Diester lipids, lipid nanoparticle containing diester lipids, and formulations thereof

Abstract

Provided is a diester lipid compound of Formula (I) or a pharmaceutically acceptable salt thereof The compound can be used to obtain lipid nanoparticles. In some embodiments, the lipid nanoparticle can comprise (a) from about 40 to about 100 mol % of the compound of Formula (I); (b) from 0 to about 10 mol % of a neutral lipid; (c) from 0 to about 50 mol % of a helper lipid; (d) from 0 to about 5 mol % of a polymer-conjugated lipid; and (e) from 0 to about 5 mol % of a hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle. (I)

Description

DIESTER LIPIDS, LIPID NANOPARTICLE CONTAINING DIESTER LIPIDS, AND FORMULATIONS THEREOFFIELD OF THE DISCLOSURE

[0001] The present disclosure relates to diester lipids, and their use for preparing systems for encapsulating cargos such as nucleic acid sequences, polypeptides or peptides. More particularly, the present disclosure relates to ionizable diester compounds useful to prepare lipid nanoparticles (LNPs). The present disclosure also relates to LNPs comprising such di ester lipids.

[0002] The present disclosure also relates to lipids nanoparticles (LNPs) for the delivery of cargos such as nucleic acid sequences, polypeptides or peptides and methods of use of these LNPs for the treatment of diseases, disorders and / or conditions.BACKGROUND

[0003] The development of systems, such as lipid nanoparticle delivery systems, for delivering therapeutics such as proteins, nucleic acid sequences, polypeptides or peptides to prevent or treat diseases has been increasing in recent years. Lipid nanoparticles (LNPs) usually contain four ingredients: an ionizable lipid, a phospholipid, cholesterol and a PEGylated lipid. A major component of LNPs is the ionizable lipid. The phospholipid supports the formation of a lipid bilayer while cholesterol can stabilize the lipid bilayer. The PEGylated lipid, being amphiphilic, remains on the surface of LNPs to provide colloidal stability by steric shielding. Designing new ionizable lipids with suitable efficacy, stability and / or biodegradability to allow the preparation of LNPs is needed.

[0004] Proteins have been the standard for therapeutics but the use of nucleic acids as therapeutic modalities for a variety of diseases and therapeutic indications has gained in prominence over the past few years. Various companies have shown that nucleic acids (e.g., siRNA, mRNA, circular RNA, DNA, etc.) can be more effective when compared to protein- based therapies. There is a need for new delivery systems, such as new LNPs, for both nucleic acid and protein therapeutics.SUMMARY

[0005] The present disclosure provides new lipid compounds, more particularly lipid compounds comprising at least two ester functions, referred to as “diester lipids” in the present disclosure.

[0006] The present disclosure also provides LNPs, more particularly LNPs formulated with diester lipid compounds. Particles, such as nanoparticles, comprising the compounds, constructs comprising the nanoparticles and cargos, wherein the cargo can be a small molecule, an antibody, a polynucleotide, or a polypeptide, methods of using the particles / constructs, and methods of preparing the compounds, particles and constructs are also provided.

[0007] Hence, according to one aspect, there is provided a compound of Formula (I): , or a pharmaceutically acceptable salt thereof,whereinR1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;X is -CH2-, -NH- or -NR7-; n is a number from 0 to 10; p is a number from 0 to 2; R5 and R6 are independently H, or an optionally substituted linear C1-C4 alkyl group;R7 is a linear or branched C1-C6 alkyl; wherein when any of R1, R2, R3 and R4 represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R1, R2, R3 and R4 represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; and wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, alkoxycarbonyl, formyl, acyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate,amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, an aromatic moiety or an heteroaromatic moiety.

[0008] According to another aspect, there is provided a compound selected from the group consisting of Compounds 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217 and 218 of Table 1 of the present disclosure, or a pharmaceutically acceptable salt thereof, preferably Compounds 201, 202, 203, 204, 205, 206, 208, 209, 210, 211, 216, 217 and 218 of Table 1, or a pharmaceutically acceptable salt thereof.

[0009] In some embodiments, the compound of the present disclosure, i.e., the di ester lipid compound of the present disclosure, or the pharmaceutically acceptable salt thereof can be in the form of any enantiomers, any diastereoisomers, any cis or trans geometric isomers, or any mixtures thereof.

[0010] According to another aspect, there is provided a lipid nanoparticle comprising at least one compound of the present disclosure, i.e., the di ester lipid compound of the present disclosure, or the pharmaceutically acceptable salt thereof.

[0011] According to another aspect, there is provided a lipid nanoparticle comprising:(a) from about 40 to about 100 mol % of an ionizable lipid;(b) from 0 to about 10 mol % of a neutral lipid;(c) from 0 to about 50 mol % of a helper lipid;(d) from 0 to about 5 mol % of a polymer-conjugated lipid; and(e) from 0 to about 5 mol % of a hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle; and wherein the ionizable lipid is at least one compound of Formula (I): whereinR1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R.2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;X is -CH2- or -NH-; n is a number from 0 to 10; p is a number from 0 to 2; R5 and R6 are independently H, or an optionally substituted linear C1-C4 alkyl group; wherein when any of R1, R2, R3 and R4 represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R1, R2, R3 and R4 represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; and wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, alkoxycarbonyl, formyl, acyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, an aromatic moiety or an heteroaromatic moiety.

[0012] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 1 to about 10 mol % of the neutral lipid; (c) from 0 to about 50 mol % of the helper lipid; (d) from 0 to about 5 mol % of the polymer- conjugated lipid; and (e) from 0 to about 5 mol % of the hydrophobic component.

[0013] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 1 to about 10 mol % of the neutral lipid; (c) from about 1 to about 50 mol % of the helper lipid; (d) from 0 to about 5 mol % of the polymer-conjugated lipid; and (e) from 0 to about 5 mol % of the hydrophobic component.

[0014] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 1 to about 10 mol % of the neutral lipid; (c) from about 1 to about 50 mol % of the helper lipid; (d) from about 1 to about 5 mol % of the polymer-conjugated lipid; and (e) from 0 to about 5 mol % of the hydrophobic component.

[0015] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 1 to about 10 mol % of the neutral lipid;(c) from about 1 to about 50 mol % of the helper lipid; (d) from about 1 to about 5 mol % of the polymer-conjugated lipid; and (e) from about 0.1 to about 5 mol % of the hydrophobic component.

[0016] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 5 to about 10 mol % of the neutral lipid; (c) from about 30 to about 50 mol % of the helper lipid; (d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and (e) from about 0.1 to about 5 mol % of the hydrophobic component.

[0017] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 5 to about 10 mol % of the neutral lipid; (c) from about 30 to about 45 mol % of the helper lipid; (d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and (e) from about 0 to about 5 mol % of the hydrophobic component.

[0018] In some embodiments, the lipid nanoparticle can comprise: (a) from about 40 to about 60 mol % of the ionizable lipid; (b) from about 5 to about 10 mol % of the neutral lipid; (c) from about 30 to about 45 mol % of the helper lipid; (d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and (e) from about 0.1 to about 5 mol % of the hydrophobic component.

[0019] According to another aspect, there is provided a pharmaceutical composition comprising a lipid nanoparticle as defined herein, and a pharmaceutical acceptable excipient.

[0020] According to another aspect, the present application relates to a method for delivering a cargo to a cell comprising contacting the cell with a lipid nanoparticle as defined herein, wherein the lipid nanoparticle comprises the cargo.

[0021] According to another aspect, the present application relates to there is provided a use of a lipid nanoparticle as defined herein, for delivering a cargo to a cell, wherein the lipid nanoparticle comprises the cargo.

[0022] According to another aspect, there is provided a vaccine comprising a lipid nanoparticle as defined herein, wherein the lipid nanoparticle comprises at least one cargo, preferably the cargo comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide, more preferably the cargo comprises at least one nucleic acid such as mRNA.

[0023] According to another aspect, there is provided a method of vaccinating a subject against an infectious agent comprising:(i) contacting the subject with a vaccine as defined herein, and(ii) eliciting an immune response.

[0024] According to another aspect, there is provided a use of a vaccine as defined herein for vaccinating a subject against an infectious agent.

[0025] According to another aspect, there is provided a method of treating cancer in a subject comprising administering a lipid nanoparticle as defined herein to the subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

[0026] According to another aspect, there is provided a lipid nanoparticle as defined herein for use in the treatment of cancer in a subject, wherein the lipid nanoparticle comprises an anti- cancer cargo or a cargo triggering an immune response against cancer cells.

[0027] According to another aspect, there is provided a use of a lipid nanoparticle as defined herein for treating cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

[0028] According to another aspect, there is provided a use of a lipid nanoparticle as defined herein for the preparation of a medicament for treating cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

[0029] According to another aspect, there is provided a lipid nanoparticle as defined herein for use in transfection of targeted cells, e.g. in transfecting human cells, including stem cells.

[0030] According to another aspect, there is provided a lipid nanoparticle as defined herein for use in gene replacing therapy.

[0031] The details of various embodiments are set forth in the description below. Other features, objects and advantages will be apparent from the description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 depicts a cryo-TEM image of LNPs-01 formulated with ionizable diester lipid 201, DSPC, cholesterol and PEG2k-DMG.

[0033] Figure 2 depicts a cryo-TEM image of LNPs-02 formulated with ionizable diester lipid 201, DSPC, cholesterol, PEG2k-DMG and squalene.

[0034] Figure 3 depicts a cryo-TEM image of LNPs-03 formulated with ionizable diester lipid 201, DSPC, cholesterol, PEG2k-DMG and cardiolipin.

[0035] Figure 4 shows a graph representing the in vitro comparison of LNPs-07 containing ionizable lipid 201 and LNPs-10 containing Dlin-MC3-DMA in Huh-7 cells. The spike protein produced is quantified using ELISA after 18 hours of DP transfection in Huh-7 cells.

[0036] Figure 5 shows a graph representing the in vitro comparison of LNPs-07, LNPs-08 and LNPs-09 containing ionizable lipid 201 and LNPs-10, LNPs-11 and LNPs-12 containing Dlin-MC3-DMA in Huh-7 cells. Four components LNPs (LNPs-07 and LNPs-10) were compared to five components LNPs containing either cardiolopin (LNPs-08 and LNPs-11) or squalene (LNPs-09 and LNPs-12) as fifth component. The spike protein produced is quantified using ELISA after 18 hours of LNPs transfection in Huh-7 cells.

[0037] Figure 6 shows a graph that represents the quantity of blood glucose over a period of time in mice that were dosed with BMDC+3gp peptides + CpG or formulation buffer on day zero and day 0+4. The blood glucose of mice is measured for 30 days from day 0. The Bone marrow dendritic cells (BMDC) were used as positive control.

[0038] Figure 7a shows a graph that represents the in vivo testing of LNPs made from ionizable lipid 201 in RIP-gp mice. The mice were dosed with LNPs-02. The blood glucose of mice is measured for 30 days from day 0. T-cell activation induced by LNPs leads to diabetes. Figure 7b shows a graph that represents the in vivo testing of LNPs made from ionizable lipid 201 in RIP-gp mice. The mice were dosed with LNPs-03. The blood glucose of mice is measured for 30 days from day 0. T-cell activation induced by LNPs leads to diabetes.

[0039] Figures 8a / 8b / 8d show graphs representing the in vivo testing of LNPs made from ionizable lipids 201 in RIP-gp mice. The mice were dosed with LNPs-02 (Figure 8a), LNPs- 03 (Figure 8b) and LNPs-13 (Figure 8d). The blood glucose of mice is measured for more than 30 days from day 0. T-cell activation induced by LNPs leads to diabetes. Figure 8c represents the diabetes incidence.

[0040] Figures 9-13 summarize the stability data of LNPs made from ionizable lipid 201. The LNPs are either four components or five components with squalene or cardiolipin. All LNPs were stored at -80 °C and samples were withdrawn on third and sixth month of manufacturing and tested for particle size (Figure 9), poly dispersity index (PDI) (Figure 10), encapsulation efficiency (EE) (Figure 11), mRNA integrity (purity) (Figure 12) and mRNA concentration (Figure 13) and compared to the same parameters at the time of manufacturing (time zero).

[0041] Figure 14 shows an in vivo comparison of LNPs made from ionizable lipid 201 and Dlin-MC3-DMA in mice. Four component LNPs (LNPs-07 and LNPs-10) were compared to five component LNPs containing either squalene (LNPs-09 and LNPs-12) or cardiolipin (LNPs-08 and LNPs-11) as fifth component. The spike antibody produced is quantified using ELISA after two weeks of second dose of LNPs.

[0042] Figure 15 depicts a cryo-TEM image of LNPs-13 formulated with ionizable diester lipid 201, DSPC, cholesterol, PEG2k-DMG and squalene.

[0043] Figure 16 depicts a cryo-TEM image of LNPs-14 formulated with ionizable di ester lipid 201, DSPC, cholesterol, PEG2k-DMG and Withaferin A.

[0044] Figure 17 depicts a cryo-TEM image of LNPs-15 formulated with ionizable di ester lipid 201, DSPC, cholesterol, PEG2k-DMG and a-tocopherol.

[0045] Figure 18 depicts a cryo-TEM image of LNPs-16 formulated with ionizable diester lipid 201, DSPC, cholesterol, PEG2k-DMG and |3-carotene.

[0046] Figure 19 depicts a cryo-TEM image of LNPs-17 formulated with ionizable di ester lipid 201, DSPC, cholesterol, PEG2k-DMG and retinol.

[0047] Figure 20A shows Spike-specific IgG antibody levels detected by using an ELISA assay 14 days after a second immunization in mice with LNPs-07, LNPs-08 and LNPs-18.

[0048] Figure 20B is a graph showing the Neutralizing Antibody (NAb) response against Wuhan-Hu-1 / D614G measured 14 days after a second immunization in mice with doses of 2.5 pg LNPs-07, LNPs-08 and LNPs-18.

[0049] Figure 21 is a graph showing the Neutralizing Antibody (NAb) response against Wuhan-Hu-1 / D614G with LNPs-18 at different mRNA doses.

[0050] Figure 22 shows level of the antigen-specific IFNy secreting cells measured in splenocytes 14 days after the second immunization in mice with LNPs-07, LNPs-08 and LNPs- 18

[0051] Figure 23 shows the level of the antigen-specific IL-4 secreting cells measured in splenocytes 14 days after the second immunization in mice with LNPs-07, LNPs-08 and LNPs- 18

[0052] Figure 24 shows the MC38gp tumor growth control after immunization in mice with LNPs-02 and LNPs-03 made from ionizable lipid 201.

[0053] Figure 25 shows the results of Day 8 CD8+ T cell induction in wild-type mice using LNPs-02, LNPs-03 and LNPs-13 made from ionizable lipid 201. (a) gp-33 tetramer strain; (b) gp-34 tetramer strain; (c) gp276 tetramer strain.

[0054] Figure 26 shows the results of Day 12 CD45+ T cell induction in wild-type mice using LNPs-02 and LNPs-03 made from ionizable lipid 201 (gp-33 tetramer strain and gp-34 tetramer strains).

[0055] Figures 27A and 27B illustrate A: representative MRI images of tumors implanted in C3H-CL1-F2 (female) mice treated with mRNA vaccine LNP (mRNA vaccine) or Control,at days 30 and 43 post-tumor implantation; and B: overall survival rate of C3H-CL1-F2 (female) mice treated with mRNA vaccine LNP (mRNA vaccine) or Control.

[0056] Figure 28 illustrates the overall survival rate of C3H-CL1-M1 (male) mice treated with mRNA vaccine LNP (mRNA vaccine) or Control.

[0057] Figure 29 illustrates representative MRI images of tumors implanted in C3H-CL1- M1 (male) mice treated with mRNA vaccine LNP (mRNA vaccine) or Control, at days 30 and 43 post-tumor implantation.

[0058] Figure 30 illustrates the mice survival rate of untreated mice (buffer), of mice treated with BMDC, of mice treated with LNPs-02 and of mice treated with LNPs-03 in MC38gp Model.

[0059] Figure 31 represents a comparison of the Wt EGFR and the EGFRvIII sequences.

[0060] Figures 32A-E shows the in vivo EGFRvIII-induced GBM mouse model and vaccination with LNPs containing EGFRvIII mRNA. Figure 32A shows the survival rate of vaccinated mice. Figure 32B shows the flow cytometry analysis of EGFRvIII-specific CTLs in the spleens from vaccinated mice. Figures 32C-32E show the quantification of EGFRvIII- specific CTLs in the spleens from vaccinated mice in frequencies and absolute numbers.

[0061] Figure 33 shows anti- EGFRvIII IgG antibody levels by ELISA in EGFRvIII- induced GBM following vaccination with LNPs.

[0062] Figure 34 shows the flow cytometry analysis of the transfection efficiency of LNPs containing tdTomato mRNA in human PBMCs.

[0063] Figure 35 shows the flow cytometry analysis of the transfection efficiency of LNPs containing tdTomato mRNA in human PBMCs preincubated with either ApoE or autologous human plasma.

[0064] Figure 36 shows the transfection efficiency of LNPs containing tdTomato mRNA in monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs) differentiated from human PBMCs with, before and after maturation / polarization.DETAILED DESCRIPTIONI. Introduction

[0065] The following description sets forth exemplary compounds, compositions, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0066] The delivery of a therapeutic agent to a subject is important for its therapeutic effects and usually it can be impeded by limited ability of the compound to reach targeted cells and tissues. Improvement of such therapeutic agents to enter the targeted cells of tissues by a variety of means of delivery is crucial. Nucleic acid therapy has emerged as the dominant method of treating various diseases and therapeutic indications given the versatility, lower immune response and higher potency as compared to traditional therapies. For example, nucleic acid therapy includes the use of small interfering (siRNA) to reduce the translation of messenger RNA (mRNA), mRNA as a way to produce a target of interest, circular RNA (oRNA) which can provide continuous production of a polypeptide or peptide or can be a sponge to compete with other RNA molecules, and viral vectors to provide a continuous production of a target of interest. However, some nucleic acids are unstable and easily degraded so they need to be formulated to prevent the degradation and to aid in the intracellular delivery of the nucleic acids.

[0067] The present disclosure relates to novel diester lipid compounds and compositions comprising the same, more particularly nanoparticles based on these diester compounds, capable of encapsulating a cargo such as a biologically active and therapeutic agent.

[0068] The present disclosure also relates to novel lipid nanoparticle compositions that may have improved stability, and / or increased efficacy such as increased immunogenicity when it is used in vaccines, and / or facilitate the intracellular delivery of biologically active and therapeutic agents. In some embodiments, the lipid nanoparticle compositions may have low or reduced toxicity. The present disclosure relates also to pharmaceutical compositions that comprise such lipid compositions, and that are useful to deliver therapeutically effective amounts of biologically active agents into the cells of patients.

[0069] Examples of biologically active agents include but are not limited to: (1) proteins including immunoglobin proteins, (2) polynucleotides such as genomic DNA, cDNA, or mRNA, (3) antisense polynucleotides, and (4) low molecular weight compounds, whether synthetic or naturally occurring, such as the peptide hormones and antibiotics.

[0070] “Lipid” means an organic compound that comprises an ester of fatty acid and is characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0071] “Lipid particle” or “lipid nanoparticle (i.e., “LNP”) means a lipid formulation that can be used to deliver a cargo, such as a therapeutic nucleic acid (e.g., mRNA) to a target siteof interest (e.g., cell, tissue, organ, and the like). In preferred embodiments, the lipid particle can be used to encapsulate a nucleic acid. In preferred embodiments, the lipid nanoparticle can be formed from an ionizable lipid, a neutral lipid (e.g., a phospholipid), a polymer- conjugated lipid that can prevent aggregation of the nanoparticle (e.g., a PEG-lipid), and optionally a helper lipid (e.g., cholesterol). In some embodiments, a therapeutic nucleic acid (e.g., mRNA) may be encapsulated in the lipid portion of the nanoparticle, thereby protecting it from enzymatic degradation. In another preferred embodiment, the lipid nanoparticle can comprise another component such as a hydrophobic component to improve LNP internalization, immune activation and / or antibody production.

[0072] Lipid nanoparticles generally comprise cholesterol (aids in stability and promotes membrane fusion), a phospholipid (which provides structure to the LNP bilayer and also may aid in endosomal escape), a polyethylene glycol (PEG) derivative (which reduces LNP aggregation and “shields” the LNP from non-specific endocytosis by immune cells), and an ionizable lipid (complexes negatively charged RNA and enhances endosomal escape), which form the LNP -forming composition.

[0073] Lipid nanoparticles typically can have a particle size, e.g., expressed as a mean diameter, ranging from 30 nm to 200 nm, from 40 nm to 180 nm, from 50 nm to 150 nm, from 60 nm to 130 nm, from 60 nm to 120 nm, from 60 nm to 110 nm, from 60 nm to 100 nm, from 60 nm to 90 nm, from 70 nm to 110 nm, from 70 nm to 100 nm, from 80 nm to 100 nm, from 90 nm to 100 nm, from 70 to 90 nm, from 80 nm to 90 nm, or from 70 nm to 80 nm. In some embodiments, the particle size can be about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or 200 nm and are substantially non-toxic. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease.II. LipidsIonizable lipids

[0074] The present disclosure relates to compounds that are ionizable lipids, more particularly ionizable diester lipids. The ionizable lipids may be cationic lipids.

[0075] In some embodiments, ionizable lipid compounds of the present disclosure comprise at least two ester bonds (-CO-O- or -O-CO-). Ester bonds present the particularity ofbeing biodegradable. In some embodiments, compounds of the present disclosure can further comprise one secondary amino group. In some embodiments, compounds of the present disclosure further comprise at least one terminal amino group, wherein the amino group may be substituted with at least one lower alkyl group (e.g., C1-C3 alky groups), which may be further substituted. In some embodiments, the terminal amino group can be NH2, a primary amino group, a secondary amino group, or a tertiary amino group. In some embodiments, the terminal amino group can be NfCHsh. -N(CM^(CH2CH3). -N(CH3)(CH2CH2OH), - N(CH2CH2OH)2, or N((CH2)2O(CO)CH3)2, to name a few examples. The ionizable lipids of the present disclosure can be characterized in that the two ester bonds are separated by two tertiary carbon atoms, a first one of the two tertiary carbon atoms being substituted with an alkyl chain and the second one of the two tertiary carbon atoms being substituted with a hydrocarbon chain bearing the terminal amino group. The hydrocarbon chain bearing the terminal amino group optionally comprises a nitrogen atom within the chain, this nitrogen atom being itself optionally substituted. These ionizable lipids may be obtained at high purity, and lipid nanoparticles made therefrom can present high stability.

[0076] In some embodiments, the ionizable lipid compound of the present disclosure can have a structure of Formula (I): or a pharmaceutically acceptable salt thereof, whereinR1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;X is -CH2-, -NH- or -NR7-; n is a number from 0 to 10; p is a number from 0 to 2;Rs and R6 are independently H, or an optionally substituted linear C1-C4 alkyl group; R7 is a linear or branched C1-C6 alkyl; wherein when any of R1, R2, R3 and R4 represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R1, R2, R3 and R4 represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds.

[0077] In some embodiments, in the structure of Formula (I), m can be an integer from 1 to 12, or m can be an integer from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or m can be 1 or 2. In some embodiments, in the structure of Formula (I), m can be 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12.

[0078] In some embodiments, in the structure of Formula (I), n can be an integer from 0 to 10, or n can be an integer from 0 to 9, or from 0 to 8, or from 0 to 7, or from 0 to 6, or from 0 to 5, or from 0 to 4, or from 0 to 3, or from 0 to 2, or n can be 0, 1 or 2. In some embodiments, in the structure of Formula (I), n can be 0, or 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10.

[0079] In some embodiments, in the structure of Formula (I), p can be an integer from 0 to 2, or p can be 0 or 1, or p can be 1 or 2, or p can be 0, or 1, or 2.

[0080] In some embodiments, in the structure of Formula (I), X is -CH2-.

[0081] In some embodiments, in the structure of Formula (I), X is -NH-.

[0082] In some embodiments, in the structure of Formula (I), X is -NR7- and R7 is a linear or branched C1-C6 alkyl.

[0083] In some embodiments, in the structure of Formula (I), X is -NR7- and R7 is a linear or branched C1-C4 alkyl.

[0084] In some embodiments, in the structure of Formula (I), X is -NR7- and R7 is C1-C2 alkyl.

[0085] In some embodiments, in the structure of Formula (I), X is -NMe-.

[0086] In some embodiments, when any of the alkyl, alkenyl and / or alkynyl groups in the substituents of the Formula (I) is substituted, these groups can independently be substituted with one or more halogen, hydroxyl, acetoxy, alkoxycarbonyl, formyl, acyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, an aromatic moiety or an heteroaromatic moiety.

[0087] In some embodiments, when any the alkyl, alkenyl and / or alkynyl groups in the substituents of the Formula (I) are substituted, these groups can independently be substituted with one or more halogen, hydroxyl, acetoxy, C1-C4alkoxy carbonyl, formyl, C1-C4acyl, C1- C4alkoxyl, amino, -(CO)NHC1-C4alkyl, -NH(CO)C1-C4alkyl, amidine, (C1-C4alkyl)2C=N-, cyano, nitro, azido, sulfhydryl, C1-C4alkylthio, sulfamoyl, -(SO2)NHC1-C4alkyl, - NH(SO2)C1-C4alkyl, -(SO2)C1-C4alkyl, 3- to 6-membered heterocyclyl, 6- to 10-membered aralkyl, or 5- to 10-membered aromatic or heteroaromatic moiety.

[0088] In some embodiments, when any alkyl, alkenyl and / or alkynyl group groups in the substituents of the Formula (I) are substituted, these groups can be independently substituted with one or more halogen, hydroxyl, acetoxy, amino, cyano, nitro, azido, or sulfhydryl.

[0089] In some embodiments, when any alkyl, alkenyl and / or alkynyl group groups in the substituents of the Formula (I) are substituted, these groups can be independently substituted with one or more hydroxyl or acetoxy.

[0090] In some embodiments, the ionizable diester lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) whereinR1 and R.4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R.2 and Rs are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;X is -CH2-, -NH- or -NR7-; n is a number from 0 to 8; p is 0 or 1 ; R5 and R6 are independently an optionally substituted C1-C4 alkyl group;R7is a linear or branched C1-C4 alkyl.

[0091] In some embodiments, the ionizable diester lipid, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) whereinR1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R22 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;X is -CH2-, -NH- or -NR7-; n is a number from 0 to 8; p is 0 or 1 ;R5 and R6 are independently an optionally substituted C1-C4 alkyl group; andR7is a linear or branched C1-C4 alkyl; wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, C1-C4alkoxy carbonyl, formyl, C1-C4acyl, C1-C4alkoxyl, amino, -(CO)NHC1-C4alkyl, -NH(CO)C1-C4alkyl, amidine, (C1-C4alkyl)2C=N-, cyano, nitro, azido, sulfhydryl, C1-C4alkylthio, sulfamoyl, - (SO2)NHC1-C4alkyl, -NH(SO2)C1-C4alkyl, -(SO2)C1-C4alkyl, 3- to 6-membered heterocyclyl, 6- to 10-membered aralkyl, or 5- to 10-membered aromatic or heteroaromatic moiety.

[0092] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:R1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H or an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 8;X is -CH2-, -NH- or -NR7-; n is a number from 0 to 8; p is 0 or 1 ;R5 and R6 are independently an optionally substituted C1-C4 alkyl group;R7is a C1-C2 alkyl.

[0093] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:R1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H or an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 8;X is -CH2-, -NH- or -NR7-; n is a number from 0 to 8; p is 0 or 1 ; R5 and R6 are independently an optionally substituted C1-C4 alkyl group;R7 is a C1-C2 alkyl; wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, amino, cyano, nitro, azido, or sulfhydryl.

[0094] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:R1 and R4 are independently linear or branched C8-C20 alkyl, linear or branched C8-C20 alkenyl, or linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, linear or branched C8-C20 alkyl, linear or branched C8- C20 alkenyl, or linear or branched C8-C20 alkynyl group; m is a number from 1 to 8;X is -CH2-, -NH- or -NMe-; n is a number from 0 to 8; p is 0 or 1 ; R5 and R6 are independently a C1-C4 alkyl group optionally substituted with hydroxyl or acetoxy.

[0095] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:R1 and R4 are independently linear C8-C20 alkyl, linear C8-C20 alkenyl, or linear C8- C20 alkynyl group;R2 and R3 are independently H, linear C8-C20 alkyl, linear C8-C20 alkenyl, or linear C8-C20 alkynyl group; m is a number from 1 to 8;X is -CH2-, -NH- or -NMe-; n is a number from 0 to 8; p is a number from 0 to 1 ; and R5 and R6 are independently a C1-C4 alkyl group.

[0096] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein when any of R1, R2, R3 and R4 is C8-C20 alkenyl, the alkenyl group comprises one to four C=C bonds, and when any ofR1, R2, R3 and R4 is C8-C20 alkynyl, the alkynyl group comprises one or two C=C bonds.

[0097] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein when any of R1, R2, R3 and R4 is C8-C20 alkenyl, the alkenyl group comprises one to three C=C bonds, and when any ofR1, R2, R3 and R4 is C8-C20 alkynyl, the alkynyl group comprises one or two c=C bonds.

[0098] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein when any of R1, R2, R3 and R4 is C8-C20 alkenyl, the alkenyl group comprises one or two C=C bonds, and when any ofR1, R2, R3 and R4 is C8-C20 alkynyl, the alkynyl group comprises one or two C=C bonds.

[0099] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein when any of R1, R2, R3 and R4 is C8-C20 alkenyl, the alkenyl group comprises one C=C bonds, and when any of R1, R2, R3 and R4 is C8-C20 alkynyl, the alkynyl group comprises one c=C bonds.

[0100] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R1, R2, R3 and R4 independently represent C8-C20 alkenyl, the alkenyl group comprising one to four C=C bonds.

[0101] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R1, R2, R3 and R4 independently represent C8-C20 alkenyl, the alkenyl group comprising one to three C=C bonds.

[0102] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri, R2, R3 and R4 independently represent C8-C20 alkenyl, the alkenyl group comprising one or two C=C bonds.

[0103] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri, R2, R3 and R4 independently represent C8-C20 alkenyl, the alkenyl group comprising one C=C bonds.

[0104] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:R1 and R4 are independently linear C8-C20 alkyl or linear C8-C20 alkenyl;R2 and R3 are independently H or linear C8-C20 alkyl or linear C8-C20 alkenyl; m is a number from 1 to 8;X is -CH2-, -NH- or -NMe-; n is a number from 0 to 8; p is a number from 0 to 1 ; and R5 and R6 are independently C1-C4 alkyl.

[0105] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R1 and R4 are identical.

[0106] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R2 and R3 are H or linear C8-C18 alkyl, preferably H or C10-C16 alkyl, more preferably H or C14 alkyl.

[0107] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R2 and R3 are H.

[0108] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R1 and R4 are identical and represent a linear C8-C18 alkyl or linear C8-C18 alkenyl, wherein the alkenyl groups comprise one to four C=C bonds.

[0109] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R1 and R4 are identical and represent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one to four C=C bonds.

[0110] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R1 and R4 are identical and represent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one to three C=C bonds.

[0111] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R1 and R4 are identical and represent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one or two C=C bonds.

[0112] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R1 and R4 are identical andrepresent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one C=C bonds.

[0113] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein anRd5 R6 are independently a C1-C2 alkyl group.

[0114] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein aRn5d R6 are identical.

[0115] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (II), (12), (13), (14), (15), (16), (17), or (18):or a pharmaceutically acceptable salt thereof, wherein m is a number from 1 to 12; n is a number from 0 to 8; p is 0 or 1; q is a number from 0 to 8; r is a number from 1 to 15; s in number from 0 to 5; t is a number from 0 to 6; X is - CH2-, -NH- or -NMe-; and LI and L2 are independently a number from 0 to 3, and wherein the C=C double bonds present in any of the Formulas (II), (12), (13), (14), (15), (16), (17), or (18) independently have the E or Z configuration.

[0116] In some embodiments, in the structures of Formulas (II), (12), (13), (14), (15) or (16), q can be a number from 0 to 11, from 0 to 10, from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or q can be 0 or 1, or q can be 1 or 2, or q can be 0, or 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11.

[0117] In some embodiments, in the structure of Formulas (17), s can be a number from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or s can be 0 or 1, or s can be 1 or 2, or s can be 0, or 1, or 2, or 3, or 4, or 5.

[0118] In some embodiments, in the structure of Formulas (18), t can be a number from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or t can be 0 or 1, or t can be 1 or 2, or t can be 2 or 3, or t can be 3 or 4, or 1, or 2, or 3, or 4, or 5, or 6.

[0119] In some embodiments, in the structures of Formulas (II), (12), (13), (14), (15), (16), (17), or (18), X is independently -CH2-.

[0120] In some embodiments, in the structures of Formulas (II), (12), (13), (14), (15), (16), (17), or (18), X is independently -NH-.

[0121] In some embodiments, in the structures of Formulas (II), (12), (13), (14), (15), (16), (17), or (18), X is independently -NMe-.

[0122] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formulas (13), (14), (15), (16), (17), or (18) wherein X is -CH2-.

[0123] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (Ila):number from 1 to 8; and q is a number from 0 to 11.

[0124] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (lib):or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.

[0125] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (lie):or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.

[0126] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (I2a):number from 1 to 8; and q is a number from 0 to 7.

[0127] In some embodiments, the ionizable diester lipid compound can have a structure ofFormula (I2b):or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 7.

[0128] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (I3a):or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.

[0129] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (I4a):number from 1 to 8; q is a number from 0 to 11; and r is a number from 1 to 13.

[0130] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (I5a):(I5a), or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.

[0131] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (I6a): number from 1 to 8; and q is a number from 0 to 8.

[0132] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (I7a):from 1 to 8; and s is a number from 0 to 5.

[0133] In some embodiments, the ionizable diester lipid compound can have a structure of number from 1 to 8; and t is a number from 0 to 6.

[0134] Each of the C=C double bonds present in the Formulas (I), (II), (12), (13), (14), (15),(16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a), or (I8a) and / or in the compounds represented in Table 1, can independently have the E or Z configuration.

[0135] In some embodiments, the compounds of Formulas (I), (II), (12), (13), (14), (15), (16),(17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a), or (I8a) and / or in the compounds represented in Table 1 can be in the form of any enantiomers, diastereoisomers, cis or trans geometric isomers, or mixtures thereof.

[0136] In some embodiments, the ionizable diester lipid compounds of the present disclosure, without being limited to, can be selected from the group consisting of Compounds 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217 and 218 of Table 1, or a pharmaceutically acceptable salt thereof.

[0137] In some embodiments, the ionizable di ester lipid compounds can be in the form of any enantiomer and / or any diastereoisomer thereof, or any mixture thereof.

[0138] Table 1: Non-Limiting Examples of Ionizable Lipid Compounds

[0139] The cis (Z) or trans (E) stereochemistry as drawn in the chemical structures of Table1 was randomly attributed. In other words, each of the C=C bonds in the chemical structures of Table 1 can independently have the cis or trans configuration. In some embodiments, the compounds of Table 1 can be in the form of mixtures of cis or trans geometric isomers.

[0140] In some embodiments, the ionizable diester lipid compounds of Table 1 can be in the form of any enantiomers, any diastereoisomers, any cis or trans geometric isomers, or any mixtures thereof.

[0141] The term “compound”, as used herein, is meant to embrace all stereoisomers, geometric isomers, tautomers, and isotopes of a depicted or described structure associated with the compound. When referring to compound features or substituents, the terms “optional” or “optionally” refer to a feature or substituent that may or may not occur. For example, “optionally substituted alkyl” encompasses both “alkyl” and “substituted alkyl” as defined below. It will be understood by those skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and / or inherently unstable.

[0142] The compounds herein described may have asymmetric centers, geometric centers (e.g., double bond), or both. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms, by synthesis from optically active starting materials, or through use of chiral auxiliaries. Geometric isomers of olefins, C=N double bonds, or other types of double bonds may be present in the compounds described herein, and all such stable isomers are included in the present disclosure. Specifically, cis and trans geometric isomers of the compounds of the present disclosure may also exist and may be isolated as a mixture of isomers or as separated isomeric forms.

[0143] Compounds described herein also embrace tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0144] Compounds described herein also embrace all the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. Thus, by way of example, each individual hydrogen atom present in formula (200) may be present as a1H,2H (deuterium) or3H (tritium) atom, preferablyJH or2H. Similarly, by way of example, each individual carbon atom present in formula (200) may be present as a12C,13C or14C atom, preferably12C.

[0145] The compounds or structures and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods. Neutral lipids

[0146] In some embodiments, the lipid nanoparticle can also include at least one neutral lipid. In some embodiments, the neutral lipids may be phospholipids, or derivatives thereof.

[0147] Examples of phospholipids suitable for use in the present disclosure include, but are not limited to: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1- myristoyl-2 -palmitoyl phosphatidylcholine (MPPC), 1-palmi toy 1-2 -myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2- diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1 -stearoyl-2 -palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof.

[0148] In some embodiments, the preferred phospholipids are distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE).

[0149] In other embodiments, the preferred phospholipids are DSPC, DOPC, DMPC and PE.Helper lipids

[0150] In some embodiments, the lipid nanoparticle can also include at least one helper lipid.

[0151] “Helper lipids” are lipids that enhance transfection, such as transfection of the lipid nanoparticle including the payloads and cargos. The mechanism by which the helper lipid enhances transfection may include enhancing particle stability and / or enhancing membrane fusogenicity. Helper lipids include steroids and alkyl resorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, cholesterol hemisuccinate, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl- 2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0152] In some embodiments, the preferred helper lipid is cholesterol.Polymer-conjugated lipids

[0153] In some embodiments, the lipid nanoparticle can further include at least one polymer- conjugated lipid.

[0154] In some embodiments, the polymer-conjugated lipid comprises a polymer conjugated to at least one lipid. In some embodiments, the polymer-conjugated lipid can comprise at least one component that reduces aggregation of particles, at least one component that decreases clearing of the lipid nanoparticle from circulation in a subject, at least component that increases the lipid nanoparticle’s ability to traverse mucus layers, at least one component that decreases a subjects immune response to administration of the lipid nanoparticle, at least one component that modifies membrane fluidity of the lipid nanoparticle, at least one component that contributes to the stability of the lipid nanoparticle, or any combination thereof. In some embodiments, the lipid nanoparticle may be essentially devoid of polymer-conjugated lipid. In some embodiments, the lipid nanoparticle may contain no amount of polymer-conjugated lipid.

[0155] In some embodiments, the polymer present in the polymer-conjugated lipid may comprise at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-oxazoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.

[0156] In some embodiments, the lipid conjugated to the polymer may be selected from, but is not limited to, at least one of the ionizable, neutral, or helper lipids listed previously.

[0157] In some embodiments, the polymer conjugated to at least one lipid is PEG and the polymer-conjugated lipid can be referred to as “PEG-lipid”. In some embodiments, the at least one PEG-lipid may be selected from, but is not limited to at least one of Siglec-IL-PEG-DSPE,R)-2,3-bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE Cl 8, PEG-DSPE, PEG- DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG Cl 4, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide Cl 6, PEG-C-DOMG, PEG- c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-Cll, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000- DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE Cl 8, PEGDMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click CIO, N(Carbonyl- methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myrj52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, 1,2-distearoyl- sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE- PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG- PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE- mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol- polyethyleneglycol, C18PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)- 2,3-bis(octadecyloxy)propyl-l-(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)- C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

[0158] In some embodiments, the preferred polymer-conjugated lipids are polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-oxazoline) (POZ), polyamide (ATTA), cationic polymer, poly sarcosine (Psar), poly glutamic acid (PGA) and 1 ,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol (PEG-DMG).

[0159] In some embodiments, the preferred polymer-conjugated lipids are PEG-lipids selected from PEG-DMG or PEG-DSG.

[0160] In some embodiments, the preferred PEG-lipid is PEG2k-DMG.

[0161] In some embodiments, the average molecular weight of the polymer moiety (e.g., PEG) of the polymer-conjugated lipid may be between 500 and 20,000 daltons. In some embodiments, the molecular weight of the polymer may be about 500 to 20,000, 1,000 to 20,000, 1,500 to 20,000, 2,000 to 20,000, 2,500 to 20,000, 3,000 to 20,000, 3,500 to 20,000,,000 to 20,000, 4,500 to 20,000, 5,000 to 20,000, 5,500 to 20,000, 6,000 to 20,000, 6,500 to0,000, 7,000 to 20,000, 7,500 to 20,000, 8,000 to 20,000, 8,500 to 20,000, 9,000 to 20,000,.500 to 20,000, 10,000 to 20,000, 10,500 to 20,000, 11,000 to 20,000, 11,500 to 20,000,2,000 to 20,000, 12,500 to 20,000, 13,000 to 20,000, 13,500 to 20,000, 14,000 to 20,000,4.500 to 20,000, 15,000 to 20,000, 15,500 to 20,000, 16,000 to 20,000, 16,500 to 20,000,7,000 to 20,000, 17,500 to 20,000, 18,000 to 20,000, 18,500 to 20,000, 19,000 to 20,000,9.500 to 20,000, 500 to 19,500, 1,000 to 19,500, 1,500 to 19,500, 2,000 to 19,500, 2,500 to9,500, 3,000 to 19,500, 3,500 to 19,500, 4,000 to 19,500, 4,500 to 19,500, 5,000 to 19,500,.500 to 19,500, 6,000 to 19,500, 6,500 to 19,500, 7,000 to 19,500, 7,500 to 19,500, 8,000 to9.500, 8,500 to 19,500, 9,000 to 19,500, 9,500 to 19,500, 10,000 to 19,500, 10,500 to 19,500,1,000 to 19,500, 11,500 to 19,500, 12,000 to 19,500, 12,500 to 19,500, 13,000 to 19,500,3.500 to 19,500, 14,000 to 19,500, 14,500 to 19,500, 15,000 to 19,500, 15,500 to 19,500,6,000 to 19,500, 16,500 to 19,500, 17,000 to 19,500, 17,500 to 19,500, 18,000 to 19,500,8.500 to 19,500, 19,000 to 19,500, 1,500 to 19,000, 2,000 to 19,000, 2,500 to 19,000, 3,000 19,000, 3,500 to 19,000, 4,000 to 19,000, 4,500 to 19,000, 5,000 to 19,000, 5,500 to 19,000,,000 to 19,000, 6,500 to 19,000, 7,000 to 19,000, 7,500 to 19,000, 8,000 to 19,000, 8,500 to9,000, 9,000 to 19,000, 9,500 to 19,000, 10,000 to 19,000, 10,500 to 19,000, 11,000 to 19,000,1.500 to 19,000, 12,000 to 19,000, 12,500 to 19,000, 13,000 to 19,000, 13,500 to 19,000,4,000 to 19,000, 14,500 to 19,000, 15,000 to 19,000, 15,500 to 19,000, 16,000 to 19,000,6.500 to 19,000, 17,000 to 19,000, 17,500 to 19,000, 18,000 to 19,000, 18,500 to 19,000,.500 to 18,500, 2,000 to 18,500, 2,500 to 18,500, 3,000 to 18,500, 3,500 to 18,500, 4,000 to8.500, 4,500 to 18,500, 5,000 to 18,500, 5,500 to 18,500, 6,000 to 18,500, 6,500 to 18,500,,000 to 18,500, 7,500 to 18,500, 8,000 to 18,500, 8,500 to 18,500, 9,000 to 18,500, 9,500 to8,500, 10,000 to 18,500, 10,500 to 18,500, 11,000 to 18,500, 11,500 to 18,500, 12,000 to8,500, 12,500 to 18,500, 13,000 to 18,500, 13,500 to 18,500, 14,000 to 18,500, 14,500 to8,500, 15,000 to 18,500, 15,500 to 18,500, 16,000 to 18,500, 16,500 to 18,500, 17,000 to8,500, 17,500 to 18,500, 18,000 to 18,500, 1,500 to 18,000, 2,000 to 18,000, 2,500 to 18,000,,000 to 18,000, 3,500 to 18,000, 4,000 to 18,000, 4,500 to 18,000, 5,000 to 18,000, 5,500 to8,000, 6,000 to 18,000, 6,500 to 18,000, 7,000 to 18,000, 7,500 to 18,000, 8,000 to 18,000,.500 to 18,000, 9,000 to 18,000, 9,500 to 18,000, 10,000 to 18,000, 10,500 to 18,000, 11,000 18,000, 11,500 to 18,000, 12,000 to 18,000, 12,500 to 18,000, 13,000 to 18,000, 13,500 to8,000, 14,000 to 18,000, 14,500 to 18,000, 15,000 to 18,000, 15,500 to 18,000, 16,000 to8,000, 16,500 to 18,000, 17,000 to 18,000, 17,500 to 18,000, 1,500 to 17,500, 2,000 to 17,500,.500 to 17,500, 3,000 to 17,500, 3,500 to 17,500, 4,000 to 17,500, 4,500 to 17,500, 5,000 to7,500, 5,500 to 17,500, 6,000 to 17,500, 6,500 to 17,500, 7,000 to 17,500, 7,500 to 17,500,,000 to 17,500, 8,500 to 17,500, 9,000 to 17,500, 9,500 to 17,500, 10,000 to 17,500, 10,500 17,500, 11,000 to 17,500, 11,500 to 17,500, 12,000 to 17,500, 12,500 to 17,500, 13,000 to7.500, 13,500 to 17,500, 14,000 to 17,500, 14,500 to 17,500, 15,000 to 17,500, 15,500 to7,500, 16,000 to 17,500, 16,500 to 17,500, 17,000 to 17,500, 1,500 to 17,000, 2,000 to 17,000,.500 to 17,000, 3,000 to 17,000, 3,500 to 17,000, 4,000 to 17,000, 4,500 to 17,000, 5,000 to7,000, 5,500 to 17,000, 6,000 to 17,000, 6,500 to 17,000, 7,000 to 17,000, 7,500 to 17,000,,000 to 17,000, 8,500 to 17,000, 9,000 to 17,000, 9,500 to 17,000, 10,000 to 17,000, 10,500 17,000, 11,000 to 17,000, 11,500 to 17,000, 12,000 to 17,000, 12,500 to 17,000, 13,000 to7,000, 13,500 to 17,000, 14,000 to 17,000, 14,500 to 17,000, 15,000 to 17,000, 15,500 to7,000, 16,000 to 17,000, 16,500 to 17,000, 1,500 to 16,500, 2,000 to 16,500, 2,500 to 16,500,,000 to 16,500, 3,500 to 16,500, 4,000 to 16,500, 4,500 to 16,500, 5,000 to 16,500, 5,500 to6.500, 6,000 to 16,500, 6,500 to 16,500, 7,000 to 16,500, 7,500 to 16,500, 8,000 to 16,500,.500 to 16,500, 9,000 to 16,500, 9,500 to 16,500, 10,000 to 16,500, 10,500 to 16,500, 11,000 16,500, 11,500 to 16,500, 12,000 to 16,500, 12,500 to 16,500, 13,000 to 16,500, 13,500 to6,500, 14,000 to 16,500, 14,500 to 16,500, 15,000 to 16,500, 15,500 to 16,500, 16,000 to6.500, 1,500 to 16,000, 2,000 to 16,000, 2,500 to 16,000, 3,000 to 16,000, 3,500 to 16,000,,000 to 16,000, 4,500 to 16,000, 5,000 to 16,000, 5,500 to 16,000, 6,000 to 16,000, 6,500 to6,000, 7,000 to 16,000, 7,500 to 16,000, 8,000 to 16,000, 8,500 to 16,000, 9,000 to 16,000,.500 to 16,000, 10,000 to 16,000, 10,500 to 16,000, 11,000 to 16,000, 11,500 to 16,000,2,000 to 16,000, 12,500 to 16,000, 13,000 to 16,000, 13,500 to 16,000, 14,000 to 16,000,4.500 to 16,000, 15,000 to 16,000, 15,500 to 16,000, 1,500 to 15,500, 2,000 to 15,500, 2,500 15,500, 3,000 to 15,500, 3,500 to 15,500, 4,000 to 15,500, 4,500 to 15,500, 5,000 to 15,500,.500 to 15,500, 6,000 to 15,500, 6,500 to 15,500, 7,000 to 15,500, 7,500 to 15,500, 8,000 to5.500, 8,500 to 15,500, 9,000 to 15,500, 9,500 to 15,500, 10,000 to 15,500, 10,500 to 15,500,1,000 to 15,500, 11,500 to 15,500, 12,000 to 15,500, 12,500 to 15,500, 13,000 to 15,500,3.500 to 15,500, 14,000 to 15,500, 14,500 to 15,500, 15,000 to 15,500, 1,500 to 15,000, 2,000 15,000, 2,500 to 15,000, 3,000 to 15,000, 3,500 to 15,000, 4,000 to 15,000, 4,500 to 15,000,,000 to 15,000, 5,500 to 15,000, 6,000 to 15,000, 6,500 to 15,000, 7,000 to 15,000, 7,500 to5,000, 8,000 to 15,000, 8,500 to 15,000, 9,000 to 15,000, 9,500 to 15,000, 10,000 to 15,000,0.500 to 15,000, 11,000 to 15,000, 11,500 to 15,000, 12,000 to 15,000, 12,500 to 15,000,3,000 to 15,000, 13,500 to 15,000, 14,000 to 15,000, 14,500 to 15,000, 1,500 to 14,500, 2,000 14,500, 2,500 to 14,500, 3,000 to 14,500, 3,500 to 14,500, 4,000 to 14,500, 4,500 to 14,500,,000 to 14,500, 5,500 to 14,500, 6,000 to 14,500, 6,500 to 14,500, 7,000 to 14,500, 7,500 to4.500, 8,000 to 14,500, 8,500 to 14,500, 9,000 to 14,500, 9,500 to 14,500, 10,000 to 14,500,0.500 to 14,500, 11,000 to 14,500, 11,500 to 14,500, 12,000 to 14,500, 12,500 to 14,500,3,000 to 14,500, 13,500 to 14,500, 14,000 to 14,500, 1,500 to 14,000, 2,000 to 14,000, 2,500 14,000, 3,000 to 14,000, 3,500 to 14,000, 4,000 to 14,000, 4,500 to 14,000, 5,000 to 14,000,.500 to 14,000, 6,000 to 14,000, 6,500 to 14,000, 7,000 to 14,000, 7,500 to 14,000, 8,000 to4,000, 8,500 to 14,000, 9,000 to 14,000, 9,500 to 14,000, 10,000 to 14,000, 10,500 to 14,000,1,000 to 14,000, 11,500 to 14,000, 12,000 to 14,000, 12,500 to 14,000, 13,000 to 14,000,3.500 to 14,000, 1,500 to 13,500, 2,000 to 13,500, 2,500 to 13,500, 3,000 to 13,500, 3,500 to3.500, 4,000 to 13,500, 4,500 to 13,500, 5,000 to 13,500, 5,500 to 13,500, 6,000 to 13,500,.500 to 13,500, 7,000 to 13,500, 7,500 to 13,500, 8,000 to 13,500, 8,500 to 13,500, 9,000 to3.500, 9,500 to 13,500, 10,000 to 13,500, 10,500 to 13,500, 11,000 to 13,500, 11,500 to3,500, 12,000 to 13,500, 12,500 to 13,500, 13,000 to 13,500, 1,500 to 13,000, 2,000 to 13,000,.500 to 13,000, 3,000 to 13,000, 3,500 to 13,000, 4,000 to 13,000, 4,500 to 13,000, 5,000 to3,000, 5,500 to 13,000, 6,000 to 13,000, 6,500 to 13,000, 7,000 to 13,000, 7,500 to 13,000,,000 to 13,000, 8,500 to 13,000, 9,000 to 13,000, 9,500 to 13,000, 10,000 to 13,000, 10,500 13,000, 11,000 to 13,000, 11,500 to 13,000, 12,000 to 13,000, 12,500 to 13,000, 1,500 to2.500, 2,000 to 12,500, 2,500 to 12,500, 3,000 to 12,500, 3,500 to 12,500, 4,000 to 12,500,.500 to 12,500, 5,000 to 12,500, 5,500 to 12,500, 6,000 to 12,500, 6,500 to 12,500, 7,000 to2.500, 7,500 to 12,500, 8,000 to 12,500, 8,500 to 12,500, 9,000 to 12,500, 9,500 to 12,500,0,000 to 12,500, 10,500 to 12,500, 11,000 to 12,500, 11,500 to 12,500, 12,000 to 12,500,.500 to 12,000, 2,000 to 12,000, 2,500 to 12,000, 3,000 to 12,000, 3,500 to 12,000, 4,000 to2,000, 4,500 to 12,000, 5,000 to 12,000, 5,500 to 12,000, 6,000 to 12,000, 6,500 to 12,000,,000 to 12,000, 7,500 to 12,000, 8,000 to 12,000, 8,500 to 12,000, 9,000 to 12,000, 9,500 to2,000, 10,000 to 12,000, 10,500 to 12,000, 11,000 to 12,000, 11,500 to 12,000, 1,500 to1.500, 2,000 to 11,500, 2,500 to 11,500, 3,000 to 11,500, 3,500 to 11,500, 4,000 to 11,500,.500 to 11,500, 5,000 to 11,500, 5,500 to 11,500, 6,000 to 11,500, 6,500 to 11,500, 7,000 to1.500, 7,500 to 11,500, 8,000 to 11,500, 8,500 to 11,500, 9,000 to 11,500, 9,500 to 11,500,0,000 to 11,500, 10,500 to 11,500, 11,000 to 11,500, 1,500 to 11,000, 2,000 to 11,000, 2,500 11,000, 3,000 to 11,000, 3,500 to 11,000, 4,000 to 11,000, 4,500 to 11,000, 5,000 to 11,000,.500 to 11,000, 6,000 to 11,000, 6,500 to 11,000, 7,000 to 11,000, 7,500 to 11,000, 8,000 to1,000, 8,500 to 11,000, 9,000 to 11,000, 9,500 to 11,000, 10,000 to 11,000, 10,500 to 11,000,.500 to 10,500, 2,000 to 10,500, 2,500 to 10,500, 3,000 to 10,500, 3,500 to 10,500, 4,000 to0.500, 4,500 to 10,500, 5,000 to 10,500, 5,500 to 10,500, 6,000 to 10,500, 6,500 to 10,500,,000 to 10,500, 7,500 to 10,500, 8,000 to 10,500, 8,500 to 10,500, 9,000 to 10,500, 9,500 to10,500, 10,000 to 10,500, 1,500 to 10,000, 2,000 to 10,000, 2,500 to 10,000, 3,000 to 10,000,3,500 to 10,000, 4,000 to 10,000, 4,500 to 10,000, 5,000 to 10,000, 5,500 to 10,000, 6,000 to 10,000, 6,500 to 10,000, 7,000 to 10,000, 7,500 to 10,000, 8,000 to 10,000, 8,500 to 10,000, 9,000 to 10,000, 9,500 to 10,000, 1,500 to 9,500, 2,000 to 9,500, 2,500 to 9,500, 3,000 to 9,500,3.500 to 9,500, 4,000 to 9,500, 4,500 to 9,500, 5,000 to 9,500, 5,500 to 9,500, 6,000 to 9,500,6.500 to 9,500, 7,000 to 9,500, 7,500 to 9,500, 8,000 to 9,500, 8,500 to 9,500, 9,000 to 9,500,1.500 to 9,000, 2,000 to 9,000, 2,500 to 9,000, 3,000 to 9,000, 3,500 to 9,000, 4,000 to 9,000,4.500 to 9,000, 5,000 to 9,000, 5,500 to 9,000, 6,000 to 9,000, 6,500 to 9,000, 7,000 to 9,000,7.500 to 9,000, 8,000 to 9,000, 8,500 to 9,000, 1,500 to 8,500, 2,000 to 8,500, 2,500 to 8,500,3,000 to 8,500, 3,500 to 8,500, 4,000 to 8,500, 4,500 to 8,500, 5,000 to 8,500, 5,500 to 8,500,6,000 to 8,500, 6,500 to 8,500, 7,000 to 8,500, 7,500 to 8,500, 8,000 to 8,500, 1,500 to 8,000,2,000 to 8,000, 2,500 to 8,000, 3,000 to 8,000, 3,500 to 8,000, 4,000 to 8,000, 4,500 to 8,000,5,000 to 8,000, 5,500 to 8,000, 6,000 to 8,000, 6,500 to 8,000, 7,000 to 8,000, 7,500 to 8,000,1.500 to 7,500, 2,000 to 7,500, 2,500 to 7,500, 3,000 to 7,500, 3,500 to 7,500, 4,000 to 7,500,4.500 to 7,500, 5,000 to 7,500, 5,500 to 7,500, 6,000 to 7,500, 6,500 to 7,500, 7,000 to 7,500,1.500 to 7,000, 2,000 to 7,000, 2,500 to 7,000, 3,000 to 7,000, 3,500 to 7,000, 4,000 to 7,000,4.500 to 7,000, 5,000 to 7,000, 5,500 to 7,000, 6,000 to 7,000, 6,500 to 7,000, 1,500 to 6,500,2,000 to 6,500, 2,500 to 6,500, 3,000 to 6,500, 3,500 to 6,500, 4,000 to 6,500, 4,500 to 6,500,5,000 to 6,500, 5,500 to 6,500, 6,000 to 6,500, 1,500 to 6,000, 2,000 to 6,000, 2,500 to 6,000,3,000 to 6,000, 3,500 to 6,000, 4,000 to 6,000, 4,500 to 6,000, 5,000 to 6,000, 5,500 to 6,000,1.500 to 5,500, 2,000 to 5,500, 2,500 to 5,500, 3,000 to 5,500, 3,500 to 5,500, 4,000 to 5,500,4.500 to 5,500, 5,000 to 5,500, 1,500 to 5,000, 2,000 to 5,000, 2,500 to 5,000, 3,000 to 5,000,3.500 to 5,000, 4,000 to 5,000, 4,500 to 5,000, 1,500 to 4,500, 2,000 to 4,500, 2,500 to 4,500,3,000 to 4,500, 3,500 to 4,500, 4,000 to 4,500, 1,500 to 4,000, 2,000 to 4,000, 2,500 to 4,000,3,000 to 4,000, 3,500 to 4,000, 1,500 to 3,500, 2,000 to 3,500, 2,500 to 3,500, 3,000 to 3,500,1.500 to 3,000, 2,000 to 3,000, 2,500 to 3,000, 1,500 to 2,500, 2,000 to 2,500, and 1,500 to 2,000 daltons.Hydrophobic components

[0162] In some embodiments, the lipid nanoparticles can also include another type of lipids, referred to as “hydrophobic components” in the present disclosure. The hydrophobic component is defined as a component, which, during the lipid nanoparticle formation, is incorporated in the lipid bilayer due to its hydrophobic nature. In some embodiments the hydrophobic component may be selected from the group consisting of cardiolipin, squalene,vitamin A and derivatives thereof, 0-carotene, withaferin A and a-tocopherol. In some embodiments the hydrophobic component may be selected from the group consisting of cardiolipin, squalene, vitamin A, retinol, 0-carotene, withaferin A and a-tocopherol.III. Lipid nanoparticlesGeneralities

[0163] In general, lipid nanoparticles can be characterized as small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-lipid nanoparticle environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space. Lipid nanoparticle membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers. In some embodiments, lipid nanoparticles may comprise a cargo or a payload into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.

[0164] The present disclosure also provides lipid nanoparticle comprising a cargo or payload. As used herein, the term “cargo” or “payload” can refer to one or more molecules or structures encompassed in a lipid nanoparticle for delivery to or into a cell or tissue. Non-limiting examples of cargo can include a nucleic acid, a polypeptide, peptide, protein, a liposome, a label, a tag, a small chemical molecule, a large biological molecule, and any combinations or fragments thereof. In the originator constructs and benchmark constructs, the region of the construct which comprises or encodes the cargo or payload is referred to as the “cargo region” or the “payload region”.

[0165] In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid of the present disclosure discussed in Section II, such as the ionizable lipid of general Formula (I), or more particularly, the compounds in Table 1. Not willing to be bound to any theory, the diester bonds of the lipids provide biodegradability and biocompatibility, such ester bonds are stable at physiological pH, but can be enzymatically hydrolyzed within tissues and cells. The length of the Rl, R2, R3 and R4 groups in Formula (I) can also be adjusted to reach the desired zeta potential, particle size or membrane rigidity.

[0166] In some embodiments, the lipid nanoparticle may comprise any lipid described in the disclosure. In some embodiments, the lipid may be any cationic lipid described in the disclosure. In some embodiments, the lipid nanoparticle may comprise neutral lipids. In some embodiments, the neutral lipid may be a phospholipid, or a derivative thereof. In some embodiments, the lipid may be any phospholipid described in the disclosure. In someembodiments, the lipid may be any cholesterol derivative described in the disclosure. In some embodiments, a polymer (e.g., PEG) may be conjugated to at least one lipid. In some embodiments, a PEG-lipid may be used in the lipid nanoparticle and can be any PEG-lipid conjugate described in the disclosure.

[0167] In some embodiments, the lipid nanoparticles can be characterized by their shape. In some embodiments, the lipid nanoparticles are essentially spherical. In some embodiments, the lipid nanoparticles are essentially rod-shaped (i.e., cylindrical). In some embodiments, the lipid nanoparticles are essentially disk shaped.

[0168] In some embodiments, the term “nanoparticle” as used herein refers to any particle ranging in size from 10-1000 nm. The nanoparticle may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250,255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345,350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440,445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535,540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630,635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725,730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820,825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915,920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 nm.

[0169] In some embodiments, a population of lipid nanoparticles, such as those resulting from the same formulation, may be characterized by measuring the uniformity of size, shape, or mass of the particles in the population, uniformity may be expressed in some embodiments as the polydispersity index (PI) of the population. In some embodiments uniformity may be expressed in some embodiments as the disparity (D) of the population. The terms “poly dispersity index” and “disparity” are understood herein to be equivalent and may be used interchangeably. In some embodiments, a population of lipid nanoparticles resulting from a given formulation can have a PI of between about 0.1 and 1. In some embodiments, a population of lipid nanoparticles resulting from a giving formulation can have a PI of less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1. In some embodiments, a population of lipid nanoparticles resulting from a given formulation can have a PI ofbetween about 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.4, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 1, 0.6 to 0.8, and 0.8 to 1. In some embodiments, the lipid nanoparticle may have PI ranging between about 0.01to 0.3, 0.02-0.3, 0.03-0.3, 0.04-0.3, 0.05-0.3, 0.06-0.3, 0.07-0.3, 0.08-0.3, 0.09-0.3, 0. 1-0.3, 0.11-0.3, 0.12-0.3, 0.13-0.3, 0.14-0.3, 0.15-0.3, 0.16-0.3, 0.17-0.3, 0.18-0.3, 0.19-0.3, 0.2-0.3, 0.21-0.3, 0.22-0.3, 0.23-0.3, 0.24-0.3, 0.25-0.3, 0.26-0.3, 0.27-0.3, 0.28-0.3, 0.29-0.3, 0.01 to 0.25, 0.02-0.25, 0.03-0.25, 0.04-0.25, 0.05-0.25, 0.06-0.25, 0.07-0.25, 0.08-0.25, 0.09-0.25, 0.1-0.25, 0.11-0.25, 0.12-0.25, 0.13-0.25, 0.14-0.25, 0.15-0.25, 0.16-0.25, 0.17-0.25, 0.18- 0.25, 0.19-0.25, 0.2-0.25, 0.21-0.25, 0.22-0.25, 0.23-0.25, 0.24-0.25, 0.01 to 0.2, 0.02-0.2, 0.03-0.2, 0.04-0.2, 0.05-0.2, 0.06-0.2, 0.07-0.2, 0.08-0.2, 0.09-0.2, 0.1-0.2, 0.11-0.2, 0.12-0.2, 0.13-0.2, 0.14-0.2, 0.15-0.2, 0.16-0.2, 0.17-0.2, 0.18-0.2, 0.19-0.2, 0.01 to 0.15, 0.02-0.15, 0.03-0.15, 0.04-0.15, 0.05-0.15, 0.06-0.15, 0.07-0.15, 0.08-0.15, 0.09-0.15, 0.1-0.15, 0.11- 0.15, 0.12-0.15, 0.13-0.15, 0.14-0.15, 0.01 to 0.1, 0.02-0.1, 0.03-0.1, 0.04-0.1, 0.05-0.1, 0.06- 0.1, 0.07-0.1, 0.08-0.1, 0.09-0.1, 0.01 to 0.05, 0.02-0.0.5, 0.03-0.0.5, or 0.04-0.05.

[0170] In some embodiments, the total mole percentage of the lipid(s) in the LNP is between about 10% to about 95%, such as between about 10% to about 20%, between about 21% to about 30%, between about 31% to about 40%, between about 41% to about 50%, between about 51% to about 60%, between about 61% to about 70%, between about 71% to about 80%, between about 81% to about 90%, or between about 91% to about 95%.Formulations

[0171] In some embodiments, the lipids of the present disclosure, including at least the ionizable lipids disclosed herein, may be incorporated into lipid nanoparticles (LNPs). In some embodiments a lipid nanoparticle may be comprised of at least one ionizable lipid, at least one neutral lipid, at least one helper lipid, at least one polymer-conjugated lipid, or any combination thereof, wherein the neutral lipid, helper lipid, polymer-conjugated lipid are as defined herein. In some embodiments, the LNP may be comprised of at least one ionizable lipid, at least one neutral lipid, and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid, at least one neutral lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid, at least one helper lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one neutral lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid and at least one polymer-conjugated lipid. In someembodiments, the LNP may be comprised of at least one helper lipid and at least one polymer- conjugated lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid. In some embodiments, a LNP may be comprised of a helper lipid. In some embodiments, the LNP may be comprised of a polymer-conjugated lipid. In some embodiments a lipid nanoparticle may be comprised of at least one ionizable lipid, at least one neutral lipid, at least one helper lipid, at least one polymer-conjugated lipid; and at least one hydrophobic component, wherein the ionizable lipid, neutral lipid, helper lipid, polymer-conjugated lipid and hydrophobic component are as defined herein.

[0172] In some embodiments, the lipid nanoparticle can further comprise an adjuvant, a cell targeting component, a fat-soluble vitamin, an immunomodulating substance or a component that promotes absorption of drugs.

[0173] In some embodiments, the adjuvant can be squalene, the cell targeting component can be cardiolipin, the fat-soluble vitamin can be vitamin A or E, the immunomodulating substance can be withaferin and the component that promote absorption of drugs can be caffeine.

[0174] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, can be from 0.1 to 100 mol%. In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is between 10%-95%, such as between about 10% to about 20%, between about 21% to about 30%, between about 31% to about 40%, between about 41% to about 50%, between about 51% to about 60%, between about 61% to about 70%, between about 71% to about 80%, between about 81% to about 90%, or between about 91% to about 95%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 40 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 20 to 60 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 50 to 85 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of less than about 20 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of more than about 60 mol%or about 85 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises an ionizable lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

[0175] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is from about 30% to 100%, from about 40% to 100%, from about 30% to about 90%, from about 30% to about 85%, from about 30% to about 80%, from about 30% to about 75%, from about 30% to about 70%, from about 30% to about 65%, or from about 30% to about 60%.

[0176] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II),(12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, or from about 40% to about 60%. In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is from about 40 to about 47 mol % of the ionizable lipid, preferably from about 40 to about 45 mol % of the ionizable lipid.

[0177] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12),(13), (14), (15), (16), (17) or (18) or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 50% to about 90%, from about 50% to about 85%, from about 50% to about 80%, from about 50% to about 75%, from about 50% to about 70%, from about 50% to about 65%, or from about 50% to about 60%.

[0178] In some embodiments, the total mole percentage of the ionizable lipid, such as thelipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) or the compounds in Table 1, in the lipid nanoparticle, is about 55% to about 90%, from about 55% to about 85%, from about 55% to about 80%, from about 55% to about 75%, from about 55% to about 70%, from about 55% to about 65%, or from about 55% to about 60%.

[0179] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 60% to about 90%, from about 60% to about 85%, from about 60% to about 80%, from about 60% to about 75%, from about 60% to about 70%, or from about 60% to about 65%.

[0180] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II),(12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 65% to about 90%, from about 65% to about 85%, from about 65% to about 80%, from about 65% to about 75%, or from about 65% to about 70%.

[0181] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12),(13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 70% to about 90%, from about 70% to about 85%, from about 70% to about 80%, or from about 70% to about 75%.

[0182] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 75% to about 90%, from about 75% to about 85%, or from about 75% to about 80%.

[0183] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib),(lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 80% to about 90%, or from about 80% to about 85%.

[0184] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 1, in the lipid nanoparticle, is about 85% to about 90%.

[0185] In some embodiments, the ionizable lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0186] In some embodiments, transfer vehicle variability between lots can be less than 15%, less than 10% or less than 5%.

[0187] In some embodiments, when the lipid nanoparticle comprises at least one neutral lipid, the neutral lipid can be present in the lipid nanoparticle in an amount of about 0.1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 5 to 35 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 5 to 25 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of less than about 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of more than about 25 mol% or about 35 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiment, the lipid nanoparticle comprises at least one neutral lipid in an amount of at most bout 10 mol%.

[0188] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 5% to about 20%, from about 5% to about 18%, from about 5% to about 16%, from about 5% to about 14%, from about 5% to about 12%, from about 5% to about 10%, or from about 5% to about 8%.

[0189] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 7% to about 20%, from about 7% to about 18%, from about 7% to about 16%, from about 7% to about 14%, from about 7% to about 12%, from about 7% to about 10%, or from about 7% to about 8%.

[0190] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 9% to about 20%, from about 9% to about 18%, from about 9% to about16%, from about 9% to about 14%, from about 9% to about 12%, or from about 9% to about 10%.

[0191] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 11% to about 20%, from about 11% to about 18%, from about 11% to about 16%, from about 11% to about 14%, or from about 11% to about 12%.

[0192] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 13% to about 20%, from about 13% to about 18%, from about 13% to about 16%, or from about 13% to about 14%.

[0193] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 15% to about 20%, or from about 17% to about 18%.

[0194] In some embodiments, the neutral lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0195] In some embodiments, when the lipid nanoparticle comprises at least one helper lipid, the helper lipid can be present in the lipid nanoparticle in an amount of about 0. 1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of at most 50 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 20 to 45 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 25 to 55 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of less than about 20 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of more than about 45 mol% or about 55 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

[0196] In some embodiments, the helper lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0197] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%, or from about 20% to about 25%.

[0198] In some embodiments, the total mole percentage of the helper lipid in the lipidnanoparticle is about 25% to about 50%, from about 25% to about 45%, from about 25% to about 40%, from about 25% to about 35%, or from about 25% to about 30%.

[0199] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 30% to about 50%, from about 30% to about 45%, from about 30% to about 40%, or from about 30% to about 35%.

[0200] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 35% to about 50%, from about 35% to about 45%, or from about 35% to about 40%. In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 40% to about 50%, or from about 40% to about 45%. In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 45% to about 50%.

[0201] In some embodiments, when the lipid nanoparticle comprises at least one polymer- conjugated lipid, the polymer-conjugated lipid can be present in the lipid nanoparticle in an amount of about 0.1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 0.5 to 15 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 15 to 40 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of less than about 0.1 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of at most about 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

[0202] In some embodiments, the polymer-conjugated lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0203] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 0.1% to about 10%, from about 0.1% to about 9%, from about 0.1% to about 8%, from about 0.1% to about 7%, from about 0.1% to about 6%, from about 0. 1% to about 5%, or from about 0. 1% to about 4%, from about 0. 1% to about 3%, from about 0. 1% to about 2%, or from about 0. 1% to about 1%.

[0204] In some embodiments, the total mole percentage of the polymer-conjugated lipid inthe lipid nanoparticle is about 0.5% to about 10%, from about 0.5% to about 9%, from about 0.5% to about 8%, from about 0.5% to about 7%, from about 0.5% to about 6%, from about 0.5% to about 5%, or from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, or from about 0.5% to about 1%.

[0205] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 1% to about 10%, from about 1% to about 9%, from about 1% to about 8%, from about 1% to about 7%, from about 1% to about 6%, from about 1% to about 5%, from about 1% to about 4%, from about 1% to about 3%, or from about 1% to about 2%.

[0206] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 2% to about 10%, from about 2% to about 9%, from about 2% to about 8%, from about 2% to about 7%, from about 2% to about 6%, from about 2% to about 5%, from about 2% to about 4%, or from about 2% to about 3%.

[0207] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 3% to about 10%, from about 3% to about 9%, from about 3% to about 8%, from about 3% to about 7%, from about 3% to about 6%, from about 3% to about 5%, or from about 3% to about 4%.

[0208] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 4% to about 10%, from about 4% to about 9%, from about 4% to about 8%, from about 4% to about 7%, from about 4% to about 6%, or from about 4% to about 5%.

[0209] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 5% to about 10%, from about 5% to about 9%, from about 5% to about 8%, from about 5% to about 7%, or from about 5% to about 6%.

[0210] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 6% to about 10%, from about 6% to about 9%, from about 6% to about 8%, or from about 6% to about 7%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 7% to about 10%, from about 7% to about 9%, or from about 7% to about 8%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 8% to about 10%, or from about 8% to about 9%. In some embodiments, the total mole percentage of the polymer- conjugated lipid in the lipid nanoparticle is about 8% to about 10%, or from about 8% to about 9%. In some embodiments, the total mole percentage of the PEG-lipid in the lipid nanoparticle is about 9% to about 10%.

[0211] In some embodiments, the lipid nanoparticle can comprise a hydrophobic component.In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle at most about 20%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, or from about 1% to about 5%. In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 3% to about 20%, from about 3% to about 15%, from about 3% to about 10%, or from about 3% to about 5%. In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is at most about 5 %.

[0212] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 5% to about 20%, from about 5% to about 15%, or from about 5% to about 10%.

[0213] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 7% to about 20%, from about 7% to about 15%, or from about 7% to about 10%.

[0214] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 9% to about 20%, from about 9% to about 15%, or from about 9% to about 10%.

[0215] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 11% to about 20%, or from about 11% to about 15%.

[0216] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 13% to about 20%, or from about 13% to about 15%.

[0217] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 15% to about 20%.

[0218] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 17% to about 20%.

[0219] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 19% to about 20%.

[0220] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-50 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0221] The at least one ionizable lipid is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie). (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof.

[0222] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0223] In some embodiments, the lipid nanoparticle is comprised of about 35-45 mol% of at least one ionizable lipid, about 25-35 mol% of at least one neutral lipid (e.g., a phospholipid), about 20-30 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0224] In some embodiments, the lipid nanoparticle is comprised of about 45-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., a phospholipid), about 25-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0225] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid, about 5-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 35-45 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-3 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0226] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 15-50 mol% of at least one helper lipid (e.g., cholesterol), and about 0.01-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0227] In some embodiments, the lipid nanoparticle is comprised of about 10-75 mol% of at least one ionizable lipid, about 0.5-50 mol% of at least one neutral lipid (e.g., a phospholipid), about 5-60 mol% of at least one helper lipid (e.g., cholesterol), and about 0.1-20 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0228] In some embodiments, the lipid nanoparticle is comprised of about 50-65 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0229] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0230] In some embodiments, the lipid nanoparticle is comprised of about 25-75 mol% of at least one ionizable lipid, about 0.1-15 mol% of at least one neutral lipid (e.g., a phospholipid),about 5-50 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-20 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0231] In some embodiments, the lipid nanoparticle is comprised of about 50-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., a phospholipid), about 25-35 mol% of at least one helper lipid (e.g., cholesterol), and about 5-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0232] In some embodiments, the lipid nanoparticle is comprised of about 20-60 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 25-55 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-15 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0233] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and / or a-tocopherol).

[0234] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and / or a-tocopherol).

[0235] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and / or a-tocopherol).

[0236] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and / or a-tocopherol).

[0237] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and / or a-tocopherol).

[0238] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and / or a-tocopherol).

[0239] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and / or a-tocopherol).

[0240] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and / or a- tocopherol).

[0241] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-25 mol% of at least one neutral (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, 0-carotene, withaferin A and / or a- tocopherol).

[0242] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of atleast one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG-lipid described herein and about 0-5 mol% of squalene, cardiolipin, withaferin A, vitamin A, retinol, [3-carotene, and / or a-tocopherol.

[0243] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1- 4 mol% of PEG-DMG and about 0-5 mol% of squalene, cardiolipin, vitamin A, retinol, f>- carotene, withaferin A and / or a-tocopherol.

[0244] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-25 mol% of at least one neutral (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, f>- carotene, withaferin A and / or a-tocopherol).

[0245] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG- lipid described herein and about 0-5 mol% of squalene, cardiolipin, withaferin A, vitamin A, retinol, [3-carotene and / or a-tocopherol.

[0246] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid selected from Table 1 herein, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1-4 mol% of PEG-DMG and about 0-5 mol% of squalene, cardiolipin, vitamin A, retinol, [3-carotene, withaferin A and / or a-tocopherol.

[0247] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 100 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I),(Il), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b) (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from 0 to about 10 mol % of the neutral lipid; from 0 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0248] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 99 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from 0 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0249] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0250] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer- conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0251] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or apharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from about 1 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0252] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from about 1 to about 5 mol % of the polymer-conjugated lipid; and from about 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0253] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of the neutral lipid; from about 30 to about 50 mol % of the helper lipid; from about 1 to about 4 mol % of the polymer-conjugated lipid; and from about 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0254] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of the neutral lipid; from about 30 to about 45 mol % of the helper lipid; from about 1 to about 4 mol % of the polymer-conjugated lipid; and from 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0255] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 1 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of a phospholipid as the neutral lipid; from about 30 to about 50 mol % of a sterol as the helper lipid; from about 1to about 4 mol % of a PEG-lipid as the polymer-conjugated lipid; and from 0. 1 to about 5 mol % of squalene, cardiolipin, a-tocopherol, withaferin A, vitamin A, or a combination thereof as the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.Encapsulation

[0256] In some embodiments, the lipid nanoparticle may fully or partially encapsulate a cargo or payload. In some embodiments, essentially 0% of the cargo present in the final formulation is exposed to the environment outside of the lipid nanoparticle (i.e., the cargo is fully encapsulated. In some embodiments, the cargo is associated with the lipid nanoparticle but is at least partially exposed to the environment outside of the lipid nanoparticle. In some embodiments, the lipid nanoparticle may be characterized by the % of the cargo not exposed to the environment outside of the lipid nanoparticle, e.g., the encapsulation efficiency. For the sake of clarity, an encapsulation efficiency of about 100% refers to a lipid nanoparticle formulation where essentially all the cargo is fully encapsulated by the lipid nanoparticle, while an encapsulation rate of about 0% refers to a lipid nanoparticle where essential none of the cargo is encapsulated in the lipid nanoparticle, such as with a lipid nanoparticle where the cargo is bound to the external surface of the lipid nanoparticle. On some embodiments, a lipid nanoparticle may have an encapsulation efficiency of less than about 100%, less than about 95%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15% less than about 10%, or less than 5%. In some embodiments, an lipid nanoparticle may have an encapsulation efficiency of between about 90 to 100%, 80 to 100%, 70 to 100%, 60 to 100%, 50 to 100%, 40 to 100%, 30 to 100%, 20 to 100%, 10 to 100%, 80 to 90%, 70 to 90%, 60 to 90%, 50 to 90%, 40 to 90%, 30 to 90%, 20 to 90%, 10 to 90%, 70 to 80%, 60 to 80%, 50 to 80%, 40 to 80%, 30 to 80%, 20 to 80%, 10 to 80%, 60 to 70%, 50 to 70%, 40 to 70%, 30 to 70%, 20 to 70%, 10 to 70%, 40 to 50%, 30 to 50%, 20 to 50%, 10 to 50%, 30 to 40%, 20 to 40%, 10 to 40%, 20 to 30%, 10 to 30%, and 10 to 20%.

[0257] In some embodiments, the weight ratio of the lipid nanoparticle (including all the lipids) and the cargo or payload is between about 100: 1 to about 1: 1, such as between about 100: 1 to about 90: 1, between about 89: 1 to about 80: 1, between about 79: 1 to about 70: 1, between about 69: 1 to about 60: 1, between about 59: 1 to about 50: 1, between about 49: 1 to about 40: 1, between about 39: 1 to about 30: 1, between about 29: 1 to about 20: 1, between about19:1 to about 10:1, and between about 9:1 to about 1:1.

[0258] In some embodiments, the lipid nanoparticle further comprises an originator construct or a benchmark construct with at least one cargo or payload. The cargo or payload can be a small molecule, an antibody, a polynucleotide or a polypeptide. The cargo or payload can comprise at least one nucleic acid, such as mRNA. The cargo or payload may be any DNA, plasmid, RNA or polypeptide described herein.

[0259] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a coding RNA.

[0260] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a non-coding RNA.

[0261] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is an oRNA.

[0262] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is an mRNA.

[0263] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a personalized vaccine mRNA. Non-limiting example includes 3-GP.

[0264] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a Covid- 19 vaccine mRNA. Non-limiting example includes PTX-CB and coronavirus spike protein.

[0265] In some embodiments, the at least one RNA compound is comprised of a functional RNA where the RNA results in at least one change in a cell, tissue, organ and / or organism. Said changes in state may include, but are not limited to, altering the expression level of a polypeptide, altering the translation level of a nucleic acid, altering the expression level of a nucleic acid, altering the amount of a polypeptide present in a cell, tissue, organ and / or organism, changing a genetic sequence of a cell, tissue, organ and / or organism, adding nucleic acids to a target genome, subtracting nucleic acids from a target genome, altering physiological activity in a cell, tissue, organ and / or organism or any combination thereof.

[0266] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is DNA.

[0267] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are DNA. The DNA may be the same DNA or different DNA. As a non-limiting example, the DNA are the same. As a non-limiting example, the DNA are different. As a non-limiting example, the DNA are different but encodethe same payload or cargo. As a non-limiting example, the DNA are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0268] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are DNA. The DNA may be the same DNA or different DNA. As a non-limiting example, the DNA are the same. As a non-limiting example, the DNA are different. As a non-limiting example, two DNA are the same and one is different. As a non-limiting example, the first DNA is different from the second and third DNA. As a non-limiting example, the first DNA, second DNA and third DNA are all different. As a non-limiting example, the first DNA is different from the second and third DNA, but they all encode the same payload or cargo. As a non-limiting example, the first DNA is different from the second and third DNA but the second and third DNA encode the same payload or cargo.

[0269] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is a polypeptide.

[0270] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are polypeptides. The polypeptides may be the same polypeptide or different polypeptides. As a non-limiting example, the polypeptides are the same. As a non-limiting example, the polypeptides are different. As a non- limiting example, the polypeptides are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0271] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are polypeptides. The polypeptides may be the same polypeptide or different polypeptides. As a non-limiting example, the polypeptides are the same. As a non-limiting example, the polypeptides are different. As a non- limiting example, two polypeptides are the same and one is different. As a non-limiting example, the first polypeptide is different from the second and third polypeptides. As a non- limiting example, the first polypeptide, second polypeptide and third polypeptide are all different. As anon-limiting example, the first polypeptide is different from the second and third polypeptides, but they all encode the same payload or cargo. As a non-limiting example, the first polypeptide is different from the second and third polypeptides but the second and third polypeptides encode the same payload or cargo.

[0272] In some embodiments, the lipid nanoparticle comprises an originator construct or abenchmark construct with at least one cargo or payload which is a peptide.

[0273] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are peptides. The peptides may be the same peptide or different peptides. As a non-limiting example, the peptides are the same. As a non-limiting example, the peptides are different. As a non-limiting example, the peptides are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0274] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are peptides. The peptides may be the same peptide or different peptides. As a non-limiting example, the peptides are the same. As a non-limiting example, the peptides are different. As a non-limiting example, two peptides are the same and one is different. As a non-limiting example, the first peptide is different from the second and third peptides. As a non-limiting example, the first peptide, second peptide and third peptide are all different. As a non-limiting example, the first peptide is different from the second and third peptides, but they all encode the same payload or cargo. As a non-limiting example, the first peptide is different from the second and third peptides but the second and third peptide encode the same payload or cargo.

[0275] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is plasmid.

[0276] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with at least one cargo or payload which is RNA.

[0277] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads which are RNAs. The RNAs may be the same RNA or different RNAs. As a non-limiting example, the RNAs are the same. As a non-limiting example, the RNAs are different. As anon-limiting example, the RNAs are different but encode the same payload or cargo. As a non-limiting example, the RNAs are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0278] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with three cargos or payloads which are RNAs. The RNAs may be the same RNA or different RNAs. As a non-limiting example, the RNAs are the same. As a non- limiting example, the RNAs are different. As a non-limiting example, two RNAs are the sameand one is different. As a non-limiting example, the first RNA is different from the second and third RNAs. As a non-limiting example, the first RNA, second RNA and third RNA are all different. As a non-limiting example, the first RNA is different from the second and third RNAs, but they all encode the same payload or cargo. As a non-limiting example, the first RNA is different from the second and third RNA but the second and third RNAs encode the same payload or cargo.

[0279] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is RNA, and one is DNA. The RNA and DNA may encode the same peptide or polypeptide or may encode different peptides or polypeptides. As a non-limiting example, the RNA and DNA may encode the same peptide or polypeptide. As a non-limiting example, the RNA and DNA may encode different peptides or polypeptides. As a non-limiting example, the RNA and DNA are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0280] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is RNA, and one is a peptide. The RNA may encode the same peptide as the peptide cargo / payload the RNA may encode a different peptide. As a non-limiting example, the RNA encodes the same peptide. As a non- limiting example, the RNA encodes a different peptide. As a non-limiting example, the RNA and peptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0281] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is RNA, and one is a polypeptide. The RNA may encode the same polypeptide as the polypeptide cargo / payload the RNA may encode a different polypeptide. As a non-limiting example, the RNA encodes the same polypeptide. As a non-limiting example, the RNA encodes a different polypeptide. As a non- limiting example, the RNA and polypeptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0282] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is DNA, and one is a peptide. The DNA may encode the same peptide as the peptide cargo / payload the DNA may encode adifferent peptide. As a non-limiting example, the DNA encodes the same peptide. As a non- limiting example, the DNA encodes a different peptide. As a non-limiting example, the DNA and peptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).

[0283] In some embodiments, the lipid nanoparticle comprises an originator construct or a benchmark construct with two cargos or payloads where one is DNA, and one is a polypeptide. The DNA may encode the same polypeptide as the polypeptide cargo / payload the DNA may encode a different polypeptide. As a non-limiting example, the DNA encodes the same polypeptide. As a non-limiting example, the DNA encodes a different polypeptide. As a non- limiting example, the DNA and polypeptide are different pieces of a larger payload or cargo (e.g., heavy chain or light chain of an antibody) that can come together using natural systems or synthetic methods known in the art to produce a functional polypeptide (e.g., antibody).Methods of preparation

[0284] The lipid nanoparticles described herein may be formed using techniques known in the art. As a non-limiting example, an organic solution containing the lipids is mixed together with an acidic aqueous solution containing the originator construct or benchmark construct in a microfluidic channel resulting in the formation of targeting system (lipid nanoparticle and the benchmark construct).

[0285] In some embodiments, a lipid nanoparticle formulation may be prepared by the methods described in International Publication No W02008103276, the content of which is herein incorporated by reference in its entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. W02019131770, the content of which is herein incorporated by reference in its entirety.

[0286] In some embodiments, a lipid nanoparticle formulation may be prepared by the methods described in International Publication No. WO2020237227, the content of which is herein incorporated by reference in its entirety.

[0287] In some embodiments, a lipid nanoparticle formulation may be prepared using the methodologies and devices described in US20240181406A1, the content of which is herein incorporated by reference in its entirety.

[0288] In further embodiments, a lipid nanoparticle formulation may be prepared using the FDmiX mixing systems developed by FDX Fluid Dynamix GmbH (Seehttps: / / www.fdx.de / en / fdmix / . the content of which is herein incorporated by reference in its entirety).

[0289] In some embodiments, a lipid nanoparticle formulation may be prepared by a so-called “Point-of-Care” mixing method, which consists in preparing separately a solution of a cargo, such as mRNA for instance, that can be frozen or lyophilized for storage, and a solution of lipid. Mixing the lipid solution and the mRNA solution via transferring the mRNA to a sealed vial containing the lipid followed by vigorous shaking of the vial provides mRNA encapsulated in LNPs ready for using / dosing. Point-of-Care mixing can produce mRNA-LNPs with comparable quality and efficacy to mRNA-LNPs obtained with conventional methods (conventional mRNA-LNPs). Manufacturing of conventional mRNA-LNPs is a sensitive process and usually involves specialized equipment and analytical techniques. The mRNA- LNPs are sensitive to minor changes in the manufacturing process. Long-term storage of mRNA-LNPs requires cryo-storage facilities. Still long-term stability of mRNA-LNPs may not be ensured. On the other hand, in Point-of-Care mixing, the LNPs are not manufactured ahead of time of administration. The mRNA and lipid solution can be manufactured, filled and released using conventional small-molecule pharmaceutical manufacturing units. Both the mRNA and lipid solution are stable under frozen conditions.IV. Cargo and Payloads

[0290] The present disclosure also provides compositions or constructs comprising the lipid nanoparticles of the present disclosure, wherein the lipid nanoparticles may comprise, encode or be conjugated to a cargo or payload to produce the constructs. In some embodiments, the cargo or payload is or encodes a biologically active molecule such as, but not limited to a therapeutic protein. As used herein, the term “biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In some embodiments, the cargo or payload is or encodes one or more prophylactically- or therapeutically-active proteins, polypeptides, or other factors. As a non-limiting example, the cargo or payload may be or encode an agent that enhances tumor killing activity such as, but not limited to, TRAIL or tumor necrosis factor (TNF), in a cancer. As another non-limiting example, the cargo or payload may be or encode an agent suitable for the treatment of conditions such as muscular dystrophy (e.g., cargo or payload is or encodes Dystrophin), cardiovascular disease (e.g., cargo or payload is or encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (e.g., cargo or payloadis or encodes NGF, BDNF, GDNF, NT-3, etc.), chronic pain (e.g., cargo or payload is or encodes GlyRal), an enkephalin, or a glutamate decarboxylase (e.g., cargo or payload is or encodes GAD65, GAD67, or another isoform), lung disease (e.g., cargo or payload is or encodes CFTR), hemophilia (e.g., cargo or pay load is or encodes Factor VIII or Factor IX), neoplasia (e.g., cargo or payload is or encodes PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, Notchl, Notch2, Notch3, Notch4, AKT, AKT2, AKT3, HIF, HI Fla, HIF3a, Met, HRG, Bcl2, PPARalpha, PPAR gamma, WT1 (Wilms Tumor), FGF Receptor Family members (5 members: 1, 2, 3, 4, 5), CDKN2a, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (Androgen Receptor), TSG101, IGF, IGF Receptor, Igfl (4 variants), Igf2 (3 variants), Igfl Receptor, Igf2 Receptor, Bax, Bcl2, caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12), Kras, Ape), age-related macular degeneration (e.g., cargo or payload is or encodes Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin D, Vldlr), schizophrenia (e.g. Neuregulin (Nrgl), Erb4 (receptor for Neuregulin), Complexin-1 (Cplxl), Tphl Tryptophan hydroxylase, Tph2 Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HIT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAO A, DTNBPI, Dao (Daol)), trinucleotide repeat disorders (e.g., HTT (Huntington's Dx), SBMA / SMAXI / AR (Kennedy's Dx), FXN / X25 (Friedrich's Ataxia), ATX3 (Machado-Joseph's Dx), ATXNI and ATXN2 (spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atnl (DRPLA Dx), CBP (Creb-BP- global instability), VLDLR (Alzheimer's), Atxn7, AtxnlO), fragile X syndrome (e.g., cargo or payload is or encodes FMR2, FXRI, FXR2, mGLUR5), secretase related disorders (e.g., cargo or payload is or encodes APH-1 (alpha and beta), Presenilin (Psenl), nicastrin (Ncstn), PEN- 2), ALS (e.g., cargo or payload is or encodes SOD1, ALS2, STEX, FUS, TARD BP, VEGF (VEGF-a, VEGF-b, VEGF-c)), autism (e.g., cargo or payload is or encodes Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1), Alzheimer's disease (e.g., cargo or payload is or encodes El, CHIP, UCH, UBB, Tau, LRP, PICALM, Clusterin, PSI, SORL1, CR1, Vldlr, Ubal, Uba3, CHIP28 (Aqpl, Aquaporin 1), Uchll, Uchl3, APP), inflammation (e.g., cargo or payload is or encodes IL-10, IL-1 (IL-Ia, IL-Ib), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL- 171), 11-23, Cx3crl, ptpn22, TNFa, NOD2 / CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cll), Parkinson's Disease (e.g., x-Synuclein, DJ-1, LRRK2, Parkin, PINK1), blood and coagulation disorders, such as, e.g., anemia, bare lymphocyte syndrome, bleeding disorders, hemophagocytic lymphohistiocytosis disorders, hemophilia A, hemophilia B, hemorrhagic disorders, leukocyte deficiencies and disorders, sickle cell anemia, and thalassemia (e.g., cargo or payload is or encodes CRAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSNI, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7,ABC7, ASAT, TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5, TBXA2R, P2RX1, P2X1, HF1, CFH, HUS, MCFD2, FANCA, FAC A, FA1, FA, FA A, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCDI, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BR1PI, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596, PRF1, HPLH2, UNC13D, MUNC13- 4, HPLH3, HLH3, FHL3, F8, FSC, PI, ATT, F5, ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4, HBB, HBA2, HBB, HBD, LCRB, HBA1), B-cell non-Hodgkin lymphoma or leukemia (e.g., cargo or payload is or encodes BCL7A, BCL7, ALI, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1AI, 1KI, LYF1, H0XD4, H0X4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AFIO, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPMI, NUP214, D9S46E, CAN, CAIN, RUNXI, CBFA2, AML1, WHS Cl LI, NSD3, FLT3, AF1Q, NPMI, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF1Q, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPNII, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABLI, NQO1, DIA4, NM0R1, NUP214, D9S46E, CAN, CAIN), inflammation and immune related diseases and disorders (e.g., cargo or payload is or encodes KIR3DL1, NKAT3, NKB1, AMB11, K1R3DS1, IFNG, CXCL12, TNFRSF6, APT1, FAS, CD95, ALPS1A, IL2RG, SCIDX1, SCIDX, IMD4, CCL5, SCYA5, D17S136E, TCP228, IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSFS, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI), inflammation (e.g., cargo or payload is or encodes IL-10, IL-1 (IL-IA, IL-IB), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2 / CARD15 forlBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cII), JAK3, JAKL, DCLREIC, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDXI, SCIDX, IMD4), metabolic, liver, kidney and protein diseases and disorders (e.g., cargo or payload is or encodes TTR, PALB, APOA1, APP, AAA, CVAP, ADI, GSN, FGA, LYZ, TTR, PALB, KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988, CFTR, ABCC7, CF, MRP7, SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM, TCF1, HNF1A, M0DY3, SCOD1, SCO1, CTNNB1, PDGFRL, PDGRL, PRLTS, AX1NI, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5, UMOD, HNFJ, FJHN, MCKD2, ADMCKD2, PAH, PKU1, QDPR, DHPR, PTS, FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63), muscular / skeletal diseases and disorders(e.g., cargo or payload is or encodes DMD, BMD, MYF6, LMNA, LMN1, EMD2, FPLD, CMDIA, HGPS, LGMDIB, LMNA, LMNI, EMD2, FPLD, CMDIA, FSHMD1A, FSHD1A, FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDCIC, LGMD21, TTN, CMD1G, TMD, LGMD2J, P0MT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTMI, GL, TCIRG1, TIRC7, OC116, OPTB1, VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1), neurological and neuronal diseases and disorders (e.g., cargo or payload is or encodes SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c), APP, AAA, CVAP, ADI, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65LI, NOS3, PLAU, URK, ACE, DCPI, ACEI, MPO, PAC1PI, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3, Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR2, FXR1, FXR2, mGLUR5, HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJI, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2, MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16,MRX79, x-Synuclein, DJ-1, Neuregulin-1 (Nrgl), Erb4 (receptor for Neuregulin), Complexin-1 (Cplxl), Tphl Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), CONT, DRD (Drdla), SLC6A , DAO A, DTNBP1, Dao (Daol), APH-l(alpha and beta), Presenilin (Psenl), Nicastrin, (Ncstn), PEN-2, Nosl, Parpl, Natl, Nat2, HTT, SBMA / SMAX1 / AR, FXN / X25, ATX3, TXN, ATXN2, DMPK, Atrophin-1, Atnl, CBP, VLDLR, Atxn7, and AtxnlO), and ocular diseases and disorders (e.g., Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin-D, Vldlr, Ccr2, CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYAI, PAX6, AN2, MGDA, CRYBAI, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQPO, CRY AB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYAI, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1, APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, Ml SI, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD, KERA, CNA2, MYOC, TIGR,GLCIA, JO AG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1BI, GLC3A, OPA1, NTG, NPG, CYP1BI, GLC3A, CRB1, RP12, CRX, CORD2, CRD, RPGRIPI, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3, ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, and VMD2).

[0291] In some embodiments, the cargo or payload is or encodes a factor that can affect the differentiation of a cell. As a non-limiting example, the expression of one or more of Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant-negative p53, Nanog, Glisl, Lin28, TFIID, mir-302 / 367, or other miRNAs can cause the cell to become an induced pluripotent stem (iPS) cell.

[0292] In some embodiments, the cargo or payload is or encodes a factor for transdifferentiating cells. Non-limiting examples of factors include: one or more of GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mespl, SMARCD3 for cardiomyocytes; Ascii, Nurrl, LmxlA, Bm2, Myth, NeuroDl, FoxA2 for neural cells; and Hnf4a, Foxal, Foxa2 or Foxa3 for hepatic cells.Polypeptides, Proteins and Peptides

[0293] The lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a polypeptide, protein or peptide. As used herein, the term “polypeptide” generally refers to polymers of amino acids linked by peptide bonds and embraces “protein and “peptides.” Polypeptides for the present disclosure include all polypeptides, proteins and / or peptides known in the art. Non-limiting categories of polypeptides include antigens, antibodies, antibody fragments, cytokines, peptides, hormones, enzymes, oxidants, antioxidants, synthetic polypeptides, and chimeric polypeptides.

[0294] As used herein, the term “peptide” generally refers to shorter polypeptides of about 50 amino acids or less. Peptides with only two amino acids may be referred to as “dipeptides.” Peptides with only three amino acids may be referred to as “tripeptides.” Polypeptides generally refer to polypeptides with from about 4 to about 50 amino acids. Peptides may be obtained via any method known to those skilled in the art. In some embodiments, peptides may be expressed in culture. In some embodiments, peptides may be obtained via chemical synthesis (e.g., solid phase peptide synthesis).

[0295] In some embodiments, the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a simple protein which upon hydrolysis yields the amino acids and occasionally small carbohydrate compounds. Non-limiting examples of simple proteins include albumins, albuminoids, globulins, glutelins, histones and protamines.

[0296] In some embodiments, the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or pay load which is a conjugated protein which may be a simple protein associated with a non-protein. Non-limiting examples of conjugated proteins include, glycoproteins, hemoglobins, lecithoproteins, nucleoproteins, and phosphoproteins.

[0297] In some embodiments, the lipid nanoparticles of the present disclosure may comprise, encode or be conjugated to a cargo or payload which is a derived protein which is a protein that is derived from a simple or conjugated protein by chemical or physical means. Non-limiting examples of derived proteins include denatured proteins and peptides.

[0298] In some embodiments, the polypeptide, protein or peptide may be unmodified.

[0299] In some embodiments, the polypeptide, protein or peptide may be modified. Types of modifications include, but are not limited to, Phosphorylation, Glycosylation, Acetylation, Ubiquitylation / Sumoylation, Methylation, Palmitoylation, Quinone, Amidation, Myristoylation, Pyrrolidone carboxylic acid, Hydroxylation, Phosphopantetheine, Prenylation, GPI anchoring, Oxidation, ADP-ribosylation, Sulfation, S-nitrosylation, Citrullination, Nitration, Gamma-carboxyglutamic acid, Formylation, Hypusine, Topaquinone (TPQ), Bromination, Lysine topaquinone (LTQ), Tryptophan tryptophylquinone (TTQ), Iodination, and Cysteine tryptophylquinone (CTQ). In some aspects, the polypeptide, protein or peptide may be modified by a post-transcriptional modification which can affect its structure, subcellular localization, and / or function.

[0300] In some embodiments, the polypeptide, protein or peptide may be modified using phosphorylation. Phosphorylation, or the addition of a phosphate group to serine, threonine, or tyrosine residues, is one of most common forms of protein modification. Protein phosphorylation plays an important role in fine tuning the signal in the intracellular signaling cascades.

[0301] In some embodiments, the polypeptide, protein or peptide may be modified using ubiquitination which is the covalent attachment of ubiquitin to target proteins. Ubiquitination- mediated protein turnover has been shown to play a role in driving the cell cycle as well as in protein-degradation-independent intracellular signaling pathways.

[0302] In some embodiments, the polypeptide, protein or peptide may be modified using acetylation and methylation which can play a role in regulating gene expression. As a non- limiting example, the acetylation and methylation could mediate the formation of chromatin domains (e.g., euchromatin and heterochromatin) which could have an impact on mediating gene silencing.

[0303] In some embodiments, the polypeptide, protein or peptide may be modified usingglycosylation. Glycosylation is the atachment of one of a large number of glycan groups and is a modification that occurs in about half of all proteins and plays a role in biological processes including, but not limited to, embryonic development, cell division, and regulation of protein structure. The two main types of protein glycosylation are N-glycosylation and O- glycosylation. For N-glycosylation the glycan is atached to an asparagine and for O- glycosylation the glycan is atached to a serine or threonine.

[0304] In some embodiments, the polypeptide, protein or peptide may be modified using Sumoylation. Sumoylation is the addition of SUMOs (small ubiquitin-like modifiers) to proteins and is a post-translational modification similar to ubiquitination.Antibodies

[0305] As used herein, the term "antibody" is referred to in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g., “functional”). Antibodies are primarily amino acid based molecules which are monomeric or multimeric polypeptides which comprise at least one amino acid region derived from a known or parental antibody sequence and at least one amino acid region derived from a non-antibody sequence. The antibodies may comprise one or more modifications (including, but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.). For the purposes herein, an "antibody" may comprise a heavy and light variable domain as well as an Fc region.

[0306] The cargo or payload may comprise or may encode polypeptides that form one or more functional antibodies.

[0307] In some embodiments, the cargo or payload may comprise or may encode polypeptides that form or function as any antibody including, but not limited to, antibodies that are known in the art and / or antibodies that are commercially available which may be therapeutic, diagnostic, or for research purposes. Additionally, the cargo or payload may comprise or may encode fragments of such antibodies or antibodies such as, but not limited to, variable domains or complementarity determining regions (CDRs).

[0308] As used herein, the term "native antibody" refers to a usually heterotetrameric glycoprotein of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Genes encoding antibody heavy and light chains are known and segments making up each have been well characterized and described (Matsuda, F. et al., 1998.The Journal of Experimental Medicine. 188(11); 2151-62 and Li, A. et al., 2004. Blood. 103(12: 4602-9, the content of each of which are herein incorporated by reference in their entirety). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. As used herein, the term "light chain" refers to a component of an antibody from any vertebrate species assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.

[0309] As used herein, the term "variable domain" refers to specific antibody domains found on both the antibody heavy and light chains that differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. Variable domains comprise hypervariable regions. As used herein, the term "hypervariable region" refers to a region within a variable domain comprising amino acid residues responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining regions (CDRs) that become part of the antigen-binding site of the antibody. As used herein, the term “CDR” refers to a region of an antibody comprising a structure that is complimentary to its target antigen or epitope. Other portions of the variable domain, not interacting with the antigen, are referred to as framework (FW) regions. The antigen-binding site (also known as the antigen combining site or paratope) comprises the amino acid residues necessary to interact with a particular antigen. The exact residues making up the antigen-binding site are typically elucidated by co- crystallography with bound antigen, however computational assessments can also be used based on comparisons with other antibodies (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p47-54, the contents of which are herein incorporated by reference in their entirety). Determining residues making up CDRs may include the use of numbering schemes including, but not limited to, those taught by Kabat [Wu,T.T. et al., 1970, JEM, 132(2):211-50 and Johnson, G. et al., 2000, Nucleic Acids Res. 28(1): 214-8, the contents of each of which are herein incorporated by reference in their entirety], Chothia [Chothia and Lesk, J. Mol. Biol. 196, 901 (1987), Chothia et al., Nature 342, 877 (1989) and Al-Lazikani, B. et al., 1997, J. Mol. Biol. 273(4):927-48, the contents of each of which are herein incorporated by reference in their entirety], Lefranc (Lefranc, M.P. et al., 2005, Immunome Res. 1 :3) and Honegger (Honegger, A. and Pluckthun, A. 2001. J. Mol. Biol. 309(3):657-70, the contents of which are herein incorporated by reference in their entirety).

[0310] VH and VL domains each have three CDRs. VL CDRs are referred to herein as CDR- Ll, CDR-L2 and CDR-L3, in order of occurrence when moving from N- to C- terminus along the variable domain polypeptide. VH CDRs are referred to herein as CDR-H1, CDR-H2, and CDR-H3, in order of occurrence when moving from N- to C-terminus along the variable domain polypeptide. Each of CDRs have favored canonical structures with the exception of the CDR- H3, which comprises amino acid sequences that may be highly variable in sequence and length between antibodies resulting in a variety of three-dimensional structures in antigen-binding domains. In some cases, CDR-H3s may be analyzed among a panel of related antibodies to assess antibody diversity.

[0311] Various methods of determining CDR sequences are known in the art and may be applied to known antibody sequences. The system described by Kabat, also referred to as “numbered according to Kabat,” “Kabat numbering,” “Kabat definitions,” and “Kabat labeling,” provides an unambiguous residue numbering system applicable to any variable domain of an antibody, and provides precise residue boundaries defining the three CDRs of each chain. (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991), the contents of which are incorporated by reference in their entirety). Kabat CDRs and comprise about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Chothia and coworkers found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. (Chothia et al. (1987) J. Mol. Biol. 196: 901-917; and Chothia et al. (1989) Nature 342: 877-883, the contents of each of which is herein incorporated by reference in its entirety). These CDRs can be referred to as “Chothia CDRs,” “Chothia numbering,” or “numbered according to Chothia,” and comprise about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26-32 (CDR1), 52-56 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Mol. Biol. 196:901-917 (1987). The system described by MacCallum,also referred to as “numbered according to MacCallum,” or “MacCallum numbering” comprises about residues 30-36 (CDR1), 46-55 (CDR2) and 89-96 (CDR3) in the light chain variable domain, and 30-35 (CDR1), 47-58 (CDR2) and 93-101 (CDR3) in the heavy chain variable domain. (MacCallum et al. ((1996) J. Mol. Biol. 262(5):732-745), the contents of which is herein incorporated by reference in its entirety). The system described by AbM, also referred to as “numbering according to AbM,” or “AbM numbering” comprises about residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain, and 26- 35 (CDR1), 50-58 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. The IMGT (INTERNATIONAL IMMUNOGENETICS INFORMATION SYSTEM) numbering of variable regions can also be used, which is the numbering of the residues in an immunoglobulin variable heavy or light chain according to the methods of the IIMGT (Lefranc, M.-P., "The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains", The Immunologist, 7, 132-136 (1999), and is herein incorporated by reference in its entirety by reference). As used herein, "IMGT sequence numbering" or “numbered according to IMTG,” refers to numbering of the sequence encoding a variable region according to the IMGT. For the heavy chain variable domain, when numbered according to IMGT, the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR3. For the light chain variable domain, when numbered according to IMGT, the hypervariable region ranges from amino acid positions 27 to 38 for CDR1, amino acid positions 56 to 65 for CDR2, and amino acid positions 105 to 117 for CDR3.

[0312] In some embodiments, the cargo or payload may comprise or may encode antibodies which have been produced using methods known in the art such as, but are not limited to immunization and display technologies (e.g., phage display, yeast display, and ribosomal display), hybridoma technology, heavy and light chain variable region cDNA sequences selected from hybridomas or from other sources,

[0313] In some embodiments, the cargo or payload may comprise or may encode antibodies which were developed using any naturally occurring or synthetic antigen. As used herein, an “antigen” is an entity which induces or evokes an immune response in an organism. An immune response is characterized by the reaction of the cells, tissues and / or organs of an organism to the presence of a foreign entity. Such an immune response typically leads to the production by the organism of one or more antibodies against the foreign entity, e.g., antigen or a portion of the antigen. As used herein, “antigens” also refer to binding partners for specific antibodies or binding agents in a display library.

[0314] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibodies, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen

[0315] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

[0316] As used herein, the term "humanized antibody" refers to a chimeric antibody comprising a minimal portion from one or more non-human (e.g., murine) antibody source(s) with the remainder derived from one or more human immunoglobulin sources. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and / or capacity.

[0317] In some embodiments, the cargo or payload may comprise or may encode antibody mimetics. As used herein, the term “antibody mimetic” refers to any molecule which mimics the function or effect of an antibody, and which binds specifically and with high affinity to their molecular targets. In some embodiments, antibody mimetics may be monobodies, designed to incorporate the fibronectin type III domain (Fn3) as a protein scaffold. In some embodiments, antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, Centyrins, DARPINSTM, fynomers, Kunitz domains, and domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide regions.Antibody Fragments and Variants

[0318] In some embodiments, the cargo or payload may comprise or may encode antibody fragments which comprise antigen binding regions from full-length antibodies. Non-limiting examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. Also produced is a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. Compounds and / or compositions of the present disclosure may comprise one or more of these fragments.

[0319] In some embodiments, the Fc region may be a modified Fc region wherein the Fc region may have a single amino acid substitution as compared to the corresponding sequence for the wild-type Fc region, wherein the single amino acid substitution yields an Fc region with preferred properties to those of the wild-type Fc region. Non-limiting examples of Fc properties that may be altered by the single amino acid substitution include bind properties or response to pH conditions

[0320] As used herein, the term “Fv” refers to an antibody fragment comprising the minimum fragment on an antibody needed to form a complete antigen binding site. These regions consist of a dimer of one heavy chain and one light chain variable domain in tight, non- covalent association. Fv fragments can be generated by proteolytic cleavage, but are largely unstable. Recombinant methods are known in the art for generating stable Fv fragments, typically through insertion of a flexible linker between the light chain variable domain and the heavy chain variable domain to form a single chain Fv (scFv) or through the introduction of a disulfide bridge between heavy and light chain variable domains.

[0321] As used herein, the term "single chain Fv" or "scFv" refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together into a single polypeptide chain by a flexible peptide linker. In some embodiments, the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding. In some embodiments, scFvs are utilized in conjunction with phage display, yeast display or other display methods where they may be expressed in association with a surface member (e.g., phage coat protein) and used in the identification of high affinity peptides for a given antigen.

[0322] As used herein, the term “antibody variant” refers to a modified antibody (in relation to a native or starting antibody) or a biomolecule resembling a native or starting antibody in structure and / or function (e.g., an antibody mimetic). Antibody variants may be altered in theiramino acid sequence, composition, or structure as compared to a native antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgGl, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.Multispecific antibodies

[0323] In some embodiments, the cargo or payload may be or may encode antibodies that bind more than one epitope. As used herein, the terms “multibody” or “multispecific antibody” refer to an antibody wherein two or more variable regions bind to different epitopes. The epitopes may be on the same or different targets. In certain embodiments, a multispecific antibody is a "bispecific antibody," which recognizes two different epitopes on the same or different antigens.

[0324] In some embodiments, multi-specific antibodies may be prepared by the methods used by BIOATLA® and described in International Patent publication WO201109726, the contents of which are herein incorporated by reference in their entirety. First a library of homologous, naturally occurring antibodies is generated by any method known in the art (i.e., mammalian cell surface display), then screened by FACSAria or another screening method, for multi- specific antibodies that specifically bind to two or more target antigens. In some embodiments, the identified multi-specific antibodies are further evolved by any method known in the art, to produce a set of modified multi-specific antibodies. These modified multi-specific antibodies are screened for binding to the target antigens. In some embodiments, the multi-specific antibody may be further optimized by screening the evolved modified multi-specific antibodies for optimized or desired characteristics.

[0325] In some embodiments, multi-specific antibodies may be prepared by the methods used by BIOATLA® and described in Unites States Publication No. US20150252119, the contents of which are herein incorporated by reference in their entirety. In one approach, the variable domains of two parent antibodies, wherein the parent antibodies are monoclonal antibodies are evolved using any method known in the art in a manner that allows a single light chain to functionally complement heavy chains of two different parent antibodies. Another approach requires evolving the heavy chain of a single parent antibody to recognize a second target antigen. A third approach involves evolving the light chain of a parent antibody so as to recognize a second target antigen. Methods for polypeptide evolution are described in International Publication W02012009026, the contents of which are herein incorporated by reference in their entirety, and include as non-limiting examples, Comprehensive PositionalEvolution (CPE), Combinatorial Protein Synthesis (CPS), Comprehensive Positional Insertion (CPI), Comprehensive Positional Deletion (CPD), or any combination thereof. The Fc region of the multi-specific antibodies described in United States Publication No. US20150252119 may be created using a knob-in-hole approach, or any other method that allows the Fc domain to form heterodimers. The resultant multi-specific antibodies may be further evolved for improved characteristics or properties such as binding affinity for the target antigen.Bispecific antibodies

[0326] In some embodiments, the cargo or payload may be or may encode bispecific antibodies. As used herein, the term “bispecific antibody” refers to an antibody capable of binding two different antigens. Such antibodies typically comprise regions from at least two different antibodies. Such antibodies typically comprise antigen-binding regions from at least two different antibodies. For example, a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies, thus allowing the BsAb to bind to two different types of antigen.

[0327] In some cases, the cargo or payload may be or may encode bispecific antibodies comprising antigen-binding regions from two different anti-tau antibodies. For example, such bispecific antibodies may comprise binding regions from two different antibodies

[0328] Bispecific antibody frameworks may include any of those described in Riethmuller, G., 2012. Cancer Immunity. 12:12-18; Marvin, J.S. et al., 2005. Acta Pharmacologica Sinica. 26(6):649-58; and Schaefer, W. et al., 2011. PNAS. 108(27): 11187-92, the contents of each of which are herein incorporated by reference in their entirety.

[0329] New generations of BsMAb, called “trifunctional bispecific” antibodies, have been developed. These consist of two heavy and two light chains, one each from two different antibodies, where the two Fab regions (the arms) are directed against two antigens, and the Fc region (the foot) comprises the two heavy chains and forms the third binding site.

[0330] Of the two paratopes that form the tops of the variable domains of a bispecific antibody, one can be directed against a target antigen and the other against a T-lymphocyte antigen like CD3. In the case of trifunctional antibodies, the Fc region may additionally bind to a cell that expresses Fc receptors, like a macrophage, a natural killer (NK) cell or a dendritic cell. In sum, the targeted cell is connected to one or two cells of the immune system, which subsequently destroy it.

[0331] Other types of bispecific antibodies have been designed to overcome certain problems, such as short half-life, immunogenicity and side-effects caused by cytokine liberation. Theyinclude chemically linked Fabs, consisting only of the Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFvs), fusion proteins mimicking the variable domains of two antibodies. The furthest developed of these newer formats are the bi- specific T-cell engagers (BiTEs) and mAb2's, antibodies engineered to contain an Fcab antigen-binding fragment instead of the Fc constant region.

[0332] Using molecular genetics, two scFvs can be engineered in tandem into a single polypeptide, separated by a linker domain, called a “tandem scFv” (tascFv). TascFvs have been found to be poorly soluble and require refolding when produced in bacteria, or they may be manufactured in mammalian cell culture systems, which avoids refolding requirements but may result in poor yields. Construction of a tascFv with genes for two different scFvs yields a “bispecific single-chain variable fragments” (bis-scFvs). Only two tascFvs have been developed clinically by commercial firms; both are bispecific agents in active early phase development by Micromet for oncologic indications and are described as “Bispecific T-cell Engagers (BiTE)”. Blinatumomab is an anti-CD19 / anti-CD3 bispecific tascFv that potentiates T-cell responses to B-cell non-Hodgkin lymphoma in Phase 2. MT110 is an anti-EP-CAM / anti- CD3 bispecific tascFv that potentiates T-cell responses to solid tumors in Phase 1. Bispecific, tetravalent “TandAbs” are also being researched by Affimed.

[0333] In some embodiments, the cargo or payload may be or may encode antibodies comprising a single antigen-binding domain. These molecules are extremely small, with molecular weights approximately one-tenth of those observed for full-sized mAbs. Further antibodies may include “nanobodies” derived from the antigen-binding variable heavy chain regions (VHHs) of heavy chain antibodies found in camels and llamas, which lack light chains.

[0334] Disclosed and claimed in PCT Publication WO2014144573 (the contents of which are herein incorporated by reference in its entirety) to Memorial Sloan-Kettering Cancer Center are multimerization technologies for making dimeric multispecific binding agents (e.g., fusion proteins comprising antibody components) with improved properties over multispecific binding agents without the capability of dimerization.

[0335] In some cases, the cargo or payload may be or may encode tetravalent bispecific antibodies (TetBiAbs as disclosed and claimed in PCT Publication WO2014144357, the contents of which are herein incorporated in its entirety). TetBiAbs feature a second pair of Fab fragments with a second antigen specificity attached to the C-terminus of an antibody, thus providing a molecule that is bivalent for each of the two antigen specificities. The tetravalent antibody is produced by genetic engineering methods, by linking an antibody heavy chain covalently to a Fab light chain, which associates with its cognate, co-expressed Fab heavychain.

[0336] In some aspects, the cargo or payload may be or may encode biosynthetic antibodies as described in U.S. Patent No. 5,091,513 (the contents of which are herein incorporated by reference in their entirety). Such antibody may include one or more sequences of amino acids constituting a region which behaves as a biosynthetic antibody binding site (BABS). The sites comprise 1) non-covalently associated or disulfide bonded synthetic VH and VL dimers, 2) VH-VL or VL-VH single chains wherein the VH and VL are attached by a polypeptide linker, or 3) individuals VH or VL domains. The binding domains comprise linked CDR and FR regions, which may be derived from separate immunoglobulins. The biosynthetic antibodies may also include other polypeptide sequences which function, e.g., as an enzyme, toxin, binding site, or site of attachment to an immobilization media or radioactive atom. Methods are disclosed for producing the biosynthetic antibodies, for designing BABS having any specificity that can be elicited by in vivo generation of antibody, and for producing analogs thereof.

[0337] In some embodiments, the cargo or payload may be or may encode antibodies with antibody acceptor frameworks taught in U.S. Patent No. 8,399,625. Such antibody acceptor frameworks may be particularly well suited accepting CDRs from an antibody of interest. In some cases, CDRs from anti-tau antibodies known in the art or developed according to the methods presented herein may be used.Miniaturized Antibody

[0338] In some embodiments, the cargo or payload may be or may encode a “miniaturized” antibody. Among the best examples of mAb miniaturization are the small modular immunopharmaceuticals (SMIPs) from Trubion Pharmaceuticals. These molecules, which can be monovalent or bivalent, are recombinant single-chain molecules containing one VL, one VH antigen-binding domain, and one or two constant “effector” domains, all connected by linker domains. Presumably, such a molecule might offer the advantages of increased tissue or tumor penetration claimed by fragments while retaining the immune effector functions conferred by constant domains. At least three “miniaturized” SMIPs have entered clinical development. TRU-015, an anti-CD20 SMIP developed in collaboration with Wyeth, is the most advanced project, having progressed to Phase 2 for rheumatoid arthritis (RA). Earlier attempts in systemic lupus erythrematosus (SLE) and B cell lymphomas were ultimately discontinued. Trubion and Facet Biotechnology are collaborating in the development of TRU- 016, an anti-CD37 SMIP, for the treatment of CLL and other lymphoid neoplasias, a projectthat has reached Phase 2. Wyeth has licensed the anti-CD20 SMIP SBI-087 for the treatment of autoimmune diseases, including RA, SLE, and possibly multiple sclerosis, although these projects remain in the earliest stages of clinical testing.Diabodies

[0339] In some embodiments, the cargo or payload may be or may encode diabodies. As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites. Diabodies comprise a heavy chain variable domain VH connected to a light chain variable domain VL in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0340] Diabodies are functional bispecific single-chain antibodies (bscAb). These bivalent antigen-binding molecules are composed of non-covalent dimers of scFvs, and can be produced in mammalian cells using recombinant methods. (See, e.g., Mack et al, Proc. Natl. Acad. Sci., 92: 7021-7025, 1995). Few diabodies have entered clinical development. An iodine-123- labeled diabody version of the anti-CEA chimeric antibody cT84.66 has been evaluated for pre-surgical immunoscintigraphic detection of colorectal cancer in a study sponsored by the Beckman Research Institute of the City of Hope (Clinicaltrials.gov NCT00647153).Unibody

[0341] In some embodiments, the cargo or payload may be or may encode a “unibody,” in which the hinge region has been removed from IgG4 molecules. While IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with one another, deletion of the hinge region prevents heavy chain-heavy chain pairing entirely, leaving highly specific monovalent light / heavy heterodimers, while retaining the Fc region to ensure stability and half- life in vivo. This configuration may minimize the risk of immune activation or oncogenic growth, as IgG4 interacts poorly with FcRs and monovalent unibodies fail to promote intracellular signaling complex formation. These contentions are, however, largely supported by laboratory, rather than clinical, evidence. Other antibodies may be “miniaturized” antibodies, which are compacted 100 kDa antibodies.Intrabodies

[0342] In some embodiments, the cargo or payload may be or may encode intrabodies. The term “intrabody” refers to a form of antibody that is not secreted from a cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies may be used to affect a multitude of cellular processes including, but not limited to intracellular trafficking,transcription, translation, metabolic processes, proliferative signaling, and cell division. In some embodiments, methods of the present disclosure may include intrabody-based therapies. In some such embodiments, variable domain sequences and / or CDR sequences disclosed herein may be incorporated into one or more constructs for intrabody-based therapy. For example, intrabodies may target one or more glycated intracellular proteins or may modulate the interaction between one or more glycated intracellular proteins and an alternative protein.

[0343] More than two decades ago, intracellular antibodies against intracellular targets were first described (Biocca, Neuberger and Cattaneo EMBO J. 9: 101-108, 1990, the contents of which are herein incorporated by reference in their entirety). The intracellular expression of intrabodies in different compartments of mammalian cells allows blocking or modulation of the function of endogenous molecules (Biocca, et al., EMBO J. 9: 101-108, 1990; Colby et al., Proc. Natl. Acad. Sci. U.S.A. 101: 17616-21, 2004, the contents of which are herein incorporated by reference in their entirety). Intrabodies can alter protein folding, protein- protein, protein-DNA, protein-RNA interactions and protein modification. They can induce a phenotypic knockout and work as neutralizing agents by direct binding to the target antigen, by diverting its intracellular trafficking or by inhibiting its association with binding partners. They have been largely employed as research tools and are emerging as therapeutic molecules for the treatment of human diseases such as viral pathologies, cancer and misfolding diseases. The fast-growing bio-market of recombinant antibodies provides intrabodies with enhanced binding specificity, stability, and solubility, together with lower immunogenicity, for their use in therapy.

[0344] In some embodiments, intrabodies have advantages over interfering RNA (iRNA); for example, iRNA has been shown to exert multiple non-specific effects, whereas intrabodies have been shown to have high specificity and affinity to target antigens. Furthermore, as proteins, intrabodies possess a much longer active half-life than iRNA. Thus, when the active half-life of the intracellular target molecule is long, gene silencing through iRNA may be slow to yield an effect, whereas the effects of intrabody expression can be almost instantaneous. Lastly, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others.

[0345] Intrabodies are often single chain variable fragments (scFvs) expressed from a recombinant nucleic acid molecule and engineered to be retained intracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum, or periplasm). Intrabodies may be used, for example, to ablate the function of a protein to which the intrabody binds. The expression of intrabodies may also be regulated through the use of inducible promoters in the nucleic acid expressionvector comprising the intrabody. Intrabodies may be produced using methods known in the art, such as those disclosed and reviewed in: Marasco et al., 1993 Proc. Natl. Acad. Sci. USA, 90: 7889-7893; Chen et al., 1994, Hum. Gene Ther. 5:595-601; Chen et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 5932-5936; Maciejewski et al., 1995, Nature Med., 1: 667-673; Marasco, 1995, Immunotech, 1: 1-19; Mhashilkar, et al., 1995, EMBO J. 14: 1542-51; Chen et al., 1996, Hum. Gene Therap., 7: 1515-1525; Marasco, Gene Ther. 4:11-15, 1997; Rondon and Marasco, 1997, Annu. Rev. Microbiol. 51:257-283; Cohen, et al., 1998, Oncogene 17:2445-56; Proba et al., 1998, J. Mol. Biol. 275:245-253; Cohen et al., 1998, Oncogene 17:2445-2456; Hassanzadeh, et al., 1998, FEBS Lett. 437:81-6; Richardson et al., 1998, Gene Ther. 5:635-44; Ohage and Steipe, 1999, J. Mol. Biol. 291:1119-1128; Ohage et al., 1999, J. Mol. Biol. 291:1129-1134; Wirtz and Steipe, 1999, Protein Sci. 8:2245-2250; Zhu et al., 1999, J. Immunol. Methods 231:207-222; Arafat et al., 2000, Cancer Gene Ther. 7:1250-6; der Maur et al., 2002, J. Biol. Chem. 277:45075-85; Mhashilkar et al., 2002, Gene Ther. 9:307-19; and Wheeler et al., 2003, FASEB J. 17: 1733-5; and references cited therein). In particular, a CCR5 intrabody has been produced by Steinberger et al., 2000, Proc. Natl. Acad. Sci. USA 97:805-810). See generally Marasco, WA, 1998, "Intrabodies: Basic Research and Clinical Gene Therapy Applications" Springer: New York; and for a review of scFvs, see Pluckthun in “The Pharmacology of Monoclonal Antibodies,” 1994, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315; the contents of each of which are each incorporated by reference in their entireties.

[0346] Sequences from donor antibodies may be used to develop intrabodies. Intrabodies are often recombinantly expressed as single domain fragments such as isolated VH and VL domains or as a single chain variable fragment (scFv) antibody within the cell. For example, intrabodies are often expressed as a single polypeptide to form a single chain antibody comprising the variable domains of the heavy and light chains joined by a flexible linker polypeptide. Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Single chain antibodies can also be expressed as a single chain variable region fragment joined to the light chain constant region.

[0347] As is known in the art, an intrabody can be engineered into recombinant polynucleotide vectors to encode sub-cellular trafficking signals at its N or C terminus to allow expression at high concentrations in the sub-cellular compartments where a target protein is located. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal. Intrabodiesintended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

[0348] There are certain technical challenges with intrabody expression. In particular, protein conformational folding and structural stability of the newly-synthesized intrabody within the cell is affected by reducing conditions of the intracellular environment.

[0349] Intrabodies may be promising therapeutic agents for the treatment of misfolding diseases, including Tauopathies, prion diseases, Alzheimer's, Parkinson's, and Huntington's, because of their virtually infinite ability to specifically recognize the different conformations of a protein, including pathological isoforms, and because they can be targeted to the potential sites of aggregation (both intra- and extracellular sites). These molecules can work as neutralizing agents against amyloidogenic proteins by preventing their aggregation, and / or as molecular shunters of intracellular traffic by rerouting the protein from its potential aggregation site.Maxibodies

[0350] In some embodiments, the cargo or payload may be or may encode a maxibody (bivalent scFV fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG.

[0351] Chimeric Antigen Receptors (CARs)

[0352] In some embodiments, the cargo or payload may be or may encode a chimeric antigen receptors (CARs) which when transduced into immune cells (e.g., T cells and NK cells), can re-direct the immune cells against the target (e.g., a tumor cell) which expresses a molecule recognized by the extracellular target moiety of the CAR.

[0353] As used herein, the term “chimeric antigen receptor (CAR)” refers to a synthetic receptor that mimics TCR on the surface of T cells. In general, a CAR is composed of an extracellular targeting domain, a transmembrane domain / region and an intracellular signaling / activation domain. In a standard CAR receptor, the components: the extracellular targeting domain, transmembrane domain and intracellular signaling / activation domain, are linearly constructed as a single fusion protein. The extracellular region comprises a targeting domain / moiety (e.g., a scFv) that recognizes a specific tumor antigen or other tumor cell- surface molecules. The intracellular region may contain a signaling domain of TCR complex (e.g., the signal region of CD3Q, and / or one or more costimulatory signaling domains, such asthose from CD28, 4-1BB (CD137) and OX-40 (CD134). For example, a “first-generation CAR” only has the CD3^ signaling domain, whereas in an effort to augment T-cell persistence and proliferation, costimulatory intracellular domains are added, giving rise to second generation CARs having a CD3ijsignal domain plus one costimulatory signaling domain, and third generation CARs having CD3^ signal domain plus two or more costimulatory signaling domains. A CAR, when expressed by a T cell, endows the T cell with antigen specificity determined by the extracellular targeting moiety of the CAR. In some aspects, one or more elements such as homing and suicide genes can be added to develop a more competent and safer architecture of CAR (so called the fourth generation CAR).

[0354] In some embodiments, the extracellular targeting domain is joined through the hinge (also called space domain or spacer) and transmembrane regions to an intracellular signaling domain. The hinge connects the extracellular targeting domain to the transmembrane domain which transverses the cell membrane and connects to the intracellular signaling domain. The hinge may need to be varied to optimize the potency of CAR transformed cells toward cancer cells due to the size of the target protein where the targeting moiety binds, and the size and affinity of the targeting domain itself. Upon recognition and binding of the targeting moiety to the target cell, the intracellular signaling domain leads to an activation signal to the CAR T cell, which is further amplified by the “second signal” from one or more intracellular costimulatory domains. The CAR T cell, once activated, can destroy the target cell.

[0355] In some embodiments, the CAR may be split into two parts, each part is linked a dimerizing domain, such that an input that triggers the dimerization promotes assembly of the intact functional receptor. Wu and Lim reported a split CAR in which the extracellular CD19 binding domain and the intracellular signaling element are separated and linked to the FKBP domain and the FRB* (T2089L mutant of FKBP-rapamycin binding) domain that heterodimerize in the presence of the rapamycin analog AP21967. The split receptor is assembled in the presence of AP21967 and together with the specific antigen binding, activates T cells (Wu et al., Science, 2015, 625(6258): aab4077, the contents of which are herein incorporated by reference in its entirety).

[0356] In some embodiments, the CAR may be designed as an inducible CAR which has an incorporation of a Tet-On inducible system to a CD19 CAR construct. The CD19 CAR is activated only in the presence of doxycycline (Dox). Sakemura reported that Tet-CD19CAR T cells in the presence of Dox were equivalently cytotoxic against CD 19+ cell lines and had equivalent cytokine production and proliferation upon CD 19 stimulation, compared with conventional CD19CAR T cells (Sakemura et al., Cancer Immuno. Res., 2016, Jun 21, Epub;the contents of which is herein incorporated by reference in its entirety). The dual systems provide more flexibility to turn-on and off of the CAR expression in transduced T cells.

[0357] In some embodiments, the cargo or payload may be or may encode a first generation CAR, or a second generation CAR, or a third generation CAR, or a fourth generation CAR. In some embodiments, the cargo or payload may be or may encode a full CAR construct composed of the extracellular domain, the hinge and transmembrane domain and the intracellular signaling region. In other embodiments, the cargo or payload may be or may encode a component of the full CAR construct including an extracellular targeting moiety, a hinge region, a transmembrane domain, an intracellular signaling domain, one or more co- stimulatory domain, and other additional elements that improve CAR architecture and functionality including but not limited to a leader sequence, a homing element and a safety switch, or the combination of such components.

[0358] In some embodiments, the cargo or payload may be or may encode a tunable CARs. The reversible on-off switch mechanism allows management of acute toxicity caused by excessive CAR-T cell expansion. The ligand conferred regulation of the CAR may be effective in offsetting tumor escape induced by antigen loss, avoiding functional exhaustion caused by tonic signaling due to chronic antigen exposure and improving the persistence of CAR expressing cells in vivo. The tunable CAR may be utilized to down regulate CAR expression to limit on target on tissue toxicity caused by tumor lysis syndrome. Down regulating the expression of the CARs following anti -tumor efficacy may prevent (1) On target off tumor toxicity caused by antigen expression in normal tissue. (2) antigen independent activation in vivo.Extracellular targeting domain / moiety

[0359] In some embodiments, the extracellular target moiety of a CAR may be any agent that recognizes and binds to a given target molecule, for example, a neoantigen on tumor cells, with high specificity and affinity. The target moiety may be an antibody and variants thereof that specifically binds to a target molecule on tumor cells, or a peptide aptamer selected from a random sequence pool based on its ability to bind to the target molecule on tumor cells, or a variant or fragment thereof that can bind to the target molecule on tumor cells, or an antigen recognition domain from native T- cell receptor (TCR) (e.g. CD4 extracellular domain to recognize HIV infected cells), or exotic recognition components such as a linked cytokine that leads to recognition of target cells bearing the cytokine receptor, or a natural ligand of a receptor.

[0360] In some embodiments, the targeting domain of a CAR may be a Ig NAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, Fv, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a unibody, a nanobody, or an antigen binding region derived from an antibody that specifically recognizes a target molecule, for example a tumor specific antigen (TSA). In one embodiment, the targeting moiety is a scFv antibody. The scFv domain, when it is expressed on the surface of a CAR T cell and subsequently binds to a target protein on a cancer cell, is able to maintain the CAR T cell in proximity to the cancer cell and to trigger the activation of the T cell. A scFv can be generated using routine recombinant DNA technology techniques and is discussed in the present disclosure.

[0361] In some embodiments, the targeting moiety of a CAR construct may be an aptamer such as a peptide aptamer that specifically binds to a target molecule of interest. The peptide aptamer may be selected from a random sequence pool based on its ability to bind to the target molecule of interest.

[0362] In some embodiments, the targeting moiety of a CAR construct may be a natural ligand of the target molecule, or a variant and / or fragment thereof capable of binding the target molecule. In some aspects, the targeting moiety of a CAR may be a receptor of the target molecule, for example, a full length human CD27, as a CD70 receptor, may be fused in frame to the signaling domain of CD3 forming a CD27 chimeric receptor as an immunotherapeutic agent for CD70-positive malignancies.

[0363] In some embodiments, the targeting moiety of a CAR may recognize a tumor specific antigen (TSA), for example a cancer neoantigen which is restrictedly expressed on tumor cells.

[0364] As non-limiting examples, the CAR of the present disclosure may comprise the extracellular targeting domain capable of binding to a tumor specific antigen selected from 5T4, 707-AP, A33, AFP (a -fetoprotein), AKAP-4 ( A kinase anchor protein 4), ALK, 0,5(31 - integrin, androgen receptor, annexin II, alpha- actinin-4, ART-4, Bl, B7H3, B7H4, BAGE (B melanoma antigen), BCMA, BCR-ABL fusion protein, beta-catenin, BKT-antigen, BTAA, CA-I (carbonic anhydrase I), CA50 (cancer antigen 50), CA125, CA15-3, CA195, CA242, calretinin, CAIX (carbonic anhydrase), CAMEL (cytotoxic T-lymphocyte recognized antigen on melanoma), CAM43, CAP-1, Caspase-8 / m, CD4, CD5, CD7, CD19, CD20, CD22, CD23, CD25, CD27 / m, CD28, CD30, CD33, CD34, CD36, CD38, CD40 / CD154, CD41, CD44v6, CD44v7 / 8, CD45,CD49f, CD56, CD68\KP1, CD74, CD79a / CD79b, CD103, CD123, CD133, CD138, CD171, cdc27 / m, CDK4 (cyclin dependent kinase 4), CDKN2A, CDS, CEA (carcinoembryonic antigen), CEACAM5, CEACAM6, chromogranin, c-Met, c-Myc, coa-1,CSAp, CT7, CT10, cyclophilin B, cyclin Bl, cytoplasmic tyrosine kinases, cytokeratin, DAM- 10, DAM-6, dek-can fusion protein, desmin, DEPDC1 (DEP domain containing 1), E2A-PRL, EBNA, EGF-R (epidermal growth factor receptor), EGP-1 (epithelial glycoprotein -1) (TROP- 2), EGP-2, EGP-40, EGFR (epidermal growth factor receptor), EGFRvIII, EF-2, ELF2M, EMMPRIN, EpCAM (epithelial cell adhesion molecule), EphA2, Epstein Barr virus antigens, Erb (ErbBl; ErbB3; ErbB4), ETA (epithelial tumor antigen), ETV6-AML1 fusion protein, FAP (fibroblast activation protein), FBP (folate-binding protein), FGF-5, folate receptor, FOS related antigen 1, fucosyl GM1, G250, GAGE (GAGE-1; GAGE-2), galectin, GD2 (ganglioside), GD3, GFAP (glial fibrillary acidic protein), GM2 (oncofetal antigen- immunogenic- 1; OFA-I-1), GnT-V, GplOO, H4-RET, HAGE (helicase antigen), HER-2 / neu, HIFs (hypoxia inducible factors), HIF-1, HIF-2, HLA-A2, HLA-A*0201-R170I, HLA-A1 1, HMWMAA, Hom / Mel-40, HSP70-2M (Heat shock protein 70), HST-2, HTgp-175, hTERT (or hTRT), human papillomavirus-E6 / human papillomavirus -E7 and E6, iCE (immune-capture EIA), IGF-1R, IGH-IGK, IL-2R, IL-5, ILK (integrin-linked kinase), IMP3 (insulin-like growth factor II mRNA-binding protein 3), IRF4 (interferon regulatory factor 4), KDR (kinase insert domain receptor), KIAA0205, KRAB-zinc finger protein (KID)-3; KID31, KSA (17-1 A), K- ras, LAGE, LCK, LDLR / FUT (LDLR-fucosyltransferaseAS fusion protein), LeY (Lewis Y), MAD-CT-1, MAGE (tyrosinase, melanoma-associated antigen) (MAGE-1; MAGE-3), melan- A tumor antigen (MART), MART-2 / Ski, MC1R (melanocortin 1 receptor), MDM2, mesothelin, MPHOSPH1, MSA(muscle-specific actin), mTOR (mammalian targets of rapamycin), MUC-1, MUC-2, MUM-1 (melanoma associated antigen (mutated) 1), MUM-2, MUM-3, Myosin / m, MYL-RAR, NA88-A, N-acetylglucosaminyltransferase, neo-PAP, NF- KB (nuclear factor-kappa B), neurofilament, NSE (neuron- specific enolase), Notch receptors, NuMa, N-Ras, NY-BR-1, NY- CO-1, NY-ESO-1, Oncostatin M, OS-9, OY-TES1, p53 mutants, pl90 minor bcr-abl, pl5(58), pl85erbB2, pl80erbB-3, PAGE (prostate associated gene), PAP (prostatic acid phosphatase), PAX3, PAX5, PDGFR (platelet derived growth factor receptor), cytochrome P450 involved in piperidine and pyrrolidine utilization (PIPA), Pml- RAR alpha fusion protein, PR-3 (proteinase 3), PSA (prostate specific antigen), PSM, PSMA (Prostate stem cell antigen), PRAME (preferentially expressed antigen of melanoma), PTPRK, RAGE (renal tumor antigen), Raf (A-Raf, B-Raf and C-Raf), Ras, receptor tyrosine kinases, RCAS1, RGSS, ROR1 (receptor tyrosine kinase-like orphan receptor 1), RU1, RU2, SAGE, SART-1, SART-3, SCP-1, SDCCAG16, SP-17 (sperm protein 17), src-family, SSX (synovial sarcoma X breakpoint)-!, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5, STAT-3, STAT-5, STAT-6, STEAD, STn, survivin, syk-ZAP70, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TACSTD1 (tumor associated calcium signal transducer 1), TACSTD2, TAG-72-4, TAGE, TARP (T cell receptor gamma alternate reading frame protein), TEL / AML 1 fusion protein, TEM1, TEM8 (endosialin or CD248), TGFβ, TIE2, TLP, TMPRSS2 ETS fusion gene, TNF-receptor (TNF-a receptor, TNF-0 receptor; or TNF-y receptor), transferrin receptor, TPS, TRP-1 (tyrosine related protein 1), TRP-2, TRP-2 / INT2, TSP-180, VEGF receptor, WNT, WT-1 (Wilm’s tumor antigen) and XAGE.

[0365] In some embodiments, the CAR of the present disclosure may comprise the extracellular targeting domain capable of binding to a tumor specific antigen such as Epidermal Growth Factor Receptor Variant III (EGFRvIII).

[0366] In some embodiments, the cargo is EGFRvIII. In this regard, one can note the differences between the EGFRvIII sequence and the Wt EGFR sequence. Figure 31 describes the EGFRvIII and the Wt EGFR sequences. On the Wt EGFR sequence: 101 represents the extracellular domain (AAs 1-621), 102 represent the transmembrane domain (AAs 622-644) and 103 represents the intracellular domain (AAs 645-1186). On the EGFRvIII sequence: 201 represents truncated extracellular domain (in-frame deletion of exons 2-7 / 268 amino acids deleted / novel glycine at exon junction), 202 represents the insertion of novel Glycine residue.

[0367] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a universal immune receptor which has a targeting moiety capable of binding to a labelled antigen.

[0368] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to a pathogen antigen.

[0369] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to non-protein molecules such as tumor- associated glycolipids and carbohydrates.

[0370] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to a component within the tumor microenvironment including proteins expressed in various tumor stroma cells including tumor associated macrophages (TAMs), immature monocytes, immature dendritic cells, immunosuppressive CD4+CD25+ regulatory T cells (Treg) and MDSCs.

[0371] In some embodiments, the cargo or payload may be or may encode a CAR which comprises a targeting moiety capable of binding to a cell surface adhesion molecule, a surface molecule of an inflammatory cell that appears in an autoimmune disease, or a TCR causing autoimmunity. As non-limiting examples, the targeting moiety of the present disclosure may be a scFv antibody that recognizes a tumor specific antigen (TSA), for example scFvs ofantibodies SS, SSI and HN1 that specifically recognize and bind to human mesothelin, scFv of antibody of GD2, a CD 19 antigen binding domain, aNKG2D ligand binding domain, human anti-mesothelin scFvs, an anti-CSl binding agent, an anti-BCMA binding domain, anti-CD19 scFv antibody, GFR alpha 4 antigen binding fragments, anti-CLL-1 (C-type lectin-like molecule 1) binding domains, CD33 binding domains, a GPC3 (glypican-3) binding domain, a GFR alpha4 (Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a -receptor 4 cell- surface receptor) binding domain, CD123 binding domains, an anti-RORl antibody or fragments thereof, scFvs specific to GPC-3, scFv for CSPG4, and scFv for folate receptor alpha.Intracellular signaling domains

[0372] The intracellular domain of a CAR fusion polypeptide, after binding to its target molecule, transmits a signal to the immune effector cell, activating at least one of the normal effector functions of immune effector cells, including cytolytic activity (e.g., cytokine secretion) or helper activity. Therefore, the intracellular domain comprises an “intracellular signaling domain" of a T cell receptor (TCR).

[0373] In some aspects, the entire intracellular signaling domain can be employed. In other aspects, a truncated portion of the intracellular signaling domain may be used in place of the intact chain as long as it transduces the effector function signal.

[0374] In some embodiments, the intracellular signaling domain may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of IT AM containing cytoplasmic signaling sequences include those derived from TCR CD3zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one example, the intracellular signaling domain is a CD3 zeta (CD3Q signaling domain.

[0375] In some embodiments, the intracellular region further comprises one or more costimulatory signaling domains which provide additional signals to the immune effector cells. These costimulatory signaling domains, in combination with the signaling domain can further improve expansion, activation, memory, persistence, and tumor-eradicating efficiency of CAR engineered immune cells (e.g., CAR T cells). In some cases, the costimulatory signaling region contains 1, 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and / or costimulatory molecules. The costimulatory signaling domain may be the intracellular / cytoplasmic domain of a costimulatory molecule, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, ICOS (CD278),GITR (glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function- associated antigen- 1), LIGHT, NKG2C, B7-H3. In one example, the costimulatory signaling domain is derived from the cytoplasmic domain of CD28. In another example, the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137). In another example, the co-stimulatory signaling domain may be an intracellular domain of GITR as taught in U.S. Pat. NO.: 9, 175, 308; the contents of which are incorporated herein by reference in its entirety.

[0376] In some embodiments, the intracellular region may comprise a functional signaling domain from a protein selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation protein (SLAM) such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, SLAMF6, SLAMF7, an activating NK cell receptor, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDlla / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, IL-15Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CD I la, LFA-1, ITGAM, CD 11b, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, NKG2C, NKD2C SLP76, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, CD270 (HVEM), GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, DAP 10, TRIM, ZAP70, Killer immunoglobulin receptors (KIRs) such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, and KIR2DP1; lectin related NK cell receptors such as Ly49, Ly49A, and Ly49C.

[0377] In some embodiments, the intracellular signaling domain of the present disclosure may contain signaling domains derived from JAK-STAT. In other embodiments, the intracellular signaling domain of the present disclosure may contain signaling domains derived from DAP-12 (Death associated protein 12) (Topfer et al., Immunol., 2015, 194: 3201-3212; and Wang et al., Cancer Immunol., 2015, 3: 815-826). DAP-12 is a key signal transduction receptor in NK cells. The activating signals mediated by DAP-12 play important roles in triggering NK cell cytotoxicity responses toward certain tumor cells and virally infected cells.The cytoplasmic domain of DAP 12 contains an Immunoreceptor Tyrosine-based Activation Motif (IT AM). Accordingly, a CAR containing a DAP12-derived signaling domain may be used for adoptive transfer of NK cells.Transmembrane domains

[0378] In some embodiments, the CAR may comprise a transmembrane domain. As used herein, the term “Transmembrane domain (TM)” refers broadly to an amino acid sequence of about 15 residues in length which spans the plasma membrane. The transmembrane domain may include at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acid residues and spans the plasma membrane. In some embodiments, the transmembrane domain may be derived either from a natural or from a synthetic source. The transmembrane domain of a CAR may be derived from any naturally membrane-bound or transmembrane protein. For example, the transmembrane region may be derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, or CD154.

[0379] Alternatively, the transmembrane domain of the present disclosure may be synthetic. In some aspects, the synthetic sequence may comprise predominantly hydrophobic residues such as leucine and valine.

[0380] In some embodiments, the transmembrane domain may be selected from the group consisting of a CD8a transmembrane domain, a CD4 transmembrane domain, a CD 28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, and a human IgG4 Fc region.

[0381] In some embodiments, the CAR may comprise an optional hinge region (also called spacer). A hinge sequence is a short sequence of amino acids that facilitates flexibility of the extracellular targeting domain that moves the target binding domain away from the effector cell surface to enable proper cell / cell contact, target binding and effector cell activation. The hinge sequence may be positioned between the targeting moiety and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. The hinge sequence may be derived from all or part of an immunoglobulin (e.g., IgGl, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge, the extracellular regions of type 1 membrane proteins such as CD8a CD4, CD28 and CD7, which may be a wild-type sequence or a derivative. Some hinge regions include an immunoglobulin CH3 domain or both a CH3domain and a CH2 domain. In certain embodiments, the hinge region may be modified from an IgGl, IgG2, IgG3, or IgG4 that includes one or more amino acid residues, for example, 1, 2, 3, 4 or 5 residues, substituted with an amino acid residue different from that present in an unmodified hinge.

[0382] In some embodiments, the CAR may comprise one or more linkers between any of the domains of the CAR. The linker may be between 1-30 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In other embodiments, the linker may be flexible.

[0383] In some embodiments, the components including the targeting moiety, transmembrane domain and intracellular signaling domains may be constructed in a single fusion polypeptide. The fusion polypeptide may be the payload.

[0384] In some embodiments, the cargo or payload may be or may encode a CD 19 specific CAR targeting different B cell malignancies and HER2-specific CAR targeting sarcoma, glioblastoma, and advanced Her2 -positive lung malignancy. Tandem CAR (TanCAR).

[0385] In some embodiments, the CAR may be a tandem chimeric antigen receptor (TanCAR) which is able to target two, three, four, or more tumor specific antigens. In some aspects, The CAR is a bispecific TanCAR including two targeting domains which recognize two different TSAs on tumor cells. The bispecific TanCAR may be further defined as comprising an extracellular region comprising a targeting domain (e.g., an antigen recognition domain) specific for a first tumor antigen and a targeting domain (e.g., an antigen recognition domain) specific for a second tumor antigen. In other aspects, the CAR is a multispecific TanCAR that includes three or more targeting domains configured in a tandem arrangement. The space between the targeting domains in the TanCAR may be between about 5 and about 30 amino acids in length, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino acids.Split CAR

[0386] In some embodiments, the CAR components including the targeting moiety, transmembrane domain and intracellular signaling domains may be split into two or more parts such that it is dependent on multiple inputs that promote assembly of the intact functional receptor. As a non-limiting example, the split CAR consists of two parts that assemble in a small molecule-dependent manner; one part of the receptor features an extracellular antigen binding domain (e.g., scFv) and the other part has the intracellular signaling domains, such as the CD3^ intracellular domain.

[0387] In other aspects, the split parts of the CAR system can be further modified to increase signal. As a non-limiting example, the second part of cytoplasmic fragment may be anchored to the plasma membrane by incorporating a transmembrane domain (e.g., CD8a transmembrane domain) to the construct. An additional extracellular domain may also be added to the second part of the CAR system, for instance an extracellular domain that mediates homo- dimerization. These modifications may increase receptor output activity, i.e., T cell activation.

[0388] In some embodiments, the two parts of the split CAR system contain heterodimerization domains that conditionally interact upon binding of a heterodimerizing small molecule. As such, the receptor components are assembled in the presence of the small molecule, to form an intact system which can then be activated by antigen engagement. Any known heterodimerizing components can be incorporated into a split CAR system. Other small molecule dependent heterodimerization domains may also be used, including, but not limited to, gibberellin-induced dimerization system (GID 1 -GAI), trimethoprim-SLF induced ecDHFR and FKBP dimerization and ABA (abscisic acid) induced dimerization of PP2C and PYL domains. The dual regulation using inducible assembly (e.g., ligand dependent dimerization) and degradation (e.g., destabilizing domain induced CAR degradation) of the split CAR system may provide more flexibility to control the activity of the CAR modified T cells.Switchable CAR

[0389] In some embodiments, the CAR may be a switchable CAR which is a controllable CARs that can be transiently switched on in response to a stimulus (e.g., a small molecule). In this CAR design, a system is directly integrated in the hinge domain that separate the scFv domain from the cell membrane domain in the CAR. Such system is possible to split or combine different key functions of a CAR such as activation and costimulation within different chains of a receptor complex, mimicking the complexity of the TCR native architecture. This integrated system can switch the scFv and antigen interaction between on / off states controlled by the absence / presence of the stimulus.Reversible CAR

[0390] In some embodiments, the CAR may be a reversible CAR system. In this CAR architecture, a LID domain (ligand-induced degradation) is incorporated into the CAR system. The CAR can be temporarily down-regulated by adding a ligand of the LID domain.Inhibitory CAR (iCAR)

[0391] In some embodiments, the CAR may be inhibitory CARs. Inhibitory CAR (iCAR) refers to a bispecific CAR design wherein a negative signal is used to enhance the tumorspecificity and limit normal tissue toxicity. This design incorporates a second CAR having a surface antigen recognition domain combined with an inhibitory signal domain to limit T cell responsiveness even with concurrent engagement of an activating receptor. This antigen recognition domain is directed towards a normal tissue specific antigen such that the T cell can be activated in the presence of first target protein, but if the second protein that binds to the iCAR is present, the T cell activation is inhibited.

[0392] As a non-limiting example, iCARs against Prostate specific membrane antigen (PMSA) based on CTLA4 and PD1 inhibitory domains demonstrated the ability to selectively limit cytokine secretion, cytotoxicity and proliferation induced by T cell activation.Chimeric switch receptor

[0393] In some embodiments, the cargo or payload may be or may encode a chimeric switch receptors which can switch a negative signal to a positive signal. As used herein, the term “chimeric switch receptor” refers to a fusion protein comprising a first extracellular domain and a second transmembrane and intracellular domain, wherein the first domain includes a negative signal region, and the second domain includes a positive intracellular signaling region. In some aspects, the fusion protein is a chimeric switch receptor that contains the extracellular domain of an inhibitory receptor on T cell fused to the transmembrane and cytoplasmic domain of a co-stimulatory receptor. This chimeric switch receptor may convert a T cell inhibitory signal into a T cell stimulatory signal.

[0394] As a non-limiting example, the chimeric switch receptor may comprise the extracellular domain of PD-1 fused to the transmembrane and cytoplasmic domain of CD28. In some aspects, Extracellular domains of other inhibitory receptors such as CTLA-4, LAG-3, TIM-3, KIRs and BTLA may also be fused to the transmembrane and cytoplasmic domain derived from costimulatory receptors such as CD28, 4-1BB, CD27, 0X40, CD40, GTIR and ICOS.

[0395] In some embodiments, chimeric switch receptors may include recombinant receptors comprising the extracellular cytokine-binding domain of an inhibitory cytokine receptor (e.g., IL-13 receptor a (IL-13Rαl), IL-10R, and IL-4Rα) fused to an intracellular signaling domain of a stimulatory cytokine receptor such as IL-2R (IL-2Rα, IL-2R0 and IL-2Rgamma) and IL- 7Rα. One example of such chimeric cytokine receptor is a recombinant receptor containing the cytokine-binding extracellular domain of IL-4Rα linked to the intracellular signaling domain ofIL-7Rα.

[0396] In some embodiments, the chimeric switch receptor may be a chimeric TGFβ receptor.The chimeric TGFβ receptor may comprise an extracellular domain derived from a TGFβ receptor such as TGFβ receptor 1, TGFβ receptor 2, TGFβ receptor 3, or any other TGFβ receptor or variant thereof; and a non- TGFβ receptor intracellular domain. The non- TGFβ receptor intracellular domain may be the intracellular domain or fragment thereof derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CD28, 4-1BB (CD137), 0X40 (CD134), CD3zeta, CD40, CD27, or a combination thereofActivation-conditional CAR

[0397] In some embodiments, the cargo or payload may be or may encode an activation- conditional chimeric antigen receptor, which is only expressed in an activated immune cell. The expression of the CAR may be coupled to activation conditional control region which refers to one or more nucleic acid sequences that induce the transcription and / or expression of a sequence e.g., a CAR under its control. Such activation conditional control regions may be promoters of genes that are upregulated during the activation of the immune effector cell e.g., IL2 promoter or NF AT binding sites.CAR targeting to tumor cells with specific proteoglycan markers

[0398] In some embodiments, the cargo or payload may be or may encode a CAR that targets specific types of cancer cells. Human cancer cells and metastasis may express unique and otherwise abnormal proteoglycans, such as polysaccharide chains (e.g., chondroitin sulfate (CS), dermatan sulfate (DS or CSB), heparan sulfate (HS) and heparin). Accordingly, the CAR may be fused with a binding moiety that recognizes cancer associated proteoglycans. In one example, a CAR may be fused with VAR2CSA polypeptide (VAR2-CAR) that binds with high affinity to a specific type of chondroitin sulfate A (CSA) attached to proteoglycans. The extracellular ScFv portion of the CAR may be substituted with VAR2CSA variants comprising at least the minimal CSA binding domain, generating CARs specific to chondroitin sulfate A (CSA) modifications. Alternatively, the CAR may be fused with a split-protein binding system to generate a spy-CAR, in which the scFv portion of the CAR is substituted with one portion of a split-protein binding system such as SpyTag and Spy-catcher and the cancer-recognition molecules (e.g., scFv and or VAR2-CSA) are attached to the CAR through the split-protein binding system.Nucleic Acids

[0399] The lipid nanoparticles of the present disclosure may comprise a payload region (which may also be referred to as a cargo region) which is a nucleic acid. The term “nucleic acid,” in its broadest sense, includes any compound and / or substance that comprise a polymerof nucleotides which may be referred to as polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof.

[0400] In some embodiments, the payload region comprises nucleic acid sequences encoding more than one cargo or payload.

[0401] In some embodiments, the payload region may be or encode a coding nucleic acid sequence.

[0402] In some embodiments, the payload region may be or encode a non-coding nucleic acid sequence.

[0403] In some embodiments, the payload region may be or encode both a coding and a non- coding nucleic acid sequence.DNA

[0404] Deoxyribonucleic acid (DNA) is a molecule that carries genetic information for all living things and consists of two strands that wind around one another to form a shape known as a double helix. Each strand has a backbone made of alternating sugar (deoxyribose) and phosphate groups. Attached to each sugar is one of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T). The two strands are held together by bonds between adenine and thymine or cytosine and guanine. The sequence of the bases along the backbones serves as instructions for assembling protein and RNA molecules.

[0405] In some embodiments, the payload region may be or encode a coding DNA.

[0406] In some embodiments, the payload region may be or encode a non-coding DNA.

[0407] In some embodiments, the payload region may be or encode both a coding and a non- coding DNA.

[0408] In some embodiments, the DNA may be modified. Types of modifications include, but are not limited to, methylation, acetylation, phosphorylation, ubiquitination, and sumoylation.Vectors

[0409] In some embodiments, the originator constructs and / or benchmark constructs described herein can be or be encoded by vectors such as plasmids or viral vectors. In some embodiments, the originator constructs and / or benchmark constructs are or are encoded by viral vectors. Viral vectors may be, but are not limited to, Herpesvirus (HSV) vectors, retroviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, and thelike. In some embodiments, the viral vectors are AAV vectors. In some embodiments, the viral vectors are lentiviral vectors. In some embodiments, the viral vectors are retroviral vectors. In some embodiments, the viral vectors are adenoviral vectors.Adeno-Associated Viral (AAVs) Vectors

[0410] Viruses of the Parvoviridae family are small non-enveloped icosahedral capsid viruses characterized by a single stranded DNA genome. Parvoviridae family viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Due to its relatively simple structure, easily manipulated using standard molecular biology techniques, this virus family is useful as a biological tool. The genome of the virus may be modified to contain a minimum of components for the assembly of a functional recombinant virus, or viral particle, which is loaded with or engineered to express or deliver a desired payload, which may be delivered to a target cell, tissue, organ, or organism.

[0411] The Parvoviridae family comprises the Dependovirus genus which includes adeno- associated viruses (AAV) capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.

[0412] The AAV vector genome is a linear, single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nts) in length. The AAV vector genome can comprise a payload region and at least one inverted terminal repeat (ITR) or ITR region. ITRs traditionally flank the coding nucleotide sequences for the non-structural proteins (encoded by Rep genes) and the structural proteins (encoded by capsid genes or Cap genes). While not wishing to be bound by theory, an AAV vector genome typically comprises two ITR sequences. The AAV vector genome comprises a characteristic T-shaped hairpin structure defined by the self- complementary terminal 145 nucleotides of the 5’ and 3’ ends of the ssDNA which form an energetically stable double stranded region. The double stranded hairpin structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the host viral replication cell.

[0413] In addition to the encoded heterologous pay load, AAV vector genomes may comprise, in whole or in part, of any naturally occurring and / or recombinant AAV serotype nucleotide sequence or variant. AAV variants may have sequences of significant homology at the nucleic acid (genome or capsid) and amino acid levels (capsids), to produce constructs which are generally physical and functional equivalents, replicate by similar mechanisms, and assemble by similar mechanisms. Chiorini et al., J. Vir. 71: 6823-33(1997); Srivastava et al., J. Vir. 45:555-64 (1983); Chiorini et al., J. Vir. 73: 1309-1319 (1999); Rutledge et al., J. Vir. 72:309-319 (1998); and Wu et al., J. Vir. 74: 8635-47 (2000), the contents of each of which are incorporated herein by reference in their entirety.

[0414] In some embodiments, the AAV vector genome comprises at least one control element which provides for the replication, transcription, and translation of a coding sequence encoded therein. Not all of the control elements need always be present as long as the coding sequence is capable of being replicated, transcribed, and / or translated in an appropriate host cell. Non- limiting examples of expression control elements include sequences for transcription initiation and / or termination, promoter and / or enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficacy (e.g., Kozak consensus sequence), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion.

[0415] AAV vector genomes may be produced recombinantly and may be based on adeno- associated virus (AAV) parent or reference sequences. As used herein, a “vector genome” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule such as the nucleic acids described herein.

[0416] In addition to single stranded AAV vector genomes (e.g., ssAAVs), the present disclosure also provides for self-complementary AAV (scAAVs) vector genomes. scAAV vector genomes contain DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.

[0417] In some embodiments, the AAV vector genome is an scAAV.

[0418] In some embodiments, the AAV vector genome is an ssAAV.

[0419] In some embodiments, the AAV vector genome may be part of an AAV particles where the serotype of the capsid may be, but is not limited to, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42- 13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2-15 / rh.62, AAV2- 3 / rh.61, AAV2-4 / rh.5O, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3- ll / rh.53, AAV4-8 / rll.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58,AAV7.3 / hu.7, AAV16.8 / hu.lO, AAV16.12 / hu.ll, AAV29.3 / bb.l, AAV29.5 / bb.2,AAV106.1 / hu.37, AAV114.3 / hu.4O, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV33.12 / hu.l7, AAV33.4 / hu.l5, AAV33.8 / hu.l6, AAV52 / hu.l9, AAV52.1 / hu.2O, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.l, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03,AAVH-l / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.ll,AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21,AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29,AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39,AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2,AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2,AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56,AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66,AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R,AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18,AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31,AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl.l, AAVhErl.5, AAVhER1.14, AAVhErl.8, AAVhErl.16, AAVhErl.18, AAVhErl.35, AAVhErl.7, AAVhErl.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV- h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.l l, AAVhu.53, AAV4- 8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24,AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr- 7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr- 7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr-E2, AAV CBr-E3, AAV CBr- E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt- P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd-B3, AAV CKd- B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd- H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd- N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg- F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-13, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8, AAV Clvl-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, PHP.B, PHP. A, G2B-26, G2B-13, TH1.1-32, and / or TH1.1-35 and variants thereof.Inverted Terminal Repeats (ITRs)

[0420] In some embodiments, the AAV vector genomes may comprise at least one ITR region and a payload region. In some embodiments, the vector genome has two ITRs. These two ITRs flank the payload region at the 5’ and 3’ ends. The ITRs function as origins of replication comprising recognition sites for replication. ITRs comprise sequence regions which can be complementary and symmetrically arranged. ITRs incorporated into vector genomes may be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0421] The ITRs may be derived from the same serotype as the capsid or a derivative thereof. The ITR may be of a different serotype than the capsid. In some embodiments, the AAV particle has more than one ITR. In a non-limiting example, the AAV particle has a vector genome comprising two ITRs. In some embodiments, the ITRs are of the same serotype as one another. In another embodiment, the ITRs are of different serotypes. Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid. In some embodiments both ITRs of the vector genome of the AAV particle are AAV2 ITRs.

[0422] Independently, each ITR may be about 100 to about 150 nucleotides in length. An ITR may be about 100-105 nucleotides in length, 106-110 nucleotides in length, 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131-135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length or 146-150 nucleotides in length. In some embodiments, the ITRs are 140-142 nucleotides in length. Non-limiting examples of ITR length are 102, 140, 141, 142, 145 nucleotides in length, and those having at least 95% identity thereto.Promoters

[0423] In some embodiments, the pay load region of the vector genome comprises at least one element to enhance the transgene target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in its entirety). Non-limiting examples of elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), CMV enhancers and introns.

[0424] In some embodiments, the promoter is efficient when it drives expression of the polypeptide(s) encoded in the pay load region of the vector genome of the AAV particle.

[0425] In some embodiments, the promoter is deemed to be efficient when it drives expression in the cell being targeted.

[0426] In some embodiments, the promoter drives expression of the payload for a period of time in targeted tissues. Expression driven by a promoter may be for a period of 1 hour, 2, hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years,4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years. Expression may be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, or 5-10 years.

[0427] In some embodiment, the promoter drives expression of the payload for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years, 60 years, 65 years, or more than 65 years.

[0428] Promoters may be naturally occurring or non-naturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters and mammalian promoters. In some embodiments, the promoters may be human promoters. In some embodiments, the promoter may be truncated.

[0429] Promoters which drive or promote expression in most tissues include, but are not limited to, human elongation factor la-subunit (EFla), cytomegalovirus (CMV) immediate- early enhancer and / or promoter, chicken [3-actin (CBA) and its derivative CAG, f> glucuronidase (GUSB), or ubiquitin C (UBC). Tissue-specific expression elements can be used to restrict expression to certain cell types such as, but not limited to, muscle specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophagepromoters, pancreatic acinar cell promoters, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or nervous system promoters which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0430] Non-limiting examples of muscle-specific promoters include mammalian muscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, mammalian troponin I (TNNI2) promoter, and mammalian skeletal alpha-actin (ASKA) promoter (see, e.g., U.S. Patent Publication US20110212529, the contents of which are herein incorporated by reference in their entirety)

[0431] Non-limiting examples of tissue-specific expression elements for neurons include neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF-0), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), Ca2+ / calmoduhn-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), 0-globin minigene nf>2. preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoters. Non-limiting examples of tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoters. A non-limiting example of a tissue-specific expression element for oligodendrocytes includes the myelin basic protein (MBP) promoter.

[0432] In some embodiments, the promoter may be less than 1 kb. The promoter may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, or more than 800 nucleotides. The promoter may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800, or 700-800.

[0433] In some embodiments, the promoter may be a combination of two or more components of the same or different starting or parental promoters such as, but not limited to, CMV and CBA. Each component may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740,750, 760, 770, 780, 790, 800, or more than 800. Each component may have a length between200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700,300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800. In some embodiments, the promoter is a combination of a 382 nucleotide CMV- enhancer sequence and a 260 nucleotide CBA-promoter sequence.

[0434] In some embodiments, the vector genome comprises a ubiquitous promoter. Non- limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc ), EF-la, PGK, UBC, GUSB (hGBp), and UCOE (promoter of HNRPA2B1-CBX3).

[0435] In some embodiments, the promoter is not cell specific.

[0436] In some embodiments, the vector genome comprises an engineered promoter.

[0437] In some embodiments, the vector genome comprises a promoter from a naturally expressed protein.Untranslated Regions (UTRs)

[0438] By definition, wild type untranslated regions (UTRs) of a gene are transcribed but not translated. Generally, the 5’ UTR starts at the transcription start site and ends at the start codon and the 3’ UTR starts immediately following the stop codon and continues until the termination signal for transcription.

[0439] Features typically found in abundantly expressed genes of specific target organs may be engineered into UTRs to enhance the stability and protein production. As a non-limiting example, a 5’ UTR from mRNA normally expressed in the liver (e.g., albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII) may be used in the vector genomes of the AAV particles to enhance expression in hepatic cell lines or liver.

[0440] While not wishing to be bound by theory, wild-type 5' untranslated regions (UTRs) include features which play roles in translation initiation. Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates translation of many genes, are usually included in 5’ UTRs. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (ATG), which is followed by another 'G1.

[0441] In some embodiments, the 5 ’UTR in the vector genome includes a Kozak sequence.

[0442] In some embodiments, the 5 ’UTR in the vector genome does not include a Kozak sequence.

[0443] While not wishing to be bound by theory, wild-type 3' UTRs are known to have stretches of Adenosines and Uridines embedded therein. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence featuresand functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995, the contents of which are herein incorporated by reference in its entirety): Class I AREs, such as, but not limited to, c-Myc and MyoD, contain several dispersed copies of an AUUUA motif within U-rich regions. Class II AREs, such as, but not limited to, GM- CSF and TNF-a, possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Class III ARES, such as, but not limited to, c-Jun and Myogenin, are less well defined. These U rich regions do not contain an AUUUA motif. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules can lead to HuR binding and thus, stabilization of the message in vivo.

[0444] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides. When engineering specific polynucleotides, e.g., pay load regions of vector genomes, one or more copies of an ARE can be introduced to make polynucleotides less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.

[0445] In some embodiments, the 3' UTR of the vector genome may include an oligo(dT) sequence for templated addition of a poly -A tail.

[0446] In some embodiments, the vector genome may include at least one miRNA seed, binding site or full sequence. microRNAs (or miRNA or miR) are 19-25 nucleotide noncoding RNAs that bind to the sites of nucleic acid targets and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. A microRNA sequence comprises a “seed” region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence of the nucleic acid.

[0447] In some embodiments, the vector genome may be engineered to include, alter or remove at least one miRNA binding site, sequence, or seed region.

[0448] Any UTR from any gene known in the art may be incorporated into the vector genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as in the gene from which they were selected, or they may be altered in orientation or location. In some embodiments, the UTR used in the vector genome of the AAV particle may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs known in the art. As used herein, the term “altered” as it relates to a UTR, means that the UTR has been changed insome way in relation to a reference sequence. For example, a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides.

[0449] In some embodiments, the vector genome of the AAV particle comprises at least one artificial UTRs which is not a variant of a wild-type UTR.

[0450] In some embodiments, the vector genome of the AAV particle comprises UTRs which have been selected from a family of transcripts whose proteins share a common function, structure, feature or property.Polyadenylation Sequence

[0451] In some embodiments, the vector genome comprises at least one polyadenylation sequence between the 3’ end of the payload coding sequence and the 5’ end of the 3TTR.

[0452] In some embodiments, the polyadenylation (poly-A) sequence may range from absent to about 500 nucleotides in length. The polyadenylation sequence may be, but is not limited to,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378. 379, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463,464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482,483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, and 500 nucleotides in length.

[0453] In some embodiments, the polyadenylation sequence is 50-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 50-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 60-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 70-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 80-200 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-100 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-150 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-160 nucleotides in length. In some embodiments, the polyadenylation sequence is 90-200 nucleotides in length.Linkers

[0454] Vector genomes may be engineered with one or more spacer or linker regions to separate coding or non-coding regions.

[0455] In some embodiments, the payload region of the vector genome may optionally encode one or more linker sequences. In some cases, the linker may be a peptide linker that may be used to connect the polypeptides encoded by the payload region (i.e., light and heavyantibody chains during expression). Some peptide linkers may be cleaved after expression to separate heavy and light chain domains, allowing assembly of mature antibodies or antibody fragments. Linker cleavage may be enzymatic. In some cases, linkers comprise an enzymatic cleavage site to facilitate intracellular or extracellular cleavage. Some payload regions encode linkers that interrupt polypeptide synthesis during translation of the linker sequence from an mRNA transcript. Such linkers may facilitate the translation of separate protein domains from a single transcript. In some cases, two or more linkers are encoded by a payload region of the vector genome.

[0456] Internal ribosomal entry site (IRES) is a nucleotide sequence (>500 nucleotides) that allows for initiation of translation in the middle of an mRNA sequence (Kim, J.H. et al., 2011. PLoS One 6(4): el 8556; the contents of which are herein incorporated by reference in its entirety). Use of an IRES sequence ensures co-expression of genes before and after the IRES, though the sequence following the IRES may be transcribed and translated at lower levels than the sequence preceding the IRES sequence.

[0457] 2A peptides are small “self-cleaving” peptides (18-22 amino acids) derived from viruses such as foot-and-mouth disease virus (F2A), porcine teschovirus-1 (P2A), Thoseaasigna virus (T2A), or equine rhinitis A virus (E2A). The 2A designation refers specifically to a region of picomavirus polyproteins that lead to a ribosomal skip at the glycyl- prolyl bond in the C-terminus of the 2A peptide (Kim, J.H. et al., 2011. PLoS One 6(4): el 8556; the contents of which are herein incorporated by reference in its entirety). This skip results in a cleavage between the 2A peptide and its immediate downstream peptide. As opposed to IRES linkers, 2A peptides generate stoichiometric expression of proteins flanking the 2A peptide and their shorter length can be advantageous in generating viral expression vectors.

[0458] Some payload regions encode linkers comprising furin cleavage sites. Furin is a calcium dependent serine endoprotease that cleaves proteins just downstream of a basic amino acid target sequence (Arg-X-(ArgZLys)-Arg) (Thomas, G., 2002. Nature Reviews Molecular Cell Biology 3(10): 753-66; the contents of which are herein incorporated by reference in its entirety). Furin is enriched in the trans-golgi network where it is involved in processing cellular precursor proteins. Furin also plays a role in activating a number of pathogens. This activity can be taken advantage of for expression of polypeptides.

[0459] In some embodiments, the payload region may encode one or more linkers comprising cathepsin, matrix metalloproteinases or legumain cleavage sites. Such linkers are described e.g., by Cizeau and Macdonald in International Publication No. W02008052322, the contents of which are herein incorporated in their entirety. Cathepsins are a family of proteases withunique mechanisms to cleave specific proteins. Cathepsin B is a cysteine protease and cathepsin D is an aspartyl protease. Matrix metalloproteinases are a family of calcium- dependent and zinc-containing endopeptidases. Legumain is an enzyme catalyzing the hydrolysis of (-Asn-Xaa-) bonds of proteins and small molecule substrates.

[0460] In some embodiments, payload regions may encode linkers that are not cleaved. Such linkers may include a simple amino acid sequence, such as a glycine rich sequence. In some cases, linkers may comprise flexible peptide linkers comprising glycine and serine residues. The linker may comprise flexible peptide linkers of different lengths, e.g., (G4S)n, where n=l- 10 ((Gly-Gly-Gly-Gly-Ser)n) and the length of the encoded linker varies between 5 and 50 amino acids. In a non-limiting example, the linker may be (G4S)5 (Gly-Gly-Gly-Gly-Ser)5. These flexible linkers are small and without side chains, so they tend not to influence secondary protein structure while providing a flexible linker between antibody segments (George, R.A., et al., 2002. Protein Engineering 15(11): 871-9; Huston, J.S. et al., 1988. PNAS 85:5879-83; and Shan, D. et al., 1999. Journal of Immunology. 162(11):6589-95; the contents of each of which are herein incorporated by reference in their entirety). Furthermore, the polarity of the serine residues improves solubility and prevents aggregation problems.

[0461] In some embodiments, payload regions may encode small and unbranched serine-rich peptide linkers, such as those described by Huston et al. in US Patent No. US5525491, the contents of which are herein incorporated in their entirety. Polypeptides encoded by the payload region, linked by serine-rich linkers, have increased solubility.

[0462] In some embodiments, payload regions may encode artificial linkers, such as those described by Whitlow and Filpula in US Patent No. US5856456 and Ladner et al. in US Patent No. US 4946778, the contents of each of which are herein incorporated by their entirety.Introns

[0463] In some embodiments, the payload region comprises at least one element to enhance the expression such as one or more introns or portions thereof. Non-limiting examples of introns include, MVM (67-97 bps), F.IX truncated intron 1 (300 bps), [3-globin SD / immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor / immunoglobin splice acceptor (500 bps), SV40 late splice donor / splice acceptor (19S / 16S) (180 bps) and hybrid adenovirus splice donor / IgG splice acceptor (230 bps).

[0464] In some embodiments, the intron or intron portion may be 100-500 nucleotides in length. The intron may have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500. The intron may have a length between 80-100, 80-120, 80-140, 80-160, 80-180, 80- 200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-500.Lenti viral Vectors

[0465] Lentiviral vectors are a type of retrovirus that can infect both dividing and nondividing cells because their viral shell can pass through the intact membrane of the nucleus of the target cell. Lentiviral vectors have the ability to deliver transgenes in tissues that had long appeared irremediably refractory to stable genetic manipulation. Lentivectors have also opened fresh perspectives for the genetic treatment of a wide array of hereditary as well as acquired disorders, and a real proposal for their clinical use seems imminent.RNA

[0466] Ribonucleic acid (RNA) is a molecule that is made up of nucleotides, which are ribose sugars attached to nitrogenous bases and phosphate groups. The nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C). Generally, RNA mostly exists in the single-stranded form but can also exists double-stranded in certain circumstances. The length, form and structure of RNA is diverse depending on the purpose of the RNA. For example, the length of an RNA can vary from a short sequence (e.g., siRNA) to a long sequences (e.g., IncRNA), can be linear (e.g., mRNA) or circular (e.g., oRNA), and can either be a coding (e.g., mRNA) or a non-coding (e.g., IncRNA) sequence.

[0467] In some embodiments, the payload region may be or encode a coding RNA.

[0468] In some embodiments, the payload region may be or encode a non-coding RNA.

[0469] In some embodiments, the payload region may be or encode both a coding and a non- coding RNA.

[0470] In some embodiments, the payload region comprises nucleic acid sequences encoding more than one cargo or payload.

[0471] In some embodiments, the payload region comprises a nucleic acid sequence to enhance the expression of a gene. As a non-limiting example, the nucleic acid sequence is a messenger RNA (mRNA). As another non-limiting example, the nucleic acid sequence is a circular RNA (oRNA).

[0472] In some embodiments, the payload region comprises a nucleic acid sequence to reduce or inhibit the expression of a gene. As a non-limiting example, the nucleic acid sequence is a small interfering RNA (siRNA) or a microRNA (miRNA).Messenger RNA (mRNA)

[0473] In some embodiments, the originator constructs and / or benchmark constructs may be mRNA. As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a target of interest and which is capable of being translated to produce the encoded target of interest in vitro, in vivo, in situ or ex vivo.

[0474] Generally, an mRNA molecule comprises at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. In some aspects, one or more structural and / or chemical modifications or alterations may be included in the RNA which can reduce the innate immune response of a cell in which the mRNA is introduced. As used herein, a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a nucleic acid without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications can result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G".

[0475] Generally, the shortest length of a region of the originator constructs and / or benchmark constructs can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the length may be sufficient to encode a peptide of 2-30 amino acids, e.g., 5-30, 10-30, 2-25, 5-25, 10-25, or 10- 20 amino acids. The length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g., no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.

[0476] Generally, the length of the region of the mRNA encoding a target of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides).

[0477] In some embodiments, the mRNA includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1 ,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1 ,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).

[0478] In some embodiments, the region or regions flanking the region encoding the target of interest may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

[0479] In some embodiments, the mRNA comprises a tailing sequence which can range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA Binding Protein binding. In this embodiment, the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein. PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.

[0480] In some embodiments, the mRNA comprises a capping sequence which comprises a single cap or a series of nucleotides forming the cap. The capping sequence may be from 1 to 10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the caping sequence is absent.

[0481] In some embodiments, the mRNA comprises a region comprising a start codon. The region comprising the start codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.

[0482] In some embodiments, the mRNA comprises a region comprising a stop codon. The region comprising the stop codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.

[0483] In some embodiments, the mRNA comprises a region comprising a restriction sequence. The region comprising the restriction sequence may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.Untranslated Regions (UTRs)

[0484] In some embodiments, the mRNA comprises at least one untranslated region (UTR) which flanks the region encoding the target of interest. UTRs are transcribed by not translated.

[0485] The 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas the 3 'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, the UTRs may have a regulatory role in terms of translation and stability of the nucleic acid.

[0486] Natural 5' UTRs usually include features which have a role in translation initiation as they tend to include Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5'UTR also have been known to form secondary structures which are involved in elongation factor binding.

[0487] 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules can lead to HuR binding and thus, stabilization of the message in vivo. Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of mRNA. For example, one or more copies of an ARE can be introduced to make mRNA less stable and thereby curtail translation and decrease production of the resultant protein.Alternatively, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.

[0488] In some embodiments, the introduction of features often expressed in genes of target organs the stability and protein production of the mRNA can be enhanced in a specific organ and / or tissue. As a non-limiting example, the feature can be a UTR. As another example, the feature can be introns or portions of introns sequences.5' Capping

[0489] The 5' cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing.

[0490] Endogenous mRNA molecules may be 5'-end capped generating a 5'-ppp-5'- triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-0-methylated. 5'- decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0491] Modifications to mRNA may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5 ' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.

[0492] Additional modified guanosine nucleotides may be used such as a-methyl- phosphonate and seleno-phosphate nucleotides.

[0493] Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and / or 5 '-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as an mRNA molecule.

[0494] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e.,endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked to a nucleic acid molecule.

[0495] For example, the Anti -Reverse Cap Analog (ARC A) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'-triphosphate-5 '-guanosine (m7G- 3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA). The N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g., mRNA).

[0496] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm- PPP-G).

[0497] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.

[0498] mRNA may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5 'cap structures are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5 'cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 '- triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation, and the 5 '-terminal nucleotide of the mRNA contains a 2'-0-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation ofcellular pro-inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5*)ppp(5*)N,pN2p (cap 0), 7mG(5*)ppp(5*)NlmpNp (cap 1), and 7mG(5*)-ppp(5')NlmpN2mp (cap 2).

[0499] In some embodiments, the 5' terminal caps may include endogenous caps or cap analogs.

[0500] In some embodiments, a 5' terminal cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro- guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2- azido-guanosine.IRES Sequences

[0501] In some embodiments, the mRNA may contain an internal ribosome entry site (IRES). First identified as a feature Picoma virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. An mRNA that contains more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes. Non-limiting examples of IRES sequences that can be used include without limitation, those from picomaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).Poly-A tails

[0502] During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the R A. The process, called polyadenylation, adds a poly-A tail of a certain length.

[0503] In some embodiments, the length of a poly-A tail is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the mRNA includes a poly-A tail from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1 ,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to1.500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to2.500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[0504] In some embodiments, the poly -A tail is designed relative to the length of the overall mRNA. This design may be based on the length of the region coding for a target of interest, the length of a particular feature or region (such as a flanking region), or based on the length of the ultimate product expressed from the mRNA.

[0505] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the mRNA or feature thereof. The poly-A tail may also be designed as a fraction of mRNA to which it belongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of mRNA for poly-A binding protein may enhance expression.

[0506] Additionally, multiple distinct mRNA may be linked together to the PABP (Poly-A binding protein) through the 3'-end using modified nucleotides at the 3 '-terminus of the poly- A tail. Transfection experiments can be conducted in relevant cell lines at, and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.

[0507] In some embodiments, the mRNA are designed to include a polyA-G Quartet. The G- quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G- rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail.Stop Codons

[0508] In some embodiments, the mRNA may include one stop codon. In some embodiments, the mRNA may include two stop codons. In some embodiments, the mRNA may include three stop codons. In some embodiments, the mRNA may include at least one stop codon. In some embodiments, the mRNA may include at least two stop codons. In some embodiments, the mRNA may include at least three stop codons. As non-limiting examples, the stop codon may be selected from TGA, TAA and TAG.

[0509] In some embodiments, the mRNA includes the stop codon TGA and one additional stop codon. In a further embodiment the addition stop codon may be TAA.Circular RNA (oRNA)

[0510] In some embodiments, the originator construct and / or the benchmark construct is a circular RNA (oRNA). As used herein, the terms "oRNA", "circRNA" or "circular RNA" are used interchangeably and can refer to an RNA that forms a circular structure through covalent or non-covalent bonds.

[0511] In some embodiments, the oRNA may be non-immunogenic in a mammal (e.g., a human, non-human primate, rabbit, rat, and mouse).

[0512] In some embodiments, the oRNA may be capable of replicating or replicates in a cell from an aquaculture animal (e.g., fish, crabs, shrimp, oysters etc.), a mammalian cell, a cell from a pet or zoo animal (e.g., cats, dogs, lizards, birds, lions, tigers and bears etc.), a cell from a farm or working animal (e.g., horses, cows, pigs, chickens etc.), a human cell, cultured cells, primary cells or cell lines, stem cells, progenitor cells, differentiated cells, germ cells, cancer cells (e.g., tumorigenic, metastatic), non-tumorigenic cells (e.g., normal cells), fetal cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any combination thereof.

[0513] In some embodiments, the oRNA has a half-life of at least that of a linear counterpart. In some embodiments, the oRNA has a half-life that is increased over that of a linear counterpart. In some embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or greater. In some embodiments, the oRNA has a half-life or persistence in a cell for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween. In some embodiments, the oRNA has a half-life or persistence in a cell for no more than about 10 mins to about 7 days, or no more than about 1 hour, 2 hours,3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days),4 days, 5 days, 6 days, or 7 days.

[0514] In some embodiments, the oRNA has a half-life or persistence in a cell while the cell is dividing. In some embodiments, the oRNA has a half-life or persistence in a cell post division. In certain embodiments, the oRNA has a half-life or persistence in a dividing cell forgreater than about 10 minutes to about 30 days, or at least about 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer or any time therebetween.

[0515] In some embodiments, the oRNA modulates a cellular function, e.g., transiently or long term. In certain embodiments, the cellular function is stably altered, such as a modulation that persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours (1 day), 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer. In certain embodiments, the cellular function is transiently altered, e.g., such as a modulation that persists for no more than about 30 mins to about 7 days, or no more than about 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours (1 day), 36 hours (1.5 days), 48 hours (2 days), 60 hours (2.5 days), 72 hours (3 days), 4 days, 5 days, 6 days, or 7 days.

[0516] In some embodiments, the oRNA is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides. In some embodiments, the oRNA may be of a sufficient size to accommodate a binding site for a ribosome.

[0517] In some embodiments, the maximum size of the oRNA may be limited by the ability of packaging and delivering the RNA to a target. In some embodiments, the size of the oRNA is a length sufficient to encode polypeptides, and thus, lengths of at least 20,000 nucleotides,at least 15,000 nucleotides, at least 10,000 nucleotides, at least 7,500 nucleotides, or at least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000 nucleotides, at least 2,000 nucleotides, at least 1,000 nucleotides, at least 500 nucleotides, at least 400 nucleotides, at least 300 nucleotides, at least 200 nucleotides, at least 100 nucleotides may be useful.

[0518] In some embodiments, the oRNA comprises one or more elements described elsewhere herein. In some embodiments, the elements may be separated from one another by a spacer sequence or linker. In some embodiments, the elements may be separated from one another by 1 nucleotide, 2 nucleotides, about 5 nucleotides, about 10 nucleotides, about 15 nucleotides, about 20 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 60 nucleotides, about 80 nucleotides, about 100 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, up to about 1 kb, at least about 1000 nucleotides.

[0519] In some embodiments, one or more elements are contiguous with one another, e.g., lacking a spacer element.

[0520] In some embodiments, one or more elements is conformationally flexible. In some embodiments, the conformational flexibility is due to the sequence being substantially free of a secondary structure.

[0521] In some embodiments, the oRNA comprises a secondary or tertiary structure that accommodates a binding site for a ribosome, translation, or rolling circle translation.

[0522] In some embodiments, the oRNA comprises particular sequence characteristics. For example, the oRNA may comprise a particular nucleotide composition. In some such embodiments, the oRNA may include one or more purine rich regions (adenine or guanosine). In some such embodiments, the oRNA may include one or more purine rich regions (adenine or guanosine). In some embodiments, the oRNA may include one or more AU rich regions or elements (AREs). In some embodiments, the oRNA may include one or more adenine rich regions.

[0523] In some embodiments, the oRNA comprises one or more modifications described elsewhere herein.

[0524] In some embodiments, the oRNA comprises one or more expression sequences and is configured for persistent expression in a cell of a subject in vivo. In some embodiments, the oRNA is configured such that expression of the one or more expression sequences in the cell at a later time point is equal to or higher than an earlier time point. In such embodiments, theexpression of the one or more expression sequences can be either maintained at a relatively stable level or can increase over time. The expression of the expression sequences can be relatively stable for an extended period of time. For instance, in some cases, the expression of the one or more expression sequences in the cell over a time period of at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days does not decrease by 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%. In some cases, in some cases, the expression of the one or more expression sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23 or more days.Regulatory Elements

[0525] In some embodiments, the oRNA comprises a regulatory element. As used herein, a “regulatory element” is a sequence that modifies expression of an expression sequence. The regulatory element may include a sequence that is located adj acent to a payload or cargo region. The regulatory element may be operatively linked operatively to a payload or cargo region.

[0526] In some embodiments, a regulatory element may increase an amount of payload or cargo expressed as compared to an amount expressed when no regulatory element exists. As a non-limiting example, one regulatory element can increase an amount of payloads or cargos expressed for multiple payload or cargo sequences attached in tandem.

[0527] In some embodiments, a regulatory element may comprise a sequence to selectively initiates or activates translation of a payload or cargo.

[0528] In some embodiments, a regulatory element may comprise a sequence to initiate degradation of the oRNA or the payload or cargo. Non-limiting examples of the sequence to initiate degradation includes, but is not limited to, riboswitch aptazymes and miRNA binding sites.

[0529] In some embodiments, a regulatory element can modulate translation of the payload or cargo in the oRNA. The modulation can create an increase (enhancer) or decrease (suppressor) in the payload or cargo. The regulatory element may be located adjacent to the payload or cargo (e.g., on one side or both sides of the payload or cargo).

[0530] In some embodiments, a translation initiation sequence functions as a regulatory element. In some embodiments, the translation initiation sequence comprises an AUG / ATG codon. In some embodiments, a translation initiation sequence comprises any eukaryotic start codon such as, but not limited to, AUG / ATG, CUG / CTG, GUG / GTG, UUG / TTG, ACG, AUC / ATC, AUU, AAG, AU A / ATA, or AGG. In some embodiments, a translation initiationsequence comprises a Kozak sequence. In some embodiments, translation begins at an alternative translation initiation sequence, e.g., translation initiation sequence other than AUG / ATG codon, under selective conditions, e.g., stress induced conditions. As anon-limiting example, the translation of the circular polyribonucleotide may begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the circular polyribonucleotide translation may begin at alternative translation initiation sequence, CUG / CTG. As another non-limiting example, the translation may begin at alternative translation initiation sequence, GUG / GTG. As yet another non-limiting example, the translation may begin at a repeat-associated non- AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g., CGG, GGGGCC, CAG, CTG.Masking Agents

[0531] Masking any of the nucleotides flanking a codon that initiates translation may be used to alter the position of translation initiation, translation efficiency, length and / or structure of the oRNA. In some embodiments, a masking agent may be used near the start codon or alternative start codon in order to mask or hide the codon to reduce the probability of translation initiation at the masked start codon or alternative start codon. Non-limiting examples of masking agents include antisense locked nucleic acids (LNA) oligonucleotides and exon junction complexes (EJCs). In some embodiments, a masking agent may be used to mask a start codon of the oRNA in order to increase the likelihood that translation initiate at an alternative start codon.Translation Initiation Sequence

[0532] In some embodiments, the oRNA encodes a polypeptide or peptide and may comprise a translation initiation sequence. The translation initiation sequence may comprise, but is not limited to a start codon, a non-coding start codon, a Kozak sequence or a Shine-Dalgamo sequence. The translation initiation sequence may be located adjacent to the payload or cargo (e.g., on one side or both sides of the payload or cargo).

[0533] In some embodiments, the translation initiation sequence provides conformational flexibility to the oRNA. In some embodiments, the translation initiation sequence is within a substantially single stranded region of the oRNA.

[0534] The oRNA may include more than 1 start codon such as, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more than 15 start codons. Translation mayinitiate on the first start codon or may initiate downstream of the first start codon.

[0535] In some embodiments, the oRNA may initiate at a codon which is not the first start codon, e.g., AUG. Translation of the circular polyribonucleotide may initiate at an alternative translation initiation sequence, such as, but not limited to, ACG, AGG, AAG, CUG / CTG, GUG / GTG, AU A / ATA, AUU / ATT, UUG / TTG. In some embodiments, translation begins at an alternative translation initiation sequence under selective conditions, e.g., stress induced conditions. As a non-limiting example, the translation of the oRNA may begin at alternative translation initiation sequence, such as ACG. As another non-limiting example, the oRNA translation may begin at alternative translation initiation sequence, CUG / CTG. As yet another non-limiting example, the oRNA translation may begin at alternative translation initiation sequence, GTG / GUG. As yet another non-limiting example, the oRNA may begin translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation initiation sequence that includes short stretches of repetitive RNA e.g., CGG, GGGGCC, CAG, CTG.IRES Sequences

[0536] I...

Claims

CLAIMS1. A compound of F ormula (I) : or a pharmaceutically acceptable salt thereof,whereinR1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;X is -CH2-, -NH- or -NR7-; n is a number from 0 to 10; p is a number from 0 to 2; R5 and R6 are independently H, or an optionally substituted linear Cl -C4 alkyl group;R7 is a linear or branched C1-C6 alkyl; wherein when any of R1, R2, R3 and R4 represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R1, R2, R3 and R4 represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; and wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, alkoxycarbonyl, formyl, acyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, an aromatic moiety or an heteroaromatic moiety.

2. The compound of claim 1, or the pharmaceutically acceptable salt thereof, whereinR1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;X is -CH2-, -NH- or -NR7-; n is a number from 0 to 8; p is 0 or 1 ; R5 and R6 are independently an optionally substituted C1-C4 alkyl group;R7 is a linear or branched C1-C4 alkyl; wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, C1-C4alkoxycarbonyl, formyl, Cl- C4acyl, C 1 -C4alkoxyl, amino, -(CO)NHC1-C4alkyl, -NH(CO)C1-C4alkyl, amidine, (Cl- C4alkyl)2C=N-, cyano, nitro, azido, sulfhydryl, Cl -C4alkylthio, sulfamoyl, -(SO2)NHC1-C4alkyl, -NH(SC>2)C1-C4alkyl, -(SO2)C1-C4alkyl, 3- to 6-membered heterocyclyl, 6- to 10-membered aralkyl, or 5- to 10-membered aromatic or heteroaromatic moiety.

3. The compound of claim 1 or 2, or the pharmaceutically acceptable salt thereof, whereinR1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 8;X is -CH2-, -NH- or -NR7-; n is a number from 0 to 8; p is 0 or 1 ; R5 and R6 are independently an optionally substituted C1-C4 alkyl group; R7 is a Cl- C2 alkyl; wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, amino, cyano, nitro, azido, or sulfhydryl.

4. The compound of any one of claims 1 to 3, or the pharmaceutically acceptable salt thereof, whereinR1 and R4 are independently linear or branched C8-C20 alkyl, linear or branched C8- C20 alkenyl, or linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, linear or branched C8-C20 alkyl, linear or branched C8- C20 alkenyl, or linear or branched C8-C20 alkynyl group; m is a number from 1 to 8;X is -CH2-, -NH- or -NMe-; n is a number from 0 to 8; p is 0 or 1 ; R5 and R6 are independently a C1-C4 alkyl group optionally substituted with one or more hydroxyl or acetoxy.

5. The compound of any one of claims 1 to 4, or the pharmaceutically acceptable salt thereof, whereinR1 and R4 are independently linear C8-C20 alkyl, linear C8-C20 alkenyl, or linear C8- C20 alkynyl group;R2 and R3 are independently H, linear C8-C20 alkyl, linear C8-C20 alkenyl, or linear C8-C20 alkynyl group; m is a number from 1 to 8;X is -CH2-, -NH- or -NMe-; n is a number from 0 to 8; p is 0 or 1 ; and R5 and R6 are independently a C1-C4 alkyl group.

6. The compound of any one of claims 1 to 5, or the pharmaceutically acceptable salt thereof, wherein when any of R1, R2, R3 and R4 is C8-C20 alkenyl, the alkenyl group independently comprises one to four C=C bonds, and when any of R1, R2, R3 and R4 is C8-C20 alkynyl, the alkynyl group independently comprises one or two C=C bonds.

7. The compound of any one of claims 1 to 6, or the pharmaceutically acceptable salt thereof, whereinR1 and R4 are independently linear C8-C20 alkyl or linear C8-C20 alkenyl;R.2 and R3 are independently H, linear C8-C20 alkyl, or linear C8-C20 alkenyl; m is a number from 1 to 8;X is -CH2-, -NH- or -NMe-; n is a number from 0 to 8; p is 0 or 1 ; and R5 and R6 are independently C 1 -C4 alkyl.

8. The compound of any one of claims 1 to 7, or the pharmaceutically acceptable salt thereof, wherein R1 and R4 are identical.

9. The compound of any one of claims 1 to 8, or the pharmaceutically acceptable salt thereof, wherein R2 and R3 are H or linear C8-C18 alkyl, preferably H or C10-C16 alkyl, more preferably H or C14 alkyl.

10. The compound of any one of claims 1 to 9, or the pharmaceutically acceptable salt thereof, wherein R2 and R3 are H.

11. The compound of any one of claims 1 to 10, or the pharmaceutically acceptable salt thereof, wherein R1 and R4 are identical and represent a linear C8-C18 alkyl or linear C8-C18 alkenyl, wherein the alkenyl groups comprise one to four C=C bonds.

12. The compound of any one of claims 1 to 11, or the pharmaceutically acceptable salt thereof, wherein R1 and R4 are identical and represent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one to four C=C bonds.

13. The compound of any one of claims 1 to 12, or the pharmaceutically acceptable salt thereof, wherein R1 and R4 are identical and represent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one or two C=C bonds, preferably one C=C bond.

14. The compound of any one of claims 1 to 13, or the pharmaceutically acceptable salt thereof, wherein R5 and R6 are independently a C1-C2 alkyl group.

15. The compound of any one of claims 1 to 14, or the pharmaceutically acceptable salt thereof, wherein R5 and R6 are identical.

16. The compound of claim 1, wherein the compound of Formula (I) has a structure ofFormula (II), (12), (13), (14), (15), (16), (17) or (18):or a pharmaceutically acceptable salt thereof, wherein m is a number from 1 to 12; n is a number from 0 to 8; p is 0 or 1; q is a number from 0 to 8; r is a number from 1 to 15; s in number from 0 to 5; t is a number from 0 to 6; X is -CH2-, - NH- or -NMe-; and LI and L2 are independently a number from 0 to 3, and wherein the C=C bonds present in any of the Formula (II), (12), (14), (15), (16), (17) and (18) independently have the E or Z configuration.

17. The compound of claim 16 or a pharmaceutically acceptable salt thereof, wherein in the compounds of Formulas (13), (14), (15), (16), (17), or (18) X is -CH2-.

18. The compound of claim 1, wherein the compound has a structure of Formula (Ila):or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11; and wherein the C=C bonds present in the Formula (Ila) independently have the E or Z configuration.

19. The compound of claim 1, wherein the compound has a structure of Formula (lib):or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11; and wherein the C=C bonds present in the Formula (lib) independently have the E or Z configuration.

20. The compound of claim 1, wherein the compound has a structure of Formula (lie):or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11; and wherein the C=C bonds present in the Formula (lie) independently have the E or Z configuration.

21. The compound of claim 1, wherein the compound has a structure of Formula (I2a):or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 7; and wherein the C=C bonds present in the Formula (I2a) independently have the E or Z configuration.

22. The compound of claim 1, wherein the compound has a structure of Formula (I2b):or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 7; and wherein the C=C bonds present in the Formula (I2b) independently have the E or Z configuration.

23. The compound of claim 1, wherein the compound has a structure of Formula (I3a):or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.

24. The compound of claim 1, wherein the compound has a structure of Formula (I4a):from 1 to 8; q is a number from 0 to 11; and r is a number from 1 to 13; and wherein the C=C bonds present in the Formula (I4a) independently have the E or Z configuration.

25. The compound of claim 1, wherein the compound has a structure of Formula (I5a):or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11; and wherein the C=C bonds present in the Formula (I5a) independently have the E or Z configuration.or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 8; and wherein the C=C bonds present in the Formula (I6a) independently have the E or Z configuration.

27. The compound of claim 1, wherein the compound has a structure of Formula (17 a):or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and s is a number from 0 to 5; and wherein the C=C bonds present in the Formula (17 a) independently have the E or Z configuration.or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and t is a number from 0 to 6; and wherein the C=C bonds present in the Formula (I8a) independently have the E or Z configuration.

29. A compound selected from the group consisting of Compounds 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217 and 218 of Table 1, or a pharmaceutically acceptable salt thereof, preferably Compounds 201, 202, 203, 204, 205, 206, 208, 209, 210, 211, 216, 217 and 218 of Table 1, or a pharmaceutically acceptable salt thereof.

30. The compound of any one of claims 1 to 29, wherein the compound or the pharmaceutically acceptable salt thereof is in the form of any enantiomers, any diastereoisomers, any cis or trans geometric isomers, or any mixtures thereof.

31. A lipid nanoparticle comprising at least one compound of any one of claims 1 to 30 or the pharmaceutically acceptable salt thereof.

32. A lipid nanoparticle comprising:(a) from about 40 to about 100 mol % of an ionizable lipid;(b) from 0 to about 10 mol % of a neutral lipid;(c) from 0 to about 50 mol % of a helper lipid;(d) from 0 to about 5 mol % of a polymer-conjugated lipid; and(e) from 0 to about 5 mol % of a hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle; and wherein the ionizable lipid is at least one compound of Formula (I):whereinR1 and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group; m is a number from 1 to 12;X is -CH2- or -NH-; n is a number from 0 to 10; p is a number from 0 to 2; R5 and R6 are independently H, or an optionally substituted linear C1-C4 alkyl group; wherein when any of R1, R2, R3 and R4 represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of R1, R2, R3 and R4 represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds; and wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, alkoxycarbonyl, formyl, acyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, an aromatic moiety or an heteroaromatic moiety.

33. The lipid nanoparticle according to claim 32, wherein the compound of Formula (I) or the pharmaceutically acceptable salt thereof, is as defined in any one of claims 1 to 30.

34. The lipid nanoparticle according to claim 32 or 33, comprising:(a) from about 40 to about 60 mol % of the ionizable lipid;(b) from about 1 to about 10 mol % of the neutral lipid;(c) from 0 to about 50 mol % of the helper lipid;(d) from 0 to about 5 mol % of the polymer-conjugated lipid; and(e) from 0 to about 5 mol % of the hydrophobic component.

35. The lipid nanoparticle according to claim 32 or 33, comprising:(a) from about 40 to about 60 mol % of the ionizable lipid;(b) from about 1 to about 10 mol % of the neutral lipid;(c) from about 1 to about 50 mol % of the helper lipid;(d) from 0 to about 5 mol % of the polymer-conjugated lipid; and(e) from 0 to about 5 mol % of the hydrophobic component.

36. The lipid nanoparticle according to claim 32 or 33, comprising:(a) from about 40 to about 60 mol % of the ionizable lipid;(b) from about 1 to about 10 mol % of the neutral lipid;(c) from about 1 to about 50 mol % of the helper lipid;(d) from about 1 to about 5 mol % of the polymer-conjugated lipid; and(e) from 0 to about 5 mol % of the hydrophobic component.

37. The lipid nanoparticle according to claim 32 or 33, comprising:(a) from about 40 to about 60 mol % of the ionizable lipid;(b) from about 1 to about 10 mol % of the neutral lipid;(c) from about 1 to about 50 mol % of the helper lipid;(d) from about 1 to about 5 mol % of the polymer-conjugated lipid; and(e) from about 0.1 to about 5 mol % of the hydrophobic component.

38. The lipid nanoparticle according to claim 32 or 33, comprising:(a) from about 40 to about 60 mol % of the ionizable lipid;(b) from about 5 to about 10 mol % of the neutral lipid;(c) from about 30 to about 50 mol % of the helper lipid;(d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and(e) from about 0.1 to about 5 mol % of the hydrophobic component.

39. The lipid nanoparticle according to claim 32 or 33, comprising:(a) from about 40 to about 60 mol % of the ionizable lipid;(b) from about 5 to about 10 mol % of the neutral lipid;(c) from about 30 to about 45 mol % of the helper lipid;(d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and(e) from about 0 to about 5 mol % of the hydrophobic component.

40. The lipid nanoparticle according to claim 33 or 33, comprising:(a) from about 40 to about 60 mol % of the ionizable lipid;(b) from about 5 to about 10 mol % of the neutral lipid;(c) from about 30 to about 45 mol % of the helper lipid;(d) from about 1 to about 4 mol % of the polymer-conjugated lipid; and(e) from about 0.1 to about 5 mol % of the hydrophobic component.

41. The lipid nanoparticle according to any one of claims 32 to 40, wherein the neutral lipid comprises at least one phospholipid.

42. The lipid nanoparticle according to any one of claims 32 to 41 , wherein the neutral lipid comprises at least one phospholipid selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl- sn-glycero-3 -phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidyl choline (DLPC), 1 -myristoyl-2 -palmitoyl phosphatidylcholine (MPPC), 1-palmitoy 1-2 -myristoyl phosphatidylcholine (PMPC), 1 -palmitoyl -2-stearoyl phosphatidylcholine (PSPC), 1 ,2-diarachidoyl-sn-glycero-3 -phosphocholine (DBPC), 1-stearoyl- 2 -palmitoyl phosphatidylcholine (SPPC), 1 ,2-dieicosenoyl-sn-glycero-3 -phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof.

43. The lipid nanoparticle according to any one of claims 32 to 42, wherein the neutral lipid comprises the phospholipid DSPC, DOPC, DMPC, PE or a combination thereof.

44. The lipid nanoparticle according to any one of claims 32 to 43, wherein the helper lipid comprises a sterol, an alkyl resorcinol or a combination thereof.

45. The lipid nanoparticle according to any one of claims 32 to 44, wherein the helper lipid comprises cholesterol, 5-heptadecylresorcinol and cholesterol hemisuccinate, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, or any combination thereof.

46. The lipid nanoparticle according to any one of claims 32 to 45, wherein the helper lipid comprises cholesterol, 5-heptadecylresorcinol, cholesterol hemisuccinate, or any combination thereof.

47. The lipid nanoparticle according to any one of claims 32 to 46, wherein the helper lipid comprises at least cholesterol.

48. The lipid nanoparticle according to any one of claims 32 to 47, wherein the polymer- conjugated lipid comprises polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2- oxazoline) (POZ), polyamide (ATTA), cationic polymer, polysarcosine (Psar), polyglutamic acid (PGA), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG), or any combination thereof.

49. The lipid nanoparticle according to any one of claims 32 to 48, wherein the polymer- conjugated lipid comprises at least one PEG-lipid.

50. The lipid nanoparticle according to any one of claims 32 to 48, wherein the polymer- conjugated lipid comprises at least one PEG2k-DMG.

51. The lipid nanoparticle according to any one of claims 32 to 50, wherein the hydrophobic component comprises squalene, cardiolipin, a-tocopherol, withaferin A, vitamin A, retinol, 0-carotene, or any combination thereof.

52. The lipid nanoparticle according to claim 32 or 33, comprising:(a) from about 40 to about 60 mol% of the ionizable lipid;(b) from about 5 to about 10 mol% of a phospholipid as the neutral lipid;(c) from about 30 to about 50 mol% of a sterol as the helper lipid;(d) from about 1 to about 4 mol% of a PEG-lipid as the polymer-conjugated lipid; and(e) from about 0.1 to about 5 mol% of squalene, cardiolipin, a-tocopherol, withaferinA, vitamin A, retinol, 0-carotene, or a combination thereof as the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

53. The lipid nanoparticle according to claim 52, wherein the phospholipid is DSPC.

54. The lipid nanoparticle according to claims 52 or 53, wherein the sterol is cholesterol.

55. The lipid nanoparticle according to any one of claims 52 to 54, wherein the hydrophobic component comprises squalene, cardiolipin, a-tocopherol, or a combination thereof.

56. The lipid nanoparticle according to any one of claims 52 to 55, comprising from about 40 to about 47 mol % of the ionizable lipid, preferably from about 40 to about 45 mol % of the ionizable lipid.

57. The lipid nanoparticle according to any one of claims 31 to 56, further comprising at least one cargo.

58. The lipid nanoparticle according to claim 57, wherein the cargo comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide.

59. The lipid nanoparticle according to claim 57, wherein the cargo comprises at least one nucleic acid, such as mRNA.

60. A pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 31 to 59, and a pharmaceutical acceptable excipient.

61. The pharmaceutical composition according to claim 60, which is formulated for intramuscular administration.

62. A method for delivering a cargo to a cell comprising contacting the cell with the lipid nanoparticle according to any one of claims 31 to 59, wherein the lipid nanoparticle comprises the cargo.

63. Use of the lipid nanoparticle according to any one of claims 31 to 59, for delivering a cargo to a cell, wherein the lipid nanoparticle comprises the cargo.

64. A vaccine comprising the lipid nanoparticle according to any one of claims 31 to 56, wherein the lipid nanoparticle comprises at least one cargo, preferably the cargo comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide, more preferably the cargo comprises at least one nucleic acid such as mRNA.

65. A method of vaccinating a subject against an infectious agent comprising:(i) contacting the subject with the vaccine according to claim 64, and(ii) eliciting an immune response.

66. The method according to claim 65, wherein the infectious agent is a virus, a bacterium, or a parasite.

67. The method according to claim 65, wherein the infectious agent is Campylobacter jejuni, Clostridium difficile, entamoeba histolytica, enterotoxin B, Norwalk virus or norovirus, Helicobacter pylori, rotavirus, Candida yeast, coronavirus including SARS-CoV, SARS-CoV-2 and MERS-CoV, Enterovirus 71, Epstein-Barr virus, Gram-Negative Bacteria including Bordetella, Gram-Positive Bacteria including Clostridium Tetani, Francisella Tularensis, Streptococcus bacteria and Staphylococcus bacteria, and Hepatitis, Human Cytomegalovirus, Human Immunodeficiency Virus, Human Papilloma Virus, Influenza, John Cunningham Virus, Mycobacterium, Poxviruses, Pseudomonas Aeruginosa, Respiratory Syncytial Virus, Rubella virus, Varicella zoster virus, Chikungunya virus, Dengue virus, Rabies virus, Trypanosoma cruzi and / or Chagas disease, Ebola virus, Plasmodium falciparum, Marburg virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile Virus, Yellow Fever virus, Bacillus anthracis, Botulinum toxin, Ricin, or Shiga toxin and / or Shiga-like toxin.

68. Use of the vaccine of claim 64 for vaccinating a subject against an infectious agent.

69. The use according to claim 68, wherein the infectious agent is a virus, a bacterium, or a parasite.

70. The use of claim 68, wherein the infectious agent is Campylobacter jejuni, Clostridium difficile, entamoeba histolytica, enterotoxin B, Norwalk virus or norovirus, Helicobacter pylori, rotavirus, Candida yeast, coronavirus including SARS-CoV, SARS-CoV-2 and MERS-CoV, Enterovirus 71, Epstein-Barr virus, Gram-Negative Bacteria including Bordetella, Gram-Positive Bacteria including Clostridium Tetani, Francisella Tularensis, Streptococcus bacteria and Staphylococcus bacteria, and Hepatitis, Human Cytomegalovirus, Human Immunodeficiency Virus, Human Papilloma Virus, Influenza, John Cunningham Virus, Mycobacterium, Poxviruses, Pseudomonas Aeruginosa, Respiratory Syncytial Virus, Rubella virus, Varicella zoster virus, Chikungunya virus, Dengue virus, Rabies virus, Trypanosoma cruzi and / or Chagas disease, Ebola virus, Plasmodium falciparum, Marburg virus, Japanese encephalitis virus, St. Louis encephalitis virus, West Nile Virus, Yellow Fever virus, Bacillus anthracis, Botulinum toxin, Ricin, or Shiga toxin and / or Shiga-like toxin.

71. A method of treating cancer in a subject comprising administering the lipid nanoparticle according to any one of claims 31 to 56 to the subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

72. The method according to claim 71, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, leukemia, central nervous system cancers (e.g., brain cancer such as glioblastoma), myeloma, or melanoma.

73. A lipid nanoparticle according to any one of claims 31 to 56 for use in the treatment of cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

74. The lipid nanoparticle for use according to claim 73, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, leukemia, central nervous system cancers, myeloma, or melanoma.

75. Use of the lipid nanoparticle according to any one of claims 31 to 56 for treating cancer in a subject, wherein the lipid nanoparticle comprises an anti-cancer cargo or a cargo triggering an immune response against cancer cells.

76. The use according to claim 75, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, leukemia, central nervous system cancers (e.g., brain cancer such as glioblastoma), myeloma, or melanoma.

77. Use of the lipid nanoparticle according to any one of claims 31 to 56 for the preparation of a medicament for treating cancer in a subject, wherein the lipid nanoparticle comprises an anti- cancer cargo or a cargo triggering an immune response against cancer cells.

78. The use according to claim 77, wherein the cancer is lung cancer, breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, leukemia, central nervous system cancers (e.g., brain cancer such as glioblastoma), myeloma, or melanoma.

79. The method of claim 71 or 72, the lipid nanoparticle for use of claim 73 or 74, or the use of any one of claims 75 to 78, wherein the cargo comprises at least one of a small molecule, an antibody, a polynucleotide or a polypeptide.

80. The method of claim 71 or 72, the lipid nanoparticle for use of claim 73 or 74, or the use of any one of claims 75 to 78, wherein the cargo comprises at least one nucleic acid, such as mRNA.

81. A lipid nanoparticle according to any one of claims 31 to 59 for use in transfection of targeted cells, e.g. in transfecting human cells, including stem cells.

82. A lipid nanoparticle according to any one of claims 31 to 59 for use in gene replacing therapy.

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