Lipid nanoparticles

Abstract

Lipid nanoparticles, compositions, and methods for their preparation and use are provided. The lipid nanoparticles contain ionizable lipids, structural lipids, PEG-coated lipids, and specific amounts of accessory lipids. The lipid nanoparticles may further contain cell-targeting groups coupled to the PEG-coated lipids. The lipid nanoparticles can carry loads, such as mRNA. The lipid nanoparticles can be used for transfection of cells, such as immune cells or hematopoietic stem cells.

Description

Technical Field

[0001] This disclosure provides lipid nanoparticles for delivering nucleic acids to hematopoietic stem cells and immune cells, as well as methods for their preparation and use. Background Technology

[0002] There is a need for safe and effective in vivo methods to target the expression of immune cell-enhancing peptides in immune cells and to perform targeted gene editing in hematopoietic stem cells (HSCs). Summary of the Invention

[0003] Lipid nanoparticles for targeted delivery of nucleic acids to target cells are provided, the lipid nanoparticles comprising ionizable lipids, structural lipids, auxiliary lipids, PEG lipids and cell-targeting groups, wherein the auxiliary lipids are present in the range of about 16 mol% to about 40 mol%.

[0004] In some respects, ionizable lipids include the structure of formula I. (I), or its salt, wherein: R 1 R 2 and R 3 Each is an independent key or C 1-3 Alkylene; R 1A R 2A and R 3A Each is an independent key or C 1-10 Alkylene; R 1A1 R 1A2 R 1A3 R 2A1 R 2A2 R 2A3 R 3A1 R 3A2 and R 3A3 H and C are independent of each other. 1-20 Alkyl, C 1-20 Alkenyl, -(CH2) 0-10 C(O)OR a1 Or -(CH2) 0-10 OC(O)R a2 ; R a1 and R a2 Each is C independently 1-20 Alkyl or C 1-20 alkenyl; R 3B yes R 3B1 It is C 1-6Alkylene; and R 3B2 and R 3B3 Each is independently H or C 1-6 alkyl.

[0005] In some respects, ionizable lipids include the structure of formula II (lipid 15). (II).

[0006] In some respects, ionizable lipids include the structure of formula III (KC2). (III).

[0007] In some respects, the structure of ionizable lipid-containing IV (KC3) (IV).

[0008] In some respects, ionizable lipids contain the structure of formula V: (V) Where R 1 and R 2 Each is a lipid independently.

[0009] In some respects, the structure of ionizable lipid-containing VI is as follows: (VI) Where R 1 and R 2 Each is a lipid independently.

[0010] In some embodiments of ionizable lipids comprising structures of formula V or VI, R 1 and R 2 Each is independently a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkenyl group. In some embodiments of ionizable lipids comprising the structure of formula V or VI, R 1 With R 2 They are the same lipids. In some embodiments of ionizable lipids comprising structures of formula V or formula VI, R 1 With R 2 These are different lipids. In some embodiments, lipids are hydrocarbons (e.g., substituted or unsubstituted, saturated or unsaturated, branched or unbranched hydrocarbons). The hydrocarbon can be an alkane, olefin, or alkyne. In some embodiments, the hydrocarbon chain is saturated or unsaturated. In some embodiments, the unsaturated hydrocarbon chain contains at least one, at least two, at least three, at least four, at least five, or at least six carbon-carbon double bonds (e.g., cis double bonds and / or trans double bonds). In some embodiments, lipids are substituted or unsubstituted C... 7-36Alkyl group. In some embodiments, the lipid is a substituted or unsubstituted C14 group. 7-36 Alkenyl. In some embodiments, the lipid is an unsubstituted C10 group. 7-36 Alkyl or unsubstituted C 7-36 Alkenyl. In some embodiments, the hydrocarbon is substituted with alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, carbonyl, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkyl thioether, thiol, urea, or combinations thereof. Each of these groups may subsequently be substituted. In some embodiments, the hydrocarbon is substituted with one, two, three, four, five, six, seven, eight, nine, ten, or more than ten substituents.

[0011] In some respects, lipid nanoparticles further contain loadings.

[0012] In some respects, the auxiliary lipids are selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and sphingomyelin.

[0013] In some respects, the assisting lipid is DSPC.

[0014] In some respects, structural lipids are selected from the group consisting of: cholesterol, coccosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, stigmasterol, and brassosterol.

[0015] In some respects, PEG lipids are selected from the group consisting of: distearylglycerol-PEG (DSG-PEG), distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), dimyristoyl-phosphatidylethanolamine-PEG (DMPE-PEG), distearyl-glycerol-phosphate-PEG (DSPG-PEG), dimyristoyl-glycerol-PEG (DMG-PEG), dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG), dipalmitoyl-glycerol-PEG (DPG-PEG), and ceramide-PEG.

[0016] In some respects, ionizable lipids exist in the range of about 30 mol% to about 70 mol%.

[0017] In some respects, ionizable lipids exist in the range of about 40 mol% to about 60 mol%.

[0018] In some respects, ionizable lipids exist in the range of about 48 mol% to about 50 mol%.

[0019] In some respects, structural lipids are present in the range of about 20 mol% to about 50 mol%.

[0020] In some respects, structural lipids are present in the range of about 30 mol% to about 40 mol%.

[0021] In some respects, structural lipids are cholesterol.

[0022] In some respects, cholesterol is present in the range of approximately 25 mol% to approximately 40 mol%.

[0023] In some respects, PEG lipids are present in the range of about 1 mol% to about 4 mol%.

[0024] In some respects, PEG lipids are DPG-PEG or DSPE-PEG.

[0025] In some respects, the PEG in DPG-PEG has a molecular weight of about 2000 Daltons (DPG-PEG2K). In other respects, the PEG in DSPE-PEG has a molecular weight of about 2000 Daltons (DSPE-PEG2K) or about 3400 Daltons (DSPE-PEG3.4K).

[0026] In some respects, DPG-PEG2K, DSPE-PEG2K, or DSPE-PEG3.4K are present in the range of about 1 mol% to about 2 mol%.

[0027] In some respects, lipid 15 was present at approximately 49.25 mol%, cholesterol at approximately 29.25 mol%, DSPC at approximately 20 mol%, and DPG-PEG2K at approximately 1.5 mol%.

[0028] In some respects, lipid nanoparticles have an average diameter of about 60 nm to about 100 nm.

[0029] In some respects, lipid nanoparticles have an average diameter of approximately 80 nm.

[0030] In some respects, lipid nanoparticles have a polydispersity index (PDI) of about 0.05 to about 1.

[0031] In some respects, lipid nanoparticles have a PDI of approximately 0.09.

[0032] In some respects, lipid nanoparticles have a zeta potential (ZP) of about -30 mV to about +5 mV.

[0033] In some respects, lipid nanoparticles have a ZP of approximately -1.4 mV.

[0034] In some respects, the payload contains one or more nucleic acids.

[0035] In some respects, one or more nucleic acids are DNA.

[0036] In some respects, one or more of these nucleic acids are RNA.

[0037] In some respects, RNA contains guide RNA and RNA encoding the Cas enzyme.

[0038] In some respects, one or more nucleic acids encode chimeric antigen receptors (CARs).

[0039] In some respects, cell-targeting groups are coupled with lipids in lipid nanoparticles to form lipid-cell-targeting group conjugates.

[0040] In some respects, cell-targeting groups are coupled with PEG-lipids.

[0041] In some respects, cell-targeting groups are coupled with DPG-PEG2K, DSPE-PEG2K, or DSPE-PEG3.4K.

[0042] In some respects, cell-targeting groups contain antibodies or fragments thereof.

[0043] In some respects, cell-targeting groups include antibodies.

[0044] In some respects, cell-targeting groups contain Fab fragments.

[0045] In some respects, cell-targeting groups contain a single variable domain.

[0046] In some respects, cell-targeting groups bind to molecules on immune cells.

[0047] In some respects, the molecules on immune cells are selected from the group consisting of CD3, CD4, CD7, and CD8.

[0048] In some respects, cell-targeting groups bind to molecules on hematopoietic stem cells.

[0049] In some respects, the molecules on hematopoietic stem cells are selected from the group consisting of CD34, CD105, and CD117.

[0050] In some respects, lipid-cell-targeting conjugates are present in lipid nanoparticles in the range of about 0.001 mol% to about 0.5 mol%.

[0051] In some aspects, a composition is provided comprising the lipid nanoparticles described herein, and one or more carriers or excipients.

[0052] In some respects, the composition is a pharmaceutical composition.

[0053] In some respects, the carrier or excipient is a pharmaceutically acceptable carrier or excipient.

[0054] In some aspects, a method for delivering nucleic acids into cells within a subject is provided, the method comprising administering to the subject the lipid nanoparticles described herein or the compositions described herein.

[0055] In some aspects, a method for delivering nucleic acids into immune cells in a subject is provided, the method comprising administering to the subject the lipid nanoparticles described herein or the compositions described herein.

[0056] In some aspects, a method for delivering nucleic acids into hematopoietic stem cells in a subject is provided, the method comprising administering to the subject the lipid nanoparticles described herein or the compositions described herein.

[0057] In some respects, polydispersity index compositions are pharmaceutical compositions.

[0058] In some respects, the use of the lipid nanoparticles or compositions described herein in the preparation of pharmaceutical agents for delivering nucleic acids to target cells is provided.

[0059] In some respects, the use of the lipid nanoparticles or compositions described herein in the preparation of pharmaceutical agents for delivering nucleic acids to immune cells is provided.

[0060] In some respects, the use of the lipid nanoparticles or compositions described herein in the preparation of pharmaceutical agents for delivering nucleic acids to hematopoietic stem cells is provided.

[0061] In some respects, the compositions used are pharmaceutical compositions.

[0062] In some aspects, lipid nanoparticles or compositions described herein are provided for use in delivering nucleic acids to target cells.

[0063] In some aspects, lipid nanoparticles or compositions described herein are provided for use in delivering nucleic acids to immune cells.

[0064] In some aspects, lipid nanoparticles or compositions described herein are provided for use in delivering nucleic acids to hematopoietic stem cells.

[0065] In some respects, compositions used in the delivery of nucleic acids to target cells are pharmaceutical compositions.

[0066] In some aspects, a method for treating a disease in a subject is provided, the method comprising administering, to the cells of a subject in need, the lipid nanoparticles described herein or the compositions described herein, or the lipid nanoparticles or compositions used for delivering nucleic acids to the cells of a subject.

[0067] In some aspects, a method for treating a disease in a subject is provided, the method comprising administering the lipid nanoparticles described herein or the compositions described herein to the immune cells of the subject in need.

[0068] In some respects, this application targets multiple immune cells.

[0069] In some respects, proteins encoded by one or more nucleic acids configured in the lipid nanoparticles were detected in at least 60% (or even 80%) of immune cells 72 hours after the application of the lipid nanoparticles.

[0070] In some respects, compared with another subject who received lipid nanoparticles containing 10 mol% of assisting lipids, the protein was detected in more immune cells of the subject 72 hours after administration of the lipid nanoparticles.

[0071] In some respects, the effector functions of one or more immune cells are enhanced by proteins encoded by the one or more nucleic acids configured in the lipid nanoparticles compared to one or more immune cells that have not been treated with the lipid nanoparticles.

[0072] In some aspects, a method for treating a disease in a subject is provided, the method comprising administering the lipid nanoparticles described herein or the compositions described herein to hematopoietic stem cells of a subject in need.

[0073] In some aspects, lipid nanoparticles were administered to multiple hematopoietic stem cells in the subjects.

[0074] In some respects, proteins encoded by one or more nucleic acids configured in lipid nanoparticles were detected in at least 80% of the multiple hematopoietic stem cells of the subject.

[0075] In some respects, proteins were detected in approximately 20% of hematopoietic stem cells in which lipid nanoparticles containing 10 mol% of assisting lipids were applied.

[0076] In some respects, the composition applied is a pharmaceutical composition.

[0077] In some respects, lipid nanoparticles or compositions described herein are provided for use in treating diseases in subjects in need.

[0078] In some respects, lipid nanoparticles or compositions described herein are provided for use in the treatment of immune cell-related diseases in subjects of need.

[0079] In some respects, lipid nanoparticles or compositions described herein are provided for use in the treatment of hematopoietic stem cell-related diseases in subjects of need.

[0080] In some respects, hematopoietic stem cell-related diseases are sickle cell diseases.

[0081] In some respects, the composition used in treating a subject’s disease, an immune cell-related disease, or a hematopoietic stem cell-related disease is a pharmaceutical composition. Attached Figure Description

[0082] Figure 1A and Figure 1B This demonstrates the sustained and efficient transfection of T cells with lipid nanoparticles containing varying amounts of DSPC within 72 hours. Figure 1A The percentage of CAR-positive T cells transfected with lipid nanoparticles containing different amounts of DSPC is shown. Figure 1B The mean fluorescence intensity (MFI) of CARs in CD8-positive T cells transfected with lipid nanoparticles containing different amounts of DSPC is shown.

[0083] Figures 2A to 2C The killing efficiency of T cells transfected with LNP containing different amounts of DSPC is shown. Figure 2A The percentage of Nam6 target cells that died after co-culturing with T cells transfected with LNP containing different amounts of DSPC is shown. Figure 2B The diagram shows LNP transfection with different amounts of DSPC and its application. Figure 2A The percentage of CAR-positive cells in T cells during the kill assay. Figure 2C The diagram shows LNP transfection with different amounts of DSPC and its application. Figure 2A The mean fluorescence intensity (MFI) of CAR in CD8-positive T cells during the killing assay.

[0084] Figures 3A to 3C The removal of Nalm6 target cells over 16 days is shown in co-cultures with CD8+ T cells transfected with LNP containing different amounts of DSPC. Figure 3AThe figure shows the mean fluorescence of Nalm6 target cells expressing red fluorescent protein over a 16-day period in the presence of CD8+ T cells transfected with LNPs containing varying amounts of DSPC. Red arrows indicate the addition of Nalm6 target cells to the co-culture; orange arrows indicate the addition of LNPs to the co-culture. Figure 3B The total count of Nalm6 target cells in the co-culture on day 16 is shown. Figure 3C The percentage of viable CD8+ T cells in the co-culture lymphocytes at day 16 is shown.

[0085] Figures 4A to 4F The transfection efficiency of lipid nanoparticles containing different amounts of DSPC in T cells from different donors is shown. Figure 4A The percentage of CAR-positive CD8+ T cells from a donor after transfection with lipid nanoparticles containing different amounts of DSPC is shown. Figure 4B The mean fluorescence intensity (MFI) of CAR in CD8-positive T cells from the same donor after transfection with lipid nanoparticles containing different amounts of DSPC is shown. Figure 4C The percentage of CAR-positive CD8+ T cells from the second donor after transfection with lipid nanoparticles containing different amounts of DSPC is shown. Figure 4D The mean fluorescence intensity (MFI) of CAR in CD8-positive T cells from a second donor is shown after transfection with lipid nanoparticles containing different amounts of DSPC. Figure 4E The mean fluorescence intensity (MFI) of CAR-positive T cells from the first donor after transfection with lipid nanoparticles containing different amounts of DSPC is shown. Figure 4F The mean fluorescence intensity (MFI) of CAR in CAR-positive T cells from a second donor is shown after transfection with lipid nanoparticles containing different amounts of DSPC. Luc-luciferase reporter gene was used as a negative control.

[0086] Figures 5A to 5C The fluorescence intensity of CAR-positive T cells transfected with lipid nanoparticles containing different amounts of DSPC and bound to immobilized CAR targets is shown. Figure 5A The MFI results are shown 24 hours after T cells were transfected with LNP containing different amounts of DSPC. Figure 5B The results of MFI are shown 48 hours after T cells were transfected with LNP containing different amounts of DSPC. Figure 5C The MFI results are shown 72 hours after T cells were transfected with LNP containing different amounts of DSPC. Figures 5A to 5C In the middle: the MFI histogram from left to right represents: no treatment, 10% DSPC, 12.5% ​​DSPC, 15% DSPC, and 20% DSPC.

[0087] Figure 6A and Figure 6B The transfection efficiency of lipid nanoparticles containing different amounts of DSPC and coated with HSC-specific Fab in human hematopoietic stem cells (HSCs) is shown. Figure 6A The percentage of mCherry-positive HSCs in the bone marrow of human HSC-transplanted NSG™ mice treated with lipid nanoparticles encapsulated with mCherry mRNA and coated with HSC-specific Fab is shown. Figure 6B This study shows off-target signals in the liver, spleen, and lungs of human HSC-transplanted NSG™ mice treated with lipid nanoparticles encapsulated with mCherry mRNA and coated with HSC-specific Fab, as measured by ELISA, and off-target signals in ovarian tissue as measured by immunohistochemistry (IHC).

[0088] Figure 7A and Figure 7B The in vitro transfection efficiency of hematopoietic stem cells is shown. Figure 7A The in vitro transfection efficiencies (expressed as % of mCherry-positive HSCs) of LNP formulation 1 (10% DSPC) and LNP formulation 2 (20% DSPC) at different concentrations (e.g., from about 0.01 μg / mL to about 1 μg / mL) are shown. Figure 7B The fluorescence intensity (MFI) after transfection with formulations 1 and 2 containing different amounts of DSPC is shown. Detailed Implementation

[0089] This disclosure provides lipid nanoparticles and lipid nanoparticle compositions comprising ionizable lipids, structural lipids, PEGylated lipids, a specified amount of cofactor lipids, and optional loading agents for efficient delivery of the loading agents to target cells. In some aspects, the lipid nanoparticles further comprise a cell-targeting group, which is optionally coupled to the lipids of the lipid nanoparticles to form a lipid-cell-targeting group conjugate. Methods for preparing lipid nanoparticles and compositions, as well as methods for using them to deliver loading agents to specified target cells, including immune cells and hematopoietic stem cells, are also provided. definition

[0090] As used herein, the term “and / or” refers to a specific disclosure of each of two specified features or components with or without the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover each of the following: 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 stated that only substitutes are referred to, or the substitutes are mutually exclusive.

[0091] Unless otherwise understood from the context and use, the expression “at least one of” as used herein includes each and every single one of the objects listed after the expression, as well as various combinations of two or more of the listed objects.

[0092] The term "approximately" or "about" applied to one or more intended values ​​means a value similar to the reference value, and within a range of values ​​falling within 10% or less (greater or less than 10%) of the reference value in either direction, unless otherwise stated or clearly apparent from the context (except where such a number would exceed 100% of the possible value). While the term "approximately" or "about" is applied herein to a particular value, values ​​without the term "approximately" or "about" are also disclosed herein. Furthermore, although not always explicitly stated, the term "about" may be added before all numerical names.

[0093] As described herein, unless otherwise specified, any concentration range, percentage range, ratio range, or integer range includes any integer value within the listed range, and, where appropriate, its fractions (e.g., tenths and hundredths of an integer). Descriptions of intervals of values ​​should be considered as specifically disclosing all possible intermediate intervals and each value within that interval. For example, a description of an interval from 1 to 6 should be considered as specifically describing each interval it includes (e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) and each value within that interval (e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6). The validity of this definition does not depend on the range of the interval.

[0094] Unless otherwise expressly stated or understood from the context, the terms “include / includes / including,” “have / has / having,” “contain / contains / containing,” and “comprise / comprises / comprising” (including their grammatical equivalents) used herein should generally be understood as open-ended and non-restrictive, e.g., not excluding additional unlisted elements or steps.

[0095] As used herein, the term "lipid nanoparticle" or "LNP" refers to nanoparticles comprising a monolayer of one or more types of lipids. In some aspects, the lipid nanoparticles have an average 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 nanoparticles have a diameter of about 200 nm to about 50 nm. In some aspects, the nanoparticles have a diameter of about 80 nm.

[0096] As used herein, the term "total lipids" refers to the collection of ionizable lipids, structural lipids, accessory lipids, and PEG lipids present in the lipid nanoparticles described herein.

[0097] As used herein, the term "ionizable lipid" refers to a lipid that can modulate its charge according to its environment. In some respects, ionizable lipids include lipids of formula I, formula II (lipid 15), formula III (KC2), formula IV (KC3), formula (V), or formula (VI). Other ionizable lipids may be used in the lipid nanoparticles described herein.

[0098] As used herein, the terms “auxiliary lipids,” “neutral lipids,” “phospholipids,” or “neutral phospholipids” refer to nonpolar lipids in lipid nanoparticles that provide stability, blood compatibility, and enhanced loading delivery.

[0099] The terms “PEG lipid” or “PEGylated lipid” are used interchangeably herein and refer to one or more lipids modified with polyethylene glycol. As described herein, PEG lipids can be free PEG lipids or PEG lipids as part of a lipid-cell targeting conjugate.

[0100] As used herein, the term "lipid-cell-targeting group conjugate" refers to a conjugate having the following 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 molecule binding group described herein. Cell-targeting groups include antibodies or fragments thereof (including Fab and scFv) and immunoglobulin single variable domains (e.g., but not limited to, VHH domains, humanized VHH domains, or camelified VH domains). The lipid in the lipid-cell-targeting group conjugate is preferably a PEG lipid.

[0101] As used herein, the term "free PEG lipid" refers to PEG lipids that are not part of a lipid-cell-targeting conjugate. For example, free PEG lipids can be part of a lipid blend of lipid nanoparticles. When, for example, a lipid-immune cell-targeting conjugate is included in lipid nanoparticles, free PEG lipids can reduce or eliminate nonspecific binding via the targeting group.

[0102] As used herein, the term "linker" or "spacer" refers to a peptide that fuses two or more polypeptides or proteins into a single molecule. The use of linkers to connect two or more (multi)peptides is well known in the art. For example, one class of peptide linkers is called "Gly-Ser" or "GS" linkers and consists essentially of glycine (G) and serine (S) residues. Linkers can also be alanine linkers.

[0103] As used in this article, the term "coupling" refers to the covalent interaction between two molecules. Therefore, "coupling" includes "covalent coupling".

[0104] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen. It should be understood that the term encompasses complete antibodies, their antigen-binding fragments, or optionally modified or engineered Fc fragments. Examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single-chain antibodies (e.g., scFv), mini-antibodies, immunoglobulin monovariable domains (e.g., VHH), and bispecific antibodies. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0105] Naturally occurring antibodies typically comprise tetramers. Each such tetramer usually consists of two pairs of identical polypeptide chains, each pair having a full-length light chain (typically with a molecular weight of about 25 kDa) and a full-length heavy chain (typically with a molecular weight of about 50–70 kDa). As used herein, the terms “heavy chain” and “light chain” refer to any immunoglobulin polypeptide having a sufficient sequence of variable domains to confer specificity against a target antigen. The N-terminal portion of each light and heavy chain typically contains a variable domain of about 100 to 110 or more amino acids, which is typically responsible for antigen recognition. The C-terminal portion of each chain typically defines a constant domain responsible for effector function. Therefore, in naturally occurring antibodies, full-length heavy chain immunoglobulin peptides include a variable domain (VH) and three constant domains (CH1, CH2, and CH3), wherein the VH domain is located at the amino terminus of the peptide and the CH3 domain is located at the carboxyl terminus, and full-length light chain immunoglobulin peptides include a variable domain (VL) and a constant domain (CL), wherein the VL domain is located at the amino terminus of the peptide and the CL domain is located at the carboxyl terminus.

[0106] Human light chains are typically classified as κ and λ light chains, and human heavy chains are typically classified as μ, δ, γ, α, or ε, with antibody isotypes defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has multiple subclasses, including but not limited to IgM1 and IgM2. IgA is similarly subdivided into multiple subclasses, including but not limited to IgA1 and IgA2. Within both the full-length light and heavy chains, variable and constant domains are typically linked by “J” regions of about 12 or more amino acids, with the heavy chain also including “D” regions of about 10 or more amino acids. See, for example, *FUNDAMENTAL IMMUNOLOGY* (edited by Paul, W., Raven Press, 2nd ed., 1989), which is incorporated herein by reference in its entirety. The variable region of each light / heavy chain pair typically forms an antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same overall structure as a relatively conserved framework region (FR) linked by three hypervariable regions (also known as complementarity-determining regions or CDRs). The CDRs of each pair of chains are usually arranged within the framework region, which allows for binding to specific epitopes. Both the light chain and heavy chain variable domains typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus to the carboxyl terminus.

[0107] The term "CDR set" refers to a group of three CDRs that appear in a single variable region capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to various systems. The system described by Kabat (Kabat et al., SEQUENCESOF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institute of Health, Bethesda, Md.) (1987) and (1991)) not only provides a definitive residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and colleagues (Chothia and Lesk, 1987, J. Mol. Biol. [Journal of Molecular Biology] 196: 901-17; Chothia et al., 1989, Nature [Nature] 342: 877-83) discovered that, despite significant diversity at the amino acid sequence level, certain sub-regions within the Kabat CDR adopt nearly identical peptide skeleton architectures. These sub-regions are designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions can be termed the Chothia CDR, whose boundaries overlap with the Kabat CDR. Other boundaries defining CDRs overlapping with the Kabat CDR have been described in the following literature: Padlan, 1995, FASEB J. [Journal of the Federation for Experimental Biology] 9: 133-39; MacCallum, 1996, J. Mol.Biol. [Journal of Molecular Biology] 262(5): 732-45; and Lefranc, 2003, Dev. Comp. Immunol. [Developmental and Comparative Immunology] 27: 55-77. There are other CDR boundary definitions that may not strictly follow one of the systems mentioned, but still overlap with the Kabat CDR, although they may be shortened or lengthened depending on whether a particular residue or group of residues or even the entire CDR significantly affects the prediction or experimental findings of antigen binding. The approach used in this paper can utilize CDRs defined according to any of these systems, but some aspects use CDRs defined by Kabat or Chothia.The use of amino acid sequence identification of predicted CDRs is well-known in the field, for example in the following literature: Martin, AC Proteinsequence and structure analysis of antibody variable domains, Antibody Engineering, Vol. 2, edited by Kontermann R., Dübel S., Springer, Berlin, pp. 33-51 (2010). The amino acid sequence of heavy chain and / or light chain variable domains can also be examined to identify CDR sequences by other conventional methods, such as by comparing with known amino acid sequences of other heavy and light chain variable regions to identify regions with sequence hypervariability. Numbered sequences can be aligned visually or by using alignment procedures (e.g., one of the CLUSTAL program suites), as described in Thompson, 1994, Nucleic Acids Res. 22: 4673-80. Molecular models are often used to correctly depict the framework and CDR regions, thereby correcting sequence-based assignments.

[0108] As used herein, the term "Fc" refers to a molecule (whether in monomeric or multimeric form) containing a sequence of a non-antigen-binding fragment produced by antibody digestion or by other means, and may contain a hinge region. The original immunoglobulin source of natural Fc is preferably human, but can be any immunoglobulin, although IgG1 and IgG2 are preferred. Fc molecules consist of monomeric polypeptides linked together by covalent (i.e., disulfide bonds) and non-covalent linkages to form dimers or multimers. Depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2), the number of intermolecular disulfide bonds between the monomeric subunits of a natural Fc molecule ranges from 1 to 4. One example of Fc is a disulfide-bonded dimer produced by papain digestion of IgG. As used herein, the term "natural Fc" is a general term for monomeric, dimer, and multimer forms.

[0109] As used herein, the term "Fab" refers to the F(ab) fragment of an antibody and typically comprises a light chain and a heavy chain with VH and CH1 domains, 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, the F(ab) fragment may also comprise a light chain containing two variable domains separated by amino acid linkers, and a heavy chain containing two variable domains and a CH1 domain separated by amino acid linkers. The F(ab') fragment typically comprises a light chain and a portion of a heavy chain containing more constant regions (between the CH1 and CH2 domains), allowing interchain disulfide bonds to form between the two heavy chains to form the F(ab')2 molecule.

[0110] As used herein, the term "immunoglobulin monovariable domain" 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 immunoglobulin monovariable domain is formed by a single VH, a single VHH, or a single VL domain. Therefore, the antigen-binding site of an immunoglobulin monovariable domain is formed by no more than three CDRs. The monovariable domain can 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 a VHH sequence) or a suitable fragment thereof; provided that it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, 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 immunoglobulin monovariable domain can be a heavy chain immunoglobulin monovariable domain, such as VH, VHH (including camelified VH or humanized VHH), or "dAb" or dAb (or an amino acid sequence suitable for use as dAb). In some respects, the immunoglobulin single variable domain is the VHH, including camel-derived VHH or humanized VHH. In some respects, the single variable domain is derived from certain shark species (e.g., the so-called "IgNAR domain"). In some respects, the immunoglobulin single variable domain is the NANOBODY® protein. [Note: NANOBODY® is a registered trademark of Ablynx NV.]

[0111] As used herein, the term "scFv" refers to a single-chain antibody containing a heavy chain variable domain and a light chain variable domain connected by a linker.

[0112] The term “VHH” is used in its broadest sense herein and is not limited to a specific biological source or a specific method of preparation. For example, VHH can be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) “humanizing” a naturally occurring VHH domain or expressing a nucleic acid encoding such a humanized VHH domain; (4) “camelizing” a naturally occurring VH domain from any animal species, particularly mammalian species (e.g., from humans), or expressing a nucleic acid encoding such a camelized VH domain; (5) “camelizing” a “domain antibody” or “dAb”, or expressing a nucleic acid encoding such a camelized VH domain; (6) using synthetic or semi-synthetic techniques for preparing proteins, peptides or other amino acid sequences; (7) preparing a nucleic acid encoding a VHH by using nucleic acid synthesis techniques and then expressing the resulting nucleic acid; and / or (8) by any combination of the foregoing.

[0113] In some respects, immunoglobulin sequences from various sources (including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences) have been used in the cell-targeting groups described herein. Furthermore, fully human, humanized, or chimeric sequences can be used in the cell-targeting groups described herein.

[0114] As used herein, the term "humanized antibody" refers to an antibody that is wholly or partially non-human in origin, and whose protein sequence has been modified to replace certain amino acids, such as amino acids appearing at one or more corresponding positions in the architecture regions of the VH and VL domains in antibody sequences derived from humans, to increase its similarity to antibodies naturally produced in humans, thereby avoiding or minimizing human immune responses. For example, using genetic engineering techniques, variable domains of a target non-human antibody can be combined with constant domains of a human antibody. In some respects, the constant domains of a humanized antibody are the human CH and CL domains.

[0115] As used herein, the term “humanized VHH” refers to an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but has been “humanized” by replacing one or more amino acid residues (particularly one or more amino acid residues in the framework sequence) of the amino acid sequence of the naturally occurring VHH with one or more amino acid residues appearing at one or more corresponding positions of the VH domain of a conventional human four-chain antibody.

[0116] As used herein, the term "camelized VH" refers to an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but has been "camelized," i.e., 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 with one or more amino acid residues appearing at one or more corresponding positions in the VHH domain of a (cameloid) heavy chain antibody. In some aspects, this "camelization" substitution is inserted into amino acid positions that form and / or exist at the VH-VL interface. In some aspects, the VH sequence used as the starting material or starting point for generating or designing camelized VHs is a VH sequence from mammals, such as human VH sequences, such as the VH3 sequence. However, it should be noted that such camelized VHs can be obtained in any suitable manner and are not strictly limited to peptides obtained using peptides containing naturally occurring VH domains as starting materials. For example, cameloid immunoglobulin sequences and humanized cameloid immunoglobulin sequences or camelized domain antibodies (e.g., camelized dAbs) may be used herein. In some respects, immunoglobulin monovariable domains are fused to form multivalent and / or multispecific constructs (for multivalent and multispecific peptides containing one or more VHH domains).

[0117] As used herein, the term "multivalent" refers to the presence of multiple immunoglobulin monovariable domains (IMDVs) in a polypeptide. In some aspects, a polypeptide is "bivalent," meaning it contains or is composed of two IMDVs. In some aspects, a polypeptide is "trivalent," meaning it contains or is composed of three IMDVs. In some aspects, a polypeptide is "tetravalent," meaning it contains or is composed of four IMDVs. Therefore, a polypeptide can be "bivalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," "heptavalent," "octavalent," "nonavalent," etc., meaning it contains or is composed of two, three, four, five, six, seven, eight, or nine IMDVs, respectively. In some aspects, multivalent IMDV polypeptides are trivalent. In some aspects, multivalent IMDV polypeptides are tetravalent. In some respects, multivalent immunoglobulin monovariable domain peptides are pentavalent. In other respects, multivalent immunoglobulin monovariable domain peptides can also be multispecific.

[0118] As used herein, the term "multispecific" refers to binding to multiple different target molecules (also called antigens). In some respects, multivalent immunoglobulin monovariable domain peptides are "bispecific," "trispecific," "quadrispecific," etc., meaning they can bind to two, three, four, or other different target molecules respectively. For example, a peptide can be bispecific-trivalent, such as a peptide containing or composed of three immunoglobulin monovariable domains, where two immunoglobulin monovariable domains bind to a first target and one immunoglobulin monovariable domain binds to a second target different from the first target. In some respects, a peptide is trispecific-quadrivalent, such as a peptide containing or composed of four immunoglobulin monovariable domains, where, for example, one immunoglobulin monovariable domain binds to a first target, two immunoglobulin monovariable domains bind to a second target different from the first target, and one immunoglobulin monovariable domain binds to a third target different from the first and second targets. In some respects, peptides are trispecific-pentavalent, for example, peptides containing or composed of five immunoglobulin monovariable domains, wherein, for example, two immunoglobulin monovariable domains bind to a first target, two immunoglobulin monovariable domains bind to a second target different from the first target, and one immunoglobulin monovariable domain binds to a third target different from the first and second targets. In some respects, multispecific immunoglobulin peptides are multicomplementary.

[0119] As used herein, the term "multiple complementary sites" refers to binding to multiple different epitopes on the same target molecule (also known as antigen). In some respects, multivalent immunoglobulin monovariable domain peptides can therefore be "double complementary sites," "triple complementary sites," etc., meaning they can bind to two, three, or other different epitopes on the same target molecule, respectively.

[0120] As used herein, the terms “subject” and “patient” refer preferably to mammalian organisms (e.g., rats, monkeys, horses, cattle, pigs, canines, felines, etc.), and more preferably to humans.

[0121] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or in vitro.

[0122] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to any standard pharmaceutical excipient, such as phosphate-buffered saline solutions, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. Compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers, and excipients, see Remington's The Science and Practice of Pharmacy, 21st edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, MD, 2006.

[0123] As used herein, the term "effective amount" refers to an amount of compound (e.g., nucleic acid) sufficient to achieve a beneficial or desired result. An effective amount may be administered in one or more applications, doses, or administrations and is not intended to be limited to a particular formulation or route of administration. The term "effective amount" may be considered to include therapeutic and / or prophylactic effective amounts of a compound.

[0124] As used herein, the phrase “therapeutic effective amount” refers to an amount of lipid nanoparticles comprising, for example, nucleic acids, or a composition comprising lipid nanoparticles, which effectively produce the desired therapeutic effect in at least one cell subpopulation in a mammal (e.g., a human) or a subject (e.g., a human subject) with a reasonable benefit / risk ratio suitable for any medical treatment.

[0125] As used herein, the phrase “preventive effective amount” refers to an amount of lipid nanoparticles, such as nucleic acids, or a composition containing lipid nanoparticles, which effectively produces the desired preventive effect in at least one cell subpopulation in a mammal (e.g., a human) or a subject (e.g., a human subject) by reducing, minimizing, or eliminating the risk of developing a condition or reducing or minimizing the severity of a condition, with a reasonable benefit / risk ratio suitable for any medical treatment.

[0126] The term "treatment" includes any action that produces improvement or relief of the symptoms of a condition, disease, ailment, etc., such as reducing, alleviating, regulating, improving, or eliminating them. lipids

[0127] This article provides lipid nanoparticles comprising lipid blends containing one or more of the following lipids: ionizable lipids, structural lipids, auxiliary lipids, and PEG lipids.

[0128] In some respects, ionizable lipids include the structure of formula I: (I), or its salt, wherein: R 1 R 2 and R 3 Each is an independent key or C 1-3 Alkylene; R 1A R 2A and R 3A Each is an independent key or C 1-10 Alkylene; R 1A1 R 1A2 R 1A3 R 2A1 R 2A2 R 2A3 R 3A1 R 3A2 and R 3A3 H and C are independent of each other. 1-20 Alkyl, C 1-20 Alkenyl, -(CH2) 0-10 C(O)OR a1 Or -(CH2) 0-10 OC(O)R a2 ;R a1 and R a2 Each is C independently 1-20 Alkyl or C 1-20 alkenyl; R 3B yes R 3B1 It is C 1-6 Alkylene; and R 3B2 and R 3B3 Each is independently H or C 1-6 alkyl.

[0129] In some respects, ionizable lipids have been engineered to deliver loads (e.g., nucleic acids) intracellularly to the cytoplasmic compartment of target cell types and rapidly degrade them into non-toxic components. The complex functions of ionizable cationic lipids are facilitated by the chemical and geometric interactions between the ionizable lipid head group, the hydrophobic "acyl tail" group, and the linker connecting the head group and the acyl tail group.

[0130] In some respects, the pKa of the ionizable amine head group is designed to be in the range of 6–8, for example, 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 to pH 5.5), neutral or weakly anionic at physiological pH (7.4), and cationic in early and late endosome compartments (e.g., pH 5.5 to pH 7).

[0131] In some respects, the acyl tail of ionizable lipids is designed to enable the fusion of lipid nanoparticles with endosome membranes and to destabilize the membrane through structural perturbation. The three-dimensional structure of the acyl tail (determined by its length, unsaturation, and site) and the relative sizes of the head and tail are thought to play a role in promoting membrane fusion and therefore in endosome escape from lipid nanoparticles (a key requirement for cytoplasmic delivery of nucleic acid loads). In some respects, ionizable lipids contain a linker connecting the head and tail groups, which is designed to be degraded by physiologically ubiquitous enzymes (e.g., esterases or proteases) or by acid-catalyzed hydrolysis.

[0132] In some respects, ionizable lipids include the structure of formula II (lipid 15): (II).

[0133] In some respects, ionizable lipids contain the structure of formula III (KC2). (III).

[0134] In some respects, the structure of ionizable lipid-containing IV (KC3) is as follows: (IV).

[0135] In some respects, ionizable lipids contain the structure of formula V: (V) Where R 1 and R 2 Each is a lipid independently.

[0136] In some respects, the structure of ionizable lipid-containing VI is as follows: (VI) Where R 1 and R 2 Each is a lipid independently.

[0137] In some embodiments of ionizable lipids comprising structures of formula V or VI, R 1 and R 2 Each is independently a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkenyl group. In some embodiments of ionizable lipids comprising the structure of formula V or VI, R 1 With R 2 They are the same lipids. In some embodiments of ionizable lipids comprising structures of formula V or formula VI, R 1 With R 2These are different lipids. In some embodiments, lipids are hydrocarbons (e.g., substituted or unsubstituted, saturated or unsaturated, branched or unbranched hydrocarbons). The hydrocarbon can be an alkane, olefin, or alkyne. In some embodiments, the hydrocarbon chain is saturated or unsaturated. In some embodiments, the unsaturated hydrocarbon chain contains at least one, at least two, at least three, at least four, at least five, or at least six carbon-carbon double bonds (e.g., cis double bonds and / or trans double bonds). In some embodiments, lipids are substituted or unsubstituted C... 7-36 Alkyl group. In some embodiments, the lipid is a substituted or unsubstituted C14 group. 7-36 Alkenyl. In some embodiments, the lipid is an unsubstituted C10 group. 7-36 Alkyl or unsubstituted C 7-36 Alkenyl. In some embodiments, the hydrocarbon is substituted with alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, carbonyl, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkyl thioether, thiol, urea, or combinations thereof. Each of these groups may subsequently be substituted. In some embodiments, the hydrocarbon is substituted with one, two, three, four, five, six, seven, eight, nine, ten, or more than ten substituents.

[0138] In some respects, ionizable lipids include one or more lipids as described in the following International PCT Publications: WO 2009028824A2, WO 2011043913A2, WO 2012091523A2, WO 2015074085A1, WO2016081029A1, WO 2017117530A1, WO 2018118102A1, WO 2018119163A1, WO 2019045897A1, WO 2020069445A1, WO 2020106903A1, WO 2020219876A1, WO 2021055892A1, WO2021163339A1, WO 2021188389A2, WO 2021202694A1, WO 2022016070A1, WO 2022119883A2, WO 2022120388A2, WO 2022159475A1, WO 2022207938A1, WO 2022218957A1, WO2022246555A1, WO 2023023410A2, WO 2023091490A1, WO 2023091787A1, WO 2023114937A2, WO 2023114944A1, WO 2023115221A1、WO 2023121970A1、WO 2023121975A1, WO2023141624A1, WO 2023144798A1, WO 2023183616A1, WO 2023196444A1, WO 2023196527A2, WO 2023207101A1, WO 2023235589A1, WO 2023240156A1, WO 2024008967A1, WO2024010330A1, WO 2024019770A1 and WO 2024035710A2. In other respects, ionizable lipids include one or more lipids as described in the following literature: Semiple, S., Akinc, A., Chen, J. et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol 28, 172-176 (2010).

[0139] In some respects, ionizable lipids are present in lipid blends of lipid nanoparticles in the range of about 20 mol% to about 70 mol%.

[0140] In some respects, ionizable lipids are present in lipid blends of lipid nanoparticles in the range of about 20 mol% to about 50 mol%. In some respects, ionizable lipids are present in lipid blends of lipid nanoparticles in the following amounts: approximately 20 mol%, approximately 21 mol%, approximately 22 mol%, approximately 23 mol%, approximately 24 mol%, approximately 25 mol%, approximately 26 mol%, approximately 27 mol%, approximately 28 mol%, approximately 29 mol%, approximately 30 mol%, approximately 31 mol%, approximately 32 mol%, approximately 33 mol%, approximately 34 mol%, approximately 35 mol%, approximately 36 mol%, approximately 37 mol%, approximately 38 mol%, approximately 39 mol%, approximately 40 mol%, approximately 41 mol%, approximately 42 mol%, approximately 43 mol%, approximately 44 mol%, approximately 45 mol%, approximately 46 mol%, approximately 47 mol%, approximately 48 mol%, approximately 49 mol%, approximately 50 mol%, approximately 51 mol%, approximately 52 mol%, approximately 53 mol%, approximately 54 mol%, approximately 55 mol%, approximately 56 mol%. mol%, approximately 57 mol%, approximately 58 mol%, approximately 59 mol%, approximately 60 mol%, approximately 61 mol%, approximately 62 mol%, approximately 63 mol%, approximately 64 mol%, approximately 65 mol%, approximately 66 mol%, approximately 67 mol%, approximately 68 mol%, approximately 69 mol%, or approximately 70 mol%.

[0141] In some aspects, ionizable lipids are present in lipid blends of lipid nanoparticles in the following ranges: 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, ionizable lipids are present in lipid blends of lipid nanoparticles in the following ranges: about 40 mol% to about 60 mol%; about 45 mol% to about 55 mol%; or about 48 mol% to about 50 mol%. In some aspects, ionizable lipids are present in lipid blends of lipid nanoparticles at about 49 mol%. In some aspects, lipid blends of lipid nanoparticles contain at least 20 mol% of ionizable lipids. In some aspects, lipid blends of lipid nanoparticles contain at least 30 mol% of ionizable lipids. In some aspects, lipid blends of lipid nanoparticles contain at least 40 mol% of ionizable lipids. In some respects, lipid blends of lipid nanoparticles contain at least 50 mol% ionizable lipids.

[0142] In some respects, lipid nanoparticles contain one or more accessory lipids. In some respects, the accessory lipids are phospholipids. In some respects, the accessory lipids are neutral phospholipids. In some respects, the accessory lipids are selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), sphingomyelin (SM), dimyristoyl-phosphatidylethanolamine (DMPE), dilinoleoyl-sn-glycerol-3-phosphate choline (SM), sphingomyelin (SM), dimyristoyl-phosphatidylethanolamine (DMPE), dilinoleoyl-sn-glycerol-3-phosphate ... Oleyl-phosphocholine (DLPC), dimyristico-glycerol-phosphocholine (DMPC), dipalmitoyl-glycerol-phosphocholine (DPPC), docosanoyl-glycerol-phosphocholine (DUPC), palmitoyl-oleoyl-glycerol-phosphocholine (POPC), octadecenyl-glycerol-phosphocholine, oleoyl-cholesterolyl-hemisuccinoyl-glycerol-phosphocholine, hexadecyl-glycerol-phosphocholine, dilinoleoyl-glycerol-phosphocholine, arachidonicoyl-glycerol-3-phosphocholine, docosahexaenoicoyl-glycerol-phosphocholine, or sphingomyelin.

[0143] In some respects, the auxiliary lipids are present in lipid blends of lipid nanoparticles in the range of about 10 mol% to about 50 mol%. In some aspects, the auxiliary lipids are present in lipid blends of lipid nanoparticles in the following amounts: about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol% or 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%. The proportions are approximately 16 mol%, about 48 mol%, about 49 mol%, or about 50 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles in the range of about 16 mol% to about 50 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles in the range of about 16 mol% to about 40 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles in the range of about 16 mol% to about 35 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles in the range of about 16 mol% to about 30 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles in the range of about 16 mol% to about 25 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles in the range of about 16 mol% to about 20 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles in the range of about 18 mol% to about 25 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 18 mol% to about 22 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 20 mol% to about 40 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 20 mol% to about 35 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 20 mol% to about 30 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 20 mol% to about 25 mol%.In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 25 mol% to about 40 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 25 mol% to about 35 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 25 mol% to about 30 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 30 mol% to about 40 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 30 mol% to about 35 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 20 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 30 mol%. In some aspects, the auxiliary lipids are present in the lipid blend of lipid nanoparticles at a concentration of about 40 mol%. In some aspects, the lipid blend of lipid nanoparticles contains at least 15 mol% of the auxiliary lipids. In some aspects, the lipid blends of lipid nanoparticles contain at least 20 mol% of auxiliary lipids. In other aspects, the lipid blends of lipid nanoparticles contain at least 30 mol% of auxiliary lipids.

[0144] In some respects, the auxiliary lipids of lipid nanoparticles are DSPCs.

[0145] In some respects, lipid nanoparticles contain one or more PEG lipids.

[0146] In some respects, PEG lipids are PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, or PEG-modified dialkylglycerol.

[0147] In some respects, PEG lipids are selected from the group consisting of: dioleoylglycerol-PEG (DOG-PEG), dimyristoylglycerol-PEG (DMG-PEG), dipalmitoylglycerol-PEG (DPG-PEG), dilinoleoylglycerol-phosphatidylethanolamine-PEG (DLPE-PEG), dimyristoylglycerol-phosphatidylethanolamine-PEG (DMPE-PEG), dipalmitoylglycerol-phosphatidylethanolamine-PEG (DPPE-PEG), distearate-glycerol-PEG (DSG-PEG), and diacylglycerol-PEG (DAG-PEG). PEG, dimyristoyl-glycerol-PEG (DMG-PEG), dipalmitoyl-glycerol (DPG-PEG), distearyl-glycerol-PEG (DSG-PEG), PEG-ceramide, distearyl-glycerol-phosphate-glycerol-PEG (DSPG-PEG), dioleoyl-glycerol-phosphate-ethanolamine-PEG (DOPE-PEG), 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide, and distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), DSPE-PEG-cysteine, or derivatives thereof. In some aspects, PEG lipids contain diacylphosphatidylethanolamine, which contains a dipalmitoyl (C16) chain or a distearyl (C18) chain.

[0148] In some aspects, PEG lipids are present in lipid blends of lipid nanoparticles in the following ranges: about 1 mol% to about 10 mol%, about 1 mol% to about 9 mol%, about 1 mol% to about 8 mol%, about 1 mol% to about 7 mol%, about 1 mol% to about 6 mol%, about 1 mol% to about 5 mol%, about 1 mol% to about 4 mol%, about 1 mol% to about 3 mol%. In some aspects, PEG lipids are present in lipid blends of lipid nanoparticles in the following ranges: about 2 mol% to about 10 mol%, about 2 mol% to about 9 mol%, about 2 mol% to about 8 mol%, about 2 mol% to about 7 mol%, about 2 mol% to about 6 mol%, about 2 mol% to about 5 mol%, about 2 mol% to about 4 mol%, about 2 mol% to about 3 mol%, or about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, or about 5 mol%. In some aspects, the PEG lipids are free PEG lipids.

[0149] In some aspects, PEG lipids are present in lipid blends of lipid nanoparticles in the following ranges: 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%. mol%, about 3 mol% to about 4 mol%, about 4 mol% to about 5 mol%, about 5 mol% to about 6 mol%. In some aspects, PEG lipids are present in lipid blends of lipid nanoparticles in the following amounts: 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, PEG lipids are free PEG lipids.

[0150] In some aspects, PEG lipids are present in lipid blends of lipid nanoparticles in the range of about 0.1 mol% to about 4 mol%. In other aspects, PEG lipids are present in lipid blends of lipid nanoparticles in the 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%, or about 1.5 mol% to about 2 mol%. In some aspects, PEG lipids are present in lipid blends of lipid nanoparticles in the following amounts: approximately 0.1 mol%, approximately 0.2 mol%, approximately 0.3 mol%, approximately 0.4 mol%, approximately 0.5 mol%, approximately 0.6 mol%, approximately 0.7 mol%, approximately 0.8 mol%, approximately 0.9 mol%, approximately 1 mol%, approximately 1.1 mol%, approximately 1.2 mol%, approximately 1.3 mol%, approximately 1.4 mol%, approximately 1.5 mol%, approximately 1.6 mol%, approximately 1.7 mol%, approximately 1.8 mol%, approximately 1.9 mol%, approximately 2 mol%, approximately 2.1 mol%, approximately 2.2 mol%, approximately 2.3 mol%, approximately 2.4 mol%, approximately 2.5 mol%, approximately 2.6 mol%, approximately 2.7 mol%, approximately 2.8 mol%, approximately 2.9 mol%, approximately 3 mol%, approximately 3.1 mol%, approximately 3.2 mol%, approximately 3.3 mol%. The PEG lipids are present in lipid blends of lipid nanoparticles at approximately 1.25 mol% to about 1.75 mol%. In some aspects, the PEG lipids are present in lipid blends of lipid nanoparticles at approximately 1.5 mol%. In some aspects, the lipid blends of lipid nanoparticles contain at least 0.5 mol% of PEG lipids. In some aspects, the lipid blends of lipid nanoparticles contain at least 1 mol% of PEG lipids. In some aspects, the lipid blends of lipid nanoparticles contain at least 2 mol% of PEG lipids.

[0151] In some respects, lipid nanoparticles contain one or more structural lipids. In some respects, the structural lipids are sterols. In some respects, one or more sterols are selected from the group consisting of: cholesterol, coprosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, stigmasterol, and brassicasterol. In some respects, the sterol is cholesterol.

[0152] In some respects, structural lipids are present in lipid blends of lipid nanoparticles in the range of about 20 mol% to about 70 mol%.

[0153] In some respects, structural lipids are present in lipid blends of lipid nanoparticles in the range of about 20 mol% to about 50 mol%. In some aspects, structural lipids are present in lipid blends of lipid nanoparticles in the following amounts: approximately 20 mol%, approximately 21 mol%, approximately 22 mol%, approximately 23 mol%, approximately 24 mol%, approximately 25 mol%, approximately 26 mol%, approximately 27 mol%, approximately 28 mol%, approximately 29 mol%, approximately 30 mol%, approximately 31 mol%, approximately 32 mol%, approximately 33 mol%, approximately 34 mol%, approximately 35 mol%, approximately 36 mol%, approximately 37 mol%, approximately 38 mol%, approximately 39 mol%, approximately 40 mol%, approximately 41 mol%, approximately 42 mol%, approximately 43 mol%, approximately 44 mol%, approximately 45 mol%, approximately 46 mol%, approximately 47 mol%, approximately 48 mol%, approximately 49 mol%, approximately 50 mol%, approximately 51 mol%, approximately 52 mol%, approximately 53 mol%, approximately 54 mol%, approximately 55 mol%, approximately 56 mol%, approximately 57 mol%. mol%, approximately 58 mol%, approximately 59 mol%, approximately 60 mol%, approximately 61 mol%, approximately 62 mol%, approximately 63 mol%, approximately 64 mol%, approximately 65 mol%, approximately 66 mol%, approximately 67 mol%, approximately 68 mol%, approximately 69 mol%, or approximately 70 mol%.

[0154] In some aspects, structural lipids are present in lipid blends of lipid nanoparticles in the following ranges: 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, structural lipids are present in lipid blends of lipid nanoparticles in the range of about 20%, about 30 mol%, about 40 mol%, or about 50 mol%. In some aspects, structural lipids are present in lipid blends of lipid nanoparticles in the range of about 29 mol%. In some aspects, structural lipids are present in lipid blends of lipid nanoparticles in the range of about 39 mol%. In some aspects, lipid blends of lipid nanoparticles contain at least 20 mol% structural lipids. In some aspects, lipid blends of lipid nanoparticles contain at least 25 mol% structural lipids. In some respects, lipid blends of lipid nanoparticles contain at least 30 mol% structural lipids.

[0155] In some aspects, the lipid nanoparticles comprise: an ionizable lipid present in a lipid blend of lipid nanoparticles in the range of about 30 mol% to about 70 mol%; an accessory lipid present in a lipid blend of lipid nanoparticles in the range of about 10 mol% to about 50 mol%; a structural lipid present in a lipid blend of lipid nanoparticles in the range of about 20 mol% to about 70 mol%; and a PEG lipid present in a lipid blend of lipid nanoparticles in the range of about 1 mol% to about 10 mol%; and the lipid nanoparticles optionally comprise a loading agent.

[0156] In some aspects, the lipid nanoparticles comprise: an ionizable lipid present in a lipid blend of lipid nanoparticles in the range of about 40 mol% to about 60 mol%; an accessory lipid present in a lipid blend of lipid nanoparticles in the range of about 16 mol% to about 40 mol%; a structural lipid present in a lipid blend of lipid nanoparticles in the range of about 20 mol% to about 50 mol%; and a PEG lipid present in a lipid blend of lipid nanoparticles in the range of about 1 mol% to about 5 mol%; and the lipid nanoparticles optionally comprise a loading agent.

[0157] In some aspects, the lipid nanoparticles comprise: an ionizable lipid present in a lipid blend of lipid nanoparticles ranging from about 48 mol% to about 50 mol%; an accessory lipid present in a lipid blend of lipid nanoparticles ranging from about 16 mol% to about 40 mol%; a structural lipid present in a lipid blend of lipid nanoparticles ranging from about 20 mol% to about 40 mol%; and a PEG lipid present in a lipid blend of lipid nanoparticles ranging from about 1 mol% to about 2 mol%; and the lipid nanoparticles optionally comprise a loading agent.

[0158] In some aspects, the lipid nanoparticles comprise: an ionizable lipid selected from the group consisting of formula II (lipid 15), formula III (KC2), formula IV (KC3), formula V and formula VI, and present in a lipid blend of lipid nanoparticles in the range of about 30 mol% to about 70 mol%; an accessory lipid, which is DSPC, and present in a lipid blend of lipid nanoparticles in the range of about 10 mol% to about 50 mol%; a structural lipid, which is cholesterol, and present in a lipid blend of lipid nanoparticles in the range of about 20 mol% to about 70 mol%; and a PEG lipid, which is DPG-PEG2K, and present in a lipid blend of lipid nanoparticles in the range of about 1 mol% to about 10 mol%, and the lipid nanoparticles optionally contain a loading.

[0159] In some aspects, the lipid nanoparticles comprise: an ionizable lipid selected from the group consisting of formula II (lipid 15), formula III (KC2), formula IV (KC3), formula V and formula VI, and present in a lipid blend of lipid nanoparticles in the range of about 40 mol% to about 60 mol%; an accessory lipid, which is DSPC, and present in a lipid blend of lipid nanoparticles in the range of about 16 mol% to about 40 mol%; a structural lipid, which is cholesterol, and present in a lipid blend of lipid nanoparticles in the range of about 20 mol% to about 50 mol%; and a PEG lipid, which is DPG-PEG2K, and present in a lipid blend of lipid nanoparticles in the range of about 1 mol% to about 5 mol%, and the lipid nanoparticles optionally contain a loading.

[0160] In some aspects, the lipid nanoparticles comprise: an ionizable lipid selected from the group consisting of formula II (lipid 15), formula III (KC2), formula IV (KC3), formula V and formula VI, and present in a lipid blend of lipid nanoparticles in the range of about 48 mol% to about 50 mol%; an accessory lipid, which is DSPC, and present in a lipid blend of lipid nanoparticles in the range of about 16 mol% to about 20 mol%; a structural lipid, which is cholesterol, and present in a lipid blend of lipid nanoparticles in the range of about 20 mol% to about 40 mol%; and a PEG lipid, which is DPG-PEG2K, and present in a lipid blend of lipid nanoparticles in the range of about 1 mol% to about 2 mol%, and the lipid nanoparticles optionally contain a loading.

[0161] In some aspects, the lipid nanoparticles comprise: an ionizable lipid selected from the group consisting of formula II (lipid 15), formula III (KC2), formula IV (KC3), formula V and formula VI, present in a lipid blend of lipid nanoparticles in an amount of about 49.24 mol%; an accessory lipid, which is DSPC, present in a lipid blend of lipid nanoparticles in an amount of about 20 mol%; a structural lipid, which is cholesterol, present in a lipid blend of lipid nanoparticles in an amount of about 29.25 mol%; a PEG lipid, which is DPG-PEG2K, present in a lipid blend of lipid nanoparticles in an amount of about 1.51 mol%; and the lipid nanoparticles optionally contain a loading agent.

[0162] In some respects, the lipid nanoparticles include lipid blends, and the mol% value refers to the mol% of the lipid blend. For example, if the nanoparticles include a lipid blend comprising ionizable lipids, structural lipids, auxiliary lipids, and PEG lipids, then the sum of the mol% of ionizable lipids, the mol% of structural lipids, the mol% of auxiliary lipids, and the mol% of PEG lipids is 100%. Cell-targeting groups

[0163] In some aspects, the lipid nanoparticles described herein include a cell-targeting group conjugated to the lipids of the lipid nanoparticles. In some aspects, the lipid nanoparticles include more than one cell-targeting group conjugated to the lipids of the lipid nanoparticles. In some aspects, the cell-targeting group includes an antibody. In some aspects, the antibody is a human antibody or a humanized antibody. In some aspects, the cell-targeting group includes an antibody fragment that does not contain an Fc component. In some aspects, the cell-targeting group includes an antigen-binding fragment selected from the group consisting of: Fab, F(ab')2, Fab'-SH, Fv, scFv fragments, or an immunoglobulin monovariable domain. In some aspects, the cell-targeting group includes Fab. In some aspects, the cell-targeting group includes an immunoglobulin monovariable domain.

[0164] In some respects, the cell-targeting group comprises one or more immunoglobulin monovariable domains. In other respects, the cell-targeting group comprises one or more antibody fragments, such as Fab, scFv, or combinations thereof.

[0165] In some respects, cell-targeting groups are monovalent, multivalent, or multispecific peptides. In other respects, cell-targeting groups comprise multivalent and multispecific peptides containing one or more antibodies, one or more immunoglobulin monovariable domains, such as one or more VHH domains and / or one or more Fab and / or one or more scFv.

[0166] In some aspects, a multivalent cell-targeting group comprises two or more immunoglobulin monovariable domains, Fab, or scFvs that target the same antigen (e.g., the same portion or epitope of the antigen, or two or more different portions or epitopes of the antigen). In some aspects, a multivalent cell-targeting group comprises two or more immunoglobulin monovariable domains, Fab, or scFvs that target different antigens. In some aspects, a multivalent cell-targeting group comprises a combination of immunoglobulin monovariable domains, Fab, or scFvs, some of which target the same antigen, while others target different antigens.

[0167] In some aspects, the bivalent cell-targeting group comprises a first immunoglobulin monovariable domain, Fab, or scFv, that targets a first portion or epitope of an antigen, and a second immunoglobulin monovariable domain, Fab, or scFv that targets the same portion or epitope of the antigen or another portion or epitope of the antigen. In some aspects, the bivalent cell-targeting group comprises a first immunoglobulin monovariable domain, Fab, or scFv that targets a first antigen, and a second immunoglobulin monovariable domain, Fab, or scFv that targets a second antigen different from the first antigen.

[0168] In some aspects, a trivalent cell-targeting group comprises three identical or different immunoglobulin monovariable domains, Fab, or scFv that target the same or different portions or epitopes of the same antigen. In some aspects, a trivalent cell-targeting group comprises two identical or different immunoglobulin monovariable domains, Fab, or scFv that target the same or different portions or epitopes of a first antigen, and a third immunoglobulin monovariable domain that targets a second antigen different from the first antigen.

[0169] In some respects, the trivalent cell-targeting group comprises a first immunoglobulin monovariable domain, Fab or scFv, targeting a first antigen, a second immunoglobulin monovariable domain, Fab or scFv, targeting a second antigen different from the first antigen, and a third immunoglobulin monovariable domain, Fab or scFv, targeting a third antigen different from the first and second antigens.

[0170] In some respects, the cell-targeting group comprises one or more immunoglobulin monovariable domains, Fab or scFv, and optionally further comprises one or more additional amino acid sequences (all amino acid sequences optionally linked by one or more suitable linkers).

[0171] In some respects, the cell-targeting group further comprises one or more other groups, residues, portions, or binding units. Such additional groups, residues, portions, binding units, or amino acid sequences may or may not provide additional functions to the immunoglobulin monovariable domain, Fab, or scFv (and / or the polypeptide in which it is located), and may or may not alter the properties of the immunoglobulin monovariable domain, Fab, or scFv.

[0172] In some aspects, such additional groups, residues, portions, or binding units may be one or more additional amino acids, such that the cell-targeting group is a (fusion) protein or a (fusion) polypeptide. In some aspects, the one or more additional groups, residues, portions, or binding units are immunoglobulins. In some aspects, the one or more additional groups, residues, portions, or binding units are domain antibodies (dAbs), amino acids suitable for use as domain antibodies, immunoglobulin monovariable domains, amino acids suitable for use as immunoglobulin monovariable domains, Fab, amino acids suitable for use as Fab, scFv, or amino acids suitable for use as scFv.

[0173] In some aspects, such groups, residues, portions, or binding units may be, for example, chemical groups, residues, or portions that may or may not have biological and / or pharmacological activity. In some aspects, such groups are linked to immunoglobulin monovariable domains, Fab, or scFv to provide “derivatives” of immunoglobulin monovariable domains, Fab, or scFv. In some aspects, the additional residues can effectively prevent or reduce the binding of so-called “pre-existing antibodies” to the polypeptide. For this purpose, the polypeptide and construct may contain a C-terminal extension (X)n, where n is 1 to 10, 1 to 5, 2, or 1; and each X is an independently selected, and preferably independently selected, amino acid residue from the group consisting of (preferably naturally occurring) alanine (A), glycine (G), valine (V), leucine (L), or isoleucine (I). In some aspects, X is not cysteine.

[0174] In the cell-targeting group, the one or more immunoglobulin monovariable domains, Fab or scFv, and the one or more groups, residues, portions, or binding units can be directly linked to each other and / or linked via one or more suitable linkers. For example, when the one or more groups, residues, portions, or binding units are amino acids, the linker can also be an amino acid, such that the resulting polypeptide is a fusion protein or fusion peptide.

[0175] In some aspects, the linker contains one or more repeats of a peptide motif (e.g., a GS motif). In some aspects, the linker contains a GGGGS motif (SEQ ID NO: 2) (e.g., having the formula (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7, or greater). In some aspects, the linker is a 9GS linker (GGGGSGGGS) (SEQ ID NO: 5), a 15GS linker (n=3), and a 35GS linker (n=7). See, for example, Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sel. 27(10): 325-330). In some aspects, the linker is a 3A linker, i.e., containing three alanine residues. In some aspects, as shown in Table 1 below, the connectors are 5GS connectors, 7GS connectors, 8GS connectors, 9GS connectors, 10GS connectors, 15GS connectors, 18GS connectors, 20GS connectors, 25GS connectors, 30GS connectors, 35GS connectors, or 40GS connectors. Table 1 Cell-targeting groups that target immune cells

[0176] In some respects, cell-targeting groups or combinations of targeting groups can be selected based on the desired localization, function, or structural characteristics of a given target cell. For example, cell-targeting groups can target T cells, T cell populations, or T cell subsets. In some respects, cell-targeting groups targeting T cells comprise one or more antibodies, immunoglobulin single variable domains, Fab, scFv, or derivatives thereof that target T cell surface antigens. Exemplary T cell surface antigens include, but are not limited to, CD3, CD4, CD7, and CD8.

[0177] Exemplary CD3 binding agents (CD3γ / δ / ε, CD3γ, CD3δ, CD3γ / ε, CD3δ / ε, or CD3ε) may be antibodies selected from the group consisting of: MEM-57 (CD3γ / δ / ε, EnzoLife Sciences), MAB100 (CD3ε, R&D Systems), CD3-H5 (CD3ε, Abnova Corporation), CD3-12 (CD3ε, Cell Signaling Technology), LE-CD3 (CD3ε, Santa Cruz Biotechnology, Inc.), NBP1-31250 (CD3γ, Novus Biologicals), 16669-1-AP (CD3δ, Invitrogen), and their antigen-binding fragments. In some respects, the binder comprises the VH and VL domains of antibodies selected from the group consisting of: MEM-57 (CD3γ / δ / ε, Enzo Life Sciences), MAB100 (CD3ε, R&D Systems), CD3-H5 (CD3ε, Anafa Biotech Inc.), CD3-12 (CD3ε, Cell Signaling Technologies Inc.), LE-CD3 (CD3ε, Santa Cruz Biotech Inc.), NBP1-31250 (CD3γ, Vibio Biotech Inc.), and 16669-1-AP (CD3δ, Ingenium Biotech Inc.).In some respects, the binder comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of the VH and VL sequences of antibodies selected from the group consisting of: MEM-57 (CD3γ / δ / ε, Enzo Life Sciences), MAB100 (CD3ε, R&D Systems), CD3-H5 (CD3ε, Anapharma Biotech Inc.), CD3-12 (CD3ε, Cell Signaling Technologies Inc.), LE-CD3 (CD3ε, Santa Cruz Biotechnology Inc.), NBP1-31250 (CD3γ, Vibio Biotechnology Co., Ltd.), and 16669-1-AP (CD3δ, Ingenium Biotech Inc.). These heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 are derived from the sequences of Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest). The CDR is determined by NIH Publication No. 91-3242, Bethesda, Chothia (see, for example, Chothia C & Lesk AM, (1987), J.MOL. BIOL. [Journal of Molecular Biology] 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J.MOL. BIOL. [Journal of Molecular Biology] 262: 732-745) or any other CDR determination method known in the art. Exemplary CD3 binding agents may also be selected from the following antibodies or antibody fragments employing cloned CDRs: hsp34, OKT-3, UCHT1, 38.1, HIT3a, RFT8, SK7, BC3, SP34-2, HU291, TRX4, Catuximab, Teplizumab, 3-106, 3-114, 3-148, 3-190, 3-271, 3-550, 4-10, 4-48, H2C F12Q, I2C, SP7, 3F3A1, CD3-12, 301, RIV9, JB38-29, JE17-74, GT0013, 4E2, 7A4, 4D10A6, SPV-T3b, M2AB, ICO-90, 30A1 or Hu38E4.v1 (US Patent Application 20200299409A1), REGN5458 (US Patent Application 20200024356A1) or blinatumomab.

[0178] Exemplary CD4 binders may be antibodies selected from the group consisting of: ipalizumab (https: / / www.genome.jp / dbget-bin / www_bget?D09575), AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Annofa Biosciences), CAL4 (Abcam), and their antigen-binding fragments. In some aspects, the binder comprises the VH and VL domains of antibodies selected from the group consisting of: AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Annofa Biosciences), and CAL4 (Abcam). In some respects, the binder comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of the VH and VL sequences of antibodies selected from the group consisting of: AF1856 (R&D Systems), MAB554 (R&D Systems), BF0174 (Affinity Biosciences), PAB31115 (Annofa Biotech, Inc.), and CAL4 (Abogen Biosciences). These heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 are derived from the work of Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest], NIH Publication No. 91-3242, Bethesda), Chothia (see, for example, Chothia C & Lesk AM, (1987), J.MOL. BIOL. [Journal of Molecular Biology] 196: The CD4 binding agent may be determined by a method known in the art, such as 901-917, MacCallum (see MacCallum RM et al., (1996) J.MOL.BIOL. [Journal of Molecular Biology] 262: 732-745), or any other CDR determination method known in the art. Exemplary CD4 binding agents may also be selected from antibodies or antibody fragments employing a cloned CDR, such as ipalizumab, OKT4, RPA-T4, S3.5, SK3, N1UG0, RIV6, OTI18E3, MEM-241, B486A1, RFT-4g, 7E14, MDX.2, MEM-115, MEM-16, ICO-86, Edu-2, or ilbalizumab.

[0179] Exemplary CD7 binders may be antibodies selected from the group consisting of: MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abogen), 1G10D8 (Proteintech), NBP2-32097 (Vibio Biotechnology), NBP2-38440 (Vibio Biotechnology), and their antigen-binding fragments. In some aspects, the binder comprises the VH and VL domains of antibodies selected from the group consisting of: MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abogen), 1G10D8 (Proteintech), NBP2-32097 (Vibio Biotechnology), and NBP2-38440 (Vibio Biotechnology). In some respects, the binder comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of the VH and VL sequences of antibodies selected from the group consisting of: MAB7579 (R&D Systems), AF7579 (R&D Systems), EPR22065 (Abogen), 1G10D8 (Protein Technologies), NBP2-32097 (Vibio Biotechnology Co., Ltd.), and NBP2-38440 (Vibio Biotechnology Co., Ltd.). These heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 are derived from the work of Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda) and Chothia (see, for example, Chothia C & Lesk AM, (1987), J.MOL). The CD7 binder may be determined by BIOL. [Journal of Molecular Biology] 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J.MOL. BIOL. [Journal of Molecular Biology] 262: 732-745), or any other CDR determination method known in the art. Exemplary CD7 binders may also be selected from antibodies or antibody fragments employing cloned CDRs such as TH-69, 3Afl1, T3-3A1, 124-1D1, 3A1f, CD7-6B7, or VHH6.

[0180] Exemplary CD8 (CD8α, CD8α / α, CD8α / β, or CD8β) binders may be antibodies selected from the group consisting of: 2.43 (Ingenieur), Du CD8-1 (CD8α, Ingenieur), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abogen), C8 / 144B (CD8α, Vibio Biotechnology), YTS105.18 (CD8α, Vibio Biotechnology), TRX2 (US 2017-0198045 A1 or US Patent No. 10,647768) and their antigen-binding fragments. In some respects, the binder comprises the VH and VL domains of antibodies selected from the group consisting of: 2.43 (Ingenie), 51.1 (ATCC HB-230), Du CD8-1 (CD8α, Ingenie), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abogen), C8 / 144B (CD8α, Vibio Biotechnology Co., Ltd.), and YTS105.18 (CD8α, Vibio Biotechnology Co., Ltd.). In some respects, the binder comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of the VH and VL sequences of antibodies selected from the group consisting of: 2.43 (Ingenieur), Du CD8-1 (CD8α, Ingenieur), 9358-CD (CD8α / β, R&D Systems), MAB116 (CD8α, R&D Systems), ab4055 (CD8α, Abogen), C8 / 144B (CD8α, Vibio Biotechnology Co., Ltd.), and YTS105.18 (CD8α, Vibio Biotechnology Co., Ltd.), these heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 are derived from Kabat (see Kabat et al., (1991) Sequences of Proteins of Immunological Interest], NIH Publication No. 91-3242, The CDR is determined by Bethesda, Chothia (see, for example, Chothia C & Lesk AM, (1987), J.MOL. BIOL. [Journal of Molecular Biology] 196: 901-917), MacCallum (see MacCallum RM et al., (1996) J.MOL. BIOL. [Journal of Molecular Biology] 262: 732-745), or any other CDR determination method known in the art.Exemplary CD8 binding agents may also be selected from the following antibodies or antibody fragments employing cloned CDRs: OKT-8, 51.1, S6F1, TRX2, and UCHT4, SP16, 3B5, C8-144B, HIT8a, RAVB3, LT8, 17D8, MEM-31, MEM-87, RIV11, DK-25, YTC141.1HL, or YTC182.20.

[0181] In some aspects, the cell-targeting group targets NK cells or NK cell populations. In some aspects, the NK cell-targeting group comprises one or more antibodies, antigen-binding fragments, or antigen-binding derivatives thereof that target NK cell surface antigens. Exemplary NK cell surface antigens include, but are not limited to, CD7, CD48, CD56, CD85a, CD85c, CD85d, CD85e, CD85f, CD85i, CD85j, CD158b2, CD161, CD244, CD16a, CD16b, IL-2 receptor, CD27, CD28, CD48, CD69, CD70, CD86, CD112, CD122, CD155, CD161, CD244, CD266, CD314 / NKG2D, CD336 / NKP44, and CD337 / NKP30.

[0182] In some aspects, the immune cell targeting group comprises a C-terminal cysteine ​​residue. In some aspects, the immune cell targeting group comprises a Fab containing a cysteine ​​residue at the C-terminus of a heavy or light chain segment. In some aspects, the Fab further comprises one or more amino acids between the heavy chain and the C-terminal cysteine ​​residue of the Fab. For example, in some aspects, the Fab comprises two or more amino acids derived from an antibody hinge region (e.g., a partial hinge sequence) between the C-terminus and the C-terminal cysteine ​​residue of the Fab. In some aspects, the Fab comprises a heavy chain variable domain linked to an antibody CH1 domain and a light chain variable domain linked to an antibody light chain constant domain, wherein the CH1 domain and the light chain constant domain are linked by one or more interchain disulfide bonds, and wherein the immune cell targeting group further comprises a single-chain variable segment (e.g., scFv) linked to the C-terminus of the light chain constant domain via an amino acid linker.

[0183] In some aspects, the immunocellular targeting group comprises an immunoglobulin monovariable domain, such as an immunoglobulin monovariable domain (e.g., VHH). In some aspects, the immunoglobulin monovariable domain contains a cysteine ​​residue at its C-terminus. In some aspects, the immunoglobulin monovariable domain further comprises a linker containing one or more amino acids between the VHH domain and the C-terminal cysteine ​​residue. In some aspects, the linker contains one or more glycine residues, such as two glycine residues. In some aspects, the immunocellular 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 contains one or more glycine and / or serine residues (e.g., one or more repeats of the sequence GGGGS). In some aspects, the immunocellular targeting group comprises a first VHH domain linked to an antibody CH1 domain and a second VHH domain linked to an antibody light chain constant domain, wherein the antibody CH1 domain and the antibody light chain constant domain are linked by one or more disulfide bonds (e.g., interchain disulfide bonds). In some respects, the immune cell targeting group includes a VHH domain linked to an antibody CH1 domain, wherein the antibody CH1 domain is linked to an antibody light chain constant domain via one or more disulfide bonds. Cell-targeting groups targeting hematopoietic stem cells

[0184] In some respects, the target cells are hematopoietic stem cells, and the cell-targeting groups are antibodies or antigen-binding fragments that bind to antigens present on hematopoietic stem cells.

[0185] In some aspects, the cell-targeting group of the lipid nanoparticles binds to molecules on the surface of hematopoietic stem cells. In some aspects, the cell-targeting group of the lipid nanoparticles is an antibody or fragment thereof that binds to molecules on the surface of hematopoietic stem cells. In some aspects, the hematopoietic stem cell molecules are CD34, CD105 (also known as endothelial glycoprotein), or CD117 (also known as c-kit, tyrosine-protein kinase KIT, or mast / stem cell growth factor receptor (SCFR)). In some aspects, the hematopoietic stem cell-targeting group of the lipid nanoparticles is an antibody or fragment thereof that binds to CD34, CD105, and / or CD117. In some aspects, the antibody is a human antibody or a humanized antibody. In some aspects, the antibody binding to CD105 and / or CD117 is covalently coupled to lipids in the lipid nanoparticles via a polyethylene glycol (PEG)-containing linker. Lipid-cell-targeting group conjugates

[0186] In some aspects, the lipid nanoparticles described herein comprise one or more lipid-cell-targeting group conjugates. In some aspects, the lipid-cell-targeting group conjugates comprise a cell-targeting peptide or a fragment thereof and a lipid. In some aspects, the cell-targeting group is covalently coupled to the lipid directly or via a linker. In some aspects, the cell-targeting group is covalently coupled to the lipid via a linker containing polyethylene glycol (PEG). In some aspects, the lipid-PEG-cell-targeting group conjugate is an insertable lipid-cell-targeting group conjugate of the lipid nanoparticles described herein.

[0187] In some respects, lipids coupled to cell-targeting groups via PEG linkers are distearylglycerol (DSG), distearyl-phosphatidylethanolamine (DSPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearyl-glycerol-phosphoglyceride (DSPG), dimyristoyl-glycerol (DMG), dipalmitoyl-phosphatidylethanolamine (DPPE), dipalmitoyl-glycerol (DPG), or ceramides.

[0188] In some respects, lipid-PEG-cell-targeting group conjugates include DSG-PEG, DSPE-PEG, DMPE-PEG, DSPG-PEG, DMG-PEG, DPPE-PEG, DPG-PEG, or ceramide-PEG. In some respects, lipid-PEG-cell-targeting group conjugates include DPG-PEG.

[0189] In some aspects, the PEG in the lipid-PEG-cell targeting conjugate is PEG 1000, PEG 2000, PEG 2100, PEG 2200, PEG 2300, PEG 2400, PEG 2500, PEG 2600, PEG 2700, PEG 2800, PEG 2900, PEG 3000, PEG 3100, PEG 3200, PEG 3300, PEG 3400, PEG 3500, PEG 3600, PEG 3700, PEG3800, PEG 3900, PEG 4000, PEG 4100, PEG 4200, PEG 4300, PEG 4400, PEG 4500, PEG 4600, PEG 4700, PEG 4800, PEG 4900 or PEG 5000. In some cases, PEG is PEG 2000 or PEG 3400. In other cases, PEG is PEG 2000 (PEG2K).

[0190] In some aspects, lipid-cell-targeting conjugates contain lipid-PEG2K-antibodies. In some aspects, lipid-cell-targeting conjugates contain lipid-PEG2K-Fab. In some aspects, lipid-cell-targeting conjugates contain lipid-PEG2K-scFv. In some aspects, lipid-cell-targeting conjugates contain lipid-PEG2K-immunoglobulin single variable domain (ISV domain). In some aspects, lipid nanoparticles contain lipid-cell-targeting conjugates containing lipid-PEG2K antibodies, lipid-cell-targeting conjugates containing lipid-PEG2K-Fab, lipid-cell-targeting conjugates containing lipid-PEG2K-scFv, and / or lipid-cell-targeting conjugates containing lipid-PEG2K-ISV domains.

[0191] In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG2K-cell-targeting peptide or a fragment thereof. In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG2K-cell-targeting antibody or a fragment thereof. In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG2K-cell-targeting Fab. In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG2K-cell-targeting scFv. In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG2K-cell-targeting ISV domain. In some aspects, the lipid nanoparticle comprises a lipid-cell-targeting conjugate containing a DSPE-PEG2K antibody, a lipid-cell-targeting conjugate containing DSPE-PEG2K-Fab, a lipid-cell-targeting conjugate containing DSPE-PEG2K-scFv, and / or a lipid-cell-targeting conjugate containing a DSPE-PEG2K-ISV domain.

[0192] In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG3.4K-cell-targeting polypeptide or a fragment thereof. In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG3.4K-cell-targeting antibody or a fragment thereof. In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG3.4K-cell-targeting Fab. In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG3.4K-cell-targeting scFv. In some aspects, the lipid-cell-targeting conjugate comprises a DSPE-PEG3.4K-cell-targeting ISV domain. In some aspects, the lipid nanoparticle comprises a lipid-cell-targeting conjugate containing a DSPE-PEG3.4K antibody, a lipid-cell-targeting conjugate containing DSPE-PEG3.4K-Fab, a lipid-cell-targeting conjugate containing DSPE-PEG3.4K-scFv, and / or a lipid-cell-targeting conjugate containing a DSPE-PEG3.4K-ISV domain.

[0193] In some aspects, lipid-cell-targeting conjugates comprise DPG-PEG2K-cell-targeting peptides or fragments thereof. In some aspects, lipid-cell-targeting conjugates comprise DPG-PEG2K-cell-targeting antibodies or fragments thereof. In some aspects, lipid-cell-targeting conjugates comprise DPG-PEG2K-cell-targeting Fab. In some aspects, lipid-cell-targeting conjugates comprise DPG-PEG2K-cell-targeting scFv. In some aspects, lipid-cell-targeting conjugates comprise a DPG-PEG2K-cell-targeting ISV domain. In some aspects, lipid nanoparticles comprise lipid-cell-targeting conjugates containing DPG-PEG2K antibodies, lipid-cell-targeting conjugates containing DPG-PEG2K-Fab, lipid-cell-targeting conjugates containing DPG-PEG2K-scFv, and / or lipid-cell-targeting conjugates containing a DPG-PEG2K-ISV domain.

[0194] In some aspects, the lipid in the lipid-cell-targeting conjugate is conjugated to any position on the N-terminus, C-terminus, or intermediate portion of the cell-targeting peptide or a fragment thereof. In some aspects, the cell-targeting peptide is an antibody, Fab, scFv, or immunoglobulin monovariable domain, and the lipid is conjugated to the N-terminus of the cell-targeting antibody, Fab, scFv, or immunoglobulin monovariable domain. In some aspects, the lipid is conjugated to the C-terminus of the cell-targeting antibody, Fab, scFv, or immunoglobulin monovariable domain. In some aspects, the lipid is conjugated at a position between the N-terminus and C-terminus of the cell-targeting antibody, Fab, scFv, or immunoglobulin monovariable domain. In some aspects, the lipid in the lipid-cell-targeting conjugate is conjugated to the cell-targeting peptide or a fragment thereof via a maleimide-thiol chemical reaction.

[0195] In some aspects, the lipid nanoparticles comprise multiple lipid-cell targeting group conjugates. In some aspects, these multiple lipid-cell targeting groups are covalently conjugated with different PEG linkers. In some aspects, these multiple lipid-cell targeting groups are present on the surface of the lipid nanoparticles, and each targeting group binds to a different molecule or molecular portion on a target cell. In some aspects, the multiple lipid-cell targeting groups each bind to a different epitope on the same antigen. In some aspects, the multiple lipid-cell targeting groups each bind to a different epitope on a different antigen. Advantageously, lipid nanoparticles having multiple lipid-cell targeting groups on their surface enhance the activity and specificity of targeting interactions with specific target cells.

[0196] In some aspects, lipid nanoparticles contain a lipid-PEG2K-CD3 targeting group. In some aspects, lipid nanoparticles contain a lipid-PEG2K-CD4 targeting group. In some aspects, lipid nanoparticles contain a lipid-PEG2K-CD8 targeting group. In some aspects, lipid nanoparticles comprise lipid-cell targeting group conjugates containing a lipid-PEG2K-CD3 targeting group, lipid-cell targeting group conjugates containing a lipid-PEG2K-CD4 targeting group, and / or lipid-cell targeting group conjugates containing a lipid-PEG2K-CD8 targeting group.

[0197] In some aspects, the lipid nanoparticles contain a DPG-PEG2K-CD3 targeting group. In some aspects, the lipid nanoparticles contain a DPG-PEG2K-CD4 targeting group. In some aspects, the lipid nanoparticles contain a DPG-PEG2K-CD8 targeting group. In some aspects, the lipid nanoparticles comprise lipid-cell targeting group conjugates containing a DPG-PEG2K-CD3 targeting group, lipid-cell targeting group conjugates containing a DPG-PEG2K-CD4 targeting group, and / or lipid-cell targeting group conjugates containing a DPG-PEG2K-CD8 targeting group.

[0198] In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD3 antibody. In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD3 Fab. In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD3 scFv. In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD3 immunoglobulin single variable domain.

[0199] In some respects, lipid nanoparticles comprise lipid-cell targeting conjugates containing DPG-PEG2K-CD3 antibodies, lipid-cell targeting conjugates containing DPG-PEG2K-CD3 Fab, lipid-cell targeting conjugates containing DPG-PEG2K-CD3 scFv, and / or lipid-cell targeting conjugates containing a single variable domain of DPG-PEG2K-CD3 immunoglobulin.

[0200] In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD4 antibody. In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD4 Fab. In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD4 scFv. In some aspects, the lipid-cell-targeting conjugate contains a single variable domain of the DPG-PEG2K-CD4 immunoglobulin.

[0201] In some respects, lipid nanoparticles comprise lipid-cell targeting conjugates containing DPG-PEG2K-CD4 antibodies, lipid-cell targeting conjugates containing DPG-PEG2K-CD4 Fab, lipid-cell targeting conjugates containing DPG-PEG2K-CD4 scFv, and / or lipid-cell targeting conjugates containing a single variable domain of DPG-PEG2K-CD4 immunoglobulin.

[0202] In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD8 antibody. In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD8 Fab. In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD8 scFv. In some aspects, the lipid-cell-targeting conjugate contains a DPG-PEG2K-CD8 immunoglobulin single variable domain.

[0203] In some respects, lipid nanoparticles comprise lipid-cell targeting conjugates containing DPG-PEG2K-CD8 antibodies, lipid-cell targeting conjugates containing DPG-PEG2K-CD8 Fab, lipid-cell targeting conjugates containing DPG-PEG2K-CD8 scFv, and / or lipid-cell targeting conjugates containing a single variable domain of DPG-PEG2K-CD8 immunoglobulin.

[0204] In some aspects, the lipid nanoparticles comprise DPG-PEG2K-CD3 antibody, DPG-PEG2K-CD4 antibody, and / or DPG-PEG2K-CD8 antibody. In some aspects, the lipid nanoparticles comprise DPG-PEG2K-CD3 Fab, DPG-PEG2K-CD4 Fab, and / or DPG-PEG2K-CD8 Fab. In some aspects, the lipid nanoparticles comprise DPG-PEG2K-CD3 scFv, DPG-PEG2K-CD4 scFv, and / or DPG-PEG2K-CD8 scFv. In some aspects, the lipid nanoparticles comprise a single variable domain of DPG-PEG2K-CD3 immunoglobulin, a single variable domain of DPG-PEG2K-CD4 immunoglobulin, and / or a single variable domain of DPG-PEG2K-CD8 immunoglobulin.

[0205] In some respects, lipid nanoparticles comprise DPG-PEG2K-CD3 antibody, DPG-PEG2K-CD3 Fab, DPG-PEG2K-CD3 scFv, DPG-PEG2K-CD3 immunoglobulin single variable domain, DPG-PEG2K-CD4 antibody, DPG-PEG2K-CD4 Fab, DPG-PEG2K-CD4 scFv, DPG-PEG2K-CD4 immunoglobulin single variable domain, DPG-PEG2K-CD8 antibody, DPG-PEG2K-CD8 Fab, DPG-PEG2K-CD8 scFv, or DPG-PEG2K-CD8 immunoglobulin single variable domain, or any combination thereof.

[0206] In some aspects, lipid nanoparticles contain a lipid-PEG2K-CD56 targeting group. In some aspects, lipid-cell-targeting group conjugates contain a lipid-PEG2K-CD56 antibody. In some aspects, lipid-cell-targeting group conjugates contain a lipid-PEG2K-CD56 Fab. In some aspects, lipid-cell-targeting group conjugates contain a lipid-PEG2K-CD56scFv. In some aspects, lipid-cell-targeting group conjugates contain a lipid-PEG2K-CD56 immunoglobulin single variable domain.

[0207] In some aspects, lipid nanoparticles contain a DPG-PEG2K-CD56 targeting group. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD56 antibody. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD56 Fab. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD56 scFv. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD56 immunoglobulin single variable domain.

[0208] In some aspects, lipid nanoparticles contain a lipid-PEG2K-CD105 targeting group. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD105 antibody. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD105 Fab. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD105 scFv. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD105 immunoglobulin monovariable domain. In some aspects, lipid nanoparticles contain lipid-cell targeting conjugates containing a lipid-PEG2K-CD105 antibody, lipid-cell targeting conjugates containing a lipid-PEG2K-CD105 Fab, lipid-cell targeting conjugates containing a lipid-PEG2K-CD105 scFv, and / or lipid-cell targeting conjugates containing a lipid-PEG2K-CD105 immunoglobulin monovariable domain.

[0209] In some aspects, lipid nanoparticles contain a DPG-PEG2K-CD105 targeting group. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD105 antibody. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD105 Fab. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD105scFv. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD105 immunoglobulin single variable domain. In some aspects, lipid nanoparticles contain lipid-cell targeting conjugates containing a DPG-PEG2K-CD105 antibody, lipid-cell targeting conjugates containing a DPG-PEG2K-CD105 Fab, lipid-cell targeting conjugates containing a DPG-PEG2K-CD105scFv, and / or lipid-cell targeting conjugates containing a DPG-PEG2K-CD105 immunoglobulin single variable domain.

[0210] In some aspects, lipid nanoparticles contain a lipid-PEG-CD117 targeting group. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD117 antibody. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD117 Fab. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD117scFv. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD117 immunoglobulin monovariable domain. In some aspects, lipid nanoparticles contain lipid-cell targeting conjugates containing a lipid-PEG2K-CD117 antibody, lipid-cell targeting conjugates containing a lipid-PEG2K-CD117 Fab, lipid-cell targeting conjugates containing a lipid-PEG2K-CD117scFv, and / or lipid-cell targeting conjugates containing a lipid-PEG2K-CD117 immunoglobulin monovariable domain.

[0211] In some aspects, lipid nanoparticles contain a DPG-PEG-CD117 targeting group. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD117 antibody. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD117 Fab. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD117scFv. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD117 immunoglobulin single variable domain. In some aspects, lipid nanoparticles contain lipid-cell targeting conjugates containing a DPG-PEG2K-CD117 antibody, lipid-cell targeting conjugates containing a DPG-PEG2K-CD117 Fab, lipid-cell targeting conjugates containing a DPG-PEG2K-CD117scFv, and / or lipid-cell targeting conjugates containing a DPG-PEG2K-CD117 immunoglobulin single variable domain.

[0212] In some aspects, lipid nanoparticles contain a lipid-PEG2K-CD34 targeting group. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD34 antibody. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD34 Fab. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD34scFv. In some aspects, lipid-cell targeting conjugates contain a lipid-PEG2K-CD34 immunoglobulin monovariable domain. In some aspects, lipid nanoparticles contain lipid-cell targeting conjugates containing a lipid-PEG2K-CD34 antibody, lipid-cell targeting conjugates containing a lipid-PEG2K-CD34 Fab, lipid-cell targeting conjugates containing a lipid-PEG2K-CD34scFv, and / or lipid-cell targeting conjugates containing a lipid-PEG2K-CD34 immunoglobulin monovariable domain.

[0213] In some aspects, lipid nanoparticles contain a DPG-PEG2K-CD34 targeting group. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD34 antibody. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD34 Fab. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD34 scFv. In some aspects, lipid-cell targeting conjugates contain a DPG-PEG2K-CD34 immunoglobulin monovariable domain. In some aspects, lipid nanoparticles contain lipid-cell targeting conjugates containing a DPG-PEG2K-CD34 antibody, lipid-cell targeting conjugates containing a DPG-PEG2K-CD34 Fab, lipid-cell targeting conjugates containing a DPG-PEG2K-CD34 scFv, and / or lipid-cell targeting conjugates containing a DPG-PEG2K-CD34 immunoglobulin monovariable domain.

[0214] In some aspects, the lipid nanoparticles contain lipid-PEG2K-CD105 targeting groups, lipid-DPG-PEG2K-CD117 targeting groups, and / or lipid-PEG2K-CD34 targeting groups.

[0215] In some respects, lipid nanoparticles comprise DPG-PEG2K-CD105 antibody, DPG-PEG2K-CD105 Fab, DPG-PEG2K-CD105 scFv, DPG-PEG2K-CD105 immunoglobulin single variable domain, DPG-PEG2K-CD117 antibody, DPG-PEG2K-CD117 Fab, DPG-PEG2K-CD117 scFv, DPG-PEG2K-CD117 immunoglobulin single variable domain, DPG-PEG2K-CD34 antibody, DPG-PEG2K-CD34 Fab, DPG-PEG2K-CD34 scFv, or DPG-PEG2K-CD34 immunoglobulin single variable domain or any combination thereof.

[0216] In some aspects, lipid-cell-targeting conjugates of lipid nanoparticles contain cell-targeting groups ranging from about 1 g to about 50 g per mole of lipid. In some aspects, lipid-cell-targeting conjugates of lipid nanoparticles contain cell-targeting groups ranging from about 10 g to about 20 g per mole of lipid. In some aspects, lipid-antibody conjugates of lipid nanoparticles contain cell-targeting groups ranging from about 12 g per mole of lipid.

[0217] In some aspects, the lipid-cell-targeting conjugate contains about 1 g to about 10 g of cell-targeting group per mole of lipid. In some aspects, the lipid-cell-targeting conjugate contains about 2 g to about 9 g of cell-targeting group per mole of lipid. In some aspects, the lipid-cell-targeting conjugate contains about 3 g to about 9 g of cell-targeting group per mole of lipid.

[0218] In some aspects, the lipid-antibody conjugate contains about 1 g to about 10 g of antibody per mole of lipid. In some aspects, the lipid-antibody conjugate contains about 2 g to about 9 g of antibody per mole of lipid. In some aspects, the lipid-antibody conjugate contains about 3 g to about 9 g of antibody per mole of lipid.

[0219] In some aspects, the lipid-Fab conjugate contains about 1 g to about 10 g of Fab per mole of lipid. In some aspects, the lipid-Fab conjugate contains about 2 g to about 9 g of Fab per mole of lipid. In some aspects, the lipid-Fab conjugate contains about 3 g to about 9 g of Fab per mole of lipid.

[0220] In some aspects, the lipid-scFv conjugate contains about 1 g to about 10 g of scFv per mole of lipid. In some aspects, the lipid-scFv conjugate contains about 2 g to about 9 g of scFv per mole of lipid. In some aspects, the lipid-scFv conjugate contains about 3 g to about 9 g of scFv per mole of lipid.

[0221] In some aspects, lipid-immunoglobulin monovariable domain conjugates contain about 1 g to about 10 g of ISV domain per mole of lipid. In some aspects, lipid-ISV domain conjugates contain about 2 g to about 9 g of ISV domain per mole of lipid. In some aspects, lipid-ISV domain conjugates contain about 3 g to about 9 g of ISV domain per mole of lipid.

[0222] In some aspects, the lipid-cell-targeting conjugate is present in the lipid nanoparticles in the range of about 0.001 mol% to about 0.5 mol%. In some aspects, the lipid-cell-targeting conjugate is present in the lipid nanoparticles in the range of about 0.002 mol% to about 0.2 mol%. In some aspects, the lipid-cell-targeting conjugate is present in the lipid nanoparticles in the 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, lipid-cell-targeting conjugates are present in lipid nanoparticles in the following ranges: 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%.

[0223] In some aspects, lipid nanoparticles containing lipid-cell targeting conjugates further comprise free PEG lipids to reduce nonspecific binding via the targeting group. In some aspects, the free PEG lipids may be the same as or different from the PEG lipids included in the conjugate. In some aspects, the free PEG lipids are selected from the group consisting of: PEG-distearyl-phosphatidylethanolamine (PEG-DSPE) or PEG-dimyristoyl-phosphatidylethanolamine (PEG-DMPE), N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE-PEG), 1,2-dimyristoyl-rac-glycerol-3-methylpolyoxyethylene (PEG-DMG), 1,2-dipalmitoyl-rac-glycerol-3-methylpolyoxyethylene The lipid nanoparticles contain PEG-DPG, 1,2-dioleoyl-rac-glycerol, methoxy polyethylene glycol (DOG-PEG), 1,2-distearate-rac-glycerol-3-methylpolyoxyethylene (PEG-DSG), N-palmitoyl-sphingosine-1-succinyl[methoxy(polyethylene glycol)] (PEG-ceramide), DSPE-PEG-cysteine, or derivatives thereof, all having an average PEG length between 2000 and 5000, or a PEG length of about 2000, 3400, or 5000. In some aspects, the lipid nanoparticles comprise a mixture of two or more PEGylated lipids. Load

[0224] In some respects, lipid nanoparticles contain a load (e.g., nucleic acid molecules configured in the lipid nanoparticles) for delivery to cells (e.g., immune cells or hematopoietic stem cells) or tissues in a subject's body.

[0225] In some aspects, lipid nanoparticles contain nucleic acids, such as DNA or RNA, such as mRNA, tRNA, microRNA, siRNA, gRNA (guide RNA), circRNA (circular RNA), ribozymes, bait RNA, or dicer substrate siRNA. In some aspects, the nucleic acids contain naturally occurring components, such as naturally occurring bases, sugars, or linker groups (e.g., phosphodiester linker groups). In some aspects, the nucleic acids contain non-naturally occurring components or modifications (e.g., thioester linker groups). For example, the nucleic acid can be synthesized to contain bases, sugars, or linker modifications known to those skilled in the art. Furthermore, the nucleic acid can be linear or circular, or have any desired conformation. In some aspects, lipid nanoparticles comprise multiple nucleic acid molecules, such as multiple RNA molecules, which can be identical or different.

[0226] In some aspects, one or more lipid nanoparticle compositions comprising one or more different mRNAs are combined and / or simultaneously contacted with cells. In some aspects, the mRNA may include one or more stem-loop, chain-terminating nucleoside, polyA sequence, polyadenylation signal, and / or 5' cap structure. In some aspects, the mRNA may encode a receptor (e.g., a chimeric antigen receptor (CAR)) for transfecting immune cells. In some aspects, the mRNA may encode a Cas nuclease. In some aspects, the RNA may contain guide RNA.

[0227] In some aspects, the wt / wt ratio of the lipid component to the loaded material (e.g., mRNA) in the lipid nanoparticles is from about 1:1 to about 50:1, for example, 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 component to the loaded material in the lipid nanoparticles is from about 5:1 to about 50:1. In some aspects, the wt / wt ratio of the lipid component to the loaded material in the lipid nanoparticles is from about 5:1 to about 40:1. In some aspects, the wt / wt ratio of the lipid component to the loaded material in the lipid nanoparticles is from about 10:1 to about 40:1. In some respects, the wt / wt ratio of lipid components to the load in lipid nanoparticles is about 15:1 to about 25:1.

[0228] In some respects, the encapsulation efficiency of the loaded material (e.g., mRNA) in the lipid nanoparticles 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 99%.

[0229] In some respects, the lipid nanoparticles described herein exhibit 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%.

[0230] In some aspects, the one or more mRNAs and lipids are selected to provide a specific N:P ratio (the ratio of positively charged lipid or polymeric 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 phosphate groups in the mRNA. Generally, a lower N:P ratio is preferred. The N:P ratio can depend on the specific lipid and its pKa. In some aspects, the relative amounts of mRNA and lipids and / or them in the lipid nanoparticles are selected to provide an N:P ratio of about 1:1 to about 30:1 or about 1:1 to about 20:1. In some aspects, the N:P ratio is, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1. In some aspects, the N:P ratio is 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 about 4:1 to about 8:1. In some respects, the N:P ratio can be approximately 4:1, approximately 4.5:1, approximately 4.6:1, approximately 4.7:1, approximately 4.8:1, approximately 4.9:1, approximately 5.0:1, approximately 5.1:1, approximately 5.2:1, approximately 5.3:1, approximately 5.4:1, approximately 5.5:1, approximately 5.6:1, approximately 5.7:1, approximately 6.0:1, approximately 6.5:1, or approximately 7.0:1. Composition

[0231] This document provides compositions comprising the lipid nanoparticles described herein. In some aspects, the compositions provided herein comprise lipid nanoparticles containing ionizable lipids. In some aspects, the ionizable lipids of the lipid nanoparticles comprise the structure of Formula I: (I), Or its salt, wherein: R 1 R 2 and R 3 Each is an independent key or C 1-3 Alkylene; R 1A R 2A and R 3A Each is an independent key or C 1-10 Alkylene; R 1A1 R 1A2 R 1A3 R 2A1 R 2A2 R 2A3 R 3A1 R 3A2 and R 3A3 H and C are independent of each other. 1-20 Alkyl, C 1-20 Alkenyl, -(CH2) 0-10C(O)OR a1 Or -(CH2) 0-10 OC(O)R a2 ;R a1 and R a2 Each is C independently 1-20 Alkyl or C 1-20 alkenyl; R 3B yes R 3B1 It is C 1-6 Alkylene; and R 3B2 and R 3B3 Each is independently H or C 1-6 alkyl.

[0232] In some respects, the lipid nanoparticles contain ionizable lipids of formula II (lipid 15): (II).

[0233] In some respects, the lipid nanoparticles contain ionizable lipids of formula III (KC2): (III)

[0234] In some respects, the structure of ionizable lipid-containing IV (KC3) lipid nanoparticles is as follows: (IV).

[0235] In some respects, the structure of ionizable lipid-containing V-shaped lipid nanoparticles is as follows: (V) Where R 1 and R 2 Each is a lipid independently.

[0236] In some respects, the structure of ionizable lipid-containing VI in lipid nanoparticles: (VI) Where R 1 and R 2 Each is a lipid independently.

[0237] In some embodiments of lipid nanoparticles containing ionizable lipids of formula V or formula VI, R 1 and R 2 Each is independently a substituted or unsubstituted alkyl group, or a substituted or unsubstituted alkenyl group. In some embodiments of ionizable lipid nanoparticles comprising structures of formula V or VI, R 1With R 2 It is the same lipid. In some embodiments of ionizable lipid nanoparticles comprising structures of formula V or formula VI, R 1 With R 2 These are different lipids. In some embodiments, lipids are hydrocarbons (e.g., substituted or unsubstituted, saturated or unsaturated, branched or unbranched hydrocarbons). The hydrocarbon can be an alkane, olefin, or alkyne. In some embodiments, the hydrocarbon chain is saturated or unsaturated. In some embodiments, the unsaturated hydrocarbon chain contains at least one, at least two, at least three, at least four, at least five, or at least six carbon-carbon double bonds (e.g., cis double bonds and / or trans double bonds). In some embodiments, lipids are substituted or unsubstituted C... 7-36 Alkyl group. In some embodiments, the lipid is a substituted or unsubstituted C14 group. 7-36 Alkenyl. In some embodiments, the lipid is an unsubstituted C10 group. 7-36 Alkyl or unsubstituted C 7-36 Alkenyl. In some embodiments, the hydrocarbon is substituted with alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, carbonyl, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkyl thioether, thiol, urea, or combinations thereof. Each of these groups may subsequently be substituted. In some embodiments, the hydrocarbon is substituted with one, two, three, four, five, six, seven, eight, nine, ten, or more than ten substituents.

[0238] In some aspects, the lipid nanoparticles of this composition further comprise accessory lipids, structural lipids, PEG lipids, and optional loading agents. In some aspects, the lipid nanoparticles of this composition further comprise lipid-cell-targeting conjugates as described herein.

[0239] In some aspects, the lipid nanoparticle composition comprises one or more pharmaceutically acceptable excipients. In some aspects, the lipid nanoparticle composition comprises one or more solvents, one or more salts, and / or one or more sugars or other cryoprotectants. In some aspects, conventional excipients and adjuvants are used in the pharmaceutical composition, except where any conventional excipient or adjuvant may be incompatible with one or more components of the lipid nanoparticle composition described herein. For example, if combining an excipient or adjuvant with a component of the lipid nanoparticle composition could result in any undesirable biological effect or otherwise harmful effect, then the excipient or adjuvant may be incompatible with that component.

[0240] In some respects, one or more excipients may constitute more than 50% of the total mass or volume of the pharmaceutical composition comprising lipid nanoparticles. For example, the one or more excipients may constitute 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical composition. method Preparation method of lipid nanoparticles

[0241] In some aspects, lipid nanoparticles are generated by rapid mixing via an orbital vortex mixer, a T-mixer, or microfluidic mixing. Orbital vortex mixing is performed by rapidly adding an ethanolic solution of lipids to an aqueous solution of nucleic acids, followed immediately by vortexing at 2,500 rpm. In some aspects, a microfluidic mixing step is used to generate lipid nanoparticles. In some aspects, microfluidic mixing is achieved by mixing water and organic streams at controlled flow rates in microfluidic channels using, for example, a NanoAssemblr device featuring optimized mixing chamber geometry and a microfluidic chip (Precision Nanosystems, Vancouver, BC). In some aspects, lipid nanoparticles are generated using a microfluidic mixing step that rapidly mixes an ethanolic lipid solution and an aqueous nucleic acid solution, encapsulating the nucleic acid within the solid lipid nanoparticles. The nanoparticle suspension is then buffer-exchanged into a full-water buffer using a selected membrane filtration device for ethanol removal and nanoparticle maturation. How to use

[0242] This disclosure provides methods for delivering a loading to target cells or tissues (e.g., target cells or tissues in a subject) and lipid nanoparticles or pharmaceutical compositions containing such lipid nanoparticles for use in such methods. Any disclosure herein relating to methods such as treating a disease or condition, or delivering nucleic acids to cells, or generating a target polypeptide in cells, should also be interpreted as a disclosure relating to lipid nanoparticles or pharmaceutical compositions containing said lipid nanoparticles for use in such methods.

[0243] In some aspects, a method for generating a target peptide in target cells is provided, as well as lipid nanoparticles or pharmaceutical compositions containing such lipid nanoparticles for use in such a method. The method for generating a peptide in such cells includes contacting the cells with lipid nanoparticles containing a load (e.g., a target nucleic acid). After contacting the cells with the lipid nanoparticles, the nucleic acid can be taken up and translated within the cells to generate the target peptide.

[0244] In some respects, the step of contacting cells with a lipid nanoparticle composition including a loading material (e.g., mRNA encoding a target polypeptide) can be performed in vivo, in vitro, or outside the body. The amount of lipid nanoparticle composition contacting the cells and / or the amount of mRNA therein can depend on the type of cells or tissues contacted, the method of application, the physicochemical characteristics of the lipid nanoparticles and the mRNA therein (e.g., size, charge, and chemical composition), and other factors.

[0245] In some respects, the step of contacting lipid nanoparticles containing a load with cells involves or causes transfection, wherein the lipid nanoparticles fuse with the cell membrane to allow delivery of the load into the cell. After introduction into the cytoplasm of the cell, the load (e.g., mRNA) is then translated into a protein or peptide via the cell's intracytoplasmic protein synthesis mechanisms.

[0246] In some respects, the efficiency of peptide production in cells can be determined, and cells can be repeatedly exposed to lipid nanoparticles until the desired target peptide production efficiency is achieved.

[0247] In some aspects, methods are provided for delivering a payload (e.g., nucleic acid) to mammalian cells or tissues (e.g., mammalian cells or tissues in a subject). In some aspects, delivering nucleic acid to such cells or tissues involves administering the lipid nanoparticle composition described herein to a subject, for example, by injection into the subject.

[0248] In some respects, after application, lipid nanoparticles can target and / or contact cells in a subject's body, such as immune cells or hematopoietic stem cells. Upon contact of cells with the lipid nanoparticle composition, nucleic acids (e.g., translatable mRNA) can be translated within the cells to produce the target peptide.

[0249] In some respects, the lipid nanoparticles described herein provide at least one of the following benefits: a. Compared with reference lipid nanoparticles, the specificity of targeted delivery to target cells is increased; b. Compared with reference lipid nanoparticles, the nucleic acid or the polypeptide encoded by the nucleic acid has an increased half-life in target cells; c. Increased transfection rate compared to reference lipid nanoparticles; and / or d. High efficiency in nucleic acid encapsulation.

[0250] In some respects, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of non-target cells are transfected with the lipid nanoparticles described herein. For example, in some respects, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of undesirable immune cells not intended to be the target of this delivery are transfected with the lipid nanoparticles. In some respects, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of undesirable hematopoietic stem cells not intended to be the target of this delivery are transfected with the lipid nanoparticles.

[0251] In some respects, the half-life of nucleic acids delivered to target cells (e.g., immune cells or hematopoietic stem cells) by the lipid nanoparticles described herein, or the half-life of peptides encoded by nucleic acids delivered by lipid nanoparticles, 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 nucleic acids delivered to said target cells by reference lipid nanoparticles or the half-life of peptides encoded by nucleic acids delivered by reference lipid nanoparticles.

[0252] In some aspects, the composition of the lipid nanoparticles differs from that of the reference lipid nanoparticles in terms of the type of ionizable cationic lipids, the relative amount of ionizable cationic lipids, the type of PEG lipids, the relative amount of PEG lipids, the type of auxiliary lipids, the relative amount of auxiliary lipids, the type of structural lipids, the relative amount of structural lipids, the type of loading material or cell-targeting group, or any combination thereof. In some aspects, the reference lipid nanoparticles are lipid nanoparticles containing less than 20 mol% of auxiliary lipids. In some aspects, the reference lipid nanoparticles are lipid nanoparticles containing less than 16 mol% of auxiliary lipids. In some aspects, the reference lipid nanoparticles are lipid nanoparticles containing about 10 mol% to about 15 mol% of auxiliary lipids. In some aspects, the reference lipid nanoparticles are lipid nanoparticles containing more than 50 mol% of auxiliary lipids.

[0253] In some aspects, the composition of the lipid nanoparticles differs from that of the reference lipid nanoparticles only in the type of ionizable cationic lipids. In some aspects, the composition of the lipid nanoparticles differs from that of the reference lipid nanoparticles in the relative amount of ionizable cationic lipids. In some aspects, the composition of the lipid nanoparticles differs from that of the reference lipid nanoparticles only in the type of accessory lipids. In some aspects, the composition of the lipid nanoparticles differs from that of the reference lipid nanoparticles in the relative amount of accessory lipids. In some aspects, the composition of the lipid nanoparticles differs from that of the reference lipid nanoparticles in the relative amount of accessory lipids, wherein the lipid nanoparticles contain about 16 mol% to about 20 mol% of accessory lipids, while the reference lipid nanoparticles contain about 10 mol% to about 15 mol% of accessory lipids, and the difference in total lipid content is compensated by the variation in the amount of structural lipids in the lipid nanoparticles; otherwise, the lipid nanoparticles and the reference lipid nanoparticles are identical.

[0254] In some respects, 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% or more of the target cells intended to be the target of this delivery are transfected with the lipid nanoparticles.

[0255] In some respects, the expression levels of nucleic acids in target cells delivered by the lipid nanoparticles described herein are 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 the expression levels of nucleic acids in the same target cells delivered by reference lipid nanoparticles.

[0256] In some respects, the use of lipid nanoparticles described herein for specific delivery can increase the amount of load delivered to the target by more than 2, 5, 10, 15, or 20 times compared to other targets (e.g., non-target cells).

[0257] In some respects, 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% or more of immune target cells are transfected with lipid nanoparticles.

[0258] In some respects, the expression levels of nucleic acids in immune target cells delivered by the lipid nanoparticles described herein are 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 the expression levels of nucleic acids in immune target cells delivered by reference lipid nanoparticles.

[0259] In some respects, the specific delivery of the lipid nanoparticles described herein can result in an increase of more than 2, 5, 10, 15, or 20 times in the amount of load delivered to immune target cells compared to immune non-target cells or other non-target cells.

[0260] In some respects, 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% or more of hematopoietic stem cell target cells were transfected with lipid nanoparticles.

[0261] In some respects, the expression levels of nucleic acids in hematopoietic cells delivered by the lipid nanoparticles described herein are at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 1.5, 2, 3, 4, 5, 10, 15, 20, or more higher than the expression levels of nucleic acids in the same hematopoietic stem cells delivered by reference lipid nanoparticles.

[0262] In some respects, the specific delivery of the lipid nanoparticles described herein can result in an increase of more than 2, 5, 10, 15, or 20 times in the amount of load delivered to target hematopoietic stem cells compared to hematopoietic non-target cells or other non-target cells.

[0263] In some respects, the lipid nanoparticle compositions described herein can be used to treat diseases, symptoms, or conditions characterized by the absence or abnormal activity of peptides. In some respects, upon delivery of lipid nanoparticles containing nucleic acids (e.g., mRNA encoding the absence or abnormal peptide) into cells, the translation of the mRNA can produce the peptide, thereby reducing or eliminating problems caused by the absence of the peptide or the abnormal activity caused by the peptide.

[0264] In some aspects, this article provides methods for targeting the delivery of nucleic acids to the immune cells of a subject. In some aspects, the method includes contacting the immune cells with the lipid nanoparticles described herein. In some aspects, the method includes contacting the immune cells with the lipid nanoparticles in vitro. In some aspects, the method includes contacting the immune cells with the lipid nanoparticles ex vivo. In some aspects, the method includes contacting the immune cells with the lipid nanoparticles in vivo.

[0265] In some aspects, methods for modulating the cellular function of immune cells in a subject are provided. In some aspects, the method includes administering lipid nanoparticles to the subject. In some aspects, the lipid nanoparticles contain immune cell targeting groups.

[0266] In some respects, immune cell targeting groups are CD3 targeting groups, CD4 targeting groups, CD7 targeting groups, or CD8 targeting groups.

[0267] In some aspects, the regulation of cellular function includes reprogramming immune cells to initiate an immune response. In some aspects, the regulation of cellular function includes expressing chimeric antigen receptors (CARs) in immune cells. In some aspects, the regulation of cellular function includes contacting T cells with lipid nanoparticles containing RNA encoding a CAR. In some aspects, the lipid nanoparticles further contain T cell targeting groups, such as CD3-targeting groups, CD4-targeting groups, and / or CD8-targeting groups. In some aspects, the regulation of cellular function includes increasing cytotoxic activity in CD8+ T cells, wherein the lipid nanoparticles for contacting T cells contain a CD8-targeting group, and the nucleic acid configured within the lipid nanoparticles encodes a CAR.

[0268] In some respects, the regulation of cellular function involves the delivery of nucleic acids encoding gene editing systems (e.g., site-directed nucleases, and optionally containing guide RNA) to T cells.

[0269] In some respects, the target group for immune cells is CD56. In some respects, the regulation of cell function includes reprogramming NK cells to initiate an immune response. In some respects, the regulation of cell function includes expressing CARs in NK cells. In some respects, the regulation of cell function includes increasing cytotoxic activity in NK cells.

[0270] In some respects, the regulation of cellular function involves the delivery of nucleic acids encoding gene editing systems (e.g., site-directed nucleases and optional guide RNAs) to NK cells.

[0271] In some aspects, this article provides methods for targeted delivery of nucleic acids to hematopoietic stem cells in a subject. In some aspects, the method includes contacting hematopoietic stem cells with lipid nanoparticles as described herein. In some aspects, the method includes contacting hematopoietic stem cells with lipid nanoparticles in vitro. In some aspects, the method includes contacting hematopoietic stem cells with lipid nanoparticles ex vivo. In some aspects, the method includes contacting hematopoietic stem cells with lipid nanoparticles in vivo.

[0272] In some respects, the lipid nanoparticles contain hematopoietic stem cell targeting groups. In some respects, the hematopoietic stem cell targeting groups are CD105 targeting groups, CD117 targeting groups, or CD34 targeting groups.

[0273] In some respects, the lipid nanoparticles provided herein are delivered to subjects suffering from diseases for in vivo gene editing and disease treatment. In some respects, the lipid nanoparticles are delivered to subjects suffering from hemoglobinopathies. In some respects, a method for treating alpha-hemoglobinopathies is provided. In some respects, a method for treating beta-hemoglobinopathies is provided. In some respects, beta-hemoglobinopathies are β-thalassemia. In some respects, beta-hemoglobinopathies are sickle cell diseases.

[0274] In some respects, the lipid nanoparticles described herein are delivered to subjects suffering from sickle cell disease or β-thalassemia for in vivo gene editing and treatment. In some respects, the lipid nanoparticles contain one or more nucleic acids encoding a gene editing system that targets a locus where gene editing increases hemoglobin F (HbF) in the subject's cells, thereby treating the disease (e.g., sickle cell disease and β-thalassemia).

[0275] In some respects, the lipid nanoparticles delivered to the subject contain one or more nucleic acids encoding a site-directed nuclease and one or more RNAs that enable the Cas nuclease to bind to a target nucleotide sequence. In some respects, the one or more RNAs that enable the Cas nuclease to bind to the target nucleotide sequence include trans-activating cRNA (tracrRNA) and CRISPR RNA (crRNA), or more commonly, guide RNA (also known as single guide RNA (sgRNA)), wherein the crRNA and tracrRNA are engineered into a single RNA molecule. In some respects, the one or more nucleic acids encoding the nuclease encode a site-directed nuclease, including CRISPR-associated (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or megaTAL. Applications of lipid nanoparticles

[0276] In some aspects, this disclosure provides lipid nanoparticles or compositions containing the same as disclosed herein for use in methods of targeted delivery of nucleic acids to target cells of a subject. In some aspects, this disclosure provides a lipid nanoparticle or composition containing the same as disclosed herein for use in methods of targeted delivery of nucleic acids to immune cells of a subject. In some aspects, the nucleic acid encodes an immune cell-enhancing polypeptide, and this nucleic acid is targeted to immune cells by the lipid nanoparticles. In some aspects, the lipid nanoparticles contain immune cell-targeting groups for targeted delivery of nucleic acids to immune cells.

[0277] In some aspects, this disclosure provides lipid nanoparticles or compositions containing the same as disclosed herein for use in methods of targeted delivery of nucleic acids to hematopoietic stem cells of a subject. In some aspects, the nucleic acid of the lipid nanoparticles encodes a polypeptide that functions in gene editing, and this nucleic acid is targeted to hematopoietic stem cells by the lipid nanoparticles. In some aspects, the lipid nanoparticles contain hematopoietic stem cell targeting groups to target the delivery of nucleic acids to hematopoietic stem cells.

[0278] In some aspects, this disclosure provides lipid nanoparticles or compositions containing them as disclosed herein for use in methods of treating a subject's disease or condition. In some aspects, the lipid nanoparticles comprise nucleic acids encoding immune cell function-enhancing peptides (e.g., CARs), and targeted immune cells expressing the CAR treat a subject's disease or condition. In some aspects, the lipid nanoparticles comprise nucleic acids providing components of a gene-editing system, and gene-edited targeted hematopoietic stem cells treat a subject's disease or condition. In some aspects, the compositions used in this method are pharmaceutical compositions. Reagent test kit

[0279] In some aspects, a kit for treating a medical condition is provided. In some aspects, the kit comprises: ionizable cationic lipids, assist lipids, structural lipids, PEG lipids, loading agents, lipid-immune cell-targeting group conjugates and / or lipid-hematopoietic stem cell-targeting group conjugates, and instructions for preparing lipid nanoparticles and using the lipid nanoparticles to treat a medical condition. In some aspects, the kit comprises a lipid nanoparticle composition and instructions for using the lipid nanoparticle composition or lipid nanoparticles to treat a medical condition, the lipid nanoparticle composition comprising lipid nanoparticles comprising ionizable cationic lipids, assist lipids, structural lipids, PEG lipids, loading agents, lipid-immune cell-targeting group conjugates and / or lipid-hematopoietic stem cell-targeting group conjugates. Example Example 1: Preparation of lipid nanoparticles

[0280] Lipid nanoparticles (LNPs) encapsulating mRNA loads were prepared by mixing an aqueous mRNA solution with an ethanol-lipid blend solution (containing ionizable lipids, DSPC, DPG-PEG, and cholesterol in lipid ratios as shown in Table 2) using an online microfluidic mixing method. The mRNA load for targeting LNPs with immune cells was eGFP mRNA, and the mRNA load for targeting LNPs with HSCs was mCherry mRNA. Several lipid nanoparticles were generated using different amounts of DSPC (e.g., 10 mol%, 12.5 mol%, 15 mol%, 20 mol%, 30 mol%, or 40 mol% DSPC).

[0281] The mRNA and lipid solutions were mixed using a NanoAssemblr Ignite microfluidic mixing device (model NIN0001) and an NxGen mixing column (model NIN0002) from Precision Nano Systems, Inc. (British Columbia, CA). Briefly, the mRNA and lipid solutions were each loaded into separate polypropylene syringes. The mixing column was inserted into the NanoAssemblr Ignite, and the syringe was attached to the Luer interface of the mixing column. The two solutions were then mixed using the NanoAssemblr Ignite at a total flow rate of 9 mL / min at a v / v ratio of 3:1 for the mRNA solution (1.5 mL) to the lipid solution (0.5 mL). The resulting suspension was held at room temperature for at least 5 minutes, followed by ethanol removal and buffer exchange.

[0282] After mixing, the resulting LNP suspension was subjected to ethanol removal and buffer exchange using a discontinuous percolation process. A centrifugal ultrafiltration apparatus (Amicon Ultra-15, MilliporeSigma, Massachusetts, USA) with a 100,000 kDa MWCO regenerated cellulose membrane was sterilized with 70% ethanol and then washed twice with HBS exchange buffer (25 mM pH 7.4 HEPES buffer with 150 mM NaCl). The LNP suspension (2 mL) was then loaded into the apparatus and centrifuged at 500 RCF until the volume was reduced by half (1 mL). The suspension was then diluted with exchange buffer (1 mL, 25 mM pH 7.4 HEPES buffer) to restore it to its original volume. This process of doubling concentration and dilution was repeated five more times, for a total of six discontinuous percolation steps. The LNP suspension was then exchanged into MBS (25 mM pH 6.5 MES buffer with 150 mL NaCl) by diluting it tenfold with MBS and centrifuging at 500 RCF until the volume was reduced by one-tenth. Repeat the steps of diluting MBS tenfold and concentrating it tenfold. Recover the LNP-containing residue in MBS from the centrifugal ultrafiltration unit and store it at 4°C until further use. Example 2: Characterization of LNP

[0283] The LNP samples generated in Example 1 were characterized to determine the average hydrodynamic diameter, zeta potential, and mRNA content (total mRNA and dye-accessible mRNA). The hydrodynamic diameter was determined by dynamic light scattering (DLS) using a Zetasizer model ZEN3600 (Malvern Pananalytical, UK). The zeta potential was measured by laser Doppler electrophoresis in 5 mM pH 5.5 MES buffer and 5 mM pH 7.4 HEPES buffer using the Zetasizer. The LNP results for formulations 1 and 2 are shown in Table 2. Table 2 Example 3: Preparation of conjugates capable of targeting T cells and hematopoietic stem cells

[0284] VHH, which binds to T-cell-specific targets (CD8) and HSC-specific targets (CD117), is conjugated to DSPE-PEG(3.4K)-maleimide and DSPE-PEG(2K)-maleimide, respectively, via covalent coupling between the maleimide group and the C-terminal cysteine ​​residue in the heavy chain (HC). In a representative reaction, a protein solution in PBS containing 5 mM EDTA was concentrated to 3–4 mg / mL using a 10 kDa MWCO regenerated cellulose centrifuge filter. The C-terminal cysteine ​​residues were selectively reduced using 2–5 mM TCEP for 1.5 h at room temperature. TCEP was removed using a 7K MWCO rotary desalting column. The reduced protein was then mixed with a mixture of DSPE-PEG-Mal and DSPE-PEG-OMe, with a molar ratio of DSPE-PEG-Mal to Fab or VHH of 1:1 and a molar ratio of DSPE-PEG-Mal:DSPE-PEG-OMe of 1:4. The reaction mixture was incubated at 37°C for 2 hours and then quenched with 15 mM cysteine ​​at 37°C for 15 minutes. The resulting DSPE-PEG-Fab or DSPE-PEG-VHH micelles were then concentrated and buffer-exchanged to HEPES buffered saline at pH 7.4 using a 50 K MWCO regenerated cellulose centrifuge filter. The concentration / buffer exchange process also removed residual cysteine ​​and unreacted Fab or VHH. The materials were characterized by SDS-PAGE and HPLC. The concentration of Fab or VHH was determined by measuring the absorbance at 280 nm. Example 4: Human T cell transfection

[0285] Human CD8+ T cells were isolated from PBMCs of healthy donors and cultured with human IL-2 for 24 hours (day 0). T cells were transfected using LNPs containing 10 mol%, 12.5 mol%, 15 mol%, 20 mol%, 30 mol%, or 40 mol% DSPC helper lipids, CAR mRNA, and GFP mRNA loadings, respectively. On day 1, CD8+ cells were transfected with LNPs at progressively increasing concentrations (0.012 µg / ml, 0.037 µg / ml, 0.11 µg / ml, 0.33 µg / ml, 1 µg / ml, or 3 µg / ml). Transfected cells were washed after 4 hours, stained, and analyzed immediately (hour 0) or at 24, 48, and 72 hours to determine the percentage of CAR-positive cells and the mean fluorescence intensity of CAR. Figure 1 shows the results of T cells transfected with 0.33 µg / ml LNPs containing between 10 mol% and 40 mol% DSPC. T cells transfected with LNP containing 20 mol%, 30 mol%, or 40 mol% DSPC showed durable transfection efficiency, with over 80% of T cells expressing CAR during the 72-hour measurement period. Figure 1A In contrast, T cells transfected with LNP containing 10 mol% DSPC were gradually lost over a 72-hour period. Figure 1A Furthermore, T cells transfected with LNPs containing 20 mol% DSPC showed significantly higher mean fluorescence intensity (MFI) than T cells transfected with LNPs containing 10 mol%, 30 mol%, or 40 mol% DSPC. Figure 1B These results demonstrate the high transfection efficiency and low toxicity of LNPs containing 20 mol% DSPC. Example 5: The therapeutic effect of LNP-treated T cells on target cell killing

[0286] Human T effector cells transfected with 0.037 µg / ml LNP containing 10 mol% to 40 mol% DSPC were incubated with Nalm6 target cells at E:T ratios of 0.01 to 20, and the percentage of Nalm6 target cells that died was determined. Effector T cells transfected with LNP containing 20 mol%, 30 mol%, and 40 mol% DSPC killed more Nalm6 target cells compared to effector T cells transfected with LNP containing 10 mol% DSPC. Figure 2AIn effector T cells, when transfected with LNP containing 20 mol% DSPC, over 50% of the cells expressed CAR, compared to only 10% in T effector cells transfected with LNP containing 10 mol% DSPC. Approximately 30% and 40% of effector T cells transfected with LNP containing 30 mol% and 40 mol% DSPC, respectively, expressed CAR. Figure 2B Furthermore, compared with T cells transfected with LNPs containing 10 mol%, 30 mol%, or 40 mol% DSPC, effector T cells transfected with LNPs containing 20 mol% DSPC showed the highest mean fluorescence intensity (MFI) of CAR. Figure 2C ).

[0287] Effector cell function was measured over a 16-day period. Effector T cells transfected with LNP containing 10 mol% to 40 mol% DSPC were co-cultured with Nalm6 target cells expressing red fluorescent protein at an E:T ratio of 2:1, and red fluorescence was measured over a 16-day period. Fresh Nalm6 target cells were added to the co-culture every 2 to 3 days, and LNP was added to the co-culture on day 8. Over the 16-day period, only effector T cells transfected with LNP containing 20 mol% DSPC effectively eliminated Nalm6 target cells (…). Figure 3A and Figure 3B In contrast, effector T cells transfected with LNP containing 10 mol% DSPC only slightly reduced the number of Nalm6 target cells. Figure 3A and Figure 3B Furthermore, in the group transfected with 20 mol% DSPC LNP, approximately 55% of the lymphocytes were viable CD8+ effector T cells on day 16, while this percentage was less than 20% in the group transfected with 10 mol% DSPC LNP. This indicates that the 20 mol% DSPC LNP group had prolonged target binding, proliferation, and survival. Figure 3C ). Example 6: Therapeutic effects of LNP targeting T cells from different human donors

[0288] T cells from two different human donors (donor 10208 and donor 11771) were transfected with 0.11 µg / ml LNP containing 10 mol%, 12.5 mol%, 15 mol%, and 20 mol% DSPC helper lipids, as well as CAR mRNA and GFP mRNA. The percentage of CAR+ cells and CAR MFI was determined immediately or at 24, 48, 72, and 144 hours. During the 72-hour measurement period, T cells from both donors transfected with LNP containing 15 mol% or 20 mol% DSPC showed approximately 80% CAR+ cells, while T cells transfected with LNP containing 10 mol% or 12.5 mol% showed a lower percentage of CAR+ cells, which decreased after 48 hours. Figure 4A and Figure 4C T cells transfected with LNP containing 20 mol% DSPC from both donors showed the highest CAR MFI, while T cells transfected with LNP containing 15 mol% DSPC showed a slightly lower CAR MFI, and T cells transfected with LNP containing 10 mol% and 12.5 mol% DSPC showed significantly lower CAR MFI. Figure 4B and Figure 4D ).

[0289] In CAR+ cells from both donors, T cells transfected with LNP containing 20 mol% DSPC showed the highest CAR MFI compared to T cells transfected with LNP containing 10 mol%, 12.5 mol%, or 15 mol% DSPC. Figure 4E and Figure 4F ).

[0290] These results indicate that LNPs containing 20 mol% DSPC helper lipids efficiently transfected human T cells from different donors and provided CAR-expressing T cells over a 72-hour period. In contrast, LNPs containing 10 mol% DSPC showed the lowest transfection efficiency and resulted in loss of transfection around 48 hours. LNPs containing 15 mol% DSPC showed similar transfection efficiency to those containing 20 mol% DSPC but with a lower overall CAR MFI. LNPs containing 12.5 mol% DSPC performed between those containing 10 mol% DSPC and 15 mol% DSPC.

[0291] The binding and expression of transfected CARs to CAR target proteins were determined by primary staining with recombinant antigen, secondary staining with FITC or APC-conjugated target antigen antibodies, and analysis by flow cytometry. T cells transfected with LNPs containing 20 mol% DSPC showed the highest binding rate to the immobilized CAR target compared to T cells transfected with 15 mol%, 12.5 mol%, or 10 mol% DSPC LNPs. Figures 5A to 5C ). Example 7: Transfection of artificial hematopoietic stem cells

[0292] Humanized NSG™ mice were generated using CD117+ / CD34+ human hematopoietic stem cells (HSCs). HSCs coated with anti-CD117 Fab (Ab1) targeting LNPs were engineered using an LNP formulation containing 10 mol% DSPC (Formulation 1) or an LNP formulation containing 20 mol% DSPC (Formulation 2) (complete LNP compositions are shown in Table 2).

[0293] Humanized NSG™ mice were treated with intravenous injection of 1 mg / kg LNP containing encapsulated mCherry mRNA and coated with anti-CD117 Fab, and transfection efficiency was measured in NSG™ bone marrow.

[0294] Using two different LNP formulations, transfection efficiency was measured in vivo in the range of 20% to approximately 80%. Figure 6A Furthermore, in vivo, the median fluorescence intensity of HSCs transfected with formulation 2 was more than 2-fold increased compared to HSCs transfected with formulation 1 (not shown). In NSG™ mice treated with 20 mol% DSPC LNP, lower off-target signals were measured in the liver and lungs compared to 10 mol% DSPC. Figure 6B ).

[0295] Overall, these results indicate that the LNP containing 20 mol% DSPC described in this article enhances the efficacy of in vivo transfection of human HSCs.

[0296] CD117+ / CD34+ human hematopoietic stem cells (HSCs) were cultured in vitro and transfected with different doses of HSCs coated with anti-CD117Fab (Ab1) targeting LNPs—using an LNP preparation containing 10 mol% DSPC (Preparation 1) or an LNP preparation containing 20 mol% DSPC (Preparation 2) (the complete LNP composition is shown in Table 2).

[0297] Using the lowest dose of LNP preparation 1 at approximately 0.01 μg / mL, the transfection efficiency was 5%, while using the same dose of LNP preparation 2, the transfection efficiency was 50%. Figure 7A ).like Figure 7B As shown, the MFI increased by approximately 4 times when using LNP formulation 2 at 1 μg / mL compared to formulation 1 at the same dose.

[0298] In summary, these results demonstrate that the LNP containing 20 mol% DSPC described in this article enhances the efficacy of in vitro transfection of human HSCs.

[0299] In this application, when an element or component is said to be included in and / or selected from the list of listed elements or components, it should be understood that the element or component can be any one of the listed elements or components, or the element or component can be selected from a group of two or more of the listed elements or components.

[0300] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways, whether explicitly stated or implied herein, without departing from the spirit and scope of the invention. For example, unless otherwise understood from the context, when a particular compound is referred to, that compound can be used in various aspects of the compositions of the invention and / or the methods of the invention. In other words, within this application, aspects have been described and depicted in a manner that enables clear and concise writing and drawing, but it is intended and will be understood that aspects can be combined or separated in various ways without departing from the teachings of the invention and one or more of the inventions. For example, it should be understood that all features described and depicted herein are applicable to all aspects of one or more of the inventions described and depicted herein. Example 1. A lipid nanoparticle for targeted delivery of nucleic acids to target cells, the lipid nanoparticle comprising a cell-targeting group and a lipid blend comprising an ionizable lipid, a structural lipid, an accessory lipid and a PEG lipid, wherein the accessory lipid is present in the lipid blend in the range of about 16 mol% to about 40 mol%. 2. The lipid nanoparticles as described in Example 1, wherein the ionizable lipid is a compound of Formula I. (I), Or its salt, wherein: R 1 R 2 and R 3 Each is an independent key or C 1-3 Alkylene; R1A R 2A and R 3A Each is an independent key or C 1-10 Alkylene; R 1A1 R 1A2 R 1A3 R 2A1 R 2A2 R 2A3 R 3A1 R 3A2 and R 3A3 H and C are independent of each other. 1-20 Alkyl, C 1-20 Alkenyl, -(CH2) 0-10 C(O)OR a1 Or -(CH2) 0-10 OC(O)R a2 ; R a1 and R a2 Each is C independently 1-20 Alkyl or C 1-20 alkenyl; R 3B yes R 3B1 It is C 1-6 Alkylene; and R 3B2 and R 3B3 Each is independently H or C 1-6 alkyl. 3. The lipid nanoparticles as described in Example 2, wherein the ionizable lipid is . 4. The lipid nanoparticles as described in any one of Examples 1 to 3, wherein the lipid nanoparticles further comprise a loading. 5. The lipid nanoparticles as described in any one of Examples 1 to 4, wherein the auxiliary lipid is selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and sphingomyelin. 6. The lipid nanoparticles as described in any one of Examples 1 to 5, wherein the auxiliary lipid is DSPC. 7. The lipid nanoparticles as described in any one of Examples 1 to 6, wherein the auxiliary lipid is present in the lipid blend in the range of about 16 mol% to about 30 mol%. 8. The lipid nanoparticles as described in Example 7, wherein the auxiliary lipid is present in the lipid blend in the range of about 16 mol% to about 25 mol%. 9. The lipid nanoparticles as described in Example 8, wherein the auxiliary lipid is present in the lipid blend at about 20 mol%. 10. The lipid nanoparticles as described in any one of Examples 1 to 9, wherein the structural lipid is selected from the group consisting of: cholesterol, coprosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, stigmasterol and brassosterol. 11. The lipid nanoparticles as described in any one of Examples 1 to 10, wherein the PEG lipid is selected from the group consisting of: distearylglycerol-PEG (DSG-PEG), distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), dimyristoyl-phosphatidylethanolamine-PEG (DMPE-PEG), distearyl-glycerol-phosphate-PEG (DSPG-PEG), dimyristoyl-glycerol-PEG (DMG-PEG), dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG), dipalmitoyl-glycerol-PEG (DPG-PEG), and ceramide-PEG. 12. The lipid nanoparticles as described in any one of Examples 1 to 11, wherein the ionizable lipid is present in the lipid blend in the range of about 30 mol% to about 70 mol%. 13. The lipid nanoparticles as described in any one of Examples 1 to 12, wherein the ionizable lipid is present in the lipid blend in the range of about 40 mol% to about 60 mol%. 14. The lipid nanoparticles as described in any one of Examples 1 to 13, wherein the ionizable lipid is present in the lipid blend in the range of about 48 mol% to about 50 mol%. 15. The lipid nanoparticles as described in any one of Examples 1 to 14, wherein the structural lipid is present in the lipid blend in the range of about 20 mol% to about 50 mol%. 16. The lipid nanoparticles as described in any one of Examples 1 to 15, wherein the structural lipid is present in the lipid blend in the range of about 30 mol% to about 40 mol%. 17. The lipid nanoparticles as described in any one of Examples 1 to 16, wherein the structural lipid is cholesterol. 18. The lipid nanoparticles as described in Example 17, wherein cholesterol is present in the lipid blend in the range of about 25 mol% to about 40 mol%. 19. The lipid nanoparticles as described in any one of Examples 1 to 18, wherein the PEG lipid is present in the lipid blend in the range of about 1 mol% to about 4 mol%. 20. The lipid nanoparticles as described in any one of Examples 1 to 19, wherein the PEG lipid is DPG-PEG or DSPE-PEG. 21. The lipid nanoparticles as described in Example 20, wherein the PEG in DPG-PEG has a molecular weight of about 2000 Daltons (DPG-PEG2K), and the PEG in DSPE-PEG has a molecular weight of about 2000 Daltons (DSPE-PEG2K) or about 3400 Daltons (DSPE-PEG3.4K). 22. The lipid nanoparticles as described in 21, wherein DPG-PEG2K, DSPE-PEG2K, or DSPE-PEG3.4K are present in the lipid blend in the range of about 1 mol% to about 2 mol%. 23. The lipid nanoparticles as described in any one of Examples 1 to 22, wherein lipid 15 is present in the lipid blend at about 49.25 mol%, cholesterol at about 29.25 mol%, DSPC at about 20 mol%, and DPG-PEG2K at about 1.5 mol%. 24. The lipid nanoparticles as described in any one of Examples 1 to 23, wherein the lipid nanoparticles have an average diameter of about 60 nm to about 100 nm. 25. The lipid nanoparticles as described in any one of Examples 1 to 24, wherein the lipid nanoparticles have an average diameter of about 80 nm. 26. The lipid nanoparticles as described in any one of Examples 1 to 25, wherein the lipid nanoparticles have a polydispersity index (PDI) of about 0.05 to about 1. 27. The lipid nanoparticles as described in Example 26, wherein the lipid nanoparticles have a PDI of about 0.09. 28. The lipid nanoparticles as described in any one of Examples 1 to 27, wherein the lipid nanoparticles have a zeta potential (ZP) of about -30 mV to about +5 mV. 29. The lipid nanoparticles as described in Example 28, wherein the lipid nanoparticles have a ZP of about -1.4 mV. 30. The lipid nanoparticles as described in any one of Examples 4 to 29, wherein the loading comprises one or more nucleic acids. 31. The lipid nanoparticles as described in Example 30, wherein the one or more nucleic acids are DNA. 32. The lipid nanoparticles as described in Example 30, wherein the one or more nucleic acids are RNA. 33. The lipid nanoparticles as described in Example 32, wherein the RNA comprises guide RNA and RNA encoding Cas enzyme. 34. Lipid nanoparticles as described in any one of Examples 30 to 32, wherein the one or more nucleic acids encode a chimeric antigen receptor (CAR). 35. The lipid nanoparticles as described in any one of Examples 1 to 34, wherein the cell-targeting group is coupled to the lipids of the lipid nanoparticles to form a lipid-cell-targeting group conjugate. 36. The lipid nanoparticles as described in Example 35, wherein the cell-targeting group is coupled to the PEG-lipid. 37. The lipid nanoparticles as described in Example 36, wherein the cell-targeting group is coupled to DPG-PEG2K, DSPE-PEG2K, or DSPE-PEG3.4K. 38. The lipid nanoparticles as described in any one of Examples 35 to 37, wherein the cell-targeting group comprises an antibody or a fragment thereof. 39. The lipid nanoparticles as described in Example 38, wherein the cell-targeting group comprises an antibody. 40. The lipid nanoparticles as described in Example 38, wherein the cell-targeting group comprises a Fab fragment. 41. The lipid nanoparticles as described in Example 38, wherein the cell-targeting group comprises a single variable domain. 42. The lipid nanoparticles as described in any one of Examples 35 to 41, wherein the cell-targeting group binds to molecules on immune cells. 43. The lipid nanoparticles as described in Example 42, wherein the molecule on the immune cell is selected from the group consisting of CD3, CD4, CD7 and CD8. 44. The lipid nanoparticles as described in any one of Examples 35 to 41, wherein the cell-targeting group binds to molecules on hematopoietic stem cells. 45. The lipid nanoparticles as described in Example 44, wherein the molecule on the hematopoietic stem cell is selected from the group consisting of CD34, CD105 and CD117. 46. ​​The lipid nanoparticles as described in any one of Examples 35 to 45, wherein the lipid-cell-targeting group conjugate is present in the lipid nanoparticles in the range of about 0.001 mol% to about 0.5 mol%. 47. A pharmaceutical composition comprising lipid nanoparticles as described in any one of Examples 1 to 46, and one or more pharmaceutically acceptable carriers or excipients. 48. A method for delivering nucleic acids into cells in a subject, the method comprising administering to the subject lipid nanoparticles as described in any one of Examples 1 to 46 or a pharmaceutical composition as described in Example 47. 49. The method as described in Example 48, wherein the cell is an immune cell. 50. The method as described in Example 48, wherein the cell is a hematopoietic stem cell. 51. Use of the lipid nanoparticles as described in any one of Examples 1 to 46 or the pharmaceutical composition as described in Example 47 in the preparation of a pharmaceutical agent for delivering nucleic acids into cells. 52. Use as described in Example 51, wherein the cell is an immune cell. 53. The use as described in Example 51, wherein the cell is a hematopoietic stem cell. 54. The lipid nanoparticles as described in any one of Examples 1 to 46, or the pharmaceutical composition as described in Example 47, for use in delivering nucleic acids into cells. 55. Lipid nanoparticles as described in Example 54, wherein the cell is an immune cell. 56. The lipid nanoparticles as described in Example 54, wherein the cell is a hematopoietic stem cell. 57. A method of treating a disease in a subject in need, the method comprising administering to the subject lipid nanoparticles as described in any one of Examples 1 to 46 or a pharmaceutical composition as described in Example 47. 58. A method of treating a disease in a subject, the method comprising administering lipid nanoparticles as described in any one of Examples 1 to 46 or a pharmaceutical composition as described in Example 47 to immune cells of a subject in need. 59. The method as described in Example 58, wherein the application is targeted at multiple immune cells. 60. The method as described in Example 59, wherein, 72 hours after application of the lipid nanoparticles, proteins encoded by the one or more nucleic acids configured in the lipid nanoparticles are detected in at least 60% (or even 80%) of immune cells. 61. The method as described in Example 60, wherein the protein was detected in more immune cells of the subject 72 hours after administration of the lipid nanoparticles compared to another subject who received the adjuvant lipid in 10 mol% of the lipid blend. 62. The method as described in Example 59, wherein the effector function of the one or more immune cells is enhanced by the protein encoded by the one or more nucleic acids configured in the lipid nanoparticles compared to one or more immune cells that have not been treated with the lipid nanoparticles. 63. A method of treating a subject's disease, the method comprising administering lipid nanoparticles as described in any one of Examples 1 to 41 and 44 to 46 or a pharmaceutical composition as described in Example 47 to hematopoietic stem cells of a subject in need. 64. The method as described in Example 63, wherein the lipid nanoparticles are administered to multiple hematopoietic stem cells of the subject. 65. The method as described in Example 63 or 64, wherein a protein encoded by the one or more nucleic acids configured in the lipid nanoparticles is detected in at least 80% of the hematopoietic stem cells of the subject. 66. The method as described in Example 65, wherein the protein is detected in approximately 20% of the hematopoietic stem cells to which the lipid nanoparticles are applied, the lipid nanoparticles comprising accessory lipids present in the lipid blend at 10 mol%. 67. The lipid nanoparticles as described in any one of Examples 1 to 46, or the pharmaceutical composition as described in Example 47, for use in treating a disease in a subject in need. 68. The lipid nanoparticles as described in any one of Examples 1 to 43 and 46, or the pharmaceutical composition as described in Example 47, for use in treating immune cell-related diseases in subjects of need. 69. The lipid nanoparticles as described in any one of Examples 1 to 41 and 44 to 46, or the pharmaceutical composition as described in Example 47, for use in treating hematopoietic stem cell-related diseases in subjects of need. 70. The lipid nanoparticles as described in Example 69, wherein the hematopoietic stem cell-related disease is sickle cell disease.

Claims

1. A lipid nanoparticle for targeted delivery of nucleic acids to target cells, the lipid nanoparticle comprising a cell-targeting group and a lipid blend comprising an ionizable lipid, a structural lipid, an accessory lipid and a PEG lipid, wherein the accessory lipid is present in the lipid blend in the range of about 16 mol% to about 40 mol%.

2. The lipid nanoparticles of claim 1, wherein the ionizable lipid is a compound of formula I. (I)， or its salt, wherein: R 1 R 2 and R 3 Each is an independent key or C 1-3 Alkylene; R 1A R 2A and R 3A Each is an independent key or C 1-10 Alkylene; R 1A1 R 1A2 R 1A3 R 2A1 R 2A2 R 2A3 R 3A1 R 3A2 and R 3A3 H and C are independent of each other. 1-20 Alkyl, C 1-20 Alkenyl, -(CH2) 0-10 C(O)OR a1 Or -(CH2) 0-10 OC(O)R a2 ; R a1 and R a2 Each is C independently 1-20 Alkyl or C 1-20 alkenyl; R 3B yes R 3B1 It is C 1-6 Alkylene; and R 3B2 and R 3B3 Each is independently H or C 1-6 alkyl.

3. The lipid nanoparticles of claim 2, wherein the ionizable lipid is 。 4. The lipid nanoparticles according to any one of claims 1 to 3, wherein the lipid nanoparticles further comprise a loading.

5. The lipid nanoparticles according to any one of claims 1 to 4, wherein the auxiliary lipid is selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and sphingomyelin.

6. The lipid nanoparticles according to any one of claims 1 to 5, wherein the auxiliary lipid is DSPC.

7. The lipid nanoparticles according to any one of claims 1 to 6, wherein the auxiliary lipid is present in the lipid blend in the range of about 16 mol% to about 30 mol%.

8. The lipid nanoparticles of claim 7, wherein the auxiliary lipid is present in the lipid blend in the range of about 16 mol% to about 25 mol%.

9. The lipid nanoparticles of claim 8, wherein the auxiliary lipid is present in the lipid blend at about 20 mol%.

10. The lipid nanoparticles according to any one of claims 1 to 9, wherein the structural lipid is selected from the group consisting of: cholesterol, coprosterol, β-sitosterol, ergosterol, campesterol, stigmasterol, stigmasterol and brassosterol.

11. The lipid nanoparticles of any one of claims 1 to 10, wherein the PEG lipid is selected from the group consisting of: distearylglycerol-PEG (DSG-PEG), distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), dimyristoyl-phosphatidylethanolamine-PEG (DMPE-PEG), distearyl-glycerol-phosphate-PEG (DSPG-PEG), dimyristoyl-glycerol-PEG (DMG-PEG), dipalmitoyl-phosphatidylethanolamine-PEG (DPPE-PEG), dipalmitoyl-glycerol-PEG (DPG-PEG), and ceramide-PEG.

12. The lipid nanoparticles according to any one of claims 1 to 11, wherein the ionizable lipid is present in the lipid blend in the range of about 30 mol% to about 70 mol%.

13. The lipid nanoparticles according to any one of claims 1 to 12, wherein the ionizable lipid is present in the lipid blend in the range of about 40 mol% to about 60 mol%.

14. The lipid nanoparticles of any one of claims 1 to 13, wherein the structural lipid is cholesterol.

15. The lipid nanoparticles of claim 14, wherein cholesterol is present in the lipid blend in the range of about 25 mol% to about 40 mol%.

16. The lipid nanoparticles according to any one of claims 1 to 15, wherein the PEG lipid is present in the lipid blend in the range of about 1 mol% to about 4 mol%.

17. The lipid nanoparticles according to any one of claims 1 to 16, wherein the PEG lipid is DPG-PEG or DSPE-PEG.

18. The lipid nanoparticles of claim 17, wherein the PEG in DPG-PEG has a molecular weight of about 2000 Daltons (DPG-PEG2K), and the PEG in DSPE-PEG has a molecular weight of about 2000 Daltons (DSPE-PEG2K) or about 3400 Daltons (DSPE-PEG3.4K).

19. The lipid nanoparticles of any one of claims 1 to 18, wherein lipid 15 is present in the lipid blend at about 49.25 mol%, cholesterol at about 29.25 mol%, DSPC at about 20 mol%, and DPG-PEG2K at about 1.5 mol%.

20. The lipid nanoparticles according to any one of claims 1 to 19, wherein the lipid nanoparticles have an average diameter of about 60 nm to about 100 nm.

21. The lipid nanoparticles according to any one of claims 1 to 20, wherein the lipid nanoparticles have a polydispersity index (PDI) of about 0.05 to about 1.

22. The lipid nanoparticles according to any one of claims 1 to 21, wherein the lipid nanoparticles have a zeta potential (ZP) of about -30 mV to about +5 mV.

23. The lipid nanoparticles of any one of claims 4 to 22, wherein the loading comprises one or more nucleic acids.

24. The lipid nanoparticles of claim 23, wherein the one or more nucleic acids are RNA.

25. The lipid nanoparticle of any one of claims 1 to 24, wherein the cell-targeting group is coupled to the lipid of the lipid nanoparticle to form a lipid-cell-targeting group conjugate.

26. The lipid nanoparticles of claim 25, wherein the cell-targeting group is coupled to the PEG lipid.

27. The lipid nanoparticle of any one of claims 25 to 26, wherein the cell-targeting group comprises an antibody or a fragment thereof.

28. The lipid nanoparticles of any one of claims 25 to 27, wherein the cell-targeting group binds to molecules on immune cells.

29. The lipid nanoparticle of claim 28, wherein the molecule on the immune cell is selected from the group consisting of CD3, CD4, CD7 and CD8.

30. The lipid nanoparticles of any one of claims 25 to 27, wherein the cell-targeting group binds to molecules on hematopoietic stem cells.

31. The lipid nanoparticles of claim 30, wherein the molecule on the hematopoietic stem cell is selected from the group consisting of CD34, CD105 and CD117.

32. The lipid nanoparticles according to any one of claims 25 to 31, wherein the lipid-cell-targeting conjugate is present in the lipid nanoparticles in the range of about 0.001 mol% to about 0.5 mol%.

33. A pharmaceutical composition comprising lipid nanoparticles as described in any one of claims 1 to 32, and one or more pharmaceutically acceptable carriers or excipients.

34. A method of delivering nucleic acids into cells in a subject, the method comprising administering to the subject lipid nanoparticles as described in any one of claims 1 to 32 or a pharmaceutical composition as described in claim 33.

35. The method of claim 34, wherein the cell is an immune cell.

36. The method of claim 34, wherein the cell is a hematopoietic stem cell.

37. A method of treating a disease in a subject in need, the method comprising administering to the subject lipid nanoparticles as described in any one of claims 1 to 32 or a pharmaceutical composition as described in claim 33.

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