Lipid nanoparticles targeting central nervous system endothelial cells

Abstract

Provided are lipid nanoparticles, compositions, and methods of making and using the same. The lipid nanoparticles are formulated to specifically target CNS endothelial cells. The lipid nanoparticles may contain a CNS endothelial cell targeting group coupled to a PEG- lipid. The lipid nanoparticles may carry a cargo, e g., a mRNA. The lipid nanoparticles are used for transfection of CNS endothelial cells.

Description

LIPID NANOPARTICLES TARGETING CENTRAL NERVOUS SYSTEM ENDOTHELIAL CELLSRELATED APPLICATIONS

[0001] This Application claims the benefit under 35 U. S. C. § 119(e) of U. S. Provisional Application No. 63 / 715188, filed on November 1, 2024, the entire contents of which are incorporated herein by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (G069670003WO00-SEQ-ACZ.xml; Size: 27,967 bytes; and Date of Creation: October 23, 2025) are herein incorporated by reference in their entirety.FIELD

[0003] The disclosure provides lipid nanoparticles for the delivery of nucleic acids to central nervous system endothelial cells, methods of making and use.BACKGROUND

[0004] Methods for delivering therapeutic molecules into the brain by inducing transcytosis of brain endothelial cells have been employed. However, no methods for specifically targeting brain endothelial cells, without also targeting intracerebral cells exist. Since brain endothelial cells play a critical role in a variety of diseases, there exists a need for safe and effective in vivo targeted transduction of brain endothelial cells.SUMMARY

[0005] Provided are lipid nanoparticles (LNP) that are designed to specifically target central nervous system (CNS) endothelial cells, including brain endothelial cells. The LNPs deliver a cargo specifically to CNS endothelial cells, including brain endothelial cells. In some aspects, the LNPs comprise: (a) a lipid-cell targeting group conjugate comprising the compound of Formula (I): [Lipid] - [optional linker] - [antibody] (Formula I), (b) an ionizable cationic lipid, and (c) a cargo. In some aspects, the cargo is encapsulated in the LNP. In some aspects, the antibody is an antibody that specifically binds to a transferrin receptor.

[0006] In some aspects, the antibody that specifically binds to a transferrin receptor is covalently coupled to the Lipid in Formula (I) via a linker comprising polyethylene glycol (PEG).

[0007] In some aspects, the Lipid in Formula (I) covalently coupled to the antibody is di stearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), or ceramide.

[0008] In some aspects, the PEG has a molecular weight of about 1.0 kDa to about 5.0 kDa.

[0009] In some aspects, the antibody is an immunoglobulin single variable domain (ISVD), a Fab, or an scFv.

[0010] In some aspects, the antibody is a Fab.

[0011] In some aspects, the antibody is a human transferrin receptor binding Fab. In some embodiments, the antibody comprises a CDR-H1 comprising the amino acid sequence of RFTFSSYAMH (SEQ ID NO: 5); a CDR-H2 comprising the amino acid sequence of VISYDGSNKYYADSVKG (SEQ ID NO: 6); a CDR-H3 comprising the amino acid sequence of DLSGYGDYPDY (SEQ ID NO: 7); a CDR-L1 comprising the amino acid sequence of GFTFSNYGMH (SEQ ID NO: 8); a CDR-L2 comprising the amino acid sequence of MIYYDSSKMNYADTVKG (SEQ ID NO: 9); and a CDR-L3 comprising the amino acid sequence of AGWDDSLTGPV (SEQ ID NO: 10). In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

[0012] In some embodiments, the antibody comprises a CDR-H1 comprising the amino acid sequence of GFTFSNYGMH (SEQ ID NO: 11); a CDR-H2 comprising the amino acid sequence of MIYYDSSKMNYADTVKG (SEQ ID NO: 12); a CDR-H3 comprising the amino acid sequence of PTSHYVVDV (SEQ ID NO: 13); a CDR-L1 comprising the amino acid sequence of QASQDIGNWLA (SEQ ID NO: 14); a CDR-L2 comprising the amino acid sequence of GATSLAD (SEQ ID NO: 15); and a CDR-L3 comprising the amino acid sequence of LQAYNTPWT (SEQ ID NO: 16). In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

[0013] In some aspects, the LNP further comprises a structural lipid, a neutral phospholipid, or a free PEG-lipid, or any combination thereof.

[0014] In some aspects, the structural lipid is a sterol.

[0015] In some aspects, the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and sphingomyelin.

[0016] In some aspects, the free PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.

[0017] In some aspects, the free PEG-lipid is PEG-dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidyl-ethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phospho-ethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide, diacylphosphatidylethanolamine comprising Dipalmitoyl (C16) chain or Distearoyl (C18) chain, or a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

[0018] In some aspects, the cargo comprises a nucleic acid molecule.

[0019] In some aspects, the nucleic acid molecule is selected from the group consisting of ssDNA, dsDNA, cDNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, CRISPR-Cas9, antagomir, antisense molecule, targeted nucleic acid, and any combination thereof.

[0020] In some aspects, the mRNA encodes a therapeutic polypeptide.

[0021] In some aspects, the cargo comprises a therapeutic polypeptide.

[0022] In some aspects, the therapeutic polypeptide is for treating familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, a rare disease or an orphan disease.

[0023] In some aspects, the rare disease is selected from the group consisting of spinal muscular atrophy, Huntington’s Disease, Rett Syndrome, Amyotrophic Lateral Sclerosis (ALS), Duchenne Type Muscular dystrophy, Friedreich’s Ataxia, progranulin (PRGN), nonAlzheimer’s cerebral degenerations, frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA) and semantic dementia.

[0024] In some aspects, the LNP selectively binds to at least one target cell of interest.

[0025] In some aspects, the target cell of interest is a central nervous system endothelial cell.

[0026] In some aspects, the ionizable cationic lipid comprises a compound of Formula (II):R1, R2, and R3are each independently a bond or C1-3 alkylene;R1A, R2A, and R3Aare each independently a bond or C1-10 alkylene;R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1-20 alkenyl, -(CH2)0-10C(O)ORa1, or -(CH2)0-10OC(O)Ra2;Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl;R3B1is C1-6 alkylene; andR3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with 1 or 2 -OH.

[0027] In some aspects, the ionizable cationic lipid is

[0028] In some aspects, the ionizable cationic lipid is selected from N, N-dimethyl-2,2-di- (9Z,12Z)-9,12-octadecadien-l-yl-l,3-dioxolane-4-ethanamine (DLin-KC2-DMA), (6Z,9Z,28Z,3 lZ)-heptatriacont-6, 9, 28, 31 -tetraene- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), N, N-dimethyl-2,3-bis[(9Z,12Z)-9,12-octadecadien-l-yloxy]-l-propanamine (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]bis-2-dodecanol (C12-200) Dioleoyl-3 -trimethylammonium propane (DOTAP), and Dioleoyl-3 -trimethylammonium propane (DOTMA).

[0029] In some aspects, the ionizable lipid is present in the lipid nanoparticle in a range of about 40 mol% to about 55 mol%.

[0030] In some aspects, the neutral phospholipid is present in the lipid nanoparticle in a range of about 5 mol% to about 25 mol%.

[0031] In some aspects, the structural lipid is present in the lipid nanoparticle in a range of about 25 mol% to about 45 mol%.

[0032] In some aspects, the PEG-lipid is present in the lipid nanoparticle in a range of about 1 mol% to about 4 mol%.

[0033] In some aspects, the cargo is presented in the LNP at a concentration of about 0.01 to 0.1 g / gLNP.

[0034] Further provided is a composition comprising a LNP as described herein and one or more carrier or excipient.

[0035] Provided is a method of delivering cargo to a CNS endothelial cell, comprising delivering a LNP described herein comprising the cargo to the cell.

[0036] Further provided is a method of modulating gene expression in a CNS endothelial cell, comprising contacting the cell with a LNP described herein.

[0037] Also provided is a method of treating a CNS dysfunction in a subject in need thereof, comprising contacting a CNS cell of the subject with a LNP described herein.

[0038] In some aspects, the dysfunction is selected from the group consisting of Alzheimer’s disease, vascular dementia, traumatic brain injury, stroke, Parkinson’s disease, neuroinflammation, advanced age, or cerebral amyloid angiopathy.

[0039] Further provided is a method of correcting a deficit in a CNS endothelial cell function in a subject due to a genetic cause, the method comprising administering to the subject an LNP described herein or a composition described herein.

[0040] In some aspects, the genetic cause of the deficit in a CSN endothelial cell function is GLUT1 deficiency syndrome (De Vivo syndrome), band-like calcification with simplified gyration and polymicrogyria (BLC-PMG), familial cerebral cavernous malformations, cerebral small vessel disease, microcephaly, or Allan-Hemdon-Dudley syndrome.BRIEF DESCRIPTION OF DRAWINGS

[0041] FIGs. 1A and IB show the percentage of cells expressing GFP and mean GFP intensity per GFP-positive cell in the Bend3 murine cell line as a function of mRNA dose, and antibody grafting density. LNPs without targeting ligands were used as a negative control. Percentage of GFP-positive cells and mean GFP expression for LNPs with incorporated Fabs was compared to the negative control. 2-6 wells of cells were treated with LNPs. GFP expression increased with grafting of mouse-specific 8D3 Fab, but not with human-specific H7-Fab. Statistical comparisons to within-dose non-targeting LNP by oneway ANOVA with Dunnett’s MCT. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.FIG. 1A shows the percentage of Bend3 murine cells expressing GFP. FIG. IB shows the mean GFP intensity (MFI) of GFP-positive Bend3 murine cells.

[0042] FIGs. 2 A and 2B show the percentage of cells expressing GFP and mean GFP intensity per GFP-positive cell in the human hCMEC cell line as a function of mRNA dose, and antibody grafting density. LNPs without targeting ligands were used as a negative control. Percentage of GFP-positive cells and mean GFP expression for LNPs with incorporated Fabs was compared to the negative control. Four wells of cells from two independent experiments were treated with LNPs. GFP expression increased with grafting of human-specific H7-Fab, but not with mouse-specific 8D3-Fab. Statistical comparisons to within-dose non-targeting LNP by one-way ANOVA with Dunnett’s MCT. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001. FIG. 2A shows the percentage of hCMEC cells expressing GFP. FIG. 2B shows the mean GFP intensity (MFI) of GFP-positive hCMEC cells.

[0043] FIG. 3 shows in-life and ex vivo images in SKH1 mice infused IV with LNPs with or without conjugation to 8D3 Fab (1 mg FireFly Luciferase RNA / kg). 6-hours post LNP treatment, animals were dosed with CycLucl (15 mg / kg) and dorsal whole-animal IVIS images were collected (FIG. 3, top). Animals were sacrificed and organs were harvested for ex vivo imaging (FIG. 3, bottom). From left to right: animal dosed with buffer (n = 1), control LNPs (without targeting ligand) (n = 1), and LNPs with anti-TfR Fab 8D3 (n = 2).

[0044] FIG. 4 shows immune-histology images of brain sections of SKH1 mice stained with antibodies to GFP and endothelial cell marker CD31. FIG. 4A shows immune-histology images of SKH1 mice treated IV with buffer. FIG. 4B shows immune-histology images of SKH1 mice treated with LNP-GFP. FIG. 4C shows immune-histology images of SKH1 mice treated with LNP with Fab 8D3 (9 g / mol). FIG. 4D shows a bar graph of GFP per totalprotein (ng / mg). LNPs contained GFP mRNA (20 pg / animal). Mice were sacrificed after 24hr. Anti-GFP in-situ hybridization (brown) and anti-CD31 immunohistochemistry (red) were performed on FFPE brain sections. Representative images of cortex are shown. GFP is present in CD31-positive microvasculature endothelial cells in the Fab-conjugated LNP group, but was not present in microvasculature from control groups (FIG. 4D). GFP protein was quantified in brain lysates from hemibrains of same animals processed for histology by ELISA. Statistical comparisons by one-way ANOVA with Tukey’s MCT. *=p<0.05.DETAILED DESCRIPTION

[0045] The disclosure provides lipid nanoparticles and lipid nanoparticle compositions comprising an ionizable lipid, a structural lipid, a PEG-lipid, and a helper lipid, and, optionally, a cargo for highly efficient targeting of CNS endothelial cells, including brain endothelial cells, and, optionally, delivery of a cargo specifically to CNS endothelial cells, such as brain endothelial cells. In some aspects, the lipid nanoparticles further comprise a cell targeting group, which optionally is coupled to a lipid of the lipid nanoparticle to form a lipid-cell targeting group conjugate. In some aspects, the cell targeting group is an antibody or fragment thereof that binds a transferrin receptor. Also provided are methods of making the lipid nanoparticles and compositions and methods of using the same to deliver cargo to specified target cells including CNS endothelial cells, such as brain microvascular endothelial cells.Definitions

[0046] The term “and / or,” as used herein is a specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). The use of the term “or” means “and / or” unless explicitly indicated to refer to alternatives only, or the alternatives are mutually exclusive.

[0047] The expression “at least one of,” as used herein, includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.

[0048] The term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value and within a range of values thatfall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). When the term “approximately” or “about” is applied herein to a particular value, the value without the term “approximately” or “about” is also disclosed herein. Further, although not always explicitly stated, all numerical designations may be preceded by the term “about.”

[0049] As described herein, any concentration range, percentage range, ratio range, or integer range includes the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The description of an interval of values should be considered as specifically disclosing all possible intermediate intervals as well as each of the values within this interval. For example, the description of an interval from 1 to 6 should be considered as specifically describing each of the intervals that it comprises, such as the intervals from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as each of the values in this interval, for example 1, 2, 2.7, 3, 4, 5, 5.3 and 6. This definition is valid independently of the scope of the interval.

[0050] The terms “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” “comprise,” “comprises,” or “comprising,” including grammatical equivalents thereof, as used herein, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0051] The term “alkyl,” as used, herein refers to a saturated straight or branched hydrocarbon, such as a straight or branched group of 1-12, 1-10, or 1-6 atoms, referred to herein as C1-C12 alkyl, C1-C10 alkyl, or Ci-Ce alkyl, respectively. In some aspects, alkyl is substituted. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-l -propyl, 2-methyl-2-propyl, 2-methyl-l -butyl, 3 -methyl- 1 -butyl, 2-methyl-3 -butyl, 2,2-dimethyl-l -propyl, 2-methyl-l -pentyl, 3 -methyl- 1 -pentyl, 4-methyl-l-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-l -butyl, 3,3-dimethyl-1 -butyl, 2-ethyl-l -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, etc. In some aspects, an alkyl is a methyl.

[0052] The term “haloalkyl,” as used herein, refers to an alkyl group that is substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like.

[0053] The term “cycloalkyl,” as used, herein refers to a monovalent saturated cyclic, bicyclic, bridged cyclic (e.g., adamantyl), or spirocyclic hydrocarbon group of 3-12, 3-10, 3-8, 4-8, or 4-6 carbons. For example, a "C4-8 cycloalkyl," derived from a cycloalkane. In some aspects, cycloalkyl is optionally substituted. Exemplary cycloalkyl groups include, but are not limited to, cyclohexanes, cyclopentanes, cyclobutanes and cyclopropanes. Unless specified otherwise, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. In some aspects, the cycloalkyl group is not substituted, i.e., it is unsubstituted.

[0054] The terms “heterocyclyl” and “heterocyclic group,” as used herein, refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3- to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. In some aspects, heterocyclyl is optionally substituted. The number of ring atoms in the heterocyclyl group can be specified using Cx-Cxnomenclature where x is an integer specifying the number of ring atoms. For example, a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The designation “C3-C7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position. One example of a C3 heterocyclyl is aziridinyl. Heterocycles may be, for example, mono-, bi-, or other multi-cyclic ring systems (e.g., fused, spiro, bridged bicyclic). A heterocycle may be fused to one or more aryl, partially unsaturated, or saturated rings. Heterocyclyl groups include, for example, biotinyl, chromenyl, dihydrofuryl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl, imidazolidinyl, isoquinolyl, isothiazolidinyl, isooxazolidinyl, morpholinyl, oxolanyl, oxazolidinyl, phenoxanthenyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrazolinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolidin-2-onyl, pyrrolinyl, tetrahydrofuryl, tetrahydroisoquinolyl, tetrahydropyranyl, tetrahydroquinolyl, thiazolidinyl, thiolanyl, thiomorpholinyl, thiopyranyl, xanthenyl, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. Unless specified otherwise, the heterocyclic ring is optionally substituted at one or more positions with substituents such as alkanoyl, alkoxy, alkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl,heterocyclyl, hydroxyl, imino, ketone, nitro, oxo, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl and thiocarbonyl. In certain aspects, the heterocyclyl group is not substituted, i.e., it is unsubstituted.

[0055] The term “aryl,” as used herein refers to a carbocyclic aromatic group. In some aspects, aryl is optionally substituted. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term “aryl” includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(O)alkyl, CChalkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CF3, -CN, or the like. In some aspects, the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In some aspects, the aromatic ring is not substituted, i.e., it is unsubstituted. In some aspects, an aryl group is a 6-to 10-membered ring structure, e.g., Ce-Cio aryl. In some aspects, an aryl group is a 6- to 14-membered ring structure, e.g., Ce-Cw aryl.

[0056] The term “heteroaryl,” as used herein, refers to aromatic groups that include at least one ring heteroatom. In some aspects, heteroaryl is optionally substituted. In some aspects, a heteroaryl group contains 1, 2, 3, or 4 ring heteroatoms. Representative examples of heteroaryl groups include pyrrolyl, furanyl, thiophenyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Unless specified otherwise, the heteroaryl ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CF3, -CN, or the like. The term “heteroaryl” also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, and / or aryls. In some aspects, the heteroaryl ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In some aspects, the heteroaryl ring is not substituted, i.e., it is unsubstituted. In some aspects, a heteroaryl group is a 5- to 10-membered ring structure, alternatively a 5- to 6-membered ring structure, whose ring structure includes 1, 2, 3, or 4 heteroatoms, such as nitrogen, oxygen, and sulfur.

[0057] The term “alkylene” as used herein refers to a diradical of an alkyl group. In some aspects, alkylene is substituted. An exemplary alkylene group is -CH2CH2-. In some aspects, alkylene is methylene.

[0058] The term “alkenyl,” as used herein, refers to an unsaturated branched or straight-chain alkyl group having the indicated number of carbon atoms (e.g., 2 to 8, or 2 to 6 carbon atoms) and at least one carbon-carbon double bond. The group may be in either the cis or trans configuration (Z or E configuration, respectively) about the double bond(s). Alkenyl groups include, but are not limited to, ethenyl, propenyl (e.g., prop-l-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl (allyl), prop-2-en-2-yl), and butenyl (e.g., but-l-en-l-yl, but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en-l-yl, but-2-en-l-yl, but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl) etc.

[0059] The terms “amine” and “amino,” as used herein, refer to both unsubstituted and substituted amines, e.g., a moiety represented by the general formula -N(R3)(R3B), wherein R3represents a bond, hydrogen, alkyl, cycloalkyl, heterocyclyl, alkenyl, or aryl. In some aspects, R3taken together with the N atom to which it is attached completes a heterocycle having from 4 to 8 atoms in the ring structure. In some aspects, R3Brepresentsome aspects, R3B1is C1-6 alkylene. In some aspects, R3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with 1 or 2 -OH. In some aspects, R3B1is C1-6 alkylene and R3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with 1 or 2 -OH. In some aspects, R3B2and R3B3are each methyl. In some aspects, R3B1is -(CH2)3-.

[0060] The terms “alkoxyl” or “alkoxy,” as used herein, refer to an alkyl group, as defined above, having an oxygen radical attached thereto. In some aspects, alkoxyl is optionally substituted. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An “ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders the alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, O-alkynyl, -O-(CH2)m-R12, where m and R12are described above. The term “haloalkoxyl” refers to an alkoxyl group that is substituted with at least one halogen. For example, -O-CH2F, -O-CHF2, -O-CF3, and the like. In someaspects, the haloalkoxyl is an alkoxyl group that is substituted with at least one fluoro group. In some aspects, the haloalkoxyl is an alkoxyl group that is substituted with from 1-6, 1-5, 1-4, 2-4, or 3 fluoro groups.

[0061] The term “oxo,” as used herein, refers to a “=O” substituent. For example, a cyclopentane substituted with an oxo group is cyclopentanone.

[0062] The term “substituted”, whether preceded by the term “optionally” or not, as used herein, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group. In some aspects, when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at each position. In some aspects, combinations of substituents are those that result in the formation of stable or chemically feasible compounds. In some aspects, “optionally substituted” is equivalent to “unsubstituted or substituted.” In some aspects, “optionally substituted” indicates that the designated atom or group is optionally substituted with one or more substituents independently selected from optional substituents provided herein. In some aspects, an optional substituent may be selected from the group consisting of: C1-6alkyl, cyano, halogen, -O-C1-6 alkyl, C1-6haloalkyl, C3-7 cycloalkyl, 3- to 7-membered heterocyclyl, 5- to 6-membered heteroaryl, and phenyl. In some aspects, an optional substituent is alkyl, cyano, halogen, halo, azide, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl, or heteroaryl. In some aspects, an optional substituent is -ORsl, -NRs2Rs3, -C(O)Rs4, -C(O)ORs5, C(O)NRS6RS7, -OC(O)RS8, -OC(O)ORS9, -OC(O)NRsl0Rn, -NRS12C(O)RS13, or -NRS14C(O)ORS15, wherein Rsl, Rs2, Rs3, Rs4, Rs5, Rs6, Rs7, Rs8, Rs9, Rsl°, Rsl1, Rsl2, Rsl3, Rsl4, and Rsl5are each independently H, C1-6 alkyl, C3-10 cycloalkyl, C6-14 aryl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocyclyl, each of which is optionally substituted.

[0063] At various places in the present specification, substituents are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. For example, the term “C1-6 alkyl” is specifically intended to individually disclose Ci, C2, C3, C4, Cs, Ce, Ci-Ce, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl. By way of other examples, an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0064] The term “dioxolane,” as used herein, refers to a heterocyclic acetal with the chemical formula (CFb^ChCFb. In some aspects, dioxolane is 1,3 -di oxolane.

[0065] The term “octadecadien,” as used herein, refers to a polyunsaturated long-chain fatty acid with an 18-carbon backbone and 2 double bonds.

[0066] The term, “ethanamine,” as used herein, refers to a compound with the chemical formula CH3CH2NH2.

[0067] The term, “propanamine,” as used herein, refers to a compound with the chemical formula CH3(CH2)2NH2.

[0068] The term “DLin-DMA,” as used herein, refers to the compound N, N-dimethyl-2,3-bis[(9Z, 12Z)-9, 12-octadecadien- 1 -yloxy]- 1 -propanamine.

[0069] The term “DLin-K-DMA,” as used herein, refers to the compound 2,2-Dilinoleyl-4-dimethylaminomethyl-[ 1,3 ]-di oxolane.

[0070] The term “C12-200,” as used herein, refers to the compound 1, 1 '-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]bis-2-dodecanol.

[0071] Ther term “DOTAP,” as used herein, refers to the compound Dioleoyl-3-trimethylammonium propane.

[0072] The term “DOTMA,” as used herein, refers to the compound Dioleoyl-3-trimethylammonium propane.

[0073] The term “DLin-KC2-DMA,” as used herein, refers to the compound N, N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-l-yl-l,3-dioxolane-4-ethanamine.

[0074] The term “DLin-MC3-DMA,” as used herein, refers to the compound (6Z,9Z,28Z,3 lZ)-heptatriacont-6,9,28,31 -tetraene- 19-yl 4-(dimethylamino)butanoate.

[0075] The term “mol%” or “mol percent,” as used herein, refers to the mol fraction of a mixture multiplied by 100, where the mol fraction is the number of moles of one ingredient in a mixture divided by the total number of mole in the mixture.

[0076] The designations “R1,” “R2,” “R3” etc., as used herein, refer to a substituent located in a central position of a molecule. For example, “R1,” “R2,” and / or “R3,” can be bonded to a carbon atom in a molecule, e.g., a lipid molecule, which carbon atom is the central carbon atom of the molecule.

[0077] The designations “R1A,” “R2A,” “R3A” etc., as used herein, refer to a substituent located at a position that is less central than the position of a “R1,” “R2,” and / or “R3” in a molecule, e.g., a lipid molecule. In some aspects, a “R1A,” “R2A, ” and / or “R3A” is bonded to a “R1,” “R2,” and / or “R3.”

[0078] The designations “R1A1,” “R2AETC as use(j herein, refer to a substituent located at a position that is less central than the position of a “R1,” “R2,” and / or “R3” and also less central than the position of a “R1A,” “R2A, ” and / or “R3A.” In some aspects, a “R1A1,” “R2A1,” and / or “R3A1” is bonded to a “R1A,” “R2A,” and / or “R3A.”

[0079] The term “pseudouridine,” as used herein, refers to the natural product which is a C-glycosyl pyrimidine that consists of uracil having a beta-D-ribofuranosyl residue attached at position 5 (i.e., 5-(beta-D-Ribofuranosyl)uracil). In some aspects, the term refers to 1-methyl-3 -(3 -amino-3 -carboxypropyl) pseudouridine. In some aspects, the term refers to 1-methylpseudouridine. In some aspects, the term refers to 2'-O- methylpseudouridine. In some aspects, the term refers to 5-methyldihydrouridine. In some aspects, the term refers to 3-methylpseudouridine. In some aspects, the term refers to a pseudouridine moiety that is not further modified. In some aspects, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In some aspects, the term refers to any other pseudouridine known in the art.

[0080] The symbol “ — ~ ” indicates a point of attachment.

[0081] The compounds of the disclosure may contain one or more chiral centers and / or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” as used herein refers to all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. In some aspects, a composition comprises various stereoisomers of a compound and / or mixture of compounds described herein. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise. Individual stereoisomers of compounds of the present invention can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary,separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Further, enantiomers can be separated using supercritical fluid chromatographic (SFC) techniques described in the literature. Still further, stereoisomers can be obtained from stereomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods. Geometric isomers can also exist in the compounds of the present invention.

[0082] The present invention encompasses the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E’ are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the “E” and “Z” isomers. Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangement of substituents around a carbocyclic ring are designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis / trans.”

[0083] The term “salts,” as used herein refers to inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, hydroiodide, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Salt may also include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemi sulfate, heptanoate, hexanoate, 2-hydroxyethanesulfonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, tartrate,thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, NH4+, and NWf (wherein W is a Ci-4 alkyl group), and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0084] Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal e.g., magnesium) hydroxides, ammonia, and compounds of formula NW4+, wherein W is Ci-4 alkyl, and the like.

[0085] The term “lipid nanoparticle” or “LNP,” as used herein, refers to a nanoparticle comprising a single layer of one or more types of lipids. In some aspects, the lipid nanoparticle has a mean diameter of about 400 nm to about 50 nm; or about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 200 nm or less, about 150 nm or less, about 100 nm or less, or about 50 nm or less. In some aspects, the nanoparticle has a diameter of about 200 nm to about 50 nm. In some aspects, the nanoparticle has a diameter of 80 nm.

[0086] The term “total lipids,” as used herein, refers to the collection of ionizable lipids, structural lipids, helper lipids, and PEG-lipids present in a lipid nanoparticle described herein.

[0087] The term “ionizable lipid,” as used herein, refers to a lipid that is capable of modulating its charge depending on the environment it is present in. In some aspects, an ionizable lipid includes a lipid of Formula II, Formula Ila (Lipid 15), Formula III (KC2), or Formula IV (KC3). Other ionizable lipids can be used in the lipid nanoparticles described herein.

[0088] The terms “helper lipid,” “neutral lipid,” “phospholipid,” or “neutral phospholipid,” as used herein, refer to a non-polar lipid of a lipid nanoparticle which lipid provides the lipid nanoparticle with stability, blood compatibility, and enhances cargo delivery.

[0089] The term “PEG-lipid,” “lipid-PEG,” or “PEGylated lipid” are used herein interchangeably and refer to one or more lipids that are modified with polyethylene glycol (PEG). As described herein, PEG-lipids can be free PEG-lipids or PEG-lipids that are part of a lipid-cell targeting conjugate. Any suitable chemistry may be used to conjugate a polypeptide to the PEG of the PEG-lipid, see, e.g., Parhiz et al., Journal of Controlled Release 291:106-115, 2018; Kolb et al., Angewandte Chemie International Edition 40(11):2004-2021, 2001; and Evans, Australian Journal of Chemistry 60(6):384-395, 2007. For example, lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, PEG-dibenzocyclooctyne (DBCO), lipid-PEG-bromo maleimide, lipid-PEG-alkynoic amide, PEG-alkanoic imide, and lipid-PEG-azide can be used to produce a Lipid-PEG-polypeptide conjugate.

[0090] The term “structural lipid,” as used herein, refers to sterols and also to lipids containing sterol moieties.

[0091] The term “lipid-cell targeting group conjugate,” as used herein, refers to a conjugate of the general formula: [Lipid] - [optional linker] - [cell targeting group], wherein the lipid can be any lipid described herein and the cell targeting group can be any cell surface moleculebinding group described herein. A cell targeting group includes an antibody or fragment thereof, including a Fab, a scFv, an immunoglobulin single variable domain (ISVD) including but not limited to a VHH domain, humanized VHH domain, or camelized VH domain. A lipid of a lipid-cell targeting group conjugate preferably is a PEG-lipid.

[0092] The term “free PEG-lipid,” as used herein, refers to a PEG-lipid that is not part of a lipid-cell targeting group conjugate. For example, a free PEG-lipid can be part of a lipid blend of a lipid nanoparticle. A free PEG-lipid can reduce or eliminate non-specific binding via a targeting group when, e.g., a lipid-cell targeting group conjugate is included in a lipid nanoparticle.

[0093] The term “reference LNP,” as used herein, refers to an LNP that does not have, e.g., a cell targeting group but is otherwise the same as the tested LNP. In some aspects, a reference LNP is an LNP that has a different ionizable cationic lipid but is otherwise the same as the tested LNP. In some aspects, a reference LNP comprises D-Lin-MC3-DMA as the ionizable cationic lipid which is different from the ionizable cationic lipid in a tested LNP, but is otherwise the same as the tested LNP.

[0094] The terms “peptide,” “polypeptide,” and “protein” are used herein interchangeably and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, and to longer chains, which generally are referred to in the art as proteins. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0095] The term “antibody,” as used herein, refers to any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) thatspecifically binds to or interacts with a particular antigen. It is understood that the term encompasses an intact antibody, antigen-binding fragment thereof, or an Fc fragment that optionally has been modified or engineered. Examples of antigen binding fragments include Fab, Fab’, (Fab’)2, Fv, single chain antibodies (e.g., scFv), minibodies, immunoglobulin single variable domains (ISVDs), and diabodies. Examples of antibodies that have been modified or engineered include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0096] Naturally occurring antibodies typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one full length light chain (typically having a molecular weight of about 25 kDa) and one full length heavy chain (typically having a molecular weight of about 50-70 kDa). The terms “heavy chain” and “light chain,” as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The aminoterminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids that typically is responsible for antigen recognition. The carboxy -terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CHI, CH2, and CH3), wherein the VH domain is at the amino-terminus of the polypeptide and the CH3 domain is at the carboxyl-terminus, and a full-length light chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), wherein the VL domain is at the amino-terminus of the polypeptide and the CL domain is at the carboxyl-terminus.

[0097] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgGl, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgMl and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgAl and IgA2. Within full-length light and heavy chains, the variable and constant domains typically are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See, e.g., FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety herein. The variable regions of each light / heavy chain pair typically form an antigen binding site. The variable domains of naturally occurringantibodies typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope. From the aminoterminus to the carboxyl-terminus, both light and heavy chain variable domains typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0098] The term “CDR set,” as used herein, refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. In some aspects, an ISVD comprises three CDRs. The exact boundaries of CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17; Chothia et al., 1989, Nature 342: 877-83) found that certain sub-portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as LI, L2, and L3 or Hl, H2, and H3 where the “L” and the “H” designates the light chain and the heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan, 1995, FASEB J. 9: 133-39; MacCallum, 1996, J. Mol. Biol. 262(5): 732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27: 55-77. Still other CDR boundary definitions may not strictly follow one of the mentioned systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although certain aspects use Kabat or Chothia defined CDRs. Identification of predicted CDRs using the amino acid sequence is well known in the field, such as in Martin, A. C. Protein sequence and structure analysis of antibody variable domains, In Antibody Engineering, Vol. 2. Kontermann R., Diibel S., eds. Springer-Verlag, Berlin, p. 33-51, 2010. The amino acid sequence of the heavy and / or light chain variable domain may be also inspected to identify the sequences of the CDRs by other conventional methods, e.g., by comparison to known amino acid sequences of other heavy and light chainvariable regions to determine the regions of sequence hypervariability. The numbered sequences may be aligned by eye, or by employing an alignment program such as one of the CLUSTAL suite of programs, as described in Thompson, Nucleic Acids Res. 22: 4673-80, 1994. Molecular models are conventionally used to correctly delineate framework and CDR regions and thus correct the sequence-based assignments.

[0099] The term “Fc,” as used herein, refers to a fragment crystallizable region, that is a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgGl and IgG2 are preferred. Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, and IgGA2). One example of a Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.

[0100] The term “Fab,” as used herein, refers to a F(ab) fragment of an antibody and typically includes one light chain and the VH and CHI domains of one heavy chain, wherein the VH-CH1 heavy chain portion of the F(ab) fragment cannot form a disulfide bond with another heavy chain polypeptide. As used herein, a F(ab) fragment can also include one light chain containing two variable domains separated by an amino acid linker and one heavy chain containing two variable domains separated by an amino acid linker and a CHI domain. A F(ab’) fragment typically includes one light chain and a portion of one heavy chain that contains more of the constant region (between the CHI and CH2 domains), such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.

[0101] The term “immunoglobulin single variable domain” or “ISVD,” as used herein, refers to an immunoglobulin domain capable of specifically binding to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an ISVD is formed by a single VH, a single VHH or single VL domain. Therefore, the antigen binding site of an ISVD is formed by no more than three CDRs. The single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence)or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (e.g., a functional antigen binding unit that essentially consists of the single CDRs, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). For example, an ISVD can be a heavy chain immunoglobulin single variable domain, such as a VH, VHH, including a camelized VH or humanized VHH or a domain antibody ("dAb" or dAb) or an amino acid sequence that is suitable for use as a dAb. In some aspects, an ISVD is a VHH, a camelized VH or a humanized VHH. In some aspects, single variable domains are derived from certain species of shark (for example, the so-called “IgNAR domains.” In some aspects, an ISVD is a Nanobody®. [Note: Nanobody® is a registered trademark of Ablynx N. V.]. An ISVD can be obtained, e.g., (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain; (3) by “humanization” of a naturally occurring VHH domain or by expression of a nucleic acid encoding a humanized VHH domain; (4) by “camelization” of a naturally occurring VH domain from any animal species, in particular a species of mammal, such as from a human, or by expression of a nucleic acid encoding a camelized VH domain; (5) by “camelization” of a “domain antibody” or “dAb” or by expression of a nucleic acid encoding a camelized VH domain; (6) using synthetic or semi -synthetic techniques for preparing proteins, polypeptides or other amino acid sequences; (7) by preparing a nucleic acid encoding an ISVD using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained; and / or (8) by any combination of the foregoing.

[0102] The term “scFv,” as used herein, refers to a single chain antibody comprising a heavy chain variable domain and a light chain variable domain linked by a linker.

[0103] The term “humanized antibody,” as used herein, refers to an antibody which is wholly or partially of non-human origin and whose protein sequence has been modified to replace certain amino acids, for instance that occur at the corresponding position(s) in the framework regions of the VH and VL domains in a sequence of an antibody from a human being, to increase its similarity to antibodies produced naturally in humans, in order to avoid or minimize an immune response in humans. For example, using techniques of genetic engineering, the variable domains of a non-human antibodies of interest may be combined with the constant domains of human antibodies. In some aspects, the constant domains of a humanized antibody are human CH and CL domains.

[0104] The term “humanized VHH,” as used herein, refers to an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that hasbeen “humanized,” i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional four-chain antibody from a human.

[0105] The term “camelized VH,” as used herein, refers to an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized,” e.g., by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional four-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a (camelid) heavy chain antibody. In some aspects, such “camelizing” substitutions are inserted at amino acid positions that form and / or are present at the VH-VL interface. In some aspects, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, such as the VH sequence of a human, such as, e.g., a VH3 sequence. However, it should be noted that such camelized VH can be obtained in any suitable manner and is not strictly limited to a polypeptide that has been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g., camelized dAb can be used herein. In some aspects, immunoglobulin single variable domains are fused forming a multivalent and / or multispecific construct (for multivalent and multispecific polypeptides containing one or more VHH domains).

[0106] The term “multivalent,” as used herein, refers to the presence of multiple immunoglobulin single variable domains in a polypeptide. In some aspects, the polypeptide is “bivalent,” e.g., comprises or consists of two immunoglobulin single variable domains. In some aspects, the polypeptide is “trivalent,” e.g., comprises or consists of three immunoglobulin single variable domains. In some aspects, the polypeptide is “tetravalent,” e.g., comprises or consists of four immunoglobulin single variable domains. The polypeptide can thus be “bivalent,” “trivalent,” “tetraval ent,” “pentavalent,” “hexavalent,” “heptavalent,” “octavalent,” “nonavalent,” etc., meaning the polypeptide comprises or consists of two, three, four, five, six, seven, eight, nine, etc., immunoglobulin single variable domains, respectively.

[0107] The term “multispecific,” as used herein, refers to binding to multiple different target molecules (also referred to as antigens). In some aspects, a multivalent immunoglobulin polypeptide is “bispecific,” “tri specific,” “tetraspecific,” etc., meaning that it can bind to two, three, four, etc., different target molecules, respectively. For example, a polypeptide may bebispecific-trivalent, such as a polypeptide comprises or consists of three immunoglobulin single variable domains, wherein two immunoglobulin single variable domains bind to a first target and one immunoglobulin single variable domain binds to a second target different from the first target. In some aspects, a polypeptide is trispecific-tetravalent, such as a polypeptide comprises or consists of four immunoglobulin single variable domains, wherein, e.g., one immunoglobulin single variable domain binds to a first target, two immunoglobulin single variable domains bind to a second target different from the first target and one immunoglobulin single variable domain binds to a third target different from the first and the second target. In some aspects, a polypeptide is trispecific-pentavalent, such as a polypeptide comprises or consists of five immunoglobulin single variable domains, wherein, e.g., two immunoglobulin single variable domains bind to a first target, two immunoglobulin single variable domains bind to a second target different from the first target and one immunoglobulin single variable domain binds to a third target different from the first and the second target. In some aspects, a multispecific immunoglobulin polypeptide is multiparatopic.

[0108] The term “multiparatopic,” as used herein, refers to binding to multiple different epitopes on the same target molecules (also referred to as antigens). In some aspects, a multivalent immunoglobulin single variable domain polypeptide can thus be “biparatopic,” “triparatopic,” etc., meaning it can bind to two, three, etc., different epitopes on the same target molecules, respectively.

[0109] The term “specifically binds,” as used herein, refers to an affinity ligand, in particular, an antibody, that recognizes and binds a specific antigen, but does not substantially recognize or bind other molecules in a sample. In some aspects, the terms “specific binding” or “specifically binding,” refers to an interaction of an antibody, a protein, or a peptide with a second protein or peptide where the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on second protein or peptide. For example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. In some aspects, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specificallybinds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.

[0110] The term “specificity of the targeted delivery” by an LNP is defined by the ratio between % of a desired cell type that receives the delivered nucleic acid (e.g., on-target delivery), and % of an undesired cell type that is not meant to be the destination of the delivery, but receives the delivered nucleic acid (e.g., off-target delivery). For example, the specificity is higher when more desired cells receive the delivered nucleic acid, while less undesired cells receive the delivered nucleic acid. Specificity of the targeted delivery by an LNP can also be defined by the ratio of amount of nucleic acid being delivered to the desired cells (e.g., on-target delivery) and amount of nucleic acid being delivered to the undesired cells (e.g., off-target delivery). Specificity of the delivery can be determined using any suitable method. As a non-limiting example, expression level of the nucleic acid in the desired cell type can be measured and compared to that of a different cell type that is not meant to be the destination of the delivery.

[0111] The term “linker” or “spacer,” as used herein, denotes a peptide that fuses together two or more polypeptides or proteins into a single molecule. The use of linkers to connect two or more (poly)peptides is well known in the art. For example, a class of peptidic linkers are known as the “Gly-Ser” or “GS” linkers and essentially consist of glycine (G) and serine (S) residues. A linker can also be an alanine linker.

[0112] The term “coupled,” as used herein refers to a covalent interaction between two molecules. Therefore, “coupled” includes “covalently coupled.”

[0113] The terms “subject” and “patient,” as used herein, refer to organisms that are preferably mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably humans.

[0114] The term “pharmaceutical composition,” as used herein, refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0115] The term “pharmaceutically acceptable carrier,” as used herein, refers to any of the standard pharmaceutical excipient, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006.

[0116] The term “effective amount,” as used herein, refers to the amount of a compound (e.g., a nucleic acid) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. The term effective amount can be considered to include therapeutically and / or prophylactically effective amounts of a compound.

[0117] The phrase “therapeutically effective amount,” as used herein, refers to an amount of a lipid nanoparticle comprising, e.g., a nucleic acid or a composition comprising a lipid nanoparticle, which is effective for producing a desired therapeutic effect in at least a subpopulation of cells in a mammal, for example, a human, or a subject (e.g., a human subject).

[0118] The phrase “prophylactically effective amount,” as used herein, refers to an amount of a lipid nanoparticle comprising, e.g., a nucleic acid or composition comprising a lipid nanoparticle, which is effective for producing a desired prophylactic effect in at least a subpopulation of cells in a mammal, for example, a human, or a subject (e.g., a human subject) by reducing, minimizing or eliminating the risk of developing a condition.

[0119] The terms “treat,” “treating,” and “treatment” include any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of a condition, disease, disorder, and the like, or ameliorating a symptom thereof.LIPIDS

[0120] Provided herein are lipid nanoparticles comprising a lipid blend comprising one or more of the following lipids: ionizable lipid, structural lipid, helper lipid, and PEG-lipid.

[0121] In some aspects, an ionizable lipid comprises a structure of Formula IEor a salt thereof, wherein:R1, R2, and R3are each independently a bond or C1-3 alkylene;R1A, R2A, and R3Aare each independently a bond or C1-10alkylene;R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1-20 alkenyl, -(CH2)o-ioC(0)ORal, or -(CH2)o-ioOC(0)Ra2; Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl;R3BisR3B1is C1-6 alkylene; and R3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with 1 or 2 -OH.

[0122] In some aspects, R1, R2, and R3are each independently a bond or methylene. In some aspects, R1Aand R2Aare each C1-10 alkylene.

[0123] In some aspects, R3Ais C1-5 alkylene.

[0124] In some aspects, R1A1, R1A2, R2A1, R2A2, R3A1, and R3A2, are each H.

[0125] In some aspects, R1A3and R2A3are each C1-20 alkylene.

[0126] In some aspects, R3A3is -C(0)0(Ci-2o alkyl).

[0127] In some aspects, R3Bis

[0128] In some aspects, R3B1is C2-4 alkylene.

[0129] In some aspects, R3B2and R3B3are each methyl.

[0130] In some aspects, R1, R2, and R3are each independently a bond or methylene; R1Aand R2Aare each C1-10 alkylene; R3Ais C1-5 alkylene; R1A1, R1A2, R2A1, R2A2, R3A1, and R3A2, are each H; R1A3and R2A3are each C1-20 alkylene; R3A3is -C(0)0(Ci-2o alkyl); R3Bisalkylene; and R3B2and R3B3are each methyl.

[0131] In some aspects, R3B1is -(CH2)3-.

[0132] In some aspects, an ionizable lipid comprises a structure of Formula (Ila)(Lipid 15).

[0133] In some aspects, an ionizable lipid comprises a structure of Formula (III) (KC2):

[0134] In some aspects, an ionizable lipid comprises a structure of Formula (IV) (KC3):

[0135] In some aspects, an ionizable lipid is DLin-KC2-DMA.

[0136] In some aspects, an ionizable lipid is DLin-MC3-DMA.

[0137] In some aspects, an ionizable lipid is DLin-DMA.

[0138] In some aspects, an ionizable lipid is DLin-K-DMA.

[0139] In some aspects, an ionizable lipid is C12-200.

[0140] In some aspects, an ionizable lipid is DOTAP.

[0141] In some aspects, an ionizable lipid is DOTMA.

[0142] In some aspects, an ionizable lipid has been designed to enable intracellular delivery of a cargo, e.g., a nucleic acid to the cytosolic compartment of a target cell type and rapidly degrade into non-toxic components. The complex functionalities of the ionizable cationic lipid are facilitated by the interplay between the chemistry and geometry of the ionizable lipid head group, the hydrophobic “acyl-tail” groups and the linkers connecting the head group and the acyl tail groups.

[0143] In some aspects, the pKa of the ionizable amine head group is designed to be in the range of 6-8, such as between 6.2-7.4, or between 6.7-7.2, such that it remains strongly cationic under acidic formulation conditions (e.g., pH 4 - pH 5.5), neutral or slightly anionic in physiological pH (7.4) and cationic in the early and late endosomal compartments (e.g., pH 5.5 - pH 7).

[0144] In some aspects, the acyl-tail groups of an ionizable lipid are designed to enable fusion of the lipid nanoparticle with endosomal membranes and membrane destabilization through structural perturbation. The three-dimensional structure of the acyl-tail (determined by its length, and degree and site of unsaturation) along with the relative sizes of the head group and tail group are thought to play a role in promoting membrane fusion, and hence lipid nanoparticle endosomal escape (a key requirement for cytosolic delivery of a nucleic acid cargo). In some aspects, an ionizable lipid comprises a linker connecting the head group and acyl tail groups, which linker is designed to degrade by physiologically prevalent enzymes (e.g., esterases, or proteases) or by acid catalyzed hydrolysis.

[0145] In some aspects, an ionizable lipid is present in the lipid blend of a lipid nanoparticle in a range of about 20 mol% to about 70 mol%.

[0146] In some aspects, an ionizable lipid is present in the lipid blend of a lipid nanoparticle in a range of about 20 mol% to about 50 mol%. In some aspects, an ionizable lipid is present in the lipid blend of a lipid nanoparticle at about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, or about 70 mol%.

[0147] In some aspects, an ionizable lipid is present in the lipid blend of a lipid nanoparticle at range of about 25 mol% to about 65 mol%; about 30 mol% to about 60 mol%; about 35 mol% to about 55 mol%; or about 40 mol% to about 50 mol%. In some aspects, an ionizable lipid is present in the lipid blend of a lipid nanoparticle at range of about 40 mol% to about 60 mol%; about 45 mol% to about 55 mol%; or about 47 mol% to about 50 mol%. In some aspects, the ionizable lipid is present in the lipid blend of a lipid nanoparticle at about 48.6 mol%.

[0148] In some aspects, a lipid nanoparticle comprises one or more lipid that is part of a lipid-cell targeting group conjugate. In some aspects, a lipid of a lipid-cell targeting group conjugate is a phospholipid. In some aspects, a lipid of a lipid-cell targeting group conjugate is a neutral phospholipid.

[0149] In some aspects, a lipid of a lipid-cell targeting group conjugate is a di stearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG), phosphatidylcholine, phosphatidylethanolamine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), hydrogenated soy phosphatidylcholine (HSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), dimyristoyl-phosphatidylethanolamine (DMPE), dilinoleoyl-glycero-phosphocholine (DLPC), dimyristoyl-glycero-phosphocholine (DMPC), dipalmitoyl-glycero-phosphocholine (DPPC), diundecanoyl-glycero-phosphocholine (DUPC), palmitoyl-oleoyl-glycero-phosphocholine (POPC), dioctadecenyl-glycero- phosphocholine, oleoyl-cholesterylhemisuccinoyl-glycero-phosphocholine, hexadecyl-glycero-phosphocholine, dilinolenoyl-glycero-phosphocholine, diarachidonoyl-glycero-3-phosphocholine, didocosahexaenoyl-glycero-phosphocholine, sphingomyelin, or ceramide.

[0150] In some aspects, the lipid of a lipid-cell targeting group conjugate is DSPC.

[0151] In some aspects, the lipid of a lipid-cell targeting group conjugate is DSPE.

[0152] In some aspects, a lipid of a lipid-cell targeting group conjugate is present in the lipid blend of a lipid nanoparticle in a range of about 5 mol% to about 50 mol%. In some aspects, a helper lipid is present in the lipid blend of a lipid nanoparticle at about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, or about 50 mol%. In some aspects, a lipid of a lipid-cell targeting group conjugate is present in the lipid blend of a lipid nanoparticle at a range of about 5 mol% to about 15 mol%. In some aspects, a lipid of a lipid-cell targeting group conjugate is present in the lipid blend of a lipid nanoparticle at about 6 mol% to about 14 mol%. In some aspects, a lipid of a lipid-cell targeting group conjugate is present in the lipid blend of a lipid nanoparticle at about 8 mol% to about 12 mol%. In some aspects, a lipid of a lipid-cell targeting group conjugate is present in the lipid blend of a lipid nanoparticle at about 10 mol%. In some aspects, a lipid of a lipid-celltargeting group conjugate is present in the lipid blend of a lipid nanoparticle at about 12 mol%. In some aspects, a lipid of a lipid-cell targeting group conjugate is present in the lipid blend of a lipid nanoparticle at about 14 mol%.

[0153] In some aspects, a lipid of the lipid-cell targeting group conjugate is covalently coupled to an antibody.

[0154] In some aspects, an antibody is covalently coupled to the lipid of the lipid-cell targeting group conjugate via a linker comprising polyethylene glycol (PEG).

[0155] In some aspects, an antibody is covalently coupled to a DSPE of a DSPE-cell targeting group conjugate via a PEG linker.

[0156] In some aspects, the PEG linker has a molecular weight of about 1.0 kDa to about 5.0 kDa.

[0157] In some aspects, the PEG linker has a molecular weight of about 3.0 kDa.

[0158] In some aspects, a lipid of the cell-targeting group conjugate is a dimyristoyl-glycerol-PEG (DMG-PEG), dioleoylgylcerol-PEG (DOG-PEG), dipalmitoyl-glycerol-PEG (DPG-PEG), dilinoleoyl-glycero-phosphatidyl ethanolamine-PEG (DLPE-PEG), dimyristoyl-phosphatidylethanolamine-PEG (DMPE-PEG), dipalmitoyl- phosphatidylethanolamine-PEG (DPPE-PEG), distearoylglycerol-PEG (DSG-PEG), diacylglycerol-PEG (DAG-PEG), dimyristoyl-glycerol-PEG (DMG-PEG), dipalmitoyl-glycerol (DPG-PEG), distearoylglycerol-PEG (DSG-PEG), PEG-ceramide, distearoyl-glycero-phosphoglycerol-PEG (DSPG-PEG), dioleoyl-glycero-phosphoethanolamine-PEG (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide, and distearoyl-phosphatidylethanolamine-PEG (DSPE-PEG), DSPE-PEG-cysteine, or a derivative thereof..

[0159] In some aspects, a lipid nanoparticle comprises one or more free PEG-lipids.

[0160] In some aspects, a free PEG-lipid is a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, or a PEG-modified dialkylglycerol.

[0161] In some aspects, a free PEG-lipid is selected from the group consisting of dimyristoyl-glycerol-PEG (DMG-PEG), dioleoylgylcerol-PEG (DOG-PEG), dipalmitoyl-glycerol-PEG (DPG-PEG), dilinoleoyl-glycero-phosphatidyl ethanolamine-PEG (DLPE-PEG), dimyristoyl-phosphatidylethanolamine-PEG (DMPE-PEG), dipalmitoyl- phosphatidylethanolamine-PEG (DPPE-PEG), distearoylglycerol-PEG (DSG-PEG), diacylglycerol-PEG (DAG-PEG), dimyristoyl-glycerol-PEG (DMG-PEG), dipalmitoyl-glycerol (DPG-PEG), distearoylglycerol-PEG (DSG-PEG), PEG-ceramide, distearoyl-glycero-phosphoglycerol-PEG (DSPG-PEG), dioleoyl-glycero-phosphoethanolamine-PEG (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide, and distearoyl-phosphatidylethanolamine-PEG (DSPE-PEG), DSPE-PEG-cysteine, or a derivative thereof. In some aspects, a free PEG-lipid comprises a diacylphosphatidylethanolamine comprising a dipalmitoyl (Cl 6) chain or a distearoyl (Cl 8) chain.

[0162] In some aspects, a free PEG-lipid is present in the lipid blend of a lipid nanoparticle in a range of about 0.01 mol% to about 10 mol%, about 0.01 mol% to about 5 mol%, about 0.01 mol% to about 4 mol%, about 0.01 mol% to about 3 mol%, about 0.01 mol% to about 2 mol%, about 0.01 mol% to about 1 mol%, about 0.1 mol% to about 10 mol%, about 0.1 mol% to about 5 mol%, about 0.1 mol% to about 4 mol%, about 0.1 mol% to about 3 mol%, about 0.1 mol% to about 2 mol%, about 0.1 mol% to about 1 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 5 mol%, about 0.5 mol% to about 4 mol%, about 0.5 mol% to about 3 mol%, about 0.5 mol% to about 2 mol%, about 0.5 mol% to about 1 mol%, about 1 mol% to about 2 mol%, about 3 mol% to about 4 mol%, about 4 mol% to about 5 mol%, about 5 mol% to about 6 mol%, or. In some aspects, a free PEG-lipid is present in the lipid blend of a lipid nanoparticle at about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol%, about 2.5 mol%, about 3 mol%, about 3.5 mol%, about 4 mol%, about 4.5 mol%, about 5 mol%, or about 5.5 mol%. In some aspects, a free PEG-lipid is present in the lipid blend of a lipid nanoparticle at about 1.5 mol%.

[0163] In some aspects, a free PEG-lipid is present in the lipid blend of a lipid nanoparticle in a range of about 0.1 mol% to about 4 mol%. In some aspects, a free PEG-lipid is present in the lipid blend of a lipid nanoparticle in a range of about 0.5 mol% to about 3.5 mol%, about 0.75 mol% to about 3 mol%, about 1 mol% to about 2.5 mol%, about 1.5 mol% to about 2 mol%. In some aspects, a free PEG-lipid is present in the lipid blend of a lipid nanoparticle in an amount of about 0.1 mol%, about 0.2 mol%, about 0.3 mol%, about 0.4 mol%, about 0.5 mol%, about 0.6 mol%, about 0.7 mol%, about 0.8 mol%, about 0.9 mol%, about 1 mol%, about 1.1 mol%, about 1.2 mol%, about 1.3 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.7 mol%, about 1.8 mol%, about 1.9 mol%, about 2 mol%, about 2.1 mol%, about 2.2 mol%, about 2.3 mol%, about 2.4 mol%, about 2.5 mol%, about 2.6 mol%, about 2.7 mol%, about 2.8 mol%, about 2.9 mol%, about 3 mol%, about 3.1 mol%, about 3.2 mol%, about 3.3 mol%, about 3.4 mol%, about 3.5 mol%, about 3.6 mol%, about 3.7 mol%, about 3.8 mol%, about 3.9 mol%, or about 4 mol%. In some aspects, a free PEG-lipid is present in the lipid blend of a lipid nanoparticle at about 1.25 mol% to about 1.75 mol%. In some aspects, a free PEG-lipid is present in the lipid blend of a lipid nanoparticles at about 1.5 mol%.

[0164] In some aspects, a lipid nanoparticle comprises one or more structural lipids. In some aspects, a structural lipid is a sterol. In some aspects, one or more sterols are selected from the group consisting of cholesterol, fecosterol, P-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, and brassicasterol. In some aspects, the sterol is cholesterol.

[0165] In some aspects, a structural lipid is present in the lipid blend of a lipid nanoparticle at a range of about 20 mol% to about 70 mol%.

[0166] In some aspects, a structural lipid is present in the lipid blend of a lipid nanoparticle at a range of about 20 mol% to about 70 mol%. In some aspects, a structural lipid is present in the lipid blend of a lipid nanoparticle at about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about 62 mol%, about 63 mol%, about 64 mol%, about 65 mol%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, or about 70 mol%.

[0167] In some aspects, a structural lipid is present in the lipid blend of a lipid nanoparticle at a range of about 20 mol% to about 65 mol%; about 20 mol% to about 60 mol%; about 20 mol% to about 55 mol%; or about 25 mol% to about 50 mol%, or about 25 mol% to about 45 mol%, or about 20 mol% to about 40 mol%. In some aspects, a structural lipid is present in the lipid blend of a lipid nanoparticle at about 20%, about 30 mol%, about 40 mol%, or about 50 mol%. In some aspects, a structural lipid is present in the lipid blend of a lipid nanoparticle at about 30 mol%. In some aspects, a structural lipid is present in the lipid blend of a lipid nanoparticle at about 39.9 mol%.

[0168] In some aspects, a lipid nanoparticle comprises a DLin-KC2-DMA ionizable lipid, a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) phospholipid, a cholesterol structural lipid, and l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) free PEG-lipid at a specific molar ratio.

[0169] In some aspects, a lipid nanoparticle comprises an ionizable lipid at a range of about 30 mol% to about 70 mol%; a phospholipid at a range of about 5 mol% to about 20 mol%; a structural lipid at a range of about 20 mol% to about 70 mol%; a free PEG-lipid at a range of about 1 mol% to about 10 mol%, and, optionally, a cargo.

[0170] In some aspects, a lipid nanoparticle comprises an ionizable lipid at a range of about 40 mol% to about 60 mol%; a phospholipid at a range of about 6 mol% to about 15 mol%; a structural lipid at a range of about 20 mol% to about 50 mol%; a free PEG-lipid at a range of about 1 mol% to about 5 mol%, and, optionally, a cargo.

[0171] In some aspects, a lipid nanoparticle comprises an ionizable lipid at a range of about 45 mol% to about 50 mol%; a phospholipid at a range of about 8 mol% to about 14 mol%; a structural lipid at a range of about 20 mol% to about 40 mol%; a free PEG-lipid at a range of about 1 mol% to about 2 mol%, and, optionally, a cargo.

[0172] In some aspects, a lipid nanoparticle comprises an ionizable lipid that is selected from the group consisting of Formula Ila (Lipid 15), Formula III (KC2), and Formula IV (KC3) and is present in the lipid nanoparticle at a range of about 30 mol% to about 70 mol%, In some aspects, the lipid nanoparticle further comprise a phospholipid that is DSPC and is present in the lipid nanoparticle at a range of about 5 mol% to about 50 mol%; a structural lipid that is cholesterol and is present in the lipid nanoparticle at a range of about 20 mol% to about 70 mol%; a free PEG-lipid that is DMG-PEG2000 (DMG-PEG2K) and is present in the lipid nanoparticle at a range of about 1 mol% to about 10 mol%, and, optionally, a cargo.

[0173] In some aspects, a lipid nanoparticle comprises an ionizable lipid that is selected from the group consisting of Formula Ila (Lipid 15), Formula III (KC2), and Formula IV (KC3) and is present in the lipid nanoparticle at a range of about 40 mol% to about 60 mol%; a phospholipid that is DSPC and is present in the lipid nanoparticle at a range of about 6 mol% to about 40 mol%; a structural lipid that is cholesterol and is present in the lipid nanoparticle at a range of about 20 mol% to about 50 mol%; a PEG-lipid that is DMG-PEG2K and is present in the lipid nanoparticle at a range of about 1 mol% to about 5 mol%, and, optionally, a cargo.

[0174] In some aspects, a lipid nanoparticle comprises an ionizable lipid that is selected from the group consisting of Formula Ila (Lipid 15), Formula III (KC2), and Formula IV (KC3) and is present in the lipid nanoparticle at a range of about 48 mol% to about 50 mol%; a phospholipid that is DSPC and is present in the lipid nanoparticle at a range of about 8 mol% to about 12 mol%; a structural lipid that is cholesterol and is present in the lipid nanoparticle at a range of about 20 mol% to about 40 mol%; a PEG-lipid that is DPG-PEG2K and is present in the lipid nanoparticle at a range of about 1 mol% to about 2 mol%, and, optionally, a cargo.

[0175] In some aspects, a lipid nanoparticle comprises an ionizable lipid that is selected from the group consisting of Formula Ila (Lipid 15), Formula III (KC2), and Formula IV (KC3)and is present in the lipid nanoparticle in an amount of about 49.24 mol%; a helper lipid that is DSPC and is present in the lipid nanoparticle in an amount of about 20 mol%; a structural lipid that is cholesterol and is present in the lipid nanoparticle in an amount of about 29.25 mol%; a PEG-lipid that is DPG-PEG2K and is present in the lipid nanoparticle in an amount of about 1.51 mol%; and, optionally, a cargo.CELL TARGETING GROUPS

[0176] In some aspects, a lipid nanoparticle described herein comprises a cell targeting group conjugated to a lipid of the lipid nanoparticle. In some aspects, a lipid nanoparticle comprises more than one cell targeting group conjugated to a lipid of the lipid nanoparticle. In some aspects, a cell targeting group comprises an antibody. In some aspects, the antibody is a human or humanized antibody. In some aspects, a cell targeting group comprises an antibody fragment without an Fc component. In some aspects, a cell targeting group comprises an antigen-binding fragment selected from the group consisting of a Fab, F(ab’)2, Fab’-SH, Fv, scFv fragment, or ISVD. In some aspects, a cell targeting group comprises a Fab. In some aspects, a cell targeting group comprises an ISVD. In some aspects, a cell targeting group comprises a scFv.

[0177] In some aspects, a cell targeting group comprises one or more ISVD. In some aspects, a cell targeting group comprises one or more antibody fragments such as Fab, scFv, ISVD, or combinations thereof.

[0178] In some aspects, a cell targeting group is a monovalent, multivalent, or multispecific polypeptide. In some aspects, a cell targeting group comprises a multivalent and multispecific polypeptide that contains one or more antibodies, one or more ISVD, such as one or more VHH domain, and / or one or more Fab, and / or one or more scFv.

[0179] In some aspects, a multivalent cell targeting group comprises two or more ISVD, Fab, or scFv that target the same antigen, e.g., the same part or epitope of said antigen or two or more different parts or epitopes of said antigen. In some aspects, a multivalent cell targeting group comprises two or more ISVD, Fab, or scFv that are directed to different antigens. In some aspects, a multivalent cell targeting group comprises a combination of ISVD, Fab, or scFv some of which target the same antigen and some of which target different antigens.

[0180] In some aspects, a bivalent cell targeting group comprises a first ISVD, Fab, or scFv targeting a first part or epitope of an antigen and a second ISVD, Fab, or scFv targeting the same part or epitope of said antigen or another part or epitope of said antigen. In some aspects, a bivalent cell targeting group comprises a first ISVD, Fab, or scFv targeting a firstantigen and a second ISVD, Fab, or scFv targeting a second antigen different from said first antigen.

[0181] In some aspects, a trivalent cell targeting group comprises three identical or different ISVD, Fab, or scFv targeting the same or different parts or epitopes of the same antigen. In some aspects, a trivalent cell targeting group comprises two identical or different ISVD, Fab, or scFv targeting the same or different parts or epitopes on a first antigen and a third ISVD, Fab, or scFv targeting a second antigen different from said first antigen.

[0182] In some aspects, a trivalent cell targeting group comprises a first ISVD, Fab, or scFv targeting a first antigen, a second ISVD, Fab, or scFv targeting a second antigen different from said first antigen, and a third ISVD, Fab, or scFv targeting a third antigen different from said first and second antigen.

[0183] In some aspects, a cell targeting group comprises one or more ISVD, Fab, or scFv and optionally further comprises one or more further amino acid sequences (all optionally linked via one or more suitable linkers).

[0184] In some aspects, a cell targeting group further comprises one or more other groups, residues, moieties or binding units. Such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the immunoglobulin single variable domain, Fab, or scFv (and / or to the polypeptide in which they are present) and may or may not modify the properties of the immunoglobulin single variable domain, Fab, or scFv.

[0185] In some aspects, such further groups, residues, moieties or binding units may be one or more additional amino acid sequences, such that the cell targeting group is a (fusion) protein or (fusion) polypeptide. In some aspects, said one or more other groups, residues, moieties or binding units are immunoglobulins. In some aspects, said one or more other groups, residues, moieties or binding units are domain antibodies (dAbs), amino acid sequences that are suitable for use as a domain antibody, ISVD, amino acid sequences that are suitable as ISVD, Fab, amino acid sequences that are suitable as Fab, scFv, or amino acid sequences that are suitable as scFv.

[0186] In some aspects, such groups, residues, moieties or binding units may be chemical groups, residues, moieties, which may or may not by themselves be biologically and / or pharmacologically active. In some aspects, such groups are linked to an ISVD, Fab, or scFv so as to provide a “derivative” of the ISVD, Fab, or scFv. In some aspects, said further residues may be effective in preventing or reducing binding of so-called “pre-existing antibodies” to the polypeptides. For this purpose, the polypeptides and constructs maycontain a C-terminal extension (X)n in which n is 1 to 10, 1 to 5, 2 or 1; and each X is an (preferably naturally occurring) amino acid residue that is independently chosen, and preferably independently chosen from the group consisting of alanine (A), glycine (G), valine (V), leucine (L) or isoleucine (I). In some aspects, X is not cysteine.

[0187] In some aspects, immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and / or camelid immunoglobulin sequences are used in the cell targeting groups described herein. Also, fully human, humanized or chimeric sequences can be used in the cell targeting groups described herein.

[0188] In a cell targeting group, the one or more ISVD, Fab, or scFv and the one or more groups, residues, moieties or binding units may be linked directly to each other and / or via one or more suitable linkers. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the linkers may also be an amino acid sequence, so that the resulting polypeptide is a fusion protein or fusion polypeptide.

[0189] In some aspect, a linker comprises one or more repeats of a peptide motif such as, e.g., a GS motif. In some aspects, a linker comprises a GGGGS (SEQ ID NO: 17) motif (for example, having the formula (Gly-Gly-Gly-Gly-Ser)n in which n may be 1, 2, 3, 4, 5, 6, 7 or more) (SEQ ID NO: 29). In some aspects, a linker is a 9GS linker (GGGGSGGGS) (SEQ ID NO: 18), 15GS linker (n=3) and 35GS linker (n=7). Reference is, e.g., made to Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sei. 27 (10): 325-330). In some aspects, a linker is a 3A linker, i.e. comprising three alanines. In some aspects, a linker is a 5GS linker, a 7GS linker, a 8GS linker, a 9GS linker, alOGS linker, a 15GS linker, a 18GS linker, a 20GS linker, a 25GS linker, a 30GS linker, a 35GS linker or a 40GS linker as shown in Table 1 below.Table 1

[0190] In some embodiments, a lipid nanoparticle comprises one or more cell targeting group (e.g., one or more antibodies or antigen-binding fragments) conjugated to a lipid of the lipid nanoparticle. The density of cell targeting groups conjugated to a nanoparticle of the present disclosure may be referred to herein as the “grafting density” of the cell targeting group. For example, the term “antibody grafting density” refers to the number of antibody (e.g., Fab) molecules attached to a lipid nanoparticle. When measured in g / mol this refers to the grams (g) of antibody to moles (mol) of LNP. In some embodiments, a lipid nanoparticle of the present disclosure has an antibody grafting density of about 2 g / mol to about 25 g / mol, of about 2 g / mol to about 5 g / mol, of about 5 g / mol to about 10 g / mol, of about 10 g / mol to about 15 g / mol, of about 15 g / mol to about 20 g / mol, of about 20 g / mol to about 25 g / mol (e.g., about 2 g / mol, about 3 g / mol, about 4 g / mol, about 5 g / mol, about 6 g / mol, about 7 g / mol, about 8 g / mol, about 9 g / mol, about 10 g / mol, about 11 g / mol, about 12 g / mol, about 13 g / mol, about 14 g / mol, about 15 g / mol, about 16 g / mol, about 17 g / mol, about 18 g / mol, about 19 g / mol, about 20 g / mol, about 21 g / mol, about 22 g / mol, about 23 g / mol, about 24 g / mol, or about 25 g / mol). Methods for quantifying grafting density (e.g., antibody grafting density) are known in the art and include, but are not limited to, UV-Vis spectroscopy, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and mass spectrometry.Cell Targeting Groups that Target CNS Endothelial Cells

[0191] In some aspects, a cell targeting group or combination of targeting groups can be selected based on the desired localization, function, or structural features of a given target cell. For example, a cell targeting group can target an endothelial cell, in general, or a CNS endothelial cell in particular. In some aspects, a cell targeting group that targets a CNS endothelial cell comprises one or more antibodies, ISVD, Fab, scFv, or derivatives thereof that target a CNS endothelial cell surface antigen. Exemplary T cell surface antigens include, but are not limited to, transferrin receptor, PECAM, or Von Willebrand Factor.

[0192] In some aspects, a CNS endothelial cell targeting group comprises an anti-TfR antibody. In some aspects, a CNS endothelial cell targeting group comprises an anti-TfR Fab. In some aspects, a CNS endothelial cell targeting group comprises an anti-TfR scFv. In some aspects, a CNS endothelial cell targeting group comprises an anti-TfR ISVD.

[0193] In some aspects, a CNS endothelial cell targeting group comprises an anti-human TfR Fab. In some embodiments, the anti-human TfR Fab comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) of a heavy chain comprising the amino acid sequence of SEQ ID NO: 1, and a light chain comprising a light chain variable region that comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) of a light chain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-human TfR Fab comprises, according to Kabat, a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-human TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-human TfR Fab comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the anti-human TfR Fab comprises a heavy chain of SEQ ID NO: 1 and a light chain of SEQ ID NO: 2 (H7 Fab-hlam3 bDS (Fab B902)).

[0194] In some aspects, a CNS endothelial cell targeting group comprises an anti-mouse TfR Fab. In some embodiments, the anti-mouse TfR Fab comprises a heavy chain variable region that comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) of a heavy chain comprising the amino acid sequence of SEQ ID NO: 3, and a light chain comprising a light chain variable region that comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) of a light chain comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-mouse TfR Fab comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:12, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-mouse TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-mouse TfR Fab comprises a light chain comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the anti-mouse TfR Fab comprises a heavy chain of SEQ ID NO: 3 and a light chain of SEQ ID NO: 4 (8D3 ChFab bDS (Fab B901)).

[0195] In some embodiments, a nanoparticle of the present disclosure comprises a grafting density of the anti-TfR Fab (e.g., Fab B902 or Fab B901) of about 2 g / mol to about 25 g / mol, of about 2 g / mol to about 5 g / mol, of about 5 g / mol to about 10 g / mol, of about 10 g / mol to about 15 g / mol, of about 15 g / mol to about 20 g / mol, of about 20 g / mol to about 25 g / mol (e.g., about 2 g / mol, about 3 g / mol, about 4 g / mol, about 5 g / mol, about 6 g / mol, about 7 g / mol, about 8 g / mol, about 9 g / mol, about 10 g / mol, about 11 g / mol, about 12 g / mol, about 13 g / mol, about 14 g / mol, about 15 g / mol, about 16 g / mol, about 17 g / mol, about 18 g / mol, about 19 g / mol, about 20 g / mol, about 21 g / mol, about 22 g / mol, about 23 g / mol, or about 24 g / mol, or about 25 g / mol).

[0196] In some aspects, a CNS endothelial cell targeting group comprises a C-terminal cysteine residue. In some aspects, a CNS endothelial cell targeting group comprises a Fab that comprises a cysteine at the C-terminus of the heavy or light chain fragment. In some aspects, the Fab further comprises one or more amino acids between the heavy chain of the Fab and the C- terminal cysteine. For example, in some aspects, a Fab comprises two or more amino acids derived from an antibody hinge region (e.g., a partial hinge sequence) between the C-terminus of the Fab and the C-terminal cysteine. In some aspects, a Fab comprises a heavy chain variable domain linked to an antibody CHI domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CHI domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and wherein the CNS endothelial cell targeting group further comprises a single chain variable fragment, e.g., a scFv linked to the C-terminus of the light chain constant domain by an amino acid linker.

[0197] In some aspects, a CNS endothelial cell targeting group comprises an immunoglobulin single variable domain, such as an ISVD (e.g., a VHH). In some aspects, the ISVD comprises a cysteine at the C-terminus. In some aspects, the ISVD further comprises a linker comprising one or more amino acids between the VHH domain and the C-terminalcysteine. In some aspects, the linker comprises one or more glycine residues, e.g., two glycine residues. In some aspects, a CNS endothelial cell targeting group comprises two or more VHH domains. In some aspects, the two or more VHH domains are linked by an amino acid linker. In some aspects, the amino acid linker comprises one or more glycine and / or serine residues (e.g., one or more repeats of the sequence GGGGS (SEQ ID NO: 17)). In some aspects, a CNS endothelial cell targeting group comprises a first VHH domain linked to an antibody CHI domain and a second VHH domain linked to an antibody light chain constant domain, and wherein the antibody CHI domain and the antibody light chain constant domain are linked by one or more disulfide bonds (e.g., interchain disulfide bonds). In some aspects, a CNS endothelial cell targeting group comprises a VHH domain linked to an antibody CHI domain, and wherein the antibody CHI domain is linked to an antibody light chain constant domain by one or more disulfide bonds.Lipid-Cell Targeting Group Conjugates

[0198] In some aspects, a lipid nanoparticle as described herein comprises one or more lipid-cell targeting group conjugates. In some aspects, a lipid-cell targeting group conjugate comprises a cell targeting polypeptide, or fragment thereof, and a lipid. In some aspects, a cell targeting group is covalently coupled to a lipid either directly or via a linker. In some aspects, a CNS endothelial cell targeting group is covalently coupled to a lipid via a polyethylene glycol (PEG) containing linker. In some aspects, a lipid-PEG-cell targeting group conjugate is an insertable lipid-cell targeting group conjugate of a lipid nanoparticle as described herein.

[0199] In some aspects, the lipid linked to a CNS endothelial cell-targeting group via a PEG linker is di stearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl- glycerol (DPG), or ceramide.

[0200] In some aspects, a lipid-PEG-CNS endothelial cell targeting group conjugate comprises a DSG-PEG, DSPE-PEG, DMPE-PEG, DSPG-PEG, DMG-PEG, DPPE-PEG, DPG-PEG or ceramide-PEG. In some aspects, a lipid-PEG-CNS endothelial cell targeting group conjugate comprises a DPG-PEG.

[0201] In some aspects, the PEG in a lipid-PEG-cell targeting conjugate is PEG 1000, PEG 2000, PEG 3000, PEG 3400, PEG 3450, PEG 4000, or PEG 5000. In some aspects, the PEG is PEG 2000 or PEG 3400. In some aspects, the PEG is PEG 2000 (PEG2K).

[0202] In some aspects, a lipid-CNS endothelial cell targeting group conjugate comprises a lipid-PEG2K-antibody. In some aspects, a lipid-CNS endothelial cell targeting group conjugate comprises a lipid-PEG2K-Fab. In some aspects, a lipid-CNS endothelial cell targeting group conjugate comprises a lipid-PEG2K-scFv. In some aspects, a lipid-CNS endothelial cell targeting group conjugate comprises a lipid-PEG2K-ISVD. In some aspects, a lipid nanoparticle comprises a lipid-CNS endothelial cell targeting group conjugate comprising a lipid-PEG2K antibody, and / or a lipid-CNS endothelial cell targeting group conjugate comprising a lipid-PEG2K-Fab, and / or a lipid-CNS endothelial cell targeting group conjugate comprising a lipid-PEG2K-scFv, and / or a lipid-CNS endothelial cell targeting group conjugate comprising a lipid-PEG2K-ISVD.

[0203] In some aspects, the lipid-CNS endothelial cell targeting group conjugate comprises a DPG-PEG2K-cell targeting polypeptide or fragment thereof. In some aspects, the lipid-CNS endothelial cell targeting group conjugate comprises a DPG-PEG2K-cell targeting antibody. In some aspects, the lipid-CNS endothelial cell targeting group conjugate comprises a DPG-PEG2K-cell targeting Fab. In some aspects, the lipid-CNS endothelial cell targeting group conjugate comprises a DPG-PEG2K-cell targeting scFv. In some aspects, the lipid-CNS endothelial cell targeting group conjugate comprises a DPG-PEG2K-cell targeting ISVD. In some aspects, a lipid nanoparticle comprises a lipid-CNS endothelial cell targeting group conjugate comprising a DPG-PEG2K antibody, and / or a lipid-cell targeting group conjugate comprising a DPG-PEG2K-Fab, and / or a lipid-cell targeting group conjugate comprising a DPG-PEG2K-scFv, and / or a lipid-cell targeting group conjugate comprising a DPG-PEG2K-ISVD.

[0204] In some aspects, the lipid of a lipid-CNS endothelial cell targeting group conjugate is conjugated to the N-terminus, C-terminus, or anywhere in the middle part of the cell targeting polypeptide or fragment thereof. In some aspects, a CNS endothelial cell targeting polypeptide is an antibody, Fab, scFv, or ISVD and the lipid is conjugated to the N-terminus of the cell targeting antibody, Fab, scFv, or ISVD. In some aspects, the lipid is conjugated to the C-terminus of a CNS endothelial cell targeting antibody, Fab, scFv, or ISVD. In some aspects, the lipid is conjugated at a position between the N- and C-terminus of a CNS endothelial cell targeting antibody, Fab, scFv, or ISVD.

[0205] In some aspects, a lipid nanoparticle comprises multiple lipid-CNS endothelial cell targeting group conjugates. In some aspects, the multiple lipid-CNS endothelial cell targeting groups are covalently conjugated to different PEG linkers. In some aspects, the multiple lipid-CNS endothelial cell targeting groups are presented at the surface of the lipidnanoparticle and each targeting group binds to a different molecule or portion of a molecule on the CNS endothelial cell. In some aspects, the multiple lipid-CNS endothelial cell targeting groups each bind to a different epitope on a same antigen. In some aspects, the multiple lipid-CNS endothelial cell targeting groups each bind to a different epitope on a different antigen. Advantageously, lipid nanoparticles with multiple lipid-CNS endothelial cell targeting groups on their surface increase the avidity and specificity of targeting interactions to a CNS endothelial cell.

[0206] In some aspects, a lipid-CNS endothelial cell targeting group conjugate of a lipid nanoparticle comprises between about 1 g and about 50 g of CNS endothelial cell-targeting group per mole lipid. In some aspects, the lipid-CNS endothelial cell-targeting group conjugate of a lipid nanoparticle comprises between about 10 g and about 20 g of CNS endothelial cell-targeting group per mole lipid. In some aspects, a lipid-antibody conjugate of a lipid nanoparticle comprises about 12 g of CNS endothelial cell-targeting group per mole lipid.

[0207] In some aspects, a lipid-CNS endothelial cell targeting group conjugate comprises about 1 g to about 10 g of cell targeting group per mole of lipid. In some aspects, a lipid-CNS endothelial cell targeting group conjugate comprises about 2 g to about 9 g of CNS endothelial cell targeting group per mole of lipid. In some aspects, a lipid-CNS endothelial cell targeting group conjugate comprises about 3 g to about 9 g of CNS endothelial cell targeting group per mole of lipid.

[0208] In some aspects, a lipid-antibody conjugate comprises about 1 g to about 10 g of antibody per mole of lipid. In some aspects, a lipid-antibody conjugate comprises about 2 g to about 9 g of antibody per mole of lipid. In some aspects, a lipid-antibody conjugate comprises about 3 g to about 9 g of antibody per mole of lipid.

[0209] In some aspects, a lipid-Fab conjugate comprises about 1 g to about 10 g of Fab per mole of lipid. In some aspects, a lipid-Fab conjugate comprises about 2 g to about 9 g of Fab per mole of lipid. In some aspects, a lipid-Fab conjugate comprises about 3 g to about 9 g of Fab per mole of lipid.

[0210] In some aspects, a lipid-scFv conjugate comprises about 1 g to about 10 g of scFv per mole of lipid. In some aspects, a lipid-scFv conjugate comprises about 2 g to about 9 g of scFv per mole of lipid. In some aspects, a lipid-scFv conjugate comprises about 3 g to about 9 g of scFv per mole of lipid.

[0211] In some aspects, a lipid-ISVD conjugate comprises about 1 g to about 10 g of ISVD per mole of lipid. In some aspects, a lipid-ISVD conjugate comprises about 2 g to about 9 gof ISVD per mole of lipid. In some aspects, a lipid-ISVD conjugate comprises about 3 g to about 9 g of ISVD per mole of lipid.

[0212] In some aspects, a lipid-CNS endothelial cell targeting group conjugate is present in a lipid nanoparticle in a range of about 0.001 mol% to about 0.5 mol%. In some aspects, a lipid-CNS endothelial cell targeting group conjugate is present in a lipid nanoparticle in a range of about 0.002 mol% to about 0.2 mol%. In some aspects, a lipid-CNS endothelial cell targeting group conjugate is present in the lipid nanoparticle in a range of about 0.001 mol% to about 0.4 mol%; about 0.001 mol% to about 0.3 mol%; about 0.001 mol% to about 0.2 mol%; about 0.001 mol% to about 0.1 mol%; or about 0.001 mol% to about 0.08 mol%.%; about 0.001 mol% to about 0.06 mol%; about 0.001 mol% to about 0.04 mol%; about 0.001 mol% to about 0.02 mol%; or about 0.001 mol% to about 0.01 mol%. In some aspects, a lipid-CNS endothelial cell targeting group conjugate is present in a lipid nanoparticle in a range of about 0.002 mol% to about 0.5 mol%; about 0.003 mol% to about 0.5 mol%; about 0.004 mol% to about 0.5 mol%; about 0.005 mol% to about 0.5 mol%; about 0.006 mol% to about 0.5 mol%; about 0.008 mol% to about 0.5 mol%; about 0.01 mol% to about 0.5 mol%; about 0.02 mol% to about 0.5 mol%; about 0.03 mol% to about 0.5 mol%; about 0.04 mol% to about 0.5 mol%; about 0.05 mol% to about 0.5 mol%; about 0.06 mol% to about 0.5 mol%; about 0.07 mol% to about 0.5 mol%; about 0.08 mol% to about 0.5 mol%; about 0.09 mol% to about 0.5 mol%; about 0.1 mol% to about 0.5 mol%; about 0.15 mol% to about 0.5 mol%; about 0.2 mol% to about 0.5 mol%; about 0.25 mol% to about 0.5 mol%; about 0.3 mol% to about 0.5 mol%; about 0.35 mol% to about 0.5 mol%; or about 0.4 mol% to about 0.5 mol%.

[0213] In some aspects, a lipid-CNS endothelial cell targeting conjugate containing lipid nanoparticle further comprises free PEG-lipid so as to reduce the amount of non-specific binding via the targeting group. In some aspects, the free PEG-lipid can be the same or different from the PEG-lipid included in the conjugate. In some aspects, the free PEG-lipid is selected from the group consisting of PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N-(Methylpolyoxyethylene oxycarbonyl)-l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG) 1,2-Dimyristoyl-rac-glycero-3 -methylpolyoxyethylene (PEG-DMG), 1,2-Dipalmitoyl-rac-glycero-3 -methylpolyoxyethylene (PEG-DPG), 1,2-Dioleoyl-rac-glycerol, methoxypolyethylene Glycol (DOG-PEG), l,2-Distearoyl-rac-glycero-3-methylpoly oxy ethylene (PEG-DSG), N-palmitoyl-sphingosine-1-succinyl [methoxy (poly ethylene glycol)] (PEG-ceramide), DSPE-PEG-cysteine, and aderivative thereof. In some aspects, a PEG of the lipid-CNS endothelial cell targeting conjugate has an average PEG length of between2000 and 5000, or a PEG length of about 2000, 3400, or 5000. In some aspects, a lipid nanoparticle comprises a mixture of two or more PEGylated lipids.Cargo

[0214] In some aspects, a lipid nanoparticle comprises a cargo, e.g., a nucleic acid molecule disposed in the lipid nanoparticle for delivery to a cell (e.g., a CNS endothelial cell) or tissue in a subject.

[0215] In some aspects, a lipid nanoparticle comprises a nucleic acid, e.g., aDNA orRNA, such as an mRNA, tRNA, microRNA, siRNA, gRNA (guide RNA), circRNA(circular RNA), ribozymes, decoy RNA or dicer substrate siRNA. In some aspects, a nucleic acid comprises naturally occurring components, such as, naturally occurring bases, sugars or linkage groups (e.g., phosphodiester linkage groups). In some aspects, a nucleic acid comprises non-naturally occurring components or modifications, (e.g., thioester linkage groups). For example, the nucleic acid can be synthesized to contain base, sugar, linker modifications known to those skilled in the art. Furthermore, the nucleic acids can be linear or circular, or have any desired configuration. In some aspects, a lipid nanoparticle includes multiple nucleic acid molecules, e.g., multiple RNA molecules, which can be the same or different.

[0216] In some aspects, one or more lipid nanoparticle compositions including one or more different mRNAs are combined, and / or simultaneously contacted with a cell. In some aspects, an mRNA may include one or more of a stem loop, a chain terminating nucleoside, a polyA sequence, a polyadenylation signal, and / or a 5' cap structure. In some aspects, the mRNA may encode a receptor, such as a chimeric antigen receptor (CAR), for transfection of a CNS endothelial cell. In some aspects, the mRNA may encode a Cas nuclease. In some aspects, the RNA may comprise a guide RNA.

[0217] In some aspects, the wt / wt ratio of the lipid components to the cargo (e.g., mRNA) in the lipid nanoparticle is from about 1:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, or 50:1. In some aspects, the wt / wt ratio of the lipid components to the cargo in the lipid nanoparticle is from about 5: 1 to about 50: 1. In some aspects, the wt / wt ratio of the lipid components to the cargo in the lipid nanoparticle is from about 5:1 to about 40:1. In some aspects, the wt / wt ratio of the lipid components to the cargo in the lipid nanoparticle is from about 10:1 to about40: 1. In some aspects, the wt / wt ratio of the lipid components to the cargo in the lipid nanoparticle is from about 15:1 to about 25:1.

[0218] In some aspects, the encapsulation efficiency of the cargo (e.g., mRNA) in the lipid nanoparticle is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99%.

[0219] In some aspects, a lipid nanoparticle as described herein exhibits dye accessible RNA of less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 7.5%, less than 5%, less than 2.5%, or less than 1%.

[0220] In some aspects, the one or more mRNAs and lipids are selected to provide a specific N: P ratio (the ratio of positively-chargeable lipid or polymer amine (N = nitrogen) groups to negatively-charged nucleic acid phosphate (P) groups). The N: P ratio of a lipid nanoparticle composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an mRNA. In general, a lower N: P ratio is preferred. A N: P ratio may be dependent on a specific lipid and its pKa. In some aspects, the mRNA and lipids in a lipid nanoparticle and / or their relative amounts are selected to provide an N: P ratio from about 1: 1 to about 30:1, or from about 1:1 to about 20:1. In some aspects, theN: P ratio is, e.g., 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1. In certain aspects, theN: P ratio is from about 2:1 to about 5:1. In some aspects, the N: P ratio is about 4: 1. In some aspects, the N: P ratio is from about 4: 1 to about 8:1. In some aspects, the N: P ratio may be about 4:1, about 4.5:1, about 4.6:1, about 4.7:1, about 4.8:1, about 4.9:1, about 5.0:1, about 5.1:1, about 5.2:1, about 5.3:1, about 5.4:1, about 5.5:1, about 5.6:1, about 5.7:1, about 6.0:1, about 6.5:1, or about 7.0:1.COMPOSITIONS

[0221] Provided herein are compositions comprising a lipid nanoparticle described herein. In some aspects, a composition provided herein comprises a lipid nanoparticle comprising an ionizable lipid. In some aspects, an ionizable lipid of the lipid nanoparticle comprises a structure of Formula II:or a salt thereof, wherein:R1, R2, and R3are each independently a bond or C1-3 alkylene;R1A, R2A, and R3Aare each independently a bond or C1-10 alkylene;R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1-20 alkenyl, -(CH2)o-ioC(0)ORal, or -(CH2)o-ioOC(0)Ra2; Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl; R3BisR3B1is C1-6 alkylene; andR3B2and R3B3are each independently H or C1-6 alkyl.

[0222] In some aspects, an ionizable lipid comprises a structure of Formula (Ila)(Lipid 15).

[0223] In some aspects, an ionizable lipid comprises a structure of Formula (III) (KC2):

[0224] In some aspects, an ionizable lipid comprises a structure of Formula (IV) (KC3):

[0225] In some aspects, an ionizable lipid is DLin-KC2-DMA.

[0226] In some aspects, an ionizable lipid is DLin-MC3-DMA.

[0227] In some aspects, an ionizable lipid is DLin-DMA.

[0228] In some aspects, an ionizable lipid is DLin-K-DMA.

[0229] In some aspects, an ionizable lipid is C12-200.

[0230] In some aspects, an ionizable lipid is DOTAP.

[0231] In some aspects, an ionizable lipid is DOTMA.

[0232] In some aspects, the lipid nanoparticle of the composition further comprises a helper lipid, a structural lipid, a PEG-lipid and, optionally, a cargo. In some aspects, the lipid nanoparticle of the composition further comprises a lipid-cell targeting group conjugate as described herein.

[0233] In some aspects, the lipid nanoparticle composition comprises one or more pharmaceutically acceptable excipient. In some aspects, conventional excipients and accessory ingredients are used in a pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more components of a lipid nanoparticle composition described herein. For example, an excipient or accessory ingredient may be incompatible with a component of a lipid nanoparticle composition if its combination with the component may result in any undesirable biological effect or otherwise deleterious effect.

[0234] In some aspects, one or more excipients may make up greater than 50% of the total mass or volume of a pharmaceutical composition including a lipid nanoparticle. For example, the one or more excipients may make up 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical composition.METHODSMethods of Making Lipid Nanoparticles

[0235] In some aspects, lipid nanoparticles are produced by using either rapid mixing by an orbital vortexer or by microfluidic mixing. Orbital vortexer mixing is accomplished by rapid addition of a lipid solution in ethanol to an aqueous solution of a nucleic acid followed immediately by vortexing at 2,500 rpm. In some aspects, a lipid nanoparticle is producedusing a microfluidic mixing step. In some aspects, microfluidic mixing is achieved mixing aqueous and organic streams at a controlled flow rate in a microfluidic channel using, e.g., a NanoAssemblr® Ignite device and microfluidic chips featuring optimized mixing chamber geometry (Precision Nanosystems, Vancouver, BC). In some aspects, a lipid nanoparticle is produced using a microfluidic mixing step to rapidly mix an ethanolic lipid solution and an aqueous nucleic acid solution, resulting in encapsulation of the nucleic acid in the lipid nanoparticles. In some aspects, the nanoparticle suspension is then buffer exchanged into an all aqueous buffer using membrane filtration device of choice for ethanol removal and nanoparticle maturation.Methods of Use

[0236] The present disclosure provides methods of delivering a cargo to a target cell or tissue, e.g., a target cell or tissue in a subject, and lipid nanoparticles or pharmaceutical compositions containing the lipid nanoparticles for use in such methods. Any disclosure herein of a method of, e.g., treating a disease or disorder or, e.g., delivering a nucleic acid to a cell or, e.g., producing a polypeptide of interest in a cell should be interpreted also as a disclosure of a lipid nanoparticle or pharmaceutical composition comprising said lipid nanoparticle for use in such methods.

[0237] In some aspects, provided is a method of producing a polypeptide of interest in a target cell, e.g., a CNS endothelial cell and lipid nanoparticles or pharmaceutical compositions containing the lipid nanoparticle for use in such methods. Methods of producing polypeptides in such a CNS endothelial cell involve contacting a CNS endothelial cell with a lipid nanoparticle comprising a cargo, e.g., a nucleic acid of interest. Upon contacting the CNS endothelial cell with the lipid nanoparticle, the nucleic acid may be taken up and translated in the CNS endothelial cell to produce the polypeptide of interest.

[0238] In some aspects, the step of contacting a cell with a lipid nanoparticle composition including a cargo, e.g., a mRNA encoding a polypeptide of interest may be performed in vivo, ex vivo, or in vitro. The amount of a lipid nanoparticle composition contacted with a cell, e.g., a CNS endothelial cell and / or the amount of mRNA therein, may depend on the means of administration, the physiochemical characteristics of the lipid nanoparticle and the mRNA (e.g., size, charge, and chemical composition) therein, and other factors.

[0239] In some aspects, the step of contacting a lipid nanoparticle including a cargo with a cell, e.g., a CNS endothelial cell, involves or causes transfection where the lipid nanoparticle fuses with the membrane of the cell to permit the delivery of the cargo into the cell. Upon introductioninto the cytoplasm of the CNS endothelial cell, the cargo, e.g., a mRNA, is then translated into a protein or peptide via the protein synthesis machinery within the cytoplasm of the CNS endothelial cell.

[0240] In some aspects, the efficiency of polypeptide production in the cell may be determined, and the cell may be re-contacted with a lipid nanoparticle repeatedly until a desired target polypeptide production efficiency is achieved.

[0241] In some aspects, provided are methods of delivering a cargo, e.g., a nucleic acid to a mammalian cell, e.g., a mammalian CNS endothelial cell in a subject. In some aspects, the delivery of a nucleic acid to such a CNS endothelial cell involves administering a lipid nanoparticle composition described herein to a subject, e.g., by injection into the subject.

[0242] In some aspects, after administration, the lipid nanoparticle can target and / or contact a cell, e.g., a CNS endothelial cell in the subject. Upon contacting the CNS endothelial cell with the lipid nanoparticle composition, a nucleic acid, e.g., a translatable mRNA, may be translated in the CNS endothelial cell to produce a polypeptide of interest.

[0243] In some aspects, a lipid nanoparticle described herein provides at least one of the following benefits:a. Increased specificity of targeted delivery to a CNS endothelial cell compared to a reference lipid nanoparticle;b. increased half-life of the nucleic acid or a polypeptide encoded by the nucleic acid in the CNS endothelial cell compared to a reference lipid nanoparticle;c. increased transfection rate compared to a reference lipid nanoparticle; and / or d. high nucleic acid encapsulation efficiency.

[0244] In some aspects, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of non-target cells, e.g., non-CNS endothelial cells and / or non-endothelial CNS cells are transfected by a lipid nanoparticle described herein. For example, in some aspects, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of undesired non-CNS endothelial cells and / or non-endothelial CNS cells that are not meant to be the destination of the delivery are transfected by the lipid nanoparticle. In some aspects, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of undesired non-CNS endothelial cells and / or non-endothelial CNS cells that are not meant to be the destination of the delivery are transfected by the lipid nanoparticle.

[0245] In some aspects, the half-life of a nucleic acid delivered by a lipid nanoparticle described herein to a target cell, e.g., a CNS endothelial cell, or of a polypeptide encoded by the nucleic acid delivered by the lipid nanoparticle is at least 5%, 10%, 15%, 20%, 25%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, or longer than the half-life of the nucleic acid delivered by a reference lipid nanoparticle to said target cell or a polypeptide encoded by the nucleic acid delivered by the reference lipid nanoparticle.

[0246] In some aspects, the composition of the lipid nanoparticle differs from the composition of a reference lipid nanoparticle in the type of ionizable cationic lipid, relative amount of ionizable cationic lipid, type of PEG-lipid, relative amount of PEG-lipid, type of helper lipid, relative amount of helper lipid, type of structural lipid, relative amount of structural lipid, cargo, or type of cell targeting group, or any combination thereof. In some aspects, a reference lipid nanoparticle is a lipid nanoparticle that comprises a helper lipid in a range of more than 10 mol%. In some aspects, a reference lipid nanoparticle is a lipid nanoparticle that comprises a helper lipid in a range of more than 11 mol%. In some aspects, a reference lipid nanoparticle is a lipid nanoparticle that comprises a helper lipid in a range of about 11 mol% to about 20 mol%. In some aspects, a reference lipid nanoparticle is a lipid nanoparticle that comprises a helper lipid in a range of about 21 mol% to about 50 mol%. In some aspects, the difference in overall lipid content in a lipid nanoparticle described herein and / or reference lipid is made up by changes in the amount of structural lipid; otherwise the lipid nanoparticle and the reference lipid nanoparticle are the same.

[0247] In some aspects, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more target CNS endothelial cells are transfected by the lipid nanoparticle.

[0248] In some aspects, expression level of a nucleic acid in a CNS endothelial target cell delivered by a lipid nanoparticle described herein is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times or more higher than expression level of the nucleic acid in the same CNS endothelial target cell delivered by a reference lipid nanoparticle.

[0249] In some aspects, specific delivery using lipid nanoparticles described herein may result in a greater than 2 fold, 5 fold, 10 fold, 15 fold, or 20 fold increase in the amount of cargo delivered to the targeted destination as compared to other destinations (a non-target cell, e.g., a non-CSN endothelial cell or a non-endothelial CNS cell).

[0250] In some aspects, specific delivery using lipid nanoparticles described herein may result in a greater than 2 fold, 5 fold, 10 fold, 15 fold, or 20 fold increase in the amount ofcargo delivered to a CNS endothelial target cell as compared to a non-CNS endothelial cell and / or a non-endothelial CNS cell.

[0251] In some aspects, expression level of a nucleic acid in a non-CNS endothelial cell and / or a non-endothelial CNS cell following administration of a lipid nanoparticle described herein to a subject in need thereof is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times or more lower than expression level of the nucleic acid in a CNS endothelial cell.

[0252] In some aspects, a lipid nanoparticle composition described herein may be useful for treating a disease, disorder, or condition characterized by a missing or aberrant polypeptide or polypeptide activity. In some aspects, upon delivery of a lipid nanoparticle comprising a nucleic acid, e.g., a mRNA encoding the missing or aberrant polypeptide to a cell, translation of the mRNA may produce the polypeptide, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide.

[0253] In some aspects, provided herein are methods of targeting the delivery of a nucleic acid to a CNS endothelial cell of a subject. In some aspects, the method comprises contacting a CNS endothelial cell with a lipid nanoparticle as described herein. In some aspects, the method comprises contacting the CNS endothelial cell in vivo with a lipid nanoparticle.

[0254] In some aspects, a method of targeting the delivery of a nucleic acid to a CNS endothelial cell of a subject comprises administering a lipid nanoparticle described herein to the subject in need thereof.

[0255] In some aspects, provided is a method of modulating cellular function of a CNS endothelial cell of a subject. In some aspects, the method comprises administering to the subject a lipid nanoparticle. In some aspects, the lipid nanoparticle comprises a CNS endothelial cell targeting group.

[0256] In some aspects, the CNS endothelial cell targeting group is a transferrin receptor targeting group. In some aspects, the CNS endothelial cell targeting group is a PEC AM targeting group. In some aspects, the CNS endothelial cell targeting group is a VWF targeting group.

[0257] In some aspects, a modulation of cell function comprises delivering a nucleic acid encoding a gene editing system (e.g., a site-directed nuclease and, optionally, comprising a guide RNA) to a CNS endothelial cell.

[0258] In some aspects, a lipid nanoparticle provided herein is administered to a subject with a disease for in vivo gene editing and treatment of the disease.

[0259] In some aspects, a method provided comprises administering a lipid nanoparticle described herein to a subject with Alzheimer’s disease, vascular dementia, traumatic brain injury, stroke, Parkinson’s disease, neuroinflammation, advanced age, or cerebral amyloid angiopathy.

[0260] In some aspects, a method provided comprises administering a lipid nanoparticle described herein to a subject with a genetic cause of a deficit in a CNS endothelial cell function. In some aspects, a deficit in a CNS endothelial cell function is a GLUT! deficiency syndrome (De Vivo syndrome), band-like calcification with simplified gyration and polymicrogyria (BLC-PMG), familial cerebral cavernous malformations, cerebral small vessel disease, microcephaly, or Allan-Hemdon-Dudley syndrome.

[0261] In some aspects, a method provided comprises administering a lipid nanoparticle described herein to a subject with a CNS endothelial cell barrier dysfunction.

[0262] In some aspects, provided is a method of repairing a CNS endothelial cell barrier dysfunction is in a subject by administering a lipid nanoparticle as described herein to a subject having a CNS endothelial cell barrier dysfunction.

[0263] In some aspects, the lipid nanoparticle that is delivered to a subject comprises one or more nucleic acids encoding a site-directed nuclease and one or more RNAs that confer binding of the Cas nuclease to the target nucleotide sequence. In some aspects, the one or more RNAs conferring binding of a Cas nuclease to the target nucleotide sequence include a transactivating cRNA (tracrRNA) and a CRISPR RNA (crRNA), or, more commonly, a guide RNA (also referred to as a single guide RNA (sgRNA)), in which crRNA and tracrRNA are engineered into one RNA molecule. In some aspects, the one or more nucleic acids encoding a nuclease encode a site-directed nuclease including a CRISPR-associated (Cas) nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a megaTAL.Uses of the Lipid Nanoparticles

[0264] In some aspects, the disclosure provides a lipid nanoparticle or a composition containing thereof, as disclosed herein, for use in a method of targeting the delivery of a nucleic acid to a CNS endothelial cell of a subject. In some aspects, the disclosure provides a lipid nanoparticle or a composition containing thereof, as disclosed herein, for use in a method of targeting the delivery of a nucleic acid to a CNS endothelial cell of a subject. In some aspects, the nucleic acid encodes a CNS endothelial cell function enhancing polypeptide and such nucleic acid is targeted to a CNS endothelial cell by the lipidnanoparticle. In some aspects, the lipid nanoparticle comprises a CNS endothelial cell targeting group to target the delivery of the nucleic acid to the CNS endothelial cell. In some aspects, the nucleic acid of the lipid nanoparticle encodes a polypeptide that functions in gene editing and such nucleic acid is targeted to a CNS endothelial cell by the lipid nanoparticle.

[0265] In some aspects, the disclosure provides a lipid nanoparticle or a composition containing thereof, as disclosed herein, for use in a method of treating a disease or condition in a subject.

[0266] In some aspects, provided is a lipid nanoparticle or a composition containing thereof, as disclosed herein, for use in a method of treating Alzheimer’s disease, vascular dementia, traumatic brain injury, stroke, Parkinson’s disease, neuroinflammation, advanced age, or cerebral amyloid angiopathy.

[0267] In some aspects, provided is a lipid nanoparticle or a composition containing thereof, as disclosed herein, for use in a method of a genetic cause of a deficit in a CNS endothelial cell function, wherein the deficit in a CNS endothelial ceil function is a GLUT1 deficiency syndrome (De Vivo syndrome), band-like calcification with simplified gyration and polymicrogyria (BLC-PMG), familial cerebral cavernous malformations, cerebral small vessel disease, microcephaly, or Allan-Hemdon-Dudley syndrome.

[0268] In some aspects, provided is a lipid nanoparticle or a composition containing thereof, as disclosed herein, for use in a method of treating a CNS endothelial cell bam er dysfunction.

[0269] In some aspects, the lipid nanoparticle comprises a nucleic acid that encodes a CNS endothelial cell function enhancing polypeptide and the targeted CNS endothelial cell expressing the polypeptide treats a disease or condition in the subject. In some aspects, the lipid nanoparticle comprises a nucleic acid that provides components of a gene editing system and the targeted CNS endothelial cell after having undergone gene editing treats a disease or condition in the subject. In some aspects, the composition used in the method is a pharmaceutical composition.KITS

[0270] In some aspects, provided is a kit for treating a disorder. In some aspects, a kit comprises: an ionizable cationic lipid, a helper lipid, a structural lipid, a PEG-lipid, a cargo, a lipid-CNS endothelial cell targeting group conjugate, and instructions for preparing a lipid nanoparticle and treating a medical disorder using a lipid nanoparticle. In some aspects, a kit comprises a lipid nanoparticle composition comprising a lipid nanoparticle comprising an ionizable cationic lipid, a helper lipid, a structural lipid, a PEG-lipid, a cargo, a lipid-CNSendothelial cell targeting group conjugate and instructions for treating a medical disorder using the lipid nanoparticle composition or lipid nanoparticle.EXAMPLESExample 1: MaterialsLNPs were formulated using DLin-KC2-DMA ionizable lipid (MedChemExpress), a phospholipid (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), Avanti Polar Lipids), a structural lipid (cholesterol, Avanti Polar Lipids), and a polyethylene glycol (PEG) lipid (1,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol-2000 (DMG-PEG2000) Avanti Polar Lipids) at a specific molar ratio shown in Table 2. CleanCap Firefly luciferase mRNA (5-methoxyuridine), or N(l)-methylpseudouridine-incorporated green fluorescence protein (GFP) reporting mRNA (Trilink Biotechnologies) were encapsulated in LNPs using microfluidic mixing. Anti-TfR Fabs or ISVD were synthesized at BioIntron (Shanghai, China). Table 2.

[0271] Anti-human TfR H7 Fab-hlam3 bDS (Fab B902) is disclosed in Poul MA et al., Journal of Molecular Biology, 301: 1149-61, 2000. The molecular weight of the Fab B902 is 47195.6 g / mol and the specific dissociation constant for binding to human TfR is KD 5 nM.

[0272] The sequence of the heavy chain amino acid sequence of anti-human TfRH7 Fab-hlam3 bDS (Fab B902) is:Q VQLQESGGGVVQPGRSLRLSC AASRFTF S S YAMHWVRQAPGKGLEWVAVIS YDGS NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSGYGDYPDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTCP AVLQ S SGL YSL S S VVTVP S S SLGTQT YICNVNHKP SNTKVDKKVEPKS SDKT HTC (SEQ IDNO: 1).

[0273] The sequence of the light chain amino acid sequence of anti-human TfR H7 Fab-hlam3 bDS (Fab B902) is:SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVMYGRNERPSG VPDRFSGSKSGTSASLAISGLQPEDEANYYCAGWDDSLTGPVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAACSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTESS (SEQ ID NO: 2).

[0274] The heavy chain complementarity determining region 1 (CDR-H1) amino acid sequence of anti-human TfR H7 Fab-hlam3 bDS (Fab B902), determined according to Kabat, is: RFTFSSYAMH (SEQ ID NO: 5).

[0275] The heavy chain complementarity determining region 2 (CDR-H2) amino acid sequence of anti-human TfR H7 Fab-hlam3 bDS (Fab B902), determined according to Kabat, is: VISYDGSNKYYADSVKG (SEQ ID NO: 6).

[0276] The heavy chain complementarity determining region 3 (CDR-H3) amino acid sequence of anti-human TfR H7 Fab-hlam3 bDS (Fab B902), determined according to Kabat, is: DLSGYGDYPDY (SEQ ID NO: 7).

[0277] The light chain complementarity determining region 1 (CDR-L1) amino acid sequence of anti-human TfR H7 Fab-hlam3 bDS (Fab B902), determined according to Kabat, is: GFTFSNYGMH (SEQ ID NO: 8).

[0278] The light chain complementarity determining region 2 (CDR-L2) amino acid sequence of anti-human TfR H7 Fab-hlam3 bDS (Fab B902), determined according to Kabat, is: MIYYDSSKMNYADTVKG (SEQ ID NO: 9).

[0279] The light chain complementarity determining region 3 (CDR-L3) amino acid sequence of anti-human TfR H7 Fab-hlam3 bDS (Fab B902), determined according to Kabat, is: AGWDDSLTGPV (SEQ ID NO: 10).

[0280] Anti-mouse TfR 8D3 ChFab bDS (Fab B901) is disclosed in Kissel et al., Histochemistry and Cell Biology, 110: 63-71, 1998. The CDRs of anti-mouse TfR 8D3 ChFab bDS (Fab B901) are described in Boado, R. J., Zhang, Y., Wang, Y., & Pardridge, W. M. (2009). Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood-brain barrier delivery in the mouse. Biotechnology and bioengineering, 102(4), 1251-1258. doi.org / 10.1002 / bit.22135. The molecular weight of the Fab B901 is 47702.6 g / mol and the specific dissociation constant for binding to murine TfR is KD 1.2 nM.

[0281] The sequence of the heavy chain amino acid sequence of anti-Mouse TfR 8D3 ChFab bDS (Fab B901) is:EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSS KMNYADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQG VSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTCPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHT C (SEQ ID NO: 3).

[0282] The sequence of the light chain amino acid sequence of Fab B901 is:

[0283] DIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSL ADGVPSRFSGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGGGTKLELKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLCSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES (SEQ ID NO: 4).

[0284] The heavy chain complementarity determining region 1 (CDR-H1) amino acid sequence of anti-Mouse TfR 8D3 ChFab bDS (Fab B901) is: GFTFSNYGMH (SEQ ID NO: 11).

[0285] The heavy chain complementarity determining region 2 (CDR-H2) amino acid sequence of anti-Mouse TfR 8D3 ChFab bDS (Fab B901) is: MIYYDSSKMNYADTVKG (SEQ ID NO: 12).

[0286] The heavy chain complementarity determining region 3 (CDR-H3) amino acid sequence of anti-Mouse TfR 8D3 ChFab bDS (Fab B901) is: PTSHYVVDV (SEQ ID NO: 13).

[0287] The light chain complementarity determining region 1 (CDR-L1) amino acid sequence of anti-Mouse TfR 8D3 ChFab bDS (Fab B901) is: QASQDIGNWLA (SEQ ID NO: 14).

[0288] The light chain complementarity determining region 2 (CDR-L2) amino acid sequence of anti-Mouse TfR 8D3 ChFab bDS (Fab B901) is: GATSLAD (SEQ ID NO: 15).

[0289] The light chain complementarity determining region 3 (CDR-L3) amino acid sequence of anti-Mouse TfR 8D3 ChFab bDS (Fab B901) is: LQAYNTPWT (SEQ ID NO: 16).

[0290] Luciferase or GFP reporting mRNA solutions were prepared in a buffer consisting of 10 mM sodium citrate (JT Baker), 150 mM sodium chloride (JT Baker), at pH 4.5, at desired mRNA concentration, and lipids were mixed in appropriate molar ratio listed in Error! Reference source not found, in pure ethyl alcohol (Sigma Aldrich). The mRNA and total lipid concentration were adjusted accordingly to produce LNPs with specific ratio of nitrogen to phosphate groups (N / P). To encapsulate luciferase or GFP reporting mRNA in LNPs, a NexGen cartridge (Precision NanoSystems) was inserted in the cartridge holder in the NanoAssemblr® Ignite instrument (Precision NanoSystems) and a slip-tip syringe (Becton Dickinson) containing the mRNA solution was placed in the left port of the cartridge. The syringe containing the lipid solution was inserted into the right port of the cartridge. mRNAand lipid solutions were mixed at the ratio of 3 v / v, at 9 mL / min injection rate. To prepare lx phosphate-buff ered saline (PBS), 1 Ox PBS (Invitrogen) was diluted 10 folds in nuclease-free water (Invitrogen). The produced LNP solutions were diluted approximately 3 folds with lx PBS. Centrifugal tubes with lOOkD MWCO membrane (MilliporeSigma) were used to thoroughly exchange the LNP solution buffer into lx PBS by spinning the LNPs at 1000g, with approximately 10 diafiltration volume. After completion of buffer exchange, LNP solutions were further concentrated to approximately 200 pg of mRNA per milliliter of solution.mRNA Concentration Measurement

[0291] To measure the concentration of the mRNA solutions prior to formulating LNPs, 2 pL of mRNA solution was loaded into NanoDrop 2000 (Thermo Scientific). A standard curve was prepared by diluting the mRNA solution in lx PBS to achieve a serial dilution of mRNA at different concentrations, starting with 1000 ng / mL, and ending with 0 ng / mL (blank). To measure the mRNA concentration in each LNP solution, LNP samples were diluted in lx PBS ensuring the theoretical mRNA concentration was within the working range of the standard curve. Ribogreen (Invitrogen) was diluted 200 times with lx PBS (RG-PBS) or 1% triton solution in PBS (RG-Tr).

[0292] To measure the total concentration of mRNA in LNPs, each diluted LNP sample was mixed with equal volume of RG-Tr. The non-encapsulated mRNA content was measured by mixing each diluted LNP sample with equal volume of RG-PBS. Similarly, each mRNA standard solution was mixed with equal volume of RG-PBS, or RG-Tr. 100 uL of each standard or test article was plated in an opaque 96-well plate (Thermo Scientific). The 96-well plate was shaken at 300 rpm for 5 minutes in dark, prior to florescence measurement at excitation / emission of 480 / 520 nm. Values obtained for the standard curve were used to generate a plot with a linear fit. The mRNA concentration for each sample was calculated from the standard curve.Size and Zeta Potential Measurements

[0293] 25 pL of each LNP solution was diluted 100-fold in lx PBS or nuclease-free water (Invitrogen) for measurement of hydrodynamic diameter and zeta potential, respectively. For size measurement, 1 mL of diluted LNP samples was loaded in a 1.5 mL clear cuvette (Fisher Scientific), and for zeta potential measurement, 0.8 mL of each sample was transferred intothe capillary cuvette (Malvern Panalytical). A Malvern Nano Zetasizer (Malvern Panalytical) was utilized to measure both parameters.Conjugation of Targeting Ligands to PEG-Lipid Linkers prior to Incorporation into the LNP

[0294] In order to incorporate targeting ligands (Fabs or, alternatively ISVD) into the LNP surface, first the targeting ligands were conjugated to a PEG-lipid linker. Each of the antitransferrin (anti-TfR) Fabs (or ISVD) were engineered with a free cysteine group in the C-terminal of the heavy chain. The PEG-lipid linkers with a headgroup of Mai eimide were added to the thiolated Fab solutions for conjugation. To perform conjugation, Fab solutions were prepared in lx PBS, containing 5mM EDTA (MilliporeSigma) at a target concentration of 3 mg / mL. To reduce the cysteine group on C terminal, TCEP reducing agent (Thermo Scientific) was added to each solution at a final concentration of 0.1 mM, and samples were incubated at room temperature for 90 minutes. After completion of the reduction reaction, each sample was washed through a Zeba desalting column (Thermo Scientific) to remove the TCEP reducing agent. 40 kD and 7 kD Zeba columns were used for Fabs and ISVD, respectively. Following the TCEP removal, DSPE-PEG2000-Maleimide (Avanti Polar Lipids) (for Fab conjugation) or DSPE-PEG3400-Maleimide (NOF America Corporation) (for ISVD conjugation) were mixed with 18:0 PEG2000 PE (Avanti Polar Lipids) at a molar ratio of 2:3 and 1:4, respectively, to form micelles. The former solution was mixed with the Fab solution at a maleimide to Fab molar ratio of 2. The latter solution was added to the ISVD solution at a maleimide to ISVD molar ratio of 1. The resulting solutions were incubated at 37°C for 2 hours. Following the incubation, a thorough buffer exchange into lx PBS was performed to remove the free antibodies (10 diafiltration volume). The resulting solution was stored at 2-8°C for up to 2 weeks.Incorporation of Targeting Ligand into LNP Surface

[0295] Values obtained for mRNA concentration for each LNP sample were translated into the total lipid quantity utilized to formulate LNPs at a defined N / P ratio. The conjugated Fab or ISVD micelles were added to LNP solutions at a specific density (grams of antibody per mole of total lipid, referred to as “grafting density herein”). The solution was mixed at 300 rpm and incubated at 37°C for 4 hours using Thermomixer C (Eppendorf). The resulting LNP solutions were used for in vitro and in vivo screenings. Grafting density for Fabs and ISVD varied in a range of 2-25 g / mol ISVD, and 0.5-15 g / mol Fabs.In Vitro Screening

[0296] Brain microvascular endothelial cells (hCMEC / D3 cells, Millipore, SCC066, lot 3597523) were grown in EndoGRO-MV complete media (Millipore, SCME004) supplemented with Ing / mL FGF-2 (Millipore, GF003). All cell culture substrates were precoated with rat tail collagen (Sigma, C3867). 24hr prior to treatment with LNPs, cells were plated at 25,000 cells per well of 96-well tissue culture plates. Cells were treated with LNPs containing 0.01 pg or 0.05 pg of GFP mRNA per well. 24hr post treatment, cells were rinsed with DPBS and lifted with 0.05% Trypsin-EDTA (5-10 minutes at 37°C). Trypsin was inactivated by the addition of FACS buffer (DPBS, 1% FBS), cells were resuspended by trituration, spun for 5 minutes at 300xg, then resuspended in lOOpl of FACS buffer. GFP expression per cell was analyzed by FACS (NovoCyte Penteon).

[0297] Mouse endothelial cells isolated from brain tissue of a mouse with endothelioma (BEND3 cells, ATCC, CRL-2299, lot 70030469) were grown in DMEM (ATCC, 302002) supplemented with lOOU / mL pen / strep (Thermo, 15140122) and 10% FBS (Thermo, 10082147). 24hr prior to treatment with LNPs, cells were plated at 25,000 cells per well of 96-well tissue culture plates. Cells were treated with LNPs containing 0.02 pg or 0.1 pg of GFP mRNA per well. 24hr post treatment, cells were rinsed with DPBS and lifted with 0.25% Trypsin-EDTA (5-10 minutes at 37°C). Trypsin was inactivated by the addition of FACS buffer (DPBS, 1% FBS), cells were resuspended by trituration, spun for 5 minutes at 300xg, then resuspended in lOOpl of FACS buffer. GFP expression per cell was analyzed by FACS (NovoCyte Penteon).

[0298] LNPs without antibodies were used as “negative control”, demonstrating the nonspecific internalization of LNPs into cells and the subsequent GFP expression. All targeted LNPs were compared to the negative control. For comparison and screening purposes, a few different antibody grafting densities were utilized.In Vivo Screening

[0299] SKH1 mice (an immune-competent, fur-free mouse strain) were dosed intravenously with LNPs (with or without anti-TfR antibodies) at a dose of approximately 1 mg of mRNA per kg of animal body mass (2 mice per test article were dosed). For the control group, 1 animal was dosed with 100 pL of Balanced Salt Solution (BSS), or lx PBS. After 6 hours, mice were dosed with CycLuc 1. (15 mg / kg dissolved in phosphate-buffered saline) for firefly luciferase imaging using the Xenogen IVIS Spectrum Imaging System (Perkin Elmer).After the substrate injection, mice were placed into the IVIS imaging chamber stage (maintained at 37°C) and imaged every 2 min up to 16 min. The imaging kinetics curves were obtained for each mouse, and peak imaging value was selected. A pseudo color scheme (generated by the living imaging software) was used to visualize the numerical contents of the acquired bioluminescent signal (superimposed onto contrast, gray-scale photographic pictures to determine the bioluminescence signal location). For whole body bioluminescence quantification, regions of interest (ROIs) were quantified as average radiance (photons / [s cm2 sr]), and the imaging signal graphic output was listed as total flux (photons s— 1 ) over ROI. Subsequently, animals were sacrificed, and organs such as liver, spleen, and brain were harvested and imaged using IVIS.

[0300] Mice dosed with LNPs encapsulating GFP mRNA were sacrificed 24 hours following the injection, and liver and brain were harvested, processed to FFPE sections, and used for anti-GFP in situ hybridization (Advanced Cell Diagnostics, 409018) and anti-CD31 immunohistochemistry (Abeam, ab 182981).Example 2 - Biophysical Properties of LNP Nanoparticles

[0301] Table 3 denotes the biophysical properties of the formulated LNPs utilized in this study. All particles had a hydrodynamic diameter <100 nm with poly dispersity index <0.2, and an encapsulation efficiency >80%. The provided range for each biophysical property is due to the inherent variability observed in different batches of formulated LNPs, and different grafting densities of antibodies used to formulate targeted LNPs. Zeta potential and encapsulation efficiency were only measured for the LNPs without any incorporated targeting ligands on the surface.Table 3Example 3 - In Vitro Screening in Human and Murine Brain Endothelial Cell Lines

[0302] Anti-TfR Fabs were incorporated into LNP surfaces and utilized for in vitro screening. The goal of in vitro testing was to assess whether utilization of anti-TfR Fab in LNP structure could facilitate the LNP delivery to brain endothelial cells via receptor-mediated internalization. FIGs. 1-2 demonstrate that the mean GFP expression in both murine Bend3 and human CMEC cell lines transfected with LNPs was dependent on the grafting density of Fab and dose of GFP mRNA. In FIG. 1, LNPs with or without incorporated anti-TfR Fabs (8D3 or H7) were screened in the murine Bend3 cell line (FIG. 1 A, B). Murinespecific 8D3 Fab showed an increase in both the number of GFP+ cells (FIG. 1 A) and the mean GFP intensity per positive cell (FIG. IB) when compared to the negative control, at Fab grafting densities of 2, 9, or 15 g / mol and doses of 0.02 mg or 0.1 mg of RNA per well of a 96-well plate. Higher levels of transfection and expression were observed at higher mRNA doses, while grafting densities of 9 g / mol or 15 g / mol led to higher expression than 2 g / mol. Due to specificity of Fab H7 to human transferrin receptor, targeted LNPs with Fab H7 failed to improve cell transfection in murine Bend3 cell line, except for a baseline GFP expression observed due to the non-specific internalization of LNPs in murine brain endothelial cells (FIG. 1A, B).

[0303] Similarly, LNPs with incorporated anti-TfR Fabs were utilized to transfect hCMEC at two different mRNA doses (0.01 mg or 0.05 mg per well of 96-well plate) and multiple grafting densities (2, 5, 9, 15, or 25 g / mol). FIG. 2 shows that H7 Fab enhanced the number of GFP positive cells (FIG. 2A) and the intensity of GFP signal (FIG. 2B) in hCMEC relative to negative control. The intensity of GFP signal was increased with both higher mRNA dose and higher Fab grafting density. The murine specific 8D3 Fab did not significantly enhance the mean GFP expression when compared to the negative control (FIG. 2 A, B).Example 4 - In Vivo Screening in Human and Murine Brain Endothelial Cell Lines

[0304] In-life and ex vivo images obtained from IVIS showed strong luciferase enzyme activity in the liver of the mouse dosed with control LNPs (without anti-TfR targeting ligand) (FIG. 3). Addition of anti-TfR Fab 8D3 into the LNP surface at a grafting density of 9 g / mol resulted in increased Luciferase signal in brain relative to unconjugated LNP (FIG. 3). To confirm the cell type that LNPs target in vivo, and due to uncertainty concerning the cell typeexpressing luciferase activity, a separate group of animals were dosed with LNPs encapsulating GFP mRNA. After 24 hours following the IV infusion, brain and liver of animals were harvested, sectioned, and stained for GFP mRNA and CD31 protein, with CD31 serving as a cell-type marker for endothelial cells. FIG. 4 demonstrates GFP+ / CD31+ cells in the brain of animal dosed with LNP-8D3 Fab (FIG. 4C), while GFP signal was not detected in CD31+ cells of LNP-GFP treated animals (FIG. 4B). GFP accumulated specifically in CD31-positive cells but not in CD31-negative cells (FIG. 4C). All three regions of brain, such as hindbrain, midbrain, and cortex showed similar results (results not shown). In addition, ELISA of hemibrains from 8D3-LNP dosed animals showed a significant increase in GFP protein relative to LNP-GFP or buffer treated mice (FIG. 4D).

[0305] Therefore, LNPs with two anti-TfR Fabs when incorporated into the LNP structure to enhance the LNP delivery to brain endothelium, showed enhanced GFP and luciferase mRNA delivery in vitro to murine Bend3 and hCMEC cells when species-specific targeting ligands were utilized.

[0306] In vivo results in SKH1 murine model confirmed the in vitro data, with murinespecific anti-TfR Fab targeting the LNPs to murine brain endothelial cells. Luciferase and GFP expression in brain were confirmed using IVIS and immunohistochemistry, respectively, with the latter showing stained (GFP positive) endothelium in different regions of the brain, such as hindbrain, midbrain, and cortex with no GFP staining in non-endothelial cells. These results demonstrated that the Fab-conjugated LNPs specifically targeted brain endothelial cells. These results further demonstrated that the Fab-conjugated LNPs described herein did not pass the blood-brain barrier, that is the Fab-conjugated LNPs did not transmigrate the brain endothelial cells to deliver GFP to non-endothelial cells in the brain.ADDITIONAL EMBODIMENTS1. A lipid nanoparticle (LNP) comprising:(a) a lipid-cell targeting group conjugate comprising the compound of Formula (I):[Lipid] - [optional linker] - [antibody],(b) an ionizable cationic lipid, and(c) a cargo, wherein the cargo is encapsulated in the LNP;wherein the antibody is an antibody that specifically binds to a transferrin receptor.2. The LNP of embodiment 1, wherein the antibody that specifically binds to a transferrin receptor is covalently coupled to the Lipid in Formula (I) via a linker comprising polyethylene glycol (PEG).3. The LNP of embodiment 1 or 2, wherein the Lipid in Formula (I) covalently coupled to the antibody is distearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), or ceramide.4. The LNP of embodiment 3, wherein the Lipid in Formula (I) covalently coupled to the antibody is DSPE.5. The LNP of any one of embodiments 2 to 4, wherein the PEG has a molecular weight of about 1.0 kDa to about 5.0 kDa.6. The LNP of embodiment 5, wherein the PEG has a molecular weight of about 3 kDa.7. The LNP of any one of embodiments 1 to 6, wherein the antibody is an immunoglobulin single variable domain (ISVD), a Fab, or an scFv.8. The LNP of embodiment 7, wherein the antibody is a Fab.9. The LNP of any one of embodiments 1 to 8, wherein the Fab is a human transferrin receptor binding Fab.10. The LNP of any one of embodiments 1 to 9, wherein the antibody comprises a heavy chain variable region that comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) of a heavy chain comprising the amino acid sequence of SEQ ID NO: 1, and a light chain comprising a light chain variable region that comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) of a light chain comprising the amino acid sequence of SEQ ID NO: 2.11. The LNP of any one of embodiments 1 to 9, wherein the antibody comprises a CDR-H1 comprising the amino acid sequence of RFTFSSYAMH (SEQ ID NO: 5); a CDR-H2 comprising the amino acid sequence of VISYDGSNKYYADSVKG (SEQ ID NO: 6); a CDR-H3 comprising the amino acid sequence of DLSGYGDYPDY (SEQ ID NO: 7); a CDR-L1 comprising the amino acid sequence of GFTFSNYGMH (SEQ ID NO: 8); a CDR-L2 comprising the amino acid sequence of MIYYDSSKMNYADTVKG (SEQ ID NO: 9); and a CDR-L3 comprising the amino acid sequence of AGWDDSLTGPV (SEQ ID NO: 10).12. The LNP of embodiment 10 or 11, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2.13. The LNP of any one of embodiments 1 to 9, wherein the antibody comprises a heavy chain variable region that comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) of a heavy chain comprising the amino acid sequence of SEQ ID NO: 3, and a light chain comprising a light chain variable region that comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) of a light chain comprising the amino acid sequence of SEQ ID NO: 4.14. The LNP of any one of embodiments 1 to 8, wherein the antibody comprises a CDR-H1 comprising the amino acid sequence of GFTFSNYGMH (SEQ ID NO: 11); a CDR-H2 comprising the amino acid sequence of MIYYDSSKMNYADTVKG (SEQ ID NO: 12); a CDR-H3 comprising the amino acid sequence of PTSHYVVDV (SEQ ID NO: 13); a CDR-L1 comprising the amino acid sequence of QASQDIGNWLA (SEQ ID NO: 14); a CDR-L2 comprising the amino acid sequence of GATSLAD (SEQ ID NO: 15); and a CDR-L3 comprising the amino acid sequence of LQAYNTPWT (SEQ ID NO: 16).15. The LNP of embodiment 13 or 14, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.16. The LNP of any one of embodiments 1 to 15, wherein the antibody grafting density is about 2 g / mol to about 25 g / mol.17. The LNP of any one of embodiments 1 to 16, wherein the LNP further comprises a structural lipid, a neutral phospholipid, or a free PEG-lipid, or any combination thereof.18. The LNP of embodiment 17, wherein the structural lipid is a sterol.19. The LNP of embodiment 18, wherein the sterol is selected from the group consisting of cholesterol, fecosterol, P-sitosterol, ergosterol, campesterol, stigmasterol, stigmastanol, and brassicasterol.20. The LNP of any one of embodiments 17 to 19, wherein the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3 -phosphocholine (DOPC), and sphingomyelin.21. The LNP of embodiment 20, wherein the neutral phospholipid is DSPC or DOPE. 22. The LNP of any one of embodiments 17 to 21, wherein the free PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.23. The LNP of any one of embodiments 17 to 22, wherein the free PEG-lipid is PEG-dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide, diacylphosphatidylethanolamine comprising dipalmitoyl (Cl 6) chain or distearoyl (Cl 8) chain, or a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.24. The LNP of embodiment 23, wherein the PEG-DAG comprises PEG-DMG, PEG-DPG, or PEG-DSG, or any combination thereof.25. The LNP of embodiment 24, wherein the free PEG-lipid comprises PEG-DPG.26. The LNP of embodiment 25, wherein the PEG-DPG comprises or is PEG 2000-DPG (DPG-PEG 2000).27. The LNP of any one of embodiments 1 to 26, wherein the cargo comprises a nucleic acid molecule.28. The LNP of embodiment 27, wherein the nucleic acid molecule is selected from the group consisting of ssDNA, dsDNA, cDNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, CRISPR-Cas9, antagomir, antisense molecule, targeted nucleic acid, and any combination thereof.29. The LNP of embodiment 28, wherein the nucleic acid molecule comprises or is RNA.30. The LNP of embodiment 29, wherein the RNA comprises or is mRNA.31. The LNP of embodiment 30, wherein the mRNA encodes a therapeutic polypeptide.32. The LNP of any one of embodiments 1 to 31, wherein cargo comprises a therapeutic polypeptide.33. The LNP of embodiment 31 or embodiment 32, wherein the therapeutic polypeptide is for treating familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, a rare disease or an orphan disease.34. The LNP of embodiment 33, wherein the rare disease is selected from the group consisting of spinal muscular atrophy, Huntington’s Disease, Rett Syndrome, Amyotrophic Lateral Sclerosis (ALS), Duchenne Type Muscular dystrophy, Friedreich’s Ataxia, progranulin (PRGN), non-Alzheimer’s cerebral degenerations, frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA) and semantic dementia.35. The LNP of embodiment 30 or 31, wherein the mRNA comprises a 5’ Cap, a 5’ untranslated region (UTR), a sequence encoding a polypeptide, a 3’ UTR, and optionally a poly A tail.36. The LNP of any one of embodiments 1 to 35, wherein the LNP selectively binds to at least one target cell of interest.37. The LNP of embodiment 36, wherein the target cell of interest is a central nervous system endothelial cell.38. The LNP of any one of embodiments 27 to 37, wherein the nucleic acid molecule comprises pseudouridine.39. The LNP of embodiment 38, wherein the pseudouridine is Nl-methyl-pseudouridine.40. The LNP of any one of embodiments 1 to 39, wherein the ionizable cationic lipid comprises a compound of Formula (II):wherein:R1, R2, and R3are each independently a bond or C1-3 alkylene;R1A, R2A, and R3Aare each independently a bond or C1-10 alkylene;R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1-20 alkenyl, -(CH2)0-10C(O)ORa1, or -(CH2)0-10OC(O)Ra2;Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl;R3B1is C1-6 alkylene; andR3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with 1 or 2 -OH.41. The LNP of embodiment 40, wherein:R1, R2, and R3are each independently a bond or methylene;R1Aand R2Aare each C1-10 alkylene;R3Ais C1-5 alkylene;R1A1, R1A2, R2A1, R2A2, R3A1, and R3A2are each H;R1A3and R2A3are each C1-20 alkenyl;R3A3is -C(0)0(Ci-2o alkyl);R3B1is C2-4 alkylene; andR3B2and R3B3are each methyl.42. The LNP of embodiment 40 or 41, wherein R3B1is -(CH2)3-.43. The LNP of any one of embodiments 1 to 42, wherein the ionizable cationic lipid is44. The LNP of any one of embodiments 1 to 42, wherein the ionizable cationic lipid is selected from N, N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-l-yl-l,3-dioxolane-4-ethanamine (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), N, N-dimethyl-2,3-bis[(9Z,12Z)-9,12-octadecadien- 1 -yloxy]- 1 -propanamine (DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3] -di oxolane (DLin-K-DMA), l,l'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]bis-2-dodecanol (C12-200) Dioleoyl-3 -trimethylammonium propane (DOTAP), and Dioleoyl-3-trimethylammonium propane (DOTMA).45. The LNP of any one of embodiments 1 to 44, wherein the ionizable lipid is present in the lipid nanoparticle in a range of about 40 mol% to about 55 mol%.46. The LNP of any one of embodiments 17 to 45, wherein the neutral phospholipid is present in the lipid nanoparticle in a range of about 5 mol% to about 25 mol%.47. The LNP of any one of embodiments 17 to 46, wherein the structural lipid is present in the lipid nanoparticle in a range of about 25 mol% to about 45 mol%.48. The LNP of any one of embodiments 17 to 47, wherein the PEG-lipid is present in the lipid nanoparticle in a range of about 1 mol% to about 4 mol%.49. The LNP of any one of embodiments 17-48, wherein the ionizable lipid is present in the lipid nanoparticle at about 45% to about 50 mol%, the neutral phospholipid is present in the lipid nanoparticle at about 8 to about 10 mol%; the structural lipid is present in the lipid nanoparticle at about 35 to about 40 mol%, and the PEG-lipid is present in the lipid nanoparticle at about 1 to about 2 mol%.50. The LNP of any one of embodiments 17 to 49, wherein the ionizable lipid is present in the lipid nanoparticle at about 45% to about 50 mol%, the neutral phospholipid is present in the lipid nanoparticle at about 15 to about 25 mol%; the structural lipid is present in the lipid nanoparticle at about 25 to about 35 mol%, and the PEG-lipid is present in the lipid nanoparticle at about 1 to about 2 mol%.51. The LNP of any one of embodiments 1 to 50, wherein the antibody is coupled to a DSPE-PEG3400.52. The LNP of any one of embodiments 1 to 51, wherein the lipid nanoparticle has a zeta potential (ZP) of about 2 mV to about 10 mV.53. The LNP of any one of embodiments 1 to 52, wherein the cargo is presented in the LNP at a concentration of about 0.01 to 0.1 g / g LNP.54. A composition comprising the LNP of any one of embodiments 1 to 53 and one or more carrier or excipient.55. The composition of embodiment 54, wherein the composition is a pharmaceutical composition.56. The composition of embodiment 54 or embodiment 55, wherein the carrier or excipient is a pharmaceutically acceptable carrier or excipient.57. A method of delivering cargo to a central nervous system (CNS) endothelial cell, comprising delivering the LNP of any one of embodiments 1 to 53 to the CNS endothelial cell.58. The method of embodiment 57, wherein the method leads to reduced off-target delivery into CNS parenchyma.59. Amethod of modulating gene expression in a central nervous system (CNS) endothelial cell, comprising contacting the cell with the LNP of any one of embodiments 1 to 53.60. A method of treating a central nervous system (CNS) dysfunction in a subject in need thereof, comprising contacting a CNS cell of the subject with the LNP of any one of embodiments 1 to 53.61. The method of embodiment 60, wherein the dysfunction is selected from the group consisting of Alzheimer’s disease, vascular dementia, traumatic brain injury, stroke, Parkinson’s disease, neuroinflammation, advanced age, or cerebral amyloid angiopathy.62. A method of correcting a deficit in a central nervous system (CNS) endothelial cell function in a subject due to a genetic cause, the method comprising administering to the subject the LNP of any one of embodiments 1 to 53 or the composition of any one of embodiments 55-57.63. The method of embodiment 62, wherein the genetic cause of the deficit in a CNS endothelial cell function is GLUT1 deficiency syndrome (De Vivo syndrome), band-like calcification with simplified gyration and polymicrogyria (BLC-PMG), familial cerebral cavernous malformations, cerebral small vessel disease, microcephaly, or Allan-Hemdon-Dudley syndrome.64. A lipid nanoparticle of any one of embodiments 1 to 53 or a composition of any one of embodiments 54 to 56 for use in delivering a cargo to a central nervous system (CNS) endothelial cell.65. A lipid nanoparticle of any one of embodiments 1 to 53 or a composition of any one of embodiments 54 to 56 for use in targeting a cargo to a central nervous system (CNS) endothelial cell and reducing off-target delivery into CNS parenchyma.66. A lipid nanoparticle of any one of embodiments 1 to 53 or a composition of any one of embodiments 54 to 56 for use in treating a central nervous system (CNS) endothelial cell dysfunction in a subject in need thereof.67. A lipid nanoparticle of any one of embodiments 1 to 53 or a composition of any one of embodiments 54 to 56 for use in a method of repairing a central nervous system (CNS) endothelial cell barrier dysfunction in a subject in need thereof.68. A lipid nanoparticle of any one of embodiments 1 to 53 or a composition of any one of embodiments 54 to 56 for use in a method of correcting a deficit in a central nervous system (CNS) endothelial cell function in a subject having a genetic cause of a deficit in a CNS endothelial cell function.

Claims

CLAIMSWhat is claimed is:

1. A lipid nanoparticle (LNP) comprising:(a) a lipid-cell targeting group conjugate comprising the compound of Formula (I):[Lipid] - [optional linker] - [antibody],(b) an ionizable cationic lipid, and(c) a cargo, wherein the cargo is encapsulated in the LNP;wherein the antibody is an antibody that specifically binds to a transferrin receptor.

2. The LNP of claim 1, wherein the antibody that specifically binds to a transferrin receptor is covalently coupled to the Lipid in Formula (I) via a linker comprising polyethylene glycol (PEG).

3. The LNP of claim 1 or 2, wherein the Lipid in Formula (I) covalently coupled to the antibody is di stearoylglycerol (DSG), distearoyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-glycero-phosphoglycerol (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoylglycerol (DPG), or ceramide.

4. The LNP of claim 2 or claim 3, wherein the PEG has a molecular weight of about 1.0 kDa to about 5.0 kDa.

5. The LNP of any one of claims 1 to 4, wherein the antibody is an immunoglobulin single variable domain (ISVD), a Fab, or an scFv.

6. The LNP of claim 5, wherein the antibody is a Fab.

7. The LNP of any one of claims 1 to 6, wherein the antibody is a human transferrin receptor binding Fab.

8. The LNP of any one of claims 1 to 7, wherein the antibody comprises a CDR-H1 comprising the amino acid sequence of RFTFSSYAMH (SEQ ID NO: 5); a CDR-H2 comprising the amino acid sequence of VISYDGSNKYYADSVKG (SEQ ID NO: 6); a CDR-H3 comprising the amino acid sequence of DLSGYGDYPDY (SEQ ID NO: 7); a CDR-L1 comprising the amino acid sequence of GFTFSNYGMH (SEQ ID NO: 8); a CDR-L2 comprising the amino acid sequence of MIYYDSSKMNYADTVKG (SEQ ID NO: 9); and a CDR-L3 comprising the amino acid sequence of AGWDDSLTGPV (SEQ ID NO: 10).

9. The LNP of claim 8, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

10. The LNP of any one of claims 1 to 7, wherein the antibody comprises a CDR-H1 comprising the amino acid sequence of GFTFSNYGMH (SEQ ID NO: 11); a CDR-H2 comprising the amino acid sequence of MIYYDSSKMNYADTVKG (SEQ ID NO: 12); a CDR-H3 comprising the amino acid sequence of PTSHYVVDV (SEQ ID NO: 13); a CDR-L1 comprising the amino acid sequence of QASQDIGNWLA (SEQ ID NO: 14); a CDR-L2 comprising the amino acid sequence of GATSLAD (SEQ ID NO: 15); and a CDR-L3 comprising the amino acid sequence of LQAYNTPWT (SEQ ID NO: 16).

11. The LNP of claim 10, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

12. The LNP of any one of claims 1 to 11, wherein the LNP further comprises a structural lipid, a neutral phospholipid, or a free PEG-lipid, or any combination thereof.

13. The LNP of claim 12, wherein the structural lipid is a sterol.

14. The LNP of claim 12 or claim 13, wherein the neutral phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and sphingomyelin.

15. The LNP of any one of claims 12 to 14, wherein the free PEG-lipid is selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.

16. The LNP of any one of claims 12 to 15, wherein the free PEG-lipid is PEG-dioleoylgylcerol (PEG-DOG), PEG-dimyristoyl-glycerol (PEG-DMG), PEG-dipalmitoyl-glycerol (PEG-DPG), PEG-dilinoleoyl-glycero-phosphatidyl ethanolamine (PEG-DLPE), PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), PEG-dipalmitoyl-phosphatidylethanolamine (PEG-DPPE), PEG-di stearoylglycerol (PEG-DSG), PEG-diacylglycerol (PEG-DAG), PEG-ceramide, PEG-distearoyl-glycero-phosphoglycerol (PEG-DSPG), PEG-dioleoyl-glycero-phosphoethanolamine (PEG-DOPE), 2-[(polyethylene glycol)-2000] -N, N -di tetradecyl acetamide, diacylphosphatidylethanolamine comprising dipalmitoyl (Cl 6) chain or distearoyl (Cl 8) chain, or a PEG-distearoyl-phosphatidylethanolamine (PEG-DSPE) lipid.

17. The LNP of any one of claims 1 to 16, wherein the cargo comprises a nucleic acid molecule.

18. The LNP of claim 17, wherein the nucleic acid molecule is selected from the group consisting of ssDNA, dsDNA, cDNA, mRNA, miRNA, siRNA, sgRNA, modified RNA, CRISPR-Cas9, antagomir, antisense molecule, targeted nucleic acid, and any combination thereof.

19. The LNP of claim 18, wherein the mRNA encodes a therapeutic polypeptide.

20. The LNP of any one of claims 1 to 19, wherein the cargo comprises a therapeutic polypeptide.

21. The LNP of claim 19 or claim 20, wherein the therapeutic polypeptide is for treating familial hypercholesterolemia, muscular dystrophy, cystic fibrosis, a rare disease or an orphan disease.

22. The LNP of claim 21, wherein the rare disease is selected from the group consisting of spinal muscular atrophy, Huntington’s Disease, Rett Syndrome, Amyotrophic Lateral Sclerosis (ALS), Duchenne Type Muscular dystrophy, Friedreich’s Ataxia, progranulin (PRGN), nonAlzheimer’s cerebral degenerations, frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA) and semantic dementia.

23. The LNP of any one of claims 1 to 22, wherein the LNP selectively binds to at least one target cell of interest.

24. The LNP of claim 23, wherein the target cell of interest is a central nervous system endothelial cell.

25. The LNP of any one of claims 1 to 24, wherein the ionizable cationic lipid comprises a compound of Formula (II):wherein:R1, R2, and R3are each independently a bond or C1-3 alkylene;R1A, R2A, and R3Aare each independently a bond or C1-10 alkylene;R1A1, R1A2, R1A3, R2A1, R2A2, R2A3, R3A1, R3A2, and R3A3are each independently H, C1-20 alkyl, C1-20 alkenyl, -(CH2)0-10C(O)ORa1, or -(CH2)0-10OC(O)Ra2;Raland Ra2are each independently C1-20 alkyl or C1-20 alkenyl;R3B1is C1-6 alkylene; andR3B2and R3B3are each independently H, unsubstituted C1-6 alkyl, or C1-6 alkyl substituted with 1 or 2 -OH.

26. The LNP of any one of claims 1 to 25, wherein the ionizable cationic lipid is27. The LNP of any one of claims 1 to 26, wherein the ionizable cationic lipid is selected from N, N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-l-yl-l,3-dioxolane-4-ethanamine (DLin-KC2-DMA), (6Z,9Z,28Z,3 lZ)-heptatriacont-6,9,28,31 -tetraene- 19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), N, N-dimethyl-2,3-bis[(9Z,12Z)-9,12-octadecadien- 1 -yloxy]- 1 -propanamine (DLin-DMA), 2, 2-Dilinoleyl-4-dimethylaminom ethylic 1,3] -di oxolane (DLin-K-DMA), l,l'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]bis-2-dodecanol (C12-200) Dioleoyl-3 -trimethylammonium propane (DOTAP), and Dioleoyl-3-trimethylammonium propane (DOTMA).

28. The LNP of any one of claims 12 to 27, wherein:(a) the ionizable lipid is present in the lipid nanoparticle in a range of about 40 mol% to about 55 mol%;(b) the neutral phospholipid is present in the lipid nanoparticle in a range of about 5 mol% to about 25 mol%;(c) the structural lipid is present in the lipid nanoparticle in a range of about 25 mol% to about 45 mol%; and / or(d) the PEG-lipid is present in the lipid nanoparticle in a range of about 1 mol% to about 4 mol%.

29. The LNP of any one of claims 1 to 28, wherein the cargo is presented in the LNP at a concentration of about 0.01 to 0.1 g / g LNP.

30. A composition comprising the LNP of any one of claims 1 to 29 and one or more carrier or excipient.

31. A method of delivering cargo to a central nervous system (CNS) endothelial cell, comprising delivering the LNP of any one of claims 1 to 29 to the CNS endothelial cell.

32. Amethod of modulating gene expression in a central nervous system (CNS) endothelial cell, comprising contacting the cell with the LNP of any one of claims 1 to 29.

33. A method of treating a central nervous system (CNS) dysfunction in a subject in need thereof, comprising contacting a CNS cell of the subject with the LNP of any one of claims 1 to 29.

34. The method of claim 33, wherein the dysfunction is selected from the group consisting of Alzheimer’s disease, vascular dementia, traumatic brain injury, stroke, Parkinson’s disease, neuroinflammation, advanced age, or cerebral amyloid angiopathy.

35. A method of correcting a deficit in a central nervous system (CNS) endothelial cell function in a subject due to a genetic cause, the method comprising administering to the subject the LNP of any one of claims 1 to 29 or the composition of claim 30.

36. The method of claim 35, wherein the genetic cause of the deficit in a CNS endothelial cell function is GLUT1 deficiency syndrome (De Vivo syndrome), band-like calcification with simplified gyration and polymicrogyria (BLC-PMG), familial cerebral cavernous malformations, cerebral small vessel disease, microcephaly, or Allan-Hemdon-Dudley syndrome.

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