Lipid nanoparticles for delivering therapeutic payloads to cells

Abstract

This disclosure provides lipid nanoparticles and a conjugate comprising a target-directed portion and lipid nanoparticles (LNPs) for encapsulating a therapeutic agent (e.g., a payload) for delivery to hematopoietic stem cells (HSCs). The conjugate can be formulated in a pharmaceutical composition and administered directly to a subject in need of treatment (e.g., via in vivo administration).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Application No. 63 / 582,456 filed on 13 September 2023, U.S. Provisional Application No. 63 / 592,196 filed on 22 October 2023, U.S. Provisional Application No. 63 / 582,457 filed on 13 September 2023, U.S. Provisional Application No. 63 / 592,195 filed on 22 October 2023, and U.S. Provisional Application No. 63 / 677,892 filed on 31 July 2024, the contents of each of these applications being incorporated herein by reference in their entirety.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (252052001340seqlist.xml, size: 430,624 bytes, and creation date: September 13, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0003] Hematopoietic stem cells (HSCs) are a rare cell population found in the bone marrow that functions as the parent cell lineage for both red and white blood cells. HSCs are uniquely capable of self-renewal and multi-lineage differentiation, and therefore, correcting defective genes at the stem cell level can lead to long-term therapeutic effects, making them an attractive target for genetic medicine therapy (Shi, Nano Letters 23:2938-2944, 2023).

[0004] In recent years, clinical success with autologous stem cell gene editing therapy has been demonstrated for diseases including β-hemoglobin disorders (Frangoul, N Engl J Med, 384:252-260, 2021), primary immunodeficiency disorders (Kohn, N Engl J Med 384:2002-2013, 2021), and metabolic disorders (Ge Engl J Med 385:1929-1940, 2021). To date, autologous HSC gene therapy and gene editing approaches for treating β-hemoglobin disorders such as sickle cell disease and beta-thalassemia have generally been performed ex vivo. This process involves HSC mobilization via G-CSF and Plerixafor, followed by HSC collection via bone marrow aspiration or apheresis. Next, bone marrow or apheresis samples are sorted to isolate a CD34+ subset containing the desired long-term HSC (LT-HSC) subpopulation. The isolated CD34+ HSCs are then edited and expanded in vitro before being returned to the patient for final adoptive transfer. To maximize the engraftment of edited CD34+ LT-HSCs, patients are typically “conditioned” with chemotherapeutic agents such as busulfan or undergo total body radiotherapy to deplete unedited LT-HSCs and their offspring from the bone marrow microenvironment. These treatments cause undesirable side effects such as short-term immunosuppression and long-term effects such as infertility or predisposition to certain neoplasms. Furthermore, the technical complexity and associated costs of ex vivo autologous stem cell gene therapy limit the geographical location where the treatment can be performed and the patient population that can afford it.

[0005] To extend the therapeutic potential of HSC therapy to a broader set of patients, one alternative is direct in vivo HSC engineering, which delivers gene therapy or gene editors directly to the bone marrow microenvironment without requiring the collection, culture, and conditioning of HSCs (Shi, Nano Letters 23:2938-2944, 2023). However, specific delivery of gene therapy or gene editors to the rare long-term HSC (LT-HSC) population in the bone marrow microenvironment can be challenging, as any systemically delivered therapeutic agent must circumvent or overcome the numerous mechanisms (e.g., hepatic uptake, phagocytic cell uptake, complement activation, opsonization, phagocytic cell uptake, etc.) that hinder its delivery to the bone marrow microenvironment. Once in the bone marrow, the therapeutic agent must be specifically delivered to the self-regenerating LT-HSC population to provide a permanent cure. Therefore, there is a therapeutic need to develop compositions that can effectively deliver therapeutic agents to HSCs (e.g., LT-HSCs) in patients. [Overview of the project]

[0006] This disclosure provides lipid nanoparticles (LNPs) or conjugates comprising lipid nanoparticles (LNPs) that encapsulate a payload, such as a therapeutic agent (e.g., a gene modification system) or a reporter construct, for delivery to hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). In some cases, the LNPs and conjugates described herein can deliver the payload to HSCs and / or HPCs more effectively than a baseline conjugate or baseline LNP. In some embodiments, this disclosure also provides conjugates comprising lipid nanoparticles (LNPs) that encapsulate a payload, such as a therapeutic agent (e.g., a gene modification system) or a reporter construct, for delivery to HSCs and / or HPCs, wherein the LNP has at least one target-directed moiety on its surface. Conjugates having at least one target-directed moiety described herein are also referred to as targeted LNPs or tLNPs.

[0007] In one embodiment, the present disclosure provides lipid nanoparticles (LNPs) for delivering therapeutic agents in vivo to HSCs and / or HPCs, wherein the LNPs comprise ionizable lipids and helper lipids, the ionizable lipids being selected from V003 or the lipids of Tables 1, 2 and 3, as shown in the following disclosure, and the therapeutic agent being encapsulated within the LNPs. Any reference to ionizable lipids in LNPs as shown herein will be understood to also include pharmaceutically acceptable salts of the lipids.

[0008] In other embodiments, the disclosure provides conjugates comprising LNPs and target-directing moieties. In some cases, the inclusion of target-directing moieties in targeted LNPs enhances the ability of the conjugate or LNP to deliver a payload to HSCs and HPCs (collectively, HSPCs). Targeted LNPs can be formulated in pharmaceutical compositions and administered directly to subjects (e.g., patients) requiring them (e.g., via in vivo administration). Once administered, the composition can deliver significant amounts of the therapeutic agent to the target HSCs (e.g., LT-HSCs). Alternatively, LNPs can be administered ex vivo to isolated HSCs (e.g., LT-HSCs). In some embodiments, the payload can be a nucleic acid encoding a reporter gene, such as green fluorescent protein (GFP). In some embodiments, the payload can be a therapeutic agent, e.g., a therapeutic peptide or protein, a nucleic acid containing the therapeutic agent, or a nucleic acid encoding the therapeutic agent. In some embodiments, the therapeutic agent can be a gene drug, which can modify, alter, or influence changes in the genomic DNA of cells (e.g., cells in a subject). In some embodiments, the therapeutic agent is a gene therapy agent or a gene editing agent. In some embodiments, the therapeutic agent is a gene modification polypeptide. In some embodiments, the therapeutic agent is a gene modification system.

[0009] In some embodiments, the target-directed moiety is an antibody, a Fab fragment or single-stranded variable fragment (scFv), DARPIN, a VHH domain antibody, an FN3 domain, a nanobody, a single-domain antibody, or centinrin. In other embodiments, the target-directed moiety is a folic acid moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, or N-acetylgalactosamine (GalNac). In some embodiments, the target-directed moiety is a peptide or protein that binds to a receptor (e.g., a receptor on the surface of an HSC or LT-HSC), such as a ligand or a part of a ligand. In some embodiments, the target-directed moiety is a peptide or protein that binds to a receptor or ligand on the surface of an HSC, HPC, or LT-HSC, and the affinity of the peptide or protein target-directed moiety to the receptor or ligand on the surface of an HSC, HPC, or LT-HSC is regulated via phage display.

[0010] In certain embodiments, the payload delivered by the LNP or targeted LNP (e.g., a therapeutic agent) may be a small molecule, a peptide or protein, non-coding RNA (ncRNA), gRNA, siRNA or miRNA, nucleic acid encoding a peptide or protein (e.g., a protein for substitution gene therapy, or a protein for modifying or altering a genome or epigenome, e.g., a protein for gene editing), one or more components of a system for modifying a genome (e.g., one or more components of a ribonucleic acid protein (RNP) complex for editing or altering a genome or epigenome (e.g., insertion deletion) The LNPs may be for introducing mutations (indels), base editing, epigenetic editing, or targeted prime reverse transcription (TPRT) by mechanisms requiring the function of a recombinase, transposase, retransposase, helicase, reverse transcriptase, polymerase, deaminase, methylase, demethylase, or ligase, or a combination thereof. In certain embodiments, the therapeutic agent delivered by the LNP or targeted LNP (i.e., payload) may be a genetically modified protein, a nucleic acid encoding a genetically modified protein, or a genetic modification system, as described herein.

[0011] In one embodiment, the disclosure provides a targeted LNP (conjugate) that encapsulates one or more nucleic acids encoding components of a system for modifying or altering DNA (e.g., genomic DNA), wherein the targeted LNP comprises one or more target-directed moieties, such as antibodies, Fab fragments, or scFvs, that are capable of binding to one or more proteins on the cell surface of hematopoietic stem cells (HSCs). In some embodiments, the targeted LNP is delivered in vivo to a subject (e.g., a human patient) that requires it. After in vivo administration, the nucleic acid encoding components of a system for modifying or altering the genome can be expressed, resulting in in vivo modification of the HSC genome (e.g., gene editing). Surprisingly, it has been found that LNPs comprising a specific combination of one or more target-directed moieties (e.g., antibodies, Fab fragments, or scFvs), ionizable lipids, and / or helper lipids can improve the delivery of a therapeutic payload (e.g., a genome-modifying system such as a gene modification system) to HSCs after in vivo administration to the subject, thereby resulting in an improved level of therapeutic activity in the HSCs.

[0012] In some embodiments, LNPs or targeted LNPs (conjugates) containing ionizable lipids as described herein can deliver payloads (e.g., therapeutic payloads) to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% more HSCs, HSPCs, or LT-HSCs than LNPs containing baseline lipids (e.g., V003). In some embodiments, LNPs containing ionizable lipids as described herein can deliver payloads (e.g., therapeutic payloads) to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% more HSCs, HSPCs, or LT-HSCs in animal models than LNPs containing baseline lipids (e.g., V003), as described herein. In some embodiments, the LNPs are targeted LNPs.

[0013] In some embodiments, the payload delivered to HSCs, HPCs, or LT-HSCs by the ionizable lipid-containing LNP described herein (e.g., a therapeutic payload) is expressed at levels at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% higher than the baseline lipid-containing payload (e.g., V003). In some embodiments, the payload delivered to HSCs, HSPCs, or LT-HSCs by LNPs containing ionizable lipids as described herein (e.g., a therapeutic payload) is expressed at levels at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% higher than the payload containing baseline lipids (e.g., V003). In some embodiments, the LNP is a targeted LNP.

[0014] In some embodiments, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% of the HSCs in the subject (e.g., LT-HSCs) include genomes that have been modified or altered after administration of LNPs described herein, such as targeted LNPs that encapsulate a therapeutic agent (e.g., a system for modifying, altering, or editing a genome). In some embodiments, about 5% to about 50%, about 5% to about 20%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, or about 20% to about 30% of the HSCs in the subject include genomes that have been modified or altered after administration of LNPs described herein, such as targeted LNPs.

[0015] High levels of in vivo gene editing following administration of the targeted LNPs of this disclosure enable the effective treatment of certain diseases, such as β-hemoglobin disorders, primary immunodeficiency disorders, and metabolic disorders. In some embodiments, the targeted LNPs of this disclosure can be used to treat sickle cell disease. In other embodiments, the targeted LNPs of this disclosure can be used to treat beta-thalassemia.

[0016] In some embodiments, LNPs or targeted LNPs are delivered ex vivo to isolated HSCs of a subject requiring them (e.g., a human patient). In some such embodiments, the HSCs are first collected from the bone marrow of the subject. Following the isolation of the desired HSC subpopulation (e.g., LT-HSCs), the targeted LNPs of this disclosure can be mixed with the HSCs, thereby resulting in effective transduction of the payload (e.g., a gene modification system) into the HSCs. The modified HSCs can then be administered to the patient for engraftment, as described in the art.

[0017] In some embodiments, the LNPs of this disclosure, such as targeted LNPs, include the ionizable lipid V003 shown below. [ka]

[0018] In some embodiments, the LNPs of this disclosure, such as targeted LNPs, include ionizable lipids from Table 1. In some embodiments, the LNP (e.g., targeted LNP) includes ionizable lipids from Table 2. In some embodiments, the LNP (e.g., targeted LNP) includes ionizable lipids from Table 3. In some embodiments, the LNPs and targeted LNPs detailed herein include any one or more ionizable lipids as detailed in Tables 1, 2, and 3. In some embodiments, the LNPs and targeted LNPs detailed herein include two or more ionizable lipids, such as any two or more of V003 and ionizable lipids from Tables 1, 2, and 3. In some embodiments, the LNPs and targeted LNPs detailed herein include only one ionizable lipid selected from the group consisting of ionizable lipids from Tables 1, 2, and 3. In some cases, LNPs containing the ionizable lipids described herein exhibit higher levels of transduction and / or higher expression of payload proteins in HSCs compared with LNPs containing V003 as the ionizable lipid. In some cases, some of the ionizable lipids described herein, when used as components of targeted LNPs for in vivo delivery to HSCs, provide higher levels of transduction and / or protein expression compared with V003. In some embodiments, the ionizable lipid has one of the structures shown below. [ka] [ka] [ka]

[0019] In some embodiments, the LNPs of this disclosure, e.g., targeted LNPs (conjugates), comprise one or more pegylated lipid molecules. In some embodiments, the target-directing moiety of the targeted LNP (e.g., an antibody, Fab fragment, or scFv) is associated with or chemically bound to at least one of the pegylated lipid molecules. In some embodiments, the targeted LNP comprises about 0.05 mol% to about 2 mol% of the pegylated lipid conjugated to the target-directing moiety. In some embodiments, the PEG spacer between the lipid and the target-directing moiety comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10 to 120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid conjugated to the target-directing moiety is about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bound to the target-directing moiety is approximately 1,000 (i.e., PEG1000) to approximately 3,000 (i.e., PEG5300). In some embodiments, the lipid moiety of the pegylated lipid bound to the target-directing moiety is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid.

[0020] As disclosed herein, different pegylated lipids in the targeted LNPs (conjugates) of this disclosure can impart different properties to the LNPs, including pharmacokinetics, distribution, and endosomal release. Surprisingly, it has been found that LNPs containing different pegylated lipids that are not covalently bonded to the target-directing moiety can exhibit different toxic properties, even if all other components of the LNP (e.g., ionizable lipids) and their relative proportions are the same. In particular, it has been found that LNPs containing PEGylated lipids having PEG lipid anchors with 16 carbon atoms (i.e., C16 PEG lipid anchors) or 18 carbon atoms (i.e., C18 PEG lipid anchors) can reduce or eliminate the toxicity associated with the same LNP other than the pegylated lipid moiety. In some embodiments, administration (in vivo or ex vivo) of LNPs containing C16 PEG lipid anchors or C18 PEG lipid anchors does not result in substantial hepatotoxicity. In some embodiments, administration of targeted LNPs does not result in an increase in serum levels of the secretory hepatic enzyme alanine aminotransferase (ALT), or results in a minimal increase.

[0021] In some embodiments, the pegylated lipid has at least one C16 (palmitoyl) PEG lipid anchor. In some embodiments, the pegylated lipid has two C16 PEG lipid anchors (i.e., dialkyl chains with a carbon length of 16). In some embodiments, the pegylated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000). In some embodiments, the pegylated lipid is 1,2-dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (DPG-PEG2000). In some embodiments, the pegylated lipid is C16 PEG ceramide. In some embodiments, targeted LNPs containing C16 pegylated lipids show significantly reduced hepatic uptake compared to other identical LNPs containing C14 pegylated lipids.

[0022] In some embodiments, the pegylated lipid has at least one C18 PEG lipid anchor. In some embodiments, the pegylated lipid has two C18 PEG lipid anchors (i.e., dialkyl chains with a carbon length of 18). In some embodiments, the C18 pegylated lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). In some embodiments, the C18 pegylated lipid is distearoyl-rac-glycerol-PEG2000 (DSG-PEG2000).

[0023] In some embodiments, the LNP of this disclosure (e.g., targeted LNP), e.g., targeted LNP, comprises one or more non-pegylated lipids (e.g., non-pegylated phospholipids). Such lipids are often referred to as helper lipids. In some embodiments, the target-directing moiety (e.g., antibody, Fab fragment, or scFv) is associated with or chemically bound to at least one of the non-pegylated lipids. In some embodiments, the non-pegylated lipid (helper lipid) is selected from POPC, DOPE, and DSPC. In some embodiments, the non-pegylated lipid (helper lipid) is a sphingolipid. In some such embodiments, the non-pegylated lipid is sphingomyelin. In some embodiments, the sphingomyelin has a head group selected from phosphocholine, phosphoethanolamine, or ceramide. In some embodiments, the sphingomyelin is egg sphingomyelin.

[0024] In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in LNPs, e.g., targeted LNPs, is about 18% to about 32%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in targeted LNPs is about 20% to about 30%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in targeted LNPs is about 22% to about 28%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in targeted LNPs is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%. As shown in the following examples, in vivo delivery of a particular payload after administration of the disclosed LNP having these percentages of helper lipids provides enhanced transduction and / or expression of the payload compared to LNPs having less or more helper lipids. It will be understood that the mol% of helper lipids used herein refers to the mol% of the total lipid components of the LNP, excluding the therapeutic agent (i.e., payload) or target-directed moiety.

[0025] In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP (i.e., excluding the target-directing portion or therapeutic agent) is about 18% to about 32%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP is about 20% to about 30%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP is about 22% to about 28%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%. As shown in the following examples, in vivo delivery of a particular payload after administration of the disclosed LNP having these percentages of helper lipids provides enhanced transduction and / or expression of the payload compared to LNPs having less or more helper lipids.

[0026] In some embodiments, this disclosure is, (i) A lipid component comprising ionizable lipids and helper lipids, wherein the ionizable lipids are present in an amount of approximately 35 mol% to approximately 60 mol% of the lipid component, and the helper lipids are present in an amount of approximately 18 mol% to approximately 32 mol% of the lipid component, (ii) One or two target-directed moieties conjugated to an LNP, wherein one or two target-directed moieties are configured to target one or more receptors on the surface of HSCs selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184, (iii) A lipid nanoparticle (LNP) is provided, comprising a therapeutic agent encapsulated within the LNP, which comprises one or more nucleic acid molecules (e.g., RNA molecules) (e.g., one, two, or three nucleic acid molecules (e.g., RNA molecules)). In some embodiments, the therapeutic agent comprises two or more nucleic acids, e.g., RNA molecules.

[0027] In some embodiments, the molar ratio of ionizable lipids to non-pegylated helper lipids (e.g., DSPC or sphingomyelin) in the targeted LNP is about 1:1 to about 7:1. In some embodiments, the molar ratio of ionizable lipids to non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 1:1 to about 4:1. In some embodiments, the molar ratio of ionizable lipids to non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 1:1 to about 3:1. In some embodiments, the molar ratio of ionizable lipids to non-pegylated helper lipids (e.g., DSPC) is about 1:1 to about 2.5:1. In some embodiments, the molar ratio of ionizable lipids to non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 1:1 to about 2:1. In some embodiments, the molar ratio between ionizable lipids and non-pegylated helper lipids (e.g., DSPC) is about 1.5:1 to about 2.5:1. In some embodiments, the molar ratio between ionizable lipids and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 2:1 to about 2.5:1.

[0028] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid O92 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid O92 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0029] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid O93 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid O93 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0030] In some embodiments, the targeted LNP comprises lipid 153 and DSPC. In some embodiments, the LNP, e.g., the targeted LNP, comprises lipid 153 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0031] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 154 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 154 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0032] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 155 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 155 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0033] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 162 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 162 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0034] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 163 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 163 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0035] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 169 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 169 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0036] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 176 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 176 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0037] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 178 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 178 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0038] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 183 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 183 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0039] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 232 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid 232 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0040] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid V003 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid V003 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin (e.g., the lipid component of the LNP) in the LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000. In some embodiments, the LNP further comprises a pegylated lipid containing at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0041] In any of the embodiments described above, the LNP or conjugate may contain one or more cholesterol molecules. In some embodiments, the molar ratio between cholesterol molecules and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 6:1 to about 0.5:1. In some embodiments, the molar ratio between cholesterol molecules and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 3:1 to about 0.5:1. In some embodiments, the molar ratio between cholesterol molecules and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 2:1 to about 0.5:1. In some embodiments, the molar ratio between cholesterol molecules and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 1.5:1 to about 0.5:1. In some embodiments, the molar ratio between cholesterol molecules and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 1:1 to about 0.5:1. In some embodiments, the molar ratio between cholesterol molecules and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 1:2 to about 0.8:1.

[0042] In some embodiments, the LNP includes a target-directed portion that targets receptors on the surface of HSCs selected from CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184, and CD338. In some embodiments, the LNP includes a target-directed portion that targets CD117. In some embodiments, the LNP includes a target-directed portion that targets CD33. In some embodiments, the LNP includes a target-directed portion that targets CD34. In some embodiments, the LNP includes a target-directed portion that targets CD38. In some embodiments, the LNP includes a target-directed portion that targets CD43. In some embodiments, the LNP includes a target-directed portion that targets CD46. In some embodiments, the LNP includes a target-directed portion that targets CD49d. In some embodiments, the LNP includes a target-directed portion that targets CD50. In some embodiments, the LNP includes a target-directional portion that targets CD59. In some embodiments, the LNP includes a target-directional portion that targets CD71. In some embodiments, the LNP includes a target-directional portion that targets CD105. In some embodiments, the LNP includes a target-directional portion that targets CD1117. In some embodiments, the LNP includes a target-directional portion that targets CD123. In some embodiments, the LNP includes a target-directional portion that targets CD164. In some embodiments, the LNP includes a target-directional portion that targets CD184. In some embodiments, the LNP includes a target-directional portion that targets CD338.

[0043] In some embodiments, the LNP includes a target-directed portion containing an array present in any of Tables S1 to S14. In some embodiments, the target-directed portion is coded by an array present in any of Tables S1 to S14. In some embodiments, the LNP includes two target-directed portions, each containing an array present in any of Tables S1 to S14. In some embodiments, each of the two target-directed portions is coded by an array present in any of Tables S1 to S14.

[0044] In some embodiments, the CD34 target-directed moiety includes a class II antibody or a fragment thereof. In some embodiments, the CD34 target-directed moiety includes a class III antibody or a fragment thereof. In some embodiments, the CD34 target-directed moiety is insensitive to or resistant to a particular proteolytic enzyme. In some embodiments, the CD34 target-directed moiety is insensitive to or resistant to cleavage by neuraminidase. In some embodiments, the CD34 target-directed moiety is insensitive to or resistant to cleavage by chymopapain. In some embodiments, the CD34 target-directed moiety is insensitive to or resistant to cleavage by glycoproteases. In some embodiments, the CD34 target-directed moiety is insensitive to or resistant to cleavage by neuraminidase, chymopapain, and glycoproteases. In some embodiments, the CD34 target-directed moiety is insensitive to or resistant to cleavage by neuraminidase, but sensitive to cleavage by chymopapain and glycoproteases.

[0045] In some embodiments, a conjugate containing a Fab fragment is prepared by a method comprising: (i) contacting a Fab fragment (e.g., a Fab fragment targeting CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184, or CD338) with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain, as well as an interchain disulfide bond linking a constant light chain domain (CL) and a constant heavy chain domain 1 (CH1), thereby causing the reducing reagent to reduce the interchain disulfide bond of the Fab fragment to produce two free cysteine ​​residues; and (ii) contacting the product of step (i) with a precursor LNP containing a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP (conjugate).

[0046] In some embodiments, the disclosure provides a conjugate comprising an LNP and a Fab fragment, wherein the LNP is covalently bonded to either or both a first cysteine ​​residue in the constant region of the heavy chain of the Fab fragment and / or a second cysteine ​​residue in the constant region of the light chain of the Fab fragment. In some embodiments, the Fab fragment does not contain a disulfide bond linking the constant region of the heavy chain of the Fab fragment to the constant region of the light chain of the Fab fragment. In some embodiments, both the constant region of the heavy chain constant region of the heavy chain and the constant region of the light chain of the Fab fragment are covalently bonded to the LNP. In some embodiments, only the constant region of the heavy chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the light chain remains associated with the covalently bonded heavy chain on the surface of the LNP. In some embodiments, only the constant region of the light chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the heavy chain remains associated with the covalently bonded light chain on the surface of the LNP. In some of the embodiments described above, the Fab fragment is linked to the LNP via a thiosuccinimide moiety. In other embodiments of the aforementioned embodiments, the Fab fragment is linked to the LNP via a dithiomalemide moiety. In yet another embodiment of the aforementioned embodiments, the Fab fragment is linked to the LNP via a maleamic acid moiety.

[0047] In some embodiments, the Fab fragment conjugated to the LNP is an IgG1 Fab fragment. In some such embodiments, the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG1 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 233 (Kabat numbering) of the heavy chain of the IgG1 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 233 (Kabat numbering) of the heavy chain of the IgG1 Fab fragment and the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG1 Fab fragment are covalently bonded to the LNP.

[0048] In some embodiments, the Fab fragment conjugated to the LNP is an IgG2 Fab fragment. In some such embodiments, the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment and the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment are covalently bonded to the LNP.

[0049] In some embodiments, the Fab fragment conjugated to the LNP is an IgG4 Fab fragment. In some such embodiments, the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment and the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment are covalently bonded to the LNP.

[0050] In some embodiments, a targeted LNP (i.e., a conjugate) is formed via a click reaction between a first click handle on a target-directing moiety (e.g., an antibody, Fab fragment, or scFv) and a second click handle on the LNP, thereby generating a click product. The second click handle may be located on one or more lipids containing the LNP. In some embodiments, the second click handle is covalently bound to a pegylated lipid containing the LNP.

[0051] In one embodiment, the click product can be formed using a copper-catalyzed click reaction. One such copper-catalyzed click reaction is the hysgene 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne. In some embodiments, the first or second click handle includes a cyclic derivative of the alkynyl group. In some embodiments, the cyclic derivative of the alkynyl group is selected from dibenzocyclooctine, cyclooctine, and difluorinated cyclooctine. In some embodiments, the click chemistry involves a strain-promoted cycloaddition of the azide. In some embodiments, the click chemistry is based on the reaction of a strained alkene. In another embodiment, the click product can be formed using a copper-free click chemistry. For example, the click product can be formed between an azide and dibenzocyclooctene (DBCO). Alternatively, the click product can be formed using a Staudinger reaction between an azide and a phosphine, thus producing an aza-ylide. In some embodiments, the click product can be formed from an inverse electron-demanded Diels-Alder reaction between a trans-cyclooctene (TCO) moiety on a first or second click handle and a tetrazine ring on a first or second click handle. In some embodiments, the first click handle comprises a tetrazine (Tz) ring, and the second click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is unsubstituted. In some such embodiments, the tetrazine ring is methyltetrazine. In some embodiments, the tetrazine ring is 6-methylsubstituted tetrazine.

[0052] In some embodiments, the click product is formed by conjugating an LNP to a target-directed moiety modified with an enzyme recognition sequence. For example, an antibody, Fab fragment, or single-stranded variable fragment (scFv) can be covalently bound to a first click handle via a linker containing an enzyme recognition sequence, and an LNP can be covalently bound to a second click handle on the LNP, thereby generating a click product. In some embodiments, the enzyme recognition sequence is a saltase recognition motif or an LplA acceptor peptide. In some embodiments, the antibody Fab fragment or scFv is directly bound to the enzyme recognition sequence. In some embodiments, the antibody Fab fragment or scFv is bound to the enzyme recognition sequence via one or more amino acid residues. Specific amino acid residues that can be covalently bound to the C-terminus of an antibody, Fab fragment, or scFv include, but are not limited to, (GGGGS)v, (G)v, (EAAAK)v, (PAPAP)v, (AP)v, and A(EAAAK)u ALEA(EAAAK)vA, where u is 1 to 10 and v is 1 to 10.

[0053] In some embodiments, as described herein, lipid nanoparticles (LNPs) for delivering a therapeutic agent to HSCs are, Ionizable lipids and helper lipids, wherein the ionizable lipid is V003, or a helper lipid selected from any of Tables 1, 2, and 3, and Multiple target-direction portions conjugated to an LNP, wherein each of the multiple target-direction portions is conjugated to at least one of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4), It includes a therapeutic agent encapsulated within the LNP.

[0054] In some embodiments, multiple target-directed moieties can be added to the surface of the LNP by the method described in Section II of the Detailed Description. In some embodiments, the targeted LNP comprises two or more different target-directed moieties, each of which may bind to the same or different targets. A targeted LNP having two different target-directed moieties that bind to the same or different targets may be referred to as a “double-binding” or “double-binding” targeted LNP. For example, in some embodiments, each of the target-directed moieties is conjugated to a lipid nanoparticle via a linker, and each linker contains a click product formed from a click reaction between a first click handle of the target-directed moiety and a second click handle of the LNP. In some such embodiments, the target-directed moiety is an antibody or its antigen-binding fragment. In other such embodiments, the target-directed moiety is an scFv. In some embodiments, the target-directed moiety is a Fab fragment.

[0055] In some embodiments, multiple target-directivity portions are coupled to two or more of the following: CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4). In some embodiments, multiple target-directivity portions are coupled to CD117 and CD34. In some embodiments, multiple target-directivity portions are coupled to CD117 and CD71. In some embodiments, multiple target-directivity portions are coupled to CD34 and CD71.

[0056] In some embodiments, as described herein, lipid nanoparticles (LNPs) for delivering a therapeutic agent to HSCs are, Multiple target-direction portions conjugated to an LNP, wherein the multiple target-direction portions are coupled to CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4), It includes a therapeutic agent encapsulated within the LNP.

[0057] In some embodiments, the Disclosure provides targeted lipid nanoparticles (LNPs) for in vivo delivery of therapeutic agents to HSCs (e.g., LT-HSCs), the targeted LNPs comprising an ionizable lipid, a helper lipid, two distinct target-directing moieties conjugated to the LNP, each of which binds to a target on the surface of an HSC (e.g., an LT-HSC), and one or more nucleic acids (e.g., two or more nucleic acids) encoding components of a system for modifying or altering genomic DNA (e.g., a gene modification system), where one or more nucleic acids (e.g., two or more nucleic acids) are RNA molecules.

[0058] In some embodiments, the double binder can be added to the surface of the LNP by the method described in Section II of the Detailed Description. For example, in some embodiments, each of the target-directed moieties is conjugated to a lipid nanoparticle via a linker, and each linker contains a click product formed from a click reaction between a first click handle of the target-directed moiety and a second click handle of the LNP. In some such embodiments, the target-directed moiety is an antibody or its antigen-binding fragment. In other such embodiments, the target-directed moiety is an scFv. In some embodiments, the target-directed moiety is a Fab fragment.

[0059] In some embodiments, one of the RNA molecules encoding components of a system for modifying or altering genomic DNA (e.g., a gene modification system) is an mRNA encoding a gene modification polypeptide, as described herein. In some embodiments, one of the components of a system for modifying or altering genomic DNA (e.g., a gene modification system) is an mRNA encoding Cas9 nickase fused to a reverse transcriptase (RT) domain. In some embodiments, one of the components of a system for modifying or altering genomic DNA is an mRNA encoding a Cas9-RT fusion protein.

[0060] In some embodiments, one of the RNA molecules encoding components of a system for modifying or altering genomic DNA (e.g., a gene modification system) is a guide RNA (gRNA). In some embodiments, one of the RNA molecules encoding components of a system for modifying or altering genomic DNA (e.g., a gene modification system) is a template RNA encoding a heterologous nucleic acid, used together with a gene modification polypeptide to insert a heterologous nucleic acid sequence into a DNA sequence, e.g., the genomic DNA of a cell. In some embodiments, the components of a system for modifying or altering genomic DNA (e.g., a gene modification system) have nuclease activity, e.g., nickase activity. In some embodiments, the components of a system for modifying or altering genomic DNA do not have nuclease activity. In some embodiments, the components of a system for modifying or altering genomic DNA (e.g., a gene modification system) do not induce double-strand breaks in genomic DNA. In such embodiments, the system for modifying or altering genomic DNA induces single-strand breaks in genomic DNA. In some embodiments, the system for modifying or altering genomic DNA induces targeted primed reverse transcription (TPRT) to insert heterologous sequences into the genomic DNA.

[0061] In some embodiments, the target on the surface of the HSC is selected from CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184 (CXCR4). In some embodiments, at least one of the target-directing portions is coupled to CD117. In some embodiments, at least one of the target-directing portions is coupled to CD34. In some embodiments, at least one of the target-directing portions is coupled to CD71. In some embodiments, at least one of the target-directing portions is coupled to CD164. In some embodiments, the targeted LNP is a double-bonded LNP. In some embodiments, two target-directing portions are coupled to CD117 and CD34. In some embodiments, two target-directing portions are coupled to CD117 and CD71.

[0062] In some embodiments, the ionizable lipid is V003, or a lipid from Table 1, Table 2, or Table 3. In some embodiments, the ionizable lipid is selected from lipids 92, 93, 95, 96, 97, 97, 110, 132, 133, 134, 140, 141, 143, 144, 147, 148, 149, 150, 151, 152, 153, 154, 155, 162, 163, 169, 170, 173, 174, 175, 176, 178, 179, 183, 184, and 232. In some embodiments, the ionizable lipid is lipid 093. In some embodiments, the ionizable lipid is lipid 092. In some embodiments, the ionizable lipid is lipid 154. In some embodiments, the ionizable lipid is lipid 232.

[0063] In some embodiments, the helper lipid of the targeted LNP is DSPC or sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin in the targeted LNP is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0064] In some embodiments, the targeted LNP further comprises a pegylated lipid. In some embodiments, the pegylated lipid comprises at least one C16 alkyl chain (e.g., two C16 alkyl chains). In some such embodiments, the pegylated lipid is DPPE-PEG2000 or DPG-PEG2000.

[0065] In some embodiments, targeted delivery of a system for modifying or altering genomic DNA (e.g., a gene modification system) using an LNP containing two or more HSC-specific binders conjugated to the LNP results in synergistic enhancement of gene editing or modification of the target HSC or LT-HSC. In some embodiments, an LNP containing a double binder can modify at least 5% of the genome of an HSC or LT-HSC. In some embodiments, an LNP containing a double binder can modify at least 10% of the genome of an HSC or LT-HSC. In some embodiments, an LNP containing a double binder can modify at least 15% of the genome of an HSC or LT-HSC. In some embodiments, an LNP containing a double binder can modify at least 20% of the genome of an HSC or LT-HSC. In some embodiments, an LNP containing a double binder can modify at least 25% of the genome of an HSC or LT-HSC. In some embodiments, an LNP containing a double binder can modify at least 30% of the genome of an HSC or LT-HSC. In some embodiments, LNPs containing a double binder can modify at least 30% of the genome of HSCs or LT-HSCs. In some embodiments, LNPs containing a double binder can modify about 10% to about 30% of the genome of HSCs or LT-HSCs. In some embodiments, LNPs containing a double binder can modify about 20% to about 30% of the genome of HSCs or LT-HSCs. In some embodiments, LNPs containing a double binder can modify about 10% to about 20% of the genome of HSCs or LT-HSCs. In any of the embodiments described above, the system for modifying or altering genomic DNA induces single-strand breaks in the genomic DNA. In some such embodiments, the system for modifying or altering genomic DNA comprises a gene modification polypeptide and template RNA for a nucleic acid sequence to be inserted at a specific position in the genomic DNA, thus resulting in modification of the genomic DNA. In some embodiments, the gene modification system comprises Cas9 nickase fused to a reverse transcriptase (RT) domain.In some embodiments, a system for modifying or altering genomic DNA induces targeted primed reverse transcription (TPRT) to insert a heterologous nucleic acid sequence into a DNA sequence, such as genomic DNA.

[0066] In some embodiments, the Disclosure provides a method for treating sickle cell disease in a subject (e.g., a human patient requiring treatment for sickle cell disease), the method comprising administering a targeted lipid nanoparticle (LNP) to the subject, comprising an ionizable lipid, a helper lipid, two different target-directed moieties conjugated to the LNP, each of which binds to a target on the surface of a HSC (e.g., LT-HSC), and one or more nucleic acids (e.g., two or more nucleic acids) encoding components of a system for modifying or altering genomic DNA (e.g., a gene modification system), wherein the two or more nucleic acids are RNA molecules.

[0067] In some embodiments, the nucleic acid component does not introduce double-strand breaks into the genomic DNA. In some embodiments, more than 5% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs. In some embodiments, more than 10% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs. In some embodiments, more than 15% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs. In some embodiments, more than 20% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs. In some embodiments, more than 30% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs. In some embodiments, approximately 5% to approximately 15% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs. In some embodiments, approximately 10% to 30% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs. In some embodiments, approximately 10% to 20% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs. In some embodiments, approximately 15% to 20% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs. In some embodiments, approximately 15% to 25% of HSCs or LT-HSCs in patients with sickle cell disease are modified after administration of targeted LNPs.

[0068] In another aspect, this disclosure is: (i) A lipid component comprising ionizable lipids and helper lipids, wherein the ionizable lipids are present in an amount of approximately 35 mol% to approximately 60 mol% of the lipid component, and the helper lipids are present in an amount of approximately 18 mol% to approximately 32 mol% of the lipid component, (ii) Means for binding to a cell surface receptor selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184, (iii) A lipid nanoparticle (LNP) comprising a therapeutic agent encapsulated within the LNP, the therapeutic agent comprising two or more RNA molecules (e.g., two or three RNA molecules).

[0069] In one modified example, means for binding to a cell surface receptor, comprising a target-directing moiety on the surface of an LNP that binds to one or more cell receptors selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184.

[0070] In some embodiments, after in vivo administration of targeted LNPs to patients with sickle cell disease, HSCs or LT-HSCs maintain their hematopoietic capacity and function to support multiseries development. In some embodiments, gene modification systems correct E6V HBB mutations in the patient's HSCs and LT-HSCs. [Brief explanation of the drawing]

[0071] [Figure 1] An exemplary conjugate containing Fab fragments and lipid nanoparticles (LNPs) is shown. [Figure 2] The image shows an assembly of an LNP encapsulating a therapeutic payload having a functional group (e.g., maleimide, 2,3-dibromomaleimide, saltase tag, etc.) that reacts with an antibody or its antigen-binding fragment. B shows an assembly of an LNP encapsulating a therapeutic payload using post-insertion techniques. [Figure 3] An illustrative schematic diagram of the reduction of interchain disulfide bonds in the Fab fragment is shown. After reduction, each cysteine ​​residue is available to react with thiol-reactive groups conjugated on the surface of the LNP. [Figure 4]A schematic diagram illustrating conjugate formation is shown. In Figure 4, a Fab fragment containing an interchain disulfide bond between the heavy and light chains is brought into contact with a reducing agent, thereby reducing the interchain disulfide to produce two free cysteine ​​residues (step (i)). In step (ii), the Fab fragment is brought into contact with an LNP containing multiple thiol-reactive groups (e.g., multiple maleimide or DBM groups) conjugated on the surface of the LNP, thereby the multiple thiol-reactive groups react with at least one of the two free cysteine ​​residues of the Fab fragment. [Figure 5] A schematic diagram illustrating conjugate formation is shown. In Figure 5, a Fab fragment containing an interchain disulfide bond between the heavy and light chains is brought into contact with a reducing agent, thereby reducing the interchain disulfide to produce two free cysteine ​​residues (step (i)). In step (ii), the Fab fragment is brought into contact with an LNP containing multiple maleimide groups conjugated on the surface of the LNP, thereby the multiple maleimide groups react with one of the two free cysteine ​​residues of the Fab fragment. [Figure 6] A schematic diagram illustrating an example of conjugate formation using DBM as a crosslinking agent is shown. [Figure 7] A schematic diagram illustrating conjugate formation is shown. In Figure 7, the first Fab fragment (Fab1) and the second Fab fragment (Fab2) are each brought into contact with a reducing agent, thereby reducing the interchain disulfide between the heavy and light chains of each Fab. After step (i), each Fab contains two free cysteine ​​residues, one on the heavy chain and one on the light chain. In step (ii), the first and second Fab fragments are brought into contact with an LNP containing multiple thiol-reactive groups (e.g., maleimide or DBM) conjugated on the surface of the LNP, thereby the multiple thiol-reactive groups react with at least one of the two free cysteine ​​residues of the first Fab fragment, and the multiple other thiol-reactive groups react with at least one of the two free cysteine ​​residues of the second Fab fragment. [Figure 8]Here are some examples of pegylated lipids bound to the maleimide moiety. [Figure 9] Here are some examples of non-pegylated lipids bound to the maleimide moiety. [Figure 10] Here are some examples of ionizable lipids bound to the maleimide moiety. [Figure 11] Here are some examples of sterols bound to the maleimide moiety. [Figure 12] A schematic diagram illustrating conjugate formation is shown. In Figure 12, the Fab fragment contains a saltase tag. The Fab fragment is brought into contact with an LNP in the presence of saltase, and the LNP contains multiple polyglycine molecules conjugated to its surface, thereby conjugating the Fab fragment to the surface of the LNP via saltase-mediated ligation of the saltase tag and multiple polyglycine molecules. [Figure 13] This demonstrates the formation of an LNP conjugated to the C-terminus of the Fab fragment via saltase-mediated ligation, followed by a click reaction. [Figure 14] This demonstrates the formation of an LNP conjugated to the C-terminus of the Fab fragment via lipoic acid ligase-mediated ligation, followed by a click reaction. [Figure 15] This paper describes an enzymatic approach to site-specifically introduce a Tz ring into the sugar portion of an antibody. [Figure 16] This example shows a photo-guided crosslinking approach that site-specifically induces a Tz ring on an antibody. [Figure 17] This example demonstrates the use of non-natural amino acids to site-specifically introduce a Tz ring onto an antibody or Fab fragment. [Figure 18]This paper presents the results of transfection experiments in human CD34+ cells using LNPs conjugated to anti-CD117 Fab fragments via saltase-mediated ligation. The transfection results are compared with unmodified (control) LNPs and LNPs modified with anti-CD117 antibody produced using a random modification method. A shows mRNA transfection quantified by flow cytometry for green fluorescent protein (GFP+%) cells. B shows transfection in terms of median fluorescence intensity (MFI). [Figure 19] Figure A shows a schematic example of producing a site-specific conjugate by reaction of an anti-CD117 Fab fragment with a site-specific saltase recognition motif with LNP. In the schematic diagram, the wavy lines represent PEG bound to the lipids of the LNP. Figure B shows a schematic diagram of producing a conjugate by reaction of a Fab fragment modified using a random approach (NHS modification) with LNP. [Figure 20] The results of transfection of Kasumi-1 cells (CD117+ cell line) with LNPs conjugated to anti-CD117 Fab fragments produced by site-specific (soltase-mediated) or random modification are shown. A shows mRNA transfection quantified by flow cytometry for green fluorescent protein (GFP+%) cells. B shows transfection in terms of median fluorescence intensity (MFI). [Figure 21]This document presents the results of in vivo experiments evaluating the transduction of LNPs conjugated with one of three tested anti-CD117 (Fab1, Fab5, or Fab6) specific Fab fragments in HSCs and early progenitor cells (HSCs + early precursors) (also referred to as HSPCs) compared to transduction using basic LNPs lacking the conjugated anti-CD117 Fab fragment. A shows that up to 58% of HSCs + early progenitor cells receive LNPs with the saltase-modified Fab fragment. B shows that up to 62% of long-term (LT)-HSCs receive LNPs with the saltase-modified Fab fragment. LNPs with the saltase-modified Fab fragment exhibited significantly higher in vivo transfection levels in both HSCs + early precursors and LT-HSCs compared to basic LNPs lacking the anti-CD117 Fab fragment. Furthermore, as shown in C, there is a good correlation between in vitro transduction of primary HSPCs and in vivo targeting in HSPCs. On the other hand, as shown in C, there is separation between nonspecific IgG and site-specifically conjugated (soltase method) Fab LNPs. [Figure 22] This study demonstrates the effect of increasing the mol% of helper lipids in targeted LNPs conjugated with a CD117-specific Fab fragment on the transduction and expression of GFP-encoding mRNA in HSCs and early progenitor cells (HSC+ early progenitor cells) (also known as HSPCs). Specifically, Figure 22 shows the results of an in vivo experiment comparing the transduction of targeted LNPs containing two different amounts of helper lipids (LNP A and LNP B). LNPs A and B consisted of 8% and 22% helper lipids (DSPC), respectively. Over 80% of HSC+ early progenitors were transduced with LNP B. [Figure 23-1]We compare the levels of transduction and MFI in humanized mouse models administered with LNPs containing helper lipids at different percentages. A and B demonstrate that targeted LNPs containing ionizable lipids and 8% DSPC provide similar levels of GFP transduction and expression in HSPCs compared to targeted LNPs containing ionizable lipids and 8% (mol) sphingomyelin. Similar to experiments using DSPC as the helper lipid, increasing the sphingomyelin content to 22% (mol) substantially increases the transduction and expression levels of the reporter protein. [Figure 23-2] We compare the levels of transduction and MFI in humanized mouse models administered with LNPs containing helper lipids at different percentages. C and D show the transduction results in LT-HSCs. [Figure 24] The results of a FACS-based screening to identify the proportion of HSPCs (Highly Sensitive Cynomolgus Monkeys) aged 3–5 years that tested positive for GFP protein expression after administration of the conjugate of this disclosure are shown. [Figure 25] A to F represent the synthesis of lipid 092. [Figure 26] A to C show the synthesis of lipid 093. [Figure 27] A to C show the synthesis of lipid 162. [Figure 28] A to C show the synthesis of lipid 153. [Figure 29] A to F represent the synthesis of lipid 154. [Figure 30] A to E represent the synthesis of lipid 155. [Figure 31] A to F represent the synthesis of lipid 176. [Figure 32-1]A shows enhanced transduction of GFP in LSK cells using LNPs containing ionizable lipid 092 or ionizable lipid 093, compared to LNPs containing ionizable lipid V003 in an in vivo mouse model. B shows enhanced expression levels of GFP in LSK cells using LNPs containing ionizable lipid 092 or ionizable lipid 093, compared to LNPs containing ionizable lipid V003 in an in vivo mouse model. [Figure 32-2] C and D show enhanced transduction and / or expression levels of GFP in LSK cells using LNPs containing ionizable lipid 154 or ionizable lipid 155 compared to LNPs containing ionizable lipid V003 in an in vivo mouse model. E and F show enhanced transduction and / or enhanced GFP expression of GFP in LT-HSCs using LNPs containing ionizable lipid 154 or ionizable lipid 155 compared to LNPs containing ionizable lipid V003. [Figure 33] A shows enhanced transduction of GFP in LT-HSCs using LNPs containing ionizable lipid 092 or ionizable lipid 093, compared to LNPs containing ionizable lipid V003 in a humanized in vivo mouse model. B shows enhanced expression levels of GFP in LT-HSCs using LNPs containing ionizable lipid 092 or ionizable lipid 093, compared to LNPs containing ionizable lipid V003 in a humanized in vivo mouse model. [Figure 34]Each of the following shows enhanced transduction of GFP in HSPCs or LT-HSPCs using LNPs containing ionizable lipid 153, lipid 154, or lipid 155, compared to LNPs containing ionizable lipid V003, in a humanized in vivo mouse model. B and D show enhanced expression of GFP in HSPCs or LT-HSCs using LNPs containing ionizable lipid 153 or ionizable lipid 154, compared to LNPs containing ionizable lipid V003, in a humanized in vivo mouse model. [Figure 35-1] The results of screening for targeted LNPs (tLNPs) in humanized NBSGW mice with increased levels of helper lipids are shown. A and B show the results of transduction and expression in HSPC, respectively. [Figure 35-2] The results of screening for targeted LNPs (tLNPs) in humanized NBSGW mice with increased levels of helper lipids are shown. C and D show the results of transduction and expression in LT-HSC mice, respectively. [Figure 36] The results of screening for targeted LNPs (tLNPs) in non-human primates are shown. [Figure 37] A and B show in vitro screening results (GFP+ cells and MFI%) for target-directing moieties that can enhance the delivery of targeted LNPs to HSPCs, normalized for cells transfected with basic LNPs lacking target-directing moieties. [Figure 38] This specification demonstrates that the site-directed conjugation process described herein can efficiently conjugate two target-directed moieties (in this case, anti-CD117 Fab and anti-CD45 Fab) to the surface of an LNP in a specific ratio. [Figure 39]This study demonstrates that tLNPs can effectively deliver gene editing payloads to human HSPCs in animal models, resulting in in vivo gene editing in HSPCs. A and B show that administration of anti-CD34 tLNPs and anti-CD117 tLNPs resulted in detectable levels of B2M indels in HSPCs and LT-HSCs, while administration of bifurcation tLNPs conjugated with anti-CD117 and anti-CD34 target-directed moieties resulted in synergistically higher levels of gene editing in HSPCs. C shows that bifurcation tLNPs conjugated with anti-CD117 and anti-CD71 target-directed moieties, or anti-CD34 and anti-CD71 target-directed moieties, can substantially increase the delivery of gene editing payloads to HSPCs and LT-HSCs, thereby resulting in increased B2M editing. D shows that doubling the amount of a single type of target-directed moiety (anti-CD117) conjugated on the surface of the tLNP did not increase the delivery of payloads to HSPCs or LT-HSCs. [Figure 40] A and B demonstrate that administered anti-CD117 tLNPs can deliver a payload suitable for gene editing to human HSPCs in an animal model, resulting in in vivo gene editing in HSPCs. A shows that tLNPs formulated with lipid 093 and wtCas9-RT mRNA and B2M gRNA payloads resulted in 8.1% indels in B2M in HSPCs. In contrast, tLNPs formulated with lipid 154 and the same payload resulted in 15.7% indels in B2M in HSPCs, representing nearly twofold increased delivery compared to lipid 093 tLNPs. B shows that tLNPs formulated with lipid 154 and nCas9-RT and B2M template RNA payloads produced higher TPRT-mediated knockout of B2M in HSPCs compared to tLNPs formulated with lipid 093 and the same payload (average 5.2% for lipid 154 and average 1.5% for lipid 93). [Figure 41]A shows that administration of anti-CD117 tLNP formulated with lipid 093 and 2mol% C16-PEG to cynomolgus monkeys (n=2) resulted in 72% GFP+ HSPCs and 33% GFP+ LT-HSCs. In comparison, administration of anti-CD117 tLNP formulated with lipid 154 and 2mol% C16-PEG to macaques (n=2) resulted in higher percentages of GFP+ HSPCs and LT-HSCs (96% GFP+ and 91% GFP+ cells, respectively). B shows that GFP mRNA expression was higher in HSPCs and nearly four times higher in LT-HSCs in NHP administered with lipid 154 tLNP compared to those administered with lipid 093 tLNP. [Figure 42] This study shows that administration of anti-CD117 / anti-CD34 bilayer tLNPs formulated with lipid 093 and C14-PEG resulted in an average of 25% B2M indels in human HSPCs in vivo. Administration of anti-CD117 / anti-CD34 bilayer tLNPs formulated with lipid 154 and C16-PEG generated higher B2M indels (over 45%) compared to lipid 093 C16-PEG tLNPs. [Figure 43] This study demonstrates that anti-CD117 / anti-CD34 bitargeted tLNPs formulated with lipid 154 and C16-PEG resulted in an average of 11.8% complete gene modification in HSPCs and an average of 14.3% complete gene modification in LT-HSCs in vivo. [Figure 44] A shows that administration of lipid-154-formulated bilayer tLNPs to cynomolgus monkeys (n=2) resulted in an average of over 90% GFP+HSPCs and LT-HSCs for anti-CD117 / anti-CD34 bilayer tLNPs, and over 80% GFP+HSPCs and over 70% GFP+LT-HSCs for anti-CD117 / anti-CD71 bilayer tLNPs. B shows that HSPCs and LT-HSCs in NHP expressed GFP(MFI) after administration of each bilayer tLNP. [Figure 45]This study demonstrates that dual-targeted LNPs (tLNPs) formulated with anti-CD117 and anti-CD34 targeting can effectively deliver gene editing systems to human HSCs and precursors in vivo, resulting in B2M knockout in a significant number of cells. [Figure 46] A and B demonstrate that targeted LNPs (tLNPs) and dual tLNPs can deliver the B2M gene editing system in vivo to human HSPCs (Figure 46A) and LT-HSCs (Figure 46B) to generate B2M indels. [Modes for carrying out the invention]

[0072] I. Definition Antibody: The term "antibody" as used herein encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0073] Antibody fragment: As used herein, the terms “antibody fragment” or “antigen-binding fragment” refer to a portion of a full-length antibody that binds to the antigen to which the full-length antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, scFab); and multispecific antibodies formed from antibody fragments.

[0074] Antigen-binding domain: As used herein, the term “antigen-binding domain” refers to the portion of an antibody or chimeric antigen receptor that binds to an antigen. In some embodiments, the antigen-binding domain binds to a cell surface antigen of a cell. In some embodiments, the antigen-binding domain binds to an antigen specific to cancer, for example, a tumor-associated antigen in a newly formed cell. In some embodiments, the antigen-binding domain binds to an antigen specific to an infectious disease, for example, a virus-associated antigen in a virus-infected cell. In some embodiments, the antigen-binding domain binds to an antigen specific to a cell targeted by the immune system in an autoimmune disease, for example, an O-antigen. In some embodiments, the antigen-binding domain is or includes an antibody or its antigen-binding portion. In some embodiments, the antigen-binding domain is or includes an scFv or Fab.

[0075] Expression Cassette: As used herein, the term "expression cassette" refers to a nucleic acid construct containing sufficient nucleic acid elements for the expression of the nucleic acid molecule of the present invention.

[0076] Fab fragment: As used herein, “Fab fragment” refers to an antibody fragment comprising a light chain fragment containing the VL domain and constant domain of the light chain (CL), and the VH domain and first constant domain (CH1) of the heavy chain.

[0077] gRNA spacer: As used herein, “gRNA spacer” refers to a portion of nucleic acid that is complementary to the target nucleic acid and, together with the gRNA scaffold, allows the Cas protein to target the target nucleic acid.

[0078] gRNA scaffold: As used herein, “gRNA scaffold” refers to a portion of nucleic acid that can bind to a Cas protein and, together with a gRNA spacer, can target the Cas protein to a target nucleic acid. In some embodiments, the gRNA scaffold includes a crRNA sequence, a tetraloop, and a tracrRNA sequence.

[0079] Genetically Modified Polypeptide: As used herein, “genetically modified polypeptide” refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with respect to a retroviral reverse transcriptase, which can incorporate a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., a mammalian host cell, or a genomic DNA molecule within a host cell). In some embodiments, the genetically modified polypeptide can incorporate the sequence substantially without relying on host mechanisms. In some embodiments, the genetically modified polypeptide incorporates the sequence into a specific target site. In some embodiments, the genetically modified polypeptide comprises one or more domains that comprehensively facilitate 1) binding of the template nucleic acid, 2) binding of the target DNA molecule, and 3) incorporation of at least a portion of the template nucleic acid into the target DNA. The genetically modified polypeptide includes both naturally occurring polypeptides and the aforementioned manipulated variants, for example, having one or more amino acid substitutions to a naturally occurring sequence. Genetically modified polypeptides also include heterologous constructs in which one or more of the domains listed above are heterologous to each other, for example, through heterologous fusion (or other conjugate) of other wild-type domains, or through fusion of modified domains via substitution or fusion of heterologous subdomains or other substitution domains. Exemplary genetically modified polypeptides that can be used in the manner provided herein, as well as systems comprising them and methods of using them, are described, for example, in PCT / US2021 / 020948, which is incorporated herein by reference with respect to genetically modified polypeptides comprising retroviral reverse transcriptase domains. In some embodiments, the genetically modified polypeptides incorporate a sequence into a gene. In some embodiments, the genetically modified polypeptides incorporate a sequence outside of a gene. As used herein, “genetic modification system” refers to a system comprising a genetically modified polypeptide and a template nucleic acid.

[0080] Genetic modification system: As used herein, “genetic modification system” means a system comprising a genetically modified polypeptide, or a nucleic acid (e.g., mRNA) encoding a genetically modified polypeptide, and a template nucleic acid.

[0081] Domain: As used herein, the term “domain” refers to a structure of a biomolecule that contributes to a particular function of that biomolecule. A domain may include a continuous region (e.g., a continuous sequence) or a distinct discontinuous region (e.g., a discontinuous sequence) of a biomolecule. Examples of protein domains, but not limited to these, include endonuclease domains, DNA-binding domains, and reverse transcriptase domains, while examples of nucleic acid domains include regulatory domains such as transcription factor-binding domains.

[0082] Exogenous: As used herein, the term “exogenous” means, when used in reference to a biomolecule (such as a nucleic acid sequence or polypeptide), that the biomolecule has been introduced into a host genome, cell, or organism by human intervention. For example, a nucleic acid that is added to an existing genome, cell, tissue, or subject using recombinant DNA technology or other methods is exogenous to the existing nucleic acid sequence, cell, tissue, or subject.

[0083] First Strand and Second Strand: When used herein, the terms “first strand” and “second strand” describe the individual DNA strands of the target DNA and distinguish the two DNA strands based on which strand the reverse transcriptase domain begins polymerization on, for example, where the targeted priming synthesis begins. The first strand refers to the strand of target DNA from which the reverse transcriptase domain begins.

[0084] Heterogeneous: When the term “heterogeneous” is used herein to describe a first element in relation to a second element, it means that the first and second elements do not exist in nature in the configuration described. For example, heterogeneous polypeptides, nucleic acid molecules, constructs, or sequences mean (a) a polypeptide, nucleic acid molecule, or a portion of a polypeptide or nucleic acid molecule sequence that is not native to the cell in which it is expressed, (b) a polypeptide or nucleic acid molecule, or a portion of a polypeptide or nucleic acid molecule that is modified or mutated from its native state, or (c) a polypeptide or nucleic acid molecule having modified expression compared to its native expression level under similar conditions. For example, heterogeneous regulatory sequences (e.g., promoters, enhancers) may be used to regulate the expression of a gene or nucleic acid molecule in a manner different from that of a gene or nucleic acid molecule that is normally expressed in nature. In another example, heterogeneous domains of a polypeptide or nucleic acid sequence (e.g., the DNA-binding domain of a polypeptide or the nucleic acid encoding the DNA-binding domain of a polypeptide) may be configured relative to other domains, or may be of a different sequence, or may be from a different source relative to other domains or portions of the polypeptide or its coding nucleic acid. In certain embodiments, heterologous nucleic acid molecules may be present in the native host cell genome but may have modified expression levels, different sequences, or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to the host cell or host genome, but may instead be introduced into the host cell by transformation (e.g., transfection, electroporation), and the added molecules may be integrated into the host genome or exist as extrachromosomal genetic material, either transiently (e.g., mRNA) or semi-stable for two or more generations (e.g., episomal viral vectors, plasmids, or other self-replicating vectors).

[0085] Mutant or Mutant: When applied to a nucleic acid sequence, the term "mutant" means that nucleotides within the nucleic acid sequence may be inserted, deleted, or altered compared to a reference (e.g., native) nucleic acid sequence. A single alteration may occur at a locus (point mutation), or multiple nucleotides may be inserted, deleted, or altered at a single locus. In addition, one or more alterations may occur at any number of loci within the nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art. In some embodiments, mutations occur naturally. In some embodiments, desired mutations can be produced by the systems described herein.

[0086] Variant region: As used herein, the term “variant region” refers to a region in template RNA that has one or more sequence differences compared to the corresponding sequence in the target nucleic acid. Sequence differences may include, for example, substitutions, insertions, frameshifts, or deletions.

[0087] Nucleic acid molecules: “Nucleic acid molecules” means both RNA molecules and DNA molecules, including complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as RNA templates, which are chemically synthesized or produced by recombination, as described herein. Nucleic acid molecules can be double-stranded or single-stranded, circular, or linear. If single-stranded, a nucleic acid molecule can be a sense strand or an antisense strand. Unless otherwise indicated, and as an example of all sequences described herein in the general format “Sequence Number” or “Nucleic Acid Containing Sequence Number 1”, a nucleic acid refers to at least a portion having either (i) the sequence of Sequence Number 1, or (ii) a sequence complementary to Sequence Number 1. The choice between the two is determined by the context in which Sequence Number 1 is used. For example, when a nucleic acid is used as a probe, the choice between the two is determined by the requirement that the probe is complementary to the desired target. The nucleic acid sequences of this disclosure may be chemically or biochemically modified, or may contain unnatural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution with one or more naturally occurring nucleotide analogs, uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelating agents, alkylating agents, and modified bonds (e.g., alpha-anomeric nucleic acids, etc.). Chemically modified bases (e.g., see Table 13), skeletons (e.g., see Tables 14 and 16), and modified caps are also included. Synthetic molecules that mimic polynucleotides in their ability to bind to specified sequences via hydrogen bonding and other chemical interactions are also included. Such molecules are known in the art, and include, for example, those in which peptide bonds are replaced by phosphate bonds within the backbone of a molecule, such as a peptide nucleic acid (PNA).Other modifications may include, for example, analogs in which the ribose ring contains other structures such as modifications found in bridged moieties or "locked" nucleic acids (LNAs). In various embodiments, the nucleic acid is operatively associated with additional genetic elements such as tissue-specific expression control sequence(s) (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements such as inverted repeats (e.g., inverted terminal repeats such as elements from or derived from viruses, e.g., AAV ITR) and tandem repeats, inverted repeats / direct repeats, regions of homology (segments with varying degrees of homology to the target DNA), untranslated regions (UTRs) (5', 3', or both 5' and 3' UTRs), and various combinations of the foregoing. The nucleic acid elements of the systems provided by the present invention can be provided in a variety of topologies, including single-stranded, double-stranded, circular, linear, linear with open ends, linear with closed ends, and specific versions thereof, such as doggybone DNA (dbDNA), and closed-ended DNA (ceDNA).

[0088] Primer Binding Sequence: As used herein, the term "primer binding site sequence" or "PBS sequence" refers to a portion of a template RNA that is capable of binding to a region comprised in a target nucleic acid sequence. In some cases, the PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases that have 100% identity with the region comprised in the target nucleic acid sequence. In some embodiments, the primer region comprises at least 5, 6, 7, 8 bases that have 100% identity with the region comprised in the target nucleic acid sequence. While not wishing to be bound by theory, in some embodiments, when the template RNA comprises a PBS sequence and a heterologous object sequence, the PBS sequence binds to a region comprised in the target nucleic acid sequence, allowing the reverse transcriptase domain to use that region as a primer for reverse transcription and the heterologous object sequence as a template for reverse transcription.

[0089] It is understood that the aspects and embodiments described herein as "comprising" include embodiments "consisting of" and "consisting essentially of".

[0090] References to an "about" value or parameter herein include (and describe) embodiments directed to that value or parameter per se. For example, a reference to "about X" includes a description of "X".

[0091] In some embodiments, references to measurable parameter values encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value.

[0092] II. Conjugates In one aspect, the present disclosure provides lipid nanoparticles (LNPs) comprising the ionizable lipids described herein (e.g., in Table 1 or Table 2), wherein the LNPs are capable of delivering a payload such as a therapeutic agent (e.g., a gene modification system) to hematopoietic stem cells (HSCs), e.g., LT-HSCs, or hematopoietic progenitor cells (HPCs). In some embodiments, the LNPs are capable of delivering a payload such as a therapeutic agent (e.g., a gene modification system) to a mixed population of hematopoietic stem and progenitor cells (HSPCs).

[0093] In some aspects, the present disclosure provides conjugates comprising an LNP and one or more targeting moieties that bind to a target molecule, e.g., a target protein such as a protein receptor, present on a target cell. In some embodiments, the targeting moiety causes the LNP conjugate to target and bind to the target cell. In some embodiments, the targeting moiety causes the LNP conjugate to target and bind to the target cell preferentially over non-target cells.

[0094] In some embodiments, the Disclosure provides a conjugate comprising an LNP and one or more target-directed moieties present on an HSC (e.g., LT-HSC) or HPC as described herein, which bind to a target molecule, such as a target protein, such as a protein receptor. In some embodiments, one or more target-directed moieties are conjugated to the surface of the LNP. In some embodiments, the LNP conjugate comprises both an ionizable lipid and target-directed moieties as described herein. In another embodiment, the Disclosure provides a conjugate comprising an LNP and one or more first target-directed moieties present on an HSC (e.g., LT-HSC) or HPC that bind to a first target molecule, such as a first target protein, such as a first protein receptor, and one or more second target-directed moieties present on an HSC (e.g., LT-HSC) or HPC that bind to a second target molecule, such as a second target protein, such as a second protein receptor. In some embodiments, one or more of the first and second target-directed moieties are conjugated to the surface of the LNP. This section describes specific conjugates of the present disclosure and methods for conjugating the target-directed portion to an LNP. Additional embodiments of the LNP component of the conjugate are described in Section III.

[0095] In one embodiment, the present disclosure provides a targeted LNP (conjugate) comprising a target-directed moiety and lipid nanoparticles (LNPs) encapsulating a payload (e.g., a therapeutic agent as described herein, such as a genetically modified polypeptide or genetic modification system), wherein the target-directed moiety binds to a protein (e.g., a protein receptor) on an HSC (e.g., LT-HSC) or HPC.

[0096] In some embodiments, the LNP includes a target-directed moiety that targets receptors on the surface of HSCs selected from CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184, and CD338. In some embodiments, the target-directed moiety on the LNP binds to CD33. In some embodiments, the target-directed moiety on the LNP binds to CD34. In some embodiments, the target-directed moiety on the LNP binds to CD38. In some embodiments, the target-directed moiety on the LNP binds to CD43. In some embodiments, the target-directed moiety on the LNP binds to CD46. In some embodiments, the target-directed moiety on the LNP binds to CD49d. In some embodiments, the target-directed moiety on the LNP binds to CD50. In some embodiments, the target-directed moiety on the LNP binds to CD59. In some embodiments, the target-directed moiety on the LNP binds to CD71. In some embodiments, the target-directed portion on the LNP is coupled to CD105. In some embodiments, the target-directed portion on the LNP is coupled to CD117. In some embodiments, the target-directed portion on the LNP is coupled to CD123. In some embodiments, the target-directed portion on the LNP is coupled to CD184. In some embodiments, the target-directed portion on the LNP is coupled to CD164. In some embodiments, the target-directed portion on the LNP is coupled to CD338.

[0097] In some embodiments, lipid nanoparticles (LNPs) for delivering the therapeutic agent described herein to HSCs include an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or any of Tables 1, 2, and 3; a plurality of target-directing moieties conjugated to the LNP, wherein each target-directing moiety is bound to at least one of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4); and a therapeutic agent encapsulated within the LNP.

[0098] In some embodiments, multiple target-directing portions are coupled to two or more of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4). In some embodiments, multiple target-directing portions are coupled to CD117 and CD34. In some embodiments, multiple target-directing portions are coupled to CD117 and CD71. In some embodiments, multiple target-directing portions are coupled to CD34 and CD71. In some embodiments, multiple target-directing portions are coupled to CD117 and CD71.

[0099] In some embodiments, two different target-directing moieties can bind to the same receptor on the surface of the HSC. For example, in some embodiments, both target-directing moieties can bind to CD117. In other embodiments, both target-directing moieties can bind to CD34. In other embodiments, both target-directing moieties can bind to CD164.

[0100] In some embodiments, the lipid nanoparticles (LNPs) for delivering a therapeutic agent to HSCs as described herein are a plurality of target-directed moieties conjugated to the LNP, the plurality of target-directed moieties comprising a plurality of target-directed moieties conjugated to CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4), and a therapeutic agent encapsulated within the LNP.

[0101] In some aspects, the present disclosure provides targeted lipid nanoparticles (LNPs) for in vivo delivery of therapeutic agents to HSCs (e.g., LT-HSCs), where the LNPs are i. Ionizable lipids, ii. Helper lipids and, iii. Two different target-directed portions conjugated to an LNP, each of which is conjugated to a target on the surface of an HSC (e.g., an LT-HSC), iv. A system comprising one or more nucleic acids (e.g., two or more nucleic acids) that encode components of a system for modifying or altering genomic DNA (e.g., a gene modification system), wherein one or more nucleic acids (e.g., two or more nucleic acids) are RNA molecules.

[0102] Targeting part The target-directed moiety may include polypeptides that specifically bind to target molecules, such as target proteins (e.g., protein receptors), present on target cells. In some embodiments, the target cells express the target molecule. In some embodiments, the target-directed moiety includes an antibody or its antigen-binding fragment (e.g., Fab, Fab', F(ab')2, Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single-domain antibody, or centinrin). In some embodiments, the target-directed moiety includes a T cell receptor (TCR). In other embodiments, the target-directed moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine.

[0103] In some embodiments, the target-directed moiety is an antibody or its antigen-binding fragment. In some embodiments, the target-directed moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the target-directed moiety is a bispecific or multispecific antibody or its fragment. In some embodiments, the bispecific or multispecific antibody or its fragment comprises two or more antigen-binding domains or fragments thereof, selected from, for example, CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4) antibody or its fragment, as provided in Tables S1 to S14. In some embodiments, the target-directed moiety is a humanized antibody or its antigen-binding fragment.

[0104] In some embodiments, the LNP includes a target-directed portion containing an array present in any of Tables S1 to S14. In some embodiments, the target-directed portion is coded by an array present in any of Tables S1 to S14. In some embodiments, the LNP includes two target-directed portions, each containing an array present in any of Tables S1 to S14. In some embodiments, each of the two target-directed portions is coded by an array present in any of Tables S1 to S14.

[0105] CD33 In some embodiments, the target molecule is CD33 (also known as Siglec-3, p67, or My9). In some embodiments, the target cells are CD33+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD33. Exemplary anti-CD33 antibodies or antigen-binding fragments include gemtuzumab (e.g., hP67.6, Pfizer, FDA approved), lintuzumab (e.g., hM195 or SGN-33, Seattle Genetics), vadasutuximab (e.g., SGN-CD33A, i.e., Seattle Genetics), PWS44 (e.g., Leica Biosystems), and E6V7H (e.g., Cell Signaling). Technology), S21002A (e.g., BioLegend), WM53, HIM3-4, P67.6, FOS, OTI1B10, OTI1C4, OTI1E10, OTI1E4, OTI2E4, OTI2F8, BLR061G, OTI2B2, OTI1G10, OTI1H8, OTI1H8, OTI2A1, OTI2C1, OTI2G8, OTI7G8, 4D3, WM-54, ZM122, 2B7C12, 2D12-G4, and RM398, as well as US9,212,225, US10,556,951, US10,711,062, US11,466,082, US11,560 Examples include anti-CD33 antibodies or antigen-binding fragments disclosed in any of the following: 428, US11,666,672, US11,174,313, US11,136,390, US2020 / 0148767, US2021 / 0017277, US2023 / 0102344, US2023 / 0365676, WO2022 / 060832, WO2021 / 138407, WO2020 / 227072, WO2019 / 006280, WO2018 / 218207, WO2019 / 028283, WO2019 / 178382, etc. (each of which is incorporated herein in its entirety by reference).

[0106] In some embodiments, the CD33 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 86, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 87. In some embodiments, the CD33 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 88, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD33 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 90, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 91. SEQ ID NOs: 86 to 91 are shown in Table S1, with complementarity determining regions (CDRs) shown in bold. [Table 1]

[0107] In some embodiments, the CD33 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 86, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 87. In some embodiments, the CD33 targeting moiety comprises CDR-H1 comprising the amino acid sequence DSNIH (SEQ ID NO: 227), CDR-H2 comprising the amino acid sequence YIYPYNGGTDYNQKFKN (SEQ ID NO: 228), CDR-H3 comprising the amino acid sequence GNPWLAY (SEQ ID NO: 229), CDR-L1 comprising the amino acid sequence RASESLDNYGIRFLT (SEQ ID NO: 230), CDR-L2 comprising the amino acid sequence AASNQGSGVPS (SEQ ID NO: 231), and CDR-L3 comprising the amino acid sequence QQTKEVPWS (SEQ ID NO: 232).

[0108] In some embodiments, the CD33 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 88, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 89. In some embodiments, the CD33 target-directing moiety includes CDR-H1 containing the amino acid sequence DYNMH (SEQ ID NO: 233), CDR-H2 containing the amino acid sequence YIYPYNGGTGYNQKFKS (SEQ ID NO: 234), CDR-H3 containing the amino acid sequence GRPAMDY (SEQ ID NO: 235), CDR-L1 containing the amino acid sequence RASESVDNYGISFMN (SEQ ID NO: 236), CDR-L2 containing the amino acid sequence AASNQGSGVPS (SEQ ID NO: 231), and CDR-L3 containing the amino acid sequence QQSKEVPWT (SEQ ID NO: 237).

[0109] In some embodiments, the CD33 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 90, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 91. In some embodiments, the CD33 target-directing moiety includes CDR-H1 containing the amino acid sequence NYDIN (SEQ ID NO: 238), CDR-H2 containing the amino acid sequence WIYPGDGSTKYNEKFKA (SEQ ID NO: 239), CDR-H3 containing the amino acid sequence GYEDAMDY (SEQ ID NO: 240), CDR-L1 containing the amino acid sequence KASQDINSYLS (SEQ ID NO: 241), CDR-L2 containing the amino acid sequence RANRLVDGVPS (SEQ ID NO: 242), and CDR-L3 containing the amino acid sequence LQYDEFPLT (SEQ ID NO: 243).

[0110] CD164 In some embodiments, the target molecule is CD164. In some embodiments, the target cells are CD164+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD164. Examples of anti-CD164 antibodies or their antigen-binding fragments include, but are not limited to, clone 5 (e.g., Invitrogen), ab238748 (e.g., AbCam), 67D2 (e.g., BioLegend), 105A5, 103B2 / 9E10, N6B6 (e.g., BD BioSciences), 96.1H5, 96.10H10, 96.12H11, 96.3F5, and 96.2D2, as well as anti-CD164 antibodies or their antigen-binding fragments disclosed in any of the following: Watt et al. (2021) Regen Med. 6:33, Zannettino et al. (1998) Blood. 92(8):2613-2628, Watt et al. (1998) Blood. 92(3):849-866, etc. (each of which is incorporated herein by reference in its entirety).

[0111] In some embodiments, the CD164 target-directing moiety includes a class I antibody, or its antigen-binding moiety, as described in Watts et al., Regenerative Medicine (2021) 6:33 (incorporated herein by reference). In some embodiments, the CD164 target-directing moiety includes a class II antibody, or its antigen-binding moiety, as described in Watts et al., Regenerative Medicine (2021) 6:33. In some embodiments, the CD164 target-directing moiety includes a class III antibody, or its antigen-binding moiety, as described in Watts et al., Regenerative Medicine (2021) 6:33. In some embodiments, the CD164 target-directing moiety includes an antibody such as 103B2 / 9E10, 105A5, N6B6, 67D2, 96.1H5, 96.2D2, 96.3F5, 96.10H10, or 96.12H11, or its antigen-binding moiety, as described in Watts et al., Regenerative Medicine (2021) 6:33.

[0112] CD117 In some embodiments, the target molecule is CD117, also referred to as c-kit, kit, or stem cell factor receptor (SCF-R). In some embodiments, the target cells are CD117+. In some embodiments, the target-directing moiety includes an antibody or antigen-binding fragment that binds to CD117. Examples of anti-CD117 antibodies or their antigen-binding fragments include briquilimab (JSP-191, e.g., Jasper Therapeutics), LOP628 (e.g., Novartis Pharmaceuticals), NEG024 (e.g., Novartis Pharmaceuticals), NEG026 (e.g., Novartis Pharmaceuticals), NEG027 (e.g., Novartis Pharmaceuticals), NEG085 (e.g., Novartis Pharmaceuticals), NEG086 (e.g., Novartis Pharmaceuticals), NEG087 (e.g., Novartis Pharmaceuticals), GZQ167 (e.g., Novartis Pharmaceuticals), MGTA-117 (e.g., Magenta Therapeutics), valzolvorimab (CDX-0159, e.g., CellDex Therapeutics), D13A2 (e.g., Cell Signaling Technology), D3W6Y (e.g., Cell Signaling Technology). Technology), 11996-R018 (e.g., Sino Biological), ab187371 (e.g., AbCam), 104D2, Ab67 (e.g., Magenta Therapeutics), Ab54 (e.g., Magenta Therapeutics), Ab55 (e.g., Magenta Therapeutics), Ab56 (e.g., Magenta Therapeutics), Ab57 (e.g., Magenta Therapeutics), Ab58 (e.g., Magenta Therapeutics), Ab61 (e.g., Magenta Therapeutics), Ab66 (e.g., Magenta Therapeutics), Ab68 (e.g., MagentaTherapeutics), Ab69 (e.g., Magenta Therapeutics), CK6, 9P3, AMG191, YB5.B8, NN2101, ACK2, SR-1, Ab1, 1C5, ST04-99, PD00-24, K45, 007, 017, HC34LC14, 2HCLC, 8D7, BLR263L, C117 / 8399R, rC117 / 9253, C117 / 8879R, OTI2B12, OTI1B6, OTI2E3, OTI1E2, C117 / 370, KIT / 982, OTI2C1D5, KIT / 2670, KIT / 2672, KIT / 2673, KIT / 2674, KIT / 983, KIT / 2669, 6F2, OTI3F9, OTI9A11, OTI14B1, UMAB216, 3A8, 1G1, 5F6, 5A11, 2C3, 1D3, X1, 2B8, 6G12, 4F19, and CL1657, as well as US 5,489,516, US 5,545,533, US 7,915,391, US 8,552,157, US 8,791,249, US 9,334,332, US 9,498,543, US 9,540,443, US 10,111,966, US 10,406,179, US 10,611,838, US 10,882,915, US 10,899,843, US 11,041,022, US 11,208,482, US 2021 / 0355212, US 2020 / 0369767, US 2022 / 0315653, US 2022 / 0177575, US 2022 / 0177578, WO 2019 / 084067, WO 2020 / 076105, WO 2020 / 242895, WO 2020 / 219770, WO 2021 / 044008, WO 2022 / 132929, WO 2023 / 204547, WO 2023 / 240272, WO 2024 / 008910, Lebron et al. (2014), Cancer Biol Ther. 15(9):1208-1218, Lerner et al. (1991) Blood. 77:1876-1883, Broudy et al. (1992) Blood. 79:338-346, Van Looy et al. (2015) Transl Oncol. 8(2):112-118, Rappold et al. (1997) Blood. 90(1):111-125, Alvarado etExamples include anti-CD117 antibodies or antigen-binding fragments disclosed in any of the following: al. (2022) Allergy 77(8):2393-2403, Abrams et al. (2018) Clin Cancer Res. 24(17):4297-4308, Kim et al. (2021) Mol Oncol. 16(6):1290-1308, etc. (each of which is incorporated herein by reference in its entirety).

[0113] In some embodiments, the CD117 target-directing portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 415 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 416. In some embodiments, the CD117 target-directing portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 417 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 418. In some embodiments, the CD117 target-directing portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 425 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 426. In some embodiments, the CD117 target-directing portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 427 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 428. SEQ ID NOs. 415-418 and 425-428 are shown in Table S2, with complementary determination regions (CDRs) shown in bold. [Table 2-1] [Table 2-2]

[0114] In some embodiments, the CD117 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 415, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 416. In some embodiments, the CD117 target-directing moiety comprises a first Fab polypeptide (i.e., VH-CH1) containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24, and / or a second Fab polypeptide (i.e., VL-CL) containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25. In some embodiments, the CD117 target-directing moiety includes CDR-H1 containing the amino acid sequence FTFSDADMD (SEQ ID NO: 32) or DADMD (SEQ ID NO: 392), CDR-H2 containing the amino acid sequence RNKAGSYTTEYAASVKG (SEQ ID NO: 33), CDR-H3 containing the amino acid sequence AREPKYWIDFDL (SEQ ID NO: 34), CDR-L1 containing the amino acid sequence RASQSISSYLN (SEQ ID NO: 35), CDR-L2 containing the amino acid sequence AASSLQS (SEQ ID NO: 36), and CDR-L3 containing the amino acid sequence QQSYIAPYT (SEQ ID NO: 37).

[0115] In some embodiments, the CD117 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 417, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 418. In some embodiments, the CD117 target-directing moiety comprises a first Fab polypeptide (i.e., VH-CH1) having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 30, and / or a second Fab polypeptide (i.e., VL-CL) having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the CD117 target-directing moiety includes CDR-H1 containing the amino acid sequence GYYMA (SEQ ID NO: 397), CDR-H2 containing the amino acid sequence NINYPGSSTYYLDSVKG (SEQ ID NO: 398), CDR-H3 containing the amino acid sequence GDYYGTTYWY (SEQ ID NO: 399), CDR-L1 containing the amino acid sequence RASQSISSYLN (SEQ ID NO: 35), CDR-L2 containing the amino acid sequence YTSRLQS (SEQ ID NO: 400), and CDR-L3 containing the amino acid sequence QQGRRLWS (SEQ ID NO: 401).

[0116] In some embodiments, the CD117 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 425, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 426. In some embodiments, the CD117 target-directing moiety includes CDR-H1 containing the amino acid sequence SYWIG (SEQ ID NO: 402), CDR-H2 containing the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO: 403), CDR-H3 containing the amino acid sequence HGRGYNGYEGA (SEQ ID NO: 404), CDR-L1 containing the amino acid sequence RASQGISSALA (SEQ ID NO: 405), CDR-L2 containing the amino acid sequence DASSLES (SEQ ID NO: 406), and CDR-L3 containing the amino acid sequence QQFNSYPLT (SEQ ID NO: 407).

[0117] In some embodiments, the CD117 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 427, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 428. In some embodiments, the CD117 target-directing moiety includes CDR-H1 containing the amino acid sequence SYNMH (SEQ ID NO: 408), CDR-H2 containing the amino acid sequence VIYSGNGDTSYNQKFKG (SEQ ID NO: 409), CDR-H3 containing the amino acid sequence RERDTR (SEQ ID NO: 410), CDR-L1 containing the amino acid sequence RASESVDIYGNSFMH (SEQ ID NO: 411), CDR-L2 containing the amino acid sequence LASNLES (SEQ ID NO: 412), and CDR-L3 containing the amino acid sequence QQNNEDPYT (SEQ ID NO: 413).

[0118] CD34 In some embodiments, the target molecule is CD34. In some embodiments, the target cells are CD34+. In some embodiments, the target-directing moiety includes an antibody or antigen-binding fragment that binds to CD34. In some embodiments, the anti-CD34 target-directing moiety includes a Fab fragment, scFv, or VHH domain. Examples of anti-CD34 antibodies or their antigen-binding fragments include S20016E (e.g., BioLegend, Inc.), 581, RAM34, 1H6, 561, 563, QBEND / 10, H4C8, L5F1, 2E10, 5B12, 4C8, 8G12, 2E9, 4H11, ICO-115, 3C8G12, BLR197J, CD34 / 4939, CD34 / 7719, CD34 / 7721, CS37, My10, OTI105C4, OTI10C8, OTI11A5, OTI11E1, OTI12F2, OTI39B1, OTI39B5, OTI51A1, OTI8F11, SPM123, SPM610, 15H1, 2C5, 4F11H3, 5A6, 5F3, 5L7E2, MEC14.7, PDM0-12, RM300, and SI16-01, as well as US8,084,033, US10,106,623, US2010 / 0311955, US2013 / 0172533, US2017 / 0320966, WO2023 / 077136, WO2023 / 141297, CN101245108, Arruda Examples include anti-CD34 antibodies or antigen-binding fragments disclosed in any of the following: et al. (2022) Haematologica 107(8):1786-1795 (each of which is incorporated herein by reference in its entirety).

[0119] In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 73 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 75 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 76. In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 77 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 78. In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 84 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 475 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 476. In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 477 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 478. In some embodiments, the CD34 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 479 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 480. In some embodiments, the CD34 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 481 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 482. SEQ ID NOs. 73-78, 84, 85, and 475-482 are shown in Table S3, with complementary determination regions (CDRs) indicated in bold. [Table 3-1] [Table 3-2]

[0120] In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 73, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 74. In some embodiments, the CD34 target-directed moiety comprises a first Fab polypeptide (i.e., VH-CH1) containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 38, and / or a second Fab polypeptide (i.e., VL-CL) containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 39. In some embodiments, the CD34 target-directing portion includes CDR-H1 containing the amino acid sequence GYTFTNYGMN (SEQ ID NO: 40), CDR-H2 containing the amino acid sequence WINTNTGEPKYAEEFKG (SEQ ID NO: 41), CDR-H3 containing the amino acid sequence GYGNYARGAWLAY (SEQ ID NO: 42), CDR-L1 containing the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 43), CDR-L2 containing the amino acid sequence QVSNRFS (SEQ ID NO: 44), and CDR-L3 containing the amino acid sequence FQGSHVPRT (SEQ ID NO: 45).

[0121] In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 75, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 76. In some embodiments, the CD34 target-directing portion includes CDR-H1 containing the amino acid sequence GYTFTNYGMN (SEQ ID NO: 40), CDR-H2 containing the amino acid sequence WINTNTGEPKYAEEFKG (SEQ ID NO: 41), CDR-H3 containing the amino acid sequence GYGNYARGAWLAY (SEQ ID NO: 42), CDR-L1 containing the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO: 43), CDR-L2 containing the amino acid sequence QVSNRFS (SEQ ID NO: 44), and CDR-L3 containing the amino acid sequence FQGSHVPRT (SEQ ID NO: 45).

[0122] In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 77, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 78. In some embodiments, the CD34 target-directed moiety includes CDR-H1 containing the amino acid sequence RYGMH (SEQ ID NO: 244), CDR-H2 containing the amino acid sequence VIWYDGRNKEYGDSVKG (SEQ ID NO: 245), CDR-H3 containing the amino acid sequence PIVGGTNY (SEQ ID NO: 246), CDR-L1 containing the amino acid sequence RASQSVSSDLA (SEQ ID NO: 247), CDR-L2 containing the amino acid sequence GASTRATGIPA (SEQ ID NO: 248), and CDR-L3 containing the amino acid sequence QQYNNWPIT (SEQ ID NO: 249).

[0123] In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 84, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CD34 target-directing moiety includes CDR-H1 containing the amino acid sequence GYFMN (SEQ ID NO: 250), CDR-H2 containing the amino acid sequence RINPYNGYTFYNQKFKG (SEQ ID NO: 251), CDR-H3 containing the amino acid sequence HFRYDGVFYYAMDY (SEQ ID NO: 252), CDR-L1 containing the amino acid sequence TLSSQHSTFTIE (SEQ ID NO: 253), CDR-L2 containing the amino acid sequence LKKDGSHSTGD (SEQ ID NO: 254), and CDR-L3 containing the amino acid sequence GVGDTIKEQFVYV (SEQ ID NO: 255).

[0124] In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 475, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 476.

[0125] In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 477, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 478.

[0126] In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 479, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 480.

[0127] In some embodiments, the CD34 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 481, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 482.

[0128] CD50 In some embodiments, the target molecule is CD50, also referred to as ICAM-3. In some embodiments, the target cells are CD50+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD50. Exemplary anti-CD50 antibodies or antigen-binding fragments include ICAM3-IB23 (e.g., Chugai Pharmaceutical), clone 002 (e.g., Invitrogen), clone 003 (e.g., Invitrogen), 1019 (e.g., NeoBiotechnologies), MEM-171, MEM-04, EPR22890-230, EPR3994-123, EPR3995, 2873R, OTI1E7, OTI1C2, OIT1F4, and OTI1 Examples include anti-CD50 antibodies or antigen-binding fragments disclosed in any of the following: F2, OTI1G4, OTI4F5, OTI4G8, OTI3A7, OTI4D4, OTI5D6, SPM505, 76203, 101, 101-1D2, 186-2G9, CG106, 2F8, MA4, BH10, JA4, ICO-60, ICAM3.1, BR-1, ICAM3.3, CBR-IC3 / 1, CBR-IC3 / 2.1.2, and US9,290,812, US2012 / 0321557, WO2011 / 105573, Skubitz et al. (1997) J Immunol. 159(2):820-828, etc. (each of which is incorporated herein by reference in its entirety).

[0129] In some embodiments, the CD50 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 328 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 329. SEQ ID NOs: 328 and 329 are shown in Table S4, with the complementary determination region (CDR) indicated in bold. [Table 4]

[0130] In some embodiments, the CD50 target-directed moiety comprises a first Fab polypeptide (i.e., VH-CH1) containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46 or 54, and / or a second Fab polypeptide (i.e., VL-CL) containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the C-terminus of the first Fab polypeptide (i.e., VH-CH1) further comprises a saltase tag as shown in SEQ ID NO: 83.

[0131] In some embodiments, the CD50 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 328, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 329. In some embodiments, the CD50 target-directing portion includes CDR-H1 containing the amino acid sequence TYWMH (SEQ ID NO: 322), CDR-H2 containing the amino acid sequence YINPNTDYTEYNQKFK (SEQ ID NO: 323), CDR-H3 containing the amino acid sequence SRDAYHGTY (SEQ ID NO: 324), CDR-L1 containing the amino acid sequence RASQSISDYLH (SEQ ID NO: 325), CDR-L2 containing the amino acid sequence YASQSIS (SEQ ID NO: 326), and CDR-L3 containing the amino acid sequence QNGHNFPLT (SEQ ID NO: 327).

[0132] CD71 In some embodiments, the target molecule is CD71, also known as the transferrin receptor. In some embodiments, the target cells are CD71+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD71. Exemplary anti-CD71 antibodies or antigen-binding fragments include CX-2029 (e.g., CytomX Therapeutics), INA03 (e.g., Inatherys), D7G9X (e.g., Cell Signaling Technology), and Ber-T9 (e.g., Santa Cruz). Biotechnology), DF1513, OKT9, H68.4, 10F11, 3B82A1, MEM-189, T56 / 14, MEM-75, RM384, JF0956, MRQ-48, ICO-92 , 3C11F11, 001, 040, 7H12, BGX.24, TFRC / 2898R, TFRC / 9106R, TFRC / 3630, TFRC / 1817, 66IG10, TFRC / 1059, TFRC / 18 Examples include anti-CD71 antibodies or antigen-binding fragments disclosed in any of the following: 18, TFRC / 1839, OTI6H9, TFRC / 1396, 1E6, R17217.1.4, TFRC / 1149, SAA1205, 7-1, 1H5, 3A12, 7C11, 2A4, 1G9, and 7B4, as well as US7,858,757, US10,179,817, etc. (each of which is incorporated herein by reference in its entirety).

[0133] In some embodiments, the CD71 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 65 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 66. In some embodiments, the CD71 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 67 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 68. In some embodiments, the CD71 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 69 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 70. In some embodiments, the CD71 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 71 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 72. SEQ ID NOs. 65 to 72 are shown in Table S5, and the complementary determination regions (CDRs) are shown in bold. [Table 5]

[0134] In some embodiments, the CD71 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 67, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 68. In some embodiments, the CD71 target-directing moiety includes CDR-H1 containing the amino acid sequence NYWLG (SEQ ID NO: 342), CDR-H2 containing the amino acid sequence DIYPGGDYPTYSEKFKV (SEQ ID NO: 343), CDR-H3 containing the amino acid sequence SGNYDEVAY (SEQ ID NO: 344), CDR-L1 containing the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 279), CDR-L2 containing the amino acid sequence KVSNRFS (SEQ ID NO: 345), and CDR-L3 containing the amino acid sequence SQSTHVPWT (SEQ ID NO: 346).

[0135] In some embodiments, the CD71 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 69, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 70. In some embodiments, the CD71 target-directing moiety includes CDR-H1 containing the amino acid sequence SYYMN (SEQ ID NO: 256), CDR-H2 containing the amino acid sequence GISGDPSNTYYADSVKG (SEQ ID NO: 257), CDR-H3 containing the amino acid sequence DLPLVYTGFAY (SEQ ID NO: 258), CDR-L1 containing the amino acid sequence SGDNLRHYYVY (SEQ ID NO: 259), CDR-L2 containing the amino acid sequence GDSKRPS (SEQ ID NO: 347) or GDSKRPSGIPE (SEQ ID NO: 260), and CDR-L3 containing the amino acid sequence QTYTGGASLV (SEQ ID NO: 261).

[0136] In some embodiments, the CD71 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with respect to the amino acid sequence of SEQ ID NO: 71, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with respect to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the CD71 target-directing moiety includes CDR-H1 containing the amino acid sequence NYWLG (SEQ ID NO: 342), CDR-H2 containing the amino acid sequence DIYPGGDYPTYSEKFKV (SEQ ID NO: 343), CDR-H3 containing the amino acid sequence SGNYDEVAY (SEQ ID NO: 344), CDR-L1 containing the amino acid sequence RSSQSLVH (SEQ ID NO: 348) or RSSQSLVHSNGNTYLH (SEQ ID NO: 279), CDR-L2 containing the amino acid sequence KVSNRFS (SEQ ID NO: 345), and CDR-L3 containing the amino acid sequence SQSTHVPWT (SEQ ID NO: 346).

[0137] In some embodiments, the CD71 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 65, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 66. In some embodiments, the CD71 target-directing moiety comprises a first Fab polypeptide (i.e., VH-CH1) containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48, and / or a second Fab polypeptide (i.e., VL-CL) containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the CD71 target-directing moiety includes CDR-H1 containing the amino acid sequence SYAMS (SEQ ID NO: 349), CDR-H2 containing the amino acid sequence YIWSGGSTDYASWA (SEQ ID NO: 350), CDR-H3 containing the amino acid sequence RYGTSYPDYGDASGFDP (SEQ ID NO: 351), CDR-L1 containing the amino acid sequence RASQSISSYLA (SEQ ID NO: 352), CDR-L2 containing the amino acid sequence RASTLAS (SEQ ID NO: 353), and CDR-L3 containing the amino acid sequence QQNYASSNVDNT (SEQ ID NO: 354).

[0138] CD133 In some embodiments, the target molecule is CD133, also referred to as PROM1, PROML1, prominin-1, AC133, CORD12, MCDR2, MSTP061, RP41, or STGD4. In some embodiments, the target cells are CD133+. In some embodiments, the target-directing moiety includes an antibody or antigen-binding fragment that binds to CD133. Examples of anti-CD133 antibodies or their antigen-binding fragments include A3G6K (e.g., Cell Signaling Technology), D2V8Q (e.g., Cell Signaling Technology), W6B3C1, W6B3, TMP4, 2F8C5, 5E3, EMK08, and BLR093G, as well as anti-CD133 antibodies or their antigen-binding fragments disclosed in any of the following: US11,098,109, US11,220,551, Glumac et al. Prostate. 2018 Sep;78(13):981-991, etc. (each of which is incorporated herein by reference in its entirety).

[0139] CD38 In some embodiments, the target molecule is CD38. In some embodiments, the target cells are CD38+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD38. Exemplary anti-CD38 antibodies or antigen-binding fragments include daratumumab (e.g., Janssen Biotech, Inc.), isatuximab (e.g., Sanofi), and ferzaltamab (MOR202, e.g., MorphoSys). AG), TAK-079 (e.g., Takeda), SAR442085 (e.g., Sanofi), CM313, 6E12D, AT1, AT2, AT13 / 5, HB7, 5C5C3, HIT2, 3C6G4, PD01-49, RM388, 1G3, 4G3, OKT10, BLR123H, 6448R, 7017R, 4247R, rCD38 / 8334, CD38 / 8075R, CD38 / 81114R, CD38 / 8335R, CD38 / 8994R, ZR351, 4328, rCD38 / 6447, r CD38 / 6982, rCD38 / 8024, clone 90, OTI1C9, THB7, HB7, OTI1H9, OTI8D5, OTI11C3, UMAB263, OTI9F8, OTI6B3, FS02, OKT10, SA0892, SAA0895, SAA1161, SAA2057, SA1116, TME-2, and ICO-20, as well as US7,829,673, US9,603,927, US10,526,417, US10,604,580; US2016 / 0376373, Kassem Examples include anti-CD38 antibodies or antigen-binding fragments disclosed in any of the following: et al. (2022) Blood. 139(8):1160-1176, Chen et al. (2024) N Engl J Med. 390:2178-2190, etc. (each of which is incorporated herein by reference in its entirety).

[0140] In some embodiments, the CD38 target-directing region includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 92 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 93. SEQ ID NOs: 92 and 93 are shown in Table S6, with complementary determination regions (CDRs) shown in bold. [Table 6]

[0141] In some embodiments, the CD38 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 92, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 93. In some embodiments, the CD38 target-directing moiety includes CDR-H1 containing the amino acid sequence SYYMN (SEQ ID NO: 256), CDR-H2 containing the amino acid sequence GISGDPSNTYYADSVKG (SEQ ID NO: 257), CDR-H3 containing the amino acid sequence DLPLVYTGFAY (SEQ ID NO: 258), CDR-L1 containing the amino acid sequence SGDNLRHYYVY (SEQ ID NO: 259), CDR-L2 containing the amino acid sequence GDSKRPSGIPE (SEQ ID NO: 260), and CDR-L3 containing the amino acid sequence QTYTGGASLV (SEQ ID NO: 261).

[0142] CD43 In some embodiments, the target molecule is CD43, also referred to as leukosialin or sialophorin. In some embodiments, the target cells are CD43+. In some embodiments, the target-directing moiety includes an antibody or antigen-binding fragment that binds to CD43. Exemplary anti-CD43 antibodies or antigen-binding fragments include AT1413, UMG-1, MT-1, S11, CD43-10G7, MEM-59, 1G10, 3G8, 84-3C1, SP55, 9D10L2, PD01-28, 4-29-5-10-21, DFT-1, 1094, 839, 1766R, 2049R, rSPN / 6563, SPN / 6562R, 2A11D6, SPN / 839, SPN / 3388, Examples include anti-CD43 antibodies or antigen-binding fragments disclosed in any of the following: DF-T1, SPN / 1094, SPM503, OTI2D4, OTI2C7, OTI3D11, OTI6A4, OTI4G8, OTI3D11, OTI4F6, Bra7G, A2F9, A2F7, and A2F8, as well as US7,674,605, US9,746,474, US10,676,531, US11,174,318, US11,524,989; KR20120081723, Gillissen et al. (2017) Blood Adv. 1(19):1551-1564, etc. (each of which is incorporated herein by reference in its entirety).

[0143] In some embodiments, the CD43 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 118 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 119. In some embodiments, the CD43 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 120 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 121. In some embodiments, the CD43 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 122 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 123. In some embodiments, the CD43 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 124 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 125. In some embodiments, the CD43 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 126 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 127. In some embodiments, the CD43 target-directed portion includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 128 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 129. In some embodiments, the CD43 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 130 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 131. In some embodiments, the CD43 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 132 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 133. SEQ ID NOs. 118-133 are shown in Table S7, with complementary determination regions (CDRs) shown in bold. [Table 7-1] [Table 7-2]

[0144] In some embodiments, the CD43 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 118, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 119. In some embodiments, the CD43 target-directing moiety includes CDR-H1 containing the amino acid sequence GYFMN (SEQ ID NO: 250), CDR-H2 containing the amino acid sequence RINPNNGDSFYNQKFKG (SEQ ID NO: 262), CDR-H3 containing the amino acid sequence EGYYGGRGYALDY (SEQ ID NO: 263), CDR-L1 containing the amino acid sequence RTSQDISNYLN (SEQ ID NO: 264), CDR-L2 containing the amino acid sequence NTSRLHSGVPS (SEQ ID NO: 265), and CDR-L3 containing the amino acid sequence QQSNMFPYT (SEQ ID NO: 266).

[0145] In some embodiments, the CD43 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 120, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 121. In some embodiments, the CD43 target-directing moiety includes CDR-H1 containing the amino acid sequence GYYMN (SEQ ID NO: 267), CDR-H2 containing the amino acid sequence RINPNSGDSFYNQKFKG (SEQ ID NO: 268), CDR-H3 containing the amino acid sequence EGYYGGRGYALDY (SEQ ID NO: 263), CDR-L1 containing the amino acid sequence RTSQDISNYLN (SEQ ID NO: 264), CDR-L2 containing the amino acid sequence NTSRLHSGVPS (SEQ ID NO: 265), and CDR-L3 containing the amino acid sequence QQSNMFPYT (SEQ ID NO: 266).

[0146] In some embodiments, the CD43 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 122, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 123. In some embodiments, the CD43 target-directing moiety includes CDR-H1 containing the amino acid sequence GYFMN (SEQ ID NO: 250), CDR-H2 containing the amino acid sequence RINPNNGDSFYNQKFQG (SEQ ID NO: 269), CDR-H3 containing the amino acid sequence EGYYGGRGYALDY (SEQ ID NO: 263), CDR-L1 containing the amino acid sequence RTSQDISNYLN (SEQ ID NO: 264), CDR-L2 containing the amino acid sequence NTSRLHSGVPS (SEQ ID NO: 265), and CDR-L3 containing the amino acid sequence QQSNMFPYT (SEQ ID NO: 266).

[0147] In some embodiments, the CD43 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 124, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 125. In some embodiments, the CD43 target-directing moiety includes CDR-H1 containing the amino acid sequence GYFMN (SEQ ID NO: 250), CDR-H2 containing the amino acid sequence RINPNNGDSFYNQKFKG (SEQ ID NO: 262), CDR-H3 containing the amino acid sequence EGYYGGRGYALDY (SEQ ID NO: 263), CDR-L1 containing the amino acid sequence RTSQDISNYLN (SEQ ID NO: 264), CDR-L2 containing the amino acid sequence NTSRLHSGVPS (SEQ ID NO: 265), and CDR-L3 containing the amino acid sequence QQSNMFPYT (SEQ ID NO: 266).

[0148] In some embodiments, the CD43 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 126, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 127. In some embodiments, the CD43 target-directing moiety includes CDR-H1 containing the amino acid sequence GYFMN (SEQ ID NO: 250), CDR-H2 containing the amino acid sequence RINPNNGDSFYNQKFQG (SEQ ID NO: 269), CDR-H3 containing the amino acid sequence EGYYGGRGYALDY (SEQ ID NO: 263), CDR-L1 containing the amino acid sequence RTSQDISNYLN (SEQ ID NO: 264), CDR-L2 containing the amino acid sequence NTSRLHSGVPS (SEQ ID NO: 265), and CDR-L3 containing the amino acid sequence QQSNMFPYT (SEQ ID NO: 266).

[0149] In some embodiments, the CD43 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 128, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 129. In some embodiments, the CD43 target-directing moiety includes CDR-H1 containing the amino acid sequence SGFMH (SEQ ID NO: 270), CDR-H2 containing the amino acid sequence YISSGSGNFYYVDTVKG (SEQ ID NO: 271), CDR-H3 containing the amino acid sequence STYYHGSRGAMDY (SEQ ID NO: 272), CDR-L1 containing the amino acid sequence SASSSVSSMY (SEQ ID NO: 273), CDR-L2 containing the amino acid sequence DTSKMASGVPI (SEQ ID NO: 274), and CDR-L3 containing the amino acid sequence QQWSSYPPIT (SEQ ID NO: 275).

[0150] In some embodiments, the CD43 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 132, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 133. In some embodiments, the CD43 target-directing moiety includes CDR-H1 containing the amino acid sequence SSPNNWWT (SEQ ID NO: 282), CDR-H2 containing the amino acid sequence EIYYGGRVSYNSALRS (SEQ ID NO: 283), CDR-H3 containing the amino acid sequence QKNIGCGYSSCFIS (SEQ ID NO: 284), CDR-L1 containing the amino acid sequence KSSQTILQRSNHLNYLA (SEQ ID NO: 285), CDR-L2 containing the amino acid sequence WASTRESGVPD (SEQ ID NO: 286), and CDR-L3 containing the amino acid sequence HQYYTTPQT (SEQ ID NO: 287).

[0151] CD45 In some embodiments, the target molecule is CD45. In some embodiments, the target cells are CD45+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD45. Examples of anti-CD45 antibodies or their antigen-binding fragments include YAML501.4, YAML568, HI30, YTH-24, YTH-54, JE03-05, 2D1, CD45-2B11, MEM-28, 4E9B2, UCHL1, BL-178-12C7, RM291, 4R0B0, 4A8A4C7A2, F10-89-4, MEM-55, 158-D3, 6O19, 1975R, 1460, 1461, 3881R, OTI4C11, UCH-L1 or UCHL-1, OTI1B7, OTI2F1, OTI3F2, PD7 / 26, OTI2E7, O Examples include anti-CD45 antibodies or antigen-binding fragments disclosed in any of the following: TI2H1, OTI1F6, OTI3C8, OTI2E7, OTI1B6, OTI3D1, 14A9, 2B11, OTI3E3, 135-4C5, Bra55, PTPRC / 1461, 136-4B5, 797, OTI1H2, OTI3B9, OTI3D8, and PTPRC / 1666, as well as US9,962,408, US11,655,452, US11,692,041, US11,406,724, etc. (each of which is incorporated herein by reference in its entirety).

[0152] In some embodiments, the CD45 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 134 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 135. In some embodiments, the CD45 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 136 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 137. In some embodiments, the CD45 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 138 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 139. SEQ ID NOs. 134 to 139 are shown in Table S8, with complementary determination regions (CDRs) shown in bold. [Table 8]

[0153] In some embodiments, the CD45 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 134, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 135. In some embodiments, the CD45 target-directing moiety includes CDR-H1 containing the amino acid sequence NYWMT (SEQ ID NO: 288), CDR-H2 containing the amino acid sequence SISSSGGSIYYPDSVKG (SEQ ID NO: 289), CDR-H3 containing the amino acid sequence DERWAGAMDA (SEQ ID NO: 290), CDR-L1 containing the amino acid sequence KASQNINKNLD (SEQ ID NO: 291), CDR-L2 containing the amino acid sequence ETNNLQTGIPS (SEQ ID NO: 292), and CDR-L3 containing the amino acid sequence YQHNSRFT (SEQ ID NO: 293).

[0154] In some embodiments, the CD45 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 136, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 137. In some embodiments, the CD45 target-directing portion includes CDR-H1 containing the amino acid sequence NFWMT (SEQ ID NO: 294), CDR-H2 containing the amino acid sequence SISSSGGSIYYPDSVKD (SEQ ID NO: 295), CDR-H3 containing the amino acid sequence LHYYSGGGDA (SEQ ID NO: 296), CDR-L1 containing the amino acid sequence KASQNINKYLD (SEQ ID NO: 297), CDR-L2 containing the amino acid sequence YTNNLHTGIPS (SEQ ID NO: 298), and CDR-L3 containing the amino acid sequence LQHSSRWT (SEQ ID NO: 299).

[0155] In some embodiments, the CD45 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 138, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 139. In some embodiments, the CD45 target-directing moiety includes CDR-H1 containing the amino acid sequence NYWMT (SEQ ID NO: 288), CDR-H2 containing the amino acid sequence SISSSGGSIYYPDSVKD (SEQ ID NO: 295), CDR-H3 containing the amino acid sequence LYYYSGGGDA (SEQ ID NO: 300), CDR-L1 containing the amino acid sequence KASQDINKYLD (SEQ ID NO: 301), CDR-L2 containing the amino acid sequence NTNNLHTGIPS (SEQ ID NO: 302), and CDR-L3 containing the amino acid sequence LQHISRWT (SEQ ID NO: 303).

[0156] CD46 In some embodiments, the target molecule is CD46. In some embodiments, the target cells are CD46+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD46. Exemplary anti-CD46 antibodies or antigen-binding fragments include those disclosed in any of the following: SB1HGNY, YS5, YS12, 8E2, MEM-258, JB25-49, 7D12, 23GB5720, ARC0428, 001, 5A8C11, M037, 05, 1E3D1, M177, 122.2, 169-1-E4.3, 3F1, 2E10, 1B6, and MCP / 931, as well as US10, 533, 056, etc. (each of which is incorporated herein by reference in whole).

[0157] In some embodiments, the CD46 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 419 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 420. In some embodiments, the CD46 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 421 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 422. In some embodiments, the CD46 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 423 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 424. SEQ ID NOs. 419 to 424 are shown in Table S9, with complementary determination regions (CDRs) shown in bold. [Table 9]

[0158] In some embodiments, the CD46 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 419, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 420. In some embodiments, the CD46 target-directing moiety includes CDR-H1 containing the amino acid sequence SYAMH (SEQ ID NO: 304), CDR-H2 containing the amino acid sequence FIRSDGSKKYYADSVKG (SEQ ID NO: 305), CDR-H3 containing the amino acid sequence HGNYFDS (SEQ ID NO: 306), CDR-L1 containing the amino acid sequence RASQGISSYLA (SEQ ID NO: 307), CDR-L2 containing the amino acid sequence AASTLQS (SEQ ID NO: 308), and CDR-L3 containing the amino acid sequence QQLASYPLT (SEQ ID NO: 309).

[0159] In some embodiments, the CD46 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 421, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 422. In some embodiments, the CD46 target-directing moiety includes CDR-H1 containing the amino acid sequence NYAMH (SEQ ID NO: 310), CDR-H2 containing the amino acid sequence VISYDGNNKYYADSVKG (SEQ ID NO: 311), CDR-H3 containing the amino acid sequence GGGYFDL (SEQ ID NO: 312), CDR-L1 containing the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 313), CDR-L2 containing the amino acid sequence GNNNRPS (SEQ ID NO: 314), and CDR-L3 containing the amino acid sequence SSYTSGTWL (SEQ ID NO: 315).

[0160] In some embodiments, the CD46 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 423, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 424. In some embodiments, the CD46 target-directing moiety includes CDR-H1 containing the amino acid sequence TYGMH (SEQ ID NO: 316), CDR-H2 containing the amino acid sequence FISYDGDEKYYADSVKG (SEQ ID NO: 317), CDR-H3 containing the amino acid sequence ASGYGMGILDY (SEQ ID NO: 318), CDR-L1 containing the amino acid sequence QGDSLRSYYVS (SEQ ID NO: 319), CDR-L2 containing the amino acid sequence GQNNRPS (SEQ ID NO: 320), and CDR-L3 containing the amino acid sequence HSRDSSGTHLRV (SEQ ID NO: 321).

[0161] CD49d In some embodiments, the target molecule is CD49d. In some embodiments, the target cells are CD49d+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD49d. Exemplary anti-CD49d antibodies or antigen-binding fragments include natalizumab (e.g., Biogen), vedolizumab (e.g., Takeda), and 9F10.

[0162] In some embodiments, the CD49d target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 94 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 95. In some embodiments, the CD49d target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 96 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 97. SEQ ID NOs. 94 to 97 are shown in Table S10, with complementary determination regions (CDRs) shown in bold. [Table 10]

[0163] In some embodiments, the CD49d target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 94, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 95. In some embodiments, the CD49d target-directing moiety includes CDR-H1 containing the amino acid sequence DTYIH (SEQ ID NO: 330), CDR-H2 containing the amino acid sequence RIDPANGYTKYDPKFQG (SEQ ID NO: 331), CDR-H3 containing the amino acid sequence EGYYGNYGVYAMDY (SEQ ID NO: 332), CDR-L1 containing the amino acid sequence KTSQDINKYMA (SEQ ID NO: 333), CDR-L2 containing the amino acid sequence YTSALQP (SEQ ID NO: 334), and CDR-L3 containing the amino acid sequence LQYDNLWT (SEQ ID NO: 335).

[0164] In some embodiments, the CD49d target-directing moiety includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 96, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 97. In some embodiments, the CD49d target-directing moiety includes CDR-H1 containing the amino acid sequence SYWMH (SEQ ID NO: 336), CDR-H2 containing the amino acid sequence EIDPSESNTNYNQKFKG (SEQ ID NO: 337), CDR-H3 containing the amino acid sequence GGYDGWDYAIDY (SEQ ID NO: 338), CDR-L1 containing the amino acid sequence RSSQSLAKSYGNTYLS (SEQ ID NO: 339), CDR-L2 containing the amino acid sequence GISNRFS (SEQ ID NO: 340), and CDR-L3 containing the amino acid sequence LQGTHQPYT (SEQ ID NO: 341).

[0165] CD49f In some embodiments, the target molecule is CD49f. In some embodiments, the target cells are CD49f+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD49f. Exemplary anti-CD49f antibodies or antigen-binding fragments include MP-4F10, GoH3, NKI-GoH3, 450-30A, 5HCLC, SR45-00, 5H14L18, 4G10, CL6957, and MAB-5A.

[0166] CD59 In some embodiments, the target molecule is CD59. In some embodiments, the target cells are CD59+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD59. Exemplary anti-CD59 antibodies or antigen-binding fragments include MEM-43, OV9A2, JM10-71, p282(H19), QA19A13, 2491C, 2E11B6, 193-27, 029, and 108.

[0167] CD90 In some embodiments, the target molecule is CD90. In some embodiments, the target cells are CD90+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD90. Exemplary anti-CD90 antibodies or antigen-binding fragments include 5E10, HIS51, 53-2.1, F15-42-1, G7, and YKIX337.217.

[0168] In some embodiments, the CD90 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 148 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 149. In some embodiments, the CD90 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 150 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 151. SEQ ID NOs. 148 to 151 are shown in Table S11, with complementary determination regions (CDRs) shown in bold. [Table 11]

[0169] In some embodiments, the CD90 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 148, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 149. In some embodiments, the CD90 target-directing portion includes CDR-H1 containing the amino acid sequence GYYVH (SEQ ID NO: 373), CDR-H2 containing the amino acid sequence WVNPNSGDTNYAQKFQG (SEQ ID NO: 374), CDR-H3 containing the amino acid sequence DGDEDWY (SEQ ID NO: 375), CDR-L1 containing the amino acid sequence RASQGISRSLV (SEQ ID NO: 376), CDR-L2 containing the amino acid sequence AASTLQS (SEQ ID NO: 308), and CDR-L3 containing the amino acid sequence LQHNTYPFT (SEQ ID NO: 377).

[0170] In some embodiments, the CD90 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 150, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 151. In some embodiments, the CD90 target-directing portion includes CDR-H1 containing the amino acid sequence SYAMS (SEQ ID NO: 349), CDR-H2 containing the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 378), CDR-H3 containing the amino acid sequence GARMDV (SEQ ID NO: 379), CDR-L1 containing the amino acid sequence KSSQSVLSSSKNKNYLA (SEQ ID NO: 380), CDR-L2 containing the amino acid sequence WASTRQS (SEQ ID NO: 381), and CDR-L3 containing the amino acid sequence QQHYSIPVT (SEQ ID NO: 382).

[0171] CD105 In some embodiments, the target molecule is CD105. In some embodiments, the target cells are CD105+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD105. Exemplary anti-CD105 antibodies or antigen-binding fragments include QA19A14, O99E5, 43A3, SN6, MJ7 / 18, 3A9, MEM-226, ARC0446, 209701, 2D5E8 JE60-59, 103, and 23GB3745.

[0172] CD110 In some embodiments, the target molecule is CD110. In some embodiments, the target cells are CD110+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD110. Exemplary anti-CD110 antibodies or antigen-binding fragments include 1D6B7, S16017A, 1H2, and ARC2257.

[0173] CD123 In some embodiments, the target molecule is CD123. In some embodiments, the target cells are CD123+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD123. Exemplary anti-CD123 antibodies or antigen-binding fragments include pibekimab (IMGN632, e.g., ImmunoGen), talakotzumab (e.g., Johnson & Johnson), S18016C, QA18A25, 6H6, 7G3, 5B11, 017, and 32703.

[0174] In some embodiments, the CD123 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 148 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 149. In some embodiments, the CD123 target-directing portion includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 150 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 151. SEQ ID NOs. 148 to 151 are shown in Table S21, with complementary determination regions (CDRs) shown in bold. [Table 12]

[0175] In some embodiments, the CD123 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with respect to the amino acid sequence of SEQ ID NO: 98, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with respect to the amino acid sequence of SEQ ID NO: 99. In some embodiments, the CD123 target-directing moiety includes CDR-H1 containing the amino acid sequence SSIMH (SEQ ID NO: 355), CDR-H2 containing the amino acid sequence YIKPYNDGTKYNEKFKG (SEQ ID NO: 356), CDR-H3 containing the amino acid sequence EGGNDYYDTMDY (SEQ ID NO: 357), CDR-L1 containing the amino acid sequence RASQDINSYLS (SEQ ID NO: 358), CDR-L2 containing the amino acid sequence RVNRLVD (SEQ ID NO: 359), and CDR-L3 containing the amino acid sequence LQYDAFPYT (SEQ ID NO: 360).

[0176] In some embodiments, the CD123 target-directed portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 100, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 101. In some embodiments, the CD123 target-directing moiety includes CDR-H1 containing the amino acid sequence DYYMK (SEQ ID NO: 361), CDR-H2 containing the amino acid sequence DIIPSNGATFYNQKFKG (SEQ ID NO: 362), CDR-H3 containing the amino acid sequence SHLLRASWFAY (SEQ ID NO: 363), CDR-L1 containing the amino acid sequence ESSQSLLNSGNQKNYLT (SEQ ID NO: 364), CDR-L2 containing the amino acid sequence WASTRES (SEQ ID NO: 365), and CDR-L3 containing the amino acid sequence QNDYSYPYT (SEQ ID NO: 366).

[0177] CD135 In some embodiments, the target molecule is CD135. In some embodiments, the target cells are CD135+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD135. Exemplary anti-CD135 antibodies or antigen-binding fragments include BV10A4H2, 4G8, A2F10, 7B7C3, BV10A4, 66903, OTI7D6, and FLT3 / 2458.

[0178] CD184 In some embodiments, the target molecule is CD184. In some embodiments, the target cells are CD184+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD184. Exemplary anti-CD184 antibodies or antigen-binding fragments include urocuplumab (e.g., Bristol-Myers Squibb), 12G5, 1D9, QA18A64, 2B11, 21H17L11, HL2424, 2F10, 2D4, ARC0381, 9D9, 4B5E4, 2G9, and 2A9.

[0179] In some embodiments, the CD184 target-directing region includes a heavy-chain variable region containing the amino acid sequence of SEQ ID NO: 146 and a light-chain variable region containing the amino acid sequence of SEQ ID NO: 147. SEQ ID NOs: 146 and 147 are shown in Table S13, with the complementary determination region (CDR) shown in bold. [Table 13]

[0180] In some embodiments, the CD184 target-directing region includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 146, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with the amino acid sequence of SEQ ID NO: 147. In some embodiments, the CD184 target-directing moiety includes CDR-H1 containing the amino acid sequence SYSMN (SEQ ID NO: 367), CDR-H2 containing the amino acid sequence YISSRSRTIYYADSVKG (SEQ ID NO: 368), CDR-H3 containing the amino acid sequence DYGGQPPYYYYYGMDV (SEQ ID NO: 369), CDR-L1 containing the amino acid sequence RASQGISSWLA (SEQ ID NO: 370), CDR-L2 containing the amino acid sequence AASSLQS (SEQ ID NO: 36), and CDR-L3 containing the amino acid sequence QQYNSYPRT (SEQ ID NO: 372).

[0181] CD201 In some embodiments, the target molecule is CD201. In some embodiments, the target cells are CD201+. In some embodiments, the target-directing moiety includes an antibody or antigen-binding fragment that binds to CD201. Exemplary anti-CD201 antibodies or antigen-binding fragments include clones 1560, 041, RCR-227, mRCR-16, 1F5C4, 011, and 016.

[0182] In some embodiments, the CD201 target-directing moiety includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 152 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 153. In some embodiments, the CD201 target-directing moiety is a single-domain antibody (sdAb) containing a VHH containing the amino acid sequence of SEQ ID NO: 102. SEQ ID NOs: 102, 152, and 153 are shown in Table S14, with complementary determination regions (CDRs) shown in bold. [Table 14]

[0183] In some embodiments, the CD201 target-directing portion includes a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 152, and / or a light chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 153. In some embodiments, the CD201 target-directing moiety includes CDR-H1 containing the amino acid sequence SYAIS (SEQ ID NO: 383), CDR-H2 containing the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO: 384), CDR-H3 containing the amino acid sequence GYSDAFDI (SEQ ID NO: 385), CDR-L1 containing the amino acid sequence RASQGIRNDLG (SEQ ID NO: 386), CDR-L2 containing the amino acid sequence AASRLQS (SEQ ID NO: 387), and CDR-L3 containing the amino acid sequence HQAYSYPLT (SEQ ID NO: 388).

[0184] In some embodiments, the CD201 target-directing moiety is an sdAb comprising a heavy chain variable region containing an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with respect to the amino acid sequence of SEQ ID NO: 102. In some embodiments, the CD201 target-directing moiety is an sdAb comprising CDR-H1 containing the amino acid sequence YYAIG (SEQ ID NO: 389), CDR-H2 containing the amino acid sequence CISSSDGSTYYADSVKG (SEQ ID NO: 390), and CDR-H3 containing the amino acid sequence SLGGAWVVAGNCPALDFNS (SEQ ID NO: 391).

[0185] CD338 In some embodiments, the target molecule is CD338. In some embodiments, the target cells are CD338+. In some embodiments, the target-directing portion includes an antibody or antigen-binding fragment that binds to CD338. Exemplary anti-CD338 antibodies or antigen-binding fragments include 5D3, 3G8, BXP-21, and EPR20080.

[0186] GPR56 In some embodiments, the target molecule is GPR56. In some embodiments, the target cells are GPR56+. In some embodiments, the target-directed portion includes an antibody or antigen-binding fragment that binds to GPR56. Exemplary anti-GPR56 antibodies or antigen-binding fragments include CG4, 4C3, 3A1D7, and 1443CT642.85.27, as well as the anti-GPR56 antibodies or antigen-binding fragments disclosed in Chatterjee et al. (2021) J Biol Chem. 296:100261 (each of which is incorporated herein by reference in its entirety).

[0187] Method for preparing targeted LNPs (conjugates) Different approaches can be used to introduce target-directed moieties to the surface of LNPs. For example, one approach relies on the functionalization of pre-formed LNPs having target-directed moieties. LNPs generally contain lipids having polyethylene glycol (PEG) spacers functionalized with reactive moieties such as thiols, amines, maleimides, or carboxylic acid groups. The functionalized lipid of the LNP reacts with complementary groups covalently bonded to the target-directed moiety, thus forming a conjugate between the LNP and the target-directed moiety.

[0188] In certain embodiments, the target-directed moieties are site-specifically conjugated onto the surface of the LNP. Conjugates prepared by site-specific methods (e.g., Figures 1-17) have a high density of target-directed moieties on the surface of the LNP. For example, a conjugate may contain two or more target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP. In some embodiments, a conjugate may contain more than 10 target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP. In some embodiments, a conjugate may contain more than 20 target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP. In some embodiments, a conjugate may contain more than 30 target-directed moieties (e.g., antibodies, Fab fragments, or scFv). In some embodiments, a conjugate may contain more than 50 target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP. In some embodiments, the conjugate may contain more than 75 target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP. In some embodiments, the conjugate may contain more than 100 target-directed moieties (e.g., antibodies, Fab fragments, or scFv). In some embodiments, the conjugate may contain about 50 to about 200 target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP. In some embodiments, the conjugate may contain about 100 to about 200 target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP. In some embodiments, the conjugate may contain about 100 to about 230 target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP. In some embodiments, the conjugate may contain about 10 to about 150 target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP. In some embodiments, the conjugate may contain about 10 to 30 target-directed moieties (e.g., antibodies, Fab fragments, or scFv) per LNP.

[0189] In some embodiments, the weight ratio between the target-directed portion on the surface of the LNP and the payload (e.g., RNA) encapsulated in the LNP can be 1:20, 1:15, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1.

[0190] In some embodiments, a conjugate prepared by a site-specific method (e.g., Figures 1-17) may have two or more different target-directing portions on the surface of the LNP, one of which is bound to CD164. For example, the conjugate may have two different target-directing portions. In some embodiments, the conjugate may have three different target-directing portions on the surface of the LNP, one of which is bound to CD164. In some embodiments, the conjugate may have two different target-directing portions, and the mass ratio of the two target-directing portions on the surface of the LNP is 20:1, 15:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, or 1:20. In some embodiments, the weight ratio between one or more target-directed regions on the surface of the LNP and the payload (e.g., RNA) encapsulated in the LNP can be 1:20, 1:15, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1.

[0191] Interchain C L -C H 1. Disulfide reduction, followed by conjugation IgG antibodies consist of four polypeptide chains linked by disulfide bonds. Two low molecular weight polypeptide chains are called light chains (L). The light chains have variable light chain domains (V). L ) and constant light chain domain (CL ). The heavy chain consists of a variable heavy domain (V H ) and three constant heavy domains (C H 1, C H 2, and C H 3). The Fab region of an antibody comprises the V L , C L , V H , and C H 1 domains. The Fc region comprises the constant heavy chain domains C H 2, and C H 3. The hinge region of an IgG antibody covalently links the C H 1 domain to the C H 2 domain. The two heavy chains of an IgG antibody are connected within the hinge region by a variable number of disulfide bonds depending on the IgG subclass. Different subclasses of IgG antibodies have varying numbers of interchain disulfide bonds. In addition, the light chain is covalently bonded to the heavy chain via a disulfide bond between the light chain and the heavy chain. Using standard IgG nomenclature, this native interchain disulfide bond is referred to as the C L -C H 1 disulfide bond to distinguish it from the disulfide bonds present in the hinge region. A therapeutic antibody of the IgG1 type has an interchain disulfide bond between Cys233 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain. A therapeutic antibody of the IgG4 type has an interchain disulfide bond between Cys127 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain. A therapeutic antibody of the IgG2 type has an interchain disulfide bond between Cys135 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.

[0192] Proteolytic cleavage of IgG antibodies results in the formation of Fab fragments known as F(ab')2 fragments. The F(ab')2 fragment comprises the C H 2 domain or C HIt does not contain 3 domains. However, the hinge region of the antibody is retained in the F(ab')2 fragment. The F(ab')2 fragment contains a disulfide bond covalently connecting the two Fab fragments. Reduction of the disulfide bond in F(ab')2 produces two F(ab') fragments. The sulfhydryl (thiol) group of F(ab') can potentially react with thiol-reactive groups on the surface of the LNP, thus producing a conjugate. However, site-specific conjugation is difficult due to the presence of multiple sulfhydryl groups in the hinge region of the F(ab') fragment. Furthermore, reduction of F(ab')2 to the F(ab') fragment is difficult. L Region and C H This can disrupt the natural interchain disulfide bonds between regions, and thus further impair site-specific conjugation.

[0193] As described herein, a Fab fragment (e.g., a Fab fragment bound to CD164) has a natural interchain disulfide bond between the heavy and light chains of the Fab fragment (i.e., C L -C H Targeted LNPs (conjugates) can be fabricated by selectively conjugating them onto the surface of precursor lipid nanoparticles (LNPs) via a disulfide bond.

[0194] The terms “precursor LNP” or “base LNP” refer to LNPs that have been functionalized with a reactive moiety (e.g., a thiol-reactive group or polyglycine) before reacting with the Fab fragment. As disclosed herein, the process for conjugating the target-directed moiety is performed on the C of the Fab fragment. L Domain and C HThis involves reducing the innate interchain disulfide bond between one domain and the other, and reacting the reduced Fab fragment with a thiol-reactive group (e.g., maleimide or DBM group) covalently bonded to the surface of the precursor LNP, thereby forming a conjugate. Alternatively, the reduced Fab fragment can be reacted with a lipid chemically modified (functionalized) with a thiol-reactive group (e.g., maleimide or DBM group). The resulting lipid can then be inserted into the existing LNP to produce a conjugate. As described herein, despite the removal of the innate interchain disulfide bond linking the heavy and light chains of the Fab fragment, the resulting conjugate can effectively target specific cell types depending on the properties of the Fab target-directing moiety. For example, specific Fab fragments for targeting immune cells or hematopoietic stem cells (HSCs) are disclosed herein. A schematic diagram of an LNP site-specifically conjugated to a Fab fragment is shown in Figure 1.

[0195] In one embodiment, the Fab fragment used for conjugation may be used by a recombinant method. In a particular embodiment, the recombinantly generated Fab fragment is designed so as not to contain a hinge region at the C-terminus. Thus, the recombinantly generated Fab fragment is C L -C H It contains only one disulfide bond between 1 and the domain. As shown herein, then C L -C H 1 can be reduced, and the resulting free thiol group can be used as an anchor to conjugate the Fab fragment onto the surface of the LNP.

[0196] In some embodiments, the Fab fragment is of the IgG class, IgM class, or IgA class. In some embodiments, the Fab fragment is of the IgG class and has an IgGl, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fab fragment is a native protein. In some embodiments, the Fab fragment is an engineered protein.

[0197] In one aspect, the present disclosure provides a method for producing a targeted LNP, the method being (i) Contacting a composition containing a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain, as well as a constant light chain domain (C L ) and constant heavy chain domain 1 (C H 1) contains interchain disulfide bonds that connect the two components, thereby allowing the reducing agent to reduce the interchain disulfide bonds of the Fab fragment, generating two free cysteine ​​residues, by contact. (ii) Contacting the product of step (i) with a precursor LNP containing multiple thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP.

[0198] In some aspects, this disclosure is, (i) Contacting a composition containing a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain, as well as a constant light chain domain (C L ) and constant heavy chain domain 1 (C H 1) contains interchain disulfide bonds that connect the two components, thereby allowing the reducing agent to reduce the interchain disulfide bonds of the Fab fragment, generating two free cysteine ​​residues, by contact. (ii) Contacting the product of step (i) with a precursor LNP containing multiple thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP, provides a conjugate produced by a method comprising these steps.

[0199] In some embodiments, a thiol-reactive group (e.g., maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl) is chemically reacted with a lipid molecule to produce a modified lipid, and the thiol-reactive group can be covalently bonded to the lipid if it can react with at least one free cysteine ​​residue (either from the heavy or light chain of the Fab fragment) of the reduced Fab fragment. The reaction between the thiol-reactive group and at least one free cysteine ​​residue can be completed before or after the formation of the LNP with the modified lipid. For example, as shown in Figure 2A, various components (e.g., lipids) including the LNP and therapeutic payload can be mixed with a lipid molecule containing one or more lipids containing a thiol-reactive group, thus generating an LNP containing multiple thiol-reactive groups (each thiol-reactive group is schematically shown as a “functional group” in Figure 2A). The thiol-reactive group can then be reacted with at least one free cysteine ​​residue of the Fab fragment, thus generating a conjugate. Alternatively, lipids modified with thiol-reactive groups can be directly reacted with at least one free cysteine ​​residue of the Fab fragment. As shown in Figure 2B, the resulting modified lipid, bound to the Fab fragment, can then be inserted into a pre-formed LNP that has not yet been surface-modified. This procedure allows the reaction to take place on individual lipid molecules rather than on the surface of the LNP.

[0200] The interchain disulfide bonds of the Fab fragment can be reduced using any suitable reducing agent. Examples of reducing agents, but not limited to, include 2-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol (DTE), and tris(carboxyethyl)phosphine (TCEP), as well as combinations thereof. In some embodiments, the reducing agent is a mild reducing agent. Examples of mild reducing agents include, for example, DTT, TCEP, and DTE. In some embodiments, the reducing agent is TCEP. Any suitable reaction conditions can be used for the reduction of the interchain disulfide bonds in step (i). In some embodiments, the reduction reaction can occur in water, aqueous buffer, or cell culture medium. In some embodiments, the reduction reaction is carried out at physiological pH (e.g., about 7.4). In some embodiments, the reduction reaction is carried out at physiological temperature (e.g., about 37°C). In some embodiments, the reduction reaction takes place at 0°C to 40°C, for example, 10°C to 35°C, 15°C to 30°C, 20°C to 30°C, or 20°C to 25°C. In some embodiments, the reduction reaction takes place at ambient temperature (e.g., about 23°C to about 25°C). In some embodiments, the reduction reaction takes place at about 0°C to about 4°C.

[0201] In some embodiments, excess reducing agent is removed after step (i) before conjugation to the precursor LNP. In some embodiments, excess reducing agent is not removed after step (i) before conjugation to the precursor LNP.

[0202] In some embodiments of the method or process, the thiol-reactive group on the LNP (or the lipid subsequently inserted into the LNP) may include, but are not limited to, any preferred reactive group, including maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl.

[0203] In some embodiments, the thiol-reactive group is maleimide. In some embodiments, maleimide reacts with one of two free cysteine ​​residues of the Fab fragment (either on the heavy or light chain) to form a thiosuccinimide moiety. In some embodiments, maleimide reacts with a free cysteine ​​residue on the heavy chain of the Fab fragment. In some embodiments, maleimide reacts with a free cysteine ​​residue on the light chain of the Fab fragment. In some embodiments, each of the two maleimide groups reacts with the Fab fragment, with one maleimide reacting with a free cysteine ​​residue on the light chain and the other maleimide reacting with a free cysteine ​​residue on the heavy chain.

[0204] Any preferred conditions can be used for the reaction between the thiol-reactive group and at least one of the two free cysteine ​​residues of the Fab fragment in step (ii). In some embodiments, the reaction may occur in water, aqueous buffer, or cell culture medium. In some embodiments, the reaction may be carried out at physiological pH (e.g., about 7.4). In some embodiments, the reaction may be carried out at physiological temperature (e.g., about 37°C). In some embodiments, the reduction reaction may be carried out at 0°C to 40°C, e.g., 10°C to 35°C, 15°C to 30°C, 20°C to 30°C, or 20°C to 25°C. In some embodiments, the reaction may be carried out at ambient temperature (e.g., about 23°C to about 25°C). In some embodiments, the reaction may be carried out at about 0°C to about 4°C.

[0205] Figure 3 shows a schematic diagram illustrating the reduction of interchain disulfide bonds in the Fab fragment. In some embodiments, the Fab fragment shown in Figure 3 can be recombinantly produced without a hinge region at the C-terminus. Therefore, in such embodiments, the Fab fragment is produced without a hinge region at the C-terminus. L -C HIt contains only a single interchain disulfide bond located at the interface. As shown in Figure 3, the interchain disulfide bond is located between the heavy and light chains of the Fab fragment. The reduction reaction breaks the covalent bond forming the disulfide bond, thereby generating two free cysteine ​​residues that can react with the thiol-reactive group in subsequent steps.

[0206] Figure 4 shows an exemplary schematic diagram of the conjugate formation described herein. In Figure 4, a Fab fragment containing an interchain disulfide bond between the heavy and light chains is brought into contact with a reducing agent, thereby reducing the interchain disulfide to produce two free cysteine ​​residues (step (i)). In step (ii), the reduced Fab fragment is reacted with an LNP containing multiple thiol-reactive groups (e.g., maleimide or DBM) conjugated on the surface of the LNP, thereby the thiol-reactive groups react with the free cysteine ​​residues of the reduced Fab fragment. The Fab fragment is site-specifically conjugated to the surface of the LNP via binding by at least one of the free cysteine ​​residues of the Fab fragment.

[0207] After the reduced Fab fragment is reacted with the thiol-reactive groups of the LNP, the heavy chain, light chain, or both of the heavy and light chains of the Fab fragment are conjugated to the surface of the LNP. Various orientations are shown in Figure 5. In some embodiments, the conjugates formed by the methods disclosed herein include at least one, at least two, or all three orientations shown in Figure 5. The concentration of the thiol-reactive group (e.g., maleimide) is likely to determine which orientation is dominant. In some embodiments, increasing the number of thiol-reactive groups on the LNP increases the number of Fab fragments conjugated to two thiol-reactive groups. In some embodiments, decreasing the number of thiol-reactive groups on the LNP decreases the number of Fab fragments conjugated to two thiol-reactive groups. Regardless of orientation, the heavy and light chains remain intact on the surface of the LNP and thus form a functional Fab fragment that can engage with receptors on targeted cells.

[0208] In some embodiments, the thiol-reactive group is maleimide, as shown in Figure 5. In some embodiments, the maleimide reacts with one of two free cysteine ​​residues of the antibody or its antigen-binding fragment. In some embodiments, the maleimide reacts with one of two free cysteine ​​residues of the Fab fragment to form a thiosuccinimide moiety. In some embodiments, the maleimide reacts with a free cysteine ​​residue on the heavy chain of the Fab fragment. In some embodiments, the maleimide reacts with a free cysteine ​​residue on the light chain of the Fab fragment. In some embodiments, each of the two maleimide groups on the LNP reacts with the Fab fragment, with one maleimide reacting with a free cysteine ​​residue on the light chain and the other maleimide reacting with a free cysteine ​​residue on the heavy chain.

[0209] In some embodiments, the thiol-reactive group is 2,3-dibromomaleimide (DBM), as shown in Figure 6. After reduction of the disulfide bond, the reduced Fab fragment is added to the DBM covalently bonded to the lipid (represented by the dashed line in Figure 6). As shown above, the lipid may be part of the LNP or may be subsequently inserted into the LNP after reaction with the Fab fragment. Both free cysteine ​​residues replace the two bromine groups of DBM, thus producing dithiomalemide. Dithiolmamemide can be converted to the corresponding maleamic acid via hydrolysis. DBM reacts with the free cysteine ​​residues on the heavy chain and light chain of the Fab fragment to form crosslinks between the cysteine ​​residues. Thus, the heavy and light chains of the Fab fragment are effectively crosslinked together after reaction with DBM.

[0210] In all embodiments discussed above, the thiol-reactive group can be introduced into any of the lipids containing the LNP. In some embodiments, the conjugate can contain one or more PEGylated lipid molecules. In some embodiments, the thiol-reactive group is covalently bonded to at least one of the PEGylated lipid molecules, thus forming a lipid-PEG. x- A thiol-reactive group structure is formed, where x is 2 to 120 ethylene glycol units. In such embodiments, at least one free cysteine ​​residue of the Fab fragment reacts with a thiol-reactive group bound to one or more pegylated lipids containing LNPs. In some embodiments, the LNPs comprise about 0.05 mol% to about 2 mol% of the pegylated lipids bound to the thiol-reactive group. In some embodiments, the PEG spacer between the lipid and the thiol-reactive group comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10 to 120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipids bound to the thiol-reactive group is about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bound to the thiol-reactive group is approximately 1,000 (i.e., PEG1000) to approximately 3,000 (i.e., PEG5300). In some embodiments, the thiol-reactive group is bound to at least one of the non-pegylated phospholipids containing LNPs. In some embodiments, the thiol-reactive group is bound to at least one of the ionizable lipids containing LNPs. In some embodiments, the thiol-reactive group is bound to at least one of the sterol molecules containing LNPs. In some embodiments, the lipid portion of the pegylated lipid bound to the thiol-reactive group is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid. Figure 8 shows some examples of pegylated lipids bound to a thiol-reactive group, in this case the maleimide portion.

[0211] In some embodiments, the thiol-reactive group is bound to at least one of the non-pegylated phospholipids (helper lipids) containing the LNP. Figure 9 shows some examples of non-pegylated lipids bound to the maleimide moiety.

[0212] In some embodiments, the thiol-reactive group is bound to at least one of the ionizable lipids containing the LNP. Figure 10 shows some examples of ionizable lipids bound to the maleimide moiety.

[0213] In some embodiments, the thiol-reactive group is bound to at least one of the sterol molecules containing the LNP. Figure 11 shows some examples of sterols bound to the maleimide moiety.

[0214] The methods of this disclosure provide a stable conjugate exhibiting excellent ability to transduce specifically targeted cells. In some embodiments, the disclosure provides a conjugate comprising an LNP and a Fab fragment, wherein the LNP is covalently bonded to either or both a first cysteine ​​residue in the constant region of the heavy chain of the Fab fragment and a second cysteine ​​residue in the constant region of the light chain of the Fab fragment. In some embodiments, the Fab fragment does not contain a disulfide bond linking the constant region of the heavy chain of the Fab fragment to the constant region of the light chain of the Fab fragment. In some embodiments, both the constant region of the heavy chain constant region of the heavy chain and the constant region of the light chain of the Fab fragment are covalently bonded to the LNP. In some embodiments, only the constant region of the heavy chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the light chain remains associated with the covalently bonded heavy chain on the surface of the LNP. In some embodiments, only the constant region of the light chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the heavy chain remains associated with the covalently bonded light chain on the surface of the LNP. In some of the embodiments described above, the Fab fragment is linked to the LNP via a thiosuccinimide moiety. In other embodiments of the embodiments described above, the Fab fragment is linked to the LNP via a dithiomalemide moiety (see Figure 6). In yet another embodiment of the embodiments described above, the Fab fragment is linked to the LNP via a maleamic acid moiety (see Figure 6).

[0215] In some embodiments, the Fab fragment conjugated to the LNP is an IgG1 Fab fragment. In some such embodiments, the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG1 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 233 (Kabat numbering) of the heavy chain of the IgG1 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 233 (Kabat numbering) of the heavy chain of the IgG1 Fab fragment and the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG1 Fab fragment are covalently bonded to the LNP.

[0216] In some embodiments, the Fab fragment conjugated to the LNP is an IgG2 Fab fragment. In some such embodiments, the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment and the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment are covalently bonded to the LNP.

[0217] In some embodiments, the Fab fragment conjugated to the LNP is an IgG4 Fab fragment. In some such embodiments, the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine ​​at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment and the cysteine ​​at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment are covalently bonded to the LNP.

[0218] The processes described herein also enable the ability to conjugate two or more different Fab fragments onto the surface of an LNP. An embodiment demonstrating the conjugation of two different Fab fragments (which may bind to the same or different antigens) onto the surface of an LNP is shown in Figure 7. In Figure 7, two Fab fragments (i.e., a first Fab fragment ("Fab1") and a second Fab fragment ("Fab2")) are reduced (step (i)) and reacted with a precursor LNP containing a thiol-reactive group (e.g., maleimide or DBM) (step (ii)). Following the reaction in step (ii), both Fab1 and Fab2 are conjugated onto the surface of the LNP. Despite the reduction of disulfide bonds in Fab1 and Fab2, the heavy and light chains in Fab1, and in Fab2, remain together on the surface of the LNP. In other words, the heavy or light chain of Fab1 does not associate with the heavy or light chain of Fab2 on the surface of the LNP.

[0219] In some embodiments, the first and second Fab fragments can be reduced in the same reaction (e.g., the first and second Fab fragments are mixed in a reaction vessel and brought into contact with the same reducing agent). In some embodiments, the first and second Fab fragments are reduced separately (e.g., the first and second Fab fragments are each brought into contact with a reducing agent in separate reaction vessels). In some embodiments, the first Fab fragment is brought into contact with the reducing agent before step (ii) (the reduced first Fab fragment is conjugated to the LNP surface). In some embodiments, the second Fab fragment is brought into contact with the reducing agent after step (ii) (the reduced second Fab fragment is conjugated to the LNP surface). In some embodiments, the reduced first and reduced second Fab fragments are brought into contact with the LNP simultaneously. In some embodiments, the reduced first Fab fragment and the reduced second Fab fragment are contacted with the LNP sequentially (in any order). It is conceivable that any number of Fab fragments can be implemented in the method or process (e.g., third, fourth, fifth, etc. Fab fragments). In some embodiments, a total of three different Fab fragments can be conjugated onto the surface of the LNP. In some embodiments, a total of four different Fab fragments can be conjugated onto the surface of the LNP.

[0220] In some embodiments, the reaction between at least one of the two free cysteine ​​residues of the Fab fragment and the thiol-reactive group of the LNP forms at least one covalent bond. In some embodiments, the formation of at least one covalent bond between at least one of the two free cysteine ​​residues of the Fab fragment and the thiol-reactive group of the LNP is reversible. In some embodiments, the formation of at least one covalent bond between at least one of the two free cysteine ​​residues of the Fab fragment and the thiol-reactive group of the LNP is irreversible. In some embodiments, the reaction efficiency between at least one of the two free cysteine ​​residues of the Fab fragment and the thiol-reactive group of the LNP is greater than 5%, greater than 10%, greater than 25%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the reaction efficiency between at least one of the two free cysteine ​​residues of the Fab fragment and the thiol-reactive group of the LNP is about 5% to about 30%, about 10% to about 20%, about 25% to about 50%, about 30% to about 40%, about 50% to about 80%, about 60% to about 70%, about 70% to about 95%, or about 80% to about 90%. In some embodiments, the conjugate product of the method of the present disclosure can be purified from the remaining intermediate product using any preferred technique such as ultrafiltration and diafiltration, but is not limited to these.

[0221] In some embodiments, the conjugate prepared by the methods or processes disclosed herein has a high density of Fab fragments on the surface of the LNP. For example, the conjugate may contain a plurality of Fab fragments conjugated on the surface of the LNP. In some embodiments, the conjugate may contain more than 10 Fab fragments per LNP. In some embodiments, the conjugate may contain more than 20 Fab fragments per LNP. In some embodiments, the conjugate may contain more than 30 Fab fragments. In some embodiments, the conjugate may contain more than 50 Fab fragments per LNP. In some embodiments, the conjugate may contain more than 75 Fab fragments per LNP. In some embodiments, the conjugate may contain more than 100 Fab fragments. In some embodiments, the conjugate may contain about 50 to about 200 Fab fragments per LNP. In some embodiments, the conjugate may contain about 100 to about 230 Fab fragments per LNP. In some embodiments, the conjugate may contain approximately 10 to 150 Fab fragments per LNP. In some embodiments, the conjugate may contain approximately 10 to 30 Fab fragments per LNP.

[0222] Conjugation via saltase-mediated enzyme ligation process In another embodiment, the disclosure provides a targeted LNP prepared by a process comprising contacting a precursor LNP containing multiple polyglycine molecules covalently bonded to the surface of the LNP with a Fab fragment functionalized with a saltase tag in the presence of saltase, thereby conjugating the Fab fragment to the surface of the precursor LNP via saltase-mediated ligation of the saltase tag and one or more polyglycine molecules, thereby forming the targeted LNP. A schematic diagram of the process is shown in Figure 12.

[0223] In some embodiments, the saltase tag of the Fab fragment is located at the C-terminus of the heavy chain of the Fab fragment. In some embodiments, the saltase tag comprises the amino acid sequence LPXTG (SEQ ID NO: 4), where X is any amino acid. In some embodiments, the saltase tag is LPETG (SEQ ID NO: 5). In some embodiments, the saltase tag is linked to the Fab fragment via an amino acid linker.

[0224] In some embodiments, the polyglycine molecule of multiple precursor LNPs contains 3 to 10 glycine residues. In some embodiments, the polyglycine molecule contains 3, 4, 5, or 6 glycine residues. In some embodiments, the polyglycine molecule is linked to the Fab fragment via an amino acid linker.

[0225] The ligation reaction can be catalyzed using any suitable saltase enzyme. In some embodiments, the saltase is saltase A5. In some embodiments, the saltase is Streptococcus pyogenes saltase A (SpSrtA WT). Any suitable conditions can be used for saltase-mediated ligation. In some embodiments, ligation can occur in water, aqueous buffer, or cell culture medium. In some embodiments, ligation is carried out at physiological pH (e.g., about 7.4). In some embodiments, ligation is carried out at physiological temperature (e.g., about 37°C). In some embodiments, ligation is carried out at ambient temperature (e.g., about 23°C to about 25°C). In some embodiments, ligation is carried out at about 0°C to about 4°C.

[0226] In some embodiments, saltase-mediated ligation forms a covalent bond. In some embodiments, saltase-mediated ligation is reversible (e.g., the Fab fragment can be released from the LNP surface). In some embodiments, saltase-mediated ligation is irreversible (e.g., saltase-mediated ligation is carried out as described in Liu et al., The Journal of Organic Chemistry 2014 79 (2), 487-492). In some embodiments, the ligation efficiency between the saltase tag of the Fab fragment and the polyglycine molecule of the LNP is greater than 5%, greater than 10%, greater than 25%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the ligation efficiency between the saltase tag of the Fab fragment and the polyglycine molecule of the LNP is about 5% to about 30%, about 10% to about 20%, about 25% to about 50%, about 30% to about 40%, about 50% to about 80%, about 60% to about 70%, about 70% to about 95%, or about 80% to about 90%. In some embodiments, the conjugate product of the method of the present disclosure can be purified from the remaining intermediate product (e.g., an antibody or its functional fragment) using any preferred technique such as ultrafiltration and diafiltration, but are not limited to these.

[0227] In some embodiments, the conjugate prepared by the methods or processes disclosed herein has a high density of Fab fragments on the surface of the LNP. For example, the conjugate may contain a plurality of Fab fragments conjugated on the surface of the LNP. In some embodiments, the conjugate may contain more than 10 Fab fragments per LNP. In some embodiments, the conjugate may contain more than 20 Fab fragments per LNP. In some embodiments, the conjugate may contain more than 30 Fab fragments. In some embodiments, the conjugate may contain more than 50 Fab fragments per LNP. In some embodiments, the conjugate may contain more than 75 Fab fragments per LNP. In some embodiments, the conjugate may contain more than 100 Fab fragments. In some embodiments, the conjugate may contain about 50 to about 200 Fab fragments per LNP. In some embodiments, the conjugate may contain about 100 to about 230 Fab fragments per LNP. In some embodiments, the conjugate may contain approximately 10 to 150 Fab fragments per LNP. In some embodiments, the conjugate may contain approximately 10 to 30 Fab fragments per LNP.

[0228] In some embodiments, the conjugate prepared by the methods or processes disclosed herein comprises two or more Fab fragments (e.g., second, third, fourth, fifth, etc.) that bind to different antigens.

[0229] In some embodiments, polyglycine molecules conjugated on the surface of an LNP can be chemically reacted with lipid molecules to obtain modified lipids that can react with the saltase tag of a Fab fragment via saltase-mediated ligation. The reaction between the polyglycine molecules and the saltase tag of the Fab fragment can be completed before or after the formation of the LNP. For example, as shown in Figure 2, various components (e.g., lipids) including an LNP can be mixed with lipid molecules, each containing a polyglycine molecule, to thus generate an LNP containing multiple polyglycine molecules. These multiple polyglycine molecules can then be reacted with the saltase tag of an antibody or its antigen-binding fragment to thus generate a conjugate. Alternatively, lipids modified with polyglycine molecules can be reacted directly with the saltase tag of an antibody or its antigen-binding fragment. The resulting modified lipids can then be inserted into a pre-formed LNP that has not yet been surface-modified. This procedure allows the reaction to take place on individual lipid molecules rather than on the surface of the LNP.

[0230] Polyglycine molecules can be introduced into any of the lipids containing LNPs. In some embodiments, the conjugate can contain one or more PEGylated lipid molecules. In some embodiments, the polyglycine molecule is covalently bonded to at least one of the PEGylated lipid molecules, thus forming a lipid-PEG. x- A polyglycine molecule structure is generated, where x is 2 to 120 ethylene glycol units. In such embodiments, the saltase tag of the Fab fragment reacts with the polyglycine molecule bound to one or more pegylated lipids containing LNPs. In some embodiments, the LNPs comprise about 0.05 mol% to about 2 mol% of the pegylated lipids bound to the polyglycine molecule. In some embodiments, the PEG spacer between the lipid and the polyglycine molecule comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10 to 120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipids bound to the polyglycine molecule is about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bound to the polyglycine molecule is approximately 1,000 (i.e., PEG1000) to approximately 3,000 (i.e., PEG5300). In some embodiments, the polyglycine molecule is bound to at least one of non-pegylated phospholipids containing LNPs. In some embodiments, the polyglycine molecule is bound to at least one of ionizable lipids containing LNPs. In some embodiments, the polyglycine molecule is bound to at least one of sterol molecules containing LNPs. In some embodiments, the lipid portion of the pegylated lipid bound to the polyglycine molecule is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid.

[0231] In some embodiments, the LNP includes a saltase tag conjugated to the surface of the LNP. In such embodiments, the Fab fragment includes a polyglycine molecule, and the Fab fragment is conjugated to the surface of the LNP via saltase-mediated ligation between the saltase tag of the LNP and the polyglycine molecule of the antibody or its antigen-binding fragment. In some embodiments, the saltase tag of the LNP includes the amino acid sequence LPXTG (SEQ ID NO: 4), where X is any amino acid. In some embodiments, the saltase tag is LPETG (SEQ ID NO: 5).

[0232] In some embodiments, a saltase tag conjugated on the surface of an LNP can be chemically reacted with a lipid molecule to obtain a modified lipid that can react with the polyglycine molecule of a Fab fragment via saltase-mediated ligation. The reaction between the saltase tag and the polyglycine molecule of the Fab fragment can be completed before or after the formation of the LNP. For example, as shown in Figure 2, various components (e.g., lipids) containing the LNP can be mixed with lipid molecules, each containing a saltase tag, thus generating an LNP containing multiple saltase tags. The multiple saltase tags can then be reacted with the polyglycine molecule of an antibody or its antigen-binding fragment, thus generating a conjugate. Alternatively, the saltase-modified lipid can be reacted directly with the polyglycine molecule of an antibody or its antigen-binding fragment. The resulting modified lipid can then be inserted into a pre-formed LNP that has not yet been surface-modified. This procedure allows the reaction to take place on individual lipid molecules rather than on the surface of the LNP.

[0233] The saltase tag can be introduced into any of the lipids containing LNPs. In some embodiments, the conjugate can contain one or more pegylated lipid molecules. In some embodiments, the saltase tag is covalently bonded to at least one of the pegylated lipid molecules, thus forming lipid-PEG. x- A structure of a saltase tag is generated, where x is 2 to 120 ethylene glycol units. In such embodiments, a polyglycine molecule of the Fab fragment reacts with a saltase tag bound to one or more pegylated lipids containing LNPs. In some embodiments, the LNPs constitute about 0.05 mol% to about 2 mol% of the pegylated lipids bound to the saltase tag. In some embodiments, the PEG spacer between the lipid and the saltase tag contains at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer contains about 10 to 120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipids bound to the saltase tag is about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bound to the saltase tag is approximately 1,000 (i.e., PEG1000) to approximately 3,000 (i.e., PEG5300). In some embodiments, the saltase tag is bound to at least one of the non-pegylated phospholipids containing LNPs. In some embodiments, the saltase tag is bound to at least one of the ionizable lipids containing LNPs. In some embodiments, the saltase tag is bound to at least one of the sterol molecules containing LNPs. In some embodiments, the lipid portion of the pegylated lipid bound to the saltase tag is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid.

[0234] Conjugate via click chemistry In some embodiments, the target-directed moiety is conjugated to lipid nanoparticles via a linker, the linker comprising a click product formed from a click reaction between a first click handle of the target-directed moiety and a second click handle of the LNP. In some such embodiments, the target-directed moiety is an antibody or its antigen-binding fragment. In other such embodiments, the target-directed moiety is an scFv. In some embodiments, the target-directed moiety is a Fab fragment.

[0235] In one embodiment, the click product can be formed using a copper-catalyzed click reaction. One such copper-catalyzed click reaction is the hysgene 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne. In some embodiments, the first or second click handle includes a cyclic derivative of the alkynyl group. In some embodiments, the cyclic derivative of the alkynyl group is selected from dibenzocyclooctin, cyclooctin, and difluorinated cyclooctin. In some embodiments, the click chemistry involves strain-promoted cycloaddition of the azide. In some embodiments, the click chemistry is based on the reaction of a strained alkene.

[0236] In another embodiment, the click product can be formed using copper-free click chemistry. For example, the click product can be formed between an azide and dibenzocyclooctene (DBCO). Alternatively, the click product can be formed using a Staudinger reaction between an azide and a phosphine, thus producing an aza-ylide.

[0237] In some embodiments, the click product can be formed from an inverse electron-demanded Diels-Alder reaction between a trans-cyclooctene (TCO) moiety on a first or second click handle and a tetrazine ring on a first or second click handle. In some embodiments, the first click handle comprises a tetrazine (Tz) ring, and the second click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is unsubstituted. In some such embodiments, the tetrazine ring is methyltetrazine. In some embodiments, the tetrazine ring is 6-methylsubstituted tetrazine.

[0238] In another embodiment, the target-directing moiety (e.g., antibody, Fab fragment, or scFv) is first selectively modified with an enzyme recognition sequence. An enzyme recognizing the enzyme recognition sequence can site-specifically introduce a first click handle to the target-directing moiety via covalent bonding. The first click handle can then react with a second click handle on the LNP to generate a targeted LNP. Thus, in one embodiment, an antibody, Fab fragment, or single-stranded variable fragment (scFv) covalently bonded to a first click handle via a linker containing the enzyme recognition sequence is reacted with an LNP containing a second click handle, thereby forming a click reaction product that conjugates the antibody, Fab fragment, or scFv to the LNP. In some embodiments, the antibody, Fab fragment, or scFv is directly bound to the enzyme recognition sequence. In some embodiments, the target-directing moiety (e.g., antibody, Fab fragment, or scFv) is bound to the enzyme recognition sequence via one or more amino acid residues. Specific amino acid residues that can be covalently bonded to the C-terminus of the target-directing moiety (e.g., antibodies, Fab fragments, or scFv) are, but are not limited to, (GGGGS). v (Sequence ID 154), (G) v、 (EAAAK) v (Sequence ID 155), (PAPAP) v (Sequence ID 156), (AP) v , and A(EAAAK) u ALEA(EAAAK)v A (sequence number 157) is given, and in the sequence, u is 1 to 10 and v is 1 to 10.

[0239] In some embodiments, the enzyme recognition sequence is a saltase recognition motif or a LplA acceptor peptide. In some embodiments in which the LNP is conjugated to an antibody, one or more C-terminuses of the heavy or light chain of the antibody are covalently bound to the enzyme recognition sequence (e.g., a saltase recognition motif or a LplA acceptor peptide) directly or via a linker comprising one or more amino acid residues as shown herein. In some embodiments in which the LNP is conjugated to a Fab fragment, the C-terminuses of the heavy or light chain of the Fab fragment are covalently bound to the enzyme recognition sequence (e.g., a saltase recognition motif or a LplA acceptor peptide). In some embodiments in which the LNP is conjugated to an scFv, the C-terminus of the scFv is covalently bound to the enzyme recognition sequence (e.g., a saltase recognition motif or a LplA acceptor peptide). In some of the embodiments described above, the first click handle comprises a tetrazine ring or TCO moiety, and the second click handle comprises a tetrazine ring or TCO moiety. In some such embodiments, the first click handle comprises a tetrazine ring, and the second click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is methyltetrazine. In some embodiments, the conjugation efficiency achieved by the method of the present disclosure is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the conjugation efficiency achieved by the method of the present disclosure is about 60% to about 95%. In some embodiments, the conjugation efficiency achieved by the disclosed method is about 70% to about 85%.

[0240] In some embodiments, the linker further includes a spacer between the target-directing moiety and the click product. The spacer may include additional functional groups that indirectly link the target-directing moiety to the Tz group. The spacer may also include additional amino acid residues that indirectly link the target-directing moiety to the Tz group.

[0241] In some embodiments, the first or second click handle is a tetrazine derivative having one of the following structures: [ka] [ka] represents the linker or connection point of the conjugate to an LNP.

[0242] In some embodiments, the first or second click handle is a TCO derivative having one of the following structures. [ka]

[0243] In some embodiments, the spacer linking the target-directing moiety to the Tz ring is an enzyme recognition sequence. Accordingly, the Disclosure provides a method for conjugating an LNP to a target-directing moiety modified with an enzyme recognition sequence, the conjugation being achieved by a click reaction between the Tz ring covalently bonded to the target-directing moiety and the TCO moiety bonded to the LNP. For example, the Disclosure provides a method for conjugating an LNP to an antibody, Fab fragment, or single-stranded variable fragment (scFv), wherein the antibody, Fab fragment, or scFv is covalently bonded to a first click handle (Tz ring) via a linker containing an enzyme recognition sequence, and the LNP is covalently bonded to a second click handle (TCO moiety), the method comprising contacting the LNP with the antibody, Fab fragment, or scFv such that the first click handle reacts with the second click handle to form a click reaction product (dihydropyridazine) that conjugates the antibody, Fab fragment, or scFv to the LNP. In some embodiments, the antibody, Fab fragment, or scFv is directly bound to the enzyme recognition sequence. In some embodiments, the antibody, Fab fragment, or scFv is bound to the enzyme recognition sequence via one or more amino acid residues. Specific amino acid residues that can be covalently bound to the C-terminus of the antibody, Fab fragment, or scFv are, but are not limited to, (GGGGS). v (Sequence ID 154), (G) v , (EAAAK) v (Sequence ID 155) 、 (PAPAP) v (Sequence ID 156), (AP) v , and A(EAAAK) u ALEA(EAAAK) v A (sequence number 157) is given, and in the sequence, u is 1 to 10 and v is 1 to 10.

[0244] In some embodiments, the enzyme recognition sequence is a saltase recognition motif or a LplA acceptor peptide. In some embodiments in which the LNP is conjugated to an antibody, one or more C-terminuses of the heavy or light chain of the antibody are covalently bound to the enzyme recognition sequence (e.g., a saltase recognition motif or a LplA acceptor peptide) directly or via a linker comprising one or more amino acid residues as shown herein. In some embodiments in which the LNP is conjugated to a Fab fragment, the C-terminuses of the heavy or light chain of the Fab fragment are covalently bound to the enzyme recognition sequence (e.g., a saltase recognition motif or a LplA acceptor peptide). In some embodiments in which the LNP is conjugated to an scFv, the C-terminus of the scFv is covalently bound to the enzyme recognition sequence (e.g., a saltase recognition motif or a LplA acceptor peptide). In some embodiments, the conjugation efficiency achieved by the method of this disclosure is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the conjugation efficiency achieved by the method disclosed is approximately 60% to approximately 95%. In some embodiments, the conjugation efficiency achieved by the disclosed method is approximately 70% to approximately 85%.

[0245] In one embodiment, lipid nanoparticles conjugated to an antibody, Fab fragment, or scFv in a site-specific manner are as follows: (i) The step of covalently attaching a peptide having a saltase recognition site to one or more C-terminuses of an antibody or Fab fragment, or to the C-terminus of scFv, (ii) In the presence of a saltase enzyme, under conditions suitable for the saltase enzyme to ligate the antibody, Fab fragment, or scFv to a first click handle, the product of step (i) is brought into contact with a first click handle containing a Tz ring covalently bonded to three or more glycine residues, (iii) A step of contacting the product of step (ii) with lipid nanoparticles (LNPs), wherein one or more lipids comprising LNPs comprises a second click handle that can react with a first click handle, and the second click handle comprises a TCO moiety, thereby producing a click product.

[0246] In some embodiments, step (i) includes covalent bonding of a linker to one or more C-terminuses of an antibody or Fab fragment, or to the C-terminus of an scFv, of a peptide having a saltase recognition site. In some embodiments, the linker comprises one or more amino acid residues. Specific amino acid residues that can be covalently bonded to the C-terminus of an antibody, Fab fragment, or scFv are, but are not limited to, (GGGGS). v (Sequence ID 154), (G) v , (EAAAK) v (Sequence ID 155) 、 (PAPAP) v (Sequence ID 156), (AP) v , and A(EAAAK) u ALEA(EAAAK) v A (sequence number 157) is given, and in the sequence, u is 1 to 10 and v is 1 to 10.

[0247] In some embodiments, the saltase recognition motif attached to the C-terminus of the antibody, Fab fragment, or scFv is bound to one or more amino acid residues. For example, in some embodiments, the glycine (G) of the LPXTG (SEQ ID NO: 4) motif can be bound to one or more (e.g., 1 to 10) histidine (H) residues (see Figure 13). The glycine and histidine residues are removed during the transpeptidation reaction.

[0248] In some embodiments, step (ii) (i.e., the saltase-catalyzed reaction) is carried out in the presence of saltase A5. Saltase A5 is a modified variant of wild-type saltase from Staphylococcus aureus that is significantly more active than wild-type saltase. See U.S. Patent No. 9,267,227. In such embodiments, the saltase recognition site binding to the antibody, Fab fragment, or scFv in step (i) has the sequence LPXTG (SEQ ID NO: 4), where X is any amino acid residue. In step (ii), saltase A catalyzes the transpeptidation reaction between the LPXTG (SEQ ID NO: 4) recognition motif and the glycine residue bound to the first click handle by cleaving the saltase recognition site between the threonine and glycine residues.

[0249] In other embodiments, step (ii) is carried out in the presence of the saltase enzyme Streptococcus pyogenes saltase A (SpSrtA WT). In such embodiments, the saltase recognition site binding to the antibody, Fab fragment, or scFv in step (i) has the sequence LPXTA (SEQ ID NO: 7), where X is any amino acid residue. In step (ii), SpSrtA catalyzes the transpeptidation reaction between the LPXTA (SEQ ID NO: 7) recognition motif and the glycine residue bound to the first click handle by cleaving the saltase recognition site between the threonine and alanine residues.

[0250] Figure 13 illustrates the formation of an LNP conjugated to the C-terminus of a Fab fragment via saltase-mediated ligation followed by a click reaction. In the schematic diagram, the C-terminus of the Fab fragment is first covalently modified with a saltase recognition motif covalently bonded to six histidine (H) residues. The saltase-modified Fab fragment is then reacted with a first click handle (click handle 1) covalently bonded to a glycine (G) residue. The resulting Fab fragment modified with the first click handle is then reacted with an LNP containing at least one lipid covalently bonded to a second click handle (click handle 2), thereby obtaining an LNP site-specifically conjugated to the Fab fragment.

[0251] In one embodiment, the conjugate comprises the aforementioned protein targeting moiety (e.g., antibody, Fab fragment, or scFv) and lipid nanoparticles (LNP), where the C-terminus of the targeting moiety is conjugated to the lipid nanoparticles via a linker, and the linker comprises a saltase recognition motif and a click product formed from a click reaction between a first click handle of the targeting moiety and a second click handle of the LNP. In some embodiments, one or more C-terminuses of the heavy or light chain of an antibody, the C-terminus of the heavy or light chain of a Fab fragment, or the C-terminus of an scFv are conjugated to the lipid nanoparticles via a linker, and the linker comprises a saltase recognition motif and a click product formed from a click reaction between a first click handle of the antibody, Fab fragment, or scFv and a second click handle of the LNP. In certain embodiments, the saltase recognition motif comprises an LPXT motif, where X is any amino acid residue. In some such embodiments, the saltase recognition motif comprises an LPET (SEQ ID NO: 79) motif. In some embodiments, the linker contains three or more glycine residues between the saltase recognition motif and the click product. In some embodiments, the linker contains 3 to 50 glycine residues. In some embodiments, the linker contains 3 to 10 glycine residues. In some such embodiments, the linker contains 3, 4, 5, or 6 glycine residues.

[0252] In some embodiments, the conjugate is antibody-LPXT(G) n - Click product - LNP, Fab fragment - LPXT(G) n - Click product - LNP, or scFv-LPXT(G) n The click product has the structure of an LNP, where X is any amino acid residue and n is between 1 and 20 (e.g., 3 to 10 or 3 to 6). In these embodiments, the leucine (L) residue of the saltase recognition motif is bound to at least one C-terminus of the antibody or Fab fragment, or to the C-terminus of the scFv. In some such embodiments, X is E. In some embodiments, the linker between the antibody (or Fab fragment or scFv) and the click includes the following amino acid residues: LPETGGG (SEQ ID NO: 106), LPETGGGG (SEQ ID NO: 107), LPETGGGGG (SEQ ID NO: 108), LPETAGGG (SEQ ID NO: 158), or LPETGGGGGG (SEQ ID NO: 159).

[0253] In some embodiments, the linker includes additional amino acid residues between the antibody, Fab fragment, or scFv and the saltase recognition motif. In such embodiments, the C-terminus of the antibody, Fab fragment, or scFv can be covalently modified with one or more amino acid residues before covalently bonding the saltase recognition motif. For example, in certain embodiments, the conjugate is antibody-Z-LPXT(G) n - Click product - LNP, Fab fragment - Z-LPXT(G) n - Click product - LNP, or scFv-Z-LPXT(G) n The structure of the click product-LNP is such that Z is a linker between the antibody (or Fab fragment or scFv) and the leucine of the saltase recognition motif. In some embodiments, Z comprises one or more amino acid residues. In some embodiments, Z is (GGGGS) v (Sequence ID 154), (G) v , (EAAAK) v (Sequence ID 155)、 (PAPAP) v (Sequence ID 156), (AP) v , and A(EAAAK) u ALEA(EAAAK) v A is sequence number 157, where u is between 1 and 10, and v is between 1 and 10. In some embodiments, Z is GG, GGG, GGGG (sequence number 160), GGGGG (sequence number 161), GGGGGG (sequence number 162), and GGGGGGG (sequence number 163) or GGGGGS (sequence number 164).

[0254] In some embodiments, the conjugate is antibody-LPXT(G) n -Dihydropyridazine-LNP, Fab fragment-LPXT(G) n -Dihydropyridazine-LNP, or scFv-LPXT(G) n -Dihydropyridazine-LNP, antibody-Z-LPXT(G) n -Dihydropyridazine-LNP, Fab fragment-Z-LPXT(G) n -Dihydropyridazine-LNP, or scFv-Z-LPXT(G) n The structure is -dihydropyridazine-LNP, where the variables n and Z are defined as above. In certain embodiments, the dihydropyridazine moiety is 1,4-dihydropyridazine.

[0255] In another embodiment, LNP lipids are (i) Covalently attaching the LplA acceptor peptide site to an antibody, Fab fragment, or scFv, (ii) Contacting the product of step (i) with a carboxylic acid compound containing a first click handle containing a Tz ring in the presence of lipoic acid ligase under conditions suitable for the lipoic acid ligase to ligate the antibody, Fab fragment, or scFv to the first click handle, (iii) contacting the product of step (ii) with a lipid nanoparticle (LNP), wherein the one or more lipids comprising the LNP comprise a second click handle capable of reacting with the first click handle, and the second click handle comprises a TCO moiety, thereby generating a click product, wherein the antibody, Fab fragment, or scFv is conjugated in a site-specific manner.

[0256] In some embodiments of step (i), the Lp1A acceptor peptide is covalently bonded to one or more C-termini of the antibody or Fab fragment. In other embodiments of step (i), the Lp1A acceptor peptide is covalently bonded to the C-terminus of the scFv.

[0257] In some embodiments, step (i) involves covalently bonding the Lp1A acceptor peptide to a linker covalently bonded to one or more C-termini of the antibody or Fab fragment, or to the C-terminus of the scFv. In some embodiments, the linker comprises one or more amino acid residues. Specific amino acid residues that can be covalently bonded to the C-terminus of an antibody, Fab fragment or scFv include, but are not limited to, (GGGGS) v (SEQ ID NO: 154), (G) v , (EAAAK) v (SEQ ID NO: 155) 、 (PAPAP) v (SEQ ID NO: 156), (AP) v , and A(EAAAK) u ALEA(EAAAK) v A(SEQ ID NO: 157), wherein in the sequences, u is 1 to 10, and v is 1 to 10.

[0258] In some embodiments, step (ii) (i.e., lipoic acid ligase-catalyzed reaction) is carried out in the presence of a mutated lipoic acid ligase, where tryptophan is mutated and located at the lipoic acid ligase site (W). Specific W37 variants are described in Cohen et al., 2012, ChemBioChem, 13, 888-894. In some embodiments, the mutant lipoic acid ligase is selected from W37V, W37I, W37T, W37L, W37C, W37A, and W37G.

[0259] In some embodiments, the carboxylic acid in step (ii) (i.e., lipoic acid ligase-catalyzed reaction) is C3-C 20 It is a carboxylic acid. In some embodiments, the carboxylic acid in step (ii) is a fatty acid, for example, a C7-C 19 These are fatty acids. In some such embodiments, the fatty acids are selected from decanoic acid, palmitic acid, lauric acid, or octanoic acid.

[0260] In other embodiments, the carboxylic acid of step (ii) has the formula COOH-R-click handle, where R comprises two lipid tails. In some such embodiments, the carboxylic acids shown below are n=1 to 120. [ka] [ka] [ka]

[0261] Figure 14 illustrates the formation of an LNP conjugated to the C-terminus of a Fab fragment via LplA ligation followed by a click reaction. In the schematic diagram, the C-terminus of the Fab fragment is first covalently modified with an LplA acceptor peptide. Specifically, a first click handle (click handle 1) containing a Tz ring is introduced by first chemically reacting a carboxylic acid with a lysine residue of the LplA acceptor peptide. The resulting Fab fragment modified with the first click handle is then reacted with an LNP containing at least one lipid covalently attached to a second click handle (click handle 2) containing a TCO moiety, thus obtaining an LNP site-specifically conjugated to the Fab fragment.

[0262] In some embodiments, the conjugate produced by the method disclosed herein comprises the above-mentioned protein targeting moiety (e.g., antibody, Fab fragment, or scFv) conjugated to a lipid nanoparticle via a linker, the linker comprising a lipoic acid ligase (LplA) acceptor peptide and a click product formed from a click reaction between a first click handle of the targeting moiety and a second click handle of the LNP. In some embodiments, the linker further comprises one or more additional amino acid residues between the protein targeting moiety (e.g., antibody, Fab fragment, or scFv) and the LplA acceptor peptide. In some embodiments, the LplA acceptor peptide has the sequence GFEDKVWYDLDA (SEQ ID NO: 165). In some embodiments, the conjugate comprises an antibody, and one or more C-terminuses of the heavy or light chain of the antibody are bound to the linker. In some embodiments, the conjugate comprises a Fab fragment, and the C-terminuses of the heavy or light chain of the Fab fragment are bound to the linker. In some embodiments, the conjugate includes an scFv, the C-terminus of which is coupled to a linker.

[0263] In some embodiments, the linker comprises an additional amino acid residue between the targeting moiety (e.g., an antibody, Fab fragment or scFv) and the LplA acceptor peptide. In such embodiments, the C-terminus of the targeting moiety (e.g., an antibody, Fab fragment, or scFv) can be covalently modified with one or more amino acid residues prior to covalently attaching the LplA acceptor peptide. For example, in certain embodiments, the conjugate has the structure of targeting moiety-Z-LplA acceptor peptide-click product-LNP (e.g., antibody-Z-LplA acceptor peptide-click product-LNP, Fab fragment-Z-LplA acceptor peptide-click product-LNP, or scFv-Z-LplA acceptor peptide-click product-LNP), wherein Z is a linker between the antibody (or Fab fragment or scFv) and the glycine residue of the LplA acceptor peptide. In some embodiments, Z comprises one or more amino acid residues. In some embodiments, Z is (GGGGS) v (SEQ ID NO: 154), (G) v , (EAAAK) v (SEQ ID NO: 155) 、 (PAPAP) v (SEQ ID NO: 156), (AP) v , and A(EAAAK) u ALEA(EAAAK) v A (SEQ ID NO: 157), wherein in the sequences, u is 1 to 10, and v is 1 to 10. In some embodiments, Z is GG, GGG, GGGG (SEQ ID NO: 160), GGGGG (SEQ ID NO: 161), GGGGGG (SEQ ID NO: 162), and GGGGGGG (SEQ ID NO: 163) or GGGGG S (SEQ ID NO: 164).

[0264] In the embodiments described above, it will be understood that the lysine (K) residue of the LplA acceptor peptide is covalently bonded to the click product which is covalently bonded to the LNP. Specifically, to generate a conjugate, the side-chain lysine group reacts with a carboxylic acid compound containing a first click handle. The resulting modified target-directed moiety (antibody, Fab fragment, or scFv) is reacted with the LNP modified with a second click handle, as disclosed herein, thereby generating a click product. The surface of the LNP modified with a Fab fragment is shown below, where R is a lipid group (e.g., C 2- C 30 It is an alkyl group. (GFEIDKVWYDLDA-SEQ ID NO: 414) [ka]

[0265] In other embodiments, the enzyme recognition sequence is a transglutaminase enzyme recognition sequence (LLQG, SEQ ID NO: 166). The transglutaminase enzyme recognition sequence (LLQG, SEQ ID NO: 166) is also referred to as the Q tag. The Q tag may be located at one or more positions of the target-directing moiety (e.g., antibody, Fab fragment, or scFv), for example, at the C-terminus, or can be inserted. The transglutaminase enzyme catalyzes the reaction between the side-chain amide group on the Q tag and the alkyl-primary amine on the component of the LNP (e.g., lipid), thereby linking the antibody to the LNP via an amide bond.

[0266] In other embodiments, the enzyme recognition sequence is a sequence recognized by formylglycinase, specifically CXPXR, where each X is any amino acid. In such embodiments, the CXPXR sequence can be inserted into one or more positions of a target-directing moiety (e.g., an antibody, Fab fragment, or scFv), for example, at the C-terminus. The formylglycinase can convert the cysteinethiol of CXPXR to an aldehyde group, which can then react with an aminooxy or hydrazine group covalently bonded to a component of the LNP.

[0267] In another embodiment, lipid nanoparticles are conjugated to a target-directed moiety, such as an antibody, Fab fragment, or scFv, in a site-specific manner via a sugar moiety on a glycosylated antibody. Figure 15 shows one specific embodiment of introducing a Tz click handle via a sugar moiety on an antibody. In step 1, an azide moiety is site-directly introduced to a sugar moiety of a target antibody catalyzed by enzymes, galactosidase and transferase. The azide functional group then reacts with dibenzocyclooctene (DBCO)-MeTz via a click reaction. Using the click reaction, the Tz ring, in this case MeTz, can be introduced into the antibody and then reacted with the TCO moiety on an LNP (not shown) in a second click reaction, thus obtaining a conjugate.

[0268] In another embodiment, the LNP is conjugated to a site-specific target moiety (e.g., antibody, Fab fragment, or scFv) via a photo-induced crosslinking reaction. Figure 16 shows one particular embodiment using oYo-linked Tz. In Figure 16, a Tz ring (e.g., MeTz) is covalently bound to a site-specific antibody label (oYo linkage) that specifically reacts with a residue on the heavy chain of the antibody. The oYo-linked tetrazine covalently bound to the antibody reacts with TCO (not shown) bound to the LNP, thus generating a surface-modified LNP.

[0269] In another embodiment, lipid nanoparticles are conjugated to a target-directed moiety (e.g., an antibody, Fab fragment, or scFv) in a site-specific manner by mutating amino acid residues on the target-directed moiety and then reacting the mutated target-directed moiety with a compound containing a Tz ring. Figure 17 shows a specific embodiment in which an azide functional group is introduced during the production of an antibody or Fab fragment. The azide group then undergoes a click reaction with a DBCO group linked to a tetrazine ring. The tetrazine ring bound to the antibody reacts with TCO (not shown) bound to the LNP, thus producing a surface-modified LNP.

[0270] In some embodiments, the click product can be formed using any suitable photo-induced click reaction. In some embodiments, the click product can be formed using a photo-induced 1,3-dipolar ring addition reaction between a tetrazole and an alkene (see, for example, Song et al., Angew. Chem., Int. Ed. 2008, 47(15), 2832-2835).

[0271] In some embodiments, the click product can be formed using oxime and hydrazone ligation. In some embodiments, the ketone or aldehyde can react with α-effect amines such as hydroxylamines, hydrazines, and hydrazides (see, for example, Agten et al., ChemBioChem 2013, 14(18), 2431-2434 and Dirksen et al., J.Am.Chem.Soc. 2006, 128(49), 15602-15603).

[0272] The following are exemplary lipid and cholesterol molecules that can be attached to click handles and used to produce targeted LNPs. [ka] [ka] [ka]

[0273] III. Lipid Nanoparticles In some embodiments, the lipid nanoparticles include one or more ionic lipids, such as noncationic lipids (e.g., neutral, anionic, or amphoteric lipids), also referred to herein as helper lipids; one or more conjugated lipids (e.g., PEG-conjugated lipids, or lipids conjugated to polymers listed in Table 5 of WO2019 / 217941 (which is incorporated herein by reference in its entirety)); one or more sterols (e.g., cholesterol); and optionally one or more target molecules (e.g., conjugated receptors, receptor ligands, antibodies); or a combination thereof.

[0274] Lipids that can be used to form nanoparticles (e.g., lipid nanoparticles) include, for example, those listed in Table 4 of WO2019 / 217941 incorporated by reference. For example, lipid-containing nanoparticles may contain one or more lipids from Table 4 of WO2019 / 217941. Lipid nanoparticles may contain additional elements such as polymers, such as those listed in Table 5 of WO2019 / 217941 incorporated by reference.

[0275] In some embodiments, the conjugated lipids, if present, include PEG-diacylglycerol (DAG) (e.g., l-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-diachyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), pegylated phosphatidylethanolamine (PEG-PE), and PEG-succinate diacylglycerol (PEGS-DAG) (4-0-(2',3'-di(tetradecane)). This may include one or more of the following: (e.g., noyloxy)propyl-l-O-(w-methoxy(polyethoxy)ethyl)butanediate (PEG-S-DMG), PEG dialkoxypropyl carbam, N-(carbonyl-methoxypolyethylethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those listed in Table 2 of WO2019 / 051289 (incorporated by reference), as well as any combination thereof.

[0276] In some embodiments, the sterols that can be incorporated into lipid nanoparticles include one or more cholesterol or cholesterol derivatives, such as W02009 / 127060 or US2010 / 0130588, which are incorporated herein by reference. Further exemplary sterols include phytosterols, including those described in Eygeris et al (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, which are incorporated herein by reference.

[0277] In some embodiments, the lipid particles include ionizable lipids, noncationic lipids, conjugated lipids that inhibit particle aggregation, and sterols. The amounts of these components can be independently varied to achieve desired properties. For example, in some embodiments, the lipid nanoparticles include about 20 mol% to about 90 mol% of the total lipids in the ionizable lipids (in other embodiments, this may be 20-70% (mol), 30-60% (mol), or 40-50% (mol); about 50 mol% to about 90 mol% of the total lipids present in the lipid nanoparticles), about 5 mol% to about 35 mol% of the total lipids in the noncationic lipids, about 0.5 mol% to about 20 mol% of the total lipids in the conjugated lipids, and about 20 mol% to about 50 mol% of the total lipids in the sterols. The ratio of total lipids to nucleic acids (e.g., including therapeutic agents and / or encoding genetically modified polypeptides, template nucleic acids, or genetically modified systems) can be varied as desired. For example, the ratio of total lipids to nucleic acids (by mass or weight) can be approximately 10:1 to 30:1.

[0278] In some embodiments, the average LNP diameter of the targeted LNP formulation may be in the range of nm from 10s to 100s, measured, for example, by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the targeted LNP formulation may be in the range of about 40nm to about 150nm, for example, about 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, or 150nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be approximately 50 nm to 100 nm, approximately 50 nm to 90 nm, approximately 50 nm to 80 nm, approximately 50 nm to 70 nm, approximately 50 nm to 60 nm, approximately 60 nm to 100 nm, approximately 60 nm to 90 nm, approximately 60 nm to 80 nm, approximately 60 nm to 70 nm, approximately 70 nm to 100 nm, approximately 70 nm to 90 nm, approximately 70 nm to 80 nm, approximately 80 nm to 100 nm, approximately 80 nm to 90 nm, or approximately 90 nm to 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be approximately 70 nm to 100 nm. In certain embodiments, the average LNP diameter of the targeted LNP formulation may be approximately 80 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be approximately 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation is in the range of approximately 1 mm to 500 mm, approximately 5 mm to 200 mm, approximately 10 mm to 100 mm, approximately 20 mm to 80 mm, approximately 25 mm to 60 mm, approximately 30 mm to 55 mm, approximately 35 mm to 50 mm, or approximately 38 mm to 42 mm.

[0279] LNPs described herein, such as targeted LNPs, may be relatively homogeneous in some cases. The polydispersity index can be used to indicate the homogeneity of LNPs, for example, the particle size distribution of lipid nanoparticles. Small polydispersity indices (e.g., less than 0.3) generally indicate a narrow particle size distribution. LNPs may have polydispersity indices ranging from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of LNPs may be about 0.10 to about 0.20.

[0280] The zeta potential of LNPs can be used to indicate the electrokinetic potential of a composition. In some embodiments, the zeta potential can account for the surface charge of the LNPs. Lipid nanoparticles with relatively low positive or negative charges are generally preferred because higher-charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of the LNP may be approximately -10mV to approximately +20mV, approximately -10mV to approximately +15mV, approximately -10mV to approximately +10mV, approximately -10mV to approximately +5mV, approximately -10mV to approximately 0mV, approximately -10mV to approximately -5mV, approximately -5mV to approximately +20mV, approximately -5mV to approximately +15mV, approximately -5mV to approximately +10mV, approximately -5mV to approximately +5mV, approximately -5mV to approximately 0mV, approximately 0mV to approximately +20mV, approximately 0mV to approximately +15mV, approximately 0mV to approximately +10mV, approximately 0mV to approximately +5mV, approximately +5mV to approximately +20mV, approximately +5mV to approximately +15mV, or approximately +5mV to approximately +10mV.

[0281] The efficiency of protein and / or nucleic acid (e.g., mRNA encoding a polypeptide) encapsulation describes the amount of protein and / or nucleic acid encapsulated in the LNP after preparation, or otherwise associated with the LNP, compared to the initial amount provided. The encapsulation efficiency is preferably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing lipid nanoparticles before and after decomposition of the lipid nanoparticles with one or more organic solvents or detergents. Anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in the solution. Fluorescence may be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in the solution. For the lipid nanoparticles described herein, the encapsulation efficiency of proteins and / or nucleic acids may be at least 50%, for example, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.

[0282] The LNPs of this disclosure may optionally include one or more coatings. In some embodiments, the LNPs may be formulated into capsules, films, or tablets having the coatings. Capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0283] Additional exemplary lipids, formulations, methods, and characterizations of LNPs are taught by WO2020 / 061457, which is incorporated herein in its entirety by reference.

[0284] In some embodiments, in vitro or ex vivo cell lipofection is performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA transfection reagent (Mirus Bio). In certain embodiments, the LNP is formulated using GenVoy_ILM ionizable lipid mixture (Precision NanoSystems). In certain embodiments, the targeted LNP of this disclosure is formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012) (the whole of which is incorporated herein by reference).

[0285] Additional specific LNP formulations useful for nucleic acid delivery are described in US8158601 and US8168775 (both incorporated by reference), which include the formulation used in patisiran, marketed under the name ONPATTRO.

[0286] Ionizable lipids The LNPs of this disclosure (e.g., targeted LNPs) comprise one or more ionizable lipids. In some embodiments, the ionizable lipid may be a cationic lipid, an ionizable cationic lipid, such as a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is, for example, a lipid that can be positively charged under physiological conditions. Exemplary cationic lipids comprise one or more positively charged amine groups. In some embodiments, the lipid particles comprise cationic lipids in formulations having one or more of the following: neutral lipids, ionizable amine-containing lipids, biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol, and polymer-conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. Exemplary cationic lipids disclosed herein may have an effective pKa greater than 6.0. In some embodiments, the lipid nanoparticles may include a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa) than the first cationic lipid. The lipid nanoparticles may include 40–60 mol percent of cationic lipids, neutral lipids, sterols, polymer-conjugated lipids, and therapeutic agents described herein (e.g., one or more nucleic acids (e.g., RNA) including gene modification systems) encapsulated within or associated with the lipid nanoparticles. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) is formulated together with the cationic lipid. The therapeutic agent (e.g., one or more nucleic acids) may be adsorbed onto the surface of an LNP, e.g., an LNP containing a cationic lipid. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) may be encapsulated within an LNP, e.g., an LNP containing a cationic lipid. In some embodiments, the lipid nanoparticles may include a target-directing moiety coated with, for example, a targeting agent. In some embodiments, the LNP formulation is biodegradable.In some embodiments, lipid nanoparticles comprising one or more lipids described herein encapsulate at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or 100% RNA molecules (e.g., mRNA molecules).

[0287] In some embodiments, the lipid-to-nucleic acid ratio (mass / mass ratio; w / w ratio) can be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipids and nucleic acids can be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the overall lipid content of the lipid nanoparticle formulation can be in the range of about 5 mg / ml to about 30 mg / mL.

[0288] In some embodiments, the LNP (e.g., targeted LNP) comprises an ionizable lipid V003, as described below. V003 is described in U.S. Patent No. 10,059,655. [ka]

[0289] In some embodiments, the LNP (e.g., targeted LNP) contains an ionizable lipid as shown in Table 1. In some cases, LNPs containing the ionizable lipids of Table 1 exhibit a higher level of transduction in HSCs and / or higher expression of the payload protein in HSCs compared to LNPs containing V003 as the ionizable lipid. In other embodiments, lipid 092 is used as the ionizable lipid for generating LNPs for delivery to HSCs. In other embodiments, lipid 093 is used as the ionizable lipid for generating LNPs for delivery to HSCs. In other embodiments, lipid 153 is used as the ionizable lipid for generating LNPs for delivery to HSCs. In other embodiments, lipid 154 is used as the ionizable lipid for generating LNPs for delivery to HSCs. In other embodiments, lipid 155 is used as the ionizable lipid for generating LNPs for delivery to HSCs. In other embodiments, lipid 162 is used as the ionizable lipid for generating LNPs for delivery to HSCs. In other embodiments, lipid 163 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, lipid 169 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, lipid 176 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, lipid 178 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, lipid 183 is used as an ionizable lipid to generate LNPs for delivery to HSCs. [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 15-5] [Table 15-6] [Table 15-7] [Table 15-8]

[0290] In some embodiments, the LNP, for example, the targeted LNP, is given by formula (I), [ka] or comprising an ionizable lipid having the structure of a pharmaceutically acceptable salt thereof, wherein the formula includes, X is either O or CH2. m is 1, 2, or 3. n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. R 1 C 4-10 It is alkyl, R 2 C 1-4 It is alkyl, R 3 and R 4 C 1-4 It is alkyl, Y is a branched or unbranched lipophilic tail.

[0291] In some embodiments of equation (I), R 1 C 6-8 It is alkyl. In some embodiments, R 1 is a C6 alkyl group. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2is a C4 alkyl group. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 4. In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4is methyl. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0292] In some embodiments, the compound of formula (I) is the compound of formula (IA), [ka] or a pharmaceutically acceptable salt thereof, in the formula, X is either O or CH2. m is 1, 2, or 3. R 1 C 4-10 It is alkyl, R 2 C 1-4 It is alkyl, R 3 and R 4 C 1-4 Alkyl or R 3 and R 4 They, together with the nitrogen atoms they bond to, form a heterocycle. Y is a branched or unbranched lipophilic tail.

[0293] In some embodiments of formula (IA), R 1 C 6-8 It is alkyl. In some embodiments, R 1 is a C6 alkyl group. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2 is a C4 alkyl group. In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0294] In some embodiments, the compound of formula (I) is the compound of formula (IB), [ka] or a pharmaceutically acceptable salt thereof, in the formula, X is either O or CH2. m is 1, 2, or 3. R 1 C 4-10 It is alkyl, R 2 C 1-4 It is alkyl, R 3 and R 4 C 1-4 Alkyl or R 3 and R 4 These atoms, together with the nitrogen atoms they bond to, form a heterocycle.

[0295] In some embodiments of formula (IB), R 1 C 6-8 It is alkyl. In some embodiments, R 1 is a C6 alkyl group. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2 is a C4 alkyl group. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0296] In some embodiments, the compound of formula (I) is the compound of formula (IC), [ka] or a pharmaceutically acceptable salt thereof, in the formula, X is either O or CH2. m is 1, 2, or 3. R 1 C 4-10 It is alkyl, R 2 C 1-4 It is alkyl, R 3 and R 4 C 1-4 Alkyl or R 3 and R 4 These atoms, together with the nitrogen atoms they bond to, form a heterocycle.

[0297] In some embodiments of formula (IC), R 1 C 6-8 It is alkyl. In some embodiments, R 1 is a C6 alkyl group. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2is a C4 alkyl group. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0298] In some embodiments, the compound of formula (I) is the compound of formula (ID), [ka] or a pharmaceutically acceptable salt thereof, in the formula, X is either O or CH2. m is 1, 2, or 3. R 1 C 4-10 It is alkyl, R 2 C 1-4 It is alkyl, R 3 and R 4 C 1-4 Alkyl or R 3 and R 4These atoms, together with the nitrogen atoms they bond to, form a heterocycle.

[0299] In some embodiments of the formula (ID), R 1 C 6-8 It is alkyl. In some embodiments, R 1 is a C6 alkyl group. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2 is a C4 alkyl group. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0300] In some embodiments, the compound of formula (I) is the compound of formula (IE), [ka] or a pharmaceutically acceptable salt thereof, in the formula, X is either O or CH2. m is 1, 2, or 3. R 1 C 4-10 It is alkyl, R 2 C 1-4 It is alkyl, R 3 and R 4 C 1-4 Alkyl or R 3 and R 4 These atoms, together with the nitrogen atoms they bond to, form a heterocycle.

[0301] In some embodiments of formula (IE), R 1 C 6-8 It is alkyl. In some embodiments, R 1 is a C6 alkyl group. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2 is a C4 alkyl group. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0302] In some embodiments, the compound of formula (I) is the compound of formula (IF), [ka] or a pharmaceutically acceptable salt thereof, in the formula, X is either O or CH2. m is 1, 2, or 3. R 1 C 4-10 It is alkyl, R 2 C 1-4 It is alkyl, R 3 and R 4 C 1-4 Alkyl or R 3 and R 4 These atoms, together with the nitrogen atoms they bond to, form a heterocycle.

[0303] In some embodiments of the expression (IF), R 1 C 6-8 It is alkyl. In some embodiments, R 1 is a C6 alkyl group. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2 is a C4 alkyl group. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0304] In some embodiments, the compound of formula (I) is the compound of formula (IG), [ka] or a pharmaceutically acceptable salt thereof, in the formula, X is either O or CH2. m is 1, 2, or 3. R 1 C 4-10 It is alkyl, R 2 C 1-4 It is alkyl, R 3 and R 4 C 1-4 Alkyl or R 3 and R 4 These atoms, together with the nitrogen atoms they bond to, form a heterocycle.

[0305] In some embodiments of formula (IG), R 1 C 6-8 It is alkyl. In some embodiments, R 1 is a C6 alkyl group. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2 is a C4 alkyl group. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0306] In some embodiments, the compound of formula (I) is the compound of formula (IH), [ka] or a pharmaceutically acceptable salt thereof, in the formula, X is either O or CH2. m is 1, 2, or 3. R 1 C 4-10 It is alkyl, R 2 C 1-4 It is alkyl, R 3 and R 4 C 1-4 Alkyl or R 3 and R 4 These atoms, together with the nitrogen atoms they bond to, form a heterocycle.

[0307] In some embodiments of formula (IH), R 1 C 6-8 It is alkyl. In some embodiments, R 1is a C6 alkyl group. In some embodiments, R 2 is a C3 alkyl group. In some embodiments, R 2 is a C4 alkyl group. In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0308] In some embodiments, the LNP, for example, the targeted LNP, is given by formula (II), [ka] or comprising an ionizable lipid having the structure of a pharmaceutically acceptable salt thereof, wherein the formula includes, X is either O or CH2. m is 1, 2, or 3. Y is a branched or unbranched lipophilic tail.

[0309] In some embodiments of equation (II), Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, X is O. In some embodiments, X is methylene. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4.

[0310] In some embodiments, the compound of formula (II) is the compound of formula (II-A), [ka] or a pharmaceutically acceptable salt thereof, in the formula, Y is a branched or unbranched lipophilic tail.

[0311] In some embodiments of equation (II-A), Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] That is the case.

[0312] In some embodiments, the LNP, for example, the targeted LNP, is given by formula (III), [ka] or comprising an ionizable lipid having the structure of a pharmaceutically acceptable salt thereof, wherein the formula includes, R 3 and R 4 C 1-4 Alkyl or R 3 and R 4 They, together with the nitrogen atoms they bond to, form a heterocycle. Y is a branched or unbranched lipophilic tail.

[0313] In some embodiments of equation (III), Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, R 3 is n-propyl. In some embodiments, R 3 is ethyl. In some embodiments, R 3 is methyl. In some embodiments, R 4 is n-propyl. In some embodiments, R 4 is ethyl. In some embodiments, R 4 is methyl. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form pyrrolidine. In some embodiments, R 3 and R 4 These, together with the N to which they are bound, form piperidine. In some embodiments, R 3 and R 4 These, together with the N atoms to which they are bound, form piperazine.

[0314] In some embodiments, the compound of formula (III) is the compound of formula (III-A), [ka] or a pharmaceutically acceptable salt thereof, in the formula, Y is a branched or unbranched lipophilic tail.

[0315] In some embodiments of equation (III-A), Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] In some embodiments, Y is [ka] That is the case.

[0316] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds in Table 2. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5]

[0317] In some embodiments, the LNP, for example, the targeted LNP, is given by formula (IV), [ka] or comprising an ionizable lipid having the structure of a pharmaceutically acceptable salt thereof, wherein the formula includes, X is -O- or -CH2-, m is 0, 1, 2, or 3. R 1 C 1-4 It is alkyl, R 2 C 1-4 It is alkyl, n is 1, 2, 3, or 4. R 3 C 4-10 It is alkyl, R 4 C 4-10 It is alkyl, p is 2, 3, 4, 5, or 6. R 5 C 4-10 It is alkyl, R 6 C 4-10 It is alkyl.

[0318] In some embodiments, the compound of formula (IV) is the compound of formula (IV-A), [ka] or a pharmaceutically acceptable salt thereof, in the formula, p is 2, 3, 4, 5, or 6. R 5 C 4-10 It is alkyl, R 6 C 4-10 It is alkyl.

[0319] In some embodiments, the compound of formula (IV) is the compound of formula (IV-B), [ka] or a pharmaceutically acceptable salt thereof, in the formula, R 5 C 4-6 It is alkyl, R 6 C 4-6 It is alkyl.

[0320] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds in Table 3. [Table 17]

[0321] In some embodiments, the ionizable lipid has one of the structures shown below. [ka] [ka] [ka]

[0322] Other exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, but are not limited to, those listed in Table 1 of WO2019 / 051289, which is incorporated herein by reference. Additional exemplary lipids include, but are not limited to, formulas: Formula X of US2016 / 0311759; Formula I of US2015 / 0376115 or US2016 / 0376224; Formula I, II, or III of US2016 / 0151284; Formula I, IA, II, or IIA of US2017 / 0210967; Formula Ic of US2015 / 0140070; Formula A of US2013 / 0178541; Formula I of US2013 / 0303587 or US2013 / 0123338; ​​US2015 / 0 Formula I of 141678; Formula II, III, IV, or V of US2015 / 0239926; Formula I of US2017 / 0119904; Formula I or II of WO2017 / 117528; Formula A of US2012 / 0149894; Formula A of US2015 / 0057373; Formula A of WO2013 / 116126; Formula A of US2013 / 0090372; Formula A of US2013 / 0274523; Formula A of US2013 / 0274504; Formula A of US2013 / 0053572; W02013 / 016058 Formula A; Formula A of W02012 / 162210; Formula I of US2008 / 042973; Formula I, II, III, or IV of US2012 / 01287670; Formula I or II of US2014 / 0200257; Formula I, II, or III of US2015 / 0203446; Formula I or III of US2015 / 0005363; Formula I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014 / 0308304; US2013 / 0338210 Formulas; Formulas I, II, III, or IV of W02009 / 132131; Formula A of US2012 / 01011478; Formula I or XXXV of US2012 / 0027796; Formula XIV or XVII of US2012 / 0058144; Formula of US2013 / 0323269; Formula I of US2011 / 0117125; Formulas I, II, or III of US2011 / 0256175; Formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012 / 0202871;Formulas I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI from US2011 / 0076335; Formula I or II from US2006 / 008378; Formula I from US2013 / 0123338; ​​Formula I or XAYZ from US2015 / 0064242; Formulas XVI, XVII, or XVIII from US2013 / 0022649; Formula I, II, or III from US2013 / 0116307; Formula I, II, or III from US2013 / 0116307; Formula I or II from US2010 / 0062967; Formula Formula IX; Formula I of US2014 / 0039032; Formula V of US2018 / 0028664; Formula I of US2016 / 0317458; Formula I of US2013 / 0195920; Formulas 5, 6, or 10 of US10,221,127; Formula III-3 of WO2018 / 081480; Formula I-5 or I-8 of WO2020 / 081938; Formula 18 or 25 of US9,867,888; Formula A of US2019 / 0136231; Formula II of WO2020 / 219876; Formula 1 of US2012 / 0027803; Formula OF-02 of US2019 / 0240349; Formula 23 of US10,086,013; Miao One or more of the following can be cited: formula cKK-E12 / A6 from et al (2020); formula C12-200 from WO2010 / 053572; 7C1 from Dahlman et al (2017); 304-O13 or 503-O13 from Whitehead et al; formula TS-P4C2 from US9,708,628; formula I from WO2020 / 106946; or formula I from WO2020 / 106946.

[0323] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetran-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA or MC3), as described in Example 9 of WO2019 / 051289A9 (the whole of which is incorporated herein by reference). In some embodiments, the ionizable lipid is lipid ATX-002, as described in Example 10 of WO2019 / 051289A9 (the whole of which is incorporated herein by reference). In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13,16-diene-l-amine (compound 32), as described in Example 11 of WO2019 / 051289A9 (which is incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is compound 6 or compound 22, as described in Example 12 of WO2019 / 051289A9 (which is incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), as described in Example 1 of US9,867,888 (which is incorporated herein by reference in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12-dienoate (LP01), as synthesized in Example 13 of WO2015 / 095340 (the whole of which is incorporated herein by reference). In some embodiments, the ionizable lipid is di((Z)-non-2-en-1-yl)9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), as synthesized in Example 7, 8, or 9 of US2012 / 0027803 (the whole of which is incorporated herein by reference).In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydododecyl)amino)ethyl)(2-hydroxydododecyl)amino)ethyl)piperazin-1-yl)ethyl)azandiyl)bis(dodecane-2-ol)(C12-200), as synthesized in Examples 14 and 16 of WO2010 / 053572 (the whole of which is incorporated herein by reference). In some embodiments, the ionizable lipid is the imidazole cholesterol ester (ICE) lipid (3S,10R,13R,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl3-(1H-imidazole-4-yl)propanoate, for example, structure (I) from WO2020 / 106946 (the whole of which is incorporated herein by reference).

[0324] In some embodiments, the mol% of ionizable lipids in LNPs, e.g., targeted LNPs, is about 25% to about 65%. In some embodiments, the mol% of ionizable lipids in LNPs, e.g., targeted LNPs, is about 35% to about 60%. In some embodiments, the mol% of ionizable lipids in LNPs, e.g., targeted LNPs, is about 40% to about 50%. In some embodiments, the mol% of ionizable lipids in LNPs, e.g., targeted LNPs, is about 45% to about 50%.

[0325] The compounds disclosed herein (e.g., the lipids in Tables 1, 2, and 3), or their pharmaceutically acceptable salts, may contain asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. This disclosure intends various stereoisomers and mixtures thereof, and includes “enantiomers” referring to two stereoisomers whose molecules are mirror images of each other and cannot be superimposed, and “diastereomers” referring to stereoisomers having at least two asymmetric atoms but not being mirror images of each other. This disclosure means including all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), or (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or may be decomposed using conventional techniques, e.g., chromatography and fractionation crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include, for example, chiral synthesis from suitable optically pure precursors or racemic decomposition using chiral high-pressure liquid chromatography (HPLC). Where the compounds described herein contain an olefinic double bond or other geometrically asymmetric center, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

[0326] Helper lipids The LNPs of this disclosure, for example, targeted LNPs, comprise one or more ionizable lipids. Examples of helper lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), and dioleoyl-phosphatidylethanolamine. Min (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanol Amines (e.g., 16-O-monomethylPE), dimethylphosphatidylethanolamine (e.g., 16-O-dimethylPE), l8-l-transPE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybean phosphatidylcholine, egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosph Phosphatidylglycerol (DSPG), Diethylphosphatidylcholine (DEPC), Palmitoyloleoylphosphatidylglycerol (POPG), Diethyloylphosphatidylethanolamine (DEPE), Lecithin, Phosphatidylethanolamine, Lysolecithin, Lysophosphatidylethanolamine, Phosphatidylserine, Phosphatidylinositol, Sphingomyelin, Egg sphingomyelin (ESM), Cephalin, Cardiolipin, Phosphatidic acid, Cerebroside, Dicetylphosphate,Examples include lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholines and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably derived from fatty acids having a C10-C24 carbon chain, such as lauroyl, myristoyl, pimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments but not limited to those described herein, are those described in Kim et al. (2020)dx.doi.org / 10.1021 / acs.nanolett.0c01386, which are incorporated herein by reference.

[0327] Other examples of noncationic lipids suitable for use in lipid nanoparticles include, but are not limited to, nonphospholipids such as stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin. Other noncationic lipids are described in WO2017 / 099823 or U.S. Patent Publication US2018 / 0028664, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the noncationic lipid is oleic acid or a compound of formula I, II, or IV of US2018 / 0028664 (the entire text of which is incorporated herein by reference).

[0328] In some embodiments, the helper lipid is a sphingolipid. In some such embodiments, the non-pegylated lipid is sphingomyelin. In some embodiments, the sphingomyelin has a head group selected from phosphocholine, phosphoethanolamine, or ceramide. In some embodiments, the sphingomyelin is egg sphingomyelin.

[0329] In some embodiments, helper lipids comprise 5–40% (mol), 8–30%, 10–28%, 20–36%, 22–32%, or 10–15% (mol) of the total lipids present in the lipid nanoparticles. In embodiments, the molar ratio of ionizable lipids to neutral lipids is in the range of about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

[0330] In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in LNPs, e.g., targeted LNPs, is about 18% to about 32%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in LNPs, e.g., targeted LNPs, is about 20% to about 30%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in LNPs, e.g., targeted LNPs, is about 22% to about 32%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in LNPs, e.g., targeted LNPs, is about 22% to about 28%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in targeted LNPs is about 21% to about 23%. In some embodiments, the mol% of helper lipids (e.g., DSPC or sphingomyelin) in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%. As shown in the following examples, in vivo delivery of a particular payload after administration of a disclosed LNP (e.g., a targeted LNP) having these percentages of helper lipids provides enhanced transduction and expression of the payload compared to a targeted LNP having less or more helper lipids.

[0331] In some embodiments, the molar ratio between ionizable lipids and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) in LNPs, e.g., targeted LNPs, is about 1:1 to about 7:1. In some embodiments, the molar ratio between ionizable lipids and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 1:1 to about 4:1. In some embodiments, the molar ratio between ionizable lipids and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 1:1 to about 3:1. In some embodiments, the molar ratio between ionizable lipids and non-pegylated helper lipids (e.g., DSPC) is about 1:1 to about 2.5:1. In some embodiments, the molar ratio between ionizable lipids and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 1:1 to about 2:1. In some embodiments, the molar ratio between ionizable lipids and non-pegylated helper lipids (e.g., DSPC) is about 1.5:1 to about 2.5:1. In some embodiments, the molar ratio between ionizable lipids and non-pegylated helper lipids (e.g., DSPC or sphingomyelin) is about 2:1 to about 2.5:1.

[0332] In some embodiments, the LNP, e.g., targeted LNP, comprises ionizable lipids and DSPC as shown in Table 1, Table 2, or Table 3. In some embodiments, the LNP, e.g., targeted LNP, comprises ionizable lipids and sphingomyelin as shown in Table 1, Table 2, or Table 3. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20% to about 25%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in LNPs, for example, targeted LNPs, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0333] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid of formula I and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid of formula I and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20% to about 25%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0334] In some embodiments, the LNP, for example, the targeted LNP, contains lipid O93 and DSPC. In some embodiments, the LNP, for example, the targeted LNP, contains lipid O93 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 25%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 20%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0335] In some embodiments, the LNP, e.g., targeted LNP, comprises lipid O92 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises lipid O92 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20% to about 25%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0336] In some embodiments, the LNP, for example, the targeted LNP, contains lipid 153 and DSPC. In some embodiments, the LNP, for example, the targeted LNP, contains lipid 153 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 25%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0337] In some embodiments, the LNP, for example, the targeted LNP, comprises lipid 154 and DSPC. In some embodiments, the LNP, for example, the targeted LNP, comprises lipid 154 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 25%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0338] In some embodiments, the LNP, for example, the targeted LNP, comprises lipid 155 and DSPC. In some embodiments, the LNP, for example, the targeted LNP, comprises lipid 155 and sphingomyelin. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 25%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, for example, the targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about ...

Claims

1. Lipid nanoparticles (LNPs), (iv) A lipid component comprising ionizable lipids and helper lipids, wherein the ionizable lipids are present in an amount of about 35 mol% to about 60 mol% of the lipid component, and the helper lipids are present in an amount of about 18 mol% to about 32 mol% of the lipid component, (v) One or two target-directed portions conjugated to the LNP, wherein the one or two target-directed portions are configured to target one or more receptors on the surface of an HSC selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184, (vi) The lipid nanoparticle (LNP) comprising the therapeutic agent encapsulated within the LNP, the therapeutic agent comprising two or more RNA molecules.

2. Lipid nanoparticles (LNPs), (iv) A lipid component comprising ionizable lipids and helper lipids, wherein the ionizable lipids are present in an amount of about 35 mol% to about 60 mol% of the lipid component, and the helper lipids are present in an amount of about 18 mol% to about 32 mol% of the lipid component, (v) Means for binding to a cell surface receptor selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184, (vi) The lipid nanoparticle (LNP) comprising the therapeutic agent encapsulated within the LNP, the therapeutic agent comprising two or more RNA molecules.

3. The LNP according to claim 2, wherein the means for binding to the cell surface receptor is present on the surface of the LNP.

4. The LNP according to any one of claims 2 to 3, wherein the LNP comprises means for binding to a first cell surface receptor and means for binding to a second cell surface receptor, the first cell surface receptor and the second cell surface receptor being different, and the first cell surface receptor and the second cell surface receptor being independently selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184.

5. The LNP according to any one of claims 1 to 4, wherein the therapeutic agent comprises two RNA molecules.

6. The LNP according to any one of claims 1 to 4, wherein the therapeutic agent comprises three RNA molecules.

7. The LNP according to any one of claims 1 to 6, wherein the helper lipid is present in an amount of about 20 mol% to about 25 mol% of the lipid component.

8. The LNP according to any one of claims 1 to 6, wherein the helper lipid is present in an amount of about 22 mol% to about 28 mol% of the lipid component.

9. The LNP according to any one of claims 1 to 8, wherein the ionizable lipid is present in an amount of about 40 mol% to about 50 mol% of the lipid component.

10. The LNP according to any one of claims 1 to 9, wherein the ionizable lipid is present in an amount of about 45 mol% to about 50 mol% of the lipid component.

11. The helper lipids are distearoyl-sn-glycerol-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (16-O-monomethylPE, etc.), dimethyl-phosphatidylethanolamine (16-O-dimethylPE, etc.), l8-l-transPE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated Soy phosphatidylcholine, egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diylcoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol (POPG), diylidoyl phosphatidylethanol An LNP according to any one of claims 1 to 10, selected from the group consisting of amine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

12. The LNP according to claim 11, wherein the helper lipid is DSPC.

13. The LNP according to claim 11, wherein the helper lipid is sphingomyelin.

14. The LNP according to any one of claims 1 to 13, further comprising cholesterol molecules.

15. The LNP according to claim 14, wherein the cholesterol molecule is selected from the group consisting of beta-sitoesterol, hydroxycholesterol, stigmamanol, and any combination thereof.

16. The LNP according to claim 14 or claim 15, wherein the cholesterol molecule is present in an amount of about 20 mol% to about 55 mol% of the lipid component.

17. The LNP according to claim 14 or claim 15, wherein the cholesterol molecule is present in an amount of about 20 mol% to about 35 mol% of the lipid component.

18. The LNP according to any one of claims 1 and 5 to 17, wherein the lipid component further comprises a PEG-modified lipid.

19. The LNP according to claim 18, wherein the one or two target-directing portions are conjugated to the LNP via the PEG-mediated lipid.

20. The LNP according to any one of claims 2 to 17, wherein the lipid component further comprises a PEG-modified lipid.

21. The LNP according to claim 20, wherein the means for binding to the cell surface receptor is conjugated to the surface of the LNP via the PEGylated lipid.

22. The LNP according to any one of claims 18 to 21, wherein the PEGylated lipid comprises a lipid portion selected from the group consisting of DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, stearic acid, and any combination thereof.

23. The LNP according to claim 22, wherein the lipid portion of the PEG-modified lipid is DSPE.

24. The LNP according to any one of claims 18 to 23, wherein the PEGylated lipid is present in an amount of about 0.05 mol% to about 2.5 mol% of the lipid component.

25. The LNP according to any one of claims 1, 5 to 19 and 22 to 24, wherein the LNP includes a target-directivity portion conjugated on the surface of the LNP.

26. The LNP according to claim 25, wherein the one target-directing portion targets the CD 117.

27. The LNP according to claim 25, wherein the one target-directing portion targets CD71.

28. The LNP according to claim 25, wherein the one target-directing portion targets the CD 34.

29. The LNP according to claim 25, wherein the one target-directing portion targets CD 50.

30. The LNP according to claim 25, wherein the one target-directing portion targets CD43.

31. The LNP according to claim 25, wherein the one target-directing portion targets the CD 338.

32. The LNP according to claim 25, wherein the one target-directing portion targets the CD 164.

33. The LNP according to claim 25, wherein the one target-directing portion targets CD33.

34. The LNP according to claim 25, wherein the one target-directing portion targets CD38.

35. The LNP according to claim 25, wherein the one target-directing portion targets CD46.

36. The LNP according to claim 25, wherein the one target-directing portion targets CD49d.

37. The LNP according to claim 25, wherein the one target-directing portion targets CD59.

38. The LNP according to claim 25, wherein the one target-directing portion targets the CD 105.

39. The LNP according to claim 25, wherein the one target-directing portion targets CD 123.

40. The LNP according to claim 25, wherein the one target-directing portion targets the CD 184.

41. The LNP according to any one of claims 1, 5 to 19 and 22 to 24, wherein the LNP includes two target-direction portions conjugated on the surface of the LNP.

42. The LNP according to claim 41, wherein the two target-directing portions are CD117 and CD71.

43. The LNP according to claim 41, wherein the two target-directing portions are CD117 and CD34.

44. The LNP according to claim 41, wherein the two target-directing portions are CD117 and CD50.

45. The ionizable lipid is a compound of formula (I), 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, in the formula, X is -O- or -CH 2 - and m is 0, 1, 2, or 3, R 1 However, C 1-4 It is alkyl, R 2 However, C 1-4 It is alkyl, n is 1, 2, 3, or 4, R 3 However, C 4-10 It is alkyl, R 4 is C 4-10 alkyl, p is 2, 3, 4, 5, or 6, R 5 However, C 4-10 It is alkyl, R 6 However, C 4-10 The LNP according to any one of claims 1 to 44, wherein the LNP is alkyl.

46. The LNP according to any one of claims 1 to 44, wherein the ionizable lipid is a compound among the structures shown below. 【Chemistry 2-1】 【Chemistry 2-2】 [Chemistry 2-3]

47. Lipid nanoparticles (LNPs), (i) Lipid components including ionizable lipids and helper lipids, (ii) Two target-directed portions conjugated to the LNP, wherein the two target-directed portions are configured to target one or more receptors on the surface of an HSC selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184, (iii) A therapeutic agent encapsulated within the LNP, comprising the therapeutic agent, and the lipid nanoparticles (LNPs).

48. The LNP according to claim 47, wherein the therapeutic agent comprises two nucleic acid molecules.

49. The LNP according to claim 47, wherein the therapeutic agent comprises three nucleic acid molecules.

50. The LNP according to any one of claims 47 to 49, wherein the nucleic acid molecule is an RNA molecule.

51. The LNP according to claim 50, wherein the therapeutic agent comprises two RNA molecules, one of which is mRNA and the other RNA molecule is a guide RNA.

52. The LNP according to any one of claims 47 to 51, wherein the two target-directing portions are CD117 and CD71.

53. The LNP according to any one of claims 47 to 51, wherein the two target-directing portions are CD117 and CD34.

54. The LNP according to any one of claims 47 to 51, wherein the two target-directing portions are CD117 and CD50.

55. The LNP according to any one of claims 47 to 54, wherein the helper lipid is present in an amount of about 20 mol% to about 25 mol% of the lipid component.

56. The LNP according to any one of claims 47 to 54, wherein the helper lipid is present in an amount of about 22 mol% to about 28 mol% of the lipid component.

57. The LNP according to any one of claims 47 to 56, wherein the ionizable lipid is present in an amount of about 40 mol% to about 50 mol% of the lipid component.

58. The LNP according to any one of claims 47 to 56, wherein the ionizable lipid is present in an amount of about 45 mol% to about 50 mol% of the lipid component.

59. The helper lipids are distearoyl-sn-glycerol-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2-Dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (e.g., 16-O-monomethylPE), dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethylPE), l8-l-transPE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soybeans Phosphatidylcholine, Egg Phosphatidylcholine (EPC), Dioleoyl Phosphatidylserine (DOPS), Sphingomyelin (SM), Dimyristoyl Phosphatidylcholine (DMPC), Dimyristoyl Phosphatidylglycerol (DMPG), Distearoyl Phosphatidylglycerol (DSPG), Diylcoyl Phosphatidylcholine (DEPC), Palmitoyloleoyl Phosphatidylglycerol (POPG), Diyloyl Phosphatidyl Ethanol LNP according to any one of claims 47 to 58, selected from the group consisting of mine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

60. The LNP according to claim 59, wherein the helper lipid is DSPC.

61. The LNP according to claim 59, wherein the helper lipid is sphingomyelin.

62. The ionizable lipid is a compound of formula (I), 【Transformation 3】 or a pharmaceutically acceptable salt thereof, in the formula, X is -O- or -CH 2 - and m is 0, 1, 2, or 3, R 1 However, C 1-4 It is alkyl, R 2 However, C 1-4 It is alkyl, n is 1, 2, 3, or 4, R 3 However, C 4-10 It is alkyl, R 4 However, C 4-10 It is alkyl, p is 2, 3, 4, 5, or 6, R 5 However, C 4-10 It is alkyl, R 6 However, C 4-10 The LNP according to any one of claims 47 to 61, wherein the LNP is alkyl.

63. The LNP according to any one of claims 47 to 61, wherein the ionizable lipid is a compound among the structures shown below. 【Chemistry 4-1】 【Chemistry 4-2】 【Chemistry 4-3】

64. Lipid nanoparticles (LNPs), (i) Lipid components including ionizable lipids and helper lipids, (ii) One or two target-directed portions conjugated to the LNP, wherein the one or two target-directed portions are configured to target one or more receptors on the surface of an HSC selected from the group consisting of CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, and CD184, A therapeutic agent encapsulated within the LNP, comprising the therapeutic agent containing two RNA molecules, wherein one nucleic acid molecule is 5' to 3', (a) gRNA spacer, (b) gRNA skeleton, (c) Arrays of heterogeneous objects, and (iv) Lipid nanoparticles (LNPs) comprising template RNA containing a primer-binding site (PBS) sequence.

65. The LNP according to claim 64, wherein the LNP includes a target-directivity portion conjugated on the surface of the LNP.

66. The LNP according to claim 65, wherein the one target-directing portion targets the CD 117.

67. The LNP according to claim 65, wherein the one target-directing portion targets CD71.

68. The LNP according to claim 65, wherein the one target-directing portion targets the CD 34.

69. The LNP according to claim 65, wherein the one target-directing portion targets CD 50.

70. The LNP according to claim 65, wherein the one target-directing portion targets CD43.

71. The LNP according to claim 65, wherein the one target-directing portion targets the CD 164.

72. The LNP according to claim 65, wherein the one target-directing portion targets CD33.

73. The LNP according to claim 65, wherein the one target-directing portion targets the CD38.

74. The LNP according to claim 65, wherein the one target-directing portion targets CD46.

75. The LNP according to claim 65, wherein the one target-directing portion targets CD49d.

76. The LNP according to claim 65, wherein the one target-directing portion targets CD59.

77. The LNP according to claim 65, wherein the one target-directing portion targets CD 105.

78. The LNP according to claim 65, wherein the one target-directing portion targets CD 123.

79. The LNP according to claim 65, wherein the one target-directing portion targets CD 184.

80. The LNP according to claim 65, wherein the one target-directing portion targets the CD 338.

81. The LNP according to claim 64, wherein the LNP includes two target-directivity portions conjugated on the surface of the LNP.

82. The LNP according to claim 81, wherein the two target-directing portions are CD117 and CD71.

83. The LNP according to claim 81, wherein the two target-directing portions are CD117 and CD34.

84. The LNP according to claim 81, wherein the two target-directing portions are CD117 and CD50.

85. A pharmaceutical composition comprising the LNP described in any one of claims 64 to 84.

86. The pharmaceutical composition according to claim 85, wherein the template RNA includes the spacers shown in Table X1.

87. The pharmaceutical composition according to claim 85, wherein the template RNA comprises the heterogeneous object sequences shown in Table 2.

88. The pharmaceutical composition according to claim 85, wherein the template RNA comprises the PBS sequence shown in Table X3.

89. The pharmaceutical composition according to claim 85, wherein the template RNA comprises the heterogeneous object sequences and PBS sequences of Table X4.

90. The pharmaceutical composition according to claim 85, wherein the template RNA comprises the sequence shown in Table X6.

91. A method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of LNP according to any one of claims 1 to 84.

92. The aforementioned diseases include adenosine deaminase severe combined immunodeficiency (ADA-SCID), adrenoleukodystrophy (CALD), alpha-mannosidosis, chronic granulomatous disease, unclassified immunodeficiency, Gaucher disease, globoid cell leukodystrophy, hemophagocytic lymphohistiocytosis, interleukin-7 receptor (IL-7R) SCID, Janus kinase 3 (JAK-3) SCID, malignant infantile osteopetrosis, metachromatic leukodystrophy, Schayet syndrome, mucopolysaccharidosis type II (MPS2), MPS7, The method according to claim 91, selected from the group consisting of mucolipidosis type II, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, paroxysmal nocturnal hemoglobinuria, Pompe disease, pyruvate kinase deficiency (PKD), recombinant activating gene (RAG) 1 / 2 deficiency, severe congenital neutropenia, sickle cell disease (SCD), Tay-Sachs disease, thalassemia, beta-thalassemia, Wiscott-Aldrich syndrome, X-linked agammaglobulinemia, and X-linked SCID.

93. The method according to claim 92, wherein the disease is sickle cell disease.

94. The method according to claim 92, wherein the disease is thalassemia.

95. The method according to claim 92, wherein the thalassemia is beta-thalassemia.

96. A method for delivering a therapeutic agent to a target, comprising administering an LNP according to any one of claims 1 to 84 to the target.

97. A method for delivering a therapeutic agent to hematopoietic stem cells (HSCs) in a subject, comprising administering an LNP according to any one of claims 1 to 84 to the subject.

98. The method according to claim 96 or 97, wherein the therapeutic agent comprises two RNA molecules, one of which is mRNA and the other RNA molecule is a guide RNA.

99. The method according to any one of claims 96 to 98, wherein the method causes modification of the genome of the HSC in the subject.

100. The method according to any one of claims 81 to 99, wherein the method includes in vivo administration to a subject.

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