Lipid nanoparticles for delivery of therapeutic payloads to cells

Abstract

The disclosure provides lipid nanoparticles (LNPS) and conjugates comprising a targeting moiety and a lipid nanoparticle (LNP) encapsulating a therapeutic agent (e.g., payload) for delivery to hematopoietic stem cells (HSCs). The conjugates can be formulated in a pharmaceutical composition and can be directly administered to a subject in need of treatment (e.g., via in vivo administration).

Description

LIPID NANOPARTICLES FOR DELIVERY OF THERAPEUTIC PAYLOADS TO CELLS CROSS REFERNCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application 63 / 582,456, filed on September 13, 2023; U.S. Provisional Application 63 / 592,196, filed on October 22, 2023; U.S. Provisional Application 63 / 582,457, filed on September 13, 2023; U.S. Provisional Application 63 / 592,195, filed on October 22, 2023; and U.S. Provisional Application 63 / 677,892, filed on July 31, 2024; the contents of each of which are hereby incorporated herein by reference in their entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (252052001340seqlist.xml; Size: 430,624 bytes; and Date of Creation: September 13, 2024) is herein incorporated by reference in its entirety. BACKGROUND

[0003] Hematopoietic stem cells (HSCs) are a rare cell population which resides in the bone marrow and serve as the parental cell lineage for both red and white blood cells. HSCs are uniquely capable of self-renewal and multi-lineage differentiation and are therefore attractive targets for genetic medicine therapies because correction of defective genes at the stem cell level can result in long-lasting therapeutic efficacy (Shi, Nano Letters 23:2938- 2944, 2023).

[0004] During recent years, clinical success with autologous stem cell gene editing therapy has been demonstrated for diseases including b-hemoglobinopathies (Frangoul, N Engl J Med, 384: 252-260, 2021), primary immunodeficiencies (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 to treat β- hemoglobinopathies such as sickle cell disease and beta thalassemia have generally been performed ex-vivo. This process involves the collection of HSCs via bone marrow aspiration or via apheresis following HSC mobilization with G-CSF and Plerixafor. The bone marrow or apheresis samples are subsequently sorted to isolate the CD34+ subset which contains the desired long-term HSC (LT-HSC) subpopulation, and the isolated CD34+ HSCs are subsequently edited and expanded in-vitro for eventual adoptive transfer back to the patient.To maximize the engraftment of the edited CD34+ LT-HSCs, patients are typically “conditioned” with chemotherapeutic agents such as busulfan or undergo whole body radiation therapy, to deplete non-edited LT-HSCs and their progeny 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. Further, the technical complexity of ex-vivo autologous stem cell gene therapy and associated cost limit the geographic locations where treatment can occur and the population of patients that can afford to pay for the therapy.

[0005] To expand the therapeutic potential of HSC therapies to a broader set of patients, one alternative is direct in vivo HSC engineering with delivery of a gene therapy or gene editor directly to the bone marrow microenvironment without the need for HSC collection, culture and conditioning (Shi, Nano Letters 23:2938-2944, 2023). However, specific delivery of a gene therapy or gene editor to the rare long-term HSC (LT-HSC) population in the bone marrow microenvironment can be difficult as any therapeutic agent delivered systemically must evade or overwhelm myriad mechanisms that will prevent its delivery to the bone marrow microenvironment (for example, liver uptake, phagocytic cell uptake, complement activation, opsonization, phagocytic cell uptake, etc.). Once in the bone marrow, the therapeutic agent must be delivered specifically to the self-renewing LT-HSCs population to provide a permanent cure. Thus, there is a therapeutic need to develop compositions capable of effectively delivering a therapeutic agent to HSCs (e.g., LT-HSCs) in a patient. SUMMARY OF THE INVENTION

[0006] The disclosure provides lipid nanoparticles (LNPs) or conjugates comprising a lipid nanoparticle (LNP) encapsulating a payload, such as a therapeutic agent (e.g., a gene modifying system) or 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 an HSC and / or HPC more effectively than a baseline conjugate or baseline LNP. In some embodiments, the disclosure also provides conjugates comprising a lipid nanoparticle (LNP) encapsulating a payload, such as a therapeutic agent (e.g., a gene modifying system) or reporter construct, for delivery to HSCs and / or HPCs wherein the LNP has at least one targeting moiety on its surface. The conjugates having atleast one targeting moiety described herein will also be referred to as targeted LNPs or tLNPs.

[0007] In one aspect, the disclosure provides a lipid nanoparticle (LNP) for delivery of a therapeutic agent to HSCs and / or HPCs in vivo, wherein the LNP comprises an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or from the lipids in Table 1, Table 2 and Table 3, as set forth in the disclosure below, and a therapeutic agent encapsulated within the LNP. It will be understood that any reference to an ionizable lipid in an LNP as set forth herein also includes pharmaceutically acceptable salts of the lipids.

[0008] In another aspect, the disclosure provides conjugates comprising an LNP and a targeting moiety. In some cases, inclusion of a targeting moiety of a targeted LNP enhances the ability of the conjugate or LNP to deliver the payload to HSCs and HPCs (collectively HSPCs). The targeted LNPs can be formulated in a pharmaceutical composition and can be directly administered to a subject (e.g., patient) in need thereof (e.g., by in vivo administration). Once administered, the compositions can deliver significant quantities of a therapeutic agent to the HSCs (e.g., LT-HSCs) of the subject. Alternatively, the LNPs can be administered to isolated HSCs (e.g., LT-HSCs) ex vivo. 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, for example, a therapeutic peptide or protein, a nucleic acid comprising a therapeutic agent, or a nucleic acid encoding a therapeutic agent. In some embodiments, the therapeutic agent can be a genetic medicine, wherein the therapeutic agent is capable of modifying, altering or effecting a change in the genomic DNA of a cell (e.g., a cell in the subject). In some embodiments, the therapeutic agent is a gene therapy agent or gene editing agent. In some embodiments, the therapeutic agent is a gene modifying polypeptide. In some embodiments, the therapeutic agent is a gene modifying system.

[0009] In some embodiments, the targeting moiety is an antibody, Fab fragment or single chain variable fragment (scFv), a DARPIN, a VHH domain antibody, a FN3 domain, a nanobody, a single domain antibody or a Centyrin. In other embodiments, the targeting moiety is a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin or a N-Acetylgalactosamine (GalNac). In some embodiments, the targeting moiety is a peptide or protein, such as a ligand or part of a ligand, that binds to a receptor (e.g., a receptor on the surface of an HSC or LT-HSC). In some embodiments, the targeting moiety is a peptide or protein that binds to a receptor or ligand on the surface of an HSC, HPC or LT-HSC, wherein the affinity of the peptide or proteintargeting moiety for the receptor or ligand on the surface of the HSC, HPC or LT-HSC is modulated via phage display.

[0010] In certain embodiments, the payload (e.g., therapeutic agent) delivered by the LNP or targeted LNP can be a small molecule, peptide or protein, non-coding RNA (ncRNA), gRNA, siRNA or miRNA, a nucleic acid (e.g., mRNA) encoding a peptide or protein (e.g., a protein for replacement gene therapy, or a protein for modifying or altering the genome or epigenome, e.g., a protein for gene editing), a nucleic acid encoding or comprising one or more components of a system for altering a genome (e.g., one or more components of a ribonucleoprotein (RNP) complex for editing or altering the genome or epigenome (e.g., for introducing insertion-deletion mutations (indels), base editing, epigenetic editing, or target-primed reverse transcription (TPRT), e.g., by a mechanism that requires a recombinase, transposase, retrotransposase, helicase, reverse transcriptase, polymerase, deaminase, methylase, demethylase, or ligase function), or combinations thereof. In certain embodiments, the therapeutic agent (i.e., payload) delivered by the LNP or targeted LNP can be a gene modifying protein, a nucleic acid encoding a gene modifying protein, or a gene modifying system, as described herein.

[0011] In one aspect, the disclosure provides a targeted LNP (conjugate) encapsulating 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 targeting moieties, such as antibodies, Fab fragments, or scFvs, capable of binding to one or more proteins on the cell surface of a hematopoietic stem cell (HSC). In some embodiments, the targeted LNP is delivered in vivo to a subject (e.g., a human patient) in need thereof. Following in vivo administration, the nucleic acids encoding components of a system for modifying or altering a genome can be expressed, resulting in in vivo altering of the genome (e.g., gene editing) of the HSC. It has surprisingly been discovered that LNPs comprising particular combinations of one or more targeting moieties (e.g., antibodies, Fab fragments or scFvs), ionizable lipids, and / or helper lipids are capable of enhancing delivery of a therapeutic payload, (e.g., a system capable of altering the genome, such as a gene modifying system) to HSCs following in vivo administration to a subject, thereby resulting in enhanced levels of therapeutic activity in the HSCs.

[0012] In some embodiments, an LNP or targeted LNP (conjugate) comprising an ionizable lipid as described herein is capable of delivering a payload (e.g., a therapeutic payload) 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 an LNP comprising abaseline lipid (such as V003). In some embodiments, an LNP comprising an ionizable lipid as described herein is capable of delivering a payload (e.g., a therapeutic payload) 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 an LNP comprising a baseline lipid (such as V003) in an animal model, as described herein. In some embodiments, the LNP is a targeted LNP.

[0013] In some embodiments, a payload (e.g., a therapeutic payload) delivered to HSCs, HPCs or LT-HSCs by an LNP comprising an ionizable lipid as described herein is expressed at levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% greater than a payload comprising a baseline lipid (such as V003). In some embodiments, a payload (e.g., a therapeutic payload) delivered to HSCs, HSPCs or LT-HSCs by an LNP comprising an ionizable lipid as described herein is expressed at levels that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% greater than a payload comprising a baseline lipid (such as 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 HSCs (e.g., LT-HSCs) in a subject comprise a genome that have been modified or altered following administration of an LNP described herein, such as a targeted LNP encapsulating a therapeutic agent (e.g., a system for modifying, altering or editing a genome). In some embodiments, from about 5% to about 50%, from about 5% to about 20%, from about 10% to about 30%, from about 20% to about 50%, from about 20% to about 40%, or from about 20% to about 30% of HSCs in a subject comprise a genome that has been modified or altered following administration of an LNP described herein, such as a targeted LNP.

[0015] The high level of in vivo gene editing following administration of the targeted LNPs of the disclosure enables effective treatment of particular diseases such as b- hemoglobinopathies, primary immunodeficiencies, and metabolic disorders. In some embodiments, the targeted LNPs of the disclosure can be used to treat Sickle Cell Disease. In other embodiments, the targeted LNPs of the disclosure can be used to treat beta thalassemia.

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

[0017] In some embodiments, an LNP of the present disclosure, such as a targeted LNP, comprises the ionizable lipid V003, depicted below.Lipid V003

[0018] In some embodiments, an LNP of the present disclosure, such as a targeted LNP, comprises an ionizable lipid in Table 1. In some embodiments, the LNP (e.g., a targeted LNP) comprises an ionizable lipid in Table 2. In some embodiments, the LNP (e.g., a targeted LNP) comprises an ionizable lipid in Table 3. The LNPs and targeted LNPs detailed herein in some embodiments comprise any one or more ionizable lipid as detailed in Tables 1, 2, and 3. In some embodiments, the LNP and targeted LNPs detailed herein comprises 2 or more ionizable lipids, such as any two or more of V003 and the ionizable lipids in Tables 1, 2 and 3. In some embodiments, the LNP and targeted LNPs as detailed herein comprise only one ionizable lipid selected from the group consisting of ionizable lipids in Tables 1, 2 and 3. In some cases, an LNP containing an ionizable lipid described herein exhibits higher levels of transduction in HSCs and / or higher expression of a payload protein in HSCs relative to LNPs that contain V003 as the ionizable lipid. In some cases, some of the ionizable lipids described herein provide higher levels of transduction and / or protein expression relative to V003 when used as a component of targeted LNP for in vivo delivery to HSCs. In some embodiments, the ionizable lipid has one of the structures depicted below:Lipid 232

[0019] In some embodiments, the LNPs of the present disclosure, e.g., targeted LNPs (conjugates), comprise one or more pegylated lipid molecules. In some embodiments, thetargeting moiety (e.g., antibody, Fab fragment or scFv) of a targeted LNP is associated with or chemically bonded to at least one of the pegylated lipid molecules. In some embodiments, the targeted LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the targeting moiety. In some embodiments, the PEG spacer between the lipid and the targeting 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 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the targeting moiety is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the targeting moiety is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300). In some embodiments, the lipid portion of the pegylated lipid bonded to the targeting 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 the disclosure can imbue different properties to the LNPS, including pharmacokinetics, distribution and endosomal release. Surprisingly, it has been discovered that LNPs comprising different pegylated lipids not covalently bound to a targeting moiety could show different toxicological properties when all other components of the LNPs (e.g., ionizable lipid) and relative proportions are the same. In particular, it has been discovered that LNPs comprising pegylated lipids with PEG lipid anchors having 16 carbon atoms (i.e., C16 PEG lipid anchors) or 18 carbon atoms (i.e., C18 PEG lipid anchors) could reduce or eliminate the toxicity associated with identical LNPs other than the pegylated lipid portion. In some embodiments, administration (in vivo or ex vivo) of the LNPS comprising C16 PEG lipid anchors or C18 PEG lipid anchors does not result in substantial liver toxicity. In some embodiments, administration of the targeted LNPS results in no or minimal increases in serum levels of the secreted liver enzyme alanine aminotransferase (ALT).

[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 of 16 carbons long). 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, the targeted LNPs comprising the C16pegylated lipids show significantly reduced liver uptake than otherwise identical LNPS comprising 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 of 18 carbons long). 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, an LNP (e.g., targeted LNP) of the present disclosure, e.g., a 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 targeting moiety (e.g., antibody, Fab fragment or scFv) is associated with or chemically bonded to at least one of the non-pegylated lipids. In some embodiments, the non-pegylated lipid (helper lipid) is selected from POPC, DOPC, DOPE, and DSPC. In some embodiments, the non-pegylated lipid (helper lipid) is a sphingolipid. In some such embodiments, the non- pegylated lipid is a 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 the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 22% to about 28%. In some embodiments, the mol% of the helper lipid (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 set forth in the examples below, in vivo delivery of certain payloads following administration of the disclosed LNPs with these percentages of helper lipids provides enhanced transduction and / or expression of the payloads relative to LNPs with smaller or larger quantities of helper lipid. It will be understood that mol% of the helper lipid as used herein refers to the mol% of the total lipid component of the LNP, which does not include the therapeutic agent (i.e., payload) or the targeting moiety.

[0025] In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP (i.e., not including the targetingmoiety or therapeutic agent) is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the lipid component of the targeted LNP is from about 22% to about 28%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the lipid component of 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 set forth in the examples below, in vivo delivery of certain payloads following administration of the disclosed LNPs with these percentages of helper lipids provides enhanced transduction and / or expression of the payloads relative to LNPs with smaller or larger quantities of helper lipid.

[0026] In some embodiments, the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, and the helper lipid is present at about 18 mol% to about 32 mol% of the lipid component; (ii) one or two targeting moieties conjugated to the LNP, wherein the one or two targeting 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; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent 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 between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 1:1 to about 7:1. In some embodiments, the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 4:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 3:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) isfrom about 1:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 2:1. In some embodiments, the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC) is from about 1.5:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 2.5:1.

[0028] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 092 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 092 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 093 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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., targeted LNP, comprises Lipid 153 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP(e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising at least one C14 alkylchain (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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP (e.g., the lipid component of the LNP) is from 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 comprising 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 comprising 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 foregoing embodiments, the LNP or conjugate can comprise one or more cholesterol molecules. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 6:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 3:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylatedhelper lipid (e.g., DSPC or sphingomyelin) is from about 1.5:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 0.5:1. In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:2 to about 0.8:1.

[0042] In some embodiments, the LNP comprises a targeting moiety that targets a receptor on the surface of the HSC selected from CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184, and CD338. In some embodiments, the LNP comprises a targeting moiety that targets CD117. In some embodiments, the LNP comprises a targeting moiety that targets CD33. In some embodiments, the LNP comprises a targeting moiety that targets CD34. In some embodiments, the LNP comprises a targeting moiety that targets CD38. In some embodiments, the LNP comprises a targeting moiety that targets CD43. In some embodiments, the LNP comprises a targeting moiety that targets CD46. In some embodiments, the LNP comprises a targeting moiety that targets CD49d. In some embodiments, the LNP comprises a targeting moiety that targets CD50. In some embodiments, the LNP comprises a targeting moiety that targets CD59. In some embodiments, the LNP comprises a targeting moiety that targets CD71. In some embodiments, the LNP comprises a targeting moiety that targets CD105. In some embodiments, the LNP comprises a targeting moiety that targets CD1117. In some embodiments, the LNP comprises a targeting moiety that targets CD123. In some embodiments, the LNP comprises a targeting moiety that targets CD164. In some embodiments, the LNP comprises a targeting moiety that targets CD184. In some embodiments, the LNP comprises a targeting moiety that targets CD338.

[0043] In some embodiments, the LNP comprises a targeting moiety that comprises a sequence present in any of Tables S1 to S14. In some embodiments, the targeting moiety is encoded by a sequence present in any of Tables S1 to S14. In some embodiments, the LNP comprises two targeting moieties, wherein each targeting moiety comprises a sequence present in any of Tables S1 to S14. In some embodiments, the two targeting moieties are each encoded by a sequence present in any of Tables S1 to S14.

[0044] In some embodiments, the CD34 targeting moiety comprises a Class II antibody or fragment thereof. In some embodiments, the CD34 targeting moiety comprises a Class III antibody or fragment thereof. In some embodiments the CD34 targeting moieity is insensitive or resistance to certain proteolytic enzymes. In some embodiments, the CD34targeting moiety is insensitive or resistant to cleavage by neuraminidase. In some embodiments, the CD34 targeting moiety is insensitive or resistant to cleavage by chymopapain. In some embodiments, the CD34 targeting moiety is insensitive or resistant to cleavage by glycoprotease. In some embodiments, the CD34 targeting moiety is insensitive or resistant to cleavage by neuraminidase, chymopapain, and glycoprotease. In some embodiments, the CD34 targeting moiety is insensitive or resistant to cleavage by neuraminidase, but sensitive to cleavage by chymopapain and glycoprotease.

[0045] In some embodiments, the conjugates comprising a Fab fragment are prepared by a method comprising: (i) contacting a Fab fragment (e.g., a Fab fragment that targets 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 and an interchain disulfide bond linking the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and (ii) contacting the product of step (i) with a precursor LNP comprising 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 aspects, the disclosure provides a conjugate comprising an LNP and a Fab fragment, wherein the LNP is covalently bonded to either or both of 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 light chain of the Fab fragment. In some embodiments, the Fab fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the Fab fragment and 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 bound 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 bound light chain on the surface of the LNP. In some of the foregoing embodiments, the Fab fragment is linked to the LNP through a thiosuccinimide moiety. In other of the foregoing embodiments, the Fab fragment is linked to the LNP through adithiomalemide moiety. In still other of the foregoing embodiments, the Fab fragment is linked to the LNP through 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, the targeted LNPs (i.e., conjugates) are formed via a Click reaction between a first Click handle on the targeting moiety (e.g., antibody, Fab fragment, or scFv) and a second Click handle on the LNP, thereby generating a Click product. The second Click handle can be on one or more of the lipids comprising the LNP. In some embodiments, the second Click handle is covalently bonded to a pegylated lipid comprising 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 a Huisgen 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne. In some embodiments, the first orsecond Click handle comprises a cyclic derivative of the alkynyl group. In some embodiments, the cyclic derivative of the alkynyl group is selected from dibenzocyclooctyne. Cyclooctyne, and difluorinated cyclooctyne. In some embodiments, the click chemistry involves strain promoted cycloaddition of azides. In some embodiments, the click chemistry is based upon reaction of strained alkenes. 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, hence producing an aza-ylide. In some embodiments, the Click product can be formed from an inverse electron demand Diels-Alder reaction between a trans-cyclooctene (TCO) moiety on the first or second Click handle and a tetrazine ring on the 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 rung is methyltetrazine. In some embodiments, the tetrazine ring is a 6-methyl substituted tetrazine.

[0052] In some embodiments, the Click product is formed by conjugating an LNP to a targeting moiety that has been modified with an enzyme recognition sequence. For instance, an antibody, Fab fragment or single chain variable fragment (scFv), can be covalently linked to a first Click handle through a linker comprising an enzyme recognition sequence and the LNP is covalently linked to a second Click handle on the LNP, thereby generating a Click product. In some embodiments, the enzyme recognition sequence is a sortase recognition motif or a LplA acceptor peptide. In some embodiments, the antibody Fab fragment or scFv is directly bonded to the enzyme recognition sequence. In some embodiments, the antibody Fab fragment or scFv is bonded to the enzyme recognition sequence via one or more amino acid residues. Particular amino acid residues added that can be covalently attached to the C- terminus of the 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, wherein u is 1- 10 and v is 1-10.

[0053] In some embodiments, a lipid nanoparticle (LNP) for delivery of a therapeutic agent to HSCs, as described herein, comprises: an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or any or the lipids in Table 1, Table 2 and Table 3;a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties binds 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.

[0054] In some embodiments, the plurality of targeting moieties, can be added to the surface of the LNP by methods described in Section II of the Detailed Description. A targeted LNP in some embodiments comprises two or more different targeting moieties, each of which may bind to the same or a different target. A targeted LNP having two different targeting moieties which bind to the same or different targets may be referred to an a “dual binder” or “dual binding” targeted LNP. For instance, in some embodiments, each of the targeting moieties is conjugated to the lipid nanoparticle through a linker, wherein each linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP. In some such embodiments, the targeting moieties are antibodies or antigen binding fragments thereof. In other such embodiments, the targeting moieties are scFvs. In some embodiments, the targeting moieties are Fab fragments.

[0055] In some embodiments, the plurality of targeting moieties binds 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, the plurality of targeting moieties binds to CD117 and CD34. In some embodiments, the plurality of targeting moieties binds to CD117 and CD71. In some embodiments, the plurality of targeting moieties binds to CD34 and CD71.

[0056] In some embodiments, a lipid nanoparticle (LNP) for delivery of a therapeutic agent to HSCs, as described herein, comprises: a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties binds 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.

[0057] In some aspects, the disclosure provides a targeted lipid nanoparticle (LNP) for in vivo delivery of a therapeutic agent to HSCs (e.g., LT-HSCs), wherein the targeted LNP comprises: an ionizable lipid; a helper lipid; two different targeting moieties conjugated to the LNP, wherein each of the two different targeting moieties binds to a target on the surface of the HSCs (e.g., LT-HSCs); 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 genemodifying system), wherein the one or more nucleic acids (e.g., two or more nucleic acids) are RNA molecules.

[0058] In some embodiments, the dual binders can be added to the surface of the LNP by methods described in Section II of the Detailed Description. For instance, in some embodiments, each of the targeting moieties is conjugated to the lipid nanoparticle through a linker, wherein each linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP. In some such embodiments, the targeting moieties are antibodies or antigen binding fragments thereof. In other such embodiments, the targeting moieties are scFvs. In some embodiments, the targeting moieties are Fab fragments.

[0059] In some embodiments, one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA (e.g., a gene modifying system) is an mRNA encoding a gene modifying polypeptide, as described herein. In some embodiments, one of the components of a system for modifying or altering genomic DNA (e.g., a gene modifying system) is an mRNA encoding a 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 a component of a system for modifying or altering genomic DNA (e.g., a gene modifying system) is a guide RNA (gRNA). In some embodiments, one of the RNA molecules encoding a component of a system for modifying or altering genomic DNA (e.g., a gene modifying system) is a template RNA encoding a heterologous nucleic acid for use with a gene modifying polypeptide to insert the 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 modifying 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 modifying system) do not elicit a double- stranded break in the genomic DNA. In such embodiments, the system for modifying or altering genomic DNA elicits a single-stranded break in the genomic DNA. In some embodiments, a system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous sequence into the genomic DNA.

[0061] In some embodiments, the targets on the surface of the HSC are 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 targeting moieties binds to CD117. In some embodiments, at least one of the targeting moieties binds to CD34. In some embodiments, at least one of the targeting moieties binds to CD71. In some embodiments, at least one of the targeting moieties binds to CD164. In some embodiments, the targeted LNP is a dual binding LNP. In some embodiments, the two targeting moieties bind to CD117 and CD34. In some embodiments, the two targeting moieties bind 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 Lipid 92, Lipid 93, Lipid 95, Lipid 96, Lipid 97, Lipid 97, Lipid 110, Lipid 132, Lipid 133, Lipid 134, Lipid 140, Lipid 141, Lipid 143, Lipid 144, Lipid 147, Lipid148, Lipid 149, Lipid 150, Lipid 151, Lipid 152, Lipid 153, Lipid 154, Lipid 155, Lipid 162, Lipid 163, Lipid 169, Lipid 170, Lipid 173, Lipid 174, Lipid 175, Lipid 176, Lipid 178, Lipid 179, Lipid 183, Lipid 184, and Lipid 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 mo1% of the DSPC or sphingomyelin in the targeted LNP is from 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 is DPPE-PEG2000 or DPG- PEG2000.

[0065] In some embodiments, the targeted delivery of a system for modifying or altering genomic DNA (e.g., a gene modifying system) with an LNP comprising two or more HSC specific binders conjugated to the LNP results in synergistic enhancement of gene editing or modification of the target HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 5% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 10% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 15% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least20% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 25% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying at least 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 10% to about 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 20% to about 30% of the genomes of the HSCs or LT-HSCs. In some embodiments, the LNPs comprising dual binders are capable of modifying from about 10% to about 20% of the genomes of the HSCs or LT-HSCs. In any of the foregoing embodiments, the system for modifying or altering genomic DNA elicits a single-stranded break in the genomic DNA. In some such embodiments, a system for modifying or altering genomic DNA includes a gene modifying polypeptide and a template RNA for a nucleic acid sequence to be inserted at a specific location of the genomic DNA, hence resulting in modification of the genomic DNA. In some embodiments, the gene modifying system comprises a Cas9 nickase fused to a reverse transcriptase (RT) domain. In some embodiments, a system for modifying or altering genomic DNA induces target-primed reverse transcription (TPRT) to insert a heterologous nucleic acid sequence into a DNA sequence, e.g., genomic DNA.

[0066] In some aspects, the disclosure provides a method of treating sickle cell disease in a subject (e.g., a human patient in need thereof), said method comprising administering to the subject a targeted lipid nanoparticle (LNP) comprising: an ionizable lipid; a helper lipid; two different targeting moieties conjugated to the LNP, wherein each of the two different targeting moieties binds to a target on the surface of the HSCs (e.g., LT-HSCs); 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 modifying system), wherein the two or more nucleic acids are RNA molecules.

[0067] In some embodiments, the nucleic acid components do not introduce double stranded breaks into the genomic DNA. In some embodiments, greater than 5% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, greater than 10% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, greater than 15% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments,greater than 20% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, greater than 30% of the HSCs or LT-HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 5% to about 15% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 10% to about 30% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 10% to about 20% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 15% to about 20% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP. In some embodiments, from about 15% to about 25% of the HSCs or LT- HSCs of the patient with sickle cell disease are altered following administration of the targeted LNP.

[0068] In another aspect, the disclosure provides a lipid nanoparticle (LNP), comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, and the helper lipid is present at about 18 mol% to about 32 mol% of the lipid component; (ii) means for binding 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; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises two or more RNA molecules (e.g., two or three RNA molecules).

[0069] In one variations, a means for binding a cell surface receptor comprising a targeting moiety on the surface of the LNP that binds to one or more of a cell receptor 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, following in vivo administration of the targeted LNPs to the patient with sickle cell disease, the HSCs or LT-HSCs maintain their hematopoietic potential and function to support multilineage development. In some embodiments, the gene modifying system corrects the E6V HBB mutation in the HSCs and LT-HSCs of the patient.BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG.1 shows an exemplary conjugate comprising a Fab fragment and a lipid nanoparticle (LNP).

[0072] FIG.2A shows assembly of an LNP encapsulating a therapeutic payload with a functional group (e.g., maleimide, 2,3-dibromomaleimide, sortase tag, etc.) to be reacted with an antibody or antigen-binding fragment thereof. FIG.2B shows assembly of an LNP encapsulating a therapeutic payload using a post-insertion technique.

[0073] FIG.3 shows an exemplary schematic of the reduction of an interchain disulfide bond in a Fab fragment. After reduction, each cysteine residue is available to react with a thiol-reactive group conjugated to the surface of the LNP.

[0074] FIG.4 shows an exemplary schematic of conjugate formation. In FIG.4, a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues (step (i)). In step (ii), the Fab fragment is contacted with an LNP comprising a plurality of thiol-reactive groups (e.g., a plurality of maleimide or DBM groups) conjugated to the surface of the LNP, whereby a thiol-reactive group of the plurality reacts with at least one of the two free cysteine residues of the Fab fragment.

[0075] FIG.5 shows an exemplary schematic of conjugate formation. In FIG.5, a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues (step (i)). In step (ii), the Fab fragment is contacted with an LNP comprising a plurality of maleimide groups conjugated to the surface of the LNP, whereby a maleimide group of the plurality reacts with one of the two free cysteine residues of the Fab fragment.

[0076] FIG.6 shows an exemplary schematic of conjugate formation using DBM as a bridging agent.

[0077] FIG.7 shows an exemplary schematic of conjugate formation. In FIG.7, a first Fab fragment (Fab1) and a second Fab fragment (Fab2) are each contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide between the heavy and light chain of each Fab. After step (i), each Fab comprises two free cysteine residues with one free cysteine residue on the heavy chain and one free cysteine residue on the light chain. In step (ii), the first Fab fragment and the second Fab fragment are contacted with an LNPcomprising a plurality of thiol-reactive groups (e.g., maleimide or DBM) conjugated to the surface of the LNP, whereby a thiol-reactive group of the plurality reacts with at least one of the two free cysteine residues of the first Fab Fragment and another thiol-reactive group of the plurality reacts with at least one of the two free cysteine residues of the second Fab Fragment.

[0078] FIG.8 shows some examples of a pegylated lipid bonded to a maleimide moiety.

[0079] FIG.9 shows some examples of non-pegylated lipid bonded to a maleimide moiety.

[0080] FIG.10 shows some examples of ionizable lipid bonded to a maleimide moiety.

[0081] FIG.11 shows some examples of sterols bonded to a maleimide moiety.

[0082] FIG.12 shows an exemplary schematic of conjugate formation. In FIG.12, a Fab fragment comprises a sortase tag. The Fab fragment is contacted with an LNP in the presence of sortase, wherein the LNP comprises a plurality of polyglycine molecules conjugated to the surface of the LNP, whereby the Fab fragment is conjugated to the surface of the LNP through a sortase mediated ligation of the sortase tag and a polyglycine molecule of the plurality.

[0083] FIG.13 shows the formation of an LNP conjugated to the C-terminus of a Fab fragment through a sortase mediated ligation followed by a Click reaction.

[0084] FIG.14 shows the formation of an LNP conjugated to the C-terminus of a Fab fragment through a lipoic acid ligase mediated ligation followed by a Click reaction.

[0085] FIG.15 shows an enzymatic approach of site-specifically introducing a Tz ring onto a sugar moiety of an antibody.

[0086] FIG.16 shows an example of a light-induced crosslinking approach of site- specifically introducing a Tz ring onto an antibody.

[0087] FIG.17 shows an example of using a non-natural amino acid to site-specifically introduce a Tz ring onto an antibody or Fab fragment.

[0088] FIG.18 shows the results of transfection experiments in human CD34+ cells using LNPs conjugated to an anti-CD117 Fab fragment through sortase-mediated ligation. The transfection results are compared to unmodified (control) LNPs and LNPs modified with an anti-CD117 antibody produced using a random modification method. FIG.18A shows mRNA transfection as quantified by flow cytometry in terms of % green fluorescent protein (%GFP+) cells. FIG.18B shows transfection in terms of Median Fluorescence Intensity (MFI).

[0089] FIG.19A shows a schematic example for producing a site-specific conjugate through the reaction of an anti-CD117 Fab fragment with site-specific sortase recognition motif and an LNP. In the schematic, the squiggly lines represent PEG bonded to a lipid of the LNP. FIG.19B shows a schematic of producing a conjugate through the reaction of a Fab fragment that has been modified using a random approach (NHS modification) and an LNP.

[0090] FIG.20 shows results of transfection into Kasumi-1 cells (CD117+ cell line) using LNPs conjugated to an anti-CD117 Fab fragment produced by site-specific (sortase- mediated) or by random modification. FIG.20A shows mRNA transfection as quantified by flow cytometry in terms of % green fluorescent protein (%GFP+) cells. FIG.20B shows transfection in terms of Median Fluorescence Intensity (MFI).

[0091] FIG.21 shows the results of in vivo experiments evaluating transduction of LNPs conjugated with one of three tested Fab fragments specific for anti-CD117 (Fab 1, Fab 5 or Fab 6) in HSCs and early progenitor cells (HSCs + early progenitors) (also referred to as HSPCs) compared to transduction using a base LNP lacking a conjugated anti-CD117 Fab fragment. FIG.21A shows that up to 58% of HSCs + early progenitors receive the LNPs with the sortase-modified Fab fragments. FIG.21B shows that up to 62% of long term (LT)- HSCs receive the LNPs with sortase-modified Fab fragments. LNPs with sortase-modified Fab fragments exhibited significantly higher in vivo transfection levels in both HSCs + early progenitors and in LT-HSCs relative to base LNPs that did not have anti-CD117 Fab fragments. Moreover, as shown in FIG.21C, there is a good correlation between in vitro transduction of primary HSPCs and in vivo targeting in HSPCs. On the other hand, as also shown in FIG.21C, there is a separation between nonspecific IgG and site-specifically conjugated (sortase method) Fab LNPs.

[0092] FIG.22 shows the effect of increasing the mol% of helper lipid in the targeted LNPs conjugated with Fab fragments specific for CD117 on the transduction and expression of an mRNA encoding GFP in HSCs and early progenitor cells (HSCs + early progenitors) (also referred to as HSPCs). Specifically, FIG.22 shows the results of in vivo experiments comparing the transduction of targeted LNPs containing two different amounts of helper lipids (LNP A and LNP B). LNPs A and B comprised 8% helper lipid (DSPC) and 22% helper lipid (DSPC), respectively. More than 80% of HSCs + early progenitors were transduced with LNP B.

[0093] FIG.23 compares the levels of transduction and MFI in a humanized mouse model administered LNPs comprising helper lipids at different percentages. FIGs.23A and 23B demonstrate that targeted LNPs comprising ionizable lipid and 8% DSPC providesimilar levels of GFP transduction and expression levels in HSPCs when compared to targeted LNPs comprising ionizable lipid and 8% (molar) sphingomyelin. Similar to experiments with DSPC as the helper lipid, increasing the sphingomyelin content to 22% (molar) results in substantially increased transduction and expression levels of the reporter protein. FIGs.23C and 23D show results for transduction in LT-HSCs.

[0094] FIG.24 shows results of FACS based sorting to identify the percentage of HSPCs that were positive for GFP protein expression following administration of a conjugate of the disclosure to Cynomolgus macaques 3-5 years of age.

[0095] FIGs.25A-25F show the synthesis of Lipid 092.

[0096] FIGs.26A-26C show the synthesis of Lipid 093.

[0097] FIGs.27A-27C show the synthesis of Lipid 162.

[0098] FIGs.28A-28C show the synthesis of Lipid 153.

[0099] FIGs.29A-29F show the synthesis of Lipid 154.

[0100] FIGs.30A-30E show the synthesis of Lipid 155.

[0101] FIGs.31A-31F show the synthesis of Lipid 176.

[0102] FIG.32A shows enhanced transduction of GFP in LSK cells using LNPs containing ionizable Lipid 092 or ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in an in vivo mouse model. FIG.32B shows enhanced expression levels of GFP in LSK cells using LNPs containing ionizable Lipid 092 or ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in an in vivo mouse model. FIGs.32C and 32D shows enhanced transduction and / or expression levels of GFP in LSK cells using LNPs containing ionizable Lipid 154 or ionizable Lipid 155 relative to LNPs containing ionizable Lipid V003 in an in vivo mouse model. FIGs.30E and 30F show enhanced transduction of GFP and / or enhanced GFP expression in in LT-HSCs using LNPs containing ionizable Lipid 154 or ionizable Lipid 155 relative to an LNPs containing ionizable Lipid V003.

[0103] FIG.33A shows enhanced transduction of GFP in LT-HSCs using LNPs containing ionizable Lipid 092 or ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model. FIG.33B shows enhanced expression of GFP in LT-HSCs using LNPs containing ionizable Lipid 092 or ionizable Lipid 093 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model.

[0104] FIG.34A and FIG.34C show enhanced transduction of GFP in HSPCs or LT- HSPCs, respectively, using LNPs containing ionizable Lipid 153, Lipid 154, or Lipid 155 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model. FIG. 34B and FIG.34D show enhanced expression of GFP in HSPCs or LT-HSCs, respectively,using LNPs containing ionizable Lipid 153 or ionizable Lipid 154 relative to LNPs containing ionizable Lipid V003 in a humanized in vivo mouse model.

[0105] FIG.35 shows results of screening of targeted LNP (tLNP) in humanized NBSGW mice with increased quantities of helper lipid. FIGs.35A and 35B show results for transduction and expression in HSPCs, respectively. FIGs.35C and 35D show results for transduction and expression in LT-HSCs, respectively.

[0106] FIG.36 shows screening results of targeted LNP (tLNP) in nonhuman primates.

[0107] FIG.37A and 37B show in vitro screening results (% GFP+ cells and MFI, respectively) for targeting moieties capable of enhancing delivery of a targeted LNP for HSPCs, normalized against cells transfected with a base LNP lacking targeting moieties.

[0108] FIG.38 shows that a site-specific conjugation process, as described herein, can efficiently conjugate two targeting moieties (anti-CD117 Fab and anti-CD45 Fab, in this case) to the surface of LNPs at specific ratios.

[0109] FIG.39 show that tLNPs can effectively deliver a gene editing payload to human HSPCs in an animal model, leading to in vivo gene editing in the HSPCs. FIG.39A and FIG.39B show that administering anti-CD34 tLNPs and anti-CD117 tLNPs resulted in detectable levels of B2M indels in HSPCs and LT-HSCs,but administering dual tLNPs conjugated to the anti-CD117 and anti-CD34 targeting moieties resulted in synergistically higher levels of gene editing in HSPCs. FIG.39C shows that dual tLNPs conjugated to anti- CD117 and anti-CD71 targeting moieties, or antiCD34 and anti-CD71 targeting moieties, can substantially increase delivery of the gene editing payload to HSPCs and LT-HSCs, thereby resulting in an increase in B2M editing. FIG.39D shows that doubling the amount of a single type of targeting moiety (anti-CD117) conjugated to the surface of tLNPs did not increase delivery of a payload to HSPCs or LT-HSCs.

[0110] FIGs.40A and 40B show that the administered anti-CD117 tLNPs can deliver payloads suitable for gene editing to human HSPCs in an animal model, leading to in vivo gene editing in HSPCs. FIG.40A shows that tLNPs formulated with Lipid093 and the wtCas9-RT mRNA and B2M gRNA payload resulted in 8.1% indels in B2M in HSPCs. By contrast, tLNPs formulated with Lipid154 and the same payload resulted in 15.7% indels in B2M in HSPCs, representing a nearly 2-fold increase in delivery over Lipid093 tLNPs. FIG. 40B shows that the tLNPs formulated with Lipid154 and the nCas9-RT and B2M template RNA payload generated higher TPRT-mediated knockout of B2M in HPSCs relative to the tLNPs formulated with Lipid093 and the same payload (mean 5.2% with Lipid154 and mean 1.5% with Lipid93).

[0111] FIG.41A shows that administration of anti-CD117 tLNPs formulated with Lipid093 and 2 mol % C16-PEG to cynomolgus macaques (n=2) resulted in 72% GFP+ HSPCs and 33% GFP+ LT-HSCs. In comparison, administration of the anti-CD117 tLNPs, formulated with Lipid154 and 2 mol % C16-PEG in the macaques (n=2) resulted in higher percentages of GFP+ HSPCs and LT-HSCs (96% GFP+ and 91% GFP+ cells, respectively). FIG.41B shows that the expression of GFP mRNA was higher in HSPCs and nearly four- fold higher in LT-HSCs in those NHPs dosed with the Lipid154 tLNPs compared to those dosed with the Lipid093 tLNPs.

[0112] FIG.42 shows that administration of the anti-CD117 / anti-CD34 dual tLNPs formulated with Lipid093 and C14-PEG led to an average of 25% B2M indels in human HSPCs in vivo. The administration of anti-CD117 / anti-CD34 dual tLNPs formulated with Lipid154 and C16-PEG generated higher B2M indels (over 45%) relative to the Lipid093 C16-PEG tLNPs.

[0113] FIG.43 shows that anti-CD117 / anti-CD34 dual-targeted tLNPs, formulated with Lipid154 and C16-PEG led to a mean 11.8% perfect gene modification in HSPCs and mean 14.3 perfect gene modification in LT-HSCs in vivo.

[0114] FIG.44A shows that administration of dual tLNPs formulated with Lipid154 to cynomolgus macaques (n=2) resulted in an average of over 90% GFP+ HSPCs and LT-HSCs for the anti-CD117 / anti-CD34 dual tLNPs, and an average of over 80% GFP+ HSPCs and over 70% GFP+ LT-HSCs for the anti-CD117 / anti-CD71 dual tLNPs. FIG.44B shows that HSPCs and LT-HSCs in the NHPs expressed GFP (MFI) following administration of each of the dual tLNPs.

[0115] FIG.45 shows that the dual targeted LNPS (tLNPs) formulated with the anti- CD117 and anti-CD34 targeting can effectively deliver a gene editing system to human HSCs and precursors in vivo, leading to B2M knockout in appreciable numbers of the cells.^

[0116] FIGs.46A and 46B show that targeted LNPs (tLNPs) and dual tLNPs could deliver the B2M gene editing system to human HSPCs (FIG.46A) and LT-HSCs (FIG.46B) in vivo to generate B2M indels.DETAILED DESCRIPTION I. DEFINITIONS

[0117] Antibody: The term “antibody” herein encompasses various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0118] Antibody fragment: The term “antibody fragment” or an “antigen binding fragment” as used herein is a portion of a full-length antibody that binds 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.

[0119] Antigen binding domain: The term “antigen binding domain” as used herein refers to that portion of antibody or a chimeric antigen receptor which binds an antigen. In some embodiments, an antigen binding domain binds to a cell surface antigen of a cell. In some embodiments an antigen binding domain binds an antigen characteristic of a cancer, e.g., a tumor associated antigen in a neoplastic cell. In some embodiments, an antigen binding domain binds an antigen characteristic of an infectious disease, e.g. a virus associated antigen in a virus infected cell. In some embodiments, an antigen binding domain binds an antigen characteristic of a cell targeted by a subject’s immune system in an autoimmune disease, e.g., a 0-antigen. In some embodiments, an antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, an antigen binding domain is or comprises an scFv or Fab.

[0120] Expression cassette: The term “expression cassette,” as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention.

[0121] Fab fragment: As used herein, a “Fab fragment” refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain.

[0122] gRNA spacer: A “gRNA spacer”, as used herein, refers to a portion of a nucleic acid that has complementarity to a target nucleic acid and can, together with a gRNA scaffold, target a Cas protein to the target nucleic acid.

[0123] gRNA scaffold: A “gRNA scaffold”, as used herein, refers to a portion of a nucleic acid that can bind a Cas protein and can, together with a gRNA spacer, target the Casprotein to the target nucleic acid. In some embodiments, the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.

[0124] Gene modifying polypeptide: A “gene modifying polypeptide”, as used herein, 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 to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell). In some embodiments, the gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery. In some embodiments, the gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate: 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) integration of the at least a portion of the template nucleic acid into the target DNA. Gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to the naturally occurring sequence. Gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains recited above are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain. Exemplary gene modifying polypeptides, and systems comprising them and methods of using them, that can be used in the methods provided herein are described, e.g., in PCT / US2021 / 020948, which is incorporated herein by reference with respect to gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene. A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide and a template nucleic acid.

[0125] Gene modifying system: A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide, or a nucleic acid (e.g., an mRNA) encoding the gene modifying polypeptide, and a template nucleic acid.

[0126] Domain: The term “domain” as used herein refers to a structure of a biomolecule that contributes to a specified function of the biomolecule. A domain may comprise acontiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non- contiguous sequences) of a biomolecule. Examples of protein domains include, but are not limited to, an endonuclease domain, a DNA binding domain, a reverse transcriptase domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain.

[0127] Exogenous: As used herein, the term “exogenous,” when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell, or organism by the hand of man. For example, a nucleic acid that is as added into an existing genome, cell, tissue, or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject.

[0128] First Strand and Second Strand: The terms “first strand” and “second strand”, as used herein, describe the individual DNA strands of target DNA, distinguish the two DNA strands based upon which strand the reverse transcriptase domain initiates polymerization, e.g., based upon where target primed synthesis initiates. The first strand refers to the strand of the target DNA upon which the reverse transcriptase domain initiates.

[0129] Heterologous: The term “heterologous”, as used herein to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to: (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous regulatory sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule is normally expressed in nature. In another example, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules maynot be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).

[0130] Mutation or Mutated: The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted, or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art. In some embodiments a mutation occurs naturally. In some embodiments a desired mutation can be produced by a system described herein.

[0131] Mutation Region: The term “mutation region,” as used herein, refers to a region in a template RNA having one or more sequence difference relative to the corresponding sequence in a target nucleic acid. The sequence difference may comprise, for example, a substitution, insertion, frameshift, or deletion.

[0132] Nucleic acid molecule: “Nucleic acid molecule” refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein. The nucleic acid molecule can be double-stranded or single-stranded, circular, or linear. If single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:,” or “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complimentary to SEQ ID NO:1. The choice between the two is dictated by the context in which SEQ ID NO:1 is used. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target. Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, inter-nucleotide modifications such as unchargedlinkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). Also included are chemically modified bases (see, for example, Table 13), backbones (see, for example 14, Table 16), and modified caps. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule, e.g., peptide nucleic acids (PNAs). Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids (LNAs). In various embodiments, the nucleic acids are in operative association 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 ITRs) and tandem repeats, inverted repeats / direct repeats, homology regions (segments with various degrees of homology to a 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 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 particular versions of these, such as doggybone DNA (dbDNA), and closed-ended DNA (ceDNA).

[0133] Primer Binding Sequence: The term “primer binding site sequence” or “PBS sequence,” as used herein, refers to a portion of a template RNA capable of binding to a region comprised in a target nucleic acid sequence. In some instances, a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. In some embodiments the primer region comprises at least 5, 6, 7, 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. Without wishing to be bound by theory, in some embodiments when a template RNA comprises a PBS sequence and a heterologous object sequence, the PBS sequence binds to a region comprised in a target nucleic acid sequence, allowing a reverse transcriptase domain to use that region as a primer for reverse transcription, and to use the heterologous object sequence as a template for reverse transcription.

[0134] It is understood that aspects and embodiments described herein as “comprising” include “consisting of” and “consisting essentially of” embodiments.

[0135] Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0136] In some embodiments, reference to about a measurable parameter value encompasses variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value. II. CONJUGATES

[0137] In one aspect, the disclosure provides a lipid nanoparticle (LNP) comprising an ionizable lipid as described herein (e.g., in Table 1 or Table 2), wherein the LNP can deliver a payload, such as a therapeutic agent (e.g., a gene modifying system) to a hematopoietic stem cell (HSC), e.g., an LT-HSC, or a hematopoietic progenitor cell (HPC). In some embodiments, the LNP can deliver a payload, such as a therapeutic agent (e.g., a gene modifying system), to mixed population of hematopoietic stem cells and progenitor cells (HSPCs).

[0138] In some aspects, the disclosure provides a conjugate comprising an LNP and one or more targeting moieties that binds 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 preferentially target and bind to a target cell over a non- target cell.

[0139] In some embodiments, the disclosure provides a conjugate comprising an LNP and one or more targeting moieties that binds to a target molecule, e.g., a target protein, such as a protein receptor, present on an HSC (e.g., a LT-HSC) or HPC, as described herein. In some embodiments, the one or more targeting moieties are conjugated to the surface of the LNP. In some embodiments, an LNP conjugate comprises both an ionizable lipid and a targeting moiety, as described herein. In another aspect, the disclosure provides a conjugate comprising an LNP and one or more of a first targeting moiety that binds to a first target molecule, a first target protein, such as a first protein receptor, present on an HSC (e.g., an LT-HSC) or HPC, and one or more of a second targeting moiety that binds to a second target molecule, e.g., a second target protein, such as a second protein receptor, present on an HSC (e.g., an LT-HSC) or HPC. In some embodiments, the one or more of the first and secondtargeting moieties are conjugated to the surface of the LNP. This section will describe specific conjugates of the disclosure and methods of conjugating targeting moieties to the LNP. Additional aspects of the LNP component of the conjugates will be described in Section III.

[0140] In one aspect, the disclosure provides targeted LNPs (conjugates) comprising a targeting moiety and a lipid nanoparticle (LNP) encapsulating a payload (e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or a gene modifying system), wherein the targeting moiety binds to a protein (e.g., protein receptor) on an HSC (e.g., LT-HSC) or HPC.

[0141] In some embodiments, the LNP comprises a targeting moiety that targets a receptor on the surface of the HSC selected from CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184, and CD338. In some embodiments, the targeting moiety on the LNP binds to CD33. In some embodiments, the targeting moiety on the LNP binds to CD34. In some embodiments, the targeting moiety on the LNP binds to CD38. In some embodiments, the targeting moiety on the LNP binds to CD43. In some embodiments, the targeting moiety on the LNP binds to CD46. In some embodiments, the targeting moiety on the LNP binds to CD49d. In some embodiments, the targeting moiety on the LNP binds to CD50. In some embodiments, the targeting moiety on the LNP binds to CD59. In some embodiments, the targeting moiety on the LNP binds to CD71. In some embodiments, the targeting moiety on the LNP binds to CD105. In some embodiments, the targeting moiety on the LNP binds to CD117. In some embodiments, the targeting moiety on the LNP binds to CD123. In some embodiments, the targeting moiety on the LNP binds to CD184. In some embodiments, the targeting moiety on the LNP binds to CD164. In some embodiments, the targeting moiety on the LNP binds to CD338.

[0142] In some embodiments, a lipid nanoparticle (LNP) for delivery of a therapeutic agent to HSCs, as described herein, comprises: an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from V003 or any or the lipids in Table 1, Table 2, and Table 3; a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties binds 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.

[0143] In some embodiments, the plurality of targeting moieties binds 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, the plurality of targetingmoieties binds to CD117 and CD34. In some embodiments, the plurality of targeting moieties binds to CD117 and CD71. In some embodiments, the plurality of targeting moieties binds to CD34 and CD71. In some embodiments, the plurality of targeting moieties binds to CD117 and CD71.

[0144] In some embodiments, two different targeting moieties can bind to the same receptor on the surface of the HSC. For instance, in some embodiments, both targeting moieties can bind to CD117. In other embodiments, both targeting moieties can bind to CD34. In other embodiments, both targeting moieties can bind to CD164.

[0145] In some embodiments, a lipid nanoparticle (LNP) for delivery of a therapeutic agent to HSCs, as described herein, comprises: a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties binds 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.

[0146] In some aspects, the disclosure provides a targeted lipid nanoparticle (LNP) for in vivo delivery of a therapeutic agent to HSCs (e.g., LT-HSCs), wherein the LNP comprises: i. an ionizable lipid; ii. a helper lipid; iii. two different targeting moieties conjugated to the LNP, wherein each of the two different targeting moieties binds to a target on the surface of the HSCs (e.g., LT-HSCs); and iv. 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 modifying system), wherein the one or more nucleic acids (e.g., two or more nucleic acids) are RNA molecules. Targeting Moieties

[0147] The targeting moiety can include a polypeptide that binds specifically to a target molecule, such as a target protein (e.g., a protein receptor), present on a target cell. In some embodiments, the target cell expresses the target molecule. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’)2, Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin. In some embodiments, the targeting moiety includes a T cell receptor (TCR). In other embodiments, the targeting moiety includes a ligand, a folate moiety, anantibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine.

[0148] In some embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. In some embodiments, the targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the targeting moiety is a bispecific or multi-specific antibody or fragment thereof. In some embodiments, the bispecific or multi-specific antibody or fragment thereof comprises two or more antigen binding domains, or fragments thereof, selected from CD33, CD34, CD38, CD43, CD59, CD105, CD123, CD164, CD338, CD71, CD117, CD50, CD49d, CD46, or CD184 (CXCR4) antibodies or fragments thereof, e.g., as provided in Tables S1-S14. In some embodiments, the targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0149] In some embodiments, the LNP comprises a targeting moiety that comprises a sequence present in any of Tables S1 to S14. In some embodiments, the targeting moiety is encoded by a sequence present in any of Tables S1 to S14. In some embodiments, the LNP comprises two targeting moieties, wherein each targeting moiety comprises a sequence present in any of Tables S1 to S14. In some embodiments, the two targeting moieties are each encoded by a sequence present in any of Tables S1 to S14. CD33

[0150] In some embodiments, the target molecule is CD33 (also known as Siglec-3, p67, or My9). In some embodiments, the target cell is CD33+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD33. Exemplary anti-CD33 antibodies or antigen-binding fragments thereof include Gemtuzumab (e.g., hP67.6, Pfizer, FDA-approved), Lintuzumab (e.g., hM195 or SGN-33, Seattle Genetics), Vadastuximab (e.g., SGN-CD33A, i.e., Seattle Genetics) PWS44 (e.g., Leica Biosystems), 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 anti-CD33 antibodies or antigen-binding fragments thereof disclosed in any of: US 9,212,225; US 10,556,951; US 10,711,062; US 11,466,082; US 11,560,428; US 11,666,672; US 11,174,313; US 11,136,390; US20200148767; US20210017277; US20230102344; US20230365676; WO2022060832; WO2021138407; WO2020227072; WO2019006280; WO2018218207; WO2019028283; WO2019178382; etc., each hereby incorporated by reference in its entirety.

[0151] 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:86-91 are shown in Table S1, with complementary determining regions (CDRs) marked in bold. Table S1: Exemplary CD33 Targeting Moiety Sequences

[0152] 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 variableregion comprising the 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 a CDR-H1 comprising an amino acid sequence DSNIH (SEQ ID NO:227), a CDR-H2 comprising an amino acid sequence YIYPYNGGTDYNQKFKN (SEQ ID NO:228), a CDR-H3 comprising an amino acid sequence GNPWLAY (SEQ ID NO:229), a CDR-L1 comprising an amino acid sequence RASESLDNYGIRFLT (SEQ ID NO:230), a CDR-L2 comprising an amino acid sequence AASNQGSGVPS (SEQ ID NO:231), and a CDR-L3 comprising an amino acid sequence QQTKEVPWS (SEQ ID NO:232).

[0153] 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:88, and / or a light chain variable region comprising the 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:89. In some embodiments, the CD33 targeting moiety comprises a CDR-H1 comprising an amino acid sequence DYNMH (SEQ ID NO:233), a CDR-H2 comprising an amino acid sequence YIYPYNGGTGYNQKFKS (SEQ ID NO:234), a CDR-H3 comprising an amino acid sequence GRPAMDY (SEQ ID NO:235), a CDR-L1 comprising an amino acid sequence RASESVDNYGISFMN (SEQ ID NO:236), a CDR-L2 comprising an amino acid sequence AASNQGSGVPS (SEQ ID NO:231), and a CDR-L3 comprising an amino acid sequence QQSKEVPWT (SEQ ID NO:237).

[0154] 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:90, and / or a light chain variable region comprising the 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:91. In some embodiments, the CD33 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NYDIN (SEQ ID NO:238), a CDR-H2 comprising an amino acid sequence WIYPGDGSTKYNEKFKA (SEQ ID NO:239), a CDR-H3 comprising an amino acidsequence GYEDAMDY (SEQ ID NO:240), a CDR-L1 comprising an amino acid sequence KASQDINSYLS (SEQ ID NO:241), a CDR-L2 comprising an amino acid sequence RANRLVDGVPS (SEQ ID NO:242), and a CDR-L3 comprising an amino acid sequence LQYDEFPLT (SEQ ID NO:243). CD164

[0155] In some embodiments, the target molecule is CD164. In some embodiments, the target cell is CD164+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD164. Exemplary anti-CD164 antibodies or antigen-binding fragments thereof 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 antigen-binding fragments thereof disclosed in any of: 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 hereby incorporated by reference in its entirety.

[0156] In some embodiments, the CD164 targeting moiety comprises a class I antibody, as described in Watts et al., Regenerative Medicine (2021)6:33, which is incorporated by reference herein, or an antigen binding portion thereof. In some embodiments, the CD164 targeting moiety comprises a class II antibody, as described in Watts et al., Regenerative Medicine (2021)6:33, or an antigen binding portion thereof. In some embodiments, the CD164 targeting moiety comprises a class III antibody, as described in Watts et al., Regenerative Medicine (2021)6:33, or an antigen binding portion thereof. In some embodiments, the CD164 targeting moiety comprises the 103B2 / 9E10 antibody, the 105A5 antibody, the N6B6 antibody, the 67D2 antibody, the 96.1H5 antibody, the 96.2D2 antibody, the 96.3F5 antibody, the 96.10H10 antibody, or the 96.12H11 antibody, as described in Watts et al., Regenerative Medicine (2021)6:33, or antigen binding portions thereof. CD117

[0157] 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 cell is CD117+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD117. Exemplary anti-CD117 antibodies or antigen-binding fragments thereof 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., NovartisPharmaceuticals), GZQ167 (e.g., Novartis Pharmaceuticals), MGTA-117 (e.g., Magenta Therapeutics), Barzolvolimab (CDX^0159, e.g., CellDex Therapeutics), D13A2 (e.g., Cell Signaling Technology), D3W6Y (e.g., Cell Signaling 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., Magenta Therapeutics), 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 anti-CD117 antibodies or antigen-binding fragments thereof disclosed in any of: 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; US20210355212; US20200369767; US20220315653; US20220177575; US20220177578; WO2019084067; WO2020076105; WO2020242895; WO2020219770; WO2021044008; WO2022132929; WO2023204547; WO2023240272; WO2024008910; 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 et 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 hereby incorporated by reference in its entirety.

[0158] In some embodiments, the CD117 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:415 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:416. In some embodiments, the CD117 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:417 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:418. In some embodiments, the CD117 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:425 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:426. In some embodiments, the CD117 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:427 and alight chain variable region comprising the amino acid sequence of SEQ ID NO:428. SEQ ID NOs:415-418 and 425-428 are shown in Table S2, with complementary determining regions (CDRs) marked in bold. Table S2: Exemplary CD117 Targeting Moiety Sequences

[0159] In some embodiments, the CD117 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:415, and / or a light chain variable region comprising the 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:416. In some embodiments, the CD117 targeting moiety comprises a first Fab polypeptide (i.e., VH-CH1) 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:24, and / or a second Fab polypeptide (i.e., VL-CL) comprising the 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 targeting moiety comprises a CDR-H1 comprising the amino acid sequence FTFSDADMD (SEQ ID NO:32) or DADMD (SEQ ID NO:392), a CDR-H2 comprising the amino acid sequence RNKAGSYTTEYAASVKG (SEQ ID NO:33), a CDR-H3 comprising the amino acid sequence AREPKYWIDFDL (SEQ ID NO:34), a CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO:35), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:36), and a CDR-L3 comprising the amino acid sequence QQSYIAPYT (SEQ ID NO:37).

[0160] In some embodiments, the CD117 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:417, and / or a light chain variable region comprising the 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:418. In some embodiments, the CD117 targeting moiety comprises a first Fab polypeptide (i.e., VH-CH1) 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:30, and / or a second Fab polypeptide (i.e., VL-CL) comprising the 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:31. In some embodiments, the CD117 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYYMA (SEQ ID NO:397), a CDR-H2 comprising the amino acid sequence NINYPGSSTYYLDSVKG (SEQ ID NO:398), a CDR-H3 comprising the amino acid sequence GDYYGTTYWY (SEQ ID NO:399), a CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO:35), a CDR-L2 comprising the amino acid sequence YTSRLQS (SEQ ID NO:400), and a CDR-L3 comprising the amino acid sequence QQGRRLWS (SEQ ID NO:401).

[0161] In some embodiments, the CD117 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:425, and / or a light chain variable region comprising the 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:426. In some embodiments, the CD117 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYWIG (SEQ ID NO:402), a CDR-H2 comprising the amino acid sequence IIYPGDSDTRYSPSFQG (SEQ ID NO:403), a CDR-H3 comprising the amino acid sequence HGRGYNGYEGA (SEQ ID NO:404), a CDR-L1 comprising the amino acid sequence RASQGISSALA (SEQ ID NO:405), a CDR-L2 comprising the amino acid sequence DASSLES (SEQ ID NO:406), and a CDR-L3 comprising the amino acid sequence QQFNSYPLT (SEQ ID NO:407).

[0162] In some embodiments, the CD117 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:427, and / or a light chainvariable region comprising the 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:428. In some embodiments, the CD117 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYNMH (SEQ ID NO:408), a CDR-H2 comprising the amino acid sequence VIYSGNGDTSYNQKFKG (SEQ ID NO:409), a CDR-H3 comprising the amino acid sequence RERDTR (SEQ ID NO:410), a CDR-L1 comprising the amino acid sequence RASESVDIYGNSFMH (SEQ ID NO:411), a CDR-L2 comprising the amino acid sequence LASNLES (SEQ ID NO:412), and a CDR-L3 comprising the amino acid sequence QQNNEDPYT (SEQ ID NO:413). CD34

[0163] In some embodiments, the target molecule is CD34. In some embodiments, the target cell is CD34+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD34. In some embodiments, the anti-CD34 targeting moiety comprises a Fab fragment, an scFv, or a VHH domain. Exemplary anti- CD34 antibodies or antigen-binding fragments thereof 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 anti-CD34 antibodies or antigen-binding fragments thereof disclosed in any of: US 8,084,033; US 10,106,623; US20100311955; US20130172533; US20170320966; WO2023077136; WO2023141297; CN101245108; Arruda et al. (2022) Haematologica 107(8): 1786-1795; etc., each hereby incorporated by reference in its entirety.

[0164] In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:73, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:74. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:75, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:76. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:77, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:78. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:84, and alight chain variable region comprising the amino acid sequence of SEQ ID NO:85. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:475, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:476. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:477, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:478. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:479, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:480. In some embodiments, the CD34 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:481, and a light chain variable region comprising 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 determining regions (CDRs) marked in bold. Table S3: Exemplary CD34 Targeting Moiety Sequences

[0165] In some embodiments, the CD34 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:73, and / or a light chain variable region comprising the 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:74. In some embodiments, the CD34 targeting moiety comprises a first Fab polypeptide (i.e., VH-CH1) comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, atleast 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) comprising the 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 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:40), a CDR-H2 comprising the amino acid sequence WINTNTGEPKYAEEFKG (SEQ ID NO:41), a CDR-H3 comprising the amino acid sequence GYGNYARGAWLAY (SEQ ID NO:42), a CDR-L1 comprising the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO:43), a CDR-L2 comprising the amino acid sequence QVSNRFS (SEQ ID NO:44), and a CDR-L3 comprising the amino acid sequence FQGSHVPRT (SEQ ID NO:45).

[0166] In some embodiments, the CD34 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:75, and / or a light chain variable region comprising the 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:76. In some embodiments, the CD34 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYTFTNYGMN (SEQ ID NO:40), a CDR-H2 comprising the amino acid sequence WINTNTGEPKYAEEFKG (SEQ ID NO:41), a CDR-H3 comprising the amino acid sequence GYGNYARGAWLAY (SEQ ID NO:42), a CDR-L1 comprising the amino acid sequence RSSQTIVHSNGNTYLE (SEQ ID NO:43), a CDR-L2 comprising the amino acid sequence QVSNRFS (SEQ ID NO:44), and a CDR-L3 comprising the amino acid sequence FQGSHVPRT (SEQ ID NO:45).

[0167] In some embodiments, the CD34 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:77, and / or a light chain variable region comprising the 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:78. In some embodiments, the CD34 targeting moiety comprises a CDR-H1 comprising the amino acid sequence RYGMH(SEQ ID NO:244), a CDR-H2 comprising the amino acid sequence VIWYDGRNKEYGDSVKG (SEQ ID NO:245), a CDR-H3 comprising the amino acid sequence PIVGGTNY (SEQ ID NO:246), a CDR-L1 comprising the amino acid sequence RASQSVSSDLA (SEQ ID NO:247), a CDR-L2 comprising the amino acid sequence GASTRATGIPA (SEQ ID NO:248), and a CDR-L3 comprising the amino acid sequence QQYNNWPIT (SEQ ID NO:249).

[0168] In some embodiments, the CD34 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:84, and / or a light chain variable region comprising the 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:85. In some embodiments, the CD34 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPYNGYTFYNQKFKG (SEQ ID NO:251), a CDR-H3 comprising the amino acid sequence HFRYDGVFYYAMDY (SEQ ID NO:252), a CDR-L1 comprising the amino acid sequence TLSSQHSTFTIE (SEQ ID NO:253), a CDR-L2 comprising the amino acid sequence LKKDGSHSTGD (SEQ ID NO:254), and a CDR-L3 comprising the amino acid sequence GVGDTIKEQFVYV (SEQ ID NO:255).

[0169] In some embodiments, the CD34 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:475, and / or a light chain variable region comprising the 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:476.

[0170] In some embodiments, the CD34 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:477, and / or a light chain variable region comprising the 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.

[0171] In some embodiments, the CD34 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:479, and / or a light chain variable region comprising the 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.

[0172] In some embodiments, the CD34 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:481, and / or a light chain variable region comprising the 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:482. CD50

[0173] In some embodiments, the target molecule is CD50, also referred to as ICAM-3. In some embodiments, the target cell is CD50+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD50. Exemplary anti-CD50 antibodies or antigen-binding fragments thereof include ICAM3-IB23 (e.g., Chugai Pharmaceuticals), 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, OTI1F2, OTI1G4, OTI4F5, OTI4G8, OTI3A7, OTI4D4, OTI5D6, SPM505, 76203, 101, 101-1D2, 186-2G9, CG106, 2F8, MA4, BH10, JA4, ICO-60, ICAM3.1, BR-1, ICAM-3.3, CBR-IC3 / 1, CBR-IC3 / 2.1.2, as well as anti-CD50 antibodies or antigen-binding fragments thereof disclosed in any of: US 9,290,812; US20120321557; WO2011105573; Skubitz et al. (1997) J Immunol.159(2):820-828; etc., each hereby incorporated by reference in its entirety.

[0174] In some embodiments, the CD50 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:328 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:329. SEQ ID NOs:328 and 329 are shown in Table S4, with complementary determining regions (CDRs) marked in bold. Table S4: Exemplary CD50 Targeting Moiety Sequences

[0175] In some embodiments, the CD50 targeting moiety comprises a first Fab polypeptide (i.e., VH-CH1) 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:46 or 54, and / or a second Fab polypeptide (i.e., VL-CL) comprising the 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 sortase tag, such as is set forth in SEQ ID NO:83.

[0176] In some embodiments, the CD50 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:328, and / or a light chainvariable region comprising the 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 targeting moiety comprises a CDR-H1 comprising the amino acid sequence TYWMH (SEQ ID NO:322), a CDR-H2 comprising the amino acid sequence YINPNTDYTEYNQKFK (SEQ ID NO:323), a CDR-H3 comprising the amino acid sequence SRDAYHGTY (SEQ ID NO:324), a CDR-L1 comprising the amino acid sequence RASQSISDYLH (SEQ ID NO:325), a CDR-L2 comprising the amino acid sequence YASQSIS (SEQ ID NO:326), and a CDR-L3 comprising the amino acid sequence QNGHNFPLT (SEQ ID NO:327). CD71

[0177] In some embodiments, the target molecule is CD71, also referred to as transferrin receptor. In some embodiments, the target cell is CD71+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD71. Exemplary anti-CD71 antibodies or antigen-binding fragments thereof include CX- 2029 (e.g., CytomX Therapeutics), INA03 (e.g., Inatherys), D7G9X (e.g., Cell Signaling Technology), 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 / 1818, TFRC / 1839, OTI6H9, TFRC / 1396, 1E6, R17217.1.4, TFRC / 1149, SAA1205, 7-1, 1H5, 3A12, 7C11, 2A4, 1G9, and 7B4, as well as anti-CD71 antibodies or antigen-binding fragments thereof disclosed in any of: US 7,858,757; US 10,179,817; etc., each hereby incorporated by reference in its entirety.

[0178] In some embodiments, the CD71 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:65, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:66. In some embodiments, the CD71 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:67, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:68. In some embodiments, the CD71 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:69, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:70. In some embodiments, the CD71 targeting moiety comprises aheavy chain variable region comprising the amino acid sequence of SEQ ID NO:71, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:72. SEQ ID NOs:65-72 are shown in Table S5, with complementary determining regions (CDRs) marked in bold. Table S5: Exemplary CD71 Targeting Moiety Sequences

[0179] In some embodiments, the CD71 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:67, and / or a light chain variable region comprising the 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:68. In some embodiments, the CD71 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYWLG (SEQ ID NO:342), a CDR-H2 comprising the amino acid sequence DIYPGGDYPTYSEKFKV (SEQ ID NO:343), a CDR-H3 comprising the amino acid sequence SGNYDEVAY (SEQ ID NO:344), a CDR-L1 comprising the amino acid sequence RSSQSLVHSNGNTYLH (SEQ ID NO:279), a CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO:345), and a CDR-L3 comprising the amino acid sequence SQSTHVPWT (SEQ ID NO:346).

[0180] In some embodiments, the CD71 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:69, and / or a light chain variable region comprising the 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:70. In some embodiments, the CD71 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYYMN (SEQ ID NO:256), a CDR-H2 comprising the amino acid sequence GISGDPSNTYYADSVKG (SEQ ID NO:257), a CDR-H3 comprising the amino acid sequence DLPLVYTGFAY (SEQ ID NO:258), a CDR-L1 comprising the amino acid sequence SGDNLRHYYVY (SEQ ID NO:259), a CDR-L2 comprising the amino acid sequence GDSKRPS (SEQ ID NO:347) or GDSKRPSGIPE (SEQ ID NO:260), and a CDR- L3 comprising the amino acid sequence QTYTGGASLV (SEQ ID NO:261).

[0181] In some embodiments, the CD71 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:71, and / or a light chain variable region comprising the 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:72. In some embodiments, the CD71 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYWLG (SEQ ID NO:342), a CDR-H2 comprising the amino acid sequence DIYPGGDYPTYSEKFKV (SEQ ID NO:343), a CDR-H3 comprising the amino acid sequence SGNYDEVAY (SEQ ID NO:344), a CDR-L1 comprising the amino acid sequence RSSQSLVH (SEQ ID NO:348) or RSSQSLVHSNGNTYLH (SEQ ID NO:279), a CDR-L2 comprising the amino acid sequence KVSNRFS (SEQ ID NO:345), and a CDR-L3 comprising the amino acid sequence SQSTHVPWT (SEQ ID NO:346).

[0182] In some embodiments, the CD71 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:65, and / or a light chain variable region comprising the 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:66. In some embodiments, the CD71 targeting moiety comprises a first Fab polypeptide (i.e., VH-CH1) 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:48, and / or a second Fab polypeptide (i.e., VL-CL) comprising the 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 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYAMS (SEQ ID NO:349), a CDR-H2 comprising the amino acid sequence YIWSGGSTDYASWA (SEQ ID NO:350), a CDR-H3 comprising the amino acid sequence RYGTSYPDYGDASGFDP (SEQ ID NO:351), a CDR-L1 comprising the amino acid sequence RASQSISSYLA (SEQ ID NO:352), a CDR-L2 comprising the amino acid sequence RASTLAS (SEQ ID NO:353), and a CDR-L3 comprising the amino acid sequence QQNYASSNVDNT (SEQ ID NO:354). CD133

[0183] 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 cell is CD133+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD133. Exemplary anti-CD133 antibodies or antigen-binding fragments thereof include A3G6K (e.g., CellSignaling Technology), D2V8Q (e.g., Cell Signaling Technology), W6B3C1, W6B3, TMP4, 2F8C5, 5E3, EMK08, and BLR093G, as well as anti-CD133 antibodies or antigen-binding fragments thereof disclosed in any of: US 11,098,109; US 11,220,551; Glumac et al. Prostate.2018 Sep; 78(13): 981–991; etc., each hereby incorporated by reference in its entirety. CD38

[0184] In some embodiments, the target molecule is CD38. In some embodiments, the target cell is CD38+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD38. Exemplary anti-CD38 antibodies or antigen-binding fragments thereof include daratumumab (e.g., Janssen Biotech, Inc.), isatuximab (e.g., Sanofi), felzartamab, (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, rCD38 / 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 anti-CD38 antibodies or antigen-binding fragments thereof disclosed in any of: US 7,829,673; US 9,603,927; US 10,526,417; US 10,604,580; US20160376373; Kassem et al. (2022) Blood.139(8):1160-1176; Chen et al. (2024) N Engl J Med.390:2178-2190; etc., each hereby incorporated by reference in its entirety.

[0185] In some embodiments, the CD38 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:92, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:93. SEQ ID NOs:92 and 93 are shown in Table S6, with complementary determining regions (CDRs) marked in bold.Table S6: Exemplary CD38 Targeting Moiety Sequences

[0186] In some embodiments, the CD38 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:92, and / or a light chain variable region comprising the 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:93. In some embodiments, the CD38 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYYMN (SEQ ID NO:256), a CDR-H2 comprising the amino acid sequence GISGDPSNTYYADSVKG (SEQ ID NO:257), a CDR-H3 comprising the amino acid sequence DLPLVYTGFAY (SEQ ID NO:258), a CDR-L1 comprising the amino acid sequence SGDNLRHYYVY (SEQ ID NO:259), a CDR-L2 comprising the amino acid sequence GDSKRPSGIPE (SEQ ID NO:260), and a CDR-L3 comprising the amino acid sequence QTYTGGASLV (SEQ ID NO:261). CD43

[0187] In some embodiments, the target molecule is CD43, also referred to as leukosialin or sialophorin. In some embodiments, the target cell is CD43+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD43. Exemplary anti-CD43 antibodies or antigen-binding fragments thereof 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, DF-T1, SPN / 1094, SPM503, OTI2D4, OTI2C7, OTI3D11, OTI6A4, OTI4G8, OTI3D11, OTI4F6, Bra7G, A2F9, A2F7, and A2F8, as well as anti-CD43 antibodies or antigen-binding fragments thereof disclosed in any of: US 7,674,605; US9,746,474; US 10,676,531; US 11,174,318; US 11,524,989; KR20120081723; Gillissen et al. (2017) Blood Adv.1(19): 1551–1564; etc., each hereby incorporated by reference in its entirety.

[0188] In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:118, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:119. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:120, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:121. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:122, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:123. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:124, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:125. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:126, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:127. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:128, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:129. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:130, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:131. In some embodiments, the CD43 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:132, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:133. SEQ ID NOs:118-133 are shown in Table S7, with complementary determining regions (CDRs) marked in bold. Table S7: Exemplary CD43 Targeting Moiety Sequences

[0189] In some embodiments, the CD43 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:118, and / or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, atleast 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 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPNNGDSFYNQKFKG (SEQ ID NO:262), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acid sequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).

[0190] In some embodiments, the CD43 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:120, and / or a light chain variable region comprising the 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:121. In some embodiments, the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYYMN (SEQ ID NO:267), a CDR-H2 comprising the amino acid sequence RINPNSGDSFYNQKFKG (SEQ ID NO:268), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acid sequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).

[0191] In some embodiments, the CD43 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:122, and / or a light chain variable region comprising the 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 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPNNGDSFYNQKFQG (SEQ ID NO:269), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acidsequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).

[0192] In some embodiments, the CD43 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:124, and / or a light chain variable region comprising the 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:125. In some embodiments, the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPNNGDSFYNQKFKG (SEQ ID NO:262), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acid sequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).

[0193] In some embodiments, the CD43 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:126, and / or a light chain variable region comprising the 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:127. In some embodiments, the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYFMN (SEQ ID NO:250), a CDR-H2 comprising the amino acid sequence RINPNNGDSFYNQKFQG (SEQ ID NO:269), a CDR-H3 comprising the amino acid sequence EGYYGGRGYALDY (SEQ ID NO:263), a CDR-L1 comprising the amino acid sequence RTSQDISNYLN (SEQ ID NO:264), a CDR-L2 comprising the amino acid sequence NTSRLHSGVPS (SEQ ID NO:265), and a CDR-L3 comprising the amino acid sequence QQSNMFPYT (SEQ ID NO:266).

[0194] In some embodiments, the CD43 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:128, and / or a light chain variable region comprising the 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:129. In some embodiments, the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SFGMH (SEQ ID NO:270), a CDR-H2 comprising the amino acid sequence YISSGSGNFYYVDTVKG (SEQ ID NO:271), a CDR-H3 comprising the amino acid sequence STYYHGSRGAMDY (SEQ ID NO:272), a CDR-L1 comprising the amino acid sequence SASSSVSSMY (SEQ ID NO:273), a CDR-L2 comprising the amino acid sequence DTSKMASGVPI (SEQ ID NO:274), and a CDR-L3 comprising the amino acid sequence QQWSSYPPIT (SEQ ID NO:275).

[0195] In some embodiments, the CD43 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:132, and / or a light chain variable region comprising the 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:133. In some embodiments, the CD43 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SSPNWWT (SEQ ID NO:282), a CDR-H2 comprising the amino acid sequence EIYYGGRVSYNSALRS (SEQ ID NO:283), a CDR-H3 comprising the amino acid sequence QKNIGCGYSSCFIS (SEQ ID NO:284), a CDR-L1 comprising the amino acid sequence KSSQTILQRSNHLNYLA (SEQ ID NO:285), a CDR-L2 comprising the amino acid sequence WASTRESGVPD (SEQ ID NO:286), and a CDR-L3 comprising the amino acid sequence HQYYTTPQT (SEQ ID NO:287). CD45

[0196] In some embodiments, the target molecule is CD45. In some embodiments, the target cell is CD45+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD45. Exemplary anti-CD45 antibodies or antigen-binding fragments thereof 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, OTI2H1, 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 anti-CD45 antibodies or antigen-binding fragments thereof disclosed in any of: US 9,962,408; US 11,655,452; US 11,692,041; US 11,406,724; etc., each hereby incorporated by reference in its entirety.

[0197] In some embodiments, the CD45 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:134, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:135. In some embodiments, the CD45 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:136, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:137. In some embodiments, the CD45 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:138, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:139. SEQ ID NOs:134-139 are shown in Table S8, with complementary determining regions (CDRs) marked in bold. Table S8: Exemplary CD45 Targeting Moiety Sequences

[0198] In some embodiments, the CD45 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, atleast 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:134, and / or a light chain variable region comprising the 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:135. In some embodiments, the CD45 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYWMT (SEQ ID NO:288), a CDR-H2 comprising the amino acid sequence SISSSGGSIYYPDSVKG (SEQ ID NO:289), a CDR-H3 comprising the amino acid sequence DERWAGAMDA (SEQ ID NO:290), a CDR-L1 comprising the amino acid sequence KASQNINKNLD (SEQ ID NO:291), a CDR-L2 comprising the amino acid sequence ETNNLQTGIPS (SEQ ID NO:292), and a CDR-L3 comprising the amino acid sequence YQHNSRFT (SEQ ID NO:293).

[0199] In some embodiments, the CD45 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:136, and / or a light chain variable region comprising the 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 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NFWMT (SEQ ID NO:294), a CDR-H2 comprising the amino acid sequence SISSSGGSIYYPDSVKD (SEQ ID NO:295), a CDR-H3 comprising the amino acid sequence LHYYSGGGDA (SEQ ID NO:296), a CDR-L1 comprising the amino acid sequence KASQNINKYLD (SEQ ID NO:297), a CDR-L2 comprising the amino acid sequence YTNNLHTGIPS (SEQ ID NO:298), and a CDR-L3 comprising the amino acid sequence LQHSSRWT (SEQ ID NO:299).

[0200] In some embodiments, the CD45 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:138, and / or a light chain variable region comprising the 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:139. In some embodiments, the CD45 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYWMT(SEQ ID NO:288), a CDR-H2 comprising the amino acid sequence SISSSGGSIYYPDSVKD (SEQ ID NO:295), a CDR-H3 comprising the amino acid sequence LYYYSGGGDA (SEQ ID NO:300), a CDR-L1 comprising the amino acid sequence KASQDINKYLD (SEQ ID NO:301), a CDR-L2 comprising the amino acid sequence NTNNLHTGIPS (SEQ ID NO:302), and a CDR-L3 comprising the amino acid sequence LQHISRWT (SEQ ID NO:303). CD46

[0201] In some embodiments, the target molecule is CD46. In some embodiments, the target cell is CD46+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD46. Exemplary anti-CD46 antibodies or antigen-binding fragments thereof include 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 anti-CD46 antibodies or antigen-binding fragments thereof disclosed in any of: US 10,533,056; etc., each hereby incorporated by reference in its entirety.

[0202] In some embodiments, the CD46 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:419, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:420. In some embodiments, the CD46 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:421, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:422. In some embodiments, the CD46 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:423, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:424. SEQ ID NOs:419-424 are shown in Table S9, with complementary determining regions (CDRs) marked in bold. Table S9: Exemplary CD46 Targeting Moiety Sequences

[0203] In some embodiments, the CD46 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:419, and / or a light chain variable region comprising the 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:420. In some embodiments, the CD46 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYAMH (SEQ ID NO:304), a CDR-H2 comprising the amino acid sequence FIRSDGSKKYYADSVKG (SEQ ID NO:305), a CDR-H3 comprising the amino acid sequence HGNYFDS (SEQ ID NO:306), a CDR-L1 comprising the amino acid sequence RASQGISSYLA (SEQ ID NO:307), a CDR-L2 comprising the amino acid sequence AASTLQS (SEQ ID NO:308), and a CDR-L3 comprising the amino acid sequence QQLASYPLT (SEQ ID NO:309).

[0204] In some embodiments, the CD46 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:421, and / or a light chain variable region comprising the 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:422. In some embodiments, the CD46 targeting moiety comprises a CDR-H1 comprising the amino acid sequence NYAMH (SEQ ID NO:310), a CDR-H2 comprising the amino acid sequence VISYDGNNKYYADSVKG (SEQ ID NO:311), a CDR-H3 comprising the amino acidsequence GGGYFDL (SEQ ID NO:312), a CDR-L1 comprising the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO:313), a CDR-L2 comprising the amino acid sequence GNNNRPS (SEQ ID NO:314), and a CDR-L3 comprising the amino acid sequence SSYTSGTWL (SEQ ID NO:315).

[0205] In some embodiments, the CD46 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:423, and / or a light chain variable region comprising the 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 targeting moiety comprises a CDR-H1 comprising the amino acid sequence TYGMH (SEQ ID NO:316), a CDR-H2 comprising the amino acid sequence FISYDGDEKYYADSVKG (SEQ ID NO:317), a CDR-H3 comprising the amino acid sequence ASGYGMGILDY (SEQ ID NO:318), a CDR-L1 comprising the amino acid sequence QGDSLRSYYVS (SEQ ID NO:319), a CDR-L2 comprising the amino acid sequence GQNNRPS (SEQ ID NO:320), and a CDR-L3 comprising the amino acid sequence HSRDSSGTHLRV (SEQ ID NO:321). CD49d

[0206] In some embodiments, the target molecule is CD49d. In some embodiments, the target cell is CD49d+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD49d. Exemplary anti-CD49d antibodies or antigen-binding fragments thereof include Natalizumab (e.g., Biogen), Vedolizumab (e.g., Takeda), and 9F10.

[0207] In some embodiments, the CD49d targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:94, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:95. In some embodiments, the CD49d targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:96, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:97. SEQ ID NOs:94-97 are shown in Table S10, with complementary determining regions (CDRs) marked in bold.Table S10: Exemplary CD49d Targeting Moiety Sequences

[0208] In some embodiments, the CD49d 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:94, and / or a light chain variable region comprising the 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:95. In some embodiments, the CD49d targeting moiety comprises a CDR-H1 comprising the amino acid sequence DTYIH (SEQ ID NO:330), a CDR-H2 comprising the amino acid sequence RIDPANGYTKYDPKFQG (SEQ ID NO:331), a CDR-H3 comprising the amino acid sequence EGYYGNYGVYAMDY (SEQ ID NO:332), a CDR-L1 comprising the amino acid sequence KTSQDINKYMA (SEQ ID NO:333), a CDR-L2 comprising the amino acid sequence YTSALQP (SEQ ID NO:334), and a CDR-L3 comprising the amino acid sequence LQYDNLWT (SEQ ID NO:335).

[0209] In some embodiments, the CD49d 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:96, and / or a light chain variable region comprising the 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:97. In some embodiments, theCD49d targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYWMH (SEQ ID NO:336), a CDR-H2 comprising the amino acid sequence EIDPSESNTNYNQKFKG (SEQ ID NO:337), a CDR-H3 comprising the amino acid sequence GGYDGWDYAIDY (SEQ ID NO:338), a CDR-L1 comprising the amino acid sequence RSSQSLAKSYGNTYLS (SEQ ID NO:339), a CDR-L2 comprising the amino acid sequence GISNRFS (SEQ ID NO:340), and a CDR-L3 comprising the amino acid sequence LQGTHQPYT (SEQ ID NO:341). CD49f

[0210] In some embodiments, the target molecule is CD49f. In some embodiments, the target cell is CD49f+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD49f. Exemplary anti-CD49f antibodies or antigen-binding fragments thereof include MP-4F10, GoH3, NKI-GoH3, 450-30A, 5HCLC, SR45-00, 5H14L18, 4G10, CL6957, and MAB-5A. CD59

[0211] In some embodiments, the target molecule is CD59. In some embodiments, the target cell is CD59+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD59. Exemplary anti-CD59 antibodies or antigen-binding fragments thereof include MEM-43, OV9A2, JM10-71, p282(H19), QA19A13, 2491C, 2E11B6, 193-27, 029, and 108. CD90

[0212] In some embodiments, the target molecule is CD90. In some embodiments, the target cell is CD90+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD90. Exemplary anti-CD90 antibodies or antigen-binding fragments thereof include 5E10, HIS51, 53-2.1, F15-42-1, G7, and YKIX337.217.

[0213] In some embodiments, the CD90 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:148, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:149. In some embodiments, the CD90 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:150, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:151. SEQ ID NOs:148-151 are shown in Table S11, with complementary determining regions (CDRs) marked in bold. Table S11: Exemplary CD90 Targeting Moiety Sequences

[0214] In some embodiments, the CD90 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:148, and / or a light chain variable region comprising the 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:149. In some embodiments, the CD90 targeting moiety comprises a CDR-H1 comprising the amino acid sequence GYYVH (SEQ ID NO:373), a CDR-H2 comprising the amino acid sequence WVNPNSGDTNYAQKFQG (SEQ ID NO:374), a CDR-H3 comprising the amino acid sequence DGDEDWY (SEQ ID NO:375), a CDR-L1 comprising the amino acid sequence RASQGISRSLV (SEQ ID NO:376), a CDR-L2 comprising the amino acid sequence AASTLQS (SEQ ID NO:308), and a CDR-L3 comprising the amino acid sequence LQHNTYPFT (SEQ ID NO:377).

[0215] In some embodiments, the CD90 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:150, and / or a light chain variable region comprising the 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:151. In some embodiments, the CD90 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYAMS (SEQ ID NO:349), a CDR-H2 comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO:378), a CDR-H3 comprising the amino acid sequence GARMDV (SEQ ID NO:379), a CDR-L1 comprising the amino acid sequence KSSQSVLSSSKNKNYLA (SEQ ID NO:380), a CDR-L2 comprising the amino acid sequence WASTRQS (SEQ ID NO:381), and a CDR-L3 comprising the amino acid sequence QQHYSIPVT (SEQ ID NO:382). CD105

[0216] In some embodiments, the target molecule is CD105. In some embodiments, the target cell is CD105+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD105. Exemplary anti-CD105 antibodies or antigen-binding fragments thereof include QA19A14, O99E5, 43A3, SN6, MJ7 / 18, 3A9, MEM-226, ARC0446, 209701, 2D5E8 JE60-59, 103, and 23GB3745. CD110

[0217] In some embodiments, the target molecule is CD110. In some embodiments, the target cell is CD110+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD110. Exemplary anti-CD110 antibodies or antigen-binding fragments thereof include 1D6B7, S16017A, 1H2, and ARC2257. CD123

[0218] In some embodiments, the target molecule is CD123. In some embodiments, the target cell is CD123+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD123. Exemplary anti-CD123 antibodies or antigen-binding fragments thereof include pivekimab (IMGN632, e.g., ImmunoGen), talacotuzumab (e.g., Johnson & Johnson), S18016C, QA18A25, 6H6, 7G3, 5B11, 017, and 32703.

[0219] In some embodiments, the CD123 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:148, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:149. In some embodiments, the CD123 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:150, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:151. SEQ ID NOs:148-151 are shown in Table S21, with complementary determining regions (CDRs) marked in bold. Table S12: Exemplary CD123 Targeting Moiety Sequences

[0220] In some embodiments, the CD123 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:98, and / or a light chain variable region comprising the 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:99. In some embodiments, the CD123 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SSIMH (SEQ ID NO:355), a CDR-H2 comprising the amino acid sequence YIKPYNDGTKYNEKFKG (SEQ ID NO:356), a CDR-H3 comprising the amino acid sequence EGGNDYYDTMDY (SEQ ID NO:357), a CDR-L1 comprising the amino acid sequence RASQDINSYLS (SEQ ID NO:358), a CDR-L2 comprising the amino acid sequence RVNRLVD (SEQ ID NO:359), and a CDR-L3 comprising the amino acid sequence LQYDAFPYT (SEQ ID NO:360).

[0221] In some embodiments, the CD123 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:100, and / or a light chain variable region comprising the amino acid sequence having at least 90% (e.g., at least 92%, atleast 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:101. In some embodiments, the CD123 targeting moiety comprises a CDR-H1 comprising the amino acid sequence DYYMK (SEQ ID NO:361), a CDR-H2 comprising the amino acid sequence DIIPSNGATFYNQKFKG (SEQ ID NO:362), a CDR-H3 comprising the amino acid sequence SHLLRASWFAY (SEQ ID NO:363), a CDR-L1 comprising the amino acid sequence ESSQSLLNSGNQKNYLT (SEQ ID NO:364), a CDR-L2 comprising the amino acid sequence WASTRES (SEQ ID NO:365), and a CDR-L3 comprising the amino acid sequence QNDYSYPYT (SEQ ID NO:366). CD135

[0222] In some embodiments, the target molecule is CD135. In some embodiments, the target cell is CD135+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD135. Exemplary anti-CD135 antibodies or antigen-binding fragments thereof include BV10A4H2, 4G8, A2F10, 7B7C3, BV10A4, 66903, OTI7D6, and FLT3 / 2458. CD184

[0223] In some embodiments, the target molecule is CD184. In some embodiments, the target cell is CD184+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD184. Exemplary anti-CD184 antibodies or antigen-binding fragments thereof include ulocuplumab (e.g., Bristol-Myers Squibb), 12G5, 1D9, QA18A64, 2B11, 21H17L11, HL2424, 2F10, 2D4, ARC0381, 9D9, 4B5E4, 2G9, and 2A9.

[0224] In some embodiments, the CD184 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:146, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:147. SEQ ID NOs:146 and 147 are shown in Table S13, with complementary determining regions (CDRs) marked in bold. Table S13: Exemplary CD184 Targeting Moiety Sequencesvariable 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:146, and / or a light chain variable region comprising the 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:147. In some embodiments, the CD184 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYSMN (SEQ ID NO:367), a CDR-H2 comprising the amino acid sequence YISSRSRTIYYADSVKG (SEQ ID NO:368), a CDR-H3 comprising the amino acid sequence DYGGQPPYYYYYGMDV (SEQ ID NO:369), a CDR-L1 comprising the amino acid sequence RASQGISSWLA (SEQ ID NO:370), a CDR-L2 comprising the amino acid sequence AASSLQS (SEQ ID NO:36), and a CDR-L3 comprising the amino acid sequence QQYNSYPRT (SEQ ID NO:372). CD201

[0226] In some embodiments, the target molecule is CD201. In some embodiments, the target cell is CD201+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD201. Exemplary anti-CD201 antibodies or antigen-binding fragments thereof include clones 1560, 041, RCR-227, mRCR-16, 1F5C4, 011, and 016.

[0227] In some embodiments, the CD201 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:152, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:153. In some embodiments, the CD201 targeting moiety is a single domain antibody (sdAb) comprising a VHH comprising the amino acid sequence of SEQ ID NO:102. SEQ ID NOs:102, 152, and 153 are shown in Table S14, with complementary determining regions (CDRs) marked in bold.Table S14: Exemplary CD201 Targeting Moiety Sequences

[0228] In some embodiments, the CD201 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:152, and / or a light chain variable region comprising the 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 targeting moiety comprises a CDR-H1 comprising the amino acid sequence SYAIS (SEQ ID NO:383), a CDR-H2 comprising the amino acid sequence GIIPIFGTANYAQKFQG (SEQ ID NO:384), a CDR-H3 comprising the amino acid sequence GYSDAFDI (SEQ ID NO:385), a CDR-L1 comprising the amino acid sequence RASQGIRNDLG (SEQ ID NO:386), a CDR-L2 comprising the amino acid sequence AASRLQS (SEQ ID NO:387), and a CDR-L3 comprising the amino acid sequence HQAYSYPLT (SEQ ID NO:388).

[0229] In some embodiments, the CD201 targeting moiety is an sdAb comprising 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:102. In some embodiments, the CD201 targeting moiety is an sdAb comprising a CDR-H1 comprising the amino acid sequence YYAIG (SEQ ID NO:389), a CDR-H2 comprising the amino acid sequence CISSSDGSTYYADSVKG (SEQ ID NO:390), and a CDR-H3 comprising the amino acid sequence SLGGAWVVAGNCPALDFNS (SEQ ID NO:391).CD338

[0230] In some embodiments, the target molecule is CD338. In some embodiments, the target cell is CD338+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD338. Exemplary anti-CD338 antibodies or antigen-binding fragments thereof include 5D3, 3G8, BXP-21, and EPR20080. GPR56

[0231] In some embodiments, the target molecule is GPR56. In some embodiments, the target cell is GPR56+. In some embodiments, the targeting moiety includes an antibody or antigen-binding fragment thereof that binds to GPR56. Exemplary anti-GPR56 antibodies or antigen-binding fragments thereof include CG4, 4C3, 3A1D7, and 1443CT642.85.27, as well as anti-GPR56 antibodies or antigen-binding fragments thereof disclosed in Chatterjee et al. (2021) J Biol Chem.296: 100261, hereby incorporated by reference in its entirety. Methods of Making Targeted LNPs (Conjugates)

[0232] Different approaches can be used to introduce a targeting moiety onto the surface of an LNP. For example, one approach relies on functionalizing a preformed LNP with a targeting moiety. The LNP generally includes a lipid that has polyethylene glycol (PEG) spacer functionalized with a reactive moiety such as a thiol, amine, maleimide or carboxylic acid group. The functionalized lipid of the LNP reacts with a complementary group that is covalently bonded to a targeting moiety, hence generating a conjugate of the LNP and the targeting moiety.

[0233] In particular embodiments, the targeting moiety is site-specifically conjugated to the surface of the LNP. Conjugates prepared by site-specific methods (e.g., FIGs.1-17) have a high density of the targeting moiety on the surface of the LNP. For instance, the conjugate can comprise more than one targeting moiety (e.g., antibody, Fab fragment, or scFv) per LNP. In some embodiments, the conjugate can comprise more than 10 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise more than 20 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise more than 30 targeting moieties (e.g., antibodies, Fab fragments, or scFvs). In some embodiments, the conjugate can comprise more than 50 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise more than 75 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate cancomprise more than 100 targeting moieties (e.g., antibodies, Fab fragments, or scFvs). In some embodiments, the conjugate can comprise from about 50 to about 200 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP. In some embodiments, the conjugate can comprise from about 10 to about 30 targeting moieties (e.g., antibodies, Fab fragments, or scFvs) per LNP.

[0234] In some embodiments, the weight ratio between a targeting moiety 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, 20:1.

[0235] In some embodiments, the conjugates prepared by site-specific methods (e.g., FIGs.1-17) can have two or more different targeting moieties on the surface of the LNP, wherein one of the targeting moieties binds to CD164. For example, the conjugate can have two different targeting moieties. In some embodiments, the conjugate can have three different targeting moieties on the surface of the LNP, wherein one of the targeting moieties binds to CD164. In some embodiments, the conjugate can have two different targeting moieties, wherein the mass ratio of the two targeting moieties 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 the one or more targeting moieties 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, 20:1. ^ Interchain CL-CH1 disulfide reduction followed by conjugation

[0236] IgG antibodies consist of four polypeptide chains linked by disulfide bonds. The two polypeptide chains of low molecular weight are call light chains (L). The light chains consist of a variable light chain domain (VL) and a constant light chain domain (CL). The heavy chains consist of a variable heavy light domain (VH) and three constant heavy chain domains (CH1, CH2, and CH3). The Fab region of the antibody includes the VL, CL, VH, and CH1 domains. The Fc region includes the constant heavy chain domains CH2, and CH3. A hinge region of the IgG antibody covalently links the CH1 domain to the CH2 domain. The two heavy chains of IgG antibodies are connected in the hinge region by a variable number ofdisulfide bonds depending on the IgG subclass. Different subclasses of IgG antibodies have varying numbers of interchain disulfide bonds. Additionally, the light chain is covalently linked to the heavy chain via a disulfide bond between the light chain and the heavy chain. Using standard IgG nomenclature, this natural interchain disulfide bond is also referred to as the CL-CH1 disulfide bond to distinguish it from disulfide bonds present in the hinge region. Therapeutic antibodies of type IgG1 possess an intermolecular disulfide bond between Cys233 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain. Therapeutic antibodies of type IgG4 possess an intermolecular disulfide bond between Cys127 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain. Therapeutic antibodies of type IgG2 possess an intermolecular disulfide bond between Cys135 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.

[0237] Proteolytic cleavage of an IgG antibody results in the formation of a Fab fragment known as a F(ab’)2 fragment. The F(ab’)2 fragment does not include the CH2 domain or the CH3 domain. However, the hinge region of the antibody is retained in a F(ab’)2 fragment. The F(ab’)2 fragment includes disulfide bonds that covalently link two Fab fragments. Reduction of the disulfide bond in the F(ab’)2generates two F(ab’) fragments. The sulfhydryl (thiol) groups of the F(ab’) could potentially react with a thiol-reactive group on the surface of an LNP, hence generating a conjugate. However, owing to the presence of multiple sulfhydryl groups in the hinge region of the F(ab’) fragment, site-specific conjugation is challenging. Moreover, the reduction of the F(ab’)2to the F(ab’) fragments could also disrupt the natural interchain disulfide bonds between the CL and CH1 regions of the Fab fragments, hence further compromising site-specific conjugation.

[0238] As set forth herein, Fab fragment (e.g., a Fab fragment that binds to CD164) can be site-selectively conjugated to the surface of a precursor lipid nanoparticle (LNP) through the natural interchain disulfide bond between the heavy chain and the light chain (i.e., the CL- CH1 disulfide bond) of the Fab fragment to make a targeted LNP (conjugate).

[0239] The term “precursor LNP” or “base LNP” refers to an LNP that has been functionalized with a reactive moiety (e.g., thiol-reactive group or polyglycine) prior to reacting with the Fab fragment. The process for conjugating a targeting moiety, as disclosed herein, involves reducing the natural interchain disulfide bond between the CL and CH1 domains of a Fab fragment, and reacting the reduced Fab fragment with a thiol-reactive group (e.g., a maleimide or DBM group) covalently bonded to the surface of a precursor LNP, thus forming a conjugate. Alternatively, the reduced Fab fragment can be reacted with a lipid thathas been chemically modified (functionalized) with a thiol-reactive group (e.g., maleimide or DBM group). The resultant lipid can then be inserted into a preexisting LNP, thus generating a conjugate. As described herein, despite the removal of the natural interchain disulfide bond linking the heavy and light chains of the Fab fragment, the resulting conjugates are able to effectively target specific cell types depending on the nature of the Fab targeting moiety. For instance, specific Fab fragments for targeting immune cells or hematopoietic stem cells (HSCs) as disclosed herein. A schematic of an LNP site-specifically conjugated to a Fab fragment is shown in FIG.1.

[0240] In one embodiment, Fab fragments used for conjugation may be used by recombinant methods. In particular embodiments, the Fab fragments generated recombinantly are designed not to include a hinge region at the C-terminus. Therefore, the recombinantly generated Fab fragments include only one disulfide bond between the CL-CH1 and domains. As set forth herein, the CL-CH1 can then be reduced and the resultant free thiol groups can be used as anchors to conjugate the Fab fragment to the surface of an LNP.

[0241] In some embodiments, the Fab fragment is of the IgG class, the IgM class, or the 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.

[0242] In one aspect, the disclosure provides methods of making a targeted LNP, said method comprising: (i) contacting a composition comprising a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain and an interchain disulfide bond linking the constant light chain domain (CL) and the constant heavy chain domain 1 (CH1), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and (ii) contacting the product of step (i) with a precursor LNP comprising a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP.

[0243] In some aspects, the disclosure provides a conjugate produced by a method comprising: (i) contacting a composition comprising a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain and an interchain disulfide bond linking the constant light chain domain (CL) and the constant heavy chaindomain 1 (CH1), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and (ii) contacting the product of step (i) with a precursor LNP comprising a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP.

[0244] In some embodiments, the thiol-reactive group (e.g., maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl) is chemically reacted with a lipid molecule to create a modified lipid wherein the thiol-reactive group is covalently attached to the lipid where it is capable of reacting with at least one free cysteine residue of the reduced Fab fragment (either on the heavy or light chain of the Fab fragment). The reaction between the thiol-reactive group and the at least one free cysteine residue can be completed prior to or after formation of the LNP with the modified lipid. For instance, as shown in FIG.2A, the various components (e.g., lipids) comprising the LNP and a therapeutic payload can be mixed with lipid molecules, including one or more lipids that comprise a thiol-reactive group, thus generating an LNP that comprises a plurality of thiol-reactive groups (each thiol-reactive group shown schematically as a “functional group” in FIG.2A). The thiol-reactive group can then be reacted with at least one free cysteine residue of the Fab fragment, hence generating a conjugate. Alternatively, a lipid that has been modified with the thiol-reactive group can be directly reacted with at least one free cysteine residue of a Fab fragment. As depicted in FIG.2B, the resultant modified lipid attached to the Fab fragment can then be inserted into a pre-formed LNP that has not yet been surface modified. This procedure allows for the reaction to be performed on an individual lipid molecule rather than on the surface of the LNP.

[0245] Any suitable reducing reagent can be used to reduce the interchain disulfide bond of the Fab fragment. Examples of reducing reagents include, but are not limited to, 2- mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol (DTE), and tris(carboxyethyl)phosphine (TCEP), and combinations thereof. In some embodiments, the reducing reagent is a mild reducing reagent. Examples of mild reducing reagents include, e.g., DTT, TCEP, and DTE. In some embodiments, the reducing reagent is TCEP. Any suitable reaction conditions can be used for the reduction of the interchain disulfide bond in step (i). In some embodiments, the reduction reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reduction reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reduction reaction is performed at physiological temperature (e.g., about 37°C). In some embodiments, the reduction reactionis performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reduction reaction is performed at ambient temperature (e.g., about 23^ to about 25°C). In some embodiments, the reduction reaction is performed at about 0°C to about 4°C.

[0246] In some embodiments, excess reducing agent is removed following step (i), prior to conjugation to the precursor LNP. In some embodiments, excess reducing agent is not removed following step (i), prior to conjugation to the precursor LNP.

[0247] In some embodiments of the method or process, the thiol-reactive groups on the LNP (or lipid to be post-inserted into an LNP) comprises any suitable reactive group, including but not limited to, maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl.

[0248] In some embodiments, the thiol-reactive group is maleimide. In some embodiments, maleimide reacts with one of the 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, two maleimide groups each react with the Fab fragment, wherein one maleimide reacts with a free cysteine residue on the light chain and the other maleimide reacts with a free cysteine residue on the heavy chain.

[0249] Any suitable 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 can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reaction is performed at physiological temperature (e.g., about 37°C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reaction is performed at ambient temperature (e.g., about 23^ to about 25°C). In some embodiments, the reaction is performed at about 0°C to about 4°C.

[0250] A schematic showing the reduction of an interchain disulfide bond in a Fab fragment is depicted in FIG.3. In some embodiments, the Fab fragment depicted in FIG.3 can be produced recombinantly without a hinge region at the C-terminus. Accordingly, in such embodiments, the Fab fragment only comprises a single interchain disulfide bond, which is located in the CL-CH1 interface of the Fab fragment. As shown in FIG.3, theinterchain disulfide bond is located between the heavy chain and the light chain of the Fab fragment. The reduction reaction breaks the covalent linkage forming the disulfide bond, thereby generating two free cysteine residues that can react in a subsequent step with a thiol- reactive group.

[0251] FIG.4 shows an exemplary schematic of conjugate formation, as described herein. In FIG.4, a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues (step (i)). In step (ii), the reduced Fab fragment is reacted with an LNP comprising a plurality of thiol-reactive groups (e.g., maleimide or DBM) conjugated to the surface of the LNP, whereby 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 a linkage through at least one of the free cysteine residues of the Fab fragment.

[0252] Following reaction of the reduced Fab fragment with the thiol-reactive group of the LNP, either the heavy chain, light chain or both the heavy chain and light chain of the Fab fragment are conjugated to the surface of the LNP. The various orientations are depicted in FIG.5. In some embodiments, the conjugates formed by methods disclosed herein include at least one, at least two or all three orientations shown in FIG.5. The concentration of the thiol-reactive group (e.g., maleimide) will likely 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 the orientation, the heavy chain and the light chain remain intact on the surface of the LNP, thus forming a functional Fab fragment that is capable of engaging with a receptor on a targeted cell.

[0253] In some embodiments, the thiol-reactive group is maleimide, as shown in FIG.5. In some embodiments, maleimide reacts with one of the two free cysteine residues of the antibody or antigen-binding fragment thereof. In some embodiments, maleimide reacts with one of the two free cysteine residues of the Fab fragment 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, two maleimide groups on the LNP each react with the Fab fragment, wherein one maleimide reacts with a free cysteineresidue on the light chain and the other maleimide reacts with a free cysteine residue on the heavy chain.

[0254] In some embodiments, the thiol-reactive group is 2,3-dibromomaleimide (DBM) as shown in FIG.6. Following reduction of the disulfide bond, the reduced Fab fragment is added to DBM covalently bonded to a lipid (represented by squiggly line in FIG.6). As set forth above, the lipid may be part of an LNP or may be post-inserted into an LNP following reaction with the Fab fragment. Both of the free cysteine residues displace the two bromine groups of DBM, hence generating a dithiomalemide. The dithiolmalemide can be converted to the corresponding maleamic acid via hydrolysis. DBM reacts with a free cysteine residue on the heavy chain and a free cysteine residue on the light chain of the Fab fragment to form a bridge between the cysteine residues. Accordingly, the heavy and light chain of the Fab fragment are effectively bridged together following reaction with DBM.

[0255] In all embodiments discussed above, the thiol-reactive group can be introduced onto any of the lipids comprising the LNP. In some embodiments, the conjugate can comprise 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, hence generating the structure Lipid-PEGx-thiol-reactive group, wherein x is 2-120 ethylene glycol units. In such embodiments, at least one free cysteine residue of a Fab fragment reacts with the thiol- reactive group bonded to the one or more of the pegylated lipids comprising the LNP. In some embodiments, the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded 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 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300). In some embodiments, the thiol-reactive group is bonded to at least one of the non-pegylated phospholipids comprising the LNP. In some embodiments, the thiol- reactive group is bonded to at least one of the ionizable lipids comprising the LNP. In some embodiments, the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP. In some embodiments, the lipid portion of the pegylated lipid bonded to the thiol-reactive group is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE,DSPE, sphingosine, sphingomyelin, and stearic acid. FIG.8 shows some examples of a pegylated lipid bonded to a thiol-reactive group, in this case a maleimide moiety.

[0256] In some embodiments, the thiol-reactive group is bonded to at least one of the non-pegylated phospholipids (helper lipids) comprising the LNP. FIG.9 shows some examples of a non-pegylated lipid bonded to a maleimide moiety.

[0257] In some embodiments, the thiol-reactive group is bonded to at least one of the ionizable lipids comprising the LNP. FIG.10 shows some examples of an ionizable lipid bonded to a maleimide moiety.

[0258] In some embodiments, the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP. FIG.11 shows some examples of sterols bonded to a maleimide moiety.

[0259] The disclosed methods provide stable conjugates that display excellent ability to transduce specific targeted cells. In some aspects, 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 light chain of the Fab fragment. In some embodiments, the Fab fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the Fab fragment and 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 bond 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 bond light chain on the surface of the LNP. In some of the foregoing embodiments, the Fab fragment is linked to the LNP through a thiosuccinimide moiety. In other of the foregoing embodiments, the Fab fragment is linked to the LNP through a dithiomalemide moiety (see FIG.6). In still other of the foregoing embodiments, the Fab fragment is linked to the LNP through a maleamic acid moiety (see FIG.6).

[0260] 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 IgG1Fab 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.

[0261] 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.

[0262] 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 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 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 is covalently to the LNP.

[0263] The processes described herein also enable the ability to conjugate two or more different Fab fragments to the surface of an LNP. An embodiment showing the conjugation of two different Fab fragments (which can bind to the same or different antigen) to the surface of an LNP is shown in FIG.7. In FIG.7, two Fab fragments (i.e., a first Fab fragment (“Fab1”) and a second Fab fragment (“Fab2”)) are reduced (step (i)) and reacted (step (ii)) with a precursor LNP comprising a thiol-reactive group (e.g., maleimide or DBM). Following the reaction in step (ii), both Fab1 and Fab2 are conjugated to the surface of the LNP. Despite reduction of the disulfide bonds in Fab1 and Fab2, the heavy chain and light chain in Fab1 and the heavy and light chain in Fab2 remain together on the surface of the LNP. In other words, neither the heavy chain or light chain of Fab1 associate with the heavy or light chain of Fab2 on the surface of the LNP.

[0264] In some embodiments involving conjugating two Fab fragments (i.e., a first Fab fragment and a second Fab fragment) to the surface of an LNP, the first Fab fragment and the second Fab fragment can be reduced in the same reaction (e.g., the first and second Fab fragments are mixed in a reaction vessel and contacted with the same reducing reagent). Insome embodiments, the first Fab fragment and the second Fab fragment are reduced separately (e.g., the first and second Fab fragments thereof are each contacted with a reducing reagent in separate reaction vessels). In some embodiments, the first Fab fragment is contacted with the reducing reagent prior to step (ii) (wherein the reduced first Fab fragment is conjugated to the LNP surface). In some embodiments, the second Fab fragment is contacted with the reducing reagent after step (ii) (wherein the reduced second Fab fragment is conjugated to the LNP surface). In some embodiments, the reduced first Fab fragment and the reduced second Fab fragment are contacted with the LNP simultaneously. In some embodiments, the reduced first Fab fragment thereof and the reduced second Fab fragment are contacted with the LNP sequentially (in either order). It can be contemplated that any number of Fab fragments thereof can be implemented in the method or process (e.g., a third, fourth, fifth, etc. Fab fragment). In some embodiments, a total of three different Fab fragments can be conjugated to the surface of the LNP. In some embodiments, a total of four different Fab fragments can be conjugated to the surface of the LNP.

[0265] 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 from 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 disclosed method can be purified from remaining intermediate product using any suitable technique such as, but not limited to, ultrafiltration and diafiltration.

[0266] In some embodiments, conjugates prepared by the method or process disclosed herein have a high density of the Fab fragment on the surface of the LNP. For instance, the conjugate can comprise a plurality of Fab fragments conjugated to the LNP surface. In someembodiments, the conjugate can comprise more than 10 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 20 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 30 Fab fragments. In some embodiments, the conjugate can comprise more than 50 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 75 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 100 Fab fragments. In some embodiments, the conjugate can comprise from about 50 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 30 Fab fragments per LNP. Conjugates through sortase-mediated enzymatic ligation process

[0267] In another aspect, the disclosure provides a targeted LNP made by a process comprising contacting a Fab fragment that has been functionalized with a sortase tag with a precursor LNP comprising a plurality of polyglycine molecules covalently bonded to the surface of the LNP in the presence of sortase, whereby the Fab fragment is conjugated to the surface of the precursor LNP through a sortase mediated ligation of the sortase tag and one or more polyglycine molecules, thereby forming a targeted LNP. A schematic of the process is depicted in FIG.12.

[0268] In some embodiments, the sortase tag of the Fab fragment is located at the C- terminus of the heavy chain of the Fab fragment. In some embodiments, the sortase tag comprises the amino acid sequence of LPXTG (SEQ ID NO:4), wherein X is any amino acid. In some embodiments, the sortase tag is LPETG (SEQ ID NO:5). In some embodiments, the sortase tag is linked to the Fab fragment through an amino acid linker.

[0269] In some embodiments, a polyglycine molecule of the plurality of the precursor LNP comprises between 3 and 10 glycine residues. In some embodiments, the polyglycine molecule comprises 3, 4, 5, or 6 glycine residues. In some embodiments, the polyglycine molecule is linked to the Fab fragment through an amino acid linker.

[0270] Any suitable sortase enzyme can be used to catalyze the ligation reaction. In some embodiments, sortase is sortase A5. In some embodiments, sortase is Streptococcus pyogenes sortase A (SpSrtA WT). Any suitable conditions can be used for the sortase mediated ligation. In some embodiments, the ligation can occur in water, aqueous buffer, or cell culture media. In some embodiments, the ligation is performed at physiological pH (e.g., about 7.4).In some embodiments, the ligation is performed at physiological temperature (e.g., about 37°C). In some embodiments, the ligation is performed at ambient temperature (e.g., about 23^ to about 25°C). In some embodiments, the ligation is performed at about 0°C to about 4°C.

[0271] In some embodiments, the sortase mediated ligation forms a covalent bond. In some embodiments, the sortase mediated ligation is reversible (e.g., the Fab fragment can be released from the LNP surface). In some embodiments, the sortase mediated ligation is irreversible (e.g., the sortase mediate ligation is performed as described in Liu et al., The Journal of Organic Chemistry 201479 (2), 487-492). In some embodiments, the ligation efficiency between the sortase 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 sortase tag of the Fab fragment and the polyglycine molecule of the LNP is from 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 disclosed method can be purified from remaining intermediate product (e.g., the antibody or functional fragment thereof) using any suitable technique such as, but not limited to, ultrafiltration and diafiltration.

[0272] In some embodiments, conjugates prepared by the method or process disclosed herein have a high density of the Fab fragment on the surface of the LNP. For instance, the conjugate can comprise a plurality of Fab fragments conjugated to the LNP surface. In some embodiments, the conjugate can comprise more than 10 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 20 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 30 Fab fragments. In some embodiments, the conjugate can comprise more than 50 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 75 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 100 Fab fragments. In some embodiments, the conjugate can comprise from about 50 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 30 Fab fragments per LNP.

[0273] In some embodiments, the conjugates prepared by the method or process disclosed herein comprise more than one Fab fragment (e.g., second, third, fourth, fifth, etc.) that bind to different antigens. In some embodiments, the conjugates prepared by the method or process disclosed herein comprise more than one Fab fragment (e.g., second, third, fourth, fifth, etc.) that bind to different epitopes on the same antigen.

[0274] In some embodiments, the polyglycine molecule conjugated to the surface of the LNP can be chemically reacted with a lipid molecule to afford a modified lipid capable of reacting with a sortase tag of a Fab fragment through a sortase mediated ligation. The reaction between the polyglycine molecule and the sortase tag of the Fab fragment can be completed prior to or after formation of the LNP. For instance, as shown in FIG.2, the various components (e.g., lipids) comprising the LNP can be mixed with lipid molecules that each comprise a polyglycine molecule, thus generating an LNP that comprises a plurality of polyglycine molecules. A polyglycine molecule of the plurality can then be reacted with the sortase tag of the antibody or antigen-binding fragment thereof, hence generating a conjugate. Alternatively, a lipid that has been modified with the polyglycine molecule can be directly reacted with the sortase tag of the antibody or antigen-binding fragment thereof. The resultant modified lipid can then be inserted into a pre-formed LNP that has not yet been surface modified. This procedure allows for the reaction to be performed on an individual lipid molecule rather than on the surface of the LNP.

[0275] The polyglycine molecule can be introduced onto any of the lipids comprising the LNP. In some embodiments, the conjugate can comprise one or more pegylated lipid molecules. In some embodiments, the polyglycine molecule is covalently bonded to at least one of the pegylated lipid molecules, hence generating the structure Lipid-PEGx-polyglycine molecule, wherein x is 2-120 ethylene glycol units. In such embodiments, the sortase tag of the Fab fragment reacts with the polyglycine molecule bonded the one or more of the pegylated lipids comprising the LNP. In some embodiments, the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded 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 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the polyglycine molecule is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the polyglycine molecule is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300). In someembodiments, the polyglycine molecule is bonded to at least one of the non-pegylated phospholipids comprising the LNP. In some embodiments, the polyglycine molecule is bonded to at least one of the ionizable lipids comprising the LNP. In some embodiments, the polyglycine molecule is bonded to at least one of the sterol molecules comprising the LNP. In some embodiments, the lipid portion of the pegylated lipid bonded to the polyglycine molecule is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid.

[0276] In some embodiments, the LNP comprises a sortase tag conjugated to the surface of the LNP. In such embodiments, a Fab fragment comprises a polyglycine molecule, wherein the Fab fragment is conjugated to the surface of the LNP through a sortase mediated ligation of the sortase tag of the LNP and the polyglycine molecule of the antibody or antigen-binding fragment thereof. In some embodiments, the sortase tag of the LNP comprises the amino acid sequence of LPXTG (SEQ ID NO:4), wherein X is any amino acid. In some embodiments, the sortase tag is LPETG (SEQ ID NO:5).

[0277] In some embodiments, the sortase tag conjugated to the surface of the LNP can be chemically reacted with a lipid molecule to afford a modified lipid capable of reacting with a polyglycine molecule of a Fab fragment through a sortase mediated ligation. The reaction between the sortase tag and the polyglycine molecule of the Fab fragment can be completed prior to or after formation of the LNP. For instance, as shown in FIG.2, the various components (e.g., lipids) comprising the LNP can be mixed with lipid molecules that each comprise a sortase tag, thus generating an LNP that comprises a plurality of sortase tags. A sortase tag of the plurality can then be reacted with the polyglycine molecule of the antibody or antigen-binding fragment thereof, hence generating a conjugate. Alternatively, a lipid that has been modified with the sortase tag can be directly reacted with the polyglycine molecule of the antibody or antigen-binding fragment thereof. The resultant modified lipid can then be inserted into a pre-formed LNP that has not yet been surface modified. This procedure allows for the reaction to be performed on an individual lipid molecule rather than on the surface of the LNP.

[0278] The sortase tag can be introduced onto any of the lipids comprising the LNP. In some embodiments, the conjugate can comprise one or more pegylated lipid molecules. In some embodiments, the sortase tag is covalently bonded to at least one of the pegylated lipid molecules, hence generating the structure Lipid-PEGx-sortase tag, wherein x is 2-120 ethylene glycol units. In such embodiments, a polyglycine molecule of a Fab fragment reacts with the sortase tag bonded the one or more of the pegylated lipids comprising the LNP. Insome embodiments, the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the sortase tag. In some embodiments, the PEG spacer between the lipid and the sortase tag 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 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the sortase tag is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the sortase tag is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300). In some embodiments, the sortase tag is bonded to at least one of the non-pegylated phospholipids comprising the LNP. In some embodiments, the sortase tag is bonded to at least one of the ionizable lipids comprising the LNP. In some embodiments, the sortase tag is bonded to at least one of the sterol molecules comprising the LNP. In some embodiments, the lipid portion of the pegylated lipid bonded to the sortase tag is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid. Conjugates through Click chemistry

[0279] In some embodiments, the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP. In some such embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. In other such embodiments, the targeting moiety is a scFv. In some embodiments, the targeting moiety is a Fab fragment.

[0280] In one embodiment, the Click product can be formed using a copper-catalyzed Click reaction. One such copper-catalyzed Click reaction is a Huisgen 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne. In some embodiments, the first or second Click handle comprises a cyclic derivative of the alkynyl group. In some embodiments, the cyclic derivative of the alkynyl group is selected from dibenzocyclooctyne. cyclooctyne, and difluorinated cyclooctyne. In some embodiments, the click chemistry involves strain promoted cycloaddition of azides. In some embodiments, the click chemistry is based upon reaction of strained alkenes.

[0281] 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, hence producing an aza-ylide.

[0282] In some embodiments, the Click product can be formed from an inverse electron demand Diels-Alder reaction between a trans-cyclooctene (TCO) moiety on the first or second Click handle and a tetrazine ring on the 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 rung is methyltetrazine. In some embodiments, the tetrazine ring is a 6-methyl substituted tetrazine.

[0283] In another embodiment, the targeting 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 the first Click handle onto the targeting moiety through covalent attachment. The first Click handle can next react with the second Click handle on the LNP to produce the targeted LNP. Hence, in one embodiment, an antibody, Fab fragment, or single chain variable fragment (scFv) that is covalently linked to a first Click handle through a linker comprising an enzyme recognition sequence is reacted with an LNP comprising 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 bonded to the enzyme recognition sequence. In some embodiments, the targeting moiety (e.g., antibody, Fab fragment, or scFv) is bonded to the enzyme recognition sequence via one or more amino acid residues. Particular amino acid residues added that can be covalently attached to the C- terminus of the targeting moiety (e.g., 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)uALEA(EAAAK)vA (SEQ ID NO:157), wherein u is 1-10 and v is 1-10.

[0284] In some embodiments, the enzyme recognition sequence is a sortase recognition motif or an LplA acceptor peptide. In some embodiments where the LNP is conjugated to an antibody, the C-terminus of one or more of the heavy or light chains of the antibody is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide) either directly or through a linker comprising one or more amino acid residues, a set forth herein. In some embodiments where the LNP is conjugated to a Fab fragment, the C-terminus of the heavy or light chain of the Fab fragment is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide). In some embodiments where the LNP is conjugated to an scFv, the C-terminus of the scFv is covalently bonded to the enzyme recognition sequence (e.g., sortase recognitionmotif or LplA acceptor peptide). In some of the foregoing embodiments, 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 a methyltetrazine. In some embodiments, the conjugation efficiency achieved by the disclosed method 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 disclosed method is from about 60% to about 95%. In some embodiments, the conjugation efficiency achieved by the disclosed methods are from about 70% to about 85%.

[0285] In some embodiments, the linker further comprises a spacer between the targeting moiety and the Click product. The spacer can include additional functional groups that indirectly link the targeting moiety to the Tz group. The spacer may also include additional amino acid residues that indirectly links the targeting moiety to the Tz group.

[0286] In some embodiments, the first or second Click handle is a tetrazine derivative having one of the following structures:wherein represents the point of attachment to the linker of the conjugate or to the LNP.

[0287] In some embodiments, the first or second Click handle is a TCO derivative having one of the following structures:

[0288] In some embodiments, the spacer that links the targeting moiety to the Tz ring is an enzyme recognition sequence. Accordingly, the disclosure provides methods of conjugating an LNP to a targeting moiety that has been modified with an enzyme recognition sequence, wherein said conjugating is accomplished via a Click reaction between a Tz ring covalently bound to the targeting moiety and a TCO moiety bound to the LNP. For instance, the disclosure provides methods of conjugating an LNP to an antibody, Fab fragment, or single chain variable fragment (scFv), wherein the antibody, Fab fragment, or scFv is covalently linked to a first Click handle (Tz ring) through a linker comprising an enzyme recognition sequence and the LNP is covalently linked to a second Click handle (TCO moiety), said method comprising contacting the LNP with an antibody, Fab fragment, or scFv such that 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 bonded to the enzyme recognition sequence. In some embodiments, the antibody, Fab fragment, or scFv is bonded to the enzyme recognition sequence via one or more amino acid residues. Particular amino acid residues added that can be covalently attached to the C-terminus of the 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)uALEA(EAAAK)vA (SEQ ID NO:157), wherein u is 1-10 and v is 1-10.

[0289] In some embodiments, the enzyme recognition sequence is a sortase recognition motif or a LplA acceptor peptide. In some embodiments where the LNP is conjugated to an antibody, the C-terminus of one or more of the heavy or light chains of the antibody is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif orLplA acceptor peptide) either directly or through a linker comprising one or more amino acid residues, a set forth herein. In some embodiments where the LNP is conjugated to a Fab fragment, the C-terminus of the heavy or light chain of the Fab fragment is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide). In some embodiments where the LNP is conjugated to an scFv, the C-terminus of the scFv is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide). In some embodiments, the conjugation efficiency achieved by the disclosed method 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 disclosed method is from about 60% to about 95%. In some embodiments, the conjugation efficiency achieved by the disclosed methods are from about 70% to about 85%.

[0290] In one embodiment, lipid nanoparticles conjugated to an antibody, Fab fragment, or scFv in a site-specific manner are produced according to the following steps: (i) covalently bonding a peptide with a sortase recognition site to one or more C- terminus of the antibody or Fab fragment, or to the C-terminus of the scFv; (ii) contacting the product of step (i) with a first Click handle comprising a Tz ring covalently bound to three or more glycine residues in the presence of a sortase enzyme under conditions suitable for the sortase enzyme to ligate the antibody, Fab fragment, or scFv to the first Click handle; and (iii) contacting the product of step (ii) with a lipid nanoparticle (LNP), wherein one or more lipids comprising the LNP include a second Click handle capable of reacting with the first Click handle, wherein the second Click handle comprises a TCO moiety, thereby generating a Click product.

[0291] In some embodiments, step (i) involves covalently bonding the peptide with the sortase recognition site to a linker covalently bonded to one or more C-terminus 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. Particular amino acid residues added that can be covalently attached to the C-terminus of the 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)uALEA(EAAAK)vA (SEQ ID NO:157), wherein u is 1-10 and v is 1-10.

[0292] In some embodiments, the sortase recognition motif bonded to the C-terminus of the antibody, Fab fragment, or scFv is bonded to one or more amino acid residues. For instance, in some embodiments, the glycine (G) of the LPXTG (SEQ ID NO:4) motif can bebonded to one or more (e.g., between 1-10) histidine (H) residues (see FIG.13). The glycine and the histidine residues are removed during the transpeptidation reaction.

[0293] In some embodiments, step (ii) (i.e., the sortase-catalyzed reaction) is carried out in the presence of sortase A5. Sortase A5 is an engineered pentamutant variant of the wild- type sortase from Staphylococcus aureus that is significantly more active than the wild-type sortase. See U.S. patent No.9,267,227. In such embodiments, the sortase recognition site bond 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), sortase A catalyzes the transpeptidation reaction between the LPXTG (SEQ ID NO:4) recognition motif and a glycine residue bound to the first Click handle by cleaving the sortase recognition site between the threonine and the glycine residues.

[0294] In other embodiments, step (ii) is carried out in the presence of the sortase enzyme Streptococcus pyogenes sortase A (SpSrtA WT). In such embodiments, the sortase recognition site bond 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 a glycine residue bound to the first Click handle by cleaving the sortase recognition site between the threonine and the alanine residues.

[0295] FIG.13 shows the formation of an LNP conjugated to the C-terminus of a Fab fragment through a sortase mediated ligation followed by a Click reaction. In the schematic, a C-terminus of the Fab fragment is first covalently modified with a sortase recognition motif covalently bonded to six histidine (H) residues. The sortase modified Fab fragment is then reacted with a first Click handle (Click handle 1) covalently bonded to glycine (G) residues. The resultant Fab fragment modified with the first Click handle is then reacted with an LNP that comprises at least one lipid that is covalently bonded to a second Click handle (Click handle 2), thus affording an LNP site-specifically conjugated to the Fab fragment.

[0296] In one embodiment, the conjugate comprises a protein targeting moiety as set forth above (e.g., antibody, Fab fragment, or scFv) and a lipid nanoparticle (LNP), wherein a C-terminus of the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a sortase recognition motif and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP. In some embodiments, the C-terminus of one or more of the heavy or light chains of the antibody, the C-terminus of the heavy or light chain of the Fab fragment, or C-terminus of the scFv is conjugated to the lipid nanoparticle through a linker, andwherein the linker comprises a sortase recognition motif and a Click product formed from a Click reaction between a first Click handle on the antibody, Fab fragment, or scFv and a second Click handle on the LNP. In particular embodiments, the sortase recognition motif comprises an LPXT motif, wherein X is any amino acid residue. In some such embodiments, the sortase recognition motif comprises an LPET (SEQ ID NO:79) motif. In some embodiments, the linker comprises 3 or more glycine residues between the sortase recognition motif and the Click product. In some embodiments, the linker comprises between 3 and 50 glycine residues. In some embodiments, the linker comprises between 3 and 10 glycine residues. In some such embodiments, the linker comprises 3, 4, 5, or 6 glycine residues.

[0297] In some embodiments, the conjugates have the structure Antibody-LPXT(G)n- Click product-LNP, Fab fragment-LPXT(G)n-Click product-LNP, or scFv-LPXT(G)n-Click product-LNP, wherein X is any amino acid residue and n is between 1 and 20 (e.g., between 3 and 10 or between 3 and 6). In these embodiments, the leucine (L) residue of the sortase recognition motif is bonded 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).

[0298] In some embodiments, the linker comprises additional amino acid residues between the antibody, Fab fragment, or scFv and the sortase recognition motif. In such embodiments, a C-terminus of the antibody, Fab Fragment, or scFv can be covalently modified with one or more amino acid residues prior to covalently linking the sortase recognition motif. For instance, in particular embodiments, the conjugates have the structure Antibody-Z-LPXT(G)n-Click product-LNP, Fab fragment-Z-LPXT(G)n-Click product-LNP, or scFv-Z-LPXT(G)n-Click product-LNP, wherein Z is a linker between the antibody (or Fab fragment or scFv) and the leucine of the sortase recognition motif. 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)vA (SEQ ID NO:157), wherein u is 1-10 and v is 1-10. 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 GGGGGS (SEQ ID NO:164).

[0299] In some embodiments, the conjugates have the structure 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-dihydropyridazine - LNP, wherein variables n and Z are defined as above. In particular embodiments, the dihydropyridazine moiety is a 1,4-dihydropyridazine.

[0300] In another embodiment, the LNP lipid is conjugated to an antibody, Fab fragment, or scFv in a site-specific manner, comprising (i) covalently bonding a LplA acceptor peptide site to the antibody, Fab fragment, or scFv; (ii) contacting the product of step (i) with a carboxylic acid compound that includes a first Click handle comprising a Tz ring in the presence of a lipoic acid ligase under conditions suitable for lipoic acid ligase to ligate the antibody, Fab fragment, or scFv to the first Click handle; and (iii) contacting the product of step (ii) with a lipid nanoparticle (LNP), wherein one or more lipids comprising the LNP includes a second Click handle capable of reacting with the first Click handle, wherein the second Click handle comprises a TCO moiety, thereby generating a Click product.

[0301] In some embodiments of step (i), the Lp1A acceptor peptide is covalently bonded to one or more C-terminus 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.

[0302] In some embodiments, step (i) involves covalently bonding the peptide with the Lp1A acceptor peptide to a linker covalently bonded to one or more C-terminus 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. Particular amino acid residues added that can be covalently attached to the C-terminus of the 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)uALEA(EAAAK)vA (SEQ ID NO:157), wherein u is 1-10 and v is 1-10.

[0303] In some embodiments, step (ii) (i.e., the lipoic acid ligase catalyzed reaction) is carried out in the presence of a mutated lipoic acid ligase, wherein the tryptophan reside (W) at position of the lipoic acid ligase is mutated. Specific W37 mutants 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.

[0304] In some embodiments, the carboxylic acid in step (ii) (i.e., the lipoic acid ligase catalyzed reaction) is a C3-C20 carboxylic acid. In some embodiments, the carboxylic acid in step (ii) is a fatty acid, for instance a C7-C19fatty acid. In some such embodiments, the fatty acid is selected from decanoic acid, palmitic acid, lauric acid or octanoic acid.

[0305] In other embodiments, the carboxylic acid in step (ii) has the formula COOH-R- Click handle, where R includes two lipid tales. In some such embodiments, the carboxylic acid as shown below, wherein n = 1—120.

[0306] FIG.14 depicts the formation of an LNP conjugated to the C-terminus of a Fab fragment through a LplA ligation followed by a Click reaction. In the schematic, a C- terminus of the Fab fragment is first covalently modified with a LplA acceptor peptide. Specifically, a first Click handle (Click handle 1) comprising a Tz ring is first introduced by chemically reacting a carboxylic acid to the lysine residue of the LplA acceptor peptide. The resultant Fab fragment modified with the first Click handle is then reacted with an LNP that comprises at least one lipid that is covalently bonded to a second Click handle comprising aTCO moiety (Click handle 2), thus affording an LNP site-specifically conjugated to the Fab fragment.

[0307] In some embodiments, the conjugate produced by methods disclosed herein comprises a protein targeting moiety as set forth above (e.g., antibody, Fab fragment, or scFv), conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a lipoic acid ligase (LplA) acceptor peptide and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on 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, wherein the C-terminus of one or more of the heavy or light chains of the antibody is bonded to the linker. In some embodiments, the conjugate comprises a Fab fragment, the C-terminus of the heavy or light chain of the Fab fragment is bonded to the linker. In some embodiments, the conjugate comprises a scFv, wherein the C-terminus of the scFv is bonded to the linker.

[0308] In some embodiments, the linker comprises additional amino acid residues between the targeting moiety (e.g., antibody, Fab fragment or scFv) and the LplA acceptor peptide. In such embodiments, a C-terminus of the targeting moiety (e.g., antibody, Fab Fragment, or scFv) can be covalently modified with one or more amino acid residues prior to covalently linking the LplA acceptor peptide. For instance, in particular embodiments, the conjugates have the structure 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)uALEA(EAAAK)vA (SEQ ID NO:157), wherein u is 1-10 and v is 1-10. 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 GGGGGS (SEQ ID NO:164).

[0309] It will be understood that in the forgoing embodiments, the lysine (K) residue of the LplA acceptor peptide is covalently linked to Click product, which is covalently linked to the LNP. Specifically, to generate conjugates, the side chain lysyl group reacts with thecarboxylic acid compound that includes the first Click handle. The resultant modified targeting moieties (antibodies or Fab fragments or scFvs) are reacted with an LNP that has been modified with a second Click handle, as disclosed herein, thereby generating a Click product. An LNP surface modified with a Fab fragment is depicted below, where R is a lipid group (e.g., C2-C30 alkyl group). (GFEIDKVWYDLDA – SEQ ID NO:414)

[0310] 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 Q-tag. The Q-tag may be present on or can be inserted at one or more locations of targeting moiety, (e.g., antibody, Fab fragment, or scFv), for instance at a C-terminus. The transglutamine enzyme catalyzes the reaction between a side-chain amide group on the Q-tag and an alkyl-primary amine on a component of the LNP (e.g., a lipid), thus linking the antibody to the LNP through an amide bond.

[0311] In other embodiments, the enzyme recognition sequence is a sequence recognized by formylglycine generating enzyme, specifically CXPXR, wherein each X is any amino acid. In such embodiments, the CXPXR sequence can be inserted at one or more locations of the targeting moiety (e.g., antibody, Fab fragment, or scFv), for instance at a C-terminus. The formylglycine generating enzyme converts the cysteine thiol of CXPXR into an aldehyde group, which can be reacted with an aminooxy or hydrazine group covalently bonded to a component of the LNP.

[0312] In another embodiment, the lipid nanoparticle is conjugated to the targeting moiety, e.g., antibody, Fab fragment, or scFv, in a site-specific manner through a sugar moiety on a glycosylated antibody. FIG.15 shows one particular embodiment of introducing a Tz Click handle through a sugar moiety on an antibody. In step 1, an azide moiety is site- specifically introduced into a sugar moiety of the targeting antibody catalyzed by the enzymes, galactosidase and transferase. The azide functionality then reacts with a dibenzocyclooctene (DBCO)-MeTz via a Click reaction. The Click reaction is used tointroduce the Tz ring, in this case MeTz onto the antibody, which can then be reacted with a TCO moiety on an LNP (not shown) in a second Click reaction, hence affording a conjugate.

[0313] In another embodiment, the LNP is conjugated to a targeting moiety (e.g., antibody, Fab fragment, or scFv) in a site-specific manner through a light-induced crosslinking reaction. FIG.16 shows one particular embodiment using oYo-Link Tz. In FIG.16, a Tz ring (e.g., MeTz) is covalently bound to a site specific antibody label (oYo link) that specifically reacts with residues on the heavy chain of the antibody. The oYo-Link tetrazine covalently bound to the antibody reacts with TCO (not shown) bound to an LNP, hence generating the surface-modified LNP.

[0314] In another embodiment, the lipid nanoparticle is conjugated to the targeting moiety (e.g., antibody, Fab fragment, or scFv) in a site-specific manner by mutating an amino acid residue on the targeting moiety and subsequently reacting the mutated targeting moiety with a compound that includes a Tz ring. FIG.17 shows particular embodiments where an azide functionality is introduced during antibody or Fab fragment production. The azide group then undergoes a Click reaction with a DBCO group that is linked to a tetrazine ring. The tetrazine ring bound to the antibody reacts with TCO (not shown) bound to an LNP, hence generating the surface modified LNP.

[0315] In some embodiments, the Click product can be formed using any suitable photo- induced Click chemistry reaction. In some embodiments, the Click product can be formed using photoinducible 1,3-dipolar cycloaddition reaction between a tetrazole and an alkene (see, e.g., Song et al., Angew. Chem., Int. Ed.2008, 47 (15), 2832-2835).

[0316] In some embodiments, the Click product can be formed using oxime and hydrazone ligations. In some embodiments, a ketone or aldehyde can react with ^ effect amine, such as hydroxylamine, hydrazine and hydrazide (see, e.g., Agten et al., ChemBioChem 2013, 14 (18), 2431-2434 and Dirksen et al., J. Am. Chem. Soc.2006, 128 (49), 15602-15603).

[0317] Exemplary lipids and cholesterol molecules bonded to Click handles that can be used to make targeted LNPs are depicted below.III. LIPID NANOPARTICLES

[0318] Lipid nanoparticles, in some embodiments, comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids), also referred to herein as helper lipids; one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO2019217941; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or combinations of the foregoing.

[0319] Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example, those described in Table 4 of WO2019217941, which is incorporated by reference—e.g., a lipid-containing nanoparticle can comprise one or more of the lipids in Table 4 of WO2019217941. Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in Table 5 of WO2019217941, incorporated by reference.

[0320] In some embodiments, conjugated lipids, when present, can include one or more of PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinatediacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypoly ethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing.

[0321] In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009 / 127060 or US2010 / 0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0322] In some embodiments, the lipid particle comprises an ionizable lipid, a non- cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticle comprises an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 35 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid (e.g., comprising the therapeutic agent and / or encoding the gene modifying polypeptide, template nucleic acid, or gene modifying system) can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1.

[0323] In some embodiments, the average LNP diameter of the targeted LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, fromabout 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the targeted LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation ranges from about l mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.

[0324] An LNP described herein, e.g., a targeted LNP, may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index 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 a LNP may be from about 0.10 to about 0.20.

[0325] The zeta potential of an LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0326] The efficiency of encapsulation of a protein and / or nucleic acid (e.g., an mRNA encoding a polypeptide), describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with an LNP after preparation, relative to the initialamount provided. The encapsulation efficiency is desirably 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 the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a protein and / or nucleic acid 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%.

[0327] An LNP of the disclosure may optionally comprise one or more coatings. In some embodiments, an LNP may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness, or density.

[0328] Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020061457, which is incorporated herein by reference in its entirety.

[0329] In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoy_ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, targeted LNPs of the disclosure are 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), incorporated herein by reference in its entirety.

[0330] Additional specific LNP formulations useful for delivery of nucleic acids are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO. Ionizable Lipids

[0331] The LNPs of the disclosure (e.g., targeted LNPs) comprise one or more ionizable lipids. In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending onpH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s), which bear the positive charge. In some embodiments, the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyn 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. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, a lipid nanoparticle may comprise a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid, and a therapeutic agent as described herein (e.g., one or more nucleic acids (e.g., RNA) comprising a gene modifying system) encapsulated within or associated with the lipid nanoparticle. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) is co-formulated with the cationic lipid. The therapeutic agent (e.g., one or more nucleic acids) may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) may be encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipid described herein encapsulates 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% of an RNA molecule (e.g., an mRNA molecule).

[0332] In some embodiments, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1 : 1 to about 25: 1, from about 10: 1 to about 14: 1, from about 3 :1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1. The amounts of lipids and nucleic acid can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg / ml to about 30 mg / mL.

[0333] In some embodiments, the LNP (e.g., targeted LNP) comprises the ionizable lipid V003, depicted below. V003 is described in U.S. Patent No.10,059,655.Lipid V003

[0334] In some embodiments, the LNP (e.g., targeted LNP) comprises the ionizable lipid shown in Table 1. In some cases, an LNP containing an ionizable lipid of Table 1 exhibits higher levels of transduction in HSCs and / or higher expression of a payload protein in HSCs relative to an LNP that contains V003 as the ionizable lipid. In other embodiments, Lipid 092 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, Lipid 093 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, Lipid 153 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, Lipid154 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, Lipid 155 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, Lipid 162 is used as an ionizable lipid to generate LNPs for delivery to HSCs. In other embodiments, Lipid163 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 1: Exemplary Ionizable Lipids

[0335] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (I):or a pharmaceutically acceptable salt thereof, wherein:

[0336] In some embodiments of the formula (I), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 4. In some embodiments, Y is. In some embodiments, Y is. , . 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0337] In some embodiments, compounds of formula (I) are compounds of formula (I-A):formula (I-A), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; R3and R4are independently C1-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle; and Y is a lipophilic tail, branched or unbranched.

[0338] In some embodiments of the formula (I-A), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. In. , .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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0339] In some embodiments, compounds of formula (I) are compounds of formula (I-B):formula (I-B), or a pharmaceutically acceptable salt thereof, wherein:R3and R4are independently C1-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0340] In some embodiments of the formula (I-B), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together withthe N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0341] In some embodiments, compounds of formula (I) are compounds of formula (I-C):formula (I-C), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0342] In some embodiments of the formula (I-C), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0343] In some embodiments, compounds of formula (I) are compounds of formula (I-D):formula (I-D), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0344] In some embodiments of the formula (I-D), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0345] In some embodiments, compounds of formula (I) are compounds of formula (I-E):formula (I-E), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0346] In some embodiments of the formula (I-E), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0347] In some embodiments, compounds of formula (I) are compounds of formula (I-F):formula (I-F), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0348] In some embodiments of the formula (I-F), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0349] In some embodiments, compounds of formula (I) are compounds of formula (I-G):formula (I-G), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0350] In some embodiments of the formula (I-G), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0351] In some embodiments, compounds of formula (I) are compounds of formula (I-H):formula (I-H), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; R1is C4-10alkyl; R2is C1-4alkyl; and R3and R4are independently C1-4 alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle.

[0352] In some embodiments of the formula (I-H), R1is C6-8alkyl. In some embodiments, R1is C6alkyl. In some embodiments, R2is C3alkyl. In some embodiments, R2is C4alkyl. 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, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0353] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (II):formula (II), or a pharmaceutically acceptable salt thereof, wherein: X is O or CH2; m is 1, 2, or 3; and Y is a lipophilic tail, branched or unbranched.

[0354] In some embodiments of the formula (II), Y is. In some embodiments, Y is. In some embodiments, Y is. , . 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.

[0355] In some embodiments, compounds of formula (II) are compounds of formula (II-or a pharmaceutically acceptable salt thereof, wherein: Y is a lipophilic tail, branched or unbranched.

[0356] In some embodiments of the formula (II-A), Y is.

[0357] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipidformula (III), or a pharmaceutically acceptable salt thereof, wherein:R3and R4are independently C1-4alkyl, or R3and R4are taken together with the nitrogen to which they are attached to form a heterocycle; and Y is a lipophilic tail, branched or unbranched.

[0358] In some embodiments of the formula (III), Y is. In some embodiments, Y is. In some embodiments, Y is. , . In some embodiments, R3is n-propyl. In some embodiments, R3is ethyl. In some embodiments, R3is methyl. In some embodiments, R4is n-propyl. In some embodiments, R4is ethyl. In some embodiments, R4is methyl. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a pyrrolidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperidine. In some embodiments, R3and R4are taken to together with the N to which they are attached to form a piperazine.

[0359] In some embodiments, compounds of formula (III) are compounds of formula (III- A):formula (III-A),or a pharmaceutically acceptable salt thereof, wherein: Y is a lipophilic tail, branched or unbranched.

[0360] In some embodiments of the formula (III-A), Y is, .

[0361] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds of Table 2.Table 2: Exemplary Ionizable Lipids

[0362] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (IV):or a pharmaceutically acceptable salt thereof, wherein: X is -O- or -CH2-; m is 0, 1, 2, or 3; R1is C1-4alkyl; R2is C1-4alkyl; n is 1, 2, 3, or 4; R3is C4-10alkyl; R4is C4-10alkyl; p is 2, 3, 4, 5, or 6; R5is C4-10alkyl; and R6is C4-10alkyl.

[0363] In some embodiments, compounds of formula (IV) are compounds of formula (IV-A):or a pharmaceutically acceptable salt thereof, wherein: p is 2, 3, 4, 5, or 6; R5is C4-10alkyl; andR6is C4-10alkyl.

[0364] In some embodiments, compounds of formula (IV) are compounds of formula (IV-B):or a pharmaceutically acceptable salt thereof, wherein: R5is C4-6alkyl; and R6is C4-6alkyl.

[0365] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds of Table 3.Table 3: Exemplary Ionizable Lipids

[0366] In some embodiments, the ionizable lipid has one of the structures depicted below:Lipid 153Lipid 176Lipid 183

[0367] Other exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016 / 0311759; I of US20150376115 or in US2016 / 0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-c of US20150140070; A of US2013 / 0178541; I of US2013 / 0303587 or US2013 / 0123338; I of US2015 / 0141678; II, III, IV, or V of US2015 / 0239926; I of US2017 / 0119904; I or II of WO2017 / 117528; A of US2012 / 0149894; A of US2015 / 0057373; A of WO2013 / 116126; A of US2013 / 0090372; A of US2013 / 0274523; A of US2013 / 0274504; A of US2013 / 0053572; A of W02013 / 016058; A of W02012 / 162210; I of US2008 / 042973; I, II, III, or IV of US2012 / 01287670; I or II of US2014 / 0200257; I, II, or III of US2015 / 0203446; I or III of US2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014 / 0308304; of US2013 / 0338210; I, II, III, or IV of W02009 / 132131; A of US2012 / 01011478; I or XXXV of US2012 / 0027796; XIV or XVII of US2012 / 0058144; of US2013 / 0323269; I of US2011 / 0117125; I, II, or III of US2011 / 0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII of US2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011 / 0076335; I or II of US2006 / 008378; I of US2013 / 0123338; I or X-A-Y-Z of US2015 / 0064242; XVI, XVII, or XVIII of US2013 / 0022649; I, II, or III of US2013 / 0116307; I, II, or III of US2013 / 0116307; I or II of US2010 / 0062967; I-X of US2013 / 0189351; I of US2014 / 0039032; V of US2018 / 0028664; I of US2016 / 0317458; I of US2013 / 0195920; 5, 6, or 10 of US10,221,127; III-3 of WO2018 / 081480; I-5 or I-8 of WO2020 / 081938; 18 or 25 of US9,867,888; A of US2019 / 0136231; II of WO2020 / 219876; 1 of US2012 / 0027803; OF-02 of US2019 / 0240349; 23 of US10,086,013; cKK-E12 / A6 of Miao et al (2020); C12-200 of WO2010 / 053572; 7C1 of Dahlman et al (2017); 304-O13 or 503-O13 of Whitehead et al; TS-P4C2 of US9,708,628; I of WO2020 / 106946; I of WO2020 / 106946.

[0368] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 lZ)- heptatriaconta- 6,9,28,3 l-tetraen-l9-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (l3Z,l6Z)-A,A-dimethyl-3- nonyldocosa-l3, l6-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein 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); e.g., as described in Example 1 of US9,867,888 (incorporated by reference herein 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)propyl octadeca-9,12-dienoate (LP01) e.g., as synthesized in Example 13 of WO2015 / 095340 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Di((Z)-non-2-en-1-yl) 9-((4- dimethylamino)butanoyl)oxy)heptadecanedioate (L319), , e.g. as synthesized in Example 7, 8, or 9 of US2012 / 0027803 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2- hydroxydodecyl) amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO2010 / 053572 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is; Imidazole cholesterolester (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-lH- cyclopenta[a]phenanthren-3-yl 3- (1H-imidazol-4-yl)propanoate, e.g., Structure (I) from WO2020 / 106946 (incorporated by reference herein in its entirety).

[0369] In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 25% to about 65%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 35% to about 60%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 40% to about 50%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 45% to about 50%.

[0370] The compounds disclosed herein (e.g., lipids in Table 1, Table 2, and Table 3), or their pharmaceutically acceptable salts, may include an asymmetric center and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another and “diastereomers,” which refers to stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The present disclosure is meant to include 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 resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Helper Lipids

[0371] The LNPs, e.g., targeted LNPs, of the disclosure comprise one or more ionizable lipids. Exemplary 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), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), l8-l-trans PE, l-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine, egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0372] Other examples of non-cationic lipids suitable for use in the lipid nanopartieles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017 / 099823 or US patent publication US2018 / 0028664, the contents of which is incorporated herein by reference in their entirety. In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018 / 0028664, incorporated herein by reference in its entirety.

[0373] In some embodiments, the helper lipid is a sphingolipid. In some such embodiments, the non-pegylated lipid is a sphingomyelin. In some embodiments, the sphingomyelin has a head group selected from, phosphocholine, phosphoethanolamine or ceramide. In some embodiments, the sphingomyelin is egg sphingomyelin.

[0374] In some embodiments, the helper lipid comprises 5-40% (mol), 8%-30%, 10%- 28%, 20%-36%, 22%-32%, or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from 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).

[0375] In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 21% to about 23%. In some embodiments, the mol% of the helper lipid (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 set forth in the examples below, in vivo delivery of certain payloads following administration of the disclosed LNPs (e.g., targeted LNPs) with these percentages of helper lipids provides enhanced transduction and expression of the payloads relative to targeted LNPs with smaller or larger quantities of helper lipid.

[0376] In some embodiments, the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 1:1 to about 7:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 4:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 3:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) is from about 1:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 2:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) is from about 1.5:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2:1 to about 2.5:1.

[0377] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid in Table 1, Table 2 or Table 3 and DSPC. In some embodiments, the LNP, e.g., targetedLNP, comprises an ionizable lipid in Table 1, Table 2 or Table 3 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from 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%.

[0378] 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from 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%.

[0379] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 093 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 20%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from 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%.

[0380] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 092 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 092 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of DSPC orsphingomyelin 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%.

[0381] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 153 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 153 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. 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%.

[0382] 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. 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%.

[0383] 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. 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%.

[0384] 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 163 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. Insome embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. 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%.

[0385] 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. 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%.

[0386] 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. 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%.

[0387] 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. 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%.

[0388] 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP,e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. 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%.

[0389] 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 a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from 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%. Sterols

[0390] In some embodiments, the LNPs, e.g., targeted LNPs, of the disclosure can comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-choiestanol, 53- coprostanol, choiesteryl-(2,-hydroxy)-ethyl ether, choiesteryl-(4'-hydroxy)-butyl ether, and 6- ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., choiesteryl-(4'-hydroxy)-buty1 ether. Exemplary cholesterol derivatives are described in PCT publication W02009 / 127060 and US patent publication US2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0391] In some embodiments, the component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20- 50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.

[0392] In some embodiments, the molar ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 6:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylatedhelper lipid (e.g., DSPC or sphingomyelin) is from about 3:1 to about 0.5...

Claims

CLAIMS 1. A lipid nanoparticle (LNP), comprising: (iv) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, and the helper lipid is present at about 18 mol% to about 32 mol% of the lipid component; (v) one or two targeting moieties conjugated to the LNP, wherein the one or two targeting 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; and (vi) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises two or more RNA molecules.

2. A lipid nanoparticle (LNP), comprising: (iv) a lipid component comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is present at about 35 mol% to about 60 mol% of the lipid component, and the helper lipid is present at about 18 mol% to about 32 mol% of the lipid component; (v) means for binding 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; and (vi) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises two or more RNA molecules.

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

4. The LNP of any one of claims 2-3, wherein the LNP comprises means for binding a first cell surface receptor and means for binding a second cell surface receptor, wherein the first cell surface receptor and the second cell surface receptor are different and wherein the first cell surface receptor and the second cell surface receptor are each 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 of any one of claims 1-4, wherein the therapeutic agent comprises two RNA molecules.

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

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

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

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

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

11. The LNP of any one of claims 1-10, wherein the helper lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), l8-l-trans PE, l- stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine, egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

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

13. The LNP of claim 11, wherein the helper lipid is sphingomeylin.

14. The LNP of any one of claims 1-13, further comprising a cholesterol molecule.

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

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

17. The LNP of claim 14 or claim 15 wherein the cholesterol molecule is present at about 20 mol% to about 35 mol% of the lipid component.

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

19. The LNP of claim 18, wherein the one or two targeting moieties are conjugated to the LNP through the PEGylated lipid.

20. The LNP of any one of claims 2-17, wherein the lipid component further comprises a PEGylated lipid.

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

22. The LNP of any one of claims 18-21, 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 of claim 22, wherein the lipid portion of the PEGylated lipid is DSPE.

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

25. The LNP of any one of claims 1, 5-19 and 22-24, wherein the LNP comprises one targeting moiety conjugated to the surface of the LNP.

26. The LNP of claim 25, wherein the one targeting moiety targets CD117.

27. The LNP of claim 25, wherein the one targeting moiety targets CD71.

28. The LNP of claim 25, wherein the one targeting moiety targets CD34.

29. The LNP of claim 25, wherein the one targeting moiety targets CD50.

30. The LNP of claim 25, wherein the one targeting moiety targets CD43.

31. The LNP of claim 25, wherein the one targeting moiety targets CD338.

32. The LNP of claim 25, wherein the one targeting moiety targets CD164.

33. The LNP of claim 25, wherein the one targeting moiety targets CD33 34. The LNP of claim 25, wherein the one targeting moiety targets CD38.

35. The LNP of claim 25, wherein the one targeting moiety targets CD46.

36. The LNP of claim 25, wherein the one targeting moiety targets CD49d.

37. The LNP of claim 25, wherein the one targeting moiety targets CD59.

38. The LNP of claim 25, wherein the one targeting moiety targets CD105.

39. The LNP of claim 25, wherein the one targeting moiety targets CD123.

40. The LNP of claim 25, wherein the one targeting moiety targets CD184.

41. The LNP of any one of claims 1, 5-19 and 22-24, wherein the LNP comprises two targeting moieties conjugated to the surface of the LNP.

42. The LNP of claim 41, wherein the two targeting moieties are CD117 and CD71.

43. The LNP of claim 41, wherein the two targeting moieties are CD117 and CD34.

44. The LNP of claim 41, wherein the two targeting moieties are CD117 and CD50.

45. The LNP of any one of claims 1-44, wherein the ionizable lipid is a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein:

46. The LNP of any one of claims 1-44, wherein the ionizable lipid is a compound of one of the structures depicted below:Lipid 232 47. A lipid nanoparticle (LNP) comprising:(i) a lipid component comprising an ionizable lipid and a helper lipid; (ii) two targeting moieties conjugated to the LNP, wherein the two targeting 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; and (iii) a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises a nucleic acid molecule.

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

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

50. The LNP of any one of claims 47-49, wherein the nucleic acid molecules are RNA molecules.

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

52. The LNP of any one of claims 47-51, wherein the two targeting moieties are CD117 and CD71.

53. The LNP of any one of claims 47-51, wherein the two targeting moieties are CD117 and CD34.

54. The LNP of any one of claims 47-51, wherein the two targeting moieties are CD117 and CD50.

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

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

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

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

59. The LNP of any one of claims 47-58, wherein the helper lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl- phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), l8-l-trans PE, l- stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine, egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

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

61. The LNP of claim 59, wherein the helper lipids is sphingomeylin.

62. The LNP of any one of claims 47-61, wherein the ionizable lipid is a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: X is -O- or -CH2-; m is 0, 1, 2, or 3;R1is C1-4alkyl; R2is C1-4alkyl; n is 1, 2, 3, or 4; R3is C4-10alkyl; R4is C4-10alkyl; p is 2, 3, 4, 5, or 6; R5is C4-10alkyl; and R6is C4-10alkyl.

63. The LNP of any one of claims 47-61, wherein the ionizable lipid is a compound of one of the structures depicted below:Lipid 232.

64. A lipid nanoparticle (LNP) comprising: (i) a lipid component comprising an ionizable lipid and a helper lipid,;(ii) one or two targeting moieties conjugated to the LNP, wherein the one or two targeting 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; and a therapeutic agent encapsulated within the LNP, wherein the therapeutic agent comprises two RNA molecules, and wherein one nucleic acid molecule comprises a template RNA, comprising from 5’ to 3’: (a) a gRNA spacer, (b) a gRNA scaffold, (c) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence.

65. The LNP of claim 64, wherein the LNP comprises one targeting moiety conjugated to the surface of the LNP.

66. The LNP of claim 65, wherein the one targeting moiety targets CD117.

67. The LNP of claim 65, wherein the one targeting moiety targets CD71.

68. The LNP of claim 65, wherein the one targeting moiety targets CD34.

69. The LNP of claim 65, wherein the one targeting moiety targets CD50.

70. The LNP of claim 65, wherein the one targeting moiety targets CD43.

71. The LNP of claim 65, wherein the one targeting moiety targets CD164.

72. The LNP of claim 65, wherein the one targeting moiety targets CD33 73. The LNP of claim 65, wherein the one targeting moiety targets CD38.

74. The LNP of claim 65, wherein the one targeting moiety targets CD46.

75. The LNP of claim 65, wherein the one targeting moiety targets CD49d.

76. The LNP of claim 65, wherein the one targeting moiety targets CD59.

77. The LNP of claim 65, wherein the one targeting moiety targets CD105.

78. The LNP of claim 65, wherein the one targeting moiety targets CD123.

79. The LNP of claim 65, wherein the one targeting moiety targets CD184.

80. The LNP of claim 65, wherein the one targeting moiety targets CD338.

81. The LNP of claim 64, wherein the LNP comprises two targeting moieties conjugated to the surface of the LNP.

82. The LNP of claim 81, wherein the two targeting moieties are CD117 and CD71.

83. The LNP of claim 81, wherein the two targeting moieties are CD117 and CD34.

84. The LNP of claim 81, wherein the two targeting moieties are CD117 and CD50.

85. A pharmaceutical composition comprising an LNP of any one of claims 64-84.

86. The pharmaceutical composition of claim 85, wherein the template RNA comprises a spacer of Table X1.

87. The pharmaceutical composition of claim 85, wherein the template RNA comprises a heterologous object sequence of Table 2.

88. The pharmaceutical composition of claim 85, wherein the template RNA comprises a PBS sequence of Table X3.

89. The pharmaceutical composition of claim 85, wherein the template RNA comprises a heterologous object sequence and a PBS sequence of Table X4.

90. The pharmaceutical composition of claim 85, wherein the template RNA comprises a sequence of Table X6.

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

92. The method of claim 91, wherein the disease is selected from the group consisting of adenosine deaminase severe combined immune deficiency (ADA-SCID), adrenoleukodystrophy (CALD), alpha-mannosidosis, chronic granulomatous disease, common variable 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, Scheie syndrome, mucopolysaccharidosis type II (MPS2), MPS7, mucolipidosis 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), recombination activating gene (RAG) 1 / 2 Deficiency, severe congenital neutropenia, Sickle Cell Disease (SCD), Tay Sachs, thalassemia, beta thalassemia, Wiskott-Aldrich syndrome, X-linked agammaglobulinemia, and X-linked SCID.

93. The method of claim 92, wherein the disease is Sickle Cell Disease.

94. The method of claim 92, wherein the disease is a thalassemia.

95. The method of claim 92, wherein the thalassemia is beta thalassemia.

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

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

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

99. The method of any one of claims 96-98, wherein the method produces a modification in the genome of an HSC in the subject.

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

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