Lipid nanoparticles comprising fatty amine polyphosphocholinated conjugates

Abstract

One or more pMPC conjugate(s) and lipid nanoparticles comprising same are provided. Pharmaceutical compositions comprising the lipid nanoparticles encapsulating an active agent are also provided. Use of the pharmaceutical composition for delivering a cargo (e.g. a nucleotide) to a subject is also provided.

Description

LIPID NANOPARTICLES COMPRISING FATTY AMINEPOLYPHOSPHOCHOLINATED CONJUGATESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 530,518, filed August 3, 2023, titled “POLY(PHOSPHOCHOLINATION) OF LIPID NANOPARTICLES AS A NEW PLATFORM FOR NUCLEIC ACID DELIVERY / RNA THERAPEUTICS”. The contents of which are all incorporated herein by reference in their entirety.FIELD OF INVENTION

[0002] The present invention is directed to polyphosphocholinated conjugates and lipid nanoparticles comprising same and use thereof in pharmaceutical compositions.BACKGROUND OF THE INVENTION

[0003] Therapeutic strategies based on RNAs offer several benefits on pathologies including cancer, infectious, immune and neurological diseases as they are able to manipulate the expression of therapeutic proteins. Among them, mRNA has convincingly demonstrated its therapeutic efficiency with the FDA-approved mRNA- based vaccine against SARS-CoV2, while others are in the stage of clinical trials. mRNA is a ribonucleic acid which carries the coding information for protein synthesis, is non-infectious, has cytosolic site of action and it does not integrate into the host genome incurring no risk of mutagenesis. mRNA has a broad spectrum of applications from cancer immunotherapies to inflammation and genome editing. Nevertheless, naked mRNA is susceptible to ribonucleases (RNases) degradation while its hydrophilicity, negative charge and interaction with proteins limit its access to its intracellular target. Different strategies have been followed to enhance RNA payloads therapeutic efficacy by increasing its stability and translational efficiency by the introduction of 5’ cap, poly(A) tail and others.

[0004] The recent successful example of mRNA vaccines emerges - among others - from the ability of the lipid nanoparticles (LNPs) to bypass the difficulties following administration and the cellular delivery of exogenous mRNA. LNPs constitute a characteristic example in the intersection of nanotechnology and biomaterials across the pharmaceutical industry proving to be promising vehicles to deliver a variety oftherapeutic agents. An ionizable cationic lipid, polyethylene glycol (PEG) conjugated with a hydrophobic moiety, cholesterol, and phospholipids, are the basic ingredients of LNPs currently used for Pfizer-BioNTech and Modema COVID-19 vaccines.

[0005] PEGylation is a common strategy applied for nanoparticles used for drug delivery, from liposomes to LNPs, as it offers “stealth” properties to the attached systems, increased dispersion steric stabilization and improved blood-circulation. However, PEG-based functionalization is still limited by multiple factors such as reduced cell uptake, reduced endosomal escape and increasing immune response activation upon repeated doses. Different studies - from PEGylated liposomes to proteins - have demonstrated that PEGylation elicits antibody responses (anti-PEG antibodies) and this immunogenic response results in the accelerated-blood clearance (ABC) effect. When the ABC phenomenon occurs, any subsequent dose of PEGylated substances accumulates rapidly in the liver reducing the therapeutic efficacy of any encapsulated drug. In addition, it has been shown that PEGylated liposomes could induce hypersensitivity reactions and even in some limited cases could lead to an anaphylactic reaction, known as Complement Activation Related Pseydoallergy (CARP A). Interestingly, the US Food and Drug Association has updated its guidelines to screen for anti-PEG antibodies during clinical trials of PEGylated therapeutics.

[0006] To this end, there is an unmet need for alternatives to non-PEGylated conjugates which are capable for increasing colloidal stability, prolonged blood retention, increased cellular uptake and reduced host immune response of LNPs comprising same.SUMMARY OF THE INVENTION

[0007] According to a first aspect, there is provided a compound, a salt of said compound or both, represented by Formula 1 :, wherein:A is a spacer;R is H or an optionally substituted C1-C10 alkyl; each Li independently is selected from an optionally substituted C5-C30 alkyl,represents a single bond, a triple bond or a double bond; each X independently comprises CH2, CHR’2, an optionally substituted C1-C10alkyl, an optionally substituted C5-C30 alkyl, an optionally substituted C5-C30 alkenyl or an optionally substituted C5-C30 alkynyl; each T independently represents NR’ 2, NH, O, S, -CONH-, -CONR’-, - C(=NH)NR’-, -C(=S)NR’-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, - NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N- , or phosphate, as allowed by valency; each R’ is independently H or comprises an optionally substituted C1-C10alkyl, an C1-C10alkyl-aryl, an C1- C10alkyl-cycloalkyl, optionally substituted C3- C10cycloalkyl, optionally substituted C3-C10heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl or a combination thereof;Y is halo; n is between 2 and 300; each k is independently between 0 and 10; and wherein at least one L is not H.

[0008] In some embodiments, R is methyl.

[0009] In some embodiments, each L is independently C10-C20 alkyl or C10-C20 alkenyl.

[0010] In some embodiments, A comprises, wherein R1 represents hydrogen, halogen, -NO2, -CN, -OH, oxo, imino, -CONH2, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -CONH- NH2, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR’, -NC(=S)OR’, - NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, Ci- C6, haloalkyl, optionally substituted C1-C6alkyl, -NH2, -NR’2, -NH(C1-C6alkyl), - N(C1-C6alkyl)2, C1-C6alkoxy, C1-C6haloalkoxy, hydroxy(C1-C6alkyl), hydroxy(C1-C6alkoxy), alkoxy(C1-C6alkyl), alkoxy(C1-C6alkoxy), C1-C6alkyl- NR’2, C1-C6alkyl-SR’, -CONH(C1-C6alkyl), -CON(C1-C6alkyl)2, -CO2H, -CO2R’, -OCOR, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, or a combination thereof; wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted C1-C10alkyl, optionally substituted C3-C10cycloalkyl, optionally substituted C3C-10heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, hydroxy, amino, -NH2, -NR’2- NH(C1-C6alkyl), -N(C1-C6alkyl)2, C1-C6alkoxy, C1-C6haloalkoxy, hydroxy(C1-C6alkyl), hydroxy(C1-C6alkoxy), alkoxy(C1-C6alkyl), alkoxy(C1-C6alkoxy), C1-C6alkyl-NR’2, C1-C6alkyl-SR’, or a combination thereof.

[0011] In some embodiments, the compound is represented by Formula 2:wherein each Li is independently C10-C20 alkyl or C10-C20 alkenyl, and wherein Y is halo.

[0012] In some embodiments, the compound has an average MW between about 1000 and about 30.000Da; and wherein each LI is independently C12-C20 saturated alkyl.

[0013] In some embodiments, each LI is selected from tetradecyl and octadecyl.

[0014] In another aspect, there is provided a lipid nanoparticle (LNP) comprising the compound of the invention and an active agent, wherein said lipid nanoparticle further comprises an ionizable lipid, a helper lipid, and a structural lipid.

[0015] In some embodiments, the lipid nanoparticle is a plurality of particles characterized by at least one of: an average size in a range between 30 and 300 nm; and an average zeta potential between 0 and -25mV at a pH of about 7.

[0016] In some embodiments, the active agent comprises a nucleic acid; and wherein said structural lipid is a sterol.

[0017] In some embodiments, a molar concentration of the compound relative to the total lipid content of the lipid nanoparticle is between 0.1 and 10 mol %.

[0018] In some embodiments, a molar concentration of any one of the ionizable lipid and the structural lipid relative to the total lipid content of the lipid nanoparticle is between 10 and 60mol%.

[0019] In some embodiments, a molar ratio between the ionizable lipid and the helper lipid within the lipid nanoparticle is between 3 : 1 and 6: 1.

[0020] In some embodiments, the lipid nanoparticle of the invention is characterized by N:P ratio between 1 and 20; and wherein the molar concentration of the compound relative to the total lipid content of the lipid nanoparticle is between 1 and 5 mol %.

[0021] In some embodiments, the structural lipid is cholesterol, wherein said ionizable lipid is ALC-0315, wherein said helper lipid is DSPC, wherein a molar concentration of the ionizable lipid and of the structural lipid is between about 40 and about 50mol%; wherein the active agent is RNA; and wherein the lipid nanoparticle further comprises between 0.5 and 10mol% of an C10-C20 alkyl amine.

[0022] In another aspect, there is provided a pharmaceutical composition comprises a plurality of the lipid nanoparticles of the present invention and a pharmaceutically acceptable carrier.

[0023] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the active agent.

[0024] In some embodiments, the lipid nanoparticles of the pharmaceutical composition of the invention are characterized by an average particle size between 50 and 150nm; and by a PDI of between 0.05 and 0.2.

[0025] In some embodiments, the pharmaceutical composition is for use in the treatment of a disease or disorder in a subject in need thereof.

[0026] In another aspect, there is provided a method for delivering an active agent to a tissue of a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of the present invention, thereby delivering the active agent to said tissue.

[0027] In some embodiments, said administering is via intravenous, intramuscular, pulmonary, intratracheal, intranasal or topical administration.

[0028] In some embodiments, the tissue comprises liver tissue, spleen tissue, or both.

[0029] In another aspect, there is provided the pharmaceutical composition of the present invention for use in a method for delivering an active agent to a tissue of a subject in need thereof.

[0030] In another aspect, there is provided use of the pharmaceutical composition of the present invention for preparation of a medicament for delivering an active agent to a tissue of a subject in need thereof.

[0031] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0032] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0033] Figure 1. 'H-NMR characterization of DTA-pMPC in CD3OD and CDCh (1 :1). The molecular weight of pMPC could be calculated from NMR (-1900), asestimated from comparing the integral intensity ratio of the1HNMR peaks attributed to the long alkyl chain at 1.26 ppm and the MPC pendant methylene protons at 3.75 ppm.

[0034] Figures 2A-2C and 2D1-2D2. Changes in hydrodynamic diameter (Size by Intensity) of functionalized LNPs (pMPC- or PEG-) with various polymer lengths at Day 1 and Day 60. (A) (C14)2 poly(A)-PEG-LNPs with 2 kDa chain length at 1.6 % grafting in the absence and presence of 5.4 % SA. (B) (C14)2 poly(A)-pMPC-LNPs with 5 kDa chain length at 1.6 % grafting in the absence and presence of 5.4 % SA. (C) (C1Q2 poly(A)-pMPC 20kDa LNPs at 1.6% grafting in the absence and presence of 5.3% SA. (DI) Size and PDI (D2) changes as function of time of (C18)2 poly(A)- PEG2kDa and C18poly(A)- pMPC20kDa in the presence of SA. Systems were stored at 4°C and measured at 25°C.

[0035] Figures 3A1-3A3 and 3B. Structural characterization of exemplary LNPs of the invention. Representative cryogenic electron micrograph of (C14)2 poly(A)-pMPC- functionalized LNPs (5 kDa, with 5.4 % SA) (3A1) and (C18)2 poly(A)-pMPC- functionalized LNPs (5 kDa (3A2) and 20 kDa (3A3), with SA). Scale bar, 50 nm. Schematic illustration of an exemplary LNP of the invention (3B).

[0036] Figures 4A-4B. Quantified cellular uptake of Dil - labeled LNPs functionalized with (C14)2 poly(A)-pMPC (5 kDa) or (C14)2 poly(A)-PEG (2 kDa) with or without positive charge (SA), obtained from ImageStream analysis. (A) Uptake by RAW 264.7 cell culture, (B) Uptake by Vero E6 cell line. The data is shown as a violin plot reflecting the data distribution from two technical repeats and an open circle indicating the median of each data set. The values are plotted on a loglO scale. The number of cells collected per tested condition is > 1000.

[0037] Figures 5A-5B. Quantified cellular uptake of Dil - labeled LNPs functionalized with (C14)2 poly(A)-pMPC (20 kDa) or (C14)2 poly(A)-PEG (2 kDa) with or without positive charge (SA), obtained from ImageStream analysis. (A) Uptake by RAW 264.7 cell culture, (B) Uptake by Vero E6 cell line. The data is shown as a violin plot reflecting the data distribution from two technical repeats, and an open circle indicating the median of each data set. The values are plotted on a loglO scale. The number of cells collected per tested condition is > 1000.

[0038] Figure 6. Representative images from ImageStream of (C14)2 poly(A)-pMPC (20 kDa)- and (C14)2 poly(A)-PEG (2kDa) - functionalized LNPs internalized by RAW264.7 cells. Bright field and fluorescent probe, Dil (LNPs membrane staining) were recorded in two channels. Scale bar, 7 μm.

[0039] Figure 7. Cytotoxicity evaluation of (C14)2 poly(A)-pMPC (5kDa)- and (C14)2 poly(A)-PEG-LNPs on Vero E6 cells culture tested at two concentrations (0.1 mM and 0.05 mM). Data represents one biological repeat with four technical repeats. Abbreviation: NT-not treated.

[0040] Figures 8A-8B. Blood clearance profile of pMPCylated and PEGylated- LNPs (C14)2 (A) and (C18)2 poly(A)-pMPCylated and PEGylated- LNPs with Dil (B) in mice upon intravenous injection. The values are expressed as mean ± SEM of minimum 3 animals per group.

[0041] Figures 9A-9C. Organ biodistribution of the (A) (C14)2 poly(A)-PEG (2kDa)- LNPS, (B) (C14)2 poly(A)-pMPC (5kDa)-LNPs and (C) pMPC (20kDa)-LNPs in CD1 mice.

[0042] Figure 10A-10C. Blood clearance profile of A: (C18)2 poly(A)-PEG 2kDa LNPs, B: (C18)2 poly(A)-pMPC5kDa LNPs and C: (C18)2poly(A)-pMPC 20kDa LNPs after the 1st and 2nd dose. The values are expressed as mean ± SEM of n> 3 animals per group.

[0043] Figures 11A-11C. LNPs size (A), poly dispersity index (PDI) (B)and zeta potential measurements (C) as obtained from dynamic light scattering (DLS) for (C18)2 poly(A)- and (C18)2 fluc-mRNA-LNPs.

[0044] Figures 12A-12D. Colloidal stability over time of the fluc-mRNA LNPs upon storage at 4°C. Size distribution of (A) (C18)2 fluc-mRNA PEG 2kDa LNPs (B) (C18)2 fluc-mRNA pMPC 5kDa LNPs and (C) (C18)2fluc-mRNA pMPC 20kDa LNPs the day of preparation (Day 1) and 25 days after (Day 25). (D) Poly dispersity index (PDI) changes for all systems in different time points.

[0045] Figures 13A-13F. (A-C) AFM topography images of (A) (C18)2 fluc-mRNA PEG 2kDa LNPs (B) (C18)2fluc-mRNA pMPC 5kDa LNPs and (C) (C18)2fluc-mRNA pMPC 20kDa LNPs on mica in lx PBS supplemented with MgSCL (LNPs concentration 0.5mM). Scale bar 400nm. (D-F) Cryo-TEM images of (D) (C18)2 fluc- mRNA PEG 2kDa LNPs (E) (C18)2fluc-mRNA pMPC 5kDa LNPs and (F) (C18)2fluc- mRNA pMPC 20kDa LNPs. Scale bar lOOnm.

[0046] Figures 14A-14B. Cell viability expressed as % for RAW 264.7 (A) and Vero E6 cells (B) upon 72 h incubation with different (C18)2 fluc-mRNA LNPs (dose: 20 ngand 100 ng / well). Data represents one biological repeat with six technical repeats.Abbreviation: NT-not treated.

[0047] Figures 15A-15B. In vitro transfection of RAW 264.7 (A) and Vero E6 cells (B) upon 48 h incubation with different (C18)2 fluc-mRNA LNPs (dose: lOOng of mRNA / well). Data represents one biological repeat with n>5 technical repeats. Abbreviation: NT-not treated.

[0048] Figures 16A-16F. (A) Body weight before and 24h after single injection of PBS and (C18)2-fluc mRNA pMPC 5kDa and 20kDa (0.15 mg / kg). (B-F) Organ to body weight ratio of internal organs (liver, spleen, kidneys, heart and lungs).

[0049] Figures 17A-17D. CD1 mice were IV injected with (C18)2 fluc-mRNA LNPs (0.15 mg / kg) and luciferease intensity was measured at 6 hr and 24h post- IV injection. (A) IVIS images at 6 hr post injection. (B) Luciferase signal intensity (p / s) at 6 hr and 24 hr post injection, presented as mean±STDEV (n=6 mice). (C) Representative organ images for in vivo luciferase assay harvested 24 h post injection. (D) Bar graphs of luciferase intensity in different organs presented as mean±STDEV (n=3 mice) 24hr post injection.

[0050] Figure 18. IgM level 5-days post-injection of intravenously administrated PBS (control), (C18)2 fluc-mRNA PEG- (2kDa) and (C18)2 fluc-mRNA pMPCylated LNPs (5kDa and 20kDa) in mice blood serum (n=4).DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention provides polyphosphocholinated (pMPC) conjugates and lipid nanoparticles comprising same and use thereof in pharmaceutical compositions. The present invention, in some embodiments thereof, is based on a surprising finding that exemplary pMPC conjugates of the invention enhanced aqueous stability of LNPs comprising same at 4°C. Moreover, upon IV injection of LNPs comprising the exemplary pMPC conjugates of the invention, the inventors observed a significantly decreased immune response (IgM production) of the host animal, as compared to PEGylated LNPs. pMPC-conjugate

[0052] According to one aspect of the invention, there is provided a compound and / or a salt thereof, comprising a polymeric moiety covalent bound to an amine moiety; wherein the polymeric moiety comprises a plurality of repeating units, each repeatingunit comprises Z moiety bound to a backbone unit, wherein Z is as described hereinbelow. In some embodiments, each repeating unit comprises phosphorylcholine bound to the backbone unit. In some embodiments, the amine moiety comprises an alkyl-amine selected from a secondary and a tertiary alkyl amine. In some embodiments, the amine moiety is represented by a general Formula: -(Ll)2-N-, wherein each LI is H or alkyl / alkenyl / alkynyl, and at least one LI is not H. In some embodiments, the amine moiety (e.g. LI) and / or the compound of the invention is devoid of a phospholipid, and wherein the polymeric moiety is devoid of PEG. In some embodiments, the polymeric moiety is a zwitterionic polymer. In some embodiments, the polymeric moiety is or comprises poly[2-(methacryloyloxy)ethyl phosphorylcholine] (pMPC).

[0053] In some embodiments, the compound of the invention is represented by Formula 1 :, wherein: m is zero or a positive integer; n is an integer which is at least 2; Y is a backbone unit which forms a polymeric backbone; L is absent or is a linking moiety; each Li independently is H, an alkyl or an alkenyl / alkynyl; wherein at least one Li is not H; and Z has the general formula:, wherein A is a substituted or unsubstituted hydrocarbon; B is an oxygen atom or is absent; and Rl, R2 and R3 are each independently hydrogen, alkyl, cycloalkyl, heteroalicyclic, aryl or heteroaryl.

[0054] In some embodiments, each Li is the same or different and is independently selected from an optionally substituted C5-C30 alkyl, H and, whereinrepresents a single bond, a triple bond or a double bond; each X independently comprises CH2, CHR’2, anoptionally substituted C1-C10 alkyl, an optionally substituted C5-C30 alkyl or an optionally substituted C5-C30 alkenyl / alkynyl; wherein each T independently represents NR’ 2, NH, O, S, -CONH-, -CONR’-, -C(=NH)NR’-, -C(=S)NR’-, -NC(=O)- , -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-, or phosphate, as allowed by valency; and each R’ is independently H or comprises an optionally substituted C1-C10alkyl, an C1-C10alkyl-aryl, an C1-C10alkyl-cycloalkyl, optionally substitutedC3- C10cycloalkyl, optionally substituted C3-C10heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl or a combination thereof.

[0055] In some embodiments, n is between 2 and 300.

[0056] As used herein, the term "backbone unit" refers to a repeating unit, wherein linkage of a plurality of the repeating unit (e.g., sequential linkage) forms a polymeric backbone. A plurality of linked repeating units per se is also referred to herein as a "polymeric backbone".

[0057] As shown in formulas I and la, L and Z together form a pendant group of at least a portion of the backbone units, which group is referred to herein for brevity simply as the "pendant group".

[0058] Each backbone unit Y with pendant group (i.e., a unit represented by Y(-L- Z), the number of which is represented by the variable n) and each backbone unit Y without a pendant group (the number of which is represented by the variable m) is also referred to herein as a "monomeric unit" .

[0059] A backbone unit may optionally be a residue of a polymerizable monomer or polymerizable moiety of a monomer. A wide variety of polymerizable monomers and moieties will be known to the skilled person, and the structure of the residues of such monomers which result upon polymerization (e.g., monomeric units) will also be known to the skilled person.

[0060] A "residue of a polymerizable monomer" refers to a modified form of a polymerizable monomer and / or a portion of a polymerizable monomer that remains after polymerization.

[0061] A portion of a polymerizable monomer may be formed, for example, by a condensation reaction, e.g., wherein at least one atom or group (e.g., a hydrogen atom or hydroxyl group) in the monomer, and optionally at least two atoms or groups (e.g., a hydrogen atom and a hydroxyl group) in the monomer, is replaced with a covalent bond with another polymerizable monomer.

[0062] A modified form of a polymerizable monomer may be formed, for example, by ring opening (wherein a covalent bond between two atoms in a ring is broken, and the two atoms optionally each become linked to another polymerizable monomer); and / or by adding to an unsaturated bond, wherein an unsaturated bond between two adjacent atoms is broken (e.g., conversion of an unsaturated double bond to a saturated bond, or conversion of an unsaturated triple bond to an unsaturated double bond) and the two atoms optionally each become linked to another polymerizable monomer.

[0063] A modified form of a polymerizable monomer may consist essentially of the same atoms as the original monomer, for example, different merely in the rearrangement of covalent bonds, or alternatively, may have a different atomic composition, for example, wherein polymerization includes a condensation reaction (e.g., as described herein). Examples of backbone units include, without limitation, substituted or unsubstituted hydrocarbons (which may form a substituted or unsubstituted hydrocarbon backbone), such as alkylene units; hydroxycarboxylic acid units (which may form a polyester backbone), e.g., glycolate, lactate, hydroxybutyrate, hydroxyvalerate, hydroxycaproate and hydroxybenzoate units; dicarboxylic acid units (which may form a polyester backbone in combination with a diol and / or a polyamide in combination with a diamine), e.g., adipate, succinate, terephthalate and naphthalene dicarboxylic acid units; diol units (which may form a polyether backbone, or form a polyester backbone in combination with a dicarboxylic acid), e.g., ethylene glycol, 1,2- propanediol, 1,3- propanediol, 1,4-butanediol, and bisphenol A units; diamine units (which may form a polyamide backbone in combination with a dicarboxylic acid), e.g., para- phenylene diamine and alkylene diamines such hexylene diamine; carbamate units (which may form a polyurethane backbone); amino acid residues (which may form a polypeptide backbone); and saccharide residues (which may form a polysaccharide backbone).

[0064] In some embodiments, Y is a substituted or unsubstituted alkylene unit.

[0065] In some embodiments, Y is a substituted or unsubstituted ethylene unit, that is, an alkylene unit 2 atoms in length.

[0066] Polymeric backbones wherein Y is a substituted or unsubstituted ethylene unit may optionally be a polymeric backbone such as formed by polymerizing ethylene (CH2=CH2) and / or substituted derivatives thereof (also referred to herein as "vinyl monomers"). Such polymerization is a very well- studied procedure, and one ofordinary skill in the art will be aware of numerous techniques for effecting such polymerization.

[0067] It is to be understood that any embodiments described herein relating to a polymeric backbone formed by a polymerization encompass any polymeric backbone having a structure which can be formed by such polymerization, regardless of whether the polymeric backbone was formed in practice by such polymerization (or any other type of polymerization).

[0068] As is well known in the art, the unsaturated bond of ethylene and substituted ethylene derivatives becomes saturated upon polymerization, such that the backbone units in a polymeric backbone are saturated, although they may be referred to as units of an unsaturated compound (e.g., a "vinyl monomer" or “olefin monomer”) to which they are analogous.

[0069] Polymers which can be formed from unsaturated monomers such as vinyl monomers and olefin monomers are also referred to by the terms "polyvinyl" and "polyolefin".

[0070] Herein, an "unsubstituted" alkylene unit (e.g., ethylene unit) refers to an alkylene unit which does not have any substituent other than the pendant group discussed herein (represented as (-L- Z)). That is, an alkylene unit attached to the aforementioned pendant group is considered unsubstituted if there are no substituents at any other positions on the alkylene unit.

[0071] In some embodiments, Y has the formula - CR4R5-CR6D-.

[0072] When Y is a backbone unit which is not attached to L or Z (i.e., to a pendant group described herein), D is R7 (an end group, as defined herein); and when Y is a backbone unit which is attached to L or Z, D is a covalent bond or a linking group attaching Y to L or Z. The linking group may optionally be -O-, -S-, arylene, sulfinyl, sulfonyl, phosphate, phosphonyl, phosphinyl, carbonyl, thiocarbonyl, urea, thiourea, O- carbamyl, N-carbamyl, O -thiocarb amyl, N-thiocarbamyl, C-amido, N-amido, C- carboxy, O-carboxy, sulfonamido, or amino.

[0073] R4-R7 are each independently hydrogen, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, azo, phosphate phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C- carboxy, O-carboxy, sulfonamido, or amino.

[0074] Herein, the phrase “linking group” describes a group (e.g., a substituent) that is attached to two or more moieties in the compound.

[0075] Herein, the phrase “end group” describes a group (e.g., a substituent) that is attached to a single moiety in the compound via one atom thereof.

[0076] When each of R4-R6 is hydrogen, and D is a covalent bond or linking group, Y is an unsubstituted ethylene group attached (via D) to a pendant group described herein.

[0077] When each of R4-R7 is hydrogen (and D is R7), Y is an unsubstituted ethylene group which is not attached to a pendant group described herein.

[0078] In some embodiments, R4 and R5 are each hydrogen. Such embodiments include polymeric backbones formed from many widely used vinyl monomers (including ethylene), including, for example, olefins (e.g., ethylene, propylene, 1- butylene, isobutylene, 4-methyl-l-pentene), vinyl chloride, styrene, vinyl acetate, acrylonitrile, acrylate and derivatives thereof (e.g., acrylate esters, acrylamides), and methacrylate and derivatives thereof (e.g., methacrylate esters, methacrylamides).

[0079] In some embodiments, Re is hydrogen. In some such embodiments, R4 and R5 are each hydrogen.

[0080] In some embodiments, R is methyl. In some such embodiments, R4 and R5 are each hydrogen. In some such embodiments, the backbone unit is a unit of methacrylate or a derivative thereof (e.g., methacrylate ester, methacrylamide). In some embodiments, the linking group represented by the variable D is -O-, -C(=O)O-, - C(=O)NH- or phenylene. In exemplary embodiments, D is -C(=O)O-.

[0081] For example, the backbone unit may optionally be a vinyl alcohol derivative (e.g., an ester or ether of a vinyl alcohol unit) when D is -O-; an acrylate or methacrylate derivative (e.g., an ester of an acrylate or methacrylate unit) when D is -C(=O)O-; an acrylamide or methacrylamide unit when D is -C(=O)NH-; and / or a styrene derivative (e.g., a substituted styrene unit) when D is phenylene.

[0082] In some embodiments, L is a substituted or unsubstituted hydrocarbon from 1 to 10 carbon atoms in length. In some embodiments, the hydrocarbon is unsubstituted. In some embodiments, the hydrocarbon is a linear, unsubstituted hydrocarbon, that is, -(CI );- wherein i is an integer from 1 to 10.

[0083] In some embodiments, L is a substituted or unsubstituted ethylene group. In some embodiments, L is an unsubstituted ethylene group (- CH2CH2-).

[0084] In some embodiments, B is an oxygen atom. In some such embodiments, L is a hydrocarbon according to any of the respective embodiments described herein (i.e., L is not absent), and Z is a phosphate group attached to L.

[0085] In some embodiments, B is absent. In some such embodiments, L is a hydrocarbon according to any of the respective embodiments described herein (i.e., L is not absent), and Z is a phosphonate group attached to L. In some embodiments, L is also absent, such that the phosphorus atom of formula II is attached directly to Y.

[0086] In some embodiments, A is a substituted or unsubstituted hydrocarbon from 1 to 4 carbon atoms in length.

[0087] In some embodiments, A is an unsubstituted hydrocarbon. In some such embodiments, the unsubstituted hydrocarbon is from 1 to 4 carbon atoms in length. In some embodiments, the hydrocarbon is a linear, unsubstituted hydrocarbon, that is, - (CH2)j- wherein j is an integer from 1 to 4.

[0088] In some embodiments, A is a substituted or unsubstituted ethylene group.

[0089] In some embodiments, A is an unsubstituted ethylene group (-CH2CH2-). In such embodiments, the moiety having general formula II (represented by the variable Z) is similar or identical to a phosphoethanolamine or phosphocholine moiety. Phosphoethanolamine and phosphocholine moieties are present in many naturally occurring compounds (e.g., phosphatidylcholines, phosphatidylethanolamines).

[0090] In some embodiments, A is an ethylene group substituted by a C-carboxy group. In some embodiments, the C-carboxy is attached to the carbon atom adjacent to the nitrogen atom depicted in formula II (rather than the carbon atom attached to the depicted oxygen atom). In such embodiments, the moiety having general formula II (represented by the variable Z) is similar or identical to a phosphoserine moiety. Phosphoserine is present in many naturally occurring compounds (e.g., phosphatidylserines).

[0091] Without being bound by any particular theory, it is believed that moieties similar or identical to naturally occurring moieties such as phosphocholine, phosphoethanolamine and / or phosphoserine may be particularly biocompatible.

[0092] In some embodiments, R1-R3 (the substituents of the nitrogen atom depicted in general formula II) are each independently hydrogen or C 1-4- alkyl. In some embodiments, R1-R3 are each independently hydrogen or methyl. In some embodiments, R1-R3 are each methyl. In some such embodiments, R1-R3 are each hydrogen.

[0093] The variable n may be regarded as representing a number of backbone units (represented by the variable Y) which are substituted by the pendant group represented by (-L-Z), and the variable m may be regarded as representing a number of backbone units which are not substituted by such a pendant group. The sum n+m may be regarded as representing the total number of backbone units in the polymeric backbone. The ratio n / (n+m) may be regarded as representing the fraction of backbone units which are substituted by the pendant group represented by (-L-Z).

[0094] The backbone unit Y substituted by the pendant group may be the same as or different than the backbone unit Y which is not substituted by the pendant group (e.g., when m is at least 1).

[0095] The plurality (indicated by the variable n) of backbone units Y substituted by the pendant group may be the same as each other or different from each other.

[0096] In addition, the plurality (indicated by the variable n) of pendant groups (-L- Z) attached to a plurality of backbone units Y may be the same as each other or different from each other (e.g., may differ in the identity of any one or more of A, B, Ri, R2, R3 and L).

[0097] In any of the embodiments described herein wherein more than one backbone unit Y is not substituted by the pendant group described herein (i.e., when m is more than 1), the plurality (indicated by the variable m) of backbone units Y which are substituted by the pendant group may be the same as each other or different from each other.

[0098] The number of types of backbone units substituted by the pendant group, the number of types of backbone units not substituted by the pendant group (if any such units are present), and / or the number of types of pendant group in the polymeric moiety may each independently be any number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more).

[0099] In some embodiments, the polymeric moiety is a copolymer moiety, that is, the polymeric moiety comprises at least two different types of monomeric unit. The different types of monomeric unit may differ in whether they comprise the pendant group (-L-Z) according to any of the respective embodiments described herein (e.g., when m is at least 1), differ in the type of backbone unit Y, and / or differ in the type of pendant group (- L-Z).

[0100] For example, In some embodiments the backbone unit Y in each of the Y(-L- Z) units may optionally be the same or different, while the L and Z moieties are the same among the Y(-L-Z) units. In some such embodiments, backbone units notsubstituted by the pendant group (if any such units are present) may optionally be the same as backbone unit Y in each of the Y(-L-Z) units. Alternatively, backbone units not substituted by the pendant group (if any such units are present) may optionally be different than backbone unit Y in each of the Y(-L-Z) units (while optionally being the same among all backbone units not substituted by the pendant group).

[0101] In some embodiments, the L moiety in each of the Y(-L-Z) units may optionally be the same or different, while the backbone units Y and the Z moieties are the same among the Y(-L-Z) units. In some such embodiments, backbone units not substituted by the pendant group (if any such units are present) may optionally be the same as backbone unit Y in each of the Y(-L-Z) units. Alternatively, backbone units not substituted by the pendant group (if any such units are present) may optionally be different than backbone unit Y in each of the Y(-L-Z) units (while optionally being the same among all backbone units not substituted by the pendant group).

[0102] In some embodiments, the Z moiety in each of the Y(-L-Z) units may optionally be the same or different, while the backbone units Y and the Z moieties are the same among the Y(-L-Z) units. In some such embodiments, backbone units not substituted by the pendant group (if any such units are present) may optionally be the same as backbone unit Y in each of the Y(-L-Z) units. Alternatively, backbone units not substituted by the pendant group (if any such units are present) may optionally be different than backbone unit Y in each of the Y(-L-Z) units (while optionally being the same among all backbone units not substituted by the pendant group).

[0103] In any of the embodiments described herein wherein the polymeric moiety is a copolymer moiety, any two or more different types of monomeric unit may be distributed randomly or non- randomly throughout the polymeric moiety. When different types of monomeric unit are distributed non-randomly, the copolymer may be one characterized by any non-random distribution, for example, an alternating copolymer, a periodic copolymer, and / or a block copolymer.

[0104] When Y is a backbone unit which is not attached to L or Z (i.e., to a pendant group as described herein), D is R7 (an end group, as defined herein); and when Y is a backbone unit which is attached to L or Z, D is a covalent bond or a linking group attaching Y to L or Z. The linking group may optionally be -O-, -S-, arylene, sulfinyl, sulfonyl, phosphate, phosphonyl, phosphinyl, carbonyl, thiocarbonyl, urea, thiourea, O- carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C- carboxy, O-carboxy, sulfonamido, or amino.

[0105] R4-R7 are each independently hydrogen, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, azo, phosphate phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C- carboxy, O-carboxy, sulfonamido, or amino.

[0106] In some embodiments, the percentage of backbone units (represented by the variable Y) which are substituted by the pendant group represented by (-L-Z) (as represented by the formula 100%*n / (n+m)) is at least 20 %. In some embodiments, the percentage of backbone units substituted by the aforementioned pendant group is at least 30 %. In some embodiments, the percentage of backbone units substituted by the aforementioned pendant group is at least 40 %. In some embodiments, the percentage of backbone units substituted by the aforementioned pendant group is at least 50 %. In some embodiments, the percentage of backbone units substituted by the aforementioned pendant group is at least 60 %. In some embodiments, the percentage of backbone units substituted by the aforementioned pendant group is at least 70 %. In some embodiments, the percentage of backbone units substituted by the aforementioned pendant group is at least 80 %. In some embodiments, the percentage of backbone units substituted by the aforementioned pendant group is at least 90 %. In some embodiments, the percentage of backbone units substituted by the aforementioned pendant group is at least 95 %. In some embodiments, the percentage of backbone units substituted by the aforementioned pendant group is at least 98 %.

[0107] In some embodiments, m is 0, such that each of the backbone units (represented by the variable Y) is substituted by the pendant group represented by (-L- Z)

[0108] In some embodiments, n is at least 5. In some embodiments, n is at least 10. In some embodiments, n is at least 15.

[0109] In some embodiments, n is in a range of from 2 to 1,000. In some embodiments, n is in a range of from 2 to 500. In some embodiments, n is in a range of from 2 to 200. In some embodiments, n is in a range of from 2 to 100. In some embodiments, n is in a range of from 2 to 50. In some such embodiments, m is 0.

[0110] In some embodiments, n is in a range of from 3 to 1,000. In some embodiments, n is in a range of from 3 to 500. In some embodiments, n is in a range of from 3 to 200. In some embodiments, n is in a range of from 3 to 100. In someembodiments, n is in a range of from 3 to 50. In some embodiments, n is in a range of from 5 to 50. In some embodiments, n is in a range of from 10 to 50. In some embodiments, n is in a range of from 10 to 25. In some such embodiments, m is 0.

[0111] In some embodiments, m is in a range of from 0 to 1,000. In some such embodiments, n is in a range of from 2 to 1,000, such that the total number of backbone units is in a range of from 2 to 2,000. In some such embodiments, n is in a range of from 3 to 1,000. In some embodiments, n is in a range of from 3 to 500. In some embodiments, n is in a range of from 3 to 200. In some embodiments, n is in a range of from 3 to 100. In some embodiments, n is in a range of from 3 to 50. In some embodiments, n is in a range of from 5 to 50. In some embodiments, n is in a range of from 10 to 50.

[0112] In some embodiments, m is in a range of from 0 to 500. In some such embodiments, n is in a range of from 2 to 1,000. In some such embodiments, n is in a range of from 3 to 1,000. In some embodiments, n is in a range of from 3 to 500. In some embodiments, n is in a range of from 3 to 200. In some embodiments, n is in a range of from 3 to 100. In some embodiments, n is in a range of from 3 to 50. In some embodiments, n is in a range of from 5 to 50. In some embodiments, n is in a range of from 10 to 50.

[0113] In some embodiments, m is in a range of from 0 to 200. In some such embodiments, n is in a range of from 2 to 1,000. In some such embodiments, n is in a range of from 3 to 1,000. In some embodiments, n is in a range of from 3 to 500. In some embodiments, n is in a range of from 3 to 200. In some embodiments, n is in a range of from 3 to 100. In some embodiments, n is in a range of from 3 to 50. In some embodiments, n is in a range of from 5 to 50. In some embodiments, n is in a range of from 10 to 50.

[0114] In some embodiments, m is in a range of from 0 to 100. In some such embodiments, n is in a range of from 2 to 1,000. In some such embodiments, n is in a range of from 3 to 1,000. In some embodiments, n is in a range of from 3 to 500. In some embodiments, n is in a range of from 3 to 200. In some embodiments, n is in a range of from 3 to 100. In some embodiments, n is in a range of from 3 to 50. In some embodiments, n is in a range of from 5 to 50. In some embodiments, n is in a range of from 10 to 50.

[0115] In some embodiments, m is in a range of from 0 to 50. In some such embodiments, n is in a range of from 2 to 1,000. In some such embodiments, n is in arange of from 3 to 1,000. In some embodiments, n is in a range of from 3 to 500. In some embodiments, n is in a range of from 3 to 200. In some embodiments, n is in a range of from 3 to 100. In some embodiments, n is in a range of from 3 to 50. In some embodiments, n is in a range of from 5 to 50. In some embodiments, n is in a range of from 10 to 50.

[0116] In some embodiments, m is in a range of from 0 to 20. In some such embodiments, n is in a range of from 2 to 1,000. In some such embodiments, n is in a range of from 3 to 1,000. In some embodiments, n is in a range of from 3 to 500. In some embodiments, n is in a range of from 3 to 200. In some embodiments, n is in a range of from 3 to 100. In some embodiments, n is in a range of from 3 to 50. In some embodiments, n is in a range of from 5 to 50. In some embodiments, n is in a range of from 10 to 50.

[0117] In some embodiments, m is in a range of from 0 to 10. In some such embodiments, n is in a range of from 2 to 1,000. In some such embodiments, n is in a range of from 3 to 1,000. In some embodiments, n is in a range of from 3 to 500. In some embodiments, n is in a range of from 3 to 200. In some embodiments, n is in a range of from 3 to 100. In some embodiments, n is in a range of from 3 to 50. In some embodiments, n is in a range of from 5 to 50. In some embodiments, n is in a range of from 10 to 50.

[0118] In another aspect, the compound of the invention is represented by Formula 1 :, wherein A is a spacer; R is H or an optionally substituted C1-C10 alkyl; each Li independently is selected from an optionally substituted C5-C30 alkyl, H andrepresents a single bond, atriple bond or a double bond; each X independently comprises CH2, an optionally substituted C1-C10 alkyl, an optionally substituted C5-C30 alkyl or an optionally substituted C5-C30 alkenyl, CHR’2; each T independently represents NR’ 2, NH, O, S, -CONH-, -CONR’-, -C(=NH)NR’-, -C(=S)NR’-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, - OC(=S)N-, or phosphate, as allowed by valency; each R’ is independently H or comprises an optionally substituted C1-C10 alkyl, an C1-C10 alkyl-aryl, an C1-C10 alkyl-cycloalkyl, optionally substitutedC3- C10 cycloalkyl, optionally substituted C3- C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl or a combination thereof; Y is selected from hydrogen, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, cyano, nitro, azide, azo, phosphate phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea, thiourea, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, C- carboxy, O-carboxy, sulfonamido, and amino; n is between 2 and 300; each k is independently between 0 and 10; and wherein at least one LI is not H.

[0119] In some embodiments, the compound is represented by Formula 1, wherein Y is halo (e.g. bromo).

[0120] In some embodiments, the compound is represented by Formula 1, wherein R is methyl or H.

[0121] In some embodiments, the compound is represented by Formula 1, wherein n is between 2 and 300, between 50 and 300, between 15 and 300, between 70 and 300, between 30 and 300, between 20 and 300, between 15 and 200, between 20 and 200, between 2 and 200, between 2 and 200, between 2 and 100, between 10 and 100, between 20 and 100, between 15 and 100, between 15 and 150, between 20 and 150, between 5 and 100, between 5 and 50, between 2 and 75, between 2 and 50, including any range between.

[0122] In some embodiments, the compound of the invention has an average molecular weight (MW) between about 1000 and about 30000 Da, between about 1000 and about 25000 Da, between about 2000 and about 30000 Da, between about 2000 and about 25000 Da, between about 3000 and about 30000 Da, between about 4000and about 30000 Da, between about 5000 and about 30000 Da, between about 5000 and about 25000 Da, between about 5000 and about 20000 Da, between about 2000 and about 18000 Da, between about 2000 and about 15000 Da, between about 2000 and about 10000 Da, including any range between.

[0123] In some embodiments, the compound is represented by any of the Formulae disclosed herein, wherein each LI is H or is selected from C10-C30 alkyl / alkenyl / alkynyl, C10-C20 alkyl / alkenyl / alkynyl, C5-C20 alkyl / alkenyl / alkynyl, and C5-C15 alkyl / alkenyl / alkynyl. In some embodiments, a total number of C-atoms in the amine moiety (i.e. the total number of C-atoms in one or two LI) is between 10 and 60, between 10 and 50, between 10 and 30, between 20 and 60, between 20 and 30, including any range between.

[0124] In some embodiments, the compound is represented by any of the Formulae disclosed herein, wherein the spacer has a linear or branched backbone and is between 1 and 100 single C-C bonds long. In some embodiments, the spacer is between 1 and 30, between 1 and 100, between 1 and 10, between 1 and 50, between 1 and 70, between 1 and 30, between 1 and 20, between 1 and 5, between 5 and 10, between 5 and 15, between 5 and 25 single C-C bonds long, including any range in between.

[0125] In some embodiments, the spacer is characterized by a molecular weight between 30 and 10,000Da, between 30 and 5,000Da, between 30 and l,000Da, between 100 and l,500Da, between 300 and l,000Da, between 300 and 5,000Da, between 300 and 2000, between 300 and 3,000Da, between 300 and 10,000Da, including any range in between. In some embodiments, the linker is characterized by a molecular weight between 300 and 3,000Da, and is between 2 and 30, between 2 and 10, between 2 and 20, C-C bonds long, including any range between.

[0126] In some embodiments, the spacer is bound to the amine moiety via a caron atom (i.e. a C-Nbond), or via a bond selected from -C(=O)-, -C(=NR’)-, -C(=NR’)NR’- ,-C(=NR’)O-, -C(=O)O-, -C(=O)S-, -C(=O)NR’-, -C(=S)-, -OC(=S)-, -NR’C(=S)- and -SO2-, wherein R’ independently represents hydrogen, or is selected from the group comprising optionally substituted Cl -CIO alkyl, optionally substitutedC3- C10 cycloalkyl, optionally substitutedC3- C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof.

[0127] In some embodiments, the spacer comprises an optionally substituted C1-C30 alkyl, a heteroatom (e.g., O, N, NH, or S), a heteroalkyl, a cycloalkyl, a heterocyclyl, a bond selected from -C(=O)-, -C(=NR’)-, -C(=NR’)NR’-, -C(=NR’)O-, -C(=O)S-, -S-S-, -C(=0)0-, -C(=O)NR’-, -C(=S)-, -OC(=S)-, -NC(=S)- , -CONR’-, -CNNR’-, - CSNR’-, -NR’C(=O)O-, -NR’C(=S)O-, -NR’C(=S)NR’-, -SO2-, -SO-, -C(=O)-, - OC(=O)-, -OC(=O)O-, -OC(=S)O-, -OC(=S)NR’- and -SO2-, a C1-C30 aminoalkyl, a C1-C30 alkoxy, a C1-C30 mercaptoalkyl, or Y-(C0-10)alkyl-X-(C0-10)alkyl-Y, including any combination thereof; wherein each X and Y is absent or is independently selected from a heteroatom, an oligomer, and the bond.

[0128] In some embodiments, the oligomer comprises between 2 and 30 repeating units. In some embodiments, the repeating unit is selected form alkylene oxide (e.g. ethylene oxide unit) , a natural or an unnatural amino acid residue, an alpha-hydroxy carboxylic acid residue, a monosaccharide, including any copolymer and any combination thereof. In some embodiments, the oligomer is characterized by an average molecular weight between 30 and lOOODa, between 30 and lOODa, between 30 and 200Da, between 30 and 300Da, between 30 and 400Da, between 30 and 500Da, between 30 and 600Da, between 30 and 700Da, between 30 and 800Da, between 30 and 900Da, between 100 and 300Da, between 50 and 350Da between 100 and 500Da between 150 and 500Da between 100 and 900Da including any range in between.

[0129] In some embodiments, the compound is represented by Formula 1, wherein A comprises:, wherein R1 represents hydrogen, halogen, -NO2, -CN, -OH, oxo, imino, -CONH2, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, - CONH-NH2, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR’, - NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, - NNR’, C1-C6haloalkyl, optionally substituted C1-C6alkyl, -NH2, -NR’2, -NH(C1-C6alkyl), -N(C1-C6alkyl)2, C1-C6alkoxy, C1-C6haloalkoxy, hydroxy(C1-C6alkyl), hydroxy(C1-C6alkoxy), alkoxy(C1-C6alkyl), alkoxy(C1-C6alkoxy), C1-C6alkyl-NR’2, Ci-C6alkyl-SR’, -CONH(C1-C6alkyl), -CON(C1-C6alkyl)2, -CO2H, -CO2R’, -OCOR, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, or a combination thereof; wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted C1-C10alkyl, optionally substituted C3- C10cycloalkyl, optionally substitutedC3- C10heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, hydroxy, amino, -NH2, -NR’2-NH(C1-C6alkyl),-N(C1-C6alkyl)2, C1-C6alkoxy, C1-C6haloalkoxy, hydroxy(C1-C6alkyl), hydroxy(Ci- Ce alkoxy), alkoxy(C1-C6alkyl), alkoxy(C1-C6alkoxy), C1-C6alkyl-NR’2, C1-C6alkyl- SR’, or a combination thereof. In some embodiments, the compound is represented by Formula 1, wherein A is as described above, and wherein R1 is H.

[0130] In some embodiments, the compound is represented by Formula 1, wherein Awherein R1 is H or is as described hereinabove.

[0131] In some embodiments, the compound is represented by Formula 2:wherein LI, n and Y are as described above. In some embodiments, each LI is selected from C10-C30 alkyl / alkenyl / alkynyl, C10-C20 alkyl / alkenyl / alkynyl, C5-C20 alkyl / alkenyl / alkynyl, and C5-C15 alkyl / alkenyl / alkynyl; and wherein Y is halo or H.

[0132] In some embodiments, the compound is represented by Formula 2, wherein Y is halo; each LI is selected from C10-C30 alkyl, C10-C30 alkenyl and C10-C30 alkynyl; and wherein n is between 5 and 100, between 5 and 200, between 5 and 300, between 10 and 100, between 10 and 70, between 10 and 150, between 10 and 200, between 20 and 100, between 20 and 150, between 30 and 200, between 30 and 100, between 30 and 150, between 20 and 70, between 5 and 50, between 5 and 75, between 5 and 50, between 5 and 30, between 5 and 40, between 5 and 20, including any range between.

[0133] In some embodiments, the compound is represented by Formula 2, wherein Y is halo, wherein each LI is C10-C15 alkyl, and wherein n is as described herein. Insome embodiments, the compound is represented by Formula 2, wherein n is between 5 and 100; wherein each LI is tetradecyl, and Y is halo.

[0134] In some embodiments, the compound is as described above, wherein the polymeric moiety (pMPC) is characterized by an average molecular weight (Mw) between 3 and 90 kDa, between 3 and 70 kDa, between 3 and 60 kDa, between 3 and 50 kDa, between 3 and 40 kDa, between 3 and 30 kDa, between 3 and 25 kDa, between 3 and 20 kDa, between 5 and 50 kDa, between 5 and 40 kDa, between 5 and 30 kDa, between 7 and 50 kDa, between 7 and 30 kDa, between 7 and 25 kDa, between 7 and 20 kDa, between 10 and 50 kDa, between 10 and 40 kDa, between 10 and 35 kDa, between 10 and 30 kDa, between 10 and 25 kDa, between 10 and 20 kDa, including any range between.

[0135] In some embodiments, pMPC is represented by Formula:, wherein R is as described above (e.g. methyl), and wherein Y is as described above (e.g. Br).

[0136] Based on experimental results (see Example 1) it is postulated that MW of the polymeric moiety being above 10, above 15, above 17, above about 20 kDa, between 15 and 50 kDa, or between 15 and 30 kDa is preferential in terms of storage stability (“stable” is as disclosed herein) of the LNPs of the invention. As shown in Fig. 12, mRNA encapsulating LNPs with 20 kDa pMPC conjugate of the invention showed colloidal stability of LNPs for at least 25 days when stored at 4°C in PBS.

[0137] Furthermore, based on experimental results (see Example 3) it postulated that MW of the polymeric moiety being above 10, above 15, above 17, above about 20 kDa, between 15 and 50 kDa, or between 15 and 30 kDa is preferential for increasing the half-life retention time of the LNPs in blood circulation following IV injection (evenafter repeated administration). Accordingly, it is expected that the LNP of the invention will have improved therapeutic efficiency compared to PEGylated counterparts.LNP

[0138] In another aspect, there is provided a carrier for an active agent(s), wherein the carrier is in a form of a nanoparticle. In some embodiments, the carrier encapsulates the active agent within the core. In some embodiments, the active agent is a small molecule and / or a biologic molecule, such as polypeptide, a polynucleotide, etc. In some embodiments, the active agent is water soluble (e.g. having water solubility of at least O. lg / L at a temperature between 20 and 30°C). In some embodiments, the active agent is selected from a therapeutic agent, a prophylactic agent and a diagnostic agent including any combination thereof. In some embodiments, the one or more active agents are selected from the group consisting of: a protein, a peptide, a nucleic acid, a small molecule, and an antibody.

[0139] In some embodiments, the carrier is in the form of a lipid nanoparticle comprising the compound of the invention (i.e., pMPC-conjugate disclosed above), and the active agent. In some embodiments, the lipid nanoparticle comprises a shell and a core (e.g. an aqueous core) comprising the active agent. In some embodiments, the shell of the lipid nanoparticle comprises the compound of the invention. In some embodiments, the core comprises the active agent and optionally an ionizable lipid. In some embodiments, the shell of the lipid nanoparticle comprises the compound of the invention and further comprises at least one of a lipid and a sterol.

[0140] In some embodiments, the carrier is in the form of a lipid nanoparticle comprising the compound of the invention, an ionizable lipid, a lipid and the active agent. In some embodiments, lipid nanoparticle comprises is in a form of a core-shell nanoparticle, wherein the shell of the nanoparticle comprises an ionizable lipid, a lipid, and at least one compound of the invention. In some embodiments, the compound of the ionizable lipid is bound (e.g., via electrostatic interactions) to the active agent (e.g., a polynucleotide).

[0141] In some embodiments, under suitable conditions the ionizable lipid and at least one compound of the invention, and optionally the lipid and / or the active agent spontaneously undergo self-assembly in an aqueous solution, so as to form the lipid nanoparticle. In some embodiments, the term "lipid nanoparticle" refers to a nanoparticle (e.g., substantially spherical particle), wherein the shell of the nanoparticle comprises one or more compounds of the invention, ionizable lipid and one or morelipids (e.g., a helper lipid, such as a cationic lipid, non-cationic lipid; and optionally a sterol). Preferably, the lipid nanoparticles are formulated to deliver one or more agents to one or more target cells and / or tissue of a subject.

[0142] In some embodiments, the nanoparticle has a spherical geometry or shape. In some embodiments, the nanoparticle has an inflated or a deflated shape. In some embodiments, a plurality of core-shell particles is devoid of any characteristic geometry or shape. In some embodiments, the nanoparticle has a spherical shape, a quasi- spherical shape, a quasi-elliptical sphere, a deflated shape, a concave shape, an irregular shape, or any combination thereof.

[0143] In some embodiments, the plurality of core-shell particles is substantially spherically shaped, wherein substantially is as described herein. In some embodiments, the plurality of core-shell particles is substantially elliptically shaped, wherein substantially is as described herein. One skilled in the art will appreciate that the exact shape of each of the plurality of core-shell particles may differ from one particle to another. Moreover, the exact shape of the nanoparticle may be derived from any of the geometric forms listed above, so that the shape of the particle does not perfectly fit a specific geometrical form. One skilled in the art will appreciate that the exact shape of the nanoparticle may have substantial deviations (such as at least 5%, at least 10%, at least 20% deviation) from a specific geometrical shape (e.g., a sphere or an ellipse).

[0144] In some embodiments, the lipid comprises a helper lipid. In some embodiments, the lipid comprises, a structural lipid, a helper lipid or both. In some embodiments, the lipid comprises a helper lipid, and a structural lipid.

[0145] The term “ionizable lipid” encompasses lipids having one, two, three, or more fatty acid or fatty alkyl / alkenyl, and / or heteroalkyl / heteroalkenyl chains (e.g. such as defined for LI above) and an ionizable amino head group. The ionizable amino head group comprises a primary amine group, a secondary amine group, a tertiary amine group (i.e. a linear or cyclic aliphatic / heteroaromatic amine group). An ionizable amino lipid is typically protonated (i.e., positively charged) at a pH below the pKa of the amino head group and is substantially not charged at a pH above the pKa. In some embodiments, the terms “ionizable lipid” and “cationic lipid” are used herein interchangeably.

[0146] The ionizable lipid is capable of undergoing ionization (positive ionization) within a solution having a pH value below the pKa value of the amino head group. In some embodiments, the ionizable lipid is capable of undergoing protonation within asolution having a pH value below the pKa value of the amine head group. In some embodiments, at least 50% by weight of the ionizable lipids are positively charged (or protonated) within a solution having a pH value below the pKa value of the amine head group.

[0147] Non-limiting example of ionizable lipids include but are not limited to [(4- hydroxybutyl)azanediyl]di(hexane-6,l-diyl)bis(2-hexyldecanoate) (ALC-315), 3- (didodecylamino)-N 1 ,N1 ,4-tridodecyl- 1 -piperazineethanamine (KL 10), N1 -[2- (didodecylamino)ethyl] N 1 ,N4,N4-tridodecyl- 1 ,4-piperazinedi ethanamine (KL22), 14,25-ditridecyl- 15,18,21 ,24-tetraaza-octatriacontane (KL25), 1 ,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]- di oxolane (DLin-K-DMA), heptatriaconta-6,9,28,3 l-tetraen-19-yl-4- (dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[l,3]-di oxolane (DLin-KC2-DMA), 1, 2-di oleyloxy -N,N- dimethylaminopropane (DODMA), 2-({8[(3β)-cholest-5-en-3-yloxy]octyl}oxy) N,N dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA (2R)), and (2S) 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA (2S)).

[0148] In some embodiments, the term “structural lipid” encompasses a nonliposome forming lipid, as described herein. In some embodiments, the structural lipid is or comprises a sterol.

[0149] In some embodiments, the helper lipid is or comprises a phospholipid. In some embodiments, the helper lipid is or comprises phosphatidylcholine. In some embodiments, the phospholipid is a phosphatidylcholine. In some embodiments, the helper lipid is or comprises a liposome forming lipid. As used herein, the term “liposome forming lipid” encompasses lipids (e.g., phospholipids) which upon dispersion or dissolution thereof in an aqueous solution at a temperature above a transition temperature (Tm), undergo self-assembly so as to form stable vesicles (e.g., liposomes). As used herein, the term Tm refers to a temperature at which the lipids undergo phase transition from solid (ordered phase, also termed as a gel phase) to a fluid (disordered phase, also termed as fluid crystalline phase). Tm also refers to a temperature (or to a temperature range) at which the maximal change in heat capacity occurs during the phase transition.

[0150] In some embodiments, the phospholipid encompasses a single phospholipid specie or a plurality of chemically distinct phospholipids.

[0151] In some embodiments, the liposome forming lipid is a phospholipid having one or two C12 to C24 hydrocarbon tails, typically, acyl, alkyl or alkenyl chain) and have varying degrees of unsaturation, from being fully saturated to being fully, partially or non-hydrogenated lipids (the level of saturation may affect rigidity of the liposome thus formed (typically liposomes formed from lipids with saturated chains are more rigid than liposomes formed from lipids of same chain length in which there are unsaturated chains, especially having cis double bonds). In some embodiments, at least one of the liposome forming lipid is a phospholipid having one or two C12 to C20, C16 to C20, or C16 to C18 hydrocarbon tails, including any value and range therebetween. In some embodiments, the liposome forming lipid is fully saturated, linear, or branched.

[0152] Further, the phospholipid may be of natural source (e.g., naturally occurring phospholipids), semi -synthetic or fully synthetic lipid, as well as electrically neutral (e.g., zwitterionic), negatively, or positively charged.

[0153] Non-limiting examples of neutral phospholipids include but are not limited to diacylphosphatidylcholines, dialkylphosphatidylcholines, sphingomyelins, and diacylphosphatidylethanolamines. Phosphatidylcholines (PC), including those obtained from egg, soybeans, or other plant sources or those that are partially or wholly synthetic, or of variable lipid chain length and unsaturation are suitable for use in the present compositions. Synthetic, semi synthetic, and natural product phosphatidylcholines including, but not limited to, POPC, DOPC, DMPC, distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), soy phosphatidylcholine (soy PC), egg phosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine (HEPC), and dipalmitoylphosphatidylcholine (DPPC) are suitable phosphatidylcholines for use in the preparation of liposomes. Charged phospholipids can include phosphatidylglycerols, cardiolipins, or headgroup modified lipids such as N-succinyl-phosphatidylethanolamines, N-glutaryl -phosphatidylethanolamines, and PEG-derivatized phosphatidylethanolamines.

[0154] Non-limiting examples of cationic lipids or ionizable cationic lipids include but are not limited to 5-carboxyspermylglycinedioctadecylamide or "DOGS," N-[l- (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or "DOTMA", 2,3- dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-pr- opanaminium or "DOSPA", l,2-Dioleoyl-3 -Dimethylammonium -Propane or "DODAP", 1,2-Dioleoyl-3-Trimethylammonium-Propane or "DOTAP". Contemplated cationic lipids also include l,2-distearyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2- dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", l,2-dilinoleyloxy-N,N- dimethyl-3 -aminopropane or "DLinDMA", l,2-dilinolenyloxy-N,N-dimethyl-3- aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylammonium bromide or "DDAB", N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12- oc- tadecadienoxy)propane or "CLinDMA", 2-[5'-(cholest-5-en-3-beta-oxy)-3'- oxapentoxy)-3-dimethy l-l-(cis,cis-9',l-2'-octadecadienoxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'- dioleylcarbamyl-3-dimethylaminopropane or "DOcarbDAP", 2,3-Dilinoleoyloxy-N,N- dimethylpropylamine or "DLinDAP", l,2-N,N'-Dilinoleylcarbamyl-3- dimethylaminopropane or "DLincarbDAP", l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane or "DLin-K-DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane or "DLin-K-XTC2-DMA", and 2-(2,2-di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)- 1,3- dioxolan-4-yl)-N,N-di- methylethanamine (DLin-KC2-DMA)).

[0155] In some embodiments, the helper lipid is or comprises a non-cationic lipid. As used herein, the term "non-cationic lipid" refers to any neutral, or zwitterionic lipid. Non-cationic lipids include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), or a mixture thereof.

[0156] In some embodiments, the helper lipid is or comprises DOPE, DSPC, POPE, or any combination thereof.

[0157] In some embodiments, the helper lipid is a cationic lipid. In some embodiments, the cationic lipid is or comprises any of DOTAP, DDAB, 1,2-dioleoyl-sn-glycero-3 -ethylphosphocholine, DPPCethyl (EPC 16:0, or 1,2-dipalmitoyl-sn- glycero-3 -ethylphosphocholine) or any combination thereof.

[0158] In some embodiments, the term “non-liposome forming lipid” is to be understood as referring to a lipid that does not spontaneously form into a vesicle when brought into an aqueous medium.

[0159] There are various types of lipids that do not spontaneously vesiculate and yet are used or can be incorporated into vesicles. In some embodiments, the non-liposome forming lipid is or comprises a sterol.

[0160] Non-limiting examples of sterols include but are not limited to: P-sitosterol, P-sitostanol, stigmasterol, stigmastanol, campesterol, campestanol, ergosterol, avenasterol, brassicasterol, fucosterol, cholesterol (Choi) , cholesteryl hemi succinate, and cholesteryl sulfate including any salt or any combination thereof.

[0161] In some embodiments, the structural lipid comprises any one of Betulin, Brassicasterol, Calcipotriol, campesterol, cholesterol, Daucosterol, DC-cholesterol, Dehydroergosterol, DMAPC-Chol, DMHAPC-Chol, ergosterol, Fucosterol, HAPC- Chol, Lupeol, MHAPC-Chol, OH-C-Chol, OH-Chol, Oleanolic acid, stigmastanol, stigmasterol, Ursolic acid, a hydrophobic vitamin (e.g. Vitamin D2, Vitamin D3, vitamin E, etc.), P-sitosterol, P- Sitosterol -Acetate, P-sitosterol-arginine, P-sitosterol- cysteine, P-sitosterol-glycine, P-sitosterol-histidine, P-sitosterol-serine, or a steroid, including any salt or any combination thereof.

[0162] In some embodiments, a molar concentration of one or more compounds of the invention within the nanoparticle is between 0.5 and 10 mol%, between 0.5 and 5 mol%, between 0.5 and 2 mol%, between 1 and 5 mol%, between 1 and 2 mol%, between 1 and 3 mol%, between 0.5 and 3 mol%, between 0.5 and 7 mol%, including any range between.

[0163] In some embodiments, a molar concentration of the helper lipid within the nanoparticle is between 5 and 30 mol%, between 5 and 10 mol%, between 5 and 20 mol%, between 5 and 15 mol%, between 8 and 15 mol%, between 10 and 30 mol%, between 8 and 30 mol%, between 8 and 20 mol%, including any range between.

[0164] In some embodiments, a molar concentration of any one of the ionizable lipid and the structural lipid within the nanoparticle is independently between 20 and 60 mol%, between 20 and 50 mol%, between 30 and 50 mol%, between 30 and 60 mol%, between about 30 and about 40 mol%, between about 30 and about 45 mol%, including any range between.

[0165] As used herein, the term “concentration” or “molar concentration” refers to a molar ratio relative to the total lipid content of the nanoparticle. In some embodiments, the total lipid content refers to the combined content of the compound of the invention, the ionizable lipid and of the lipid, wherein the lipid encompasses a liposome forming lipid, and a non-liposome forming lipid. A skilled artisan will appreciate that a molar ratio of any one of the essential constituents (i.e., the compound of the invention, the helper lipid, the structural lipid, and the ionizable lipid) within the LNP is identical to a molar ratio of the same constituent within the composition of the invention. Thus, the molar concentrations and molar ratios disclosed herein for example with respect to LNP also encompass the corresponding molar concentrations and molar ratios within the composition of the invention and vice versa.

[0166] In some embodiments, a molar ratio of the helper lipid and the ionizable lipid ranges between 1 :3 and 1 :6, between 1 :4 and 1 :6, between 1 :3 and 1 :5, including any range in between.

[0167] In some embodiments, a molar ratio of the compound of the invention and the ionizable lipid ranges between 1 : 10 and 1 :50, between 1 : 10 and 1 :40, between 1 : 10 and 1 :30, between 1 :20 and 1 :50, between 1 :20 and 1 :40, between 1 :20 and 1 :30, including any range in between.

[0168] In some embodiments, a molar ratio of the structural lipid and the ionizable lipid ranges between 1.5: 1 and 1 : 1.5, between 1 : 1 and 1 : 1.5 , between 1.5: 1 and 1 : 1, between 1.2: 1 and 1 : 1.2, including any range in between.

[0169] In some embodiments, the LNP is as described herein, wherein the helper lipid is a phosphatidylcholine, the structural lipid is a sterol, and wherein a molar concentration of the compound of the invention within the LNP is between 0.5 and 5 mol%; wherein a molar concentration of the helper lipid within the nanoparticle is between 5 and 20 mol%; and wherein a molar concentration of any one of the ionizable lipid and the structural lipid within the nanoparticle is independently between 35 and 50 mol%.

[0170] In some embodiments, the LNP further comprises an alkyl amine. In some embodiments, the alkyl amine is selected from primary-, secondary and tertiary amine. In some embodiments, the alkyl amine is a primary amine. In some embodiments, a total number of C atoms within the alkyl amine is between 10 and 30, between 8 and 30, between 12 and 30, between 15 and 30, between 12 and 20, between 15 and 20,including any range between. In some embodiments, the alkyl amine is stearylamine (SA).

[0171] In some embodiments, the LNP is substantially devoid of PEG-lipid. In some embodiments, the LNP is substantially devoid of pMPC conjugated to a phospholipid.

[0172] In some embodiments, a molar concentration of the alkyl amine within the LNP is between 0.5 and 10, between 1 and 10, between 0.5 and 8, between 2 and 8, between 4 and 10, between 4 and 6mol%, including any range between.

[0173] In some embodiments, the active agent is a polynucleotide, and wherein aN:P ratio within the lipid nanoparticle or within the composition of the invention ranges between 1 and 20, between 3 and 20, between 3 and 5, between 4 and 8, between 6 and 8, between 8 and 10, between 10 and 12, between 10 and 20, between 8 and 20, between 8 and 15, and between 12 and 14, including any range in between. The term “N:P ratio” refers to a ratio between N atoms of the compound of the invention and P atoms of the polynucleotide within the lipid nanoparticles or within the composition of the invention.

[0174] In some embodiments, the nanoparticles within the composition are characterized by an average particle size of less than 500 nm to facilitate its entrance through the extracellular matrix to a cell and / or to penetrate the cell membrane. In some embodiments, the carrier is characterized by an average particle size of less than 300 nm in diameter to facilitate its entrance through the extracellular matrix to a cell and / or to penetrate the cell membrane.

[0175] In some embodiments, the nanoparticles within the composition are characterized by an average particle size of less than 300 nm, less than 250 nm, less than 200 nm, or less than 150 nm, including any range between.

[0176] In some embodiments, the nanoparticles within the composition are characterized by an average particle size of between about 30 and about 300nm, between about 50 and about 300, between about 50 and about 250nm, between about 50 and about 200nm, between about 50 and about 150nm, between about 100 and about 300nm, between about 50 and about lOOnm, between about 100 and about 200nm, including any range between.

[0177] In some embodiments, the nanoparticles are characterized by an average particle size as disclosed herein and are further characterized by size distribution (poly dispersity index, PDI) of between 0.05 and 0.4, between 0.05 and about 0.3, between 0.05 and about 0.2, between about 0.1 and about 0.3, about 0.1, about 0.2, about 0.3, including any range between. In some embodiments, the average particlesize and / or PDI refer to a size / PDI value in solution and is / are measured by Dynamic Light Scattering, Nanoparticle Tracking Analysis etc.

[0178] In some embodiments, the nanoparticles within the composition are characterized an average particle size between 50 and 150nm, and by a PDI between 0.05 and 0.25, between 0.05 and about 0.2, or between 0.1 and 0.3. In some embodiments, at least 80%, at least 90% at least 95% of the nanoparticles within the composition are monodispersed.

[0179] In some embodiments, the nanoparticle is characterized by a negative average zeta potential. In some embodiments, the nanoparticle is characterized by a negative average zeta potential ranging between -0.1 and -40m V, between 0 and -30m V, between 0 and -25mV, between 0 and -20m V, between -1 and -30m V, between -1 and -20m V, between -1 and -25m V, including any range between, wherein the zeta potential is measured at pH of about 7.

[0180] In some embodiments, the nanoparticle is stable for a time period ranging between 1 day and 1 year, or more, including any range between. In some embodiments, the term “stable” refers to physical and chemical stability of a dry nanoparticle (such as being substantially devoid of phase separation, agglomeration, disintegration, and / or substantially retaining the initial loading of the active agent) under appropriate storage conditions. In some embodiments, the term “stable” refers to physical and chemical stability of the nanoparticle within an aqueous solution (e.g., dispersion stability). In some embodiments, the term “stable” refers to physical and chemical stability of the lyophilized or dry LNPs of the invention. In some embodiments, the term “stable” refers to a PDI value of the LNPs being up to 0.3, up to 0.25, up to 0.21, or up to 0.2, or between 0.05 and 0.3, including any range between, when measured in an aqueous solution after storage at 4°C or at 25°C.

[0181] In some embodiments, the stable LNPs substantially retain the average particle size within an aqueous composition (i.e. an aqueous solution consisting essentially of the LNP) after storage at 4°C or at 25°C. In some embodiments, “substantially retains” encompasses that the PDI varies by not more than by 30%, or not more than by 20%, and / or remains below 0.3, below 0.25, or below 0.21 for at least 72h, at least 5d, at least 10 d, at least 20 d, at least 30 d, at least 40 d, at least 50 d, at least 60 d upon preparation of the aqueous composition.

[0182] In some embodiments, the term “polynucleic acid” and the term “polynucleotide” are used herein interchangeably. In some embodiments, thepolynucleotide comprises 60 to 15000 nucleobases, 15000 to 10000, 10000 to 4700, 200 to 5000 nucleobases, 300 to 5000 nucleobases, 400 to 5000 nucleobases, 400 to 2500 nucleobases, 200 to 3000 nucleobases, 400 to 2000 nucleobases, 400 to 1000 nucleobases, including any range between.

[0183] In some embodiments, the polynucleotide comprises at least 20 nucleobases, at least 250 nucleobases, at least 300 nucleobases, at least 350 nucleobases, at least 400 nucleobases, at least 450 nucleobases, at least 475 nucleobases, or at least 500 nucleobases. Each possibility represents a separate embodiment of the invention.

[0184] In some embodiments, the polynucleotide comprises 500 nucleobases at most, 750 nucleobases at most, 1,000 nucleobases at most, 1,250 nucleobases at most, 1,750 nucleobases at most, 2,500 nucleobases at most, 3000 nucleobases at most, 4000 nucleobases at most, or 5000 nucleobases at most. Each possibility represents a separate embodiment of the invention.

[0185] In some embodiments, the polynucleotide comprises a plurality of polynucleotide types. In some embodiments, the nanoparticle comprises a plurality of polynucleotide types. In some embodiments, the composition comprises a plurality of nanoparticle types, each type of nanoparticle comprises a specific polynucleotide.

[0186] In some embodiments, a specific polynucleotide comprises a plurality of polynucleotide molecules harboring the same or an identical nucleic acid sequence. In some embodiments, a specific polynucleotide comprises a plurality of polynucleotide molecules harboring essentially the same nucleic acid sequence.

[0187] As used herein, the term “plurality” encompasses any integer equal to or greater than 2. In some embodiments, a plurality comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0188] As used herein, the term “polynucleotide types” refers to a plurality of polynucleotides each of which comprises a nucleic acid sequence differing from any one of the other polynucleotides of the plurality of polynucleotides by at least 1 nucleobase, at least 2 nucleobases, at least 10 nucleobases, at least 20 nucleobases, at least 5 nucleobases, or at least 50 nucleobases, or any value and range there between. Each possibility represents a separate embodiment of the invention.

[0189] In some embodiments, a polynucleotide comprises RNA, DNA, a synthetic analog of RNA, circular RNA (circRNA), a synthetic analog of DNA, DNA / RNAhybrid, or any combination thereof. In some embodiments, a nanoparticle of the invention comprises a polynucleotide selected from: RNA, DNA, a synthetic analog of RNA, a synthetic analog of DNA, DNA / RNA hybrid, or any combination thereof.

[0190] In some embodiments, the polynucleotide comprises or consists of RNA. The polynucleotide comprises or consists of a messenger RNA (mRNA). "Messenger RNA" (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally occurring, non-naturally occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. Polynucleotides may function as mRNA but can be distinguished from wild-type mRNA in their functional and / or structural design features which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.

[0191] The mRNA, as provided herein, comprises at least one (one or more) ribonucleic acid (RNA) polynucleotide having an open reading frame encoding at least one polypeptide of interest. In some embodiments, an RNA polynucleotide of an mRNA encodes 1-2, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 polypeptides. In some embodiments, an RNA polynucleotide of an mRNA encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 polypeptides. In some embodiments, an RNA polynucleotide of an mRNA encodes at least 100 or at least 200 polypeptides.

[0192] In some embodiments, the nucleic acids are therapeutic mRNAs. As used herein, the term "therapeutic mRNA" refers to an mRNA that encodes a therapeutic protein or an antigen. Therapeutic proteins mediate a variety of effects in a host cell or a subject in order to treat a disease or ameliorate the signs and symptoms of a disease. For example, a therapeutic protein can replace a protein that is deficient or abnormal, augment the function of an endogenous protein, provide a novel function to a cell (e.g., inhibit or activate an endogenous cellular activity, or act as a delivery agent for another therapeutic compound (e.g., an antibody-drug conjugate). Therapeutic mRNA may be useful for the treatment of the following diseases and conditions: bacterial infections, viral infections, parasitic infections, cell proliferation disorders, genetic disorders, and autoimmune disorders.

[0193] Thus, the structures of the invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine. For example, the mRNA of the structures described herein can be administered to a subject, wherein the polynucleotides are translated in vivo to produce a peptide / protein or a therapeutic peptide.

[0194] In some embodiments, the polynucleotide comprises an inhibitory nucleic acid. In some embodiments, the polynucleotide comprises an antisense oligonucleotide.

[0195] As used herein, an "antisense oligonucleotide" refers to a nucleic acid sequence that is reversed and complementary to a DNA or RNA sequence.

[0196] As referred to herein, a "reversed and complementary nucleic acid sequence" is a nucleic acid sequence capable of hybridizing with another nucleic acid sequence comprised of complementary nucleotide bases. By "hybridize" is meant pair to form a double-stranded molecule between complementary nucleotide bases (e.g., adenine (A) forms a base pair with thymine (T) (or uracil (U) in the case of RNA), and guanine (G) forms a base pair with cytosine (C)) under suitable conditions of stringency. (See, e.g., Wahl, G. M., and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). For the purposes of the present methods, the inhibitory nucleic acid need not be complementary to the entire sequence, only enough of it to provide specific inhibition; for example, in some embodiments the sequence is 100% complementary to at least nucleotides (nts) 2-7 or 2-8 at the 5' end of the microRNA itself (e.g., the 'seed sequence'), e.g., nts 2-7 or 20.

[0197] In some embodiments of the inhibitory nucleic acid has one or more chemical modifications to the backbone or side chains. In some embodiments, the inhibitory nucleic acid has at least one locked nucleotide, and / or has a phosphorothioate backbone.

[0198] Non-limiting examples of inhibitory nucleic acids useful according to the herein disclosed invention include, but are not limited to: antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases / locked nucleic acids (LNAs), antagomirs, peptide nucleic acids (PNAs), ribozymes (catalytic RNA molecules capable to cut other specific sequences of RNA molecules) and other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target nucleic acid and modulate its function. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA(siRNA); a micro-RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.

[0199] In some embodiments, the inhibitory nucleic acid is an RNA interfering molecule (RNAi). In some embodiments, the RNAi is or comprises double stranded RNA (dsRNA).

[0200] As used herein "an interfering RNA" refers to any double stranded or single stranded RNA sequence, capable-either directly or indirectly (i.e., upon conversion)-of inhibiting or down regulating gene expression by mediating RNA interference. Interfering RNA includes but is not limited to small interfering RNA ("siRNA") and small hairpin RNA ("shRNA"). "RNA interference" refers to the selective degradation of a sequence-compatible messenger RNA transcript.

[0201] In some embodiments, the polynucleotide is chemically modified. In some embodiments, the chemical modification is a modification of a backbone of the polynucleotide. In some embodiments, the chemical modification is a modification of a sugar of the polynucleotide. In some embodiments, the chemical modification is a modification of a nucleobase of the polynucleotide. In some embodiments, the chemical modification increases stability of the polynucleotide in a cell. In some embodiments, the chemical modification increases stability of the polynucleotide in vivo. In some embodiments, the chemical modification increases the stability of the polynucleotide in vitro, such as, in the open air, field, on a surface exposed to air, etc. In some embodiments, the chemical modification increases the polynucleotide’s ability to induce silencing of a target gene or sequence, including, but not limited to an RNA molecule derived from a pathogen or an RNA derived from a plant cell, as described herein. In some embodiments, the chemical modification is selected from: a phosphateribose backbone, a phosphate-deoxyribose backbone, a phosphorothioate-deoxyribose backbone, a 2'-O-methyl-phosphorothioate backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethyl phosphorothioate backbone, a constrained ethyl backbone, an alternating locked nucleic acid backbone, a phosphorothioate backbone, N3'-P5' phosphoroamidates, 2'- deoxy-2'-fluoro-P-d-arabino nucleic acid, cyclohexene nucleic acid backbone nucleic acid, tricyclo-DNA (tcDNA) nucleic acid backbone, ligand-conjugated antisense, and a combination thereof.Pharmaceutical composition

[0202] In another aspect, there is provided a pharmaceutical composition comprising the lipid nanoparticles of the invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is also referred to as an excipient or adjuvant. As used herein, the term “carrier,” “excipient,” or “adjuvant” refers to any component of a pharmaceutical composition that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Officeof Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful in the present compositions include distilled water, physiological saline, Hartmann solution, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergam on Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety.

[0203] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0204] In some embodiments, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition is formulated for administration to a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a laboratory animal. Examples of laboratory animals include, but are not limited to, mice, rats, rabbits, hamsters, dogs, cats, and monkeys. In some embodiments, the mammal is a mouse or rat. In some embodiments, the subject is in need of the composition of the invention. In some embodiments, the subject is in need of treatment. In some embodiments, the subject is a volunteer for a diagnostic method. In some embodiments, the subject is in need of diagnosis.

[0205] In some embodiments, the pharmaceutical composition is for use in a therapeutic method. In some embodiments, a therapeutic method is a method of treatment. In some embodiments, the pharmaceutical composition is for use in a diagnostic method. In some embodiments, the method comprises administering the composition of the invention to a subject. In some embodiments, the pharmaceutical composition is for use in treatment or prevention of a disease or condition in humans and other mammals. The active therapeutic agents of the invention include the nanoparticles, or polypeptides translated from the polynucleotides contained in the nanoparticles.

[0206] In some embodiments, the pharmaceutical composition of the invention (i.e., comprising the pMPC conjugates provided herein) induces a decreased immune response (IgM production) as compared to PEGylated LNPs. In some embodiments, a decreased immune response is decreased IgM production (such as following IV injection).

[0207] As used herein, the terms “administering,” “administration,” and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of a composition of the present subject matter to a patient in need thereof. Other suitable routes of administration can include intravenous, subcutaneous, oral, pulmonary, intramuscular, intrathecal, ocular, intra-articular, via inhalation, parenteral, intratracheal, intranasal, intracerebroventricular, intravitreal, transdermal, or intraperitoneal, or any combination thereof.

[0208] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0209] By another aspect, there is provided a method of treating a subject in need thereof, the method comprising administering to the subject a therapeutic composition of the invention.

[0210] In some embodiments, there is provided a method for delivering an active agent into a cell of a subject, comprising contacting the cell with the nanoparti cle(s) of the invention, wherein each nanoparticle comprises the active agent (e.g. wherein the active agent is encapsulated within the nanoparticle). In some embodiments, the active agent is described hereinabove. In some embodiments, the active agent is an impermeable cell. Cell delivery (i.e. intracellular delivery) can be determined by quantifying the amount of the active agent inside the cell (e.g. by fluorescent labeling of the compound, or in the case of a polynucleotide, by determining the expression level of the polynucleotide).

[0211] In some embodiments, delivering is so as to obtain an increased concentration of the active agent within the cell of a subject, as compared to a control formulation comprising the same active agent and lipofectamine as the cell internalizing agent instead of the lipid nanoparticles of the invention. In some embodiments, increased concentration is a therapeutically effective concentration. In some embodiments,increased concentration is referred to at least 10 times, at least 50 times, at least 100 times, at least 200 times greater concentration, as compared to the control.

[0212] In some embodiments, the cell is a tissue cell. In some embodiments, the method is for delivering the active agent to one or more tissues of the subject. In some embodiments, the method is for selectively delivering the active agent to one or more tissue of the subject. In some embodiments, selectively delivering encompasses a concentration of the active gent in the target tissue after administration (e.g. iv- administration) being at least 2 times, at least 5times, or at least 10 times greater in the target tissue compared to a non-target tissue.

[0213] In some embodiments, the tissue is selected from lung, liver, spleen, heart and kidney tissue, including any combination thereof. In some embodiments, delivering is to obtain a therapeutically effective concentration of the active agent within the tissue. In some embodiments, delivering is to obtain an increased amount of the active agent within the tissue, wherein increased is as compared to the control, as disclosed herein.Definitions

[0214] As used herein, the term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0215] As used herein, the term “substantially” refers to at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or between 60 and 99.9%, between 70 and 80%, between 70 and 90%, between 80 and 90%, between 90 and 95%, between 95 and 99.9%, including any range or value therebetween.

[0216] As used herein, the term “substituent” encompasses hydrogen, halogen, -NO2, -CN, -OH, oxo, imino, -CONH2, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, - CONH-NH2, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR’, - NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NR’2, -NH(C1- C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl - NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -CO2H, - CO2R’, -OCOR, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, or a combination thereof; wherein each R’ independently represents hydrogen, or isselected from the group comprising optionally substituted Cl -CIO alkyl, optionally substitutedC3- C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, hydroxy, amino, -NH2, -NR’2- NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl- C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(C1-C6 alkyl), alkoxy(Cl-C6 alkoxy), Cl- C6 alkyl-NR’2, C1-C6 alkyl-SR’, or a combination thereof.

[0217] As used herein, the term "alkyl" describes an aliphatic hydrocarbon including straight chain and branched chain groups. The term "alkyl", as used herein, also encompasses saturated or unsaturated hydrocarbon, hence this term further encompasses alkenyl and alkynyl.

[0218] The term "alkenyl" describes an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

[0219] The term "alkynyl", as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

[0220] The term "cycloalkyl" describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted, as indicated herein.

[0221] The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. The aryl group may be substituted or unsubstituted, as indicated herein.

[0222] The term "alkoxy" describes both an O-alkyl and an -O-cycloalkyl group, as defined herein. The term "aryloxy" describes an -O-aryl, as defined herein.

[0223] Each of the alkyl, cycloalkyl and aryl groups in the general formulas herein may be substituted by one or more substituents, whereby each substituent group can independently be, for example, halide, alkyl, alkoxy, cycloalkyl, nitro, amino, hydroxyl, thiol, thioalkoxy, carboxy, amide, aryl and aryloxy, depending on the substituted group and its position in the molecule. Additional substituents are also contemplated.

[0224] The term "halide", "halogen" or “halo” describes fluorine, chlorine, bromine, or iodine. The term “haloalkyl” describes an alkyl group as defined herein, furthersubstituted by one or more halide(s). The term “haloalkoxy” describes an alkoxy group as defined herein, further substituted by one or more halide(s). The term “hydroxyl” or "hydroxy" describes a -OH group. The term "mercapto" or “thiol” describes a -SH group. The term "thioalkoxy" describes both an -S-alkyl group, and a -S-cycloalkyl group, as defined herein. The term "thioaryloxy" describes both an -S-aryl and a -S- heteroaryl group, as defined herein. The term “amino” describes a -NR’R” group, or a salt thereof, with R’ and R’ ’ as described herein.

[0225] The term "heterocyclyl" describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen, and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino and the like.

[0226] The term "carboxy" describes a -C(O)OR' group, or a carboxylate salt thereof, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon) or heterocyclyl (bonded through a ring carbon) as defined herein, or "carboxylate".

[0227] The term “carbonyl” describes a -C(O)R' group, where R' is as defined hereinabove. The above-terms also encompass thio-derivatives thereof (thiocarboxy and thiocarbonyl).

[0228] The term “thiocarbonyl” describes a -C(S)R' group, where R' is as defined hereinabove. A "thiocarboxy" group describes a -C(S)OR' group, where R' is as defined herein. A "sulfinyl" group describes an -S(O)R' group, where R' is as defined herein. A "sulfonyl" or “sulfonate” group describes an -S(O)2R' group, where R' is as defined herein.

[0229] A "carbamyl" or “carbamate” group describes an -OC(O)NR'R" group, where R' is as defined herein and R" is as defined for R'. A "nitro" group refers to a -NO2 group. The term "amide" as used herein encompasses C-amide and N-amide. The term "C-amide" describes a -C(O)NR'R" end group or a -C(O)NR'-linking group, as these phrases are defined hereinabove, where R' and R" are as defined herein. The term "N- amide" describes a -NR"C(O)R' end group or a -NR'C(O)- linking group, as these phrases are defined hereinabove, where R' and R" are as defined herein.

[0230] A "cyano" or "nitrile" group refers to a -CN group. The term "azo" or "diazo" describes an -N=NR' end group or an -N=N- linking group, as these phrases are defined hereinabove, with R' as defined hereinabove. The term "guanidine" describes a -R'NC(N)NR"R"' end group or a -R'NC(N) NR"- linking group, as these phrases are defined hereinabove, where R', R" and R'" are as defined herein. As used herein, the term “azide” refers to a -N3 group. The term “sulfonamide” refers to a -S(0)2NR'R" group, with R' and R" as defined herein.

[0231] The term “phosphonyl” or “phosphonate” describes an -OP(O)-(OR')2 group, with R' as defined hereinabove. The term “phosphinyl” describes a -PR'R" group, with R' and R" as defined hereinabove. The term “alkylaryl” describes an alkyl, as defined herein, which substituted by an aryl, as described herein. An exemplary alkylaryl is benzyl.

[0232] The term "heteroaryl" describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. As used herein, the term “heteroaryl” refers to an aromatic ring in which at least one atom forming the aromatic ring is a heteroatom. Heteroaryl rings can be foamed by three, four, five, six, seven, eight, nine and more than nine atoms. Heteroaryl groups can be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C3- 8 heterocyclic groups containing one oxygen or sulfur atom, or two oxygen atoms, or two sulfur atoms or up to four nitrogen atoms, or a combination of one oxygen or sulfur atom and up to two nitrogen atoms, and their substituted as well as benzo- and pyrido-fused derivatives, for example, connected via one of the ring-forming carbon atoms. In certain embodiments, heteroaryl is selected from among oxazolyl, isoxazolyl, oxadi azolyl, thiazolyl, isothiazolyl, pyridinyl, pyridazinyl, pyrimidinal, pyrazinyl, indolyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinazolinyl or quinoxalinyl.

[0233] In some embodiments, a heteroaryl group is selected from among pyrrolyl, furanyl (furyl), thiophenyl (thienyl), imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4- triazolyl, 1,3-oxazolyl (oxazolyl), 1,2-oxazolyl (isoxazolyl), oxadiazolyl, 1,3-thiazolyl (thiazolyl), 1,2-thiazolyl (isothiazolyl), tetrazolyl, pyridinyl (pyridyl)pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, 1, 2,4,5- tetrazinyl, indazolyl, indolyl, benzothiophenyl, benzofuranyl, benzothiazolyl, benzimidazolyl, benzodioxolyl, acridinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, thi enothiophenyl, 1,8-naphthyridinyl, other naphthyridinyls, pteridinyl or phenothiazinyl. Where the heteroaryl group includes more than one ring, each additional ring is the saturated form (perhydro form) or thepartially unsaturated form (e.g., the dihydro form or tetrahydro form) or the maximally unsaturated (nonaromatic) form. The term heteroaryl thus includes bicyclic radicals in which the two rings are aromatic and bicyclic radicals in which only one ring is aromatic. Such examples of heteroaryl are include 3H-indolinyl, 2(lH)-quinolinonyl, 4-oxo- 1 ,4-dihydroquinolinyl, 2H- 1 -oxoisoquinolyl, 1 ,2-dihydroquinolinyl, (2H)quinolinyl N-oxide, 3,4-dihydroquinolinyl, 1,2-dihydroisoquinolinyl, 3,4-dihydro- isoquinolinyl, chromonyl, 3,4-dihydroiso-quinoxalinyl, 4-(3H)quinazolinonyl, 4H- chromenyl, 4-chromanonyl, oxindolyl, 1,2,3,4-tetrahydroisoquinolinyl, 1, 2,3,4- tetrahydro-quinolinyl, lH-2,3-dihydroisoindolyl, 2,3-dihydrobenzo[f]isoindolyl,1.2.3.4-tetrahydrobenzo-[g]isoquinolinyl, l,2,3,4-tetrahydro-benzo[g]isoquinolinyl, chromanyl, isochromanonyl, 2,3-dihydrochromonyl, 1,4-benzo-dioxanyl, 1, 2,3,4- tetrahydro-quinoxalinyl, 5,6-dihydro-quinolyl, 5,6-dihydroiso-quinolyl, 5,6- dihydroquinoxalinyl, 5,6-dihydroquinazolinyl, 4,5-dihydro-lH-benzimidazolyl, 4,5- dihydro-benzoxazolyl, 1,4-naphthoquinolyl, 5,6,7,8-tetrahydro-quinolinyl, 5, 6,7,8- tetrahydro-isoquinolyl, 5,6,7,8-tetrahydroquinoxalinyl, 5,6,7,8-tetrahydroquinazolyl,4.5.6.7-tetrahydro-lH-benzimidazolyl, 4,5,6,7-tetrahydro-benzoxazolyl, lH-4-oxa- l,5-diaza-naphthalen-2-onyl, l,3-dihydroimidizolo-[4,5]-pyridin-2-onyl, 2,3-dihydro-1.4-dinaphtho-quinonyl, 2,3-dihydro-lH-pyrrol[3,4-b]quinolinyl, 1, 2,3,4- tetrahydrobenzo[b]-[ 1 ,7]naphthyridinyl, 1 ,2,3 ,4-tetra-hydrobenz[b] [1,6]- naphthyridinyl, l,2,3,4-tetrahydro-9H-pyrido[3,4-b]indolyl, l,2,3,4-tetrahydro-9H- pyrido[4,3-b]indolyl, 2,3-dihydro-lH-pyrrolo-[3,4-b]indolyl, 1 H-2, 3,4,5 -tetrahydro- azepino[3,4-b]indolyl, lH-2,3,4,5-tetrahydroazepino-[4,3-b]indolyl, lH-2,3,4,5- tetrahydro-azepino[4,5-b]indolyl, 5,6,7,8-tetrahydro[l,7]napthyridinyl, 1, 2,3,4- tetrahydro-[2,7]-naphthyridyl, 2,3-dihydro[l,4]dioxino[2,3-b]pyridyl, 2,3- dihydrof 1 ,4]-dioxino[2,3 -b ] pry i dy 1 , 3 ,4-dihydro-2H- 1 -oxa[4,6]diazanaphthalenyl,4.5.6.7-tetrahydro-3H-imidazo-[4,5-c]pyridyl, 6,7-dihydro[5,8]diazanaphthalenyl,1.2.3.4-tetrahydro[l,5]-napthyridinyl, l,2,3,4-tetrahydro[l,6]napthyridinyl, 1, 2,3,4- tetrahydro[l,7]napthyridinyl, l,2,3,4-tetrahydro-[l,8]napthyridinyl or 1, 2,3,4- tetrahydro[2,6]napthyridinyl. In some embodiments, heteroaryl groups are optionally substituted. In some embodiments, the one or more substituents are each independently selected from among halo, hydroxy, amino, cyano, nitro, alkylamido, acyl, Cl-6-alkyl, Cl-6-haloalkyl, Cl-6-hydroxy alkyl, Cl-6-aminoalkyl, Cl-6-alkylamino, alkylsulfenyl, alkylsulfinyl, alkyl sulfonyl, sulfamoyl, or trifluorom ethyl.

[0234] Examples of heteroaryl groups include, but are not limited to, unsubstituted and mono- or di -substituted derivatives of furan, benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, triazole, benzotri azole, pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline and quinoxaline. In some embodiments, the substituents are halo, hydroxy, cyano, O — Cl-6-alkyl, Cl-6-alkyl, hydroxy-Cl-6-alkyl, and amino-Cl-6-alkyl.

[0235] As used herein, the terms "halo" and "halide", which are referred to herein interchangeably, describe an atom of a halogen, that is fluorine, chlorine, bromine, or iodine, also referred to herein as fluoride, chloride, bromide, and iodide.General

[0236] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[0237] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0238] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or claims, should be understood to contemplate the possibilities of including one of theterms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0239] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0240] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0241] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0242] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531;5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Materials and methodsMaterials

[0243] Benzoyl acetic acid (98%), 1 -(3 -Dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride (EDC*HC1, 98%), 1 -Hydroxybenzotriazole (HOBt, 98%), and diisopropylethylamine (DIPEA, 99%) were purchased from Alfa (Israel). Ditetradecyl amine (DTA, 95%) was purchased from Ambeed (US). Dioctadecylamine (DSA, 99%) was obtained from IPlusChem LLC (San Diego, USA). mPEG-COOH MW 2K was purchased from Advanced BioChemicals (Lawrenceville, GA, USA). Dichloromethane (anhydrous), ethyl acetate, ethanol, acetone, triethylamine, palladium carbon, a-bromoisobutyryl bromide, chloroform (anhydrous), 2-Methacryloyloxyethyl phosphorylcholine (MPC, 97%), l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC, Mw 790.15), cholesterol (C27H46O, Mw 386.65), stearylamine (SA, CH3(CH2)I?NH2, Mw 269.509), sodium acetate buffer solution pH 5.2 for molecular biology, XTT cell proliferation assay and dialysis bag Float- A-Lyzers (MW cutoff 100 kDa) were obtained from Sigma-Aldrich (Israel). ((4-hydroxybutyl)azanediyl)di(hexane-6,l-diyl) bis(2 -hexyldecanoate) (ALC-0315) was provided by DC Chemicals (China). Methoxypoly ethylenegly col oxy(2000)-N,N-ditetradecylacetamide (ALC-0159) provided by Avanti Polar Lipids. Poly (A) (lyophilized, suitable for PCR, mol wt 700- 3500 kDa) was purchased from Roche Diagnostics (Mannheim, Germany). CleanCap™ FireFly Luciferase mRNA (Catalog # L-7602) was obtained from TriLink Biotechnologies (CA, USA). RPMI and DMEM medium (Gibco), fetal bovine serum (FBS; Gibco), penicillin-streptomycin, Dil dye (DiIC18(3)) powder was purchased from Thermo Fisher Scientific Inc (Israel). Holey carbon grid (C flat 3 / 2200 mesh) was purchased from Electron Microscopy Science (Hatfield, PA, USA). Water used was purified using a Barnstead NanoPure system (ThermoFisher Scientific, Waltham, MA,USA) to 18.2 M cm resistance (at 25°C) and total organic content < 1 ppb. RNase free water was obtained from Bio-Lab Ltd. (Israel). D-luciferin potassium salt was purchased from GoldBio (USA). Gel serum separator tubes were obtained from KABE- Labortechnik GmbH (Germany). Qubit™ RNA High Sensitivity (HS) was purchased from ThermoFisher Scientific (USA).Synthesis and characterization of the atom transfer radical polymerization (ATRP) initiator

[0244] After benzoyl acetic acid (10 mmol) was dissolved in 20 mL dry chloroform, 1.5 eq EDC, 1.5 eq HOBt, 3 eq DIPEA were added in the flask under ice-water bath. With 1 h stirring, DTA 7. 3mmol and 30 mL dry chloroform were added to the mixture. After overnight stirring at RT (12-16 h), the condensation product of DTA and Benzoyl acetic acid (Scheme 1, compound 1, yield: 50%) was purified through the column with a mobile phase di chloromethane: ethyl acetate = between 10 to l5: 1. 2 g compound 1 and 100 mg 10% palladium carbon were mixed with 8 mL ethanol, and the mixture was stirred at RT for about 8 h at 3 bar hydrogen pressure. After the filtration, all the solvent was removed to obtain Scheme 1, compound 3 (1.4 g). 1.4 g Scheme 1, compound 4 and 2 eq of triethylamine was dissolved in 10 mL of anhydrous di chloromethane, and 2 eq a-bromoisobutyryl bromide was added dropwise. The salt was removed by extraction. Compound 4 (ATRP initiator, Scheme 1), was reprecipitated as a white solid in methanol three times (yield: 63%).

[0245] Similar protocol was followed for the synthesis of DSA-based initiator using DS A instead of DTA.Synthesis and characterization of DTA or DSA-pMPC

[0246] DTA-pMPC orDSA-PEG / pMPC (Scheme 1, compound 5, for example DTA- pMPC 2kDa) were synthesized by atom transfer radical polymerization (ATRP) using compound 4 as the ATRP initiator (shown in Scheme 1). Compound 4 (62 mg, 0.1 mmol) was dissolved in dichloromethane (1 mL), and MPC (150 mg, 0.5 mmol) was dissolved in absolute ethanol (2 mL). The mixture and CuBr (14 mg, 0.1 mmol) were added to a Schlenk flask with a magnetic stirrer bar. The flask was degassed by nitrogen for 20 min. Then, PMDETA (40 pL, 0.2 mmol) was injected quickly. The flask was stirred overnight (12 h) at RT. Then, the solution was dialyzed against ethanol and water for 48 h, respectively (CelluSep membrane, MWCO 1000). The polymer DTA-pMPC2kDa was obtained after freeze-drying (yield: 72%). 'H NMR chemicalshift (ppm, 300 MHz, CDC13 / CD3OD 1: 1): 0.70-1.20 (3H,CH3-CR-CH2-), 1.26 (22H,-CH2-(CH2)11-CH3), 1.45-2.2O(2H,-CH2-CR-CH3), 3.30(9H ,-N+(CH3)3), 3 75(2H ,-O-CH2-CH2-N+(CH3)3), 4.15(2H ,-C-Q-CH2-),4.25(2H,-CH2-O-P),4.35(2H,-CH2-CH2-N+(CH3)3). The molecular weight of pMPC could be calculated from NMR (-1900), as estimated from comparing the integral intensity ratio of the 'H NMR peaks attributed to the long alkyl chain at 1.26 ppm and the MPC pendant methylene protons at 3.75 ppm (Figure 1). DTA-pMPC lOkDa (Mw 10,059), DTA-pMPC 20kDa (Mw 20,417), DSA-PEG 2kDa (Mw 2,040), DSA-pMPC 5kDa (Mw 5,399), DSA-pMPC 20kDa (Mw 20,178) have been also prepared following the same procedure. The initiator synthesized directly from DTA and 2-Bromo-2-methylpropionyl bromide (BIBB) was found to have no control capability on polymer molecular weight, which might be due to its low initiation efficiency.Scheme 1. Synthesis procedure of DTA-pMPC through ATRP.Preparation of LNPs

[0247] LNPs with poly(A) were prepared by mixing an aqueous phase (2.7 mg / mL poly(A) in sodium acetate buffer 25mM, pH 4) and a lipid mixture of ionizable lipid:phospholipid: cholesterol: PEG / pMPC conjugate dissolved in ethanol (46.3: 9.4: 42.7: 1.6 mol ratio) with a total concentration of 15 mM. In the case of LNPs including stearylamine (SA) the molar fraction of the lipid mixture ionizable lipid: phospholipid: cholesterol: PEG / pMPC conjugate modified accordingly: 40.9: 9.4: 42.7: 5.4: 1.6.

[0248] For Fluc-mRNA LNPs (flue = firefly luciferase), 12 mM ethanolic lipid mixture with SA (same component ratios as in case of poly(A) LNPs) was mixed with flue mRNA (O. lmg / mL) in 25 mM sodium acetate buffer pH 4 for N / P ratio 6 (ration between the amine groups of the ionizable lipid to the phosphate groups of the cargo). The ethanol and aqueous phases were mixed by using the NanoAssemblr® Ignite ™ microfluidic mixing system (Precision Nanosystems Inc., Vancouver, Canada) at a flow rate ratio (FRR) of 1 :3, respectively, using total flow rate (TFR) of 12 mL / min. The mixture was dialyzed against 0.5X PBS for 4 h, following overnight dialysis against IX PBS in Float-A-Lyzers dialysis bag (MWCO 100 kDa) at 4°C and protected from light. After dialysis the encapsulation efficiency (EE%) of mRNA was measured using the Quant-iT RiboGreen RNA assay kit (Life Technologies). In brief 5uL of LNPs were diluted in lOOul of 1XTE buffer in the absence or presence of Triton X-100 (0.5%). The samples upon incubation at RT for 15-20 min were loaded in a 96-well black opaque plate (Costar, Corning). IOOUL of 0.5% v / v RiboGreen reagent in TE IX buffer were added to each well and the fluorescence was detected using a microplate reader according to the manufacturer’s protocol. After subtracting the blank measurements, the encapsulation efficiency (EE%) was calculated based on was calculated as (1 - (non lysed LNPs / lysed LNPs)) x 100. Various systems were developed by varying (a) the carbon chain length of the secondary fatty amines, DTA (C14)2 and DSA (C18)2, and (b) the MW of the conjugated polymers: PEG 2 kDa and pMPC ranging from 2 kDa to 20 kDa. Henceforth, the systems will be denoted by the fatty amine chain length (C14)2 or (C18)2, the encapsulated cargo (poly(A) or fluc-mRNA), and the polymer along with its corresponding molecular weight.Dynamic Light Scattering (DLS)-Size Distribution and Stability Studies

[0249] LNPs were characterized using a Zetasizer Nano-ZS instrument (Malvern Instruments, Malvern, U.K.) equipped with a red laser at 633 nm set at a scattering angle of 173°. Size distribution measurements were carried out at 0.1 mM LNP concentration in 1 x PBS (25 °C). The particle size and the size distribution of both PEGylated and pMPCylated LNPs were measured at specific time intervals while thesamples were stored at 4°C. ξ-potential was measured at the day of preparation at 0.3 mM LNP concentration in ultra-pure water (25 °C).Cryo-TEM imaging

[0250] A Vitrobot Mark IV plunger system was used to prepare the samples for cryo- TEM. Humidity was kept close to 80% for all experiments and the temperature was set at 24 °C. 3 pL of the sample were placed onto a holey carbon grid (C flat 3 / 2200 mesh) which was rendered hydrophilic via glow discharge (Evactron Plasma Cleaning, Evactron, USA). Excess sample was removed by blotting with filter paper and the sample grid was vitrified by rapid plunging into liquid ethane (-180 °C). The grids were kept in liquid nitrogen before being transferred into a Gatan 626 cryo-holder. Cryo- TEM imaging was performed on a FEI Tecnai T12 TEM (120 kV) with a with a TVIPS F244HD CCD digital camera or a Talos Arctica S / TEM microscope from Thermo Fisher Scientific equipped with a Gatan OneView camera.AFM imaging

[0251] Freshly cleaved pieces of mica (ca. 4 cm2) were glued on glass slides and 0.5 mM of (C18)2 flue- mRNA LNPs were added for a liquid meniscus between the tip and the sample to be formed. Following 30 min of sample deposition, MgSCE in water (lOmM) was added to facilitate absorption and AFM measurements were recorded. An Asylum MFP-3D™ stand-alone AFM (Oxford Instruments, Santa Barbara, USA) was used. The surfaces were scanned in non-contact mode under lx PBS using a silicon probe (SNL-10, Bruker) with a V-shaped cantilever. The AFM tip holder was irradiated in a ProCleaner™ Plus UV-ozone cleaning (BioForce Nanosciences, Utah, USA) for 20 min prior to use to remove molecular levels of contamination.Cell culture

[0252] Vero E6 cells were cultured in RPMI medium, supplemented with 1% Pen / Strep and 10% fetal bovine serum (FBS), and were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. RAW 264.7 cells were cultured in DMEM medium, supplemented with 2 mM glutamine and 10% FBS, and were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2.Cytotoxicity studies

[0253] Prior cytotoxicity experiment, cells were cultured in 96-well plates at 1 x 105cells / mL and allowed to attach for 24 h. Poly(A)-LNPs cytotoxicity was evaluated byadding pMPC-, PEG- LNPs at final concentration of 0.1 mM and 0.05 mM to 100 pL well containing cultured cells and incubated for 72 h at 37 °C (95% air and 5% CO2). In the case of (C18)2 fluc-mRNA LNPs the systems were tested in two different concentrations of mRNA (20 ng and 100 ng / well). Cell viability was determined by the production of the yellow formazan product upon cleavage of XTT by mitochondrial dehydrogenases. Following the 72 h incubation period with LNPs, the cells were incubated with 50 pL of XTT solution for 4 h at 37 °C (95% air and 5% CO2). Absorbance values were later measured with ClarioStar microplate reader (BMG LAB TECH GmbH, Germany) at a wavelength of 450 nm. Background absorbance was measured at 620 nm and subtracted from the 450 nm measurement. The absorbance of a solution in cell-free media with XTT was subtracted from all samples. The potential toxic effect of the different LNPs formulations tested was expressed as a viability percentage calculated using the following formula:Where ODtest was the optical density of those wells with LNPs, and ODcwas the optical density of cells in media only (no LNPs added).In vitro transfection

[0254] Vero E6 and RAW 264.7 cells were seeded (1 x 104cells / well) in 96-well white / clear bottom culture plates (Greiner Bio-one, Germany) and allowed to adhere. At 24 h post-seeding, cells were treated with (C 18)2 fluc-mRNA LNPs (100 ng dose per well) for 48 h supplemented in culture medium (in the presence of FBS). Luciferase activity was measured using Promega ® VivoGlow™ Luciferin according to the manufacturer’s protocol. In brief, cell medium was aspirated and was replaced with ImM of Promega ® VivoGlow™ Luciferin. The plate was incubated for a period of 20 min at 37 °C before measuring luminescence with ClarioStar microplate reader (BMG LABTECH GmbH, Germany). Untreated cells and treated with non-encapsulated mRNA at the same dose were used as controls.ImageStream measurements

[0255] Raw 264.7 and Vero E6 cells were seeded into 6-well plates with density of 1 x 106cells / well. At 24 h post-seeding, medium was changed and poly(A) LNPs in fresh cell culture medium were added (final concentration 0.2 mM), and samples were incubated for 4 h at 37 °C (95% air and 5% CO2). Following incubation, cells werewashed with sterile PBS and 0.25% trypsin was added (3 min, 37 °C (95% air and 5% CO2)). After incubation, cells were detached and resuspended in medium by gentle pipetting. Suspension was centrifuge at 400 g for 5 min, supernatant was discarded, and pellets were resuspended in fresh medium following another round of centrifuge. Final pellets were resuspended in 35 pL sterile PBS. Cells were imaged using multispectral imaging flow cytometry (ImageStreamX mark II imaging flow-cytometer; Amnis Corp, Seattle, WA, Luminex). Approximately 1.5 - 2.0 x 104cells were collected from each sample and data were analyzed using image analysis software (IDEAS 6.3; Amnis Corp). Data were acquired using a 60 lens, and lasers used were 405 nm (120mW) and 561 nm (100 mW). Data were analyzed using the manufacturer’s software IDEAS 6.2 (AMNIS corp.). Cells were first gated according to their area (in μm2) and aspect ratio (the Minor Axis divided by the Major Axis of the best-fit ellipse) of the iRFP staining. Cropped cells were eliminated using the bright-field Area and Centroid X (the number of pixels in the horizontal axis from the upper, left corner of the image to the center of the mask) features. Cells were further gated for Dil according to its corresponding intensity. Plotting these features on a bi-variate plot gave a clear distinction of the LNPs internalization.In vivo experimentsAnimal care

[0256] Ten to eleven-week-old female Hsd:ICR (CD1) mice were purchased from Envigo (Israel). All mice were housed in groups of 2-5 mice per micro-isolator cage in a room with a 12 h light / dark schedule with ad libitum access to food and water. All procedures on animals were approved by the IACUC committee (IACUC Number: 02900322-2, 00130124-2).Intravenous (IV) administration of LNPs, blood collection and organ biodistribution

[0257] Dil- labeled (C14)2 and (C18)2 poly(A)-LNPs suspension (100 pL, 5 mM) was administered intravenously (IV) in the lateral tail vein and at well-defined time points blood was collected from facial vein into test tubes for serum collection. Blood was stored at RT and protected from light for 30 min and centrifuged at 3000 g for 10 min at 4°C. 10 pL of serum was collected and examined for Dil presence in a 384-well black plate using microplate reader (BMG LABTECH GmbH, Germany).Alternatively, 24 h post administration, mice were sacrificed, and the spleen, liver, kidneys, heart, and lungs were harvested and imaged using the IVIS imaging system. To evaluate the effect of repeated doses on blood retention time (ABC phenomenon induced by the initial dose of (C18)2 poly(A)-LNPs), mice were administered two doses at a 7-day interval. Blood samples were collected and analyzed using the same protocol.In vivo toxicity study

[0258] Mice were intravenously injected with (C18)2 fluc-mRNA LNPs containing (mRNA dose: 0.15 mg / kg) for toxicity studies. 24 h post-injection, blood was collected from the facial vein of the mice. Blood was stored at RT for 30 min and centrifuged at 3000 g for 10 min at 4°C. Serum was kept in ice and analyzed with the VetScan VS2 Chemistry Analyzer (Zoetis, USA) and the Comprehensive Profile Rotors VS2 (Abaxis, USA) the day of collection. Before loading the sample, the serum equilibrated in RT. The weight of the animals was recorded before the inj ection and before the blood withdrawn.In vivo luciferase assay & Organ distribution study

[0259] Mice were intravenously injected with (C18)2 fluc-mRNA LNPs with DSA- pMPC 5kDa and DSA-pMPC 20kDa at a dose of fluc-mRNA 0.15 mg / kg. At 6 h and 24 h post injection, mice were intraperitoneally (IP) injected with D-luciferin potassium salt solution dissolved in 1 x PBS (150 mg / kg). 5 minutes following the IP injection, mice were anesthetized using the rodent anesthesia system with isoflurane. Mice were imaged for luminescence 8 min post D-luciferin injection with the in vivo imaging system (IVIS) LagoX (Spectral Instruments Imaging, USA). After measuring the 24 h point, the mice were sacrificed in their home cages with CO2 exposure, dissected and the major organs (heart, lungs, liver, spleen and kidneys) were collected and imaged by the IVIS system.Immunogenicity study

[0260] Mice were intravenously injected with a single lOOul injection of 6mM (C18)2 fluc-mRNA LNPs containing DSA-polymer conjugates- PEG2kDa, pMPC5kDa and pMPC20kDa and blood from the facial vein was collected in serum gel separator tubes 5-days post injection. IgM protein levels in the blood serum in Hsd:ICR (CD1) mice were determined using the ELISA kit from Abeam (ab-133047) according to the recommendations of the manufacturer.EXAMPLE 1

[0261] We functionalized LNPs either with the novel zwitterionic polymer- conjugated fatty amines, poly(2-methacryloyloxyethyl phosphorylcholine) (DTA / DSA-pMPC), as described above with pMPC molecular weights between 5kDa and 20kDa; or by PEGylation as for the standard functionalized LNPs with 2kDa PEG (using ALC-0159) or synthesized DSA-PEG 2kDa. We prepared LNPs using the established microfluidics technique; in all cases the polymer surface functionalization (either PEG or pMPC) density was kept at 1.6% (mol / mol). The behaviour and properties of our novel pMPCylated LNPs are described below with poly(A) or fluc- mRNA encapsulated.Determination of pMPC-LNP structure and size distribution

[0262] Scheme 1 shows the new pMPC conjugate (compound 5) that was synthesized. For comparison with PEG-stabilized LNPs (as used for example in Pfizer Coronavirus COVID19-vaccine), therefore, the molecule for stabilizing LNPs by pMPCylation is made by conjugating an amide ((C14ji) with a pMPC moiety. We functionalize the DTA with a bromine initiator and then polymerize the MPC via ATRP and the resulting molecule is a poly-phosphocholinated analogue of ALC-0159 (DTA- PEG). These molecules were then incorporated into LNPs forming pMPCylated LNPs with different molecular weight pMPC tails (designated as pMPC-LNPs).

[0263] To investigate whether pMPC-LNPs induce changes in cellular uptake, blood retention and toxicity relative to PEG-LNPs, we prepared three types of LNPs differing in the pMPC length, and with or without added SA (at 5% (mol / mol) level as described earlier). All LNPs were composed of ALC-0315 ionizable lipid, DSPC, cholesterol, which comprise the basic LNP composition, while fatty amine conjugated polymers DTA-PEG 2kDa (as used for the Pfizer vaccine nanodelivery vehicles) and DTA-pMPC (5kDa and 20kDa) were used for the nanoparticle stabilization. Higher M values were used for the pMPC moieties as the MPC-monomer molecular weight is substantially higher than that of the PEG monomer. For introducing a positive charge into LNPs 5.4 % (mol / mol) of SA was added. However, since LNPs interact with various biological molecules, leading to surface modifications and significant polymer shedding, (C18)2- PEG / pMPC conjugates were also developed and tested for their potential advantages by slowing the shedding kinetics from the LNPs.

[0264] LNP stability is a property that can impact their clinical application. Particles of greater size are generally taken up rapidly by the reticuloendothelial system (RES) resulting in their rapid clearance and shortened half-life. Dynamic light scattering (DLS) measurements were used to characterize and validate the LNPs hydrodynamic diameter and the poly dispersity index (PDI) of all the LNPs. As shown in Table 1, the LNPs size was in the range of 70-100 nm in diameter with a relatively narrow size distribution. LNPs with SA added have a slightly increased diameter -in the cases of lower polymer length. The incorporation of polymers of different length resulted in increased hydrodynamic diameter as implied from the differences between 5 kDa and 20 kDa pMPC systems- independently to the fatty amine carbon chain length- attributed to the increased polymer chain length extending from the LNPs surface.

[0265] (C14)2 and (C18)2 poly(A)-pMPCylated-LNPs were studied in comparison with PEGylated-LNPs to assess their colloidal stability upon storage time. Interestingly, we observed that (C14)2 short chain pMPC (5 kDa) compared to (C14)2 PEG 2kDa has greater stability. In the case of (C14)2 PEGylated-LNP on day 60 we can observe 3 different populations and a change of PDI from 0.131 ±0.01 to 0.301±0.01 while on (644)2 pMPC 5kDa 3 populations were observed later on Day 90 (Figure 2 A).

[0266] The addition of SA in the case of PEG-LNPs slows down the appearance of a second population and as a result the increase of the PDI for about 30 days, while in case of (C14)2 pMPC 5kDa in Day 90 the system still has a comparatively low increase of PDI (Figure 2B). In the case of (C18)2 poly(A) systems where (C18)2 poly(A)-pMPC 5kDa and pMPC 20kDa systems have better colloidal stability upon storage at 4°C compared to (C18)2 -poly(A) PEG2kDa, which at 55 days resulted in increased diameter (130.1 ± 3.8 nm) and increased PDI (0.27±0.02), respectively (Figures 2D1-2). In the case of (C18)2 poly(A)-PEG2kDa system, no additional peaks were observed; instead, a widening in the size distribution was noted. This can be attributed to slower aggregation phenomena, likely caused by the gradual shedding of polymers from the surface of the lipid nanoparticles (LNPs). This extended stability should facilitate the handling of the pMPC-LNPs relative to PEG-LNPs, especially in case of their use in vaccination.Table 1. Hydrodynamic diameter (d,nm) and PDI of PEG- and pMPC-functionalized poly(A) LNPs by Dynamic Light Scattering (DLS, Size by intensity).

[0267] Cryo-transmission electron microscopy (Cryo-TEM) further confirms the pMPC-LNP structure (Figure 3A1-A3), with particles possessing a spherical morphology, and displaying an electron-dense core morphology of nanoparticle. The sizes of the Cryo-TEM images reported slightly reduced compared to the DLS analysis with more pronounced differences in the case of 20 kDa due to the extended and highly hydrated polymer which is considered to the hydrodynamic diameter calculations of DLS in contrast to cryo-TEM.

[0268] In the case of (C18)2 poly(A)-PEG 2kDa SA and (C18)2 poly(A)-pMPC 5kDa SA LNPs the number of particles presenting bleb structures (Figure 3 A2, black arrow) is higher, as compared to the corresponding (C18)2 poly(A)-pMPC 20kDa SA system.EXAMPLE 2Cell uptake and biocompatibility of pMPC-LNPs

[0269] The interaction and cell uptake of pMPC-LNPs were studied in two different cell line systems, RAW 264.7 and Vero E6 cell line. RAW 264.7 (murine macrophage cells) were selected as a model system for studying the mRNA delivery systems in cancer research, while Vero E6 were chosen as a common model for viral infections and antiviral treatment.

[0270] To quantify the total cellular uptake of functionalized LNPs, cells were incubated for 4 h with the nanoparticles (final concentration: 0.2 mM), subsequently thoroughly washed to remove non-internalized nanoparticles, and then analyzed by ImageStream flow cytometry. Fluorescence staining of LNPs (Dil) was used to estimate the number of internalized targets and intracellular localization of LNPs was performed on a per-cell basis. By quantifying fluorescence signals from internalized (C14)2 poly(A)-pMPC 5kDa LNPs and (C14)2poly(A)-PEG 2kDa LNPs by RAW 264.7 cells we observed similar values of fluorescence intensity for both particles containing and lacking positively charged lipids present in the nanoparticles’ membrane (Figure 4A). Additionally, uptake of RAW cells appears not affected by the choice of surface functionalization as well. In contrast, the LNPs uptake by Vero E6 cells instead shows a clear dependency on the amount of positive charge (Figure 4 B), with both pMPC- and PEG-LNPs displaying improved uptake for higher positively charged lipids content. Interestingly, enhancement of the uptake in the presence of SA appears larger for pMPC-LNPs compared to PEGylated nanoparticles. Similar patterns were followed in the case of (C18)2-poly (A)-pMPC5kDa SA which also showed a similar uptake at 4h incubation for RAW cells and increased compared to (C14)2counterparts for Vero E6 cells (data not shown).

[0271] Increasing the polymer length for pMPC (20 kDa), we observed similar results in case of RAW cell line, with comparable fluorescence intensity values across all 4 formulations (Figure 5 A). However, we observed a different trend for Vero E6 cells’ uptake (Figure 5 B), with an overall increase in uptake of pMPC particles compared to shorter chain length (Figure 4 B) and lower uptake difference between pMPC-and PEG- LNPs (Figure 5 B), alongside with comparable internalization values independently of positive charge.

[0272] We further validated the internalization of LNPs, as the ImageStream measurements allow visualization of the acquired cells and the fluorescence distribution arising from nanoparticles’ uptake (Figure 6). We observed that for both pMPC-and PEG-LNPs, the fluorescence signal is located inside the cell (not bound or fused to thecell membrane) in a punctuated vesicular pattern, in line with reported endosomal intracellular uptake.

[0273] To complement the LNPs internalization measurement, we evaluated the nanoparticles’ cytotoxicity by exposing monolayers of Vero E6 cells to different LNPs. As shown in Fig. 7, (C14)2 poly (A)-LNPs formulations after 72 hours of incubation showed no negative impacts on cell viability, with >80% cell survival upon exposure to LNPs indicating low toxicity to cells and biocompatibility, profile that was also followed by the (C18)2poly (A)-LNPs.EXAMPLE 3In vivo circulation time and biodistribution of pMPC- functionalized LNPs

[0274] To evaluate the in vivo properties of pMPC functionalization, LNPs were fluorescently labeled and injected into mice via tail vein injection. At specific time points, blood was collected and the amount of LNPs in the bloodstream was determined from the fluorescence signal arising from LNPs’ encapsulated Dil. Figs. 8A-8B show that (C14)2 poly (A)- 5 kDa pMPC, 20 kDa pMPC and 2 kDa PEG-LNPs all supplemented with SA have comparable blood clearance kinetics with - 11-16 minutes persistence half-time in blood. These findings could be attributed to the similar shedding profile of PEG and pMPC from the LNPs which is an important factor for the effective delivery of nucleic acids but at the same time leads to rapid immunogenic clearance of the LNP from the blood. To increase the blood retention time of the LNPs in the bloodstream, (C18)2-poly(A) LNPs with SA were also tested with the higher molecular weight 20kDa pMPC (- 51 min) to have comparable half retention time to its PEGylated counterpart (-68 min respectively) in contrast to shorter pMPC chains (-29 min for 5 kDa).

[0275] Following blood retention time studies, we also measured the Dil signal in different organs to evaluate any difference in the organ distribution of the LNPs. LNPs are well-known for their short half-lives within the blood circulation and their rapid accumulation (within hours) in target tissues or organs. To verify this biodistribution, 100 pL, 5 mM LNPs were injected into the tail vein of CD1 mice (n=3) and allowed to circulate for 24 hours. Organs were collected separately for closer examinations on NP biodistribution. IVIS images revealed that LNPs, regardless of their polymer length, were present in the same organs, including lungs, liver, spleen, and kidneys (Figures 9A-C).

[0276] Upon second injection of (C18)2-poly(A) LNPs rapid blood circulation clearance of the nanoparticles was observed, a well- known phenomenon known as ABC phenomenon (Figs. 10A-10C) attributed to the triggering of immune system upon repeated doses. (C18)2-poly(A) 2kDa PEG LNPs and 5kDa pMPC LNPs showed decreased retention time in blood after the second injection (ti / 2 <10min), while as the pMPC length was increased to 20 kDa there was a still significant but slower reduction. This observation indicates that replacing PEG with pMPC while fine tuning the molecular weight of the polymer could increase blood circulation and reduce immune response, resulting in the reduction of the ABC phenomenon (Fig.lOA-C). mRNA Encapsulation

[0277] Upon successful development of pMPCylated LNPs, of different lipid carbon chain and pMPC length, fluc-mRNA (1929 nucleotides, MW 622,225) was selected as a model nucleic acid for testing the ability of the pMPCylated LNPs to encapsulate and deliver effectively their cargo. (C18)2-polymer conjugate LNPs with SA were used for further experimentation, as they proved to have higher blood retention time compared to the LNPs with (C14)2 polymer-conjugates, potentially increasing their overall efficiency. Encapsulation of fluc-mRNA led to increased size LNPs (95.5-138.3 nm) compared to their poly(A) counterparts, while retaining low PDI values (<0.11) (Fig. 11A-C, Table 2).

[0278] The surface charge of the systems was close-to-neutral as seen by zeta potential values, making them appropriate for in vivo applications. Encapsulation efficiency for the (C18)2-fluc PEG2kDa and (C18)2- flue pMPC5kDa LNPs was 79.1 % and 79.4 % respectively.Table 2. Physicochemical properties of (C18)2 fluc-mRNA LNPs. § | §

[0279] Stability study of the LNPs encapsulating fluc-mRNA demonstrates colloidal stability for at least 25 days when stored at 4°C in PBS, without the addition of any cryoprotectant (Fig. l2A-D). Throughout the study period, no additional populations or significant increase in size distribution were observed, proving that all three formulations are suitable for mRNA encapsulation. The poly dispersity index (PDI) values remain below 0.2 for all formulations, indicating good homogeneity of the mRNA loaded nanoparticles.

[0280] AFM was used to evaluate the properties of lipid nanoparticles (LNPs) deposit on mica substrate. As observed by the inventors, the (C18)2 fluc-mRNA PEG 2kDa LNPs were not able to be absorbed when using mica as a substrate. However, the pMPCylated LNPs were efficiently deposited on mica, indicating a differential interaction with the negatively charged mica due to the zwitterionic nature of the polymer, favorably compared to PEG. AFM showed spherical structures of pMPCylated mRNA-LNPs (Fig. 13A-13C).

[0281] Cryo-TEM analysis (Fig. 13D-13F) reveals that all the mRNA encapsulated formulations exhibit spherical structure, with the anticipated presence of mRNA-rich areas / protrusions, known as blebs, observed in the literature. Bleb formation has been correlated with improved transfection efficiencies as they provide a specific environment for mRNA, potentially protecting it against degradation and increasing its efficiency towards cellular uptake.In vitro toxicity and transfection studies

[0282] The in vitro toxicity of the systems was evaluated in 2 different doses of mRNA (20 ng and 100 ng). In all cases, the systems did not exhibit cytotoxicity, with values > 80% (Figs. 14A-14B), allowing their use for further in vitro assays without compromising the cell viability.

[0283] The above-mentioned cell types of macrophages and epithelial origin were used for the transfection of flue- mRNA upon 48 h incubation period. As can be seenfrom Figs.l5A-15B, the lipid nanoparticles successfully facilitated mRNA transfection in the dose of 100 ng. Fluc-mRNA in the same dose has been used as a negative control as the absence of any transfection agent leads to low efficiency due to instability of the mRNA in the cell medium. Interestingly, the pMPCylated LNPs showed significantly higher efficiency compared to the PEG2kDa LNPs.

[0284] The differences are more pronounced in the VeroE6 cells as RAW 264.7 cells are known for their efficient antigen degradation capability, which explains the decreased transfection efficiency.In vivo toxicity studies

[0285] The in vivo toxicity of (C18)2-fluc mRNA LNPs was evaluated by quantifying body weight changes, serum biochemistry levels and organ-to-body weight ratios. After a single IV injection of (C18)2-fluc mRNA LNPs (0.15 mg / kg) and 24 h post-injection, no significant differences were observed in body weight and no visual signs of toxicity were reported suggesting that neither of the LNPs was associated with substantial toxicity (Fig. 16A-16F).

[0286] In general, changes in blood biochemistry can indicate specific organ dysfunction caused by LNPs, while changes in organ-to-body weight ratios can provide a sensitive indicator of treatment-related effects on target organs. When quantifying the various bioanalytes in serum, an increase in ALT levels was observed compared to PBS group. Increased ALT levels are in accordance with previous studies dealing with the administration of LNP loaded with mRNA, or empty LNPs in rodents with transient effect.

[0287] The levels of other liver related bioanalytes such as alkaline phosphatase (ALP), total bilirubin (TBIL) and blood urea nitrogen (BUN) were not significantly changed from those of PBS control in all cases, indicating no additional implications (Table 3).

[0288] Additionally, no changes in organ-to-body weight ratios were observed between the groups, indicating the safety of the administered formulations. Toxicity tests upon repeated doses of (C18)2-poly(A) pMPC 20kDa LNPs (2 doses / week for 3 weeks) did not lead to mortality and did not decrease the bodyweight during the experiment period (n=5).Table 3. Serum biochemistry values obtained 24h post injection of PBS, (C18)2-fluc mRNA 5kDa and (C18)2-fluc mRNA 20kDa (0.15 mg / kg) analyzed by VetScan Analyzer. Data are presented as mean ±STDEV, n=3 mice per group.EXAMPLE 4In vivo screening of pMPCylated LNPs for mRNA delivery

[0289] To evaluate the ability of the (C18)2 fluc-mRNA LNPs to deliver efficiently their cargo, CD1 mice were intravenously injected with a single dose of LNPs, and the mRNA expression was measured and compared between the two different pMPCylated groups. At 6 h post injection the mice had same bioluminescence intensity while 24 hr post injection we observed lower firefly luciferase expression in 5k Da pMPCylated carriers compared to the 20kDa. At 24 hr post injection the organs of the animals were also measured with the use of IVIS and the luciferase signal was detected mainly on the liver and spleen, which are the major organs of drug metabolism and excretion, along with the increased hepatotropism of LNPs. Proving their ability to deliver and retain the properties of the encapsulated cargo, pMPCylated systems can prove beneficial in nucleic acid delivery (Figs. 17A-17D).Immune response against LNPs

[0290] Administration of PEGylated LNPs (intravenously, intramuscularly, subcutaneously) can trigger formation of anti-PEG IgM antibodies, which are relatedto the ABC phenomenon and the reduced effectiveness of repeated doses. To test the immune response and the IgM production towards the developed systems, we injected mice with the same concentration of LNPs and measured the production of IgM level 5 days post injection in serum (Figure 18).

[0291] Blood serum from control mice (PBS IV injection) established the physiological level of antibodies, which were found to be 210 pg / mL. The injection of (C18)2 fluc-mRNA PEG 2kDa LNPs shows IgM levels of 443 pg / mL (range of IgM levels: 403-521 pg / mL), while for the IV administrated LNPs with 5kDa and 20kDa pMPC functionalization the values were 379 pg / mL (range of IgM levels: 386-391 pg / mL) and 360 pg / mL (range of IgM levels: 314-432 pg / mL) respectively (Fig. 18). The results show a trend to significantly better IgM response when pMPC- functionalized LNPs are IV administrated compared to their PEGylated counterpart, paving the way for their use in clinic for improved outcomes.

[0292] In summary, we demonstrate for the first time a successful (pMPC) functionalization of LNPs with the conjugates of the invention, for the replacement of PEGylated LNPs, which are known to be associated with undesirable in vivo effects, among others, with immune response activation by the PEG moieties. The pMPC functionalization of the LNPs disclosed herein provides comparable or better particle dispersion stability over time compared with the PEGylated LNPs (as monitored by detection of larger aggregates), while having the same macrophage uptake.

[0293] As a result, the LNPs disclosed herein avoid macrophage recognition at the same level as the PEGylated ones, and at the same time show no cytotoxicity in culture. LNPs disclosed herein efficiently encapsulate mRNA, while presenting increased transfection efficiency to epithelial cells. Initial in vivo studies suggest that the LNPs disclosed herein are efficient in mRNA expression (in the liver and spleen), while being well tolerated by animals upon single injection.

[0294] Surprisingly, upon IV injection of the LNP of the invention the inventors observed a significantly decreased immune response (IgM production), as compared to PEGylated counterparts. In addition, LNPs with the conjugate of the invention having pMPC of about 20kD showed even more decreased immune response compared to pMPC of about 5kD (Figure 18). Accordingly, the LNPs disclosed herein can be used for example as carriers of biological drugs (e.g. RNA), thus providing an alternative for the conventional PEGylated-LNPs (which are known to generate anti-PEG antibodies and contribute to a reduced therapeutic efficacy of biological drugs).

[0295] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMS1. A compound, a salt of said compound or both, wherein the compound is represented by Formula 1 :wherein:A is a spacer;R is H or an optionally substituted Cl -CIO alkyl; each Li independently is selected from an optionally substituted C5-C30 alkyl, H andrepresents a single bond, a triple bond or a double bond;each X independently comprises CH2, CHR’2, an optionally substituted C1-C10 alkyl, an optionally substituted C5-C30 alkyl, an optionally substituted C5-C30 alkenyl or an optionally substituted C5-C30 alkynyl; each T independently represents NR’2, NH, O, S, -CONH-, -CONR’-, -C(=NH)NR’-, - C(=S)NR’-, -NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, - C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-, or phosphate, as allowed by valency; each R’ is independently H or comprises an optionally substituted C1-C10alkyl, an Ci- C10 alkyl-aryl, an C1-C10alkyl-cycloalkyl, optionally substituted C3-C10cycloalkyl, optionally substituted C3-C10heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl or a combination thereof;Y is halo; n is between 2 and 300; each k is independently between 0 and 10; and wherein at least one L is not H.

2. The compound of claim 1, wherein R is methyl.

3. The compound of claim 1 or 2, each L is independently C10-C20 alkyl or C10-C20 alkenyl.

4. The compound of any one of claims 1 to 3, wherein A comprises , wherein R1 represents hydrogen, halogen, -NO2, -CN, -OH, oxo, imino, -CONH2, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -CONH-NH2, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR’, -NC(=S)OR’, - NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, Ci-C6haloalkyl, optionally substituted C1-C6alkyl, -NH2, -NR’2, -NH(C1-C6alkyl), -N(Ci- C6, alkyl)2, C1-C6alkoxy, C1-C6haloalkoxy, hydroxy(C1-C6alkyl), hydroxy(C1-C6alkoxy), alkoxy(C1-C6alkyl), alkoxy(C1-C6alkoxy), C1-C6alkyl-NR’2, C1-C6alkyl- SR’, -CONH(C1-C6alkyl), -CON(C1-C6alkyl)2, -CO2H, -CO2R’, -OCOR, -OCOR’, - OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, or a combination thereof; wherein each R’ independently represents hydrogen, or is selected from the group comprising optionally substituted C1-C10alkyl, optionally substitutedC3- C10cycloalkyl, optionally substitutedC3- C10heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, hydroxy, amino, -NH2, -NR’2-NH(C1-C6alkyl), -N(C1-C6alkyl)2, C1-C6alkoxy, C1-C6haloalkoxy, hydroxy(C1-C6alkyl), hydroxy(Ci- Ce alkoxy), alkoxy(C1-C6alkyl), alkoxy(C1-C6alkoxy), C1-C6alkyl-NR’2, C1-C6alkyl- SR’, or a combination thereof.

5. The compound of any one of claims 1 to 4, wherein the compound is represented by Formula 2:wherein each Li is independently C10-C20 alkyl or C10-C20 alkenyl, and wherein Y is halo.

6. The compound of claim 5, having an average MW between about 1000 and about 30.000Da; and wherein each Li is independently C12-C20 saturated alkyl.

7. The compound of any one of claims 1 to 6, wherein each Li is selected from tetradecyl and octadecyl.

8. A lipid nanoparticle (LNP) comprising the compound of any one of claims 1 to 7 and an active agent, wherein said lipid nanoparticle further comprises an ionizable lipid, a helper lipid, and a structural lipid.

9. The lipid nanoparticle of claim 8, wherein said lipid nanoparticle is a plurality of particles characterized by at least one of: an average size in a range between 30 and 300 nm; and an average zeta potential between 0 and -25mV at a pH of about 7.

10. The lipid nanoparticle of claim 8 or 9, wherein said active agent comprises a nucleic acid; and wherein said structural lipid is a sterol.

11. The lipid nanoparticle of any one of claims 8 to 10, wherein a molar concentration of the compound relative to the total lipid content of the lipid nanoparticle is between 0.1 and 10 mol %.

12. The lipid nanoparticle of any one of claims 8 to 11, wherein a molar concentration of any one of the ionizable lipid and the structural lipid relative to the total lipid content of the lipid nanoparticle is between 10 and 60mol%.

13. The lipid nanoparticle of any one of claims 8 to 12, wherein a molar ratio between the ionizable lipid and the helper lipid within the lipid nanoparticle is between 3: 1 and 6: 1.

14. The lipid nanoparticle of any one of claims 8 to 13, characterized by N:P ratio between 1 and 20; and wherein the molar concentration of the compound relative to the total lipid content of the lipid nanoparticle is between 1 and 5 mol %.

15. The lipid nanoparticle of any one of claims 8 to 14, wherein said structural lipid is cholesterol, wherein said ionizable lipid is ALC-0315, wherein said helper lipid is DSPC, wherein a molar concentration of the ionizable lipid and of the structural lipid is between about 40 and about 50mol%; wherein the active agent is RNA; and wherein the lipid nanoparticle further comprises between 0.5 and 10mol% of an C10-C20 alkyl amine.

16. A pharmaceutical composition comprises a plurality of the lipid nanoparticles of any one of claims 8 to 15 and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, comprising a therapeutically effective amount of the active agent.

18. The pharmaceutical composition of claim 16 or 17, wherein said lipid nanoparticles are characterized by an average particle size between 50 and 150nm; and by a PDI of between 0.05 and 0.2.

19. The pharmaceutical composition of any one of claims 16 to 18, for use in the treatment of a disease or disorder in a subject in need thereof.

20. A method for delivering an active agent to a tissue of a subject, the method comprising administering to said subject an effective amount of the pharmaceutical composition of any one of claims 16 to 18, thereby delivering the active agent to said tissue.

21. The method of claim 20, wherein said administering is via intravenous, intramuscular, pulmonary, intratracheal, intranasal or topical administration.

22. The method of claim 20 or 21, wherein said tissue comprises liver tissue, spleen tissue, or both.

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