product eluted at 30% Ethyl acetate and lOOmg (48% yield) was obtained as a clear pale-yellow oil. ’H NMR (400 MHz, CDCh): 55.35(m, 8H), 4.20 (t, 8H), 3.6 (s, 4H), 3.5 (t, 4H), 2.29 (t, 12H), 2.10 (t,8H), 2.0(m,15H), 1.79-1.67 (m, 13H), 1.06 (s, 80H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 6A)

[0583] ESI-MS: m / z 691.16 [M2‘], 1382.21 [M+H], 1404.19 [M+Na] (Figure 6B).

[0584] Synthesis of Lipid 20 (also referred to as NV5-015):

[0585] Synthetic scheme:

[0586] 1 brunicd JiitH / irii? Molecular Weiyht J49 74

[0587] KI; KaCO3

[0588] '■ 1 ACN ' ■ II

[0589] NV05-015

[0590] ■V > V-::.ethan?-1,2-diyibis i coy c i er’.ane-2 ■’ -cl yl i ids:

[0591]

[0592] ::c:::e d.-d:-: a- ’ urirn? ■

[0593] Molesu'ar We ght: 8 1.50

[0594] Experimental procedure:

[0595] To a clean flask, 0.1g2,2'-(ethane-l,2-diylbis(oxy))bis(ethan-l-amine) (1 eq., 7x10-4mmol), 1.34g 1 -Bromododecane (8 eq., 5.4x10-3mmol), 0.46g potassium iodide (5 eq., 3.3 x10-3mmol), and 2.24g potassium carbonate (20 eq., 1.35 x 10-2mmol) was dissolved in lOmL ACN and lOmL THF. The reaction mixture was stirred at 60 °C for two days. Reaction mixture was quenched with 50mL water and diluted with 50mL ethyl acetate. The phases were separated and aqueous layer was washed with 3 x 30mL ethyl acetate. The organic layers were washed with 50mL brine, dried with Na₂SO₄ and filtered over paper. The filtrate was concentrated under reduced pressure, giving 1,2g of dark orange oil as a crude product. The crude mixture was dry loaded and purified using a 40g Silica Select Cartridge and Methanol: Chloroform as the mobile phase in a Flash Pure System machine. The product eluted at 1% Methanol in Chloroform. 130mg (23% yield) of pure product was isolated as a white semisolid. ’H NMR (400 MHz, DMSO): 53.7(t, 4H), 3.6 (s, 4H), 2.8(s, 4H), 2.6(s, 6H), 1.6-1.49 (s, 8H), 1.3-1.2 (s, 75H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 7A).

[0596] ESI-MS: m / z 412.1 [M2‘], 822.5 [M+H] (Figure 7B).

[0597] Synthesis of Lipid 19 (also referred to as NV5-016):

[0598] Synthetic scheme:

[0599] Ki; K2CO3l-Broinotfodecane

[0600] ACN I l li

[0601] fv V i: ynisi:: IIM'K.-? I cl:nr>i. T ■>: mis p-oT-ru::i 1

[0602] d.y;:b DJiltn-ylduileudl' I dini'ie;

[0603]

[0604] Mnle-i. ar Wt' r]P r?’

[0605] Experimental procedure:

[0606] To a clean flask, 0.2g 3,3'-((oxybis(ethane-2,l-diyl))bis(oxy))bis(propan-l-amine) (1 eq., 9x10-4mmol), 2.0g 1 -Bromododecane (8 eq., 8x10-3mmol), 0.7g potassium iodide (5 eq., 5 x10-3mmol), and 3.3g potassium carbonate (20 eq., 1.9 x 10-2mmol) was dissolved in 15mL ACN and 15mL THF. The reaction mixture was stirred at 60 °C for 1 day. Reaction mixture was then quenched with 50mL water and diluted with lOOmL ethyl acetate. The phases were separated and aqueous layer was washed with 3 x 50mL ethyl acetate. The organic layers were washed with 50mL brine, dried with Na₂SO₄ and filtered over paper. The filtrate was concentrated under reduced pressure, giving 2.6 g of dark orange oil as a crude product. The crude mixture was dry loaded and purified using a 80g Silica Select Cartridge and Methanol: Chloroform as the mobile phase in a Flash Pure System machine. The product elutedat 1% Methanol in Chloroform. 60mg (8% yield) of pure product was isolated as a yellow oil.

[0607] ’H NMR (400 MHz, CHCh): 53,7(t, 4H), 3.6 (s, 4H), 3.5 (t, 4H), 2.5 (m, 4H), 2.4 (m, 8H), 1.7- 1.6 (t, 6H), 1.4 (s, 8H) 1.3-1.2 (s, 72H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 8A). ESI-MS: m / z 447.4 [M2‘], 893.9 [M+H] (Figure 8B).

[0608] Synthesis of Lipid 13 (also referred to as NV5-017):

[0609] Synthetic scheme:

[0610] " br jri L JjduLd’i-'

[0611] Molecular Weight 249.24

[0612] r ACN i i iiH.iN

[0613] 2 ( dcjd[?ryfan>iriti pllum ' cl

[0614] MulecuJdt 'Weight 229 41

[0615] KJCOJ; M

[0616] AhN I HI

[0617] Heat

[0618]

[0619] NV05-017

[0620] 3, 12-didodecy I-6, 9-dioxa-3, 12-diazatetradecane- 1s14-diol

[0621] nk? E ul.ir Weigh! / cJEi

[0622] Experimental procedure:

[0623] Synthesis of 2-(dodecylamino)ethan-l-ol:

[0624] 2-:l e:. • *c:eh ’ ‘- -c l

[0625]

[0626] r: ■:..!• ■! ■■ I

[0627] To a clean flask, 1-Bromo dodecane (4.0g, 16.048mmol, leq.) and 2-aminoethanol-l-ol (9.7 mL, 160.48 mmol, 10.0 equiv.) were charged, dissolved in 40mL ACN: THF and stirred at room temperature for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh) and LCMS. After completion of SM, the solvent was removed diluted with EtOAc (100 mL) and washed with water (2x100 mL). The organic layer washed with brine solution and dried over anhydrous Na₂SO₄, filtered over a silica 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.

[0628] ’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).

[0629] ESI-MS analysis: m / z 230.6 [M+H],

[0630] Synthesis of 3,12-didodecyl-6,9-dioxa-3,12-diazatetradecane-l,14-diol (Lipid 13, NV5-017)

[0631]

[0632] NV05-Q17

[0633] 3 ^-cidcc&cyi-o. S-diox -l 12-diaz?etrarjecane-1 U-dioi

[0634] I olm-uldr Weigh? 6 / 7 06

[0635] To a clean flask, Br PEG2 (0.25g, 0.9 mmol, 1 eq.), 2-(dodecylamino) ethan-l-ol (0.7g, 2.4mmol, 2.5 eq.), K2CO3 (0.62g, 3.6mmol, 5 eq.) and KI (0.38g, 2.3mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70-80°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of starting material. The reaction mixture was then quenched with H2O and diluted with ethyl acetate. The phases were separated and aqueous layer was washed with ethyl acetate 3 x 50 mL. The organic layers were combined, washed with 50mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.95g crude yellow oil (small traces of DMF present). The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 25g Silica Select Cartridge and CHCh and MeOH as a mobile phase. The compound eluted with 3-5% MeOH as 60mg (12% yield) yellow oil.

[0636] ’H NMR (400 MHz, CDCh): 53 65(t, 4H), 3.55 (t, 15H), 2.7 (dt, J = 4.9 Hz, 8H), 2.5(t, 4H), 1.4- 1.5 (m, 4H), 1.31-1.25 (m, 37H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 9A).

[0637] ESI-MS: m / z 574.0 [M + H]+, 595.9 [M + Na]+, 287.9 [M-2]+(Figure 9B).

[0638] Synthesis of Lipid 18 (also referred to as NV5-018):

[0639] Synthetic scheme:

[0640] I'.

[0641] ACN: THF

[0642] OH OH I T J

[0643] i r

[0644] HlJ

[0645] ■. ■: -. li:. ’:-!!. ’;:l.;< ~s: - I <l>r;

[0646] rn* "’r h"

[0647] NV05-018

[0648]

[0649] M:-:., -■.:-i! l.-!>.

[0650] Experimental procedure:

[0651] Synthesis of 3, 17-bis(2 -hydroxy ethyl)-7, 10,13 -trioxa-3, 17-diazanonadecane- 1, 19-diol:

[0652] r. y l'. >.:i.1..iir. i t,.r. ’ V -I..

[0653]

[0654] To a clean flask, 1g 3,3'-((oxybis(ethane-2,l-diyl))bis(oxy))bis(propan-l-amine) (4.5 mmol, 1.0 eq.), 3.2mL 2-Bromoethanol (4.5 x 10-2mmol, 10.0 eq.), 3.73g KI (2.25x10-2mmol, 5.0 eq.), 3.73g K₂CO₃ (2.7x10-2mmol, 6.0 eq.) were charged. These were dissolved in 15mL ACN and 15mL THF. The reaction mixture was stirred at 80°C for 24h. The reaction progress was monitored using TLC and MS direct injection analysis. After 24h, the SM was fully consumed. The reaction mixture was then filtered over a celite plug, and this was washed several times with ethyl acetate. The ethyl acetate filtrate solution was evaporated under reduced pressure, and the crude mixture was obtained as a yellow semisolid (m = 3.0 g). The crude mixture was purified using Buchi Flash Pure machine with an 12g Silica select column using 0-20% Methanol in Chloroform. The product eluted at 10% Methanol in Chloroform and 0.25g yellow gummy liquid was obtained (14% yield) as a single spot by TLC. ESI-MS: m / z 397.6 [M+H], 419.6 [M+Na],

[0655] Synthesis of Lipid 18 (NV5-018):

[0656] o

[0657] NVfl54M8

[0658]

[0659] To a clean flask, 0.25g tetra alcohol from the previous step (6xl0‘4mmol, 1.0 eq.), 0.85g dodecanoic acid (4.9xl0‘3mmol, 8.0 eq.), 1.32g EDC HC1 (6.9x10-3mmol, 15.0 eq.), and 56mg DMAP (6xl0‘4mmol, 1.0 eq.) were dissolved lOmL anhydrous DCM and stirred at 30-40°C for 24h. The reaction mixture was monitored using MS analysis and the mass of the desired product was observed. The reaction mixture was then quenched with 20mL NaHCCh water and diluted with another 20mL DCM. Phases were separated and the aqueous layer was washed with 3 x 30mL DCM. DCM layers were then combined aqnd washed with brine, dried with Na₂SO₄ and filtered over paper. The filtrate was evaporated under reduced pressure and 860mg of white solid was obtained. This was then purified using Buchi Flash Pure System using a 40g Silica Select column and ethyl acetate in petroleum ether as a mobile phase. Two purifications were necessary to isolate the product as 60mg (8% yield) yellow oil, which eluted at 40% Ethyl acetate.

[0660] ’H NMR (400 MHz, CDCI3): 54.20 (t, 8H), 3.7(m,4H), 3.6 (m, 4H), 3.5 (t, 4H),2.75(t,8H), 2.6(t,4H), 2.29 (t, 8h), 1.79-1.67 (m, 18H), 1.06 (s, 66H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 10A).

[0661] ESI-MS: m / z 562 [M2‘], 1125.7 [M+H], 1148.1 [M+Na] (Figure 10B). Synthesis of Lipid 23 (also referred to as NV5-019):

[0662] Synthetic scheme:

[0663] ' ' -iLN h-!’; N

[0664] 4-(dods:,.''aiT' rc niitar-1 -al

[0665] VlrJi.-cuIri! '■A'Hiqhl?!:.•'.1b

[0666] .-n -r'.n. ■'

[0667] OMF

[0668] heat I

[0669] ♦

[0670] NIW5-0W

[0671] 5 11 d nnnnr.'vl R 11 nia-.a 5 1 d'.^an.-tan’ccare 1 1R d d

[0672]

[0673] Malecdljr VS't <jl'1 iZV C.‘

[0674] Synthesis of 4-(dodecylamino)butan-l-ol

[0675] . OH

[0676]

[0677] M D e:.f:■?.‘e j < 257SC

[0678] To a clean flask, 1-Bromo dodecane (3.0g, 12.0mmol, leq.) and 4-aminobutan-l-ol (10.1 mL, 113.3 mmol, 10.0 eq.) were dissolved in 24mL ACN: THF and stirred at room temperature for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh) and LCMS. After completion of SM, the solvent was removed and crude product was diluted with EtOAc (100 mL) and washed with water (2x50 mL). The organic layer was washed with brine solution and dried over anhydrous Na₂SO₄, filtered over silica 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 4-(dodecylamino)butan-l-ol as a white solid. Product eluted at 2% MeOH. ’H NMR (400 MHz, CDCh): 53.65 (t, J = 5.0, 3.3 Hz, 2H), 2.75 (t, 2H), 2.65 (t, 2H), 1.7-1 6(dt, 5H), 1.47 (q, J = 7.0 Hz, 2H), 1.31-1.25 (m, 18H), 0.88 (t, J = 6.8 Hz, 3H).

[0679] ESI-MS analysis: m / z 258.8 [M+H],

[0680] Synthesis of 5,14-didodecyl-8,l l-dioxa-5,14-diazaoctadecane-l,18-diol (Lipid 23, NV5-019):

[0681] Nvns.

[0682] S 1 - d ‘ S 1:l,-i "ijrMr.rfjrf. 1 12 d r.l

[0683]

[0684] \k:li.c.ik ‘-■•i.:jl l:<1f.

[0685] To a clean flask, Br PEG2 (0.4g, 1.4 mmol, 1 eq.), 4-(dodecylamino)butan-l-ol (0.8g, 3.1 mmol, 2.5 eq.), K2CO3 (0.96g, 6.9mmol, 5 eq.) and KI (0.58g, 3.5mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70-80°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of the starting material. The reaction mixture was then quenched with H2O and diluted with ethyl acetate. The phases were separated and the aqueous layer was washed with ethyl acetate 3 x 50 mL. The organic layers were combined, washed with 50mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 1,2g crude yellow oil (small traces of DMF present). The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 25g Silica Select Cartridge and CHCI3 and MeOH as a mobile phase. The compound eluted with 1-2% MeOH as 400g (45% yield) yellow oil.

[0686] ’H NMR (400 MHz, CDCI3): 5 3.65(s, 6H), 3.55 (t, 4H), 2.7 (dt, J = 4.9 Hz, 5H), 2.5(t, 7H), 1.7(s,7H), 1.5-1.4 (m, 4H), 1.31-1.25 (m, 37H), 0.88 (t, J = 6.8 Hz, 6H) (Figure HA).

[0687] ESI-MS: m / z 629.8 [M + H]+, 651.9 [M + Na]+(Figure 11B).

[0688] HPLC-CAD purity: 95.7% Synthesis of Lipid 24 (also referred to as NV5-020):

[0689] Synthetic scheme:

[0690] "■ ‘Br

[0691] 1 -bromo-dodecane

[0692] 11 ACIOHF ■,;i i

[0693] u

[0694] zX / X / X / X / X / ''N

[0695] H

[0696] 3-(dudecylaniiino)>propan-1-o>l

[0697] Q _ g

[0698] ')MI (ludt Br" 'O'

[0699] I

[0700]

[0701] NV05-020

[0702] 4.13-didodecy I-7.10-dioxa-4.13-dazatiexadecane- 1, 16-diol

[0703] Molecular Weight 601 0’

[0704] Experimental procedure:

[0705] Synthesis of 3-(dodecylamino)propan-l-ol:

[0706] 3 -* d: r t:> p-;

[0707]

[0708] To a clean flask, 1-Bromo dodecane (4.0g, 16.0mmol, leq.) and 3 -aminopropan- l-ol (12.07 mL, 160.7 mmol, 10.0 equiv.) were charged, dissolved in 40mL ACN: THF and stirred at room temperature for 24h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh) and LCMS. After completion of SM, the solvent was removed and the crude product was diluted with DCM (100 mL) and washed with water (2x50 mL). The organic layer was washed with brine solution, dried over anhydrous Na₂SO₄, filtered over silica plug and the solvent evaporated, affording 3.86g crude white solid. The crude was recrystallized using the minimum amount of acetonitrile, yielding 1g of white needles. The filtrate of the crude was purified by Buchi Flash system using 0-10% MeOH in CHCI3 to give the 1.2g of 3-(dodecylamino)propan-l-ol as a white solid. Product eluted at 2% MeOH.

[0709] Synthesis of 4,13 -didodecyl-7,10-dioxa-4, 13 -diazahexadecane- 1,16-diol (Lipid 24, NV5-020):

[0710] To a clean flask, Br PEG2 (0.25g, 0.9 mmol, 1 eq.), 2-(dodecylamino)propan-l-ol (0.7g, 4.5mmol, 2.5 eq.), K2CO3 (0.62g, 3.6mmol, 5 eq.) and KI (0.37g, 2.3mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70-80°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of the starting material. The reaction mixture was quenched with H2O and diluted with petroleum ether. The phases were separated and the aqueous layer was washed with petroleum ether 3 x 50 mL. The organic layers were combined, washed with 50mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.97g crude yellow oil (small traces of DMF present). The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 12g Silica Select Cartridge and CHCI3 and IP A as a mobile phase. The compound eluted with 0-10% MeOH as 158mg (29% yield) pale- yellow translucent oil.

[0711] 'H NMR (400 MHz, CDCI3): 8 3.65(s, 3H), 3.55 (t, 6H), 2.7 (dt, J = 4.9 Hz, 6H), 2.5(t, 3H), 1.7(t,4H), 1.5 (s, 8H), 1.4 (s, 4H), 1.31-1.25 (m, 30H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 12A). ESI-MS: m / z 601.8 [M + H]+, 623.7 [M + Na]+(Figure 12B).

[0712] HPC-CAD purity: 96%

[0713] Synthesis of Lipid 27 (also referred to as NV5-021):

[0714] Synthetic scheme: 1. brumo elade can®

[0715] '• 1 ■ r.'.. I < W= Q T Z 4

[0716] I I / .' "'•■■'■"■"'UH

[0717] 2-sc sc e 1,

[0718] a- ‘.'V:- r ■! ” ■■ -i f

[0719] h K. C”..

[0720] r:.r

[0721] NV05-021

[0722] 3 did.ndfic / l-6 fi Vdr nxv^d ^-d a’inf'pfidecanf’ * 1 •’ -mni

[0723]

[0724] IM deed Al ‘A'eldti! u' ■

[0725] Experimental procedure:

[0726] Synthesis of 2-(dodecylamino)ethan-l-ol:

[0727] 2 -:l: c -:.? -c:eCi> •- ’ <

[0728]

[0729] L: ■:.!■ A ■: I?!• I

[0730] To a clean flask, 1-Bromo dodecane (4.0g, 16.048mmol, leq.) and 2-aminoethanol-l-ol (9.7 mL, 160.48 mmol, 10.0 equiv.) were charged, dissolved in 40mL ACN: THF and stirred at room temperature for 24h-48h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh) and LCMS. After completion of SM, the solvent was removed and the crude product was diluted with EtOAc (100 mL)and washed with water (2x100 mL). The organic layer was washed with brine solution, dried over anhydrous Na₂SO₄, filtered over silica 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.

[0731] ’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). ESI-MS: m / z 230.6 [M+H],

[0732] Synthesis of 3,15-didodecyl-6,9,12-trioxa-3,15-diazaheptadecane-l,17-diol (Lipid 27, NV5-021)

[0733] OH OH

[0734] IMV05-021

[0735] .:irl:ir|f'r--,l r:f. ’ 1( r. < i 1f::l.-i ■ i v'.r vk'.-.n-f. ‘ 1 ■’ r.m

[0736]

[0737] Vi-b..i, i I'K.rr.

[0738] To a clean flask, Br PEG3 (0.25g, 0.8 mmol, 1 eq.), 2-(dodecylamino) ethan-l-ol (0.45g, 2.0mmol, 2.5 eq.), K2CO3 (0.55g, 4.0mmol, 5 eq.) and KI (0.33g, 2.0mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70-80°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of starting material. The reaction mixture was then quenched with H2O and diluted with petroleum ether. The phases were separated and aqueous layer was washed with petroleum ether 3 x 30 mL. The organic layers were combined, washed with 20mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.55g crude yellow oil (small traces of DMF present). The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 25g Silica Select Cartridge and CHCI3 and MeOH as a mobile phase. The compound eluted with 0-1% MeOH as 155mg (31% yield) dark yellow semi-solid.

[0739] ’H NMR (400 MHz, CDCI3): 5 3.65 (t, 7H), 3.55 (t, 7H), 2.75 (t, 4H) 2.7 (dt, J = 4.9 Hz, 8H), 2.5(t, 4H), 1.5-1.4 (m, 4H), 1.31-1.25 (s, 37H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 13A).

[0740] ESI-MS: m / z 618.1 [M + H]+, 639.8 [M + Na]+(Figure 13B).

[0741] HPLC CAD purity: 94%

[0742] Synthesis of Lipid 28 (also referred to as NV5-022):

[0743] Synthetic scheme: JDH

[0744] .::l> ■: i 1 ':

[0745] k'-j pj.,r.1 ■*'

[0746] ; ' ■

[0747] DMF, h<eat:

[0748]

[0749] 3,18-didc 54:..'c;:

[0750] Molecular Weight: 6B1.07

[0751] Experimental procedure:

[0752] Synthesis of 3,18-didodecyl-6,9,12,15-tetraoxa-3,18-diazaicosane-l,20-diol (Lipid 28, NV5-022):

[0753] OH

[0754] rJ

[0755] NV05-022

[0756]

[0757] 3, 18-didcs; f: ■ a- 3 £:,z:•:: s.ve - ’ 13-3:

[0758] Molecular Weight: BB1.07

[0759] To a clean flask, Br PEG4 (0.25g, 0.7 mmol, 1 eq.), 2-(dodecylamino) ethan-l-ol (0.41g, 1.8mmol, 2.5 eq.), K2CO3 (0.48g, 3.5mmol, 5 eq.) and KI (0.29g, 1.7mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70-80°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of starting material. The reaction mixture was then quenched with H2O and diluted with petroleum ether. The phases were separated and the aqueous layer was washed with petroleum ether 3 x 30 mL. The organic layers were combined, washed with 20mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.48g crude yellow semi-solid (small traces of DMF present). The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 12g Silica Select Cartridge and CHCI3 and MeOH as a mobile phase. The compound eluted with 0-1% MeOH as 52mg (12% yield) dark yellow semi-solid. ’H NMR (400 MHz, CDCk): 5 3.65 (t, 8H), 3.55 (t, 5H), 2.75 (t, 2H) 2.7 (dt, J = 4.9 Hz, 2H), 2.5(t, 2H), 1.5-1.4 (m, 3H), 1.31-1.25 (s, 28H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 14A).

[0760] ESI-MS: m / z 662.0 [M + H]+, 683.9 [M + Na]+(Figure 14B).

[0761] HPLC CAD Purity: 90%

[0762] Synthesis of Lipid 21 (also referred to as NV5-024):

[0763] Synthetic scheme:

[0764] HaN

[0765] Mo rri la.,'F.

[0766] KI: NCOj

[0767] ACK I H‘ Ibfomodocfeeane

[0768] 'It'rll T

[0769] X / Sv^ / XZX / S^N x / \ / \ZKZX / X /

[0770] NV05 924

[0771] .'■,'. V-i.;c<'.'Disie1nane-2.1-dryl;i3tsi c.v.,?i3'5ie1,iane-2.1-d:yl;i3tsi '•.'-dodecyldc'cecan-l-anin®:1

[0772]

[0773] Mt:lt:r:.iMi S:ij Sb

[0774] Experimental procedure:

[0775] To a clean flask, 0.2g 2,2'-((oxybis(ethane-2,l-diyl))bis(oxy))bis(ethan-l -amine) (1 eq., lOmmol), 2.0g 1 -Bromododecane (8 eq., 8 mmol), 0.7g potassium iodide (5 eq., 5 mmol), and 3.3g potassium carbonate (20 eq., 19.0 mmol) were dissolved in 15mL ACN and 15mL THF. The reaction mixture was stirred at 60 °C for 1 day. Reaction mixture was quenched with 50mL water and diluted with lOOmL ethyl acetate. The phases were separated and the aqueous layer was washed with 3 x 50mL ethyl acetate. The organic layers were washed with 50mL brine, dried with Na₂SO₄ and filtered over paper. The filtrate was concentrated under reduced pressure, giving 2.6 g of dark orange oil as a crude product. The crude mixture was dry loaded and purified using an 80g Silica Select Cartridge and Methanol: Chloroform as a mobile phase in a Flash Pure System machine. The product eluted at 1% Methanol in Chloroform. 72mg (9% yield) of pure product was isolated as a yellow oil.

[0776] ’H NMR (400 MHz, CHCh): 53,7(t, 8H), 3.6 (t, 4H), 3.5 (t, 4H), 2.7 (s,4H), 2.5 (s, 8H), 2.4 (s, 8H), 1.4 (s, 8H) 1.3-1.2 (s, 72H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 15A). ESI-MS: m / z 865.7 [M2‘], 888.5 [M+H] (Figure 15B).

[0777] HPLC CAD purity: 97%

[0778] Synthesis of Lipid 22 (also referred to as NV5-025):

[0779] Synthetic scheme:

[0780] “ =1 <■ 7 k-=.-; 14-d»mine

[0781] K‘- fiij'j- ‘A'i- yr.'J d1

[0782] ACM THF 1 bnsmotiodeca-ne

[0783] ■V',> VJ'at -2 e f ' 2-tet'c-oj.'-tr: at eoane- 1, 14-diarnine

[0784]

[0785] Vu U- ‘A'l‘ y - J 1

[0786] Experimental procedure:

[0787] Synthesis of N1, N1, N14, N14-tetradodecyl-3,6,9,12-tetraoxatetradecane-l,14-diamine (Lipid 22, NV5-025).

[0788] To a clean flask, 0.2g 3,6,9, 12-tetraoxatetradecane-l,14-diamine (1 eq., 0.8 mmol), 1.5g 1-Bromododecane (8 eq., 6 mmol), 0.7g potassium iodide (5 eq., 5 mmol), and 3.3g potassium carbonate (20 eq., 19.0 mmol) were dissolved in 15mL ACN and 15mL THF. The reaction mixture was stirred at 70 °C for 1 day. Reaction mixture was then quenched with 50mL water and diluted with lOOmL ethyl acetate. The phases were separated and the aqueous layer was washed with 3 x 50mL ethyl acetate. The organic layers were washed with 50mL brine, dried with Na₂SO₄ and filtered over paper. The filtrate was concentrated under reduced pressure, giving 1.6 g of dark orange oil as a crude product. The crude mixture was dry loaded and purified using an 40g Silica Select Cartridge and Methanol: Chloroform as a mobile phase in a Flash Pure System machine. The product eluted at 0-1% Methanol in Chloroform. 150mg (21% yield) of pure product were isolated as a yellow oil. ’H NMR (400 MHz, CHCh): 53.7(t, 8H), 3.6 (t, 4H), 3.5 (t, 4H), 2.7 (s,4H), 2.5 (s, 8H), 2.4 (s, 8H), 1.4 (s, 8H) 1.3-1.2 (s, 76H), 0.88 (t, J = 6.8 Hz, 12H) (Figure 16A).

[0789] ESI-MS: m / z 455.9 [M2’]+, 910.4 [M + H]+(Figure 16B).

[0790] HPLC CAD purity: 97.2%

[0791] Synthesis of Lipid 29 (also referred to as NV5-026):

[0792] Synthetic scheme:

[0793] X / XZ W Xz ’,-DH

[0794] ■4

[0795] fctateeular Weight: 22Sj1

[0796] N VO 5-026

[0797] :i

[0798]

[0799] jf =■ c 'M 7 K.12

[0800] Experimental procedure:

[0801] Synthesis of 3,21-didodecyl-6,9,12,15,18-pentaoxa-3,21-diazatricosane-l,23-diol (Lipid 29, NV5-026):

[0802] OH OH L J

[0803] H. Zx ZX / X

[0804] N VO 5-026

[0805]

[0806] c n, 7 K.12

[0807] To a clean flask, Br2 PEGs (0.25g, 0.6 mmol, 1 eq.), 2-(dodecylamino) ethan-l-ol (0.34g, 1.5mmol, 2.5 eq.), K2CO3 (0.42g, 3.0mmol, 5 eq.) and KI (0.25g, 3.8mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70-80°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of the starting material. The reaction mixture was then quenched with H2O and diluted with petroleum ether. The phases were separated and the aqueous layer was washed with petroleum ether 3 x 30 mL. The organic layers were combined, washed with 20mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.54g crude yellow semi-solid (small traces of DMF present). The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 12g Silica Select Cartridge and CHCh and MeOH as a mobile phase. The compound eluted with 1-2% MeOH as 87mg (13% yield) yellow semi-solid.

[0808] ’H NMR (400 MHz, CDCh): 53.7(s, 9H), 3.65 (t, 5H), 3.55 (t, 7H), 2.75 (t, 4H) 2.7 (dt, J = 4.9 Hz,4H), 2.5(t, 4H), 1.5-1.4 (m, 4H), 1.31-1.25 (s, 35H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 17A). ESI-MS: m / z 355.8 [M2’]+, 705.6 [M + H]+, 728.0 [M + Na]+(Figure 17B).

[0809] HPLC-CAD purity: 93.2%

[0810] Synthesis of Lipid 30 (also referred to as NV5-027):

[0811] Synthetic scheme:

[0812] T' l- -I I -’ I

[0813] KI, K2CO3DMF. heat

[0814] x*o L.

[0815] NVOS O? Z ft. tB-ditfSZ, 12Z)-otaadeoa-9, 12-dien- t-y I J-3,fl.12, 15„21 -penta® a-ft, tfi- diazatf eoisane- i,23-diei

[0816]

[0817] ’‘ If I..! I? | I, ■>.

[0818] Synthesis of 2-(2-(((9Z,12Z)-octadeca-9,12-dien-l-yl) amino) ethoxy) ethan-l-ol

[0819] 2-i 2-i 1 ( '2Z -zctar'eta-!11.’ 2-:!ie_-'-y ■: ef z -.yieran-l-ol

[0820]

[0821] : e:. i ■. e ■ • ■.5:: I

[0822] To a stirred solution of (6Z,9Z)-18-bromooctadeca-6,9-diene (4.0 g, 12.144 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added 2-(2-aminoethoxy) ethan-l-ol (12.16mL, 121.43 mmol, 10.0 equiv.) at room temperature and then stirred for 24h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh). After completion of SM, the solvent was removed and the crude product was diluted with EtOAc (100 mL) and washed with water (2x100 mL). The organic layer was washed with brine solution, dried over anhydrous Na₂SO₄ and the solvent evaporated. The crude was purified by Buchi flash using 0-10% MeOH in CHCI3 to give the 3.2 g of 2-(2- (((9Z,12Z)-octadeca-9,12-dien-l-yl) amino) ethoxy) ethan-l-ol as pale-yellow liquid.

[0823] ’H NMR (400 MHz, CDCh): 35.44 - 5.27 (m, 4H), 3.76 - 3.69 (m, 2H), 3.67-3.57 (m,4H), 2.85 - 2.73 (m, 4H), 2.66 - 2.58 (m, 2H), 2.10-2.00 (m,4H), 1.50 (q, J = 7.2 Hz, 2H), 1.42 - 1.28 (m, 18H), 0.94-0.84(m, 3H).

[0824] ESI-MS: m / z 354.7 [M+H]+.

[0825] Synthesis of 6,18 -di((9Z, 12Z)-octadeca-9, 12 -di en- 1 -y 1 ) -3, 9, 12, 15,21 -pentaoxa-6, 18- diazatricosane- 1,23 -diol (Lipid 30, NV5-027):

[0826] NVO&-O27

[0827]

[0828] To a clean flask, l-bromo-2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethane (0.2g, 0.6 mmol, 1 eq.), 2-(2-(((9Z,12Z)-octadeca-9,12-dien-l-yl) amino) ethoxy) ethan-l-ol (0.7g, 2.0mmol, 2.5 eq ), K2CO3 (0.43g, 3.1mmol, 5 eq.) and KI (0.26g, 1.6mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of the starting material. The reaction mixture was then quenched with H2O and diluted with petroleum ether. The phases were separated and the aqueous layer was washed with petroleum ether 3 x 40 mL. The organic layers were combined, washed with 30mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.89g crude yellow semi-solid (small traces of DMF present). The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 12g Silica Select Cartridge and CHCI3 and MeOH as a mobile phase. The compound eluted with 0-1% MeOH as 90mg (12% yield) yellow oil. ’H NMR (400 MHz, CDCI3): 5 3.65 (t, 8H), 3.55 (t, 5H), 2.75 (t, 2H) 2.7 (dt, J = 4.9 Hz, 2H), 2.5(t, 2H), 1.5-1.4 (m, 3H), 1.31-1.25 (s, 28H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 18A).

[0829] ESI-MS: m / z 433.8 [M2’]+, 866.2 [M + H]+, 888.3 [M + Na]+(Figure 18B).

[0830] HPLC-CAD purity: 89.8%

[0831] Synthesis of Lipid 25 (also referred to as NV5-028):

[0832] Synthetic scheme:

[0833] ......r... - -... ■.

[0834] Kjeo, - ■ -J fF i‘ J- ’> - I

[0835] ,’i b i-w 1 f- - <-■

[0836] • - - >....... ■...v

[0837]

[0838] :: r Synthesis of (9Z,9'Z, 12Z,12'Z)-N, N'-(((oxybis(ethane-2, l-diyl))bis(oxy))bis(ethane-2, 1- diyl))bis(N-((9Z,12Z)-octadeca-9,12-dien-l-yl)octadeca-9,12-dien-l-amine) (Lipid 25, NV5- 028)

[0839] NMB-028

[0840] I II I! ' Ji? ■: ■. r. ■

[0841]

[0842] ■ -7::

[0843] To a clean flask, 0.2g NH2 PEG3 2,2'-((oxybis(ethane-2,l-diyl))bis(oxy))bis(ethan-l-amine) (1 eq., 1.3 mmol), 2.63g (6Z,9Z)-18-bromooctadeca-6,9-diene (8 eq., 8.0 mmol), 1.10g potassium iodide (8.0 eq., 8.0 mmol), and 2.23g potassium carbonate (12 eq., 19.0 mmol) were dissolved in 15mL DMF. The reaction mixture was stirred at 80 °C for 4h. Reaction mixture was quenched with 20mL water and diluted with lOOmL petroleum ether. The phases were separated and the aqueous layer was washed with 3 x 60mL petroleum ether, the organic layers were washed with 50mL brine, dried with Na₂SO₄ and filtered over paper. The filtrate was concentrated under reduced pressure, giving 2.7g of dark yellow oil as a crude product. The crude mixture was dry loaded and purified using an 12g Silica Select Cartridge and Methanol: Chloroform as a mobile phase in a Flash Pure System machine. The product eluted at 0% Methanol in Chloroform. 186mg of pure product was isolated as a yellow oil after three purifications.

[0844] ’H NMR (400 MHz, CHCk): 5.45-5.35 (m, 16H), 3.6 (s, 12H), 2.75 (t,8H), 2.05 (m, 18H), 1.4 (br, 8H) 1.3-1.2 (s, 75H), 0.88 (t, J = 6.8 Hz, 13H) (Figure 19A).

[0845] ESI-MS: m / z 593.1 [M2’]+, 1186.07 [M + H]+(Figure 19B).

[0846] HPLC-CAD purity: 77.4%

[0847] Synthesis of Lipid 31 (also referred to as NV5-029):

[0848] Synthetic scheme:

[0849] * feat

[0850] NW5-029

[0851] Synthesis of 6,18 -di((9Z, 12Z)-octadeca-9, 12 -di en- 1 -y 1 ) -3, 9, 12, 15,21 -pentaoxa-6, 18-diazatricosane- 1,26-diol (NV05-029):

[0852]

[0853] To a clean flask, l,14-dibromo-3,6,9,12-tetraoxatetradecane (0.25g, 0.7 mmol, 1 eq.), 2-(2-(((9Z,12Z)-octadeca-9,12-dien-l-yl) amino) ethoxy) ethan-l-ol (0.63g, 1.8 mmol, 2.5 eq ), K2CO3 (0.63g, 3.4 mmol, 5 eq.) and KI (0.23g, 1.2 mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of starting material. The reaction mixture was quenched with H2O and diluted with petroleum ether. The phases were separated and the aqueous layer was washed with petroleum ether 3 x 40 mL. The organic layers were combined, washed with 30mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.85g crude yellow semi-solid. The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 40g Silica Select Cartridge and CHCI3 and MeOH as a mobile phase. The compound eluted with 0-1% MeOH as 121mg (27 % yield) yellow oil.

[0854] ’H NMR (400 MHz, CDCI3): 53.65 (t, 4H), 3.55 (m, 24H), 2.75 (t, 2H) 2.7 (dt, J = 4.9 Hz, 13H), 2.5(t, 4H), 2.05 (t, 8H), 1.5-1.4 (m, 4H), 1.31-1.25 (s, 37H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 20A). ESI-MS: m / z 455.4 [M2’]+, 910.4 [M + H]+, 932.2 [M + Na]+(Figure 20B).

[0855] HPLC-CAD purity: 90 5%

[0856] Synthesis of Lipid 33 (also referred to as NV5-034):

[0857] Synthetic scheme: c-=-:an-1-ot

[0858] Vi,h-,>iLt

[0859] - i. V heat

[0860] OH OH

[0861] NVW0341

[0862]

[0863] Experimental procedure:

[0864] Synthesis of 4-(((9Z,12Z)-octadeca-9,12-dien-l-yl) amino) butan-l-ol:

[0865] !,.■. =■!, >.ui -i ’ \ i::::i T,i'-!::

[0866]

[0867] ■■ ■■ i".■

[0868] To a stirred solution of (6Z,9Z)-18-bromooctadeca-6,9-diene 1 (4.0 g, 12.144 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added butanol amine (10.82g, 121.44 mmol, 10.0 equiv.) at room temperature and then stirred for 24h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh). After completion of SM, the solvent was removed and the crude product was diluted with EtOAc (100 mL) and washed with water (2x100 mL). The organic layer was washed with brine solution, dried over anhydrous Na₂SO₄, and the solvent evaporated. The crude was triturated with acetonitrile, solids were precipitated and collected by filtration to afford 2.2 g of 4- (((9Z,12Z)-octadeca-9,12-dien-l-yl) amino) butan-l-ol (8) as pale-yellow liquid.

[0869] ’H NMR (400 MHz, CDCh): 3 5.44 - 5.27 (m, 4H), 3.70 (s, 1H), 3.62 - 3.51 (m, 2H), 2.83 -2.73 (m, 2H), 2.73 - 2.59 (m, 4H), 2.10 - 1.98 (m, 4H), 1.74 - 1.61 (m, 4H), 1.53 (s, 2H), 1.42 - 1.22 (m, 17H), 0.95 - 0.84 (m, 3H). ESI-MS: m / z 338.8 [M+H]+.

[0870] Synthesis of 5-((8Z,llZ)-octadeca-8,ll-dien-l-yl)-17-((9Z,12Z)-octadeca-9,12-dien-l-yl)- 8,ll,14-trioxa-5,17-diazahenicosane-l,21-diol (Lipid 33, NV05-034)

[0871] OH OH

[0872] k J

[0873] dWS-034

[0874] •3’> r - ad- d,-r ',-|i ‘ i iT ' 2

[0875] -'ll 'I,■ > i ■ M. du.. I v I---.. I ■. ■ -.

[0876]

[0877] V::!-::nl.ir:■?

[0878] To a clean flask, Br PEG3 (0.2g, 0.6 mmol, 1 eq.), 4-(((9Z,12Z)-octadeca-9,12-dien-l-yl) amino) butan-l-ol (0.63g, 2.1 mmol, 2.2 eq.), K2CO3 (0.93g, 5 mmol, 5 eq.) and KI (0.41g, 3.0 mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of starting material. The reaction mixture was quenched with H2O and diluted with petroleum ether. The phases were separated and aqueous was washed with petroleum ether 3 x 40 mL. The organic layers were combined, washed with 30mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.54g crude yellow oil. The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 25g Silica Select Cartridge and DCM and MeOH as a mobile phase. The compound eluted with 2% MeOH as 181mg (40% yield) yellow oil. A second purification was performed using reversed phase chromatography. The solvents used was Solvent A: Water with 0.01% TFA and Solvent B:

[0879] 70% IPA 30% ACN with 0.01% TFA. The product eluted with 15-36% solvent B. This afforded 70mg of compound (15% yield) with better purity.

[0880] ’H NMR (400 MHz, CDCh): 55.45-5.35(m, 8), 3.65 (t, 8H), 3.35 (s, 5H), 3.1 (t, 2H), 2.75 (t, 2H) 2.05 (t, 10H), 1.75(s,3H), 1.5-1.4 (m, 4H), 1.31-1.25 (s, H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 21 A). ESI-MS: m / z 417.7.7 [M2’]+, 834.2 [M + H]+(Figure 21B).

[0881] HPLC-CAD purity: 84 1%

[0882] Synthesis of Lipid 34 (also referred to as NV5-036):

[0883] Synthetic scheme: H XZ X^X«ZX«Z *-xX XjH:■?r 12 ■3;:3-::eca-t 2-^le-- ’-'■?■•■ ic s:i3"- -3

[0884] -.'.■i r!..-::.:■! L;

[0885] OH

[0886] s -XZXZ\ / X / =\ / =^^ NWS-D36 1H.- ■-. ■ ~::,- -.<■ ■- - ■■ ■ ' ■ lekSSsXS-l 1 S^!^0£O5M^-l JO-^! Ct: ■• '' ’» ■ ■ jf i:::

[0887] Experimental procedure:

[0888] Synthesis of 3,18-di((9Z,12Z)-octadeca-9,12-dien-l-yl)-6,9,12,15-tetraoxa-3,18-diazaicosane- 1,20-diol (NV05-036).

[0889] OH

[0890] s

[0891] 05434 bln.- =. ■?,’■ -I.-,.-,?.-, r '?^€- ' ■. -5 2 ’■- ietraaKa-3, 1 Mlazalco6»ne-t:.2Mia

[0892]

[0893] ■:■<!,11-:|l,l ■!, ■.:.,

[0894] To a clean flask, l,14-dibromo-3,6,9,12-tetraoxatetradecane (0.25g, 0.7 mmol, 1 eq.), 2-(((9Z,12Z)-octadeca-9,12-dien-l-yl) amino) ethan-l-ol (0.46g, 1.5 mmol, 2.2 eq ), K2CO3 (0.63g, 3.4 mmol, 5 eq.) and KI (0.23g, 3.0 mmol, 1.2 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of starting material. The reaction mixture was quenched with H2O and diluted with petroleum ether. The phases were separated and the aqueous layer was washed with petroleum ether 3 x 40 mL. The organic layers were combined, washed with 30mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.54g crude yellow oil. The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 25g Silica Select Cartridge and CHCI3 and MeOH as a mobile phase. Two purifications were needed to isolate the product. The compound eluted with 1% MeOH as 45mg (11 % yield) yellow oil. ’H NMR (400 MHz, CDCk): 5 5.4 (m, 6H), 3.65 (t, 15H), 2.75 (dd, 9H) 2.7 (t, J = 4.9 Hz, 3H), 2.65 (t,4H), 2.05 (t, 8H), 1.5-1.4 (m, 4H), 1.31-1.25 (s, 30H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 22A).

[0895] ESI-MS: m / z 411.8 [M2’]+, 821.6 [M + H]+, 844.2[M + Na]+(Figure 22B).

[0896] HPLC CAD purity: 83 9%

[0897] Synthesis of Lipid 35 (also referred to as NV5-037):

[0898] Synthetic scheme:

[0899] H

[0900] s ’ ’ ■* r-.

[0901] ji ■« CLh'Jrrr

[0902] i

[0903] OH OH

[0904] NV& M3F

[0905] vi ■, s- pentac«a-3,2t-dHzaWcosane-i,23-aBI

[0906]

[0907] Experimental procedure:

[0908] Synthesis of 3,21-di((9Z,12Z)-octadeca-9,12-dien-l-yl)-6,9,12,15,18-pentaoxa-3,21- diazatricosane- 1,23 -diol (Lipid 35, NV5-037)

[0909] NV05-03?

[0910] ' 7 ■- V; i n ■, - r - pentac®a-3,2MBzaHao83ffle-t,23-a»l

[0911]

[0912] -.; 1 '

[0913] To a clean flask, l,17-dibromo-3,6,9,12,15-pentaoxaheptadecane (0.35g, 0.9 mmol, 1 eq.), 2- (((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethan-l-ol (0.65g, 2.0mmol, 2.2 eq.), K2CO3 (0.83g, 4.5mmol, 5 eq.) and KI (0.31g, 2.3mmol, 2.2 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 70-80°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of the starting material. The reaction mixture was quenched with H2O and diluted with petroleum ether. The phases were separated and the aqueous layer was washed with petroleum ether 3 x 50 mL. The organic layers were combined, washed with 50mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.9g crude yellow oil. The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 12g C18 Silica Select Cartridge and Water (0.1% TFA) and 70:30 IPA: ACN (0.1%TFA) as a mobile phase. The compound eluted with 7- 15% IPA: ACN as 44mg (6% yield) yellow oil.

[0914] ’H NMR (400 MHz, CDCh): 5 5.35 (m, 4H), 3.65(t, 11H), 3.55 (t, 4H), 2.9 (t, 3H), 2.8 (t, 3H), 2.7 (dt, 8H), 2.5(t, 4H), 2.0 (t, 5H), 1.4-1.5 (s, 3H), 1.4-1.3 (s, 27H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 23A).

[0915] ESI-MS: m / z 866.2 [M + H]+, 868.3 [M + Na]+, 431.6 [M-2]+(Figure 23B).

[0916] HPLC CAD purity: 91 5%

[0917] Synthesis of Lipid 36 (also referred to as NV5-038):

[0918] Synthetic scheme:

[0919] ♦ • ’ I-

[0920] 1

[0921] NVllb (MU

[0922] " ‘: <■ -- >< I

[0923]

[0924] '’. Vuk - Experimental procedure:

[0925] Synthesis of 4-(((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)butan-l-ol , / -■.:.i I - ■ >kr r... r.:..,- ■ ■.

[0926]

[0927] :■. A:?2i."

[0928] To a stirred solution of (6Z,9Z)-18-bromooctadeca-6,9-diene (4.0 g, 12.144 mmol, 1.0 equiv.) in MeCN: THF (1:1, 30 mL) was added 3 -aminopropan- l-ol (9.0mL, 117.71 mmol, 10.0 equiv.) at room temperature and then stirred for 24h. The progress of the reaction was monitored by TLC (10% MeOH in CHCh). After completion of SM, the solvent was removed and the crude product diluted with EtOAc (100 mL) and washed with water (2x100 mL). The organic layer was washed with brine solution, dried over anhydrous Na₂SO₄ and the solvent evaporated. The crude was purified by Buchi flash using 0-1% MeOH in CHCI3 to give the 1.34 g (34% yield) of 3- (((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)propan-l-olas pale-yellow liquid.

[0929] ’H NMR (400 MHz, CDCI3): d 5.35 (m, 4H), 3.79 (t, 2H), 2.88 (t, 2H), 2.60 (m, 2H), 2.10-2.00 (q, 4H), 1.69 (m, J = 7.2 Hz, 2H), 1.42 - 1.28 (m, 16H), 0.94-0.84(m, 3H).

[0930] ESI-MS: m / z 324.1 [M+H]+.

[0931] Synthesis of 4-((8Z,llZ)-octadeca-8,ll-dien-l-yl)-16-((9Z,12Z)-octadeca-9,12-dien-l-yl)- 7,10,13-trioxa-4,16-diazanonadecane-l,19-diol (Lipid 36, NV5-038).

[0932] ., -j,

[0933] NVHb o:iu

[0934] ■>!«•»... '■: • J 1! <’ '1'.1..1 Iw'h >,•

[0935] I’ ' I.. I ■'!. S'X..".. Jr- ' '« I:

[0936]

[0937] ‘Wnh> ' ■ '

[0938] To a clean flask, Br PEG3 (0.3g, 0.9 mmol, 1 eq.), 3-(((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)propan-l-ol (0.66g, 2.0mmol, 2.2 eq.), K2CO3 (0.87g, 4.7mmol, 5 eq.) and KI (0.32g, 2.3mmol, 2.5 eq.) were added and dissolved in lOmL DMF. The reaction mixture was heated at 80°C for 12h. After overnight reaction, TLC and MS was performed on the reaction mixture, showing full consumption of the starting material. The reaction mixture was then quenched with H2O and diluted with petroleum ether. The phases were separated and the aqueous layer was washed with petroleum ether 3 x 40 mL. The organic layers were combined, washed with 30mL brine, dried with Na₂SO₄, filtered over a cotton plug and concentrated under reduced pressure, yielding 0.78g crude yellow semi-solid. The product was dissolved in DCM and dry loaded with silica gel. The crude was purified using a 12g Silica Select Cartridge and CHCI3 and MeOH as a mobile phase. The compound eluted with 0-1% MeOH as 14mg (6% yield) yellow oil.

[0939] ’H NMR (400 MHz, CDCh): 55.35 (q, 7H), 3.65 (t, 4H), 3.55 (t, 11H), 2.75 (t, 10H), 2.5(t, 3H), 1.5-1.4 (m, 4H), 1.31-1.25 (s, 37H), 0.88 (t, J = 6.8 Hz, 6H) (Figure 24A).

[0940] ESI-MS: m / z 403.7 [M2’]+, 805.7[M + H]+, 828.3 [M + Na]+(Figure 24B).

[0941] HPLC CAD purity: 84 8%

[0942] EXAMPLE 2: Lipid nanoparticle (LNP) formulation and physico-chemical properties Various formulations of LNPs using lipids of the present invention were prepared and their physico-chemical properties assessed.

[0943] Table 1: LNP formulations.

[0944] Formulation name Molar ratio Composition

[0945] F1.2 40:09:49.5:1.5 lipid: DSPC: Cholesterol: DMG-PEG

[0946] F2.3 40:09:49.5:1.5 lipid: DOPE: Cholesterol: DMG-PEG

[0947] F2.9 30:09:59.0:2.0 lipid: DOPE: Cholesterol: DMG-PEG

[0948]

[0949] Table 1 shows the formulations that were prepared. The LNPs were formulated using the microfluidic mixing device (NanoAssemblr Ignite, Cytiva) according to the molar ratios of Table 1. 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.

[0950] 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. Shown in Table 2 are the physico-chemical properties of the formulated LNPs using the lipids of the present invention that encapsulate firefly Luciferase mRNA.

[0951] Table 2: physico-chemical properties of LNPs.

[0952] Lipid ormulation nar Size (d.nm) oly-dispersion ind< eta Potential (m EE* (%)

[0953] (PDI)

[0954] Lipid 11 F1.2 97 0.14 14.9 95

[0955] (NV5-009) F2.3 77 0.08 20.2 98

[0956] Lipid 15 F2.3 94.9 0.08 - 21.3 94

[0957] (NV5-012)

[0958] Lipid 3 F2.3 123.6 0.17 - 44.9 42

[0959] (NV5-013)

[0960] Lipid 16 F2.3 108.5 0.11 - 30.3 78

[0961] (NV5-014)

[0962] Lipid 20 F2.3 112.8 0.21 - 11.8 96

[0963] (NV5-015) F2.9 119 0.20 - 1.7 98

[0964] Lipid 19 F2.3 93.6 0.14 - 5.3 98

[0965] (NV5-016) F2.9 84.4 0.12 - 0.89 99

[0966] Lipid 13 F2.3 85.6 0.05 10.5 96

[0967] (NV5-017) F2.9 66.4 0.13 13.9 95

[0968] Lipid 18 F2.3 100.5 0.15 - 31 90

[0969] (NV5-018)

[0970] Lipid 23 F2.3 137.6 0.13 20.3 90

[0971] (NV5-019)

[0972] F2.9 67.4 0.16 13.4 96

[0973] Lipid 24 F2.3 77.6 0.11 13.0 97

[0974] (NV5-020)

[0975] Lipid 27 F2.3 175.7 0.13 14.6 83

[0976] (NV5-021)

[0977] F2.9 67.5 0.10 15.1 98

[0978] Lipid 28 F2.3 238.8 0.21 21.3 72

[0979] (NV5-022)

[0980] F2.9 82.9 0.11 14.6 96

[0981]

[0982] Lipid 21 F2.3 99.7 0.03 2.6 99 (NV5-024)

[0983] Lipid 25 F2.3 122.7 0.01 - 3.03 100

[0984] (NV5-028)

[0985] Lipid 31 F1.2 179.5 0.08 25.4 70

[0986] (NV5-029)

[0987] Lipid 32 F2.3 64.3 0.13 16.4 98

[0988] (NV5-032)

[0989]

[0990] *EE= Encapsulation efficiency

[0991] As shown in Table 2, most of the mRNA-LNP formulations were uniformly distributed with more than 90% mRNA encapsulation.

[0992] EXAMPLE 3: Lipid nanoparticle (LNP) formulation and physico-chemical properties The transfection efficiency of the formulations was tested in a human hepatocarcinoma cell line (HepG2) and a human neuroblastoma cell line (HTB11) by encapsulating firefly Luciferase mRNA (mLuc) as a reporter gene.

[0993] Cells were cultured according to producer’s instructions. HepG2 (14000 cells / well) cells were treated with the following mRNA-LNP formulations (formulations as defined in Table 1): Lipid 11 (Formulation F2.9), Lipid 20 (Formulation F2.3), Lipid 20 (Formulation F2.9), Lipid 19 (Formulation F2.3), Lipid 19 (Formulation F2.9), Lipid 13 (Formulation F2.9), Lipid 23 (Formulation F2.9), Lipid 24 (Formulation F2.9), Lipid 27 (Formulation F2.9), Lipid 21 (Formulation F2.3), Lipid 21 (Formulation Fl.2), Lipid 32 (Formulation Fl.2), Lipid 32 (Formulation F2.3), and Lipid 32 (Formulation F2.9). The mLuc mRNA concentration of 25ng / ml, 50ng / ml, 100ng / ml, and 200ng / ml.

[0994] HTB11 (16000 cells / well) cells were treated with the following mRNA-LNP formulations: Lipid 11 (Formulation F2.9), Lipid 20 (Formulation F2.3), Lipid 20 (Formulation F2.9), Lipid 19 (Formulation F2.3), Lipid 19 (Formulation F2.9), Lipid 13 (Formulation F2.9), Lipid 21 (Formulation F2.3), Lipid 31 (Formulation Fl.2), Lipid 32 (Formulation Fl.2), Lipid 32 (Formulation F2.3), and Lipid 32 (Formulation F2.9). The mLuc mRNA concentration of 25ng / ml, 50ng / ml, 100ng / ml, and 200ng / ml. After 24hrs, cells were lysed and analysed for luciferase expression using a Promega plate reader.

[0995] Results are shown in Figures 25A-B. As shown in Figure 25A, Lipids 20 (NV5-015), 19 (NV5-016), 21 (NV5-024), and 32 (NV5-032) were efficient in delivering the mRNA into HepG2 cells. As shown in Figure 25B, Lipids 20 (NV5-015), 19 (NV5-016), and 21 (NV5-024) were efficient in delivering the mRNA into HTB 11 cells.

[0996] EXAMPLE 4: LNPs in vitro toxicity testing

[0997] Luciferase mRNA encapsulated LNPs were transfected in HepG2 and HTB 11 cell lines at 1mg / mL mRNA concentration. After 24hrs, cells were analyzed for viability using Alamar Blue [Invitrogen, cat# A50100],

[0998] HepG2 cells were treated with the following mRNA-LNP formulations (formulations as defined in Table 1): Lipid 11 (Formulation F2.9), Lipid 20 (Formulation F2.3), Lipid 20 (Formulation F2.9), Lipid 19 (Formulation F2.3), Lipid 19 (Formulation F2.9), Lipid 13 (Formulation F2.9), Lipid 23 (Formulation F2.9), Lipid 24 (Formulation F2.9), Lipid 27 (Formulation F2.9), Lipid 21 (Formulation F2.3), Lipid 21 (Formulation Fl.2), Lipid 32 (Formulation Fl.2), Lipid 32 (Formulation F2.3), and Lipid 32 (Formulation F2.9).

[0999] HTB 11 cells were treated with the following mRNA-LNP formulations: Lipid 11 (Formulation F2.9), Lipid 20 (Formulation F2.3), Lipid 20 (Formulation F2.9), Lipid 19 (Formulation F2.3), Lipid 19 (Formulation F2.9), Lipid 13 (Formulation F2.9), Lipid 18 (Formulation F2.3), Lipid 21 (Formulation F2.3), Lipid 31 (Formulation Fl.2), Lipid 32 (Formulation Fl.2), Lipid 32 (Formulation F2.3), and Lipid 32 (Formulation F2.9).

[1000] As shown in Figure 26A, except Lipid 24 (NV5-020), all other tested mRNA-LNPs were nontoxic to HepG2 cells, demonstrating their overall safety.

[1001] As shown in Figure 26B, except Lipid 13 (NV5-017), all other tested mRNA-LNPs were nontoxic to HTB11 cells, demonstrating their overall safety.

[1002] EXAMPLE 5: In vivo biodistribution

[1003] 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.

[1004] The experiment was performed with LNPs formulated with Lipid 20. The same experiment was performed independently with LNPs formulated with Lipid 21, Lipid 11, and Lipid 32. All LNPs were formulated according to Formulation F2.3 as described in Table 1. In both sets of these experiments, untreated mice served as the control (naive).

[1005] Luciferase expression in the liver, spleen, lungs, heart, and kidneys with LNPs formulated with Lipid 20 are shown in Figures 27A, C, E, G, and I, respectively. Luciferase expression in the liver, spleen, lungs, heart, and kidneys with LNPs formulated with Lipid 21, Lipid 11, and Lipid 32 are shown in Figures 27B, D, F, H, and J, respectively. The results indicate that the tested firefly luciferase-mRNA-LNPs formulated with Lipid 20 and Lipid 21 facilitated efficient in vivo delivery of Luciferase mRNA to the liver in mice.

Claims

CLAIMSWhat is claimed is:

1. A lipid represented by the structure of Formula (I) or salt thereof:Formula (I)whereineach one of xa, Xb, xc, and xa is independently selected from the group consisting of: Co-12 alkylene, C2-12 alkenylene, C2-12 alkynylene and (CH₂CH₂O)n3;each one of Za, Zb, Zc, and Za is independently selected from the group consisting of: absent,each one of La, Lb, Lc, and La is 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-NR1R2, Co-12 alkylene-N(Co-i2 alkylene-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; andeach one of nl and n2 and m is, independently, 1, 2, 3, 4 or 5;and n3 is an integer in the range of 1 to 45.

2. The lipid of claim 1, wherein the lipid is represented by Formula (la):I — Za 7 I^“3 a XFormula (la)wherein each one of na, nb, nc, and nd is independently an integer in the range of 1 to 20.The lipid of claim 1, wherein the lipid is represented by Formula (lb):Formula (lb).

4. The lipid of claim 1 or 2, wherein xais an unsubstituted Co-12 alkylene.

5. The lipid of claim 4, wherein xais selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, -CH2(CH2)4CH2- and -CH2(CH2)7CH2-.

6. The lipid of claim 1 or 2, wherein Xb is an unsubstituted Co-12 alkylene.

7. The lipid of claim 6, wherein Xb is selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, -CH2(CH2)4CH2- and -CH2(CH2)7CH2-.

8. The lipid of claim 1 or 2, wherein xcis an unsubstituted Co-12 alkylene.

9. The lipid of claim 8, wherein xcis selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2- and -CH2(CH2)4CH2-.

10. The lipid of claim 1 or 2, wherein xa is an unsubstituted Co-12 alkylene.

11. The lipid of claim 10, wherein xa is selected from the group consisting of: absent, -CH2CH2-, -CH2CH2CH2CH2-, and -CH2(CH2)4CH2-.

12. The lipid of any one of claims 1 to 11, wherein Zais selected from the group consisting of: absent, - OC(O)-, -OC(O)NH-, -NHC(O)O-, and -C(O)O-.

13. The lipid of any one of claims 1 to 12, wherein Zb is selected from the group consisting of: absent, - OC(O)-, -OC(O)NH-, -NHC(O)O-, and -C(O)O-.

14. The lipid of any one of claims 1 to 13, wherein Zcis selected from the group consisting of: absent, - O-, -OC(O)-, -OC(O)NH-, -NHC(O)O-, and -C(O)O-.

15. The lipid of any one of claims 1 to 14, wherein Za is selected from the group consisting of: absent, - O-, -OC(O)-, -OC(O)NH-, -NHC(O)O-, and -C(O)O-.

16. The lipid of any one of claims 1 to 15, wherein Zeis selected from the group consisting of: absent, - O-, -OC(O)-, and -C(O)O-.

17. The lipid of any one of claims 1 to 16, wherein Lais selected from the group consisting of: C4-i8 alkyl, C4-i8 alkenyl, and Co-6 alkylene-Ze-N(Ci-i2 alkyl)2.

18. The lipid of claim 17, wherein Lais selected from the group consisting of:-(CH2)IOCH3, -(CH2)IICH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -CH2CH2-N((CH2)7CH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -N((CH2)7CH3)2, -(CH2)4-N((CH2)7CH3)2, and -(CH2)7- CH=CH-(CH2)7CH3.

19. The lipid of any one of claims 1 to 18, wherein Lb is selected from the group consisting of: C4-18 alkyl, C4-i8 alkenyl, and Co-6 alkylene-Ze-N(Ci-i2 alkyl)2.

20. The lipid of claim 19, wherein Lb is selected from the group consisting of: -(CH2)IOCH3, -(CH2)IICH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -CH2CH2. N((CH2)7CH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, -N((CH2)7CH3)2, -(CH2)4-N((CH2)7CH3)2, and -(CH2)7- CH=CH-(CH2)7CH3.

21. The lipid of any one of claims 1 to 20, wherein Lcis selected from the group consisting of: Co-6 alkylene-Ze-R3, C4-i8 alkyl, C4-i8 alkenyl, and Co-6 alkylene-Ze-N(Ci-i2alkyl)2.

22. The lipid of claim 21, wherein Lcis selected from the group consisting of: Cs-i4alkyl, C15-18 alkenyl, Co-4alkylene-N(Ce-i2 alkyl)2, and Co-6 alkylene-OH.

23. The lipid of claim 21 or 22, wherein Lcis selected from the group consisting of:-CH2CH2-OH, -(CH2)4OH, -(CH2)3OH, -(CH2)10CH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)IICH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8CH3, -CH2CH2-N((CH2)7CH3)2, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, and -(CH2)7-CH=CH-(CH2)7CH3.

24. The lipid of any one of claims 1 to 23, wherein La is selected from the group consisting of: Co-6 alkylene-Ze-R3, C4-i8alkyl, C4-i8alkenyl, and Co-6 alkylene-Ze-N(Ci-i2alkyl)2.

25. The lipid of claim 24, wherein Lais selected from the group consisting of: Co-6 alkylene-OH, C8-i4alkyl, Ci5-i8alkenyl, and Co-4alkylene-N(Ce-i2alkyl)2.

26. The lipid of claim 24 or 25, wherein La is selected from the group consisting of:-CH2CH2-OH, -(CH2)4OH, -(CH2)3OH, -(CH2)10CH3, -(CH2)7-CH=CH-CH2-CH=CH-(CH2)4CH3, - CH2CH2-N((CH2)7CH3)2, -(CH2)11CH3, -(CH2)8-CH=CH-CH2-CH=CH-(CH2)4CH3, -(CH2)8CH3, -CH[CH2(CH2)4CH3]CH2(CH2)3CH3, and -(CH2)7-CH=CH-(CH2)7CH3.

27. The lipid of claim 1, wherein m is 1, 2, or 3 and each one of nl and n2 is independently 1 or 2.

28. The lipid of claim 27, wherein xais absent; Zais absent; and Lais selected from the group consisting of: Ce-i8alkyl, Ce-i8alkynyl, and C2.4alkylene-Ze-R3, wherein Zeis selected from -O-, -OC(O)-, and -C(O)O- and R3is Ce-18 alkyl.

29. The lipid of claim 27 or 28, wherein xb is absent; Zb is absent; and Lb is selected from the group consisting of: Ce-18 alkyl, Ce-18 alkynyl, and C2-4 alkylene-Ze-R3, wherein Zeis selected from -O-, - OC(O)-, and -C(O)O- and R3is Ce-18 alkyl.

30. The lipid of claim 27, wherein xcis absent; Zcis absent; and Lcis selected from the group consisting of: Ce-i8 alkyl, C2-6 alkylene-Ze-R3, and (CH2CH2O)n3R3, wherein n3 is 1-4, wherein Zeis selected from -O-, -OC(O)-, and -C(O)O-, and R3is H.

31. The lipid of claim 27 or 28, wherein xa is absent; Za is absent; and Ld is selected from the group consisting of: Ce-18 alkyl, C2-6 alkylene-Ze-R3, and (CH2CH2O)n3R3, wherein n3 is 1-4, wherein Zeis selected from -O-, -OC(O)-, and -C(O)O-, and R3is H.

32. The lipid according to claim 1, which is selected from the group consisting of:NV5-033Lipid 1DLipid 2NV05-013Lipid 3Lipid 4Lipid 5Lipid 6Lipid 7 Lipid 8Lipid 10NV05-009Lipid 11NV05-010 Lipid 12NV05-017Lipid 13NV5-011Lipid 14 OLipid 15NV05-014 Lipid 16Lipid 17NV05-018 Lipid 18 NV05-016 Lipid 19 NV05-015 Lipid 20 NV05 024 Lipid 21NV05-025Lipid 22NV05-019Lipid 23NV05-020 Lipid 24NV5-028 Lipid 25Lipid 26NV05-021Lipid 27NV05-022 Lipid 29NV05-026 Lipid 30Lipid 31NV05-027Lipid 32 anr,‘vv- jLipid 33Lipid 34 NV05-036 NV05-037 NV05-038 Lipid 35 Lipid 36Lipid 37Lipid 38Lipid 39Lipid 40Lipid 41Lipid 42Lipid 43Lipid 44Lipid 45Lipid 46Lipid 47 Lipid 48Lipid 49Lipid 50Lipid 51Lipid 52Lipid 53Lipid 54Lipid 55Lipid 56Lipid 57Lipid 58and salts thereof.

33. The lipid according to claim 32, which is selected from the group consisting of: Lipid 11, Lipid 20, Lipid 19, Lipid 13, Lipid 18, Lipid 21, Lipid 31, Lipid 32, Lipid 23, Lipid 24, Lipid 27, and salts thereof.

34. A particle comprising the lipid according to any one of claims 1 to 33 and a membrane stabilizing lipid.

35. The particle comprising the according to claim 34, comprising the membrane stabilizing lipid and a lipid membrane comprising the lipid.5 36. The particle according to claim 34 or 35, wherein the membrane stabilizing lipid is selected from the group consisting of cholesterol, phospholipids, cephalins, sphingolipids and glycoglycerolipids.

37. The particle according to any one of claims 34 to 36, wherein the membrane stabilizing lipid comprises cholesterol.

38. The particle according to any one of claims 34 to 37, further comprising one or more additional 0 components selected from the group consisting of a PEG-lipid conjugate, a neutral lipid and a charged lipid.

39. The particle according to claim 38, wherein the additional component comprises 1,2-Dioleoyl-sn- glycero-3 -phosphoethanolamine (DOPE) or 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC).

40. The particle according to claim 38 or 39, wherein the additional component comprises 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG).

41. The particle according to any one of claims 34 to 40, comprising the lipid, cholesterol, 1,2-Dioleoyl- sn-glycero-3 -phosphoethanolamine (DOPE) and 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG).

42. The particle according to any one of claims 34 to 41, comprising the lipid, cholesterol, 1,2-Distearoyl-0 sn-glycero-3 -phosphocholine (DSPC) and 1,2-Dimyristoyl-sn-glyceryl-methoxy polyethylene glycol (DMG-PEG).

43. The particle according to any one of claims 34 to 42, which is conjugated to a targeting moiety.

44. The particle according to any one of claims 34 to 43, further comprising a nucleic acid encapsulated within a particle comprising the lipid.5 45. The particle according to claim 44, 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.

46. The particle according to any one of claims 34 to 45, further comprising a therapeutic agent, wherein the therapeutic agent is encapsulated within a particle comprising the lipid.

47. The particle according to claim 46, 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.

48. A pharmaceutical composition comprising a plurality of particles according to any one of claims 34 to 47 and a pharmaceutically acceptable carrier, diluent or excipient.

49. A method of gene silencing, comprising contacting a cell with the pharmaceutical composition according to claim 48.

50. A method for administering a therapeutic agent, the method comprising administering the pharmaceutical composition of claim 48 to a subject in need thereof.

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