Lipid nanoparticles for delivery of therapeutic payloads to cells

Abstract

The disclosure provides ionizable lipids for constructing lipid nanopartides. The disclosure further provides conjugates comprising a targeting moiety conjugated to a lipid nanoparticle (LNP) encapsulating a. therapeutic agent (i.e., payload) for delivery to cells, including immune cells, hematopoietic stem cells (HSCs), or liver cells. The conjugates can be formulated in a pharmaceutical composition and can be directly administered to a patient in need thereof, for example, via in vivo administration.

Description

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

[0001] The application claims priority to U.S. Provisional Patent Application No. 63 / 564,392, filed March 12, 2024, which is herein incorporated by reference in its entirety. BACKGROUND

[0002] Significant advances have been made in the development of lipid nanoparticles (LNPs) for intracellular delivery of payloads such as nucleic acids (e.g., mRNA or siRNA). LNPs are generally comprised of multiple components including an ionizable lipid, a PEGylated lipid, a helper lipid and cholesterol, all of which play important roles in enabling effective delivering of a payload to diseased tissue.

[0003] Despite advances in the development of LNPs, substantial challenges remain. For example, the creation of LNPs capable of delivering a sufficient quantity of larger payloads or more complex payloads, such as those useful for gene editing, to target cells has been challenging. Other challenges include overcoming often unfavorable toxicological profiles of LNP components, including the ionizable lipid, and selective targeting of the LNPs to particular tissues and cell types in vivo.

[0004] Accordingly, there exists a need to develop LNPs with favorable safety profiles that can be used to deliver complex payloads to therapeutically relevant cell types in vivo, including hepatocytes, hematopoietic stem cells, and immune cells, such as T cells. SUMMARY OF THE INVENTION

[0005] In one aspect, the disclosure provides lipid nanoparticles (LNPs) 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 cells. In some embodiments, the cells are liver cells (e.g., hepatocytes). In some embodiments, the cells are immune cells (e.g., T cells). In some embodiments, the cells are hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs). In some embodiments, the cells are long -term hematopoietic stem cells (LT- HSCs).

[0006] In one aspect, the disclosure provides an ionizable lipid that is a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: L1is C1-8alkylene, wherein one or more alkylene units is optionally replaced with a group selected from -O-, -NRy-, and -S-; L2is -C(=O)O- or -C(=O)OCH2-; R1is C1-10alkyl; R2is C4-10alkyl or -CH2S-(C4-10alkyl); m is 1, 2, 3, 4 or 5; R7is H or C1-4alkyl; Z is NR5R6or a 5- to 10-membered heteroaryl group; R5and R6are each independently C1-4alkyl or R5and R6are taken together to form a 4- to 12-membered heterocyclyl; X is -O- or -CH2-; each Ryis H or C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0007] In some embodiments of the compound of Formula (I), R7is H. In some embodiments of the compound of Formula (I), R7is H C1-4alkyl

[0008] In another aspect, the disclosure provides an ionizable lipid that is a compound of formula (I’):or a pharmaceutically acceptable salt thereof, wherein: L1is C1-8alkylene, wherein one or more alkylene units is optionally replaced with a group selected from -O-, -NRy-, and -S-; L2is -C(=O)O- or -C(=O)OCH2-; R1is C1-10alkyl; R2is C4-10alkyl or -CH2S-(C4-10alkyl); m is 1, 2, 3, 4 or 5; R7is H or C1-4alkyl; Z is NR5R6or a 5- to 10-membered heteroaryl group; R5and R6are each independently C1-4alkyl or R5and R6are taken together to form a 4- to 12-membered heterocyclyl;each Ryis H or C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0009] Any embodiments provided herein of a compound of formula (I) or formula (I’), or a pharmaceutically acceptable salt thereof, apply where applicable to any other formula detailed herein, the same as if each and every embodiment were specifically and individually listed. Thus, it is understood and described that each embodiment provided herein of a compound of formula (I) or a pharmaceutically acceptable salt thereof, such as embodiments related to L1, L2, R1, R2, m, R5, R6, R7, X, Y, L3, L4, R3, and R4apply to formula (IA), (IA’), (II), (II-A), (II- B), (II-C), (II-C-1), (II-C-2), (III), (III-A), or (III-A-1), or a pharmaceutically acceptable salt thereof, the same as if each and every embodiment were specifically and individually listed.

[0010] In one embodiment, the disclosure provides an ionizable lipid that is a compound of formula (IA):or a pharmaceutically acceptable salt thereof, wherein: L1is C1-8alkylene, wherein one or more alkylene units is optionally replaced with a group selected from -O-, -NRy-, and -S-; L2is -C(=O)O- or -C(=O)OCH2-; R1is C1-10alkyl;10alkyl); m is 1, 2, 3, 4 or 5; R7is H or C1-4alkyl; Z is NR5R6or a 5- to 10-membered heteroaryl group; R5and R6are each independently C1-4alkyl or R5and R6are taken together to form a 4- to 12-membered heterocyclyl; X is -O- or -CH2-; each Ryis H or C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0011] In some embodiments of the compound of Formula (IA), R7is H. In some embodiments of the compound of Formula (I), R7is H C1-4alkyl

[0012] In one embodiment, the ionizable lipid that is a compound of formula (IA’):(IA’), or a pharmaceutically acceptable salt thereof, wherein: L1is C1-8alkylene, wherein one or more alkylene units is optionally replaced with a group selected from -O-, -NRy-, and -S-; L2is -C(=O)O- or -C(=O)OCH2-; R1is C1-10alkyl; R2is C4-10alkyl or -CH2S-(C4-10alkyl); m is 1, 2, 3, 4 or 5; R5and R6are each independently C1-4alkyl or R5and R6are taken together to form a 4- to 12-membered heterocyclyl; R7is C1-4alkyl; X is -O- or -CH2-; each Ryis H or C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0013] In some embodiments of a compound of formula (IA) or (IA’), L1is C1-8alkylene. In some embodiments of a compound of formula (IA) or (IA’), L1is C3-8alkylene. In some embodiments of a compound of formula (IA) or (IA’), L1is C5-7alkylene. In some embodiments of a compound of formula (IA) or (IA’), L1is C6 alkylene. In some embodiments of a compound of formula (IA) or (IA’), L1is C7 alkylene. In some embodiments of the compound of formula (IA) or (IA’), L1has an S-S linkage. In some embodiments, L1is (CH2)2- S-S-(CH2)2.

[0014] In some embodiments of a compound of formula (I) or (I’), the compound is a compound of formula (II):herein.

[0015] In some embodiments of the compound of Formula (II), R7is H. In some embodiments of the compound of Formula (II), R7is H C1-4alkyl

[0016] In some embodiments of a compound of formula (I) or (I’), the compound is a compound of formula (II-C):pharmaceutically acceptable salt thereof, wherein L2, R1, R2, R5, R6, R7, X, L4, R3, and R4are defined as elsewhere herein.

[0017] In some embodiments of the compound of Formula (II-C), R7is H. In some embodiments of the compound of Formula (II-C), R7is H C1-4alkyl.

[0018] In some embodiments, the compound of the formula (I’) has a structure as set forth in Table 1, as disclosed herein.

[0019] In some embodiments, the ionizable lipid has one of the structures depicted below, or a pharmaceutically acceptable salt thereof:Lipid 343

[0020] In another aspect, the disclosure provides a lipid nanoparticle (LNP) for delivery of a therapeutic agent encapsulated in the LNP (to, e.g., liver cells, T cells, or HSCs), wherein the LNP comprises an ionizable lipid and a helper lipid, wherein the ionizable lipid is a compound of formula (I). In some embodiments, the lipid is selected from Lipid 167, Lipid 196, Lipid 199 and Lipid 200. In some embodiments, the ionizable lipid is Lipid 167. In some embodiments, the ionizable lipid is Lipid 196. In some embodiments, the ionizable lipid is Lipid 199. In some embodiments, the ionizable lipid is Lipid 200. In some embodiments, the ionizable lipid is Lipid 254. In some embodiments, the ionizable lipid is Lipid 343.

[0021] In some embodiments, the LNP is delivered in vivo, e.g., to a patient in need thereof. In some embodiments, the LNPs are delivered ex vivo, e.g., to the isolated cells of a subject (e.g., human patient) in need thereof.

[0022] In another aspect, the disclosure provides conjugates (targeted LNPs) comprising an LNP and a targeting moiety. In some cases, inclusion of a targeting moiety of a targeted LNP enhances the ability of the LNP to deliver the payload to cells. For example, inclusion of a targeting moiety enhances the specificity of delivery to particular target cells while reducing delivery to other, nontargeted cell types. In some embodiments, the cells are liver cells, suchas hepatocytes. In some embodiments, the cells are immune cells, such as T cells. In some embodiments, the cells are HSCs and / or HPCs (collectively HSPCs). The conjugates (targeted LNPs) can be formulated in a pharmaceutical composition and can be directly administered to a subject (e.g., patient) in need thereof (i.e., by in vivo administration). Once administered, the compositions can deliver significant quantities of a therapeutic agent to the targeted cells (e.g. T cells, LT- HSCs, or liver cells of the subject). Alternatively, the conjugates can be administered to isolated cells such as T cells or HSCs (e.g., LT-HSCs) ex vivo.

[0023] In some embodiments, the targeting moiety of the conjugate is an antibody, Fab fragment, 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 a cell, such as a T cell, HSC, or liver cell). In some embodiments, the targeting moiety is a peptide or protein that binds to a receptor or ligand on the surface of a T cell, HSC, HPC, LT-HSC, or liver cell, wherein the affinity of the peptide or protein targeting moiety for the receptor or ligand on the surface of the T cell, HSC, HPC, LT-HSC, or liver cell is modulated via phage display.

[0024] In some embodiments, the conjugate comprises an ionizable lipid and a helper lipid, wherein the ionizable lipid is a compound of formula (I). In some embodiments, the lipid is selected from Lipid 167, Lipid 196, Lipid 199, and Lipid 200. In some embodiments, the lipid is selected from Lipid 167, Lipid 196, Lipid 199, Lipid 200, Lipid 254, and Lipid 343. In some embodiments, the lipid is Lipid 167. In some embodiments, the lipid is Lipid 196. In some embodiments, the lipid is Lipid 199. In some embodiments, the lipid is Lipid 200. In some embodiments, the lipid is Lipid 254. In some embodiments, the lipid is Lipid 343.

[0025] In some embodiments where the cells are T cells, the cells can be activated prior to ex vivo delivery of the LNPs or conjugates. In other embodiments, the T cells are not activated prior to the delivery of the LNPs or conjugates ex vivo to T cells.

[0026] The disclosure further provides delivery of particular payloads to cells, wherein the payloads are encapsulated within LNPs or conjugates of the disclosure. 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 system. In some embodiments, the therapeutic agent is a heterologous gene modifying system.

[0027] In certain embodiments, the payload (e.g., therapeutic agent) delivered by the LNP or conjugates of the disclosure 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 gene modifying system, or a heterologous gene modifying system, as described herein.

[0028] In some embodiments, an LNP or 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 liver cells (e.g., hepatocytes) in a subject. In some embodiments, an LNP or 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, HPSCs, and / or LT-HSCs in a subject. In some embodiments, an LNP or 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 T cells in a subject.

[0029] In some embodiments, an LNP or 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 T cells, HSCs, HPSCs, LT-HSCs, or liver cells than an LNP comprising a baseline 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 T cells, 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 conjugate.

[0030] In some embodiments, a payload (e.g., a therapeutic payload) delivered to T cells, HSCs, HPSCs, LT-HSCs, or liver cells 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 conjugate.

[0031] 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 T cells in a subject comprise a genome that have been modified or altered following administration of an LNP described herein, such as a targeted LNP (conjugate) 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 T cells in a subject comprise a genome that has been modified or altered following administration of an LNP described herein, such as a conjugate.

[0032] 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 (conjugate) 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 about20% 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 conjugate.

[0033] 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%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% of liver cells (e.g., hepatocytes) in a subject comprise a genome that have been modified or altered following administration of an LNP described herein, such as a targeted LNP (conjugate) 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 50%, from about 10% to about 30%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, or from about 20% to about 30% of liver cells in a subject comprise a genome that has been modified or altered following administration of an LNP described herein, such as a conjugate.

[0034] In some embodiments, the LNPs or conjugates of the present disclosure comprise one or more pegylated lipid molecules. In some embodiments, the mol% of the one or more pegylated lipid in the LNP or conjugate is from about 0.05 mol% to about 3.0 mol% of the lipid component of the LNP or conjugate. In some embodiments, the mol% of the one or more pegylated lipid in the LNP or conjugate is from about 0.5 mol% to about 2.5 mol% of the lipid component of the LNP or conjugate. In some embodiments, the mol% of the one or more pegylated lipid in the LNP or conjugate is about 2.5 mol% of the lipid component. In some embodiments, the lipid component 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. In some embodiments of conjugates, the targeting moiety (e.g., antibody, Fab fragment, VHH domain, or scFv) of a conjugate is associated with or chemically bonded to at least one of the pegylated lipid molecules. In some embodiments of conjugates, the targeting moiety is a GalNAc that is associated with or chemically bonded to at least one of the pegylated lipid molecules. In some embodiments, the conjugate comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the targeting moiety, e.g., from about 0.5 mol% to about 1.0 mol%. In some embodiments, the PEG spacer between the lipid and the targeting moiety comprises at least about 5, 10, 20, 30, 40, 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 lipidbonded 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 component 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.

[0035] In some embodiments, an LNP of the present disclosure comprises one or more PEGylated lipids, wherein at least one of the pegylated lipids has one or two C16 (palmitoyl) PEG lipid anchors. In some embodiments, the pegylated lipid that has one or two C16 (palmitoyl) PEG lipid anchors is dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (DPPE-PEG2000) or 1,2-Dipalmitoyl-rac-glycero-3- methylpolyoxyethylene (DPG-PEG2000). In some embodiments, the pegylated lipid that has one or two C16 (palmitoyl) PEG lipid anchors is present at about 0.05 mol% to about 2.5 mol% of the lipid component.

[0036] In some embodiments, the LNPs or conjugates of the present disclosure comprise a first PEGylated lipid and a second PEGylated lipid. In some embodiments, the first 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. In some embodiments, the first PEGylated lipid comprises DSCP. In some embodiments, the first PEGylated lipid is present at about 0.025 mol% to about 1.0 mol% of the lipid component of the LNP or conjugate, e.g., about 0.5 mol% to about 1.0 mol%. In some embodiments, the first and the second PEGylated lipid are the same PEGylated lipid. In some embodiments, the second 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. In some embodiments, the second PEGylated lipid is present at about 0.05 mol% to about 3.0 mol% of the lipid component of the LNP or conjugate, e.g., about 0.05 mol% to about 2.5 mol% of the lipid component or about 1.0 mol% to about 2.0 mol% of the lipid component. In some embodiments, the first and the second PEGylated lipids are the same. In some embodiments, the first and the second PEGylated lipids are different. In some embodiments, the second PEGylated lipid has one or two C16 (palmitoyl) PEG lipid anchors. In some embodiments, the pegylated lipid that has one or two C16 (palmitoyl) PEG lipid anchors is dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000) or 1,2- Dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (DPG-PEG2000). the pegylated lipids that has one or two C16 (palmitoyl) PEG lipid anchors is present at about 0.05 mol% to about 2.5 mol% of the lipid component of the LNP or conjugate. In some embodiments, the pegylated lipid that has one or two C16 (palmitoyl) PEG lipid anchors is present at about 1.0 mol% to about 2.0 mol% of the lipid component of the LNP or conjugate. In some embodiments, an LNP or conjugate of the disclosure comprises one or more non-pegylated lipids (e.g. non- pegylated phospholipids). Such lipids are often referred to as helper lipids. In some embodiments of conjugates, 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.

[0037] In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP or conjugate is from about 6% to about 12%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP or conjugate is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP or conjugate is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP or conjugate is from about 22% to about 28%. In some embodiments, the mol% of the helper in the LNP or conjugate is about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, 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%.

[0038] In some embodiments, the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) in the LNP or conjugate 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, themolar 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.

[0039] In some embodiments, the LNP or conjugate comprises Lipid 167 and DSPC. In some embodiments, the LNP or conjugate comprises Lipid 167 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP or conjugate is from about 8% to about 12%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP or conjugate is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP or conjugate is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0040] In some embodiments, the LNP or conjugate comprises Lipid 196 and DSPC. In some embodiments, the LNP or conjugate comprises Lipid 196 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP or conjugate is from about 8% to about 12%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP or conjugate is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP or conjugate is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0041] In some embodiments, the LNP or conjugate comprises Lipid 199 and DSPC. In some embodiments, the LNP or conjugate comprises Lipid 199 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP or conjugate is from about 8% to about 12%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP or conjugate is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP or conjugate is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0042] In some embodiments, the LNP or conjugate comprises Lipid 200 and DSPC. In some embodiments, the LNP or conjugate comprises Lipid 200 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP or conjugate is from about 8% to about 12%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP or conjugate is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP or conjugate is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0043] 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-pegylated helper 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0045] FIGs. 1A-1D are schematics showing the synthesis of Lipid 200.

[0046] FIGs. 2A-2D are schematics showing the synthesis of Lipid 167.

[0047] FIGs. 3A-3C are schematics showing the synthesis of Lipid 196.

[0048] FIGs. 4A and 4B are schematics showing the synthesis of Lipid 199.

[0049] FIGs. 5A-5C are schematics showing the synthesis of Lipid 254.

[0050] FIGs. 6A and 6B are schematics showing the synthesis of Lipid 199.

[0051] FIGs.7A and 7B provide bar graphs showing the percentage of LSK+ cells and LT- HSCs that express GFP (GFP%) , respectively, following administration of LNPs containing Lipid 167 or Lipid V003 to mice. FIGs. 7C and 7D are bar graphs showing GFP expression levels (MFI) in LSK+ cells and LT-HSCs, respectively, following administration of LNPs containing Lipid 167 or Lipid V003 to mice.

[0052] FIGs. 8A and 8B provide bar graphs showing the percentage of LSK+ cells that express GFP (GFP%) and the GFP expression levels (MFI), respectively, following administration of LNPs containing Lipid 185, Lipid 196, Lipid 197, or Lipid V003 to mice.

[0053] FIGs. 9A and 9B provide bar graphs showing the percentage of LSK+ cells that express GFP (GFP%) and the GFP expression levels (MFI), respectively, following administration of LNPs containing Lipid 199, Lipid 200, or Lipid V003 to mice.

[0054] FIGs. 10A and 10C provide bar graphs showing the percentage of human HSPCs and LT-HSCs that express GFP (GFP%), respectively, following administration of tLNPs containing Lipid 167 or Lipid 093 to mice. FIGs. 10B and 10D are bar graphs showing GFP expression levels (MFI) in human HSPCs and LT-HSCs, respectively, following administration of tLNPs containing Lipid 167 or Lipid 093 to mice.

[0055] FIG.11A is a bar graph showing the percentages of human HSPCs expressing GFP (%GFP) following administration of tLNPs comprising Lipid 196, Lipid 200, or Lipid 093 to mice. FIG. 11B is a bar graph showing the GFP expression level (MFI) in human HSPCs following administration of tLNPs comprising Lipid 196, Lipid 200, or Lipid 093 to mice.

[0056] FIG. 12 is a bar graph showing the level of editing (% editing by AmpSeq) at the TTR locus in hepatocytes following administration of LNPs formulated with Lipid 167, Lipid 196, Lipid 199, or Lipid 200 and a gene editing system to mice.

[0057] FIG. 13 is a bar graph showing the level of TPRT-mediated rewriting (% rewriting by AmpSeq) at the human PAH locus in hepatocytes following administration of LNPs formulated with Lipid 167, Lipid 196, or Lipid 200 and a gene modifying system to mice.

[0058] FIG. 14 is a bar graph showing the level of TPRT-mediated rewriting (% rewriting by AmpSeq) at the human PAH locus in hepatocytes following administration of LNPs formulated with Lipid 200, Lipid 233, Lipid 237, or Lipid 248 and a gene modifying system to mice.

[0059] FIG. 15 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.

[0060] FIG. 16 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.

[0061] FIG.17 shows an enzymatic approach of site-specifically introducing a Tz ring onto a sugar moiety of an antibody.

[0062] FIG. 18 shows an example of a light-induced crosslinking approach of site- specifically introducing a Tz ring onto an antibody.

[0063] FIG. 19 shows an example of using a non-natural amino acid to site-specifically introduce a Tz ring onto an antibody or Fab fragment.

[0064] FIG.20 shows an example of using a cysteine-maleimide reaction to site-specifically introduce a first Click handle onto a Fab fragment, which subsequently reacts with an LNP bonded to a second Click handle, thereby generating a targeted LNP (conjugate).

[0065] FIG. 21 shows a Fab fragment modified with a free cysteine designed to react with a maleimide group.

[0066] FIG.22A 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. 22B 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.

[0067] FIG. 23 shows an exemplary conjugate comprising a Fab fragment and a lipid nanoparticle (LNP).

[0068] FIG. 24A 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. 24B shows assembly of an LNP encapsulating a therapeutic payload using a post-insertion technique.

[0069] FIG. 25 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.

[0070] FIG. 26 shows an exemplary schematic of conjugate formation. In FIG. 26, 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.

[0071] FIG. 27 shows an exemplary schematic of conjugate formation. In FIG. 27, 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.

[0072] FIG. 28 shows an exemplary schematic of conjugate formation using DBM as a bridging agent.

[0073] FIG. 29 shows an exemplary schematic of conjugate formation. In FIG. 29, 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 onefree 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 LNP comprising 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. DETAILED DESCRIPTION I. DEFINITIONS

[0074] 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.

[0075] 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 a contiguous 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.

[0076] 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.

[0077] 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

[0078] 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.

[0079] 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 Cas protein to the target nucleic acid. In some embodiments, the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.

[0080] 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 random position in a genome, and 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) facilitate 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, whichis 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.

[0081] 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.

[0082] 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.

[0083] Heterologous: The term “heterologous”, when used 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 may not 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). In some embodiments, a domain is heterologous relative to another domain, if the first domain is not naturally comprised in the same polypeptide as the other domain (e.g., a fusion between two domains of different proteins from the same organism).

[0084] Heterologous gene modifying polypeptide: As used herein, the term “heterologous gene modifying polypeptide” refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity 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 heterologous gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery. In some embodiments, the heterologous gene modifying polypeptide integrates a sequence into a random position in a genome, and in some embodiments, the heterologous gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, the sequence that is integrated comprises a deletion, substitution, or insertion relative to the target DNA molecule. In some embodiments, a heterologous 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) facilitate integration of the at least a portion of the template nucleic acid into the target DNA. Heterologous 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. Heterologous 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 heterologous 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 heterologous gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a heterologous gene modifyingpolypeptide integrates a sequence into a gene. In some embodiments, a heterologous gene modifying polypeptide integrates a sequence into a sequence outside of a gene. A “heterologous gene modifying system,” as used herein, refers to a system comprising a heterologous gene modifying polypeptide and a template nucleic acid.

[0085] 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.

[0086] 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 uncharged linkages (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 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), closed-ended DNA (ceDNA).

[0087] 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.

[0088] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.

[0089] It is understood that aspects and embodiments described herein as “comprising” include “consisting of” and “consisting essentially of” embodiments. II. LIPID NANOPARTICLES

[0090] 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.

[0091] 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.

[0092] 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 succinate diacylglycerol (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.

[0093] 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.

[0094] 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 thesecomponents 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.

[0095] In some embodiments, the average LNP diameter of the targeted LNP formulation may be between 10 nm and 150 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 70 nm to about 150 nm, from about 80 nm to about 120 nm, from about 80 nm to about 110 nm, 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, from about 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.

[0096] 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. An 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.

[0097] 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 a 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.

[0098] 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 initial amount 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 someembodiments, 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%.

[0099] 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.

[0100] Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020061457, which is incorporated herein by reference in its entirety. Ionizable Lipids

[0101] The LNPs of the disclosure (e.g., targeted LNPs) comprise one or more ionizable lipids as set forth below. The LNPs are capable of encapsulating a therapeutic agent (e.g., one or more nucleic acids encoding or comprising a therapeutic agent). The therapeutic agent (e.g., one or more nucleic acids) may be adsorbed to the surface of an LNP, e.g., an LNP comprising the ionizable lipid. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) may be encapsulated in an LNP. In some embodiments, the lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting moiety. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipids 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 a therapeutic agent, such as an RNA molecule (e.g., an mRNA molecule) comprising or encoding a therapeutic agent.

[0102] 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.

[0103] In some embodiments, the disclosure provides an ionizable lipid that is a compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: L1is C1-8alkylene, wherein one or more alkylene units is optionally replaced with a group selected from -O-, -NRy-, and -S-; L2is -C(=O)O- or -C(=O)OCH2-; R1is C1-10alkyl; R2is C4-10alkyl or -CH2S-(C4-10alkyl); m is 1, 2, 3, 4 or 5; R7is H or C1-4alkyl; Z is NR5R6or a 5- to 10-membered heteroaryl group; R5and R6are each independently C1-4alkyl or R5and R6are taken together to form a 4- to 12-membered heterocyclyl; X is -O- or -CH2-; each Ryis H or C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0104] In some embodiments, the disclosure provides an ionizable lipid that is a compound of formula (I’):or a pharmaceutically acceptable salt thereof, wherein: L1is C1-8alkylene, wherein one or more alkylene units is optionally replaced with a group selected from -O-, -NRy-, and -S-;R1is C1-10alkyl; R2is C4-10alkyl or -CH2S-(C4-10alkyl); m is 1, 2, 3, 4 or 5; R7is C1-4alkyl; Z is NR5R6or a 5- to 10-membered heteroaryl group; R5and R6are each independently C1-4alkyl or R5and R6are taken together to form a 4- to 12-membered heterocyclyl;each Ryis H or C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0105] In some embodiments of the compound of formula (I) or (I’), Z is NH2. In some embodiments of the compound of formula (I) or (I’), Z is N(CH3)2. In some embodiments of the compound of formula (I) or (I’), Z is N(Et)2.In some embodiments of the compound of formula (I) or (I’), Z is a 5- to 10- membered heteroaryl group selected from pyrazyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, isoindolyl, indolyl, piperidinyl, piperazinyl, indolinyl, morpholinyl, thiomorpholinyl, and pyrrolidinyl. In some embodiments, Z is N(Et)2.In some embodiments, Z is a 5- membered heteroaryl group. In some embodiments, Z is imidazolyl. In some embodiments, Z is pyridyl.

[0106] In some embodiments of the compound of formula (I) or (I’), R1is C4-10alkyl. In some embodiments of the compound of formula (I) or (I’), R1is C4-8alkyl. In some embodiments of the compound of formula (I) or (I’), R1is C4-8alkyl.

[0107] In some embodiments of the compound of formula (I) or (I’), R1is C4-10alkyl and R2is C4-10alkyl. In some embodiments of the compound of formula (I) or (I’), R1is C4-8alkyl and R2is C4-8alkyl.

[0108] In some embodiments of the compound of formula (I’), R7is methyl. In some embodiments of the compound of formula (I’), R7is ethyl.

[0109] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (IA):or a pharmaceutically acceptable salt thereof, wherein: L1is C1-8alkylene, wherein one or more alkylene units is optionally replaced with a group selected from -O-, -NRy-, and -S-; L2is -C(=O)O-, or -C(=O)OCH2-; R1is C1-10alkyl; R2is C4-10alkyl or -CH2S-(C4-10alkyl); m is 1, 2, 3, 4 or 5; R5and R6are each independently C1-4alkyl or R5and R6are taken together to form a 4- to 12-membered heterocyclyl; R7is H or C1-4alkyl; X is -O- or -CH2-;each Ryis H or C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0110] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (IA’):or a pharmaceutically acceptable salt thereof, wherein: L1is C1-8alkylene, wherein one or more alkylene units is optionally replaced with a group selected from -O-, -NRy-, and -S-; L2is -C(=O)O-, or -C(=O)OCH2-; R1is C1-10alkyl; R2is C4-10alkyl or -CH2S-(C4-10alkyl); m is 1, 2, 3, 4 or 5; R5and R6are each independently C1-4alkyl or R5and R6are taken together to form a 4- to 12-membered heterocyclyl; R7is C1-4alkyl; X is -O- or -CH2-; each Ryis H or C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

[0111] In some embodiments of a compound of formula (IA) or (IA’), L1is C1-8alkylene. In some embodiments of a compound of formula (IA) or (IA’), L1is C3-8alkylene. In some embodiments of a compound of formula (IA) or (IA’), L1is C5-7alkylene. In some embodiments of a compound of formula (IA) or (IA’), L1is C6 alkylene. In some embodimentsof a compound of formula (IA) or (IA’), L1is C7alkylene. In some embodiments of the compound of formula (IA) or (IA’), L1has an S-S linkage. In some embodiments, L1is (CH2)2- S-S-(CH2)2.

[0112] In some embodiments of a compound of formula (I’), or a pharmaceutically acceptable salt thereof: L1is C1-8alkylene;R1is C4-10alkyl; R2is C4-10alkyl or -CH2S-(C4-10alkyl); m is 1, 2, or 3; R5and R6are each independently C1-4alkyl; or R5and R6are taken together to form a 4- to 12-membered heterocyclyl; R7is C1-4alkyl; X is -O- or -CH2-; andL3is C1-8alkylene; L4is -C(=O)O-, or -C(=O)OCH2-; R3is C4-10alkyl; and R4is C2-10alkyl or -CH2S-(C4-10alkyl).

[0113] In some embodiments of a compound of formula (I) or (I’), or a pharmaceutically acceptable salt thereof, X is -CH2-. In some embodiments, X is -O-.

[0114] In some embodiments of a compound of formula (I) or (I’), or a pharmaceutically acceptable salt thereof, R7is H or C1-2alkyl. In some embodiments, R7is H, -CH3, or -CH2CH3. In some embodiments, R7is H. In some embodiments, R7is -CH3. In some embodiments, R7is -CH2CH3.

[0115] In some embodiments of a compound of formula (I) or (I’), or a pharmaceutically acceptable salt thereof, m is 2 or 3. In some embodiments, m is 2. In some embodiments, m is 3.

[0116] In some embodiments of a compound of formula (I) or (I’), or a pharmaceutically acceptable salt thereof, R1is C4-8alkyl. In some embodiments, R1is C6-8alkyl. In some embodiments, R1is -CH2CH2CH2CH2CH2CH3.

[0117] In some embodiments of a compound of formula (I) o (I’), or a pharmaceutically acceptable salt thereof, R2is C6-10alkyl or -S-(C4-8alkyl). In some embodiments, R2is C6-8alkyl or -S-(C4-6alkyl). In some embodiments, R2is -CH2CH2CH2CH2CH2CH3, - CH2CH2CH2CH2CH2CH2CH2CH3, or -SCH2CH2CH2CH3. In some embodiments, R2is - CH2CH2CH2CH2CH2CH3. In some embodiments, R2is -SCH2CH2CH2CH3.

[0118] In some embodiments of a compound of formula (I) or (I’), or a pharmaceutically acceptable salt thereof, R3is C6-10alkyl. In some embodiments, R3is C6-8alkyl. In some embodiments, R3is -CH2CH2CH2CH2CH2CH3or -CH2CH2CH2CH2CH2CH2CH2CH3.

[0119] In some embodiments of a compound of formula (I) or (I’), or a pharmaceutically acceptable salt thereof, R4is C2-8alkyl or -S-(C4-8alkyl). In some embodiments, R4is C2-6alkylsome embodiments, R4is -CH2CH2CH2CH2CH2CH3.

[0120] In some embodiments of a compound of formula (I) or (I’), or a pharmaceutically acceptable salt thereof, R5and R6are each independently C1-4alkyl; or R5and R6are taken together to form a 4- to 7-membered heterocyclyl. In some embodiments, R5and R6are each independently C1-4alkyl; or R5and R6are taken together to form a 5- to 6-membered heterocyclyl. In some embodiments, R5and R6are each independently C1-4alkyl. In some embodiments, R5and R6are each independently C1-2alkyl. In some embodiments, R5and R6are both -CH3 or -CH2CH3. In some embodiments, R5and R6are both -CH3. In some embodiments, R5and R6are both -CH2CH3.

[0121] In some embodiments of a compound of formula (I) or (I’), the compound is a compound of formula (II):pharmaceutically acceptable saltthereof, wherein L1, L2, R1, R2, m, R7, X, L3, L4, R3, and R4are defined as elsewhere herein.

[0122] In some embodiments of the compound of formula (II), Z is NH2.In some embodiments of the compound of formula (II), Z is N(CH3)2.In some embodiments of the compound of formula (II), Z is N(Et)2. In some embodiments of the compound of formula (II), Z is a 5- to 10- membered heteroaryl group selected from pyrazyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, isoindolyl, indolyl, piperidinyl, piperazinyl, indolinyl, morpholinyl, thiomorpholinyl, and pyrrolidinyl. In some embodiments, Z is N(Et)2. In some embodiments, Z is a 5- membered heteroaryl group. In some embodiments, Z is imidazolyl. In some embodiments, Z is pyridyl.

[0123] In some embodiments of the compound of formula (II), R1is C4-10alkyl. In some embodiments of the compound of formula (II), R1is C4-8alkyl. In some embodiments of the compound of formula (II), R1is C4-8alkyl.

[0124] In some embodiments of the compound of formula (II), R1is C4-10alkyl and R2is C4-10alkyl. In some embodiments of the compound of formula (II), R1is C4-8alkyl and R2is C4-8alkyl.

[0125] In some embodiments of the compound of formula (II), R7is methyl. In some embodiments of the compound of formula (I), R7is ethyl.

[0126] In some embodiments of the compound of formula (II), R7is hydrogen.

[0127] In some embodiments of a compound of formula (IA) or (IA’), the compound is a compound of formula (IIA):pharmaceutically acceptable saltthereof, wherein L1, L2, R1, R2, m, R5, R6, R7, X, L3, L4, R3, and R4are defined as elsewhere herein.

[0128] In some embodiments of a compound of formula (I) or (I’), the compound is a compound of formula (II-A):

[0129] In some embodiments of a compound of formula (I) or (I’), the compound is a compound of formula (II-B):O Oas elsewhere herein.

[0130] In some embodiments of a compound of formula (I) or (I’), the compound is a compound of formula (II-C):pharmaceutically acceptable salt thereof, wherein L2, R1, R2, R5, R6, R7, X, L4, R3, and R4are defined as elsewhere herein.

[0131] In some embodiments of a compound of formula (I) or (I’), the compound is a compound of formula (II-C-1):pharmaceutically acceptable salt thereof, wherein L2, R1, R2, R7, X, L4, R3, and R4are defined as elsewhere herein.

[0132] In some embodiments of a compound of formula (I) or (I’), the compound is a compound of formula (II-C-2):(II-C-2), or a pharmaceutically acceptable salt thereof, wherein X1and X2are independently CH2 or S; n and p are independently 0 or 1; and R7and X are defined as elsewhere herein.

[0133] In some embodiments of a compound of formula (I’), the compound is a compound of formula (III):pharmaceutically acceptable salt thereof, wherein L1, L2, R1, R2, R5, R6, and Y are defined as elsewhere herein.

[0134] In some embodiments of a compound of formula (I’), the compound is a compound of formula (III-A):pharmaceutically acceptable salt thereof, wherein L1, L2, R1, R2, R5, R6, and Y are defined as elsewhere herein.

[0135] In some embodiments of a compound of formula (I’), the compound is a compound of formula (III-A-1):(III-A-1), or a pharmaceutically acceptable salt thereof, wherein X2is CH2or S; n is 0 or 1; and R5, R6, and Y are defined as elsewhere herein.

[0136] In some embodiments, the compound of formula (I’) is a compound selected from the exemplary compounds of Table 1.Table 1: Exemplary Ionizable Lipids

[0137] In some embodiments, the compound of formula (I’) is a compound selected from theexemplary compounds of Table 2. Table 2: Additional Exemplary Ionizable Lipids

[0138] In some embodiments, the ionizable lipid is selected from the group consisting of Lipid 167, Lipid 196, Lipid 199, and Lipid 200 from Table 1. In some embodiments, theionizable lipid is selected from the group consisting of Lipid 167, Lipid 196, Lipid 199, Lipid 200, Lipid 254 and Lipid 343 from Table 1.

[0139] In some embodiments, the ionizable lipid has the structure depicted below:.

[0140] In some embodiments, the ionizable lipid has the structure depicted below:.

[0141] In some embodiments, the ionizable lipid has the structure depicted below:.

[0142] In some embodiments, the ionizable lipid has the structure depicted below:.

[0143] In some embodiments, the ionizable lipid has the structure depicted below:.

[0144] In some embodiments, the ionizable lipid has the structure depicted below:.

[0145] In some cases, an LNP containing an ionizable lipid of formula (I), formula (I’), or an ionizable lipid shown in Table 1 or Table 2 exhibits similar levels of transduction in HSCs and / or LT-HSCs and / or similar expression of a payload protein in HSCs and / or in LT-HSCs relative to an LNP that contains a benchmark ionizable lipid, such as V003 as the ionizable lipid. In some cases, an LNP containing an ionizable lipid of formula (I), formula (I’), or an ionizable lipid shown in Table 1 or Table 2 exhibits higher levels of transduction in HSCs and / or LT-HSCs and / or higher expression of a payload protein in HSCs and / or LT-HSCs relative to an LNP that contains a benchmark ionizable lipid, such as V003 as the ionizable lipid.

[0146] In some cases, an LNP containing an ionizable lipid of formula (I), formula (I’), or anionizable lipid shown in Table 1 or Table 2 exhibits similar levels of transduction in T cellsand / or similar expression of a payload protein in T cells relative to an LNP that contains abenchmark ionizable lipid, such as V003 as the ionizable lipid. In some cases, an LNPcontaining an ionizable lipid of formula (I) or an ionizable lipid shown in Table 1 or Table 2exhibits higher levels of transduction in T cells and / or higher expression of a payload protein in T cells relative to an LNP that contains a benchmark ionizable lipid, such as V003 as the ionizable lipid.

[0147] In some cases, an LNP containing an ionizable lipid of formula (I), formula (I’), or anionizable lipid shown in Table 1 or Table 2 exhibits similar levels of transduction in liver cells(e.g., hepatocytes) and / or similar expression of a payload protein in liver cells (e.g.,hepatocytes) relative to an LNP that contains a benchmark ionizable lipid, such as V003 as theionizable lipid. In some cases, an LNP containing an ionizable lipid of formula (I), formula(I’), or an ionizable lipid shown in Table 1 or Table 2 exhibits higher levels of transductionin liver cells (e.g., hepatocytes) and / or higher expression of a payload protein in liver cells(e.g., hepatocytes) relative to an LNP that contains a benchmark ionizable lipid, such as V003 as the ionizable lipid.

[0148] V003 is depicted below and described in U.S. Patent No. 10,059,655.Lipid V003

[0149] In some embodiments, Lipid 167 is used as an ionizable lipid to generate LNPs fordelivery to cells (e.g., T cells, HSCs, or liver cells). In some embodiments, Lipid 196 is usedas an ionizable lipid to generate LNPs for delivery to cells (e.g., T cells, HSCs, or liver cells). In some embodiments, Lipid 199 is used as an ionizable lipid to generate LNPs for delivery to cells (e.g., T cells, HSCs, or liver cells). In some embodiments, Lipid 200 is used as an ionizable lipid to generate LNPs for delivery to cells (e.g., T cells, HSCs, or liver cells). In some embodiments, Lipid 254 is used as an ionizable lipid to generate LNPs for delivery to cells (e.g., T cells, HSCs, or liver cells). In some embodiments, Lipid 343 is used as an ionizable lipid to generate LNPs for delivery to cells (e.g., T cells, HSCs, or liver cells).

[0150] 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%.

[0151] The compounds disclosed herein (e.g., lipids in Table 1 or Table 2), 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

[0152] The LNPs, e.g., targeted LNPs, of the disclosure comprise one or more helper 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.

[0153] Other examples of non-cationic lipids suitable for use in the lipid nanoparticles 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 acompound of Formula I, II, or IV of US2018 / 0028664, incorporated herein by reference in its entirety.

[0154] 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.

[0155] 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 some embodiments, the helper lipid comprises from about 5% to about 12% of the total lipid present in the lipid nanoparticle. In some embodiments, the helper lipid comprises from about 6% to about 10% 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).

[0156] In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 6% to about 15%, e.g., 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 8% to about 12%, e.g., 8%, 9%, 10%, 11%, or 12%. 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.

[0157] 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.

[0158] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid in Table 1 or Table 2 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid in Table 1 or Table 2 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 8% to about 12%. 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%.

[0159] 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 8% to about 12%. 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 fromabout 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%.

[0160] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 167 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 167 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 8% to about 12%. 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 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%.

[0161] In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 196 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 196 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 8% to about 12%. 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 targeted LNP is about 8%, is about 9%, is about 10%, is about 11%, is about 12%, is about 13%, is about 14%, is about 15%, is about 16%, is about 17%, is about 18%, is about 19%, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0162] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 199 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 199 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 8% to about 12%. 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 fromabout 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 8%, is about 9%, is about 10%, is about 11%, is about 12%, is about 13%, is about 14%, is about 15%, is about 16%, is about 17%, is about 18%, is about 19%, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0163] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 200 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 200 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 8% to about 12%. 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 8%, is about 9%, is about 10%, is about 11%, is about 12%, is about 13%, is about 14%, is about 15%, is about 16%, is about 17%, is about 18%, is about 19%, 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

[0164] 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.

[0165] 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 totallipid 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.

[0166] 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-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 3:1 to about 0.5:1. In some embodiments, the 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 ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1.5:1 to about 0.5:1. In some embodiments, the 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 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. Pegylated Lipids

[0167] In some embodiments, the LNPs, e.g., targeted LNPs, of the disclosure can comprise a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.

[0168] Exemplary PEG-lipid conjugates include, but are not limited to, 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), 1,2-dimyristoyl-sn-glycerol, methoxypoly ethylene glycol (DMG-PEG-2K), PEG succinate diacylglycerol (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-methoxypolyethylene glycol 2000)-l,2-distearoyl- sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in US5,885,6l3, US6,287,59l, US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058, US2011 / 0117125,US2010 / 0130588, US2016 / 0376224, US2017 / 0119904, and US / 099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formulathe content of which is incorporated herein by reference in its entirety. In some embodiments, a PEG-lipid is of Formula II of US20150376115 or US2016 / 0376224, the content of both of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG- dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG- DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG- dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG- disterylglycamide, PEG-cholesterol (l-[8'-(Cholest-5-en-3[beta]- oxy)carboxamido-3',6'- dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG- DMB (3,4- Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1,2- dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2- dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from: ,,.

[0169] In some embodiments, lipids conjugated with a molecule other than a PEG can alsobe used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide- lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.

[0170] Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipidconjugates and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289A9 and in WO2020106946A1, the contents of all of which are incorporated herein by reference in their entirety.

[0171] In some embodiments, the PEG or the conjugated lipid can comprise 0-20% (mol) ofthe total lipid present in the lipid nanoparticle. In some embodiments, the PEG or the conjugated lipid can comprise 0.5-10% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the PEG or the conjugated lipid can comprise 1-10% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the PEG or the conjugated lipid can comprise 1-3% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle.

[0172] In some embodiments, the pegylated lipid has at least one C16 (palmitoyl) PEG lipidanchor. 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 C16 pegylated lipids show reduced liver uptake than otherwise identical LNPs comprising C14 pegylated lipids. In some embodiments, the targeted LNPs comprising the C16 pegylated lipids show increasedtransduction and / or expression of the delivered payload in HSCs or T cells than otherwise identical LNPs comprising C14 pegylated lipids.

[0173] In other embodiments, the pegylated lipid has at least one C14 PEG lipid anchor.

[0174] In some embodiments, the PEG lipid has at least one C16 (palmitoyl) PEG lipid anchor. or the conjugated lipid can comprise 0.025-5% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG lipid has at least one C16 (palmitoyl) PEG lipid anchor can comprise 0.05-3.5% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG lipid has at least one C16 (palmitoyl) PEG lipid anchor can comprise 0.05-2.5% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG lipid has at least one C16 (palmitoyl) PEG lipid anchor can comprise 1-2.5% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG lipid has at least one C16 (palmitoyl) PEG lipid anchor can comprise 1.5-2.5% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG lipid has at least one C16 (palmitoyl) PEG lipid anchor can comprise 1.5-2.0% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG lipid has at least one C16 (palmitoyl) PEG lipid anchor can comprise 2.0- 2.5% (mol) of the total lipid present in the lipid nanoparticle.

[0175] In some embodiments, the LNPs disclosed herein comprise more than one pegylated lipid molecule. In some such embodiments, one of the pegylated lipid molecules is conjugated to a binder targeting a receptor (e.g., a binder targeting HSCS or T cells) and the other pegylated lipid is not conjugated to a binder. In one embodiment, the pegylated lipid conjugated to a binder is DSPE-PEG2000-malemide and the pegylated lipid not conjugated to the binder is PEG lipid has at least one C16 (palmitoyl) PEG lipid anchor, as set forth above. Additional components

[0176] In some embodiments, one or more additional compounds can also be included in the LNPs, e.g., targeted LNPs, of the disclosure. Those compounds can be administered separately or the additional compounds can be included in the lipid nanoparticles of the invention. In other words, the lipid nanoparticles can contain other compounds, such as a payload, e.g., a therapeutic agent, as described herein. In some embodiments, the lipid nanoparticles can contain one or more nucleic acids. In some embodiments, the lipid nanoparticles can contain a first nucleic acid and a second nucleic acid, wherein the second nucleic acid is different than the first nucleic acid. Without limitations, other additional compounds can be selected from thegroup consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.

[0177] In some embodiments, a lipid nanoparticle (or a formulation comprising lipid nanoparticles) lacks reactive impurities (e.g., aldehydes or ketones), or comprises less than a preselected level of reactive impurities (e.g., aldehydes or ketones). While not wishing to be bound by theory, in some embodiments, a lipid reagent is used to make a lipid nanoparticle formulation, and the lipid reagent may comprise a contaminating reactive impurity (e.g., an aldehyde or ketone). A lipid regent may be selected for manufacturing based on having less than a preselected level of reactive impurities (e.g., aldehydes or ketones). Without wishing to be bound by theory, in some embodiments, aldehydes can cause modification and damage of RNA, e.g., cross-linking between bases and / or covalently conjugating lipid to RNA (e.g., forming lipid-RNA adducts). This may, in some instances, lead to failure of a reverse transcriptase reaction and / or incorporation of inappropriate bases, e.g., at the site(s) of lesion(s), e.g., a mutation in a newly synthesized target DNA.

[0178] In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, the lipid nanoparticle formulation is produced using a plurality of lipid reagents, and each lipid reagent of the plurality independently meets one or more criterion described in this paragraph. In some embodiments, each lipid reagent of the plurality meets the same criterion, e.g., a criterion of this paragraph.

[0179] In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity(e.g., aldehyde) content. In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, the lipid nanoparticle formulation comprises: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.

[0180] In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.

[0181] In some embodiments, total aldehyde content and / or quantity of any single reactive impurity (e.g., aldehyde) species is determined by liquid chromatography (LC), e.g., coupled with tandem mass spectrometry (MS / MS), e.g., according to the method described in Example 34. In some embodiments, reactive impurity (e.g., aldehyde) content and / or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleic acid molecule (e.g., an RNA molecule, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents. In some embodiments, reactive impurity (e.g., aldehyde) content and / or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleotide or nucleoside (e.g., a ribonucleotide or ribonucleoside, e.g., comprised in or isolated from a template nucleic acid, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents. In embodiments, chemical modifications of a nucleic acid molecule, nucleotide, or nucleoside are detected bydetermining the presence of one or more modified nucleotides or nucleosides, e.g., using LC- MS / MS analysis. III. CONJUGATES

[0182] In one aspect, the disclosure provides an LNP (conjugate) 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., one or more RNA molecules comprising or encoding a therapeutic agent) to a cell. In some embodiments, the cell is an HSC (e.g., a LT-HSC) or HPC. In other embodiments, the cell is a T cell. In some embodiments, the cell is a liver cell, such as a hepatocyte. In some embodiments, the payload (e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or nucleic acid encoding a gene modifying system) is capable of modifying one or more genes of the target cell. In some embodiments, the payload (e.g., a therapeutic agent, as described herein, such as a heterologous gene modifying polypeptide or nucleic acid encoding a heterologous gene modifying system) is capable of modifying one or more genes of the target cell.

[0183] In another aspect, the LNP (conjugate) comprises a targeting moiety that binds to a protein (e.g., a protein receptor or ligand) on a cell such as an HSC (e.g., a LT-HSC), HPC, T cell, or liver cell (e.g., hepatocyte), as described herein. In some embodiments, an LNP (conjugate) comprises both an ionizable lipid and a targeting moiety, as described herein.

[0184] 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), wherein the targeting moiety binds to a protein (e.g., protein receptor or ligand) on an HSC (e.g., a LT-HSC) HPC, T cell, or liver cell (e.g., hepatocyte). In some embodiments, the targeting moiety is an antibody, Fab fragment, Fab’ fragment, 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, HPC, LT-HSC, T cell, or hepatocyte). 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, LT-HSC, T cell, or hepatocyte, wherein the affinity of thepeptide or protein targeting moiety for the receptor or ligand on the surface of the HSC, HPC, LT-HSC, T cell or hepatocyte is modulated via phage display.

[0185] In some embodiments, the targeting moiety component of a conjugate of the disclosure can target a cell surface antigen or receptor. In some embodiments, the targeting moiety component of a conjugate of the disclosure binds to a T cell receptor or ligand such as CD2, CD3, CD4, CD5, CD6, CD7, CD8 or CD28. In other embodiments, the targeting moiety component of a conjugate binds to TCR alpha, TCR beta, CD10, CD33, CD34, CD68, CD19, CCR7, CD62L, CD25, CXCR3, CCR2, CCR3, CCR4, CCR5, CCR, or CCR7. In some embodiments, the targeting moiety on the LNP binds to CD2. In some embodiments, the targeting moiety on the LNP binds to CD3. In some embodiments, the targeting moiety on the LNP binds to CD4. In some embodiments, the targeting moiety on the LNP binds to CD5. In some embodiments, the targeting moiety on the LNP binds to CD6. In some embodiments, the targeting moiety on the LNP binds to CD7. In some embodiments, the targeting moiety on the LNP binds to CD8. In some embodiments, the targeting moiety on the LNP binds to CD28.

[0186] In certain embodiments, the targeting moiety binds to CD4+ or CD8+ T cell. In other embodiments, the targeting moiety binds to a natural killer (NK) cell. In other embodiments, the targeting moiety binds to a hematopoietic stem cell. In other embodiments, the targeting moiety binds to a lymphoid progenitor cell. In other embodiments, the targeting moiety binds to a myeloid cell. In other embodiments, the targeting moiety binds to a macrophage. In other embodiments, the targeting moiety binds to a B cell.

[0187] In other embodiments, the targeting moiety component of a conjugate of the disclosure binds to an antigen (e.g., receptor or ligand) present on the surface of an HSC and / or LT-HSC. In some embodiments, the targeting moiety component of a conjugate of the disclosure binds to an HSC receptor or ligand such as CD90 or CD117. In some embodiments, the targeting moiety component of a conjugate binds to CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184 (CXCR4), or CD338. 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 someembodiments, 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.

[0188] In some embodiments, the targeting moiety component of a conjugate of the disclosure binds to a receptor or ligand on the surface of a liver cell, such as a hepatocyte. In some embodiments, the targeting moiety binds to the asialoglycoprotein receptor (ASGPR). In some embodiments, the targeting moiety is an N-acetylgalactosamine or GalNAc.

[0189] 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.

[0190] 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, e.g., a Fab fragment. In other such embodiments, the targeting moiety is a scFv. In some embodiments, the targeting moiety is a Fab fragment. In some embodiments, the targeting moiety is a VHH domain.

[0191] 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 cyclicderivative 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.

[0192] 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.

[0193] 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.

[0194] 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, (G)v, (EAAAK)v, (PAPAP)v, (AP)vand A(EAAAK)uALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10.

[0195] 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 s 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 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%.

[0196] 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.

[0197] 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.

[0198] In some embodiments, the first or second Click handle is a TCO derivative having one of the following structures:

[0199] 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 orsScFv 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, (G)v, (EAAAK)v,(PAPAP)v, (AP)vand A(EAAAK)uALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10.

[0200] 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 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 s 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%.

[0201] 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; (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.

[0202] 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, (G)v, (EAAAK)v,(PAPAP)v, (AP)vand A(EAAAK)uALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10.

[0203] 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 motif can be bonded to one or more (e.g., between 1-10) histidine (H) residues (see FIG. 12). The glycine and the histidine residues are removed during the transpeptidation reaction.

[0204] 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, where X is any amino acid residue. In step (ii), sortase A catalyzes the transpeptidation reaction between the LPXTG 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.

[0205] 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, where X is any amino acid residue. In step (ii), SpSrtA catalyzes the transpeptidation reaction between the LPXTA 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.

[0206] FIG. 15 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.

[0207] 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 aClick 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, 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 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 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.

[0208] 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, LPETGGGG, LPETGGGGG, LPETAGGG or LPETGGGGGG.

[0209] 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, (G)v, (EAAAK)v, (PAPAP)v, (AP)v andA(EAAAK)uALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10. In some embodiments, Z is GG, GGG, GGGG, GGGGG, GGGGGG, and GGGGGGG or GGGGGS.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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, (G)v, (EAAAK)v,(PAPAP)v, (AP)vand A(EAAAK)uALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10.

[0214] 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.

[0215] In some embodiments, the carboxylic acid in step (ii) (i.e., the lipoic acid ligase catalyzed reaction) is a C3-C20carboxylic 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.

[0216] 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.

[0217] FIG. 16 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 atleast one lipid that is covalently bonded to a second Click handle comprising a TCO moiety (Click handle 2), thus affording an LNP site-specifically conjugated to the Fab fragment.

[0218] 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. 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.

[0219] 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, (G)v, (EAAAK)v,(PAPAP)v, (AP)vand A(EAAAK)uALEA(EAAAK)vA, wherein u is 1-10 and v is 1-10. In some embodiments, Z is GG, GGG, GGGG, GGGGG, GGGGGG, and GGGGGGG or GGGGGS.

[0220] 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 the carboxylicacid 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-C30alkyl group). Fab-Z-

[0221] In other embodiments, the enzyme recognition sequence is a transglutaminase enzyme recognition sequence (LLQG). The transglutaminase enzyme recognition sequence (LLQG) 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.

[0222] 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.

[0223] 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. 17 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)-MeTzvia a Click reaction. The Click reaction is used to introduce 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.

[0224] 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. 18 shows one particular embodiment using oYo-Link Tz. In FIG. 18, 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.

[0225] 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. 19 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.

[0226] 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 using a cysteine-maleimide reaction to introduce the first Click handle onto the targeting moiety (antibody, Fab fragment or scFv). FIG. 20 shows one particular embodiment of introducing a first Click handle (e.g., Tz) onto a Fab fragment. In FIG. 20, a hinge with a free cysteine is first introduced at a C- terminus of the Fab fragment. In some such embodiments, an engineered inter-chain disulfide bound away from the C-terminus and buried in the CL-CH1 interface is introduced for improved stability (FIG.21). The resultant modified Fab fragment is reacted with a maleimide moiety with a first Click handle (Click handle 1) covalently bonded to the maleimide nitrogen. The first Click handle than reacts with a second Click handle (Click handle 2) on the LNP, hence generating the surface modified targeted LNP. In some embodiments, Click handle 1 is a TZ and Click handle 2 is a TCO moiety,

[0227] 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).

[0228] In some embodiments, the Click product can be formed using oxime and hydrazone ligations. In some embodiments, a ketone or aldehyde can react with 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).

[0229] Exemplary lipids and cholesterol molecules bonded to Click handles that can be used to make targeted LNPs are depicted below.

[0230] Conjugates prepared by site-specific methods (e.g., FIGs.15-29) 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 can comprise 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 orscFvs) 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.

[0231] 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. Interchain CL-CH1 disulfide reduction followed by conjugation

[0232] 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 of disulfide 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.

[0233] 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’)2fragment includes disulfide bonds that covalently link two Fab fragments. Reduction of the disulfide bond in the F(ab’)2 generates two F(ab’) fragments. The sulfhydryl (thiol) groups of the F(ab’) could potentially react with a thiol-reactive group on the surface of anLNP, 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’)2 to the F(ab’) fragments could also disrupt the natural interchain disulfide bonds between the CLand CH1 regions of the Fab fragments, hence further compromising site-specific conjugation.

[0234] 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).

[0235] 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 that has 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. 23.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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 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.

[0240] 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. 24A, 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. 24A). 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.24B, 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.

[0241] 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 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 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] A schematic showing the reduction of an interchain disulfide bond in a Fab fragment is depicted in FIG. 25. In some embodiments, the Fab fragment depicted in FIG. 25 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. 25, the interchain 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.

[0247] FIG. 26 shows an exemplary schematic of conjugate formation, as described herein. In FIG. 26, 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.

[0248] 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. 27. In some embodiments, the conjugates formed by methods disclosed herein include at least one, at least two or all three orientations shown in FIG. 27. 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.

[0249] In some embodiments, the thiol-reactive group is maleimide, as shown in FIG.27. 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 cysteine residue on the light chain and the other maleimide reacts with a free cysteine residue on the heavy chain.

[0250] In some embodiments, the thiol-reactive group is 2,3-dibromomaleimide (DBM) as shown in FIG. 28. 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. 28). 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 abridge between the cysteine residues. Accordingly, the heavy and light chain of the Fab fragment are effectively bridged together following reaction with DBM.

[0251] 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.28). In still other of the foregoing embodiments, the Fab fragment is linked to the LNP through a maleamic acid moiety (see FIG. 28).

[0252] 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.

[0253] 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 suchembodiments, 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.

[0254] 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.

[0255] 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. 29. In FIG. 29, two Fab fragments (Fab1 and 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.

[0256] 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). In some 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 LNPsurface). 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.

[0257] 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.

[0258] 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, theconjugate 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. IV. PAYLOADS

[0259] The LNPs, e.g., conjugates (targeted LNPs), of the disclosure can be used to deliver payloads to cells, e.g., T cells, HSCs, HSC progenitors, or liver cells (e.g., hepatocytes). In some embodiments, the LNPs can be used to deliver a payload into long-term HSC (LT-HSC) subpopulations. In some embodiments, a targeted LNP as described herein can be used to deliver a payload to cells expressing cell-surface receptors targeted by the targeting moiety, e.g., antibody, Fab fragment, scFv, VHH domain, or nanobody component, of the targeted LNP (conjugate).

[0260] The conjugates disclosed herein can be used to deliver a therapeutic of interest to a cell. For instance, in some embodiments the conjugates disclosed herein can be used to deliver a nucleic acid (e.g., mRNA) encoding a vaccine. In other embodiments, the conjugates disclosed herein can be used to deliver a nucleic acid (e.g., mRNA) encoding an enzyme. In other embodiments, the conjugates disclosed herein can be used to deliver a nucleic acid encoding a chimeric antigen receptor (CAR) to T cells.

[0261] In some embodiments, the payload is one or more nucleic acids. In some embodiments, the payload is one or more RNA molecules. In some embodiments, the payload is an mRNA (e.g., an mRNA encoding an enzyme). In some embodiments, the RNA molecule is a non-coding RNA (ncRNA). In some embodiments, the payload is an RNA template (for example, an RNA template for reverse transcription, e.g., Target Primed Reverse Transcription (TPRT)). In other embodiments the payload is a siRNA or a microRNA (miRNA). In other embodiments, the payload is a guide RNA. In other embodiments, the payload is a tRNA. In other embodiments, the payload is an antisense oligonucleotide (ASO). In other embodiments, the payload is a DNA molecule, for example, a DNA plasmid, closed-ended DNA (ceDNA), or a small circular DNA (e.g., a minicircle or nanoplasmid). Nucleic acid payloads can be linear, circular, covalently closed, single-stranded, double-stranded, or hybrid RNA / DNAmolecules. In other embodiments, the payload is a small molecule. In some embodiments, the payload is a peptide or protein. In some embodiments, the conjugates disclosed herein can include two or more payloads, for example, selected from mRNA, ncRNA, guide RNA, siRNA, miRNA, ASO, DNA vector, small molecule, peptide, or protein.

[0262] The LNPs described herein, e.g., targeted LNPs, can be used to deliver a therapeutic of interest to a cell, e.g., an HSC or HSC progenitor, a liver cell such as a hepatocyte, or a T cell. In some embodiments, the LNP, e.g., targeted LNP, contains a payload that is a therapeutic agent. In some embodiments, the therapeutic agent can be 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 (e.g., for gene therapy or gene editing), 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 a 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, as described herein. In some embodiments, the therapeutic agent is a gene modifying system, as described herein.

[0263] In some embodiments, the therapeutic agent can be a peptide or protein, such as an enzyme, or a nucleic acid (e.g., mRNA or DNA) encoding the peptide or protein (e.g., an enzyme). In some embodiments, the enzyme can be a nuclease, recombinase, integrase, transposase, retrotransposase, helicase, transcriptase, polymerase, reverse transcriptase, deaminase, methylase, demethylase, or ligase, or can have a combination of enzymatic activities thereof. In some embodiments, the therapeutic agent can be a peptide or protein, or a nucleic acid encoding the peptide or protein, for use as a replacement gene therapy. In some embodiments, the therapeutic agent can be a peptide or protein, or a nucleic acid encoding the peptide or protein, for use in modifying or altering the genome or epigenome of a cell, e.g., a T cell, an HSC, an HSC progenitor, or a liver cell (e.g., hepatocyte) of a subject. In some embodiments, the therapeutic agent can comprise one or more components of a system for modifying or altering the genome or epigenome of a cell, e.g., a T cell, an HSC, an HSC progenitor, or a liver cell (e.g., hepatocyte) of a subject. In some embodiments, the system for modifying or altering the genome or epigenome of a cell of a subject comprises one or more proteins, one or more nucleic acids (e.g., RNA and / or DNA), or combinations thereof.

[0264] In some embodiments, the therapeutic agent can be one or more components of a ribonucleoprotein (RNP) complex for modifying or altering the genome or epigenome of a cell,e.g., a T cell, an HSC, an HSC progenitor, or a liver cell (e.g., hepatocyte). For example, in some cases the therapeutic agent can be a protein, or a nucleic acid (e.g., mRNA) encoding the protein, and / or an RNA molecule (e.g., a gRNA) for guiding the protein to a particular location in the genome or epigenome, wherein the protein is capable of modifying or altering the genome or epigenome as a nuclease, recombinase, integrase, transposase, retrotransposase, helicase, transcriptase, polymerase, reverse transcriptase, deaminase, methylase, demethylase, or ligase, or combinations thereof. In certain embodiments, the therapeutic agent is a nuclease. In some embodiments, the nuclease cleaves DNA (e.g., both strands of the DNA), thereby introducing insertion and / or deletion (indel) mutations in DNA, e.g., genomic DNA. In certain embodiments, the nuclease is a nickase (i.e., it cleaves a single stand of DNA). In certain embodiments, the nuclease is mutated such that it is inactive or comprises reduced nuclease activity. In certain embodiments, the therapeutic agent can be used to introduce a substitution in DNA, e.g., genomic DNA, via base editing. In certain embodiments, the therapeutic agent can be used for epigenome editing. In certain embodiments, the therapeutic agent can be used to introduce an indel or a substitution in DNA, e.g., genomic DNA, by inducing target-primed reverse transcription (TPRT). In certain embodiments, the therapeutic agent (i.e., payload) delivered by the LNP, e.g., targeted LNP, can be a gene modifying protein, a nucleic acid encoding a gene modifying protein, or a gene modifying system, as described herein. In certain embodiments, the therapeutic agent can be a small molecule. In certain embodiments, the therapeutic agent can be an siRNA or miRNA.

[0265] In some embodiments, the LNPs, e.g., targeted LNPs, of the disclosure can be used to deliver gene editing components into T cells, HSCs, HSC progenitors, or liver cells (e.g., hepatocytes). In some embodiments, the LNPs, e.g., targeted LNPs, can be used to deliver a CRISPR-Cas system into T cells, HSCs, or liver cells (e.g., hepatocytes). In some embodiments, the LNPs, e.g., targeted LNPs, can be used to deliver a Class 1 (type I, type III, or type IV) CRISPR system into T cells, HSCs, or liver cells (e.g., hepatocytes). In some embodiments, the LNPs, e.g., targeted LNPs, can be used to deliver a Class 2 (type II, type V, or type VI) CRISPR system into T cells, HSCs, or liver cells (e.g., hepatocytes). In some embodiments, the LNPs, e.g., targeted LNPs, can be used to deliver a CRISPR-Cas9 system, or a nucleic acid encoding one or more components of the CRISPR-Cas9 system, into T cells, HSCs (e.g., LT-HSCs), or liver cells (e.g., hepatocytes). In some embodiments, the LNPs, e.g., targeted LNPs, can be used to deliver a CRISPR-Cas12 system (e.g., a Cas12a system), or a nucleic acid encoding one or more components of the CRISPR-Cas12 system, into T cells,HSCs (e.g., LT-HSCs), or liver cells (e.g., hepatocytes). In some such embodiments, the LNPs, e.g., targeted LNPs, can comprise two RNA molecules, such as an RNA comprising a guide RNA (gRNA) and an mRNA encoding the Cas protein. In some embodiments, the Cas is Cas9 or Cas12a. In some embodiments, the Cas is Cas9. In some embodiments, the Cas is Cas12a. In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the LNPs, e.g., targeted LNPs, comprise a payload consisting of or comprising a Cas9 or an mRNA encoding a Cas9. In some embodiments, the LNPs, e.g., targeted LNPs, comprise a payload consisting of or comprising a Cas12 (e.g., Cas12a) or an mRNA encoding a Cas12 (e.g., Cas12a). In some embodiments, the payload further consists of or comprises a gRNA.

[0266] In some embodiments, the mRNA component of a gene modifying system comprises a recombinant nuclease, or a nucleic acid encoding the nuclease, for example a restriction endonuclease, meganuclease, homing endonuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

[0267] In some embodiments, multiple components of a gene modifying system may be prepared as a single LNP formulation, e.g., an LNP formulation comprises mRNA encoding the gene modifying polypeptide and an RNA template. Ratios of nucleic acid components may be varied in order to maximize the properties of a therapeutic. In some embodiments, the ratio of RNA template to mRNA encoding a gene modifying polypeptide is about 1:1 to 100:1, e.g., about 1:1 to 20:1, about 20:1 to 40:1, about 40:1 to 60:1, about 60:1 to 80:1, or about 80:1 to 100:1, by molar ratio. In other embodiments, a system of multiple nucleic acids may be prepared by separate formulations, e.g., one LNP formulation comprising a template RNA and a second LNP formulation comprising an mRNA encoding a gene modifying polypeptide. In some embodiments, the system may comprise more than two nucleic acid components formulated into LNPs. In some embodiments, the system may comprise a protein, e.g., a gene modifying polypeptide, and a template RNA formulated into at least one LNP formulation.

[0268] In some embodiments, the gene modifying system can be used to introduce a regulatory edit. In some embodiments, the regulatory edit is introduced to a regulatory sequence of a gene, for example, a gene promoter, gene enhancer, gene repressor, or a sequence that regulates gene splicing. In some embodiments, the regulatory edit increases or decreases the expression level of a target gene. In some embodiments, the target gene is the same as the gene containing a disease-causing mutation. In some embodiments, the target gene is different from the gene containing a disease-causing mutation. For example, the systems or methodsprovided herein can be used to upregulate the expression of fetal hemoglobin by introducing a regulatory edit at the promoter of bcl11a, thereby treating sickle cell disease.

[0269] Table 3 provides exemplary indications (column 1), genes (column 2), and regulatory edits that can be introduced using the systems or methods described herein (column 3). Table 3. Indications, genes, and compensatory regulatory edits.#: See J T den Dunnen and S E Antonarakis, Hum Mutat.2000;15(1):7-12, herein incorporated by reference in its entirety, for details of the nomenclatures of gene mutations

[0270] The conjugates described herein may be used to target and modify immune cells. In some embodiments, the conjugates may be used to modify T cells. In some embodiments, T- cells may include any subpopulation of T-cells, e.g., CD4+, CD8+, gamma-delta, naïve T cells, stem cell memory T cells, central memory T cells, or a mixture of subpopulations. In some embodiments, the conjugates may be used to deliver or modify a T-cell receptor (TCR) in a T cell. In some embodiments, the conjugates may be used to deliver at least one chimeric antigen receptor (CAR) to T-cells. For instance, in specific embodiments, the conjugates can be used to deliver mRNA encoding a CAR to T-cells. In some embodiments, the conjugates may be used to deliver at least one CAR to natural killer (NK) cells. In some embodiments, the conjugates be used to deliver at least one CAR to natural killer T (NKT) cells. In some embodiments, the conjugates may be used to deliver at least one CAR to a progenitor cell, e.g., a progenitor cell of T, NK, or NKT cells. In some embodiments, cells modified with at least one CAR (e.g., CAR-T cells, CAR-NK cells, CAR-NKT cells), or a combination of cells modified with at least one CAR (e.g., a mixture of CAR-NK / T cells) are used to treat a condition as identified in the targetable landscape of CAR therapies in MacKay, et al. Nat Biotechnol 38, 233-244 (2020), incorporated by reference herein in its entirety. In some embodiments, the immune cells comprise a CAR specific to a tumor or a pathogen antigen selected from a group consisting of AChR (fetal acetylcholine receptor), ADGRE2, AFP (alpha fetoprotein), BAFF-R, BCMA, CAIX (carbonic anhydrase IX), CCR1, CCR4, CEA (carcinoembryonic antigen), CD3, CD5, CD8, CD7, CD10, CD13, CD14, CD15, CD19, CD20, CD22, CD30, CD33, CLLI, CD34, CD38, CD41, CD44, CD49f, CD56, CD61, CD64, CD68, CD70,CD74, CD99,CD117, CD123, CD133, CD138, CD44v6, CD267, CD269, CDS, CLEC12A, CS1, EGP-2 (epithelial glycoprotein-2), EGP-40 (epithelial glycoprotein-40), EGFR(HER1), EGFR-VIII, EpCAM (epithelial cell adhesion molecule), EphA2, ERBB2 (HER2, human epidermal growth factor receptor 2), ERBB3, ERBB4, FBP (folate-binding protein), Flt3 receptor, folate receptor-a, GD2 (ganglioside G2), GD3 (ganglioside G3), GPC3(glypican-3), GPI00, hTERT (human telomerase reverse transcriptase), ICAM-1, integrin B7, interleukin 6 receptor, IL13Ra2 (interleukin-13 receptor 30 subunit alpha-2), kappa-light chain, KDR (kinase insert domain receptor), LeY (Lewis Y), L1CAM (LI cell adhesion molecule), LILRB2 (leukocyte immunoglobulin like receptor B2), MARTI, MAGE-A1 (melanoma associated antigen Al), MAGE- A3, MSLN (mesothelin), MUC16 (mucin 16), MUCI (mucin I), KG2D ligands, NY-ESO-1 (cancer-testis antigen), PRI (proteinase 3), TRBCI, TRBC2, TFM-3, TACI, tyrosinase, survivin, hTERT, oncofetal antigen (h5T4), p53, PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), hRORl, TAG-72 (tumor- associated glycoprotein 72), VEGF-R2 (vascular endothelial growth factor R2), WT-1 (Wilms tumor protein), and antigens of HIV (human immunodeficiency virus), hepatitis B, hepatitis C, CMV (cytomegalovirus), EBV (Epstein-Barr virus), HPV (human papilloma virus).

[0271] In some embodiments, immune cells, e.g., T-cells, NK cells, NKT cells, or progenitor cells are modified ex vivo and then delivered to a patient. In some embodiments, a nucleic acid (e.g., mRNA) is delivered by one of the methods mentioned herein, and immune cells, e.g., T- cells, NK cells, NKT cells, or progenitor cells are modified in vivo in the patient.

[0272] In certain embodiments, the targeting moiety is a T-cell targeting moiety, for example, an antibody, Fab fragment or ScFv that binds to a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD28, CD137, CD45, T-cell receptor -- TCR- CTLA4, TIM3, LAG3, CD18, IL-2 receptor,CD11a, TLR2, TLR4, TLR5, IL-7 receptor, or IL-15 receptor. A. Gene Modifying Systems

[0273] The LNPs described herein can be formulated to comprise one or more components of a gene modifying system or one or more nucleic acids encoding said components. Accordingly, in some embodiments, the payload comprises a gene modifying system, or one or more nucleic acids encoding the components of the gene modifying system. For instance, in some embodiments, the payload comprises a template RNA and an mRNA encoding the gene modifying polypeptide. In some embodiments, conjugates prepared in accordance with this disclosure are used to deliver to target cells systems that are capable of inserting a heterologous object sequence (e.g., a sequence encoding a CAR) into the genome of the cell, e.g., an immune cell, such as a T cell. In some embodiments, the system comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises: (i) an endonuclease and / or DNA binding domain; and(ii) a reverse transcriptase (RT) domain, where (i) and (ii) are both derived from a retrotransposon (e.g., from the same retrotransposon or different retrotransposons); and (B) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds the polypeptide and (ii) a heterologous object sequence. A gene modifying polypeptide, in some embodiments, acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell), substantially without relying on host machinery. The heterologous object sequence may include, e.g., a coding sequence, a regulatory sequence, a gene expression unit.

[0274] In some embodiments, systems described herein can have a number of advantages relative to various earlier systems. For instance, the disclosure describes retrotransposases capable of inserting long sequences of heterologous nucleic acid into a genome. In addition, retrotransposases described herein can insert heterologous nucleic acid in an endogenous site in the genome, such as the rDNA locus. This is in contrast to Cre / loxP systems, which require a first step of inserting an exogenous loxP site before a second step of inserting a sequence of interest into the loxP site. (i) Gene modifying polypeptides

[0275] Non-long terminal repeat (LTR) retrotransposons are a type of mobile genetic elements that are widespread in eukaryotic genomes. They include, for example, the apurinic / apyrimidinic endonuclease (APE)-type, the restriction enzyme-like endonuclease (RLE)-type, and the Penelope-like element (PLE)-type.

[0276] The APE class retrotransposons are comprised of two functional domains: an endonuclease / DNA binding domain, and a reverse transcriptase domain. Examples of APE- class retrotransposons can be found, for example, in Table 1 of PCT Application No. PCT / US2019 / 048607, US 2023 / 0235358, US 2023 / 0242899, and US 2020 / 0109398, the disclosures of which are incorporated herein by reference in their entireties, including the sequence listing and sequences referred to in Table 1 in PCT / US2019 / 048607 and US 2020 / 0109398.

[0277] The RLE class are comprised of three functional domains: a DNA binding domain, a reverse transcription domain, and an endonuclease domain. Examples of RLE-class retrotransposons can be found, for example, in Table 2 of PCT Application No.PCT / US2019 / 048607, US 2023 / 0235358, US 2023 / 0242899, and US 2020 / 0109398, the disclosures of which are incorporated herein by reference in their entireties, including the sequence listing and sequences referred to in Table 2 in in PCT / US2019 / 048607 and US 2020 / 0109398.

[0278] The reverse transcriptase domain of non-LTR retrotransposon functions by binding an RNA sequence template and reverse transcribing it into the host genome’s target DNA. The RNA sequence template has a 3’ untranslated region which is specifically bound to the retrotransposase, and a variable 5’ region generally having Open Reading Frame(s) (“ORF”) encoding retrotransposase proteins. The RNA sequence template may also comprise a 5’ untranslated region which specifically binds the retrotransposase.

[0279] Penelope-like elements (PLEs) are distinct from both LTR and non-LTR retrotransposons. PLEs generally comprise a reverse transcriptase domain distinct from that of APE and RLE elements, but similar to that of telomerases and Group II introns, and an optional GIY-YIG endonuclease domain.

[0280] Other exemplary classes of retrotransposon include, without limitation, RTE (e.g., RTE-1_MD, RTE-3_BF, and RTE-25_LMi), CR1 (e.g., CR1-1_PH), Crack (e.g., Crack- 28_RF), L2 (e.g., L2-2_Dre and L2-5_GA), and Vingi (e.g., Vingi-1_Acar) retrotransposons.

[0281] As described herein, the elements of such retrotransposons can be functionally modularized and / or modified to target, edit, modify or manipulate a target DNA sequence, e.g., to insert an object (e.g., heterologous) nucleic acid sequence into a target genome, e.g., a mammalian genome, by reverse transcription. In some embodiments, a gene modifying system comprises: (A) a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a retrotransposase reverse transcriptase domain, and (ii) a retrotransposase endonuclease domain that contains DNA binding functionality; and (B) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds the polypeptide and (ii) a heterologous object sequence. The RNA template element of a gene modifying system is typically heterologous to the polypeptide element and provides an object sequence to be inserted (reverse transcribed) into the host genome.

[0282] In some embodiments, the gene modifying system comprises a retrotransposase sequence of an element listed in any one of Table 10, Table 11, Table X, Table Z1 Table 3A,or 3B of PCT Pub. No.: WO / 2021 / 178717, US 2023 / 0235358, and US 2023 / 0242899, which are incorporated herein by reference as they relate to domains from retrotransposons.

[0283] In some embodiments, an amino acid sequence encoded by an element of Table 4 is an amino acid sequence encoded by the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the full-length sequence of an element listed in Table 4 may comprise one or more (e.g., all of) of a 5’ UTR, polypeptide-encoding sequence, or 3’ UTR of a retrotransposon as described herein. In some embodiments, an amino acid sequence of Table 4 is an amino acid sequence encoded by the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, a 5’ UTR of an element of Table 4 comprises a 5’ UTR of the full length sequence of an element listed in Table 7, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, a 3’ UTR of an element of Table 4 comprises a 3’ UTR of the full length sequence of an element listed in Table 4, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0284] Also indicated in Table 4 are the host organisms from which the nucleic acid sequences were obtained and a listing of domains present within the polypeptide encoded by the open reading frame of the nucleic acid sequence.

[0285] In certain embodiments, the gene modifying polypeptide further comprises a heterologous protein domain.

[0286] Table 4 provides gene modifying polypeptides comprising retrotransposon elements, altered for improved efficiency of integration into the human genome. Retrotransposase polypeptides were improved through consensus mapping to re-derive the optimal amino acid sequence. Template molecules for use with cognate retrotransposase enzymes were mapped back to their host genomes and flanking genomic DNA used to elucidate target site motifs. When detectable, conserved sequence motifs from the flanking genomic DNA of endogenous occurrences of an element were aligned to the human genome, and new sequences were derived from the human genome as 5’ or 3’ “Human Homology Arms.” In some embodiments, a template RNA described herein comprises one or both of a first homology domain comprising a sequence of a 5' Human Homology Arm of Table 4 (or a sequence having at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identity thereto) and a second homology domain comprising a sequence of a 3' Human Homology Arm of Table 4 (or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto).W(ii) Retrotransposon discovery tools

[0287] As the result of repeated mobilization over time, transposable elements in genomic DNA often exist as tandem or interspersed repeats (Jurka Curr Opin Struct Biol 8, 333-337 (1998)). Tools capable of recognizing such repeats can be used to identify new elements from genomic DNA and for populating databases, e.g., Repbase (Jurka et al Cytogenet Genome Res 110, 462-467 (2005)). One such tool for identifying repeats that may comprise transposable elements is RepeatFinder (Volfovsky et al Genome Biol 2 (2001)), which analyzes the repetitive structure of genomic sequences. Repeats can further be collected and analyzed using additional tools, e.g., Censor (Kohany et al BMC Bioinformatics 7, 474 (2006)). The Censor package takes genomic repeats and annotates them using various BLAST approaches against known transposable elements. An all-frames translation can be used to generate the ORF(s) for comparison.

[0288] Other exemplary methods for identification of transposable elements include RepeatModeler2, which automates the discovery and annotation of transposable elements in genome sequences (Flynn et al bioRxiv (2019)). In addition to accomplishing this via available packages like Censor, one can perform an all-frames translation of a given genome or sequence and annotate with a protein domain tool like InterProScan, which tags the domains of a given amino acid sequence using the InterPro database (Mitchell et al. Nucleic Acids Res 47, D351- 360 (2019)), allowing the identification of potential proteins comprising domains associated with known transposable elements.

[0289] Retrotransposons can be further classified according to the reverse transcriptase domain using a tool such as RTclass1 (Kapitonov et al Gene 448, 207-213 (2009)). (iii) Polypeptide component of gene modifying system a) RT domain

[0290] In certain aspects, the reverse transcriptase domain of the gene modifying system is based on a reverse transcriptase domain of an APE-type or RLE-type non-LTR retrotransposon, or of a PLE-type retrotransposon. A wild-type reverse transcriptase domain of an APE-type, RLE-type, or PLE-type retrotransposon can be used in a gene modifying system or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) to alter the reverse transcriptase activity for target DNA sequences. In some embodiments, the reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g. improved forhuman cells. In some embodiments, the reverse transcriptase domain is a heterologous reverse transcriptase from a different LTR-retrotransposon, non-LTR retrotransposon, or other source. In certain embodiments, a gene modifying system includes a polypeptide that comprises a reverse transcriptase domain of a RTE (e.g., RTE-1_MD, RTE-3_BF, and RTE-25_LMi), CR1 (e.g., CR1-1_PH), Crack (e.g., Crack-28_RF), L2 (e.g., L2-2_Dre and L2-5_GA), and Vingi (e.g., Vingi-1_Acar) retrotransposon.

[0291] In certain embodiments, a gene modifying system includes a polypeptide that comprises a reverse transcriptase domain of a retrotransposon listed in Table 10, Table 11, Table X, Table Z1, Table Z2, or Table 3A or 3B of PCT Pub. No.: WO / 2021 / 178717.

[0292] In certain embodiments, a gene modifying system includes a polypeptide that comprises a reverse transcriptase domain of a retrotransposon listed in Table 4. In some embodiments, the amino acid sequence of the reverse transcriptase domain of a gene modifying system is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of a reverse transcriptase domain of a retrotransposon whose DNA sequence is referenced in Table 4. Reverse transcriptase domains can be identified, for example, based upon homology to other known reverse transcription domains using routine tools as Basic Local Alignment Search Tool (BLAST). In some embodiments, reverse transcriptase domains are modified, for example by site-specific mutation. In some embodiments, the reverse transcriptase domain is engineered to bind a heterologous template RNA.

[0293] In some embodiments, a polypeptide (e.g., RT domain) comprises an RNA-binding domain, e.g., that specifically binds to an RNA sequence. In some embodiments, a template RNA comprises an RNA sequence that is specifically bound by the RNA-binding domain.

[0294] In some embodiments, the RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, the RT domain is monomeric. In some embodiments, an RT domain naturally functions as a monomer or as a dimer (e.g., heterodimer or homodimer). In some embodiments, an RT domain naturally functions as a monomer. Naturally heterodimeric RT domains may, in some embodiments, also be functional as homodimers. In some embodiments, dimeric RT domains are expressed as fusion proteins, e.g., as homodimeric fusion proteins or heterodimeric fusion proteins. In some embodiments, the RT function of thesystem is fulfilled by multiple RT domains (e.g., as described herein). In further embodiments, the multiple RT domains are fused or separate, e.g., may be on the same polypeptide or on different polypeptides.

[0295] In some embodiments, a gene modifying polypeptide described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of the RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain, e.g., an endogenous RNAse H domain or a heterologous RNase H domain. In some embodiments, an RT domain (e.g., as described herein) lacks an RNase H domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain that has been added, deleted, mutated, or swapped for a heterologous RNase H domain. In some embodiments, mutation of an RNase H domain yields a polypeptide exhibiting lower RNase activity, e.g., as determined by the methods described in Kotewicz et al. Nucleic Acids Res 16(1):265-277 (1988), e.g., lower by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to an otherwise similar domain without the mutation. In some embodiments, RNase H activity is abolished.

[0296] In some embodiments, an RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation. For instance, in some embodiments, a YADD (SEQ ID NO: 39) or YMDD (SEQ ID NO: 40) motif in an RT domain (e.g., in a reverse transcriptase) is replaced with YVDD (SEQ ID NO: 41). In embodiments, replacement of the YADD (SEQ ID NO: 39) or YMDD (SEQ ID NO: 40) or YVDD (SEQ ID NO: 41) results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011.) b) Endonuclease domain

[0297] In some embodiments, the polypeptide comprises an endonuclease domain (e.g., a heterologous endonuclease domain). In certain embodiments, the endonuclease / DNA binding domain of an APE-type retrotransposon, the endonuclease domain of an RLE-type retrotransposon, or the endonuclease domain of a PLE-type retrotransposon can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein. In some embodiments, the endonuclease domain or endonuclease / DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments, the endonuclease element is aheterologous endonuclease element. The amino acid sequence of an endonuclease domain of a gene modifying system described herein may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of an endonuclease domain of a retrotransposon whose DNA sequence is referenced in Table X, Z1, Z2, 3A, or 3B of PCT Pub. No: WO / 2021 / 178717.

[0298] In certain embodiments, a gene modifying system includes a polypeptide that comprises an endonuclease domain of a retrotransposon listed in Table 4. In some embodiments, the amino acid sequence of the endonuclease domain of a gene modifying system is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of a endonuclease domain of a retrotransposon whose DNA sequence is referenced in Table 4. Endonuclease domains can be identified, for example, based upon homology to other known endonuclease domains using tools as Basic Local Alignment Search Tool (BLAST).

[0299] In some embodiments, a gene modifying polypeptide possesses the function of DNA target site cleavage via an endonuclease domain. In some embodiments, the endonuclease domain is also a DNA-binding domain. In some embodiments, the endonuclease domain is also a template nucleic acid (e.g., template RNA) binding domain. In certain embodiments, the endonuclease / DNA binding domain of an APE-type retrotransposon or the endonuclease domain of an RLE-type retrotransposon can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein. c. Template nucleic acid binding domain

[0300] A gene modifying polypeptide typically contains regions capable of associating with the template nucleic acid (e.g., template RNA). In some embodiments, the template nucleic acid binding domain is an RNA binding domain. In some embodiments, the RNA binding domain is a modular domain that can associate with RNA molecules containing specific signatures, e.g., structural motifs, e.g., secondary structures present in the 3’ UTR in non-LTR retrotransposons. In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) RNA binding domain is contained within the reverse transcription domain,e.g., the reverse transcriptase-derived component has a known signature for RNA preference, e.g., secondary structures present in the 3’ UTR in non-LTR retrotransposons. d. DNA binding domain

[0301] In certain aspects, the DNA-binding domain of a gene modifying polypeptide described herein is selected, designed, or constructed for binding to a desired host DNA target sequence. In certain embodiments, the DNA-binding domain of the engineered retrotransposon is a heterologous DNA-binding protein or domain relative to a native retrotransposon sequence. In certain embodiments, the heterologous DNA-binding domain is a DNA binding domain of a retrotransposon described in Table 4 herein or in Table X, Table Z1, Table Z2, or Table 3A or 3B of PCT Pub. No.: WO / 2021 / 178717. In some embodiments, DNA binding domains can be identified based upon homology to other known DNA binding domains using tools as Basic Local Alignment Search Tool (BLAST). In still other embodiments, DNA-binding domains are modified, for example by site-specific mutation. In some embodiments, the DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells.

[0302] In embodiments, the DNA binding domain comprises one or more modifications relative to a wild-type DNA binding domain, e.g., a modification via directed evolution, e.g., phage-assisted continuous evolution (PACE).

[0303] In certain aspects of the present invention, the host DNA-binding site integrated into by the gene modifying system can be in a gene, in an intron, in an exon, an ORF, outside of a coding region of any gene, in a regulatory region of a gene, or outside of a regulatory region of a gene. In other aspects, the engineered retrotransposon may bind to one or more than one host DNA sequence. In other aspects, the engineered retrotransposon may have low sequence specificity, e.g., bind to multiple sequences or lack sequence preference.

[0304] In some embodiments, a gene modifying system is used to edit a target locus in multiple alleles. In some embodiments, a gene modifying system is designed to edit a specific allele. For example, a gene modifying polypeptide may be directed to a specific sequence that is only present on one allele, e.g., comprises a template RNA with homology to a target allele, e.g., an annealing domain, but not to a second cognate allele. In some embodiments, a gene modifying system can alter a haplotype-specific allele. In some embodiments, a genemodifying system that targets a specific allele preferentially targets that allele, e.g., has at least a 2, 4, 6, 8, or 10-fold preference for a target allele. e. Localization sequences for gene modifying systems

[0305] In certain embodiments, a gene modifying system RNA further comprises an intracellular localization sequence, e.g., a nuclear localization sequence.

[0306] The nuclear localization sequence may be an RNA sequence that promotes the import of the RNA into the nucleus. In certain embodiments, the nuclear localization signal is located on the template RNA. In certain embodiments, the retrotransposase polypeptide is encoded on a first RNA, and the template RNA is a second, separate, RNA, and the nuclear localization signal is located on the template RNA and not on an RNA encoding the retrotransposase polypeptide. While not wishing to be bound by theory, in some embodiments, the RNA encoding the retrotransposase is targeted primarily to the cytoplasm to promote its translation, while the template RNA is targeted primarily to the nucleus to promote its retrotransposition into the genome. In some embodiments, the nuclear localization signal is at the 3’ end, 5’ end, or in an internal region of the template RNA. In some embodiments the nuclear localization signal is 3’ of the heterologous sequence (e.g., is directly 3’ of the heterologous sequence) or is 5’ of the heterologous sequence (e.g., is directly 5’ of the heterologous sequence). In some embodiments, the nuclear localization signal is placed outside of the 5’ UTR or outside of the 3’ UTR of the template RNA. In some embodiments the nuclear localization signal is placed between the 5’ UTR and the 3’ UTR, wherein optionally the nuclear localization signal is not transcribed with the transgene (e.g., the nuclear localization signal is an anti-sense orientation or is downstream of a transcriptional termination signal or polyadenylation signal). In some embodiments, the nuclear localization sequence is situated inside of an intron. In some embodiments a plurality of the same or different nuclear localization signals are in the RNA, e.g., in the template RNA. In some embodiments, the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 bp in length. Various RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature 555 (107-111), 2018 describe RNA sequences, which drive RNA localization into the nucleus. In some embodiments, the nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal. In some embodiments, the nuclear localization signal binds a nuclear-enriched protein. In some embodiments, the nuclear localization signal binds the HNRNPK protein. In some embodiments the nuclear localization signal is rich inpyrimidines, e.g., is a C / T rich, C / U rich, C rich, T rich, or U rich region. In some embodiments, the nuclear localization signal is derived from a long non-coding RNA. In some embodiments, the nuclear localization signal is derived from MALAT1 long non-coding RNA or is the 600 nucleotide M region of MALAT1 (described in Miyagawa et al., RNA 18, (738-751), 2012). In some embodiments, the nuclear localization signal is derived from BORG long non-coding RNA or is a AGCCC motif (described in Zhang et al., Molecular and Cellular Biology 34, 2318-2329 (2014). In some embodiments, the nuclear localization sequence is described in Shukla et al., The EMBO Journal e98452 (2018). In some embodiments, the nuclear localization signal is derived from a non-LTR retrotransposon, an LTR retrotransposon, retrovirus, or an endogenous retrovirus.

[0307] In some embodiments, a polypeptide described herein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example, a nuclear localization sequence (NLS), e.g., as described herein. In some embodiments, the NLS is a bipartite NLS. In some embodiments, an NLS facilitates the import of a protein comprising an NLS into the cell nucleus. In some embodiments, the NLS is fused to the N-terminus of a gene modifying polypeptide described herein. In some embodiments, the NLS is fused to the C-terminus of the gene modifying polypeptide. In some embodiments, a linker sequence is disposed between the NLS and the neighboring domain of the gene modifying polypeptide. In some embodiments, an NLS comprises the amino acid sequence of an NLS described herein.

[0308] In some embodiments, a nucleic acid described herein (e.g., an RNA encoding a gene modifying polypeptide, or a DNA encoding the RNA) comprises a microRNA binding site. In some embodiments, the microRNA binding site is used to increase the target-cell specificity of a gene modifying system. For instance, the microRNA binding site can be chosen on the basis that is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when the RNA encoding the gene modifying polypeptide is present in a non-target cell, it would be bound by the miRNA, and when the RNA encoding the gene modifying polypeptide is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by theory, binding of the miRNA to the RNA encoding the gene modifying polypeptide may reduce production of the gene modifying polypeptide, e.g., by degrading the mRNA encoding the polypeptide or by interfering with translation. Accordingly, the heterologous object sequence would be inserted into the genomeof target cells more efficiently than into the genome of non-target cells. A system having a microRNA binding site in the RNA encoding the gene modifying polypeptide (or encoded in the DNA encoding the RNA) may also be used in combination with a template RNA that is regulated by a second microRNA binding site.

[0309] In some embodiments, a polypeptide for use in any of the systems described herein can be a molecular reconstruction or ancestral reconstruction based upon the aligned polypeptide sequence of multiple retrotransposons. In some embodiments, a 5’ or 3’ untranslated region for use in any of the systems described herein can be a molecular reconstruction based upon the aligned 5’ or 3’ untranslated region of multiple retrotransposons. Based on the Accession numbers, polypeptides or nucleic acid sequences can be aligned, e.g., by using routine sequence analysis tools as Basic Local Alignment Search Tool (BLAST) or CD-Search for conserved domain analysis. Molecular reconstructions can be created based upon sequence consensus, e.g. using approaches described in Ivics et al., Cell 1997, 501 – 510 ; Wagstaff et al., Molecular Biology and Evolution 2013, 88-99. In some embodiments, the retrotransposon from which the 5’ or 3’ untranslated region or polypeptide is derived is a young or a recently active mobile element, as assessed via phylogenetic methods such as those described in Boissinot et al., Molecular Biology and Evolution 2000, 915-928.

[0310] In some embodiments, the gene modifying system comprises an intein. Generally, an intein comprises a polypeptide that has the capacity to join two polypeptides or polypepide fragments together via a peptide bond. In some embodiments, the intein is a trans-splicing intein that can join two polypeptide fragments, e.g., to form the polypeptide component of a system as described herein. (iv) Promoters

[0311] In some embodiments, one or more promoter or enhancer elements are operably linked to a nucleic acid encoding a gene modifying protein or a template nucleic acid, e.g., that controls expression of the heterologous object sequence. In certain embodiments, the one or more promoter or enhancer elements comprise cell-type or tissue specific elements. In some embodiments, the promoter or enhancer is the same or derived from the promoter or enhancer that naturally controls expression of the heterologous object sequence.

[0312] In some embodiments, a gene modifying system is capable of producing a substitution into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides. In some embodiments, the substitution is a transition mutation. In some embodiments, the substitution is a transversion mutation. In some embodiments, the substitution converts an adenine to a thymine, an adenine to a guanine, an adenine to a cytosine, a guanine to a thymine, a guanine to a cytosine, a guanine to an adenine, a thymine to a cytosine, a thymine to an adenine, a thymine to a guanine, a cytosine to an adenine, a cytosine to a guanine, or a cytosine to a thymine.

[0313] In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases).

[0314] In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g. transcription or translation) of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof, increases ordecreases expression (e.g. transcription or translation) of a gene by altering, adding, or deleting sequences in a promoter or enhancer, e.g. sequences that bind transcription factors. In some embodiments, an insertion, deletion, substitution, or combination thereof alters translation of a gene (e.g. alters an amino acid sequence), inserts or deletes a start or stop codon, alters or fixes the translation frame of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof alters splicing of a gene, e.g. by inserting, deleting, or altering a splice acceptor or donor site. In some embodiments, an insertion, deletion, substitution, or combination thereof alters transcript or protein half-life. In some embodiments, an insertion, deletion, substitution, or combination thereof alters protein localization in the cell (e.g. from the cytoplasm to a mitochondria, from the cytoplasm into the extracellular space (e.g. adds a secretion tag)). In some embodiments, an insertion, deletion, substitution, or combination thereof alters (e.g. improves) protein folding (e.g. to prevent accumulation of misfolded proteins). In some embodiments, an insertion, deletion, substitution, or combination thereof, alters, increases, decreases the activity of a gene, e.g. a protein encoded by the gene.

[0315] In some embodiments, a system or method described herein results in “scarless” insertion of the heterologous object sequence, while in some embodiments, the target site can show deletions or duplications of endogenous DNA as a result of insertion of the heterologous sequence. The mechanisms of different retrotransposons could result in different patterns of duplications or deletions in the host genome occurring during retrotransposition at the target site. In some embodiments, the system results in a scarless insertion, with no duplications or deletions in the surrounding genomic DNA. In some embodiments, the system results in a deletion of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA upstream of the insertion. In some embodiments, the system results in a deletion of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA downstream of the insertion. In some embodiments, the system results in a duplication of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA upstream of the insertion. In some embodiments, the system results in a duplication of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA downstream of the insertion.

[0316] In some embodiments, a gene modifying system described herein, or a DNA-binding domain thereof, binds to its target site specifically, e.g., as measured using an assay of Example 21 of PCT Application No. PCT / US2019 / 048607. In some embodiments, the gene modifying polypeptide or DNA-binding domain thereof binds to its target site more strongly than to any other binding site in the human genome. For example, in some embodiments, in an assay ofExample 21 of PCT Application No. PCT / US2019 / 048607, the target site represents more than 50%, 60%, 70%, 80%, 90%, or 95% of binding events of the gene modifying polypeptide or DNA-binding domain thereof to human genomic DNA. In some embodiments, the DNA binding domain of the gene modifying polypeptide is heterologous to the remainder of the gene modifying polypeptide, e.g., such that the gene modifying polypeptide targets a different target site that the endogenous DNA binding domain associated with the remainder of the gene modifying polypeptide. (v) Genetically engineered, e.g., dimerized gene modifying polypeptides

[0317] Some non-LTR retrotransposons utilize two subunits to complete retrotransposition (Christensen et al PNAS 2006). In some embodiments, a retrotransposase described herein comprises two connected subunits as a single polypeptide. For instance, two wild-type retrotransposases could be joined with a linker to form a covalently “dimerized” protein. In some embodiments, the nucleic acid coding for the retrotransposase codes for two retrotransposase subunits to be expressed as a single polypeptide. In some embodiments, the subunits are connected by a peptide linker. Based on mechanism, not all functions are required from both retrotransposase subunits. In some embodiments, the fusion protein may consist of a fully functional subunit and a second subunit lacking one or more functional domains. In some embodiments, one subunit may lack reverse transcriptase functionality. In some embodiments, one subunit may lack the reverse transcriptase domain. In some embodiments, one subunit may possess only endonuclease activity. In some embodiments, one subunit may possess only an endonuclease domain. In some embodiments, the two subunits comprising the single polypeptide may provide complimentary functions.

[0318] In some embodiments, one subunit may lack endonuclease functionality. In some embodiments, one subunit may lack the endonuclease domain. In some embodiments, one subunit may possess only reverse transcriptase activity. In some embodiments, one subunit may possess only a reverse transcriptase domain. In some embodiments, one subunit may possess only DNA-dependent DNA synthesis functionality. (vi) Evolved Variants of Gene Modifying Polypeptides

[0319] In some embodiments, the invention provides evolved variants of gene modifying(vii) Template RNA component of gene modifying system

[0320] The gene modifying systems described herein can transcribe an RNA sequence template into host target DNA sites by target-primed reverse transcription. By writing DNA sequence(s) via reverse transcription of the RNA sequence template directly into the host genome, the gene modifying system can insert an object sequence into a target genome without the need for exogenous DNA sequences to be introduced into the host cell (unlike, for example, CRISPR systems), as well as eliminate an exogenous DNA insertion step. Therefore, the gene modifying system provides a platform for the use of customized RNA sequence templates containing object sequences, e.g., sequences comprising heterologous gene coding and / or function information.

[0321] In some embodiments, the template RNA encodes a gene modifying protein in cis with a heterologous object sequence. Various cis constructs were described, for example, in Kuroki-Kami et al (2019) Mobile DNA 10:23 (incorporated by reference herein in its entirety), and can be used in combination with any of the embodiments described herein. For instance, in some embodiments, the template RNA comprises a heterologous object sequence, a sequence encoding a gene modifying protein (e.g., a protein comprising (i) a reverse transcriptase domain and (ii) an endonuclease domain, e.g., as described herein), a 5’ untranslated region, and a 3’ untranslated region. The components may be included in various orders. In some embodiments, the gene modifying protein and heterologous object sequence are encoded in different directions (sense vs. anti-sense), e.g., using an arrangement shown in Figure 3A of Kuroki-Kami et al, Id. In some embodiments, the gene modifying protein and heterologous object sequence are encoded in the same direction. In some embodiments, the nucleic acid encoding the polypeptide and the template RNA or the nucleic acid encoding the template RNA are covalently linked, e.g., are part of a fusion nucleic acid, and / or are part of the same transcript. In some embodiments, the fusion nucleic acid comprises RNA or DNA.

[0322] The nucleic acid encoding the gene modifying polypeptide may, in some instances, be 5’ of the heterologous object sequence. For example, in some embodiments, the template RNA comprises, from 5’ to 3’, a 5’ untranslated region, a sense-encoded gene modifying polypeptide, a sense-encoded heterologous object sequence, and 3’ untranslated region. In some embodiments, the template RNA comprises, from 5’ to 3’, a 5’ untranslated region, a sense-encoded gene modifying polypeptide, anti-sense-encoded heterologous object sequence, and 3’ untranslated region.

[0323] It is understood that, when a template RNA is described as comprising an open reading frame or the reverse complement thereof, in some embodiments the template RNA must be converted into double stranded DNA (e.g., through reverse transcription) before the open reading frame can be transcribed and translated.

[0324] In certain embodiments, customized RNA sequence template can be identified, designed, engineered and constructed to contain sequences altering or specifying host genome function, for example by introducing a heterologous coding region into a genome; affecting or causing exon structure / alternative splicing; causing disruption of an endogenous gene; causing transcriptional activation of an endogenous gene; causing epigenetic regulation of an endogenous DNA; causing up- or down-regulation of operably liked genes, etc. In certain embodiments, a customized RNA sequence template can be engineered to contain sequences coding for exons and / or transgenes, provide for binding sites to transcription factor activators, repressors, enhancers, etc., and combinations of thereof. In other embodiments, the coding sequence can be further customized with splice acceptor sites, poly-A tails. In certain embodiments the RNA sequence can contain sequences coding for an RNA sequence template homologous to the retrotransposase, be engineered to contain heterologous coding sequences, or combinations thereof.

[0325] The template RNA may have some homology to the target DNA. In some embodiments the template RNA has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of exact homology to the target DNA at the 3’ end of the RNA. In some embodiments the template RNA has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 175, 180, or 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the target DNA, e.g., at the 5’ end of the template RNA. In some embodiments, the template RNA has a 3’ untranslated region derived from a retrotransposon, e.g. a retrotransposons described herein. In some embodiments the template RNA has a 3’ region of at least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the 3’ sequence of a retrotransposon, e.g., a retrotransposon described herein, e.g. a retrotransposon in Table 4. In some embodiments, the template RNA has a 5’ untranslated region derived from a retrotransposon, e.g. a retrotransposons described herein. In some embodiments the template RNA has a 5’ region ofat least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, or 200 or more bases of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater homology to the 5’ sequence of a retrotransposon, e.g., a retrotransposon described herein, e.g. a retrotransposon described in Table 4.

[0326] The template RNA component of a gene modifying system described herein typically is able to bind the gene modifying protein of the system. In some embodiments, the template RNA has a 3’ region that is capable of binding a gene modifying genome editing protein. The binding region, e.g., 3’ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying protein of the system.

[0327] The template RNA component of a gene modifying system described herein typically is able to bind the gene modifying protein of the system. In some embodiments, the template RNA has a 5’ region that is capable of binding a gene modifying protein. The binding region, e.g., 5’ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying protein of the system. In some embodiments, the 5’ untranslated region comprises a pseudoknot, e.g., a pseudoknot that is capable of binding to the gene modifying protein.

[0328] In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a stem-loop sequence. In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a hairpin. In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a helix. In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a psuedoknot. In some embodiments, the template RNA comprises a ribozyme. In some embodiments the ribozyme is similar to a hepatitis delta virus (HDV) ribozyme, e.g., has a secondary structure like that of the HDV ribozyme and / or has one or more activities of the HDV ribozyme, e.g., a self-cleavage activity. See, e.g., Eickbush et al., Molecular and Cellular Biology, 2010, 3142-3150.

[0329] In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 3’ untranslated region) comprises one or more stem-loops or helices. Exemplary structures of R2 3’ UTRs are shown, for example, in Ruschak et al. “Secondary structure –987,e.g., at Figure 3, therein, and in Eikbush and Eikbush, “R2 and R2 / R1 hybrid non-autonomous retrotransposons derived by internal deletions of full-length elements” Mobile DNA (2012) 3:10; e.g., at Figure 3 therein, which articles are hereby incorporated by reference in their entirety.

[0330] In some embodiments, a template RNA described herein comprises a sequence that is capable of binding to a gene modifying protein described herein. For instance, in some embodiments, the template RNA comprises an MS2 RNA sequence capable of binding to an MS2 coat protein sequence in the gene modifying protein. In some embodiments, the template RNA comprises an RNA sequence capable of binding to a B-box sequence. In some embodiments, in addition to or in place of a UTR, the template RNA is linked (e.g., covalently) to a non-RNA UTR, e.g., a protein or small molecule.

[0331] In some embodiments, the template RNA has a poly-A tail at the 3’ end. In some embodiments, the template RNA does not have a poly-A tail at the 3’ end.

[0332] In some embodiments the template RNA has a 5’ region of at least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200 or more bases of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater homology to the 5’ sequence of a retrotransposon, e.g., a retrotransposon described herein.

[0333] The template RNA of the system typically comprises an object sequence for insertion into a target DNA. The object sequence may be coding or non-coding.

[0334] In some embodiments, a system or method described herein comprises a single template RNA. In some embodiments, a system or method described herein comprises a plurality of template RNAs.

[0335] In some embodiments, the object sequence may contain an open reading frame. In some embodiments, the template RNA has a Kozak sequence. In some embodiments, the template RNA has an internal ribosome entry site. In some embodiments, the template RNA has a self-cleaving peptide such as a T2A or P2A site. In some embodiments, the template RNA has a start codon. In some embodiments, the template RNA has a splice acceptor site. In some embodiments, the template RNA has a splice donor site. Exemplary splice acceptor and splice donor sites are described in US10435677, incorporated herein by reference in its entirety. Exemplary splice acceptor site sequences are known to those of skill in the art and include, byway of example only, CTGACCCTTCTCTCTCTCCCCCAGAG (SEQ ID NO: 42) (from human HBB gene) and TTTCTCTCCCACAAG (SEQ ID NO: 43) (from human immunoglobulin-gamma gene). In some embodiments the template RNA, has a microRNA binding site downstream of the stop codon. In some embodiments, the template RNA has a polyA tail downstream of the stop codon of an open reading frame. In some embodiments, the template RNA comprises one or more exons. In some embodiments, the template RNA comprises one or more introns. In some embodiments, the template RNA comprises a eukaryotic transcriptional terminator. In some embodiments, the template RNA comprises an enhanced translation element or a translation enhancing element. In some embodiments, the RNA comprises the human T-cell leukemia virus (HTLV-1) R region. In some embodiments, the RNA comprises a posttranscriptional regulatory element that enhances nuclear export, such as that of Hepatitis B Virus (HPRE) or Woodchuck Hepatitis Virus (WPRE). In some embodiments, in the template RNA, the heterologous object sequence encodes a polypeptide and is coded in an antisense direction with respect to the 5’ and 3’ UTR. In some embodiments, in the template RNA, the heterologous object sequence encodes a polypeptide and is coded in a sense direction with respect to the 5’ and 3’ UTR.

[0336] In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a microRNA binding site. In some embodiments, the microRNA binding site is used to increase the target-cell specificity of a gene modifying system. For instance, the microRNA binding site can be chosen on the basis that is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when the template RNA is present in a non-target cell, it would be bound by the miRNA, and when the template RNA is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by theory, binding of the miRNA to the template RNA may interfere with insertion of the heterologous object sequence into the genome. Accordingly, the heterologous object sequence would be inserted into the genome of target cells more efficiently than into the genome of non-target cells. A system having a microRNA binding site in the template RNA (or DNA encoding it) may also be used in combination with a nucleic acid encoding a gene modifying polypeptide, wherein expression of the gene modifying polypeptide is regulated by a second microRNA binding site, e.g., as described herein, e.g., in the section entitled “Polypeptide component of gene modifying system.”

[0337] In some embodiments, the object sequence may contain a non-coding sequence. For example, the template RNA may comprise a promoter or enhancer sequence. In some embodiments, the template RNA comprises a tissue specific promoter or enhancer, each of which may be unidirectional or bidirectional. In some embodiments, the promoter is an RNA polymerase I promoter, RNA polymerase II promoter, or RNA polymerase III promoter. In some embodiments, the promoter comprises a TATA element. In some embodiments, the promoter comprises a B recognition element. In some embodiments, the promoter has one or more binding sites for transcription factors. In some embodiments, the non-coding sequence is transcribed in an antisense-direction with respect to the 5’ and 3’ UTR. In some embodiments, the non-coding sequence is transcribed in a sense direction with respect to the 5’ and 3’ UTR.

[0338] In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a promoter sequence, e.g., a tissue specific promoter sequence. In some embodiments, the tissue-specific promoter is used to increase the target-cell specificity of a gene modifying system. For instance, the promoter can be chosen on the basis that it is active in a target cell type but not active in (or active at a lower level in) a non-target cell type. Thus, even if the promoter integrated into the genome of a non-target cell, it would not drive expression (or only drive low-level expression) of an integrated gene. A system having a tissue-specific promoter sequence in the template RNA may also be used in combination with a microRNA binding site, e.g., in the template RNA or a nucleic acid encoding a gene modifying protein, e.g., as described herein. A system having a tissue-specific promoter sequence in the template RNA may also be used in combination with a DNA encoding a gene modifying polypeptide, driven by a tissue-specific promoter, e.g., to achieve higher levels of gene modifying protein in target cells than in non-target cells.

[0339] In some embodiments, a heterologous object sequence comprised by a template RNA (or DNA encoding the template RNA) is operably linked to at least one regulatory sequence. In some embodiments, the heterologous object sequence is operably linked to a tissue-specific promoter, such that expression of the heterologous object sequence, e.g., a therapeutic protein, is upregulated in target cells, as above. In some embodiments, the heterologous object sequence is operably linked to a miRNA binding site, such that expression of the heterologous object sequence, e.g., a therapeutic protein, is downregulated in cells with higher levels of the corresponding miRNA, e.g., non-target cells, as above.

[0340] In some embodiments, the template RNA comprises a microRNA sequence, a siRNA sequence, a guide RNA sequence, a piwi RNA sequence.

[0341] In some embodiments, the template RNA comprises a non-coding heterologous object sequence, e.g., a regulatory sequence. In some embodiments, integration of the heterologous object sequence thus alters the expression of an endogenous gene. In some embodiments, integration of the heterologous object sequence upregulates expression of an endogenous gene. In some embodiments, integration of the heterologous object sequence downregulated expression of an endogenous gene.

[0342] In some embodiments, the template RNA comprises a site that coordinates epigenetic modification. In some embodiments, the template RNA comprises an element that inhibits, e.g., prevents, epigenetic silencing. In some embodiments, the template RNA comprises a chromatin insulator. For example, the template RNA comprises a CTCF site or a site targeted for DNA methylation.

[0343] In order to promote higher level or more stable gene expression, the template RNA may include features that prevent or inhibit gene silencing. In some embodiments, these features prevent or inhibit DNA methylation. In some embodiments, these features promote DNA demethylation. In some embodiments, these features prevent or inhibit histone deacetylation. In some embodiments, these features prevent or inhibit histone methylation. In some embodiments, these features promote histone acetylation. In some embodiments, these features promote histone demethylation. In some embodiments, multiple features may be incorporated into the template RNA to promote one or more of these modifications. CpG dinucleotides are subject to methylation by host methyl transferases. In some embodiments, the template RNA is depleted of CpG dinucleotides, e.g., does not comprise CpG nucleotides or comprises a reduced number of CpG dinucleotides compared to a corresponding unaltered sequence. In some embodiments, the promoter driving transgene expression from integrated DNA is depleted of CpG dinucleotides.

[0344] In some embodiments, the template RNA comprises a gene expression unit composed of at least one regulatory region operably linked to an effector sequence. The effector sequence may be a sequence that is transcribed into RNA (e.g., a coding sequence or a non-coding sequence such as a sequence encoding a micro RNA).

[0345] In some embodiments, the object sequence of the template RNA is inserted into a target genome in an endogenous intron. In some embodiments, the object sequence of the template RNA is inserted into a target genome and thereby acts as a new exon. In some embodiments, the insertion of the object sequence into the target genome results in replacement of a natural exon or the skipping of a natural exon.

[0346] In some embodiments, the object sequence of the template RNA is inserted into the target genome in a genomic safe harbor site, such as AAVS1, CCR5, or ROSA26. In some embodiments, the object sequence of the template RNA is inserted into the albumin locus. In some embodiments, the object sequence of the template RNA is inserted into the TRAC locus. In some embodiments, the object sequence of the template RNA is added to the genome in an intergenic or intragenic region. In some embodiments, the object sequence of the template RNA is added to the genome 5’ or 3’ within 0.1 kb, 0.25 kb, 0.5 kb, 0.75, kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 7.5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50, 75 kb, or 100 kb of an endogenous active gene. In some embodiments, the object sequence of the template RNA is added to the genome 5’ or 3’ within 0.1 kb, 0.25 kb, 0.5 kb, 0.75, kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 7.5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50, 75 kb, or 100 kb of an endogenous promoter or enhancer. In some embodiments, the object sequence of the template RNA can be, e.g., 50-50,000 base pairs (e.g., between 50- 40,000 bp, between 500-30,000 bp between 500-20,000 bp, between 100-15,000 bp, between 500-10,000 bp, between 50-10,000 bp, between 50-5,000 bp. In some embodiments, the heterologous object sequence is less than 1,000, 1,300, 1500, 2,000, 3,000, 4,000, 5,000, or 7,500 nucleotides in length.

[0347] The template nucleic acid (e.g., template RNA) component of a gene modifying system described herein typically is able to bind the gene modifying protein of the system. In some embodiments, the template nucleic acid (e.g., template RNA) has a 3’ region that is capable of binding a gene modifying protein. The binding region, e.g., 3’ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying protein of the system. The binding region may associate the template nucleic acid (e.g., template RNA) with any of the polypeptide modules. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with an RNA-binding domain in the polypeptide. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with the reverse transcription domain of the polypeptide (e.g., specifically bind to the RT domain). For example, where the reversetranscription domain is derived from a non-LTR retrotransposon, the template nucleic acid (e.g., template RNA) may contain a binding region derived from a non-LTR retrotransposon, e.g., a 3’ UTR from a non-LTR retrotransposon. In some embodiments a system or method described herein comprises a single template nucleic acid (e.g., template RNA). In some embodiments a system or method described herein comprises a plurality of template nucleic acids (e.g., template RNAs). In some embodiments, when the system comprises a plurality of nucleic acids, each nucleic acid comprises a conjugating domain. In some embodiments, a conjugating domain enables association of nucleic acid molecules, e.g., by hybridization of complementary sequences.

[0348] In some embodiments, the template nucleic acid may comprise one or more UTRs (e.g., a 5’ UTR or a 3’ UTR, e.g., from an R2-type retrotransposon). In some embodiments, the UTR facilitates interaction of the template with the reverse transcriptase domain of the polypeptide. In some embodiments, the template possesses one or more sequences aiding in association of the template with the gene modifying polypeptide. In some embodiments, these sequences may be derived from retrotransposon UTRs. In some embodiments, the UTRs may be located flanking the desired insertion sequence. In some embodiments, a sequence with target site homology may be located outside of one or both UTRs. In some embodiments, the sequence with target site homology can anneal to the target sequence to prime reverse transcription. In some embodiments, the 5’ and / or 3’ UTR may be located terminal to the target site homology sequence. In some embodiments, the gene modifying system may result in the insertion of a desired payload without any additional sequence (e.g., a gene expression unit without UTRs used to bind the gene modifying protein).

[0349] The template nucleic acid (e.g., template RNA) can be designed to result in insertions, mutations, or deletions at the target DNA locus. In some embodiments, the template nucleic acid (e.g., template RNA) may be designed to cause an insertion in the target DNA. For example, the template nucleic acid (e.g., template RNA) may contain a heterologous sequence, wherein the reverse transcription will result in insertion of the heterologous sequence into the target DNA. In other embodiments, the RNA template may be designed to write a deletion into the target DNA. For example, the template nucleic acid (e.g., template RNA) may match the target DNA upstream and downstream of the desired deletion, wherein the reverse transcription will result in the copying of the upstream and downstream sequences from the template nucleic acid (e.g., template RNA) without the intervening sequence, e.g., causing deletion of theintervening sequence. In other embodiments, the template nucleic acid (e.g., template RNA) may be designed to write an edit into the target DNA. For example, the template RNA may match the target DNA sequence with the exception of one or more nucleotides, wherein the reverse transcription will result in the copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations.

[0350] In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). (viii) Methods and Compositions for Modified RNA (e.g., template RNA)

[0351] In some embodiments, an RNA component of the system (e.g., a template RNA, as described herein) comprises one or more nucleotide modifications. In some embodiments, the modification pattern of the template RNA can significantly affect in vivo activity compared to unmodified or end-modified guides. Without wishing to be bound by theory, this process may be due, at least in part, to a stabilization of the RNA conferred by the modifications. Non-limiting examples of such modifications may include 2'-O-methyl (2'-O-Me), 2'-O-(2- methoxyethyl) (2'-O-MOE), 2'- fluoro (2'-F), phosphorothioate (PS) bond between nucleotides, G-C substitutions, and inverted basic linkages between nucleotides and equivalents thereof.

[0352] In some embodiments, the template RNA (e.g., at the portion thereof that binds a target site) comprises a 5' terminus region. In some embodiments, the template RNA does not comprise a 5' terminus region. In some embodiments, the 5' terminus region comprises a 5' end modification. In some embodiments, the template RNA comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the template RNA comprises a 2'-O-(2-methoxy ethyl) (2'-O-moe) modified nucleotide. In some embodiments, the template RNA comprises a 2'-fluoro (2'- F) modified nucleotide. In some embodiments, the template RNA comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the template RNA comprises a 5' end modification, a 3' end modification, or 5' and 3' end modifications. In some embodiments, the 5' end modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the 5' end modification comprises a 2'-O-methyl (2'-O- Me), 2'-O-(2-methoxy ethyl) (2'-O-MOE), and / or 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the 5' end modification comprises at least one phosphorothioate (PS) bond and one or more of a 2'-O-methyl (2'-O- Me), 2'-O-(2-methoxyethyl) (2'-O-MOE), and / or 2'-fluoro (2'-F) modified nucleotide. The end modification may comprise a phosphorothioate (PS), 2'- O-methyl (2'-O-Me), 2'-O-(2- methoxyethyl) (2'-O-MOE), and / or 2'-fluoro (2'-F) modification. Equivalent end modifications are also encompassed by embodiments described herein. In some embodiments, the template RNA comprises an end modification in combination with a modification of one or more regions of the template RNA. In some embodiments, structure- -OMe- -F-RNA, and PS modifications) to a template RNA, for example, as described in Mir et al. Nat Commun 9:2641 (2018) (incorporated by reference herein in its entirety). In some -F-RNAs increases thermal and nuclease stability ofRNA -endo sugar- -OMe at positions where-OH is important for RNA:DNA duplex stability. In some embodiments, structure-guided and systematic approaches (e.g., as described in Mir et al. Nat Commun 9:2641 (2018); incorporated herein by reference in its entirety) are employed to find modifications for the template RNA. In some embodiments, a structure of polypeptide bound to template RNA is used to determine non-protein-contacted nucleotides of the RNA that may then be selected formodifications, e.g., with lower risk of disrupting the association of the RNA with the polypeptide. Secondary structures in a template RNA can also be predicted in silico by software tools, e.g., the RNAstructure tool available at rna.urmc.rochester.edu / RNAstructureWeb (Bellaousov et al. Nucleic Acids Res 41:W471-W474 (2013); incorporated by reference herein in its entirety), e.g., to determine secondary structures for selecting modifications, e.g., hairpins, stems, and / or bulges.

[0353] It is contemplated that it may be useful to employ circular and / or linear RNA states during the formulation, delivery, or gene modifying reaction within the target cell. Thus, in some embodiments of any of the aspects described herein, a gene modifying system comprises one or more circular RNAs (circRNAs). In some embodiments of any of the aspects described herein, a gene modifying system comprises one or more linear RNAs. In some embodiments, a nucleic acid as described herein (e.g., a template nucleic acid, a nucleic acid molecule encoding a gene modifying polypeptide, or both) is a circRNA. In some embodiments, a circular RNA molecule encodes the gene modifying polypeptide. In some embodiments, the circRNA molecule encoding the gene modifying polypeptide is delivered to a host cell. In some embodiments, the circRNA molecule encoding the gene modifying polypeptide is linearized (e.g., in the host cell, e.g., in the nucleus of the host cell) prior to translation. Circular RNAs are described, e.g., at p. 1215-1218 of PCT Pub. No. WO / 2021 / 178720.

[0354] Further included here are compositions and methods for the assembly of full or partial template RNA molecules. Methods of making template RNAs are described, e.g., at p. 1150- 1155 of PCT Pub. No. WO / 2021 / 178720. (ix) Additional Template Features

[0355] In some embodiments, the template (e.g., template RNA) comprises certain structural features, e.g., determined in silico. In embodiments, the template RNA is predicted to have minimal energy structures between -280 and -480 kcal / mol (e.g., between -280 to -300, -300 to -350, -350 to -400, -400 to -450, or -450 to -480 kcal / mol), e.g., as measured by RNAstructure, e.g., as described in Turner and Mathews Nucleic Acids Res 38:D280-282 (2009) (incorporated herein by reference in its entirety).

[0356] In some embodiments, the template (e.g., template RNA) comprises certain structural features, e.g., determined in vitro. In embodiments, the template RNA is sequence optimized, e.g., to reduce secondary structure as determined in vitro, for example, by SHAPE-MaP (e.g.,as described in Siegfried et al. Nat Methods 11:959-965 (2014); incorporated herein by reference in its entirety).In some embodiments, the template (e.g., template RNA) comprises certain structural features, e.g., determined in cells. In embodiments, the template RNA is sequence optimized, e.g., to reduce secondary structure as measured in cells, for example, by DMS-MaPseq (e.g., as described in Zubradt et al. Nat Methods 14:75-82 (2017); incorporated by reference herein in its entirety). (x) Additional Functional Characteristics and Features of gene modifying systems

[0357] A gene modifying system as described herein may, in some instances, be characterized by one or more functional measurements or characteristics. In some embodiments, the DNA binding domain has one or more of the functional characteristics described below. In some embodiments, the RNA binding domain has one or more of the functional characteristics described below. In some embodiments, the endonuclease domain has one or more of the functional characteristics described below. In some embodiments, the reverse transcriptase domain has one or more of the functional characteristics described below. In some embodiments, the template (e.g., template RNA) has one or more of the functional characteristics described below. In some embodiments, the target site bound by the gene modifying polypeptide has one or more of the functional characteristics described below. (xi) Gene modifying Polypeptide a) DNA Binding Domain

[0358] In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with greater affinity than a reference DNA binding domain. In some embodiments, the reference DNA binding domain is a DNA binding domain from R2_BM of B. mori. In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with an affinity between 100 pM – 10 nM (e.g., between 100 pM-1 nM or 1 nM – 10 nM).

[0359] In some embodiments, the affinity of a DNA binding domain for its target sequence (e.g., dsDNA target sequence) is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety).

[0360] In embodiments, the DNA binding domain is capable of binding to its target sequence (e.g., dsDNA target sequence), e.g, with an affinity between 100 pM – 10 nM (e.g., between 100 pM-1 nM or 1 nM – 10 nM) in the presence of a molar excess of scrambled sequence competitor dsDNA, e.g., of about 100-fold molar excess.

[0361] In some embodiments, the DNA binding domain is found associated with its target sequence (e.g., dsDNA target sequence) more frequently than any other sequence in the genome of a target cell, e.g., human target cell, e.g., as measured by ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated herein by reference in its entirety). In some embodiments, the DNA binding domain is found associated with its target sequence (e.g., dsDNA target sequence) at least about 5-fold or 10-fold, more frequently than any other sequence in the genome of a target cell, e.g., as measured by ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010), supra.

[0362] In some embodiments, a gene modifying polypeptide comprises a modification to a DNA-binding domain, e.g., relative to the wild-type polypeptide. In some embodiments, the DNA-binding domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original DNA-binding domain. In some embodiments, the DNA- binding domain is modified to include a heterologous functional domain that binds specifically to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the functional domain replaces at least a portion (e.g., the entirety of) the prior DNA-binding domain of the polypeptide. In some embodiments, a gene modifying polypeptide comprises a modification to an endonuclease domain, e.g., relative to the wild-type polypeptide. In some embodiments, the endonuclease domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original endonuclease domain. In some embodiments, the endonuclease domain is modified to include a heterologous functional domain that binds specifically to and / or induces endonuclease cleavage of a target nucleic acid (e.g., DNA) sequence of interest. b) RNA Binding Domain

[0363] In some embodiments, the RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain. In some embodiments, the reference RNA binding domain is an RNA binding domain from R2_BM of B. mori. In someembodiments, the RNA binding domain is capable of binding to a template RNA with an affinity between 100 pM – 10 nM (e.g., between 100 pM-1 nM or 1 nM – 10 nM). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in cells (e.g., by FRET or CLIP-Seq).

[0364] In some embodiments, the RNA binding domain is associated with the template RNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11):5490-5501 (incorporated by reference herein in its entirety). In some embodiments, the RNA binding domain is associated with the template RNA in cells (e.g., in HEK293T cells) at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019), supra. c) Endonuclease Domain

[0365] In some embodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled dsDNA. In some embodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled dsDNA, e.g., in a cell (e.g., a HEK293T cell). In some embodiments, the frequency of association between the endonuclease domain and the target DNA or scrambled DNA is measured by ChIP-seq, e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated by reference herein in its entirety).

[0366] In some embodiments, the endonuclease domain can catalyze the formation of a nick at a target sequence, e.g., to an increase of at least about 5-fold or 10-fold relative to a non- target sequence (e.g., relative to any other genomic sequence in the genome of the target cell). In some embodiments, the level of nick formation is determined using NickSeq, e.g., as described in Elacqua et al. (2019) bioRxiv doi.org / 10.1101 / 867937 (incorporated herein by reference in its entirety).

[0367] In some embodiments, the endonuclease domain is capable of nicking DNA in vitro. In embodiments, the nick results in an exposed base. In embodiments, the exposed base can be detected using a nuclease sensitivity assay, e.g., as described in Chaudhry and Weinfeld (1995) Nucleic Acids Res 23(19):3805-3809 (incorporated by reference herein in its entirety). In embodiments, the level of exposed bases (e.g., detected by the nuclease sensitivity assay) is increased by at least 10%, 50%, or more relative to a reference endonuclease domain. In some embodiments, the reference endonuclease domain is an endonuclease domain from R2_BM of B. mori.

[0368] In some embodiments, the endonuclease domain is capable of nicking DNA in a cell. In embodiments, the endonuclease domain is capable of nicking DNA in a HEK293T cell. In embodiments, an unrepaired nick that undergoes replication in the absence of Rad51 results in increased NHEJ rates at the site of the nick, which can be detected, e.g., by using a Rad51 inhibition assay, e.g., as described in Bothmer et al. (2017) Nat Commun 8:13905 (incorporated by reference herein in its entirety). In embodiments, NHEJ rates are increased above 0-5%. In embodiments, NHEJ rates are increased to 20-70% (e.g., between 30%-60% or 40-50%), e.g., upon Rad51 inhibition.

[0369] In some embodiments, the endonuclease domain releases the target after cleavage. In some embodiments, release of the target is indicated indirectly by assessing for multiple turnovers by the enzyme, e.g., as described in Yourik at al. RNA 25(1):35-44 (2019) (incorporated herein by reference in its entirety) and shown in Figure 2. In some embodiments, the kexpof an endonuclease domain is 1 x 10-3– 1 x 10-5 min-1 as measured by such methods.

[0370] In some embodiments, the endonuclease domain has a catalytic efficiency (kcat / Km) greater than about 1 x 108s-1M-1in vitro. In embodiments, the endonuclease domain has a catalytic efficiency greater than about 1 x 105, 1 x 106, 1 x 107, or 1 x 108, s-1M-1in vitro. In embodiments, catalytic efficiency is determined as described in Chen et al. (2018) Science 360(6387):436-439 (incorporated herein by reference in its entirety). In some embodiments, the endonuclease domain has a catalytic efficiency (kcat / Km) greater than about 1 x 108s-1M-1in cells. In embodiments, the endonuclease domain has a catalytic efficiency greater than about 1 x 105, 1 x 106, 1 x 107, or 1 x 108s-1M-1in cells.(xii) Reverse Transcriptase Domain a) Target Site and Integration

[0371] In some embodiments, after gene editing, the target site surrounding the integrated sequence contains a limited number of insertions or deletions, for example, in less than about 50% or10% of integration events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. (2020) bioRxiv doi.org / 10.1101 / 645903 (incorporated by reference herein in its entirety). In some embodiments, the target site does not show multiple insertion events, e.g., head-to-tail or head-to-head duplications, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org / 10.1101 / 645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains an integrated sequence corresponding to the template RNA. In some embodiments, the target site does not contain insertions resulting from endogenous RNA in more than about 1% or10% of events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org / 10.1101 / 645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains the integrated sequence corresponding to the template RNA.

[0372] In some embodiments, the target site contains an integrated sequence corresponding to the template RNA. In embodiments, the target site does not comprise sequence outside of the template, e.g., as determined by long-read amplicon sequencing of the target site (for example, as described in Karst et al. bioRxiv doi.org / 10.1101 / 645903 (2020); incorporated herein by reference in its entirety). (xiii) DNA Damage Response

[0373] In some embodiments, modifying a genome of a cell (e.g., a primary cell, e.g., a T cell) using a gene modifying system does not result in activation of the endogenous DNA damage response (DDR) pathway. In some embodiments, modifying a genome of a cell (e.g., a primary cell) using a gene modifying system results in activation of the cell’s endogenous DDR pathway less than in an otherwise similar cell treated with Cas9.

[0374] In some embodiments, modifying a genome of a cell (e.g., a primary cell, e.g., a T cell) using a gene modifying system does not result in activation of the endogenous interferon response. In some embodiments, modifying a genome of a cell using a gene modifying systemresults in activation of the cell’s interferon response less than in an otherwise similar cell treated with a gene modifying system comprising elements from a LINE-1 retrotransposase.

[0375] In some embodiments, the gene modifying polypeptide systems described herein includes a self-inactivating module. The self-inactivating module leads to a decrease of expression of the gene modifying polypeptide, the gene modifying template, or both. Self- inactivating modules are described, e.g., at p. 1200-1201 of PCT Pub. No. WO / 2021 / 178720.

[0376] In some embodiments a polypeptide described herein (e.g., a gene modifying polypeptide) is controllable via a small molecule. In some embodiments, the polypeptide is dimerized via a small molecule. Polypeptides of this type are described, e.g., at p. 1201-1203 of WO / 2021 / 178720. B. Heterologous Gene Modifying Systems

[0377] The conjugates (targeted LNPs) described herein can be formulated to comprise one or more components of a heterologous gene modifying system, or one or more nucleic acids encoding said components. Accordingly, in some embodiments, the payload comprises a heterologous gene modifying system, or one or more nucleic acids encoding the components of the heterologous gene modifying polypeptide. For instance, in some embodiments, the payload comprises a template RNA and an mRNA encoding the heterologous gene modifying polypeptide.

[0378] A heterologous gene modifying system may comprise a heterologous gene modifying polypeptide and a template RNA. The heterologous gene modifying polypeptide may comprise an endonuclease domain, a DNA binding domain, a linker, and a reverse transcriptase domain derived from a retrovirus. The heterologous gene modifying polypeptide may comprise a Cas domain, a linker, and a reverse transcriptase domain derived from a retrovirus. The template RNA compatible with the heterologous gene modifying polypeptide may comprise (e.g., from heterologous gene modifying polypeptide, e.g., the Cas domain of the heterologous gene modifying polypeptide, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence. These components are now described in more detail.

[0379] In some aspects, a heterologous gene modifying polypeptide described herein comprises (e.g., a system described herein comprises a gene modifying polypeptide thatcomprises): 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain, wherein the RT domain is C-terminal of the Cas domain; and a linker disposed between the RT domain and the Cas domain.

[0380] In some embodiments, a heterologous gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 59 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 59 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the N-terminal end of the heterologous gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 59 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the N-terminal end of the heterologous gene modifying polypeptide.

[0381] In some embodiments, a heterologous gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 60 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 60 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the C-terminal end of the heterologous gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 60 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the C-terminal end of the heterologous gene modifying polypeptide. Exemplary C-terminal sequence comprising an NLS AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 60)

[0382] In some embodiments, the heterologous gene modifying polypeptide comprises an amino acid sequence according to SEQ ID NO: 61, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. Exemplary benchmarking sequenceExemplary N-terminal NLS-Cas9 domainYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRID LSQLGGDGG (SEQ ID NO: 44) Exemplary C-terminal sequence comprising an NLS

[0384] AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 45)

[0385] In some embodiments, a heterologous gene modifying polypeptide described herein comprises an RT domain having an amino acid sequence according to Table 6 of International Application WO / 2023 / 039440 (which Table is incorporated herein by reference in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0386] In some embodiments, a heterologous gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a heterologous gene modifying polypeptide comprises an amino acid sequence according to Table 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. Table 5: Selection of exemplary gene modifying polypeptidesTable 6: Full length amino acid sequence corresponding to Table

[0387] In some embodiments, the heterologous gene modifying polypeptide has a sequence disclosed in International Application WO / 2023 / 039424 (which is incorporated by reference herein in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. For instance, in some embodiments, the heterologous gene modifying polypeptide has an RT domain as described in Table 6 of International Application WO / 2023 / 039424 (which table is incorporated by reference herein in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. In some embodiments, a heterologous gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 10 of International Application WO / 2023 / 039424 (which Table is incorporated herein by reference in its entirety). In some embodiments, the heterologous gene modifying polypeptide has a sequence according to Table T2 of International Application WO / 2023 / 039424 (which table is incorporated by reference herein in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. In some embodiments, the heterologous gene modifying polypeptide has a sequence according to Table A1 of International Application WO / 2023 / 039424 (which table is incorporated by reference herein in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0388] In some embodiments, a gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., an M-MLV RT, e.g., comprising the following sequence: TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTP VSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLR EVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWR DPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAAT SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKE55), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto.

[0389] In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, one or more mutations, e.g., selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, e.g., a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and W313F. In some embodiments, an M-MLV RT used herein comprises the mutations D200N, L603W, T330P, T306K and W313F. In embodiments, the mutant M-MLV RT comprises the following amino acid sequence:

[0390] Exemplary gene modifying system comprises mutant M-MLV RT region:

[0391] In some embodiments, a template RNA molecule for use in the system comprises, primer binding site (PBS) sequence. In some embodiments: (1) Is a gRNA spacer of ~18-22 nt, e.g., is 20 nt (2) Is a gRNA scaffold comprising one or more hairpin loops, e.g., 1, 2, of 3 loops for associating the template with a Cas domain, e.g., a nickase Cas9 domain. In some(3) In some embodiments, the heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20- 30, 30-40, 40-50, 50-60, 60-70, or 70-80 nt or, 80-90 nt in length. (4) In some embodiments, the PBS sequence that binds the target priming sequence after nicking occurs is e.g., 3-20 nt, e.g., 7-15 nt, e.g., 12-14 nt. In some embodiments, the PBS sequence has 40-60% GC content. V. METHODS OF TREATMENT

[0392] In some embodiments, an LNP described herein is used to treat a disease, disorder, or condition. In some embodiments, an LNP described herein, or component or portion thereof, is used to treat a disease, disorder, or condition listed in any of Tables 3-8. In some embodiments, an LNP described herein is used to treat a hematopoietic stem cell (HSC) disease, disorder, or condition, e.g., as listed in Table 7. In some embodiments, an LNP described herein is used to treat a kidney disease, disorder, or condition, e.g., as listed in Table 8. In some embodiments, an LNP described herein is used to treat a liver disease, disorder, or condition, e.g., as listed in Table 9. In some embodiments, an LNP described herein is used to treat a lung disease, disorder, or condition, e.g., as listed in Table 10. In some embodiments, an LNP described herein is used to treat a skeletal muscle disease, disorder, or condition, e.g., as listed in Table 11. In some embodiments, an LNP described herein is used to treat a skin disease, disorder, or condition, e.g., as listed in Table 12.Table 7: Exemplary diseases treated with LNPs of the disclosureTable 8: KidneyTable 9: LiverTable 10: LungPrimary ciliary dyskinesia DNAI1Table 11: Skeletal muscleTable 12: Skin

[0393] In some embodiments, a conjugate described herein is delivered to a tissue or cell from the cerebrum, cerebellum, adrenal gland, ovary, pancreas, parathyroid gland, hypophysis, testis, thyroid gland, breast, spleen, tonsil, thymus, lymph node, bone marrow, lung, cardiac muscle, esophagus, stomach, small intestine, colon, liver, salivary gland, kidney, prostate, blood, or other cell or tissue type. In some embodiments, a conjugate described herein is used to treat a disease, such as a cancer, inflammatory disease, infectious disease, genetic defect, or other disease. A cancer can be cancer of the cerebrum, cerebellum, adrenal gland, ovary,pancreas, parathyroid gland, hypophysis, testis, thyroid gland, breast, spleen, tonsil, thymus, lymph node, bone marrow, lung, cardiac muscle, esophagus, stomach, small intestine, colon, liver, salivary gland, kidney, prostate, blood, or other cell or tissue type, and can include multiple cancers.

[0394] In some embodiments, an LNP, e.g., targeted LNP, disclosed herein is delivered ex vivo to isolated cells (e.g., T cells, HSCs, or liver cells, e.g., hepatocytes) of a subject (e.g., human patient) in need thereof. In some such embodiments, T cells or HSCS are first collected from the bone marrow of a subject. Following isolation of the desired cell population, the LNPs of the disclosure can be mixed with the cells, thereby resulting in effective transduction of the payload (e.g., gene modifying system) into the T cells or HSCs. The modified T cells or HSCs can be administered to the patient.

[0395] In some embodiments where the cells are T cells, the cells can be activated prior to ex vivo delivery of the LNPs or conjugates. In other embodiments, the T cells are not activated prior to the delivery of the LNPs or conjugates ex vivo to T cells.

[0396] In other embodiments, an LNP, e.g., targeted LNP, as disclosed herein can be introduced into cells, tissues and / or multicellular organisms. In other embodiments, the LNPs, e.g., targeted LNPs, are delivered to the cells via mechanical means or physical means.

[0397] Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

[0398] In some embodiments, the LNP described herein, e.g., targeted LNPs, are administered in vivo to a patient in need thereof. The high level of transduction and protein expression following in vivo administration allows for sufficient levels of transduction and expression of proteins (e.g., gene modifying proteins), and hence can be used to treat various diseases including but not limited to the diseases listed in Tables 7-12.

[0399] In some embodiments, an LNP, e.g., targeted LNP, described herein is delivered to a tissue or cell from or in the bone marrow. In some embodiments, an LNP, e.g., targeted LNP, described herein is delivered to a tissue or cell from or in the blood or lymph nodes. In some embodiments, an LNP, e.g., targeted LNP, described herein is delivered to a tissue or cell from or in the liver. In some such embodiments, the LNP, e.g., targeted LNP, is administered byparenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, epidural, intracerebral, intracerebroventricular, epicutaneous, nasal, intra-arterial, intra- articular, intracavernous, intraocular, intraosseous infusion, intraperitoneal, intrathecal, intrauterine, intravaginal, intravesical, perivascular, or transmucosal administration).

[0400] In some embodiments, an LNP, e.g., targeted LNP, of the disclosure delivers a therapeutic agent, as described above, to T cells, to HSCs (e.g., LT-HSCs), or to liver cells (e.g., hepatocytes) of a patient following in vivo administration. In some embodiments, an LNP, e.g., targeted LNP, of the disclosure delivers a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, to T cells, to HSCs (e.g., LT-HSCs), or to liver cells (e.g., hepatocytes) of a patient following in vivo administration. In some embodiments, an LNP, e.g., targeted LNP, of the disclosure delivers a gene modifying system to T cells, to HSCs (e.g., LT-HSCs), or to liver cells (e.g., hepatocytes) of a patient following in vivo administration. In some embodiments, an LNP, e.g., targeted LNP, of the disclosure delivers gene editing components to T cells, to HSCs (e.g., LT-HSCs), or to liver cells (e.g., hepatocytes) of a patient following in vivo administration. Exemplary gene editing systems are described above. 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 gene editing of the T cells, HSCs (e.g., LT-HSCs), or liver cells (e.g., hepatocytes).

[0401] The high level of in vivo gene editing following administration of the LNPs, e.g., targeted LNPs, of the disclosure enables effective treatment of particular diseases such as, e.g., cancer, -hemoglobinopathies, primary immunodeficiencies, and metabolic disorders. In some embodiments, the LNPs, e.g., targeted LNPs, of the disclosure can be used to treat sickle cell diseases. In other embodiments, the LNP, e.g., targeted LNPs, of the disclosure can be used to treat beta thalassemia. Targeting Moieties for treating treat a hematopoietic stem cell (HSC) disease, disorder, or condition

[0402] 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, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine.

[0403] 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.

[0404] 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

[0405] 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.

[0406] 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

[0407] In some embodiments, the CD33 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:86, and / or a light chain variable region comprising 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).

[0408] 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).

[0409] 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%) sequenceidentity 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 acid sequence 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

[0410] 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.

[0411] 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

[0412] 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 thereofthat 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., Novartis Pharmaceuticals), GZQ167 (e.g., Novartis Pharmaceuticals), MGTA-117 (e.g., MagentaTherapeutics), Barzolvolimab ( , e.g., CellDex Therapeutics), D13A2 (e.g., CellSignaling 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.

[0413] 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 sequenceof 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 a light 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

[0414] 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:500, 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:501. In some embodiments, the CD117 targeting moiety comprises a CDR-H1 comprising the amino acid sequence FTFSDADMD (SEQ ID NO:504) or DADMD (SEQ ID NO:392), a CDR-H2 comprising the amino acid sequence RNKAGSYTTEYAASVKG (SEQ ID NO:505), a CDR-H3 comprising the amino acid sequence AREPKYWIDFDL (SEQ ID NO:506), a CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO:507), a CDR-L2 comprising the amino acid sequenceAASSLQS (SEQ ID NO:508), and a CDR-L3 comprising the amino acid sequence QQSYIAPYT (SEQ ID NO:509).

[0415] 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:502, 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:503. 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).

[0416] 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 ...

Claims

CLAIMS 1. A compound of formula (IA’):or a pharmaceutically acceptable salt thereof, wherein: L1is C1-8alkylene, wherein one or more alkylene units is optionally replaced with a group selected from -O-, -NRy-, and -S-; L2is -C(=O)O-, or -C(=O)OCH2-; R1is C1-10alkyl; R2is C4-10alkyl or -CH2S-(C4-10alkyl); m is 1, 2, 3, 4 or 5; R5and R6are each independently C1-4alkyl or R5and R6are taken together to form a 4- to 12-membered heterocyclyl; R7is C1-4alkyl; X is -O- or -CH2-; each Ryis H or C1-4alkyl; and Y is a lipophilic tail, branched or unbranched.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein:L3is C1-8alkylene; L4is -C(=O)O-, or -C(=O)OCH2-; R3is C4-10alkyl; and R4is C2-10alkyl or -CH2S-(C4-10alkyl).

3. The compound of claim 1 or 2, wherein the compound is a compound of formula (II):pharmaceutically acceptablesalt thereof.

4. The compound of any of claims 1-3, wherein the compound is a compound of formula (III):pharmaceutically acceptablesalt thereof.

5. The compound of any of claims 1-3, wherein the compound is a compound of formula (IV):pharmaceutically acceptable salt thereof.

6. A compound having the structure:.

7. A compound having the structure:.

8. A compound having the structure:.

9. A compound having the structure:.

10. A compound having the structure:.

11. A compound having the structure:

12. A lipid nanoparticle (LNP) comprising a compound of any one of claims 1-11.

13. The LNP of claim 12, further comprising a targeting moiety.

14. The LNP of claim 13, wherein the targeting moiety is an antibody.

15. The LNP of claim 13, wherein the targeting moiety is an scFv.

16. The LNP of claim 13, wherein the targeting moiety is a VHH domain.

17. The LNP of claim 13, wherein the targeting moiety is a nanobody.

18. The LNP of claim 13, wherein the targeting moiety is a Fab fragment.

19. The LNP of any one of claims 13-18, wherein the targeting moiety binds to an antigen present on a hematopoietic stem cell (HSC).

20. The LNP of any one of claims 13-18, wherein the targeting moiety binds to an antigen present on an immune cell.

21. The LNP of claim 20, wherein the immune cell is a T cell.

22. The LNP of any one of claims 13-18, wherein the targeting moiety binds to an antigen on a liver cell (e.g., a hepatocyte).

23. The LNP of claim 22, wherein the targeting moiety is a N-acetylgalactosamine (GalNAc).

24. A lipid nanoparticle (LNP) for delivery of a therapeutic agent to cells in vivo, the LNP comprising: an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from the lipids in Table 1; and a therapeutic agent encapsulated within the LNP.

25. The LNP of claim 24, wherein the ionizable lipid has the structure:.

26. The LNP of claim 24, wherein the ionizable lipid has the structure:.

27. The LNP of claim 24, wherein the ionizable lipid has the structure:.

28. The LNP of claim 24, wherein the ionizable lipid has the structure:.

29. The LNP of claim 24, wherein the ionizable lipid has the structure:.

30. The LNP of claim 24, wherein the ionizable lipid has the structure:.

31. The LNP of any one of claims 12-30, wherein the therapeutic agent comprises at least one nucleic acid molecule.

32. The LNP of claim 31, wherein the nucleic acid molecule comprises an mRNA molecule or a DNA molecule.

33. The LNP of claim 31, wherein the nucleic acid molecule comprises an mRNA molecule.

34. The LNP of claim 31, wherein the nucleic acid molecule comprises a siRNA or miRNA molecule.

35. The LNP of claim 31, wherein the therapeutic agent comprises two nucleic acid molecules.

36. The LNP of claim 35, wherein the two nucleic acid molecules are RNA molecules.

37. The LNP of claim 35, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises an RNA molecule.

38. The LNP of claim 35, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises a guide RNA.

39. The LNP of claim 35, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises a template RNA.

40. The LNP of claim 35, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises a DNA molecule.

41. The LNP of any one of claims 31-40, wherein the at least one nucleic acid molecules encode at least one component for altering a genome.

42. The LNP of claim 35, wherein the at least one component comprises an mRNA encoding a CRISPR-associated nuclease (Cas) and a guide RNA.

43. The LNP of claim 42, wherein the Cas is a Cas9 (e.g., spCas9) or a Cas12 (e.g., Cas12a).

44. The LNP of any one of claims 24-43, wherein the therapeutic agent comprises a gene modifying system.

45. The LNP of any one of claims 24-43, wherein the therapeutic agent comprises a heterologous gene modifying system.

46. The LNP any one of claims 24-45, wherein the mol% of ionizable lipids in the LNP ranges from about 35% to about 60%.

47. The LNP of claim 46, wherein the mol% of ionizable lipids in the LNP ranges from about 40% to about 50%.

48. The LNP of claim 46, wherein the mol% of ionizable lipids in the LNP ranges from about 45% to about 50%.

49. The LNP of any one of claims 24-48, wherein the helper lipid is selected from 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.

50. The LNP of claim 49, wherein the helper lipid is DSPC.

51. The LNP of claim 49, wherein the helper lipid is sphingomeylin.

52. The LNP of claim 51, wherein the sphingomeylin has a head group selected from phosphocholine, phosphoethanolamine and ceramide.

53. The LNP of claim 52, wherein the sphingomeylin is egg sphingomeylin.

54. The LNP of any of claims 24-53, wherein the mol% of helper lipid is from about 5% to about 12% of the lipid component.

55. The LNP of any of claims 24-53, wherein the mol% of helper lipid is from about 6% to about 10% of the lipid component.

56. The LNP of any one of claims 24-53, wherein the mol% of helper lipid is from about 18% to about 32% of the lipid component.

57. The LNP of any one of claims 13-56, further comprising a PEGylated lipid., 58. The LNP of claim 57, 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.

59. The LNP of claim 57 or 58, wherein the pegylated lipid is present at about 0.05 mol% to about 2.5 mol% of the lipid component.

60. A method of treating and / or delaying the progression of a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an LNP of any one of claims 24-59.

61. The method of claim 60, wherein the disease is selected from any one of the diseases in Tables 7-12.

62. The method of claim 61, wherein the disease is Phenylketonuria (PKU).

63. The method of claim 61, wherein the disease is alpha-1 antitrypsin deficiency (AATD)or sickle cell disease (SCD).

64. A conjugate for delivery of a therapeutic agent to cells in vivo, the conjugate comprising: a lipid nanoparticle (LNP) comprising an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from the lipids in Table 1; a plurality of targeting moieties conjugated to the LNP; and a therapeutic agent encapsulated within the LNP.

65. The conjugate of clam 64, wherein the ionizable lipid has the structure:.

66. The conjugate of clam 64, wherein the ionizable lipid has the structure:.

67. The conjugate of clam 64, wherein the ionizable lipid has the structure:.

68. The conjugate of clam 64, wherein the ionizable lipid has the structure:.

69. The conjugate of clam 64, wherein the ionizable lipid has the structure:.

70. The conjugate of clam 64, wherein the ionizable lipid has the structure:.

71. The conjugate of any one of claims 64-70, wherein the plurality of targeting moieties bind to an antigen on the surface of an HSC.

72. The conjugate of claim 71, wherein the antigen is selected from CD33, CD34, CD38, CD43, CD46, CD49d, CD50, CD59, CD71, CD105, CD117, CD123, CD164, CD184.

73. The conjugate of claim 72, wherein the plurality of targeting moieties target CD33.

74. The conjugate of claim 72, wherein the plurality of targeting moieties target CD34.

75. The conjugate of claim 72, wherein the plurality of targeting moieties target CD38.

76. The conjugate of claim 72, wherein the plurality of targeting moieties target CD43.

77. The conjugate of claim 72, wherein the plurality of targeting moieties target CD46.

78. The conjugate of claim 72, wherein the plurality of targeting moieties target CD49d.

79. The conjugate of claim 72, wherein the plurality of targeting moieties target CD50.

80. The conjugate of claim 72, wherein the plurality of targeting moieties target CD59.

81. The conjugate of claim 72, wherein the plurality of targeting moieties target CD71.

82. The conjugate of claim 72, wherein the plurality of targeting moieties target CD105.

83. The conjugate of claim 72, wherein the plurality of targeting moieties target CD117.

84. The conjugate of claim 72, wherein the plurality of targeting moieties target CD123.

85. The conjugate of claim 72, wherein the plurality of targeting moieties target CD164.

86. The conjugate of claim 72, wherein the plurality of targeting moieties target CD184.

87. The conjugate of any one of claims 64-70, wherein the plurality of targeting moieties bind to an antigen on the surface of a T cell.

88. The conjugate of claim 87, wherein the antigen is selected from CD2, CD3, CD4, CD5, CD7, CD8 and CD28.

89. The conjugate of claim 88, wherein the plurality of targeting moieties target CD2.

90. The conjugate of claim 88, wherein the plurality of targeting moieties target CD3.

91. The conjugate of claim 88, wherein the plurality of targeting moieties target CD4.

92. The conjugate of claim 88, wherein the plurality of targeting moieties target CD5.

93. The conjugate of claim 88, wherein the plurality of targeting moieties target CD7.

94. The conjugate of claim 88, wherein the plurality of targeting moieties target CD8.

95. The conjugate of claim 88, wherein the plurality of targeting moieties target CD28.

96. The conjugate of any one of claims 64-70, wherein the plurality of targeting moieties bind to an antigen on the surface of liver cells.

97. The conjugate of claim 96, wherein the plurality of targeting moieties are N- acetylgalactosamines (GalNAcs).

98. The conjugate of claim 96 or claim 97, wherein the antigen on the surface of the liver cells is asialoglycoprotein receptor (ASGPR).

99. The conjugate of any one of claims 64-98, wherein the plurality of targeting moieties comprises antibodies or fragments thereof.

100. The conjugate of any one of claims 64-98, wherein the plurality of targeting moieties comprises Fab fragments.

101. The conjugate of any one of claims 64-98, wherein the plurality of targeting moieties comprises VHH domains.

102. The conjugate of any one of claims 64-98, wherein the plurality of targeting moieties comprises single chain variable fragments (scFv).

103. The conjugate of any one of claims 64-102, wherein the therapeutic agent comprises at least one nucleic acid molecule.

104. The conjugate of claim 103, wherein the nucleic acid molecule comprises an mRNA molecule or a DNA molecule.

105. The conjugate of claim 103, wherein the nucleic acid molecule comprises an mRNA molecule.

106. The conjugate of claim 103, wherein the nucleic acid molecule comprises a siRNA or miRNA molecule.

107. The conjugate of claim 103, wherein the therapeutic agent comprises two nucleic acid molecules.

108. The conjugate of claim 107, wherein the two nucleic acid molecules are RNA molecules.

109. The conjugate of claim 108, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises an RNA molecule.

110. The conjugate of claim 108, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises a guide RNA.

111. The conjugate of claim 108, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises a template RNA.

112. The conjugate of claim 103, wherein one nucleic acid comprises an mRNA molecule and one nucleic acid comprises a DNA molecule.

113. The conjugate of any one of claims 107-112, wherein the at least one nucleic acid molecules encode at least one component for altering a genome.

114. The conjugate of claim 108, wherein the at least one component comprises an mRNA encoding a CRISPR-associated nuclease (Cas) and a guide RNA.

115. The conjugate of claim 114, wherein the Cas is a Cas9 (e.g., spCas9) or a Cas12 (e.g., Cas12a).

116. The conjugate of any one of claims 107-115, wherein the therapeutic agent comprises a gene modifying system.

117. The conjugate of any one of claims 107-115, wherein the therapeutic agent comprises a heterologous gene modifying system.

118. The conjugate of any one of claims 64-117, wherein the mol% of ionizable lipids in the LNP ranges from about 35% to about 60% of the lipid component.

119. The conjugate of claim 118, wherein the mol% of ionizable lipids in the LNP ranges from about 40% to about 50% of the lipid component.

120. The conjugate of claim 118, wherein the mol% of ionizable lipids in the LNP ranges from about 45% to about 50% of the lipid component.

121. The conjugate of any one of claims 64-120, wherein the helper lipid is selected from 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.

122. The conjugate of claim 121, wherein the helper lipid is DSPC.

123. The conjugate of claim 121, wherein the helper lipids is sphingomeylin.

124. The conjugate of claim 123, wherein the sphingomeylin has a head group selected from phosphocholine, phosphoethanolamine and ceramide.

125. The conjugate of claim 124, wherein the sphingomeylin is egg sphingomeylin.

126. The conjugate of any of claims 64-125, wherein the mol% of helper lipid in the conjugate is from about 5% to about 12% of the lipid component.

127. The conjugate of any one of claims 64-126, wherein the LNP further comprises at least one a PEGylated lipid.

128. The conjugate of claim 127, 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.

129. The conjugate of claim 127 or claim 128, wherein the PEGylated lipid is DSPE.

130. The conjugate of any of claims 127-129, wherein the PEGylated lipid is present at about 0.05 mol% to about 2.5 mol% of the lipid component of the LNP.

131. The conjugate of any of claims 127-130, wherein the plurality of targeting moieties are conjugated to the LNP through the at least one PEGylated lipid .

132. The conjugate of claim 127, wherein the LNP comprises a first PEGylated lipid and a second PEGylated lipid.

133. The conjugate of claim 132, wherein the first 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.

134. The conjugate of claim 132 or claim 133, wherein the first PEGylated lipid is present at about 0.25 mol% to about 1.0 mol% of the lipid component of the LNP..

135. The conjugate of claim 133 or claim 134, wherein plurality of targeting moieties are conjugated to the LNP through the first PEGylated lipid.

136. The conjugate of any of claims 132-135, wherein the second PEGylated lipid comprises one or two C16 (palmitoyl) PEG lipid anchors.

137. The conjugate of any one of claims 64-136, further comprising one or more cholesterol molecules.

138. The conjugate of claim 137, wherein the cholesterol molecules are selected from beta- sitoesterol, hydroxycholesterol, stigmastanol, and any combination thereof.

139. The conjugate of claim 137 or claim 138, wherein the molar ratio of cholesterol molecules in the LNP ranges from about 35% to about 55% of the lipid component.

140. A method of treating and / or delaying the progression of a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate of any one of claims 64-139.

141. The method of claim 140, wherein the disease is selected from any one of the diseases in Tables 7-12.

142. The method of claim 141, wherein the disease is PKU.

143. The method of claim 140, wherein the disease is alpha-1 antitrypsin deficiency (AATD).

144. The method of claim 140, wherein the disease is sickle cell disease (SCD).